Expanding Indications For The New Macrolides, Azalides, and Streptogramins (Infectious Disease and Therapy)

Expanding Indications for the New Macrolides, Azalides, and Streptogramins edited by Stephen H. Zinner Brown Univenity Rhode Island Hospital Roger Williams Medical Center Providence, Rhode Island

Lowell S. Young Kuzell Institutefor Arthritis and Infctious Diseases California Pacific Medical Center San Francisco, California

Jacques F. Acar Fondation HSpital Saint-Joseph Paris, France

Harold C. Neu College Physicians &' Strtgeons Columbia Univenity N m York, Neprlr York

MARCELDEKKER, INC. D E K K E R

NEWYORK BASEL HONGKONG

Library of Congress Cataloging-in-Publication Data Expanding indications for the new macrolides, azalides, and streptogramins / edited by Stephen H. Zinner... [et al.]. p. cm. - (Infectious disease and therapy ;v. 21) (Infectious disease and therapy ;v. 21) “The Third International Conference on Macrolides, Azalides, and Streptogramins (ICMAS111) was held January 24-26,1996, in Lisbon, Portugal”-Pref. Includes index. ISBN 0-8247-0056-2 (hardcover :alk.paper), ISBN 0-8247-0141-0 (soficover .:alk. paper) 1. Macrolide antibiotics-Congresses.2. Antibiotics-Congresses. I. Zinner, StephenH. 11. International Conference on the Macrolides,halides, and Streptogramins (3rd: 1996: Lisbon, Portugal) 111. Series. IV. Series: Infectious disease and therapy; v. 21. [DNLM: 1. Antibiotics, Macrolide-congresses. 3. Virginiamycincongresses. W1IN406HMNv.2119971 RM666.M25E97 1997 615’.3294c21 DNLMDLC for Libraryof Congress 97-12233 CIP The publisher offers discountson this book when ordered in bulk quantities. For more information, write to Special SalesProfessional Marketing at the address below. This book is printed on acid-free paper. Copyright

1997 by Marcel Dekker, Inc. All Rights Reserved.

Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher. Marcel Dekker, Inc. 270 Madison Avenue, New York, New York 10016 Current printing (last digit): 10987654321 PRINTED IN THE UNITED STATES OF AMERICA

Series Introduction

Marcel Dekker, Inc., has for many years specialized in the publication of high-quality monographs in tightly focused areas in a variety of medical disciplines. These have been great valueto both the practicing physician and the research scientist as sources of detailed andup-to-date information presented in an attractive format. During the last decade,there has been a veritable explosion in knowledge inthe various fields relatedto infectious diseases and clinical microbiology. Antimicrobial resistance, antibacterial andantiviralagents,AIDS, Lyme disease,infectionsinimmunocompromised patients, and parasitic diseases are but a fewof the areas in which an enormous amount significant work has been published. The Infectious Disease and Therapy series covers carefully chosen topics that should be of interest and valueto the practicing physician,the clinical microbiologist, and the research scientist. Brian E. Scully, M . B., B.Ch. Harold C. Neu, M.D.

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Preface

The Third International Conference on Macrolides, Azalides, and Streptogramins (ICMAS 111) was held January 24-26, 1996, in Lisbon, Portugal. This meetingattracted over 1300 scientists, physicians, and clinical investigators from all over the world. The program included state of the art lectures, roundtable discussions, eight workshop symposia, research updates fromindustry,andover 300 poster presentations. Since the first ICMAS meeting in January, 1992, enormous progress has been made the in research and development ofnew compoundsandtheirapplication to clinical medicine. This volume includes plenary papers, summariesof the workship symposia, and selectedextended.abstractsfrom the meeting. The macrolide antibiotics were amongthe earliest antibioticalternatives to penicillin. Erythromycin has certainly maintained an important place in the worldwide therapeutic armamentarium. Certain therapeutic disadvantages of erythromycin were overcome by the introduction of the new macrolides and azalides, clarithromycin, roxithromycin, and azithromycin.Theseagentshaveprovidedexcellentintracellularantibacterial activity-with a broader bacterial spectrum than erythromycin-and considerably fewer unwanted side effects. The streptogramins, currently used extensively in some countries, haveattracted a tremendous levelof interest. Quinupristiddalfopristin,the newest streptogramin, has important activity against staphylococci resistant to methicillin andEnferococcwsfueciurnresistant to vancomycin. The latest clinical and microbiological data on this and related agentsare summarized in this volume. As resistant bacteria continue to challenge clinicians worldwide, additional antibioticswill surely be needed. The ketolide antibiotics, introduced in this volume, provide activity against penicillin-resistant pneumococci as V

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Preface

well asother important gram-positive and gram-negative pathogens. Several other uses for macrolide and azalide type antibiotics have been discovered in recent years. The nonbacterial effectsof macrolideshzalides on immunity and on theproduction of mucoid bacterial products were discussed at this conference andare included in this book. This conference provided an opportunityfor the exchange of scientific informationabout laboratory and clinical research among the pharmaceutical industry and clinical and laboratory investigators. Many new aspects of the use of macrolides, azalides, and streptogramins were presented and are discussed. Through the active discussion at this meeting we recognize the major accomplishments with these compounds and also the new problems on the horizon to challenge basic and clinical investigators in industry and academe. Interest in the macrolide, azalide, streptogramin, and new ketolide compounds undoubtedlywill increase as we struggle to maintain our advantage over bacterial pathogens. Many of these challenges are set forth within these pages and we look forward to pursuing their solutions in future, similar meetings.The organizers are grateful formajor contributions fromAbbott Laboratories, Hoechst Marion Roussel, Pfizer, Inc., and Rh6ne-Poulenc Rorer, which made possible the organization of ICMAS 111. Other support from Pliva Pharmaceuticals and Eli Lilly is greatly appreciated. The editors are grateful to our General Secretariat, Ann Wallace, for her style, substance, and tireless efforts on behalf ICMAS. Stephen H. Zinner Lowell S. Young. Jacques F. Acar Harold C. Neu

Contents

Series Introduction Preface Contributors to Plenary Sessions and Workshops

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PLENARY SESSIONS 1. Overview of the Clinical Use of Macrolides and Streptogramins Roger G. Finch 2. Postantibiotic Effects and the Dosing of Macrolides, Azalides, and Streptogramins William A . Craig Ketolides:NewSemisynthetic14-MemberedRing Macrolides Andrt! Bryskier, Constantin Agouridas, and Jean-FranCois Chantot 4. Streptogramins: From Parenteral to Oral Daniel H . Bouanchaud

51

Azalides: Basic and Clinical Research Michael W; Dunne

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7. Nonantibiotic Effects of Macrolide Antibiotics: Suppression of the Bacterial Glycocalyx of Pseudomonas aeruginosa Isolates from Patientswith Cystic Fibrosis Charles W. Stratton

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8. Effects of Macrolides on Leukocytes and Inflammation Marie-Thdr2se Labro

101

9. Animal Uses of Macrolides and Related Antibiotics Jean-Pierre Lafont, Elisabeth Chaslus-Dancla, and Jean-Louh Martel

117

10. The New Macrolides for Treatment of Pediatric Infections: Roundtable Discussion George H . Mecracken, Jr., Urs B. Schaad, and M. J. Tarlow The Molecular Mechanism of Action of Streptogramins and Related Antibiotics C. Cocito, M. Di Giambattista, E. Nyssen, and l? Vannuflel

12. Early Clinical Results withQuinupristinlDalfopristin for the Therapy of Bacteremia Due to Resistant Gram-positive Bacteria Robert C. Moellering, Jr., and Sharon L. Cenvinka 13. Drug Interactions of Macrolides and halides Ethan Rubinstein and Shlomo Segev 14. Macrolides, halides, and Streptogramins in Treatment of Opportunistic Infections in Immunocompromised Patients Jack S. Remington

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WORKSHOPS

15. Legionella and Spirochetes I. M. Hoepelman and Daniel M. Musher

207

16. Mycoplasma and Chlamydia J. Thomas Grayston and Pentti Huovinen

219

17. Staphylococci, Streptococci, and Pneumococci Jacques E Acar and Josd Melo-Cristino

225

18. Pharmacokinetics and Pharmacodynamics Charles H . Nightingale and Fritz Sorgel

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Contents Campylobacter and Helicobacter pylori Jean-Paul Butzler and Francis Mkgraud

Haemophilus influenzae, Enterococci, and Anaerobes Vincent T. Andriole and Ian Phillips The Newer Macrolides, Azalides, and Streptogramins for the Management of HIV-Associated Opportunistic Infections Kenneth H. Mayer and J. Allen McCutchan 22. Special Pathogens John E. McGowan, Jr., and Joel Ruskin EXTENDED ABSTRACTS

I. NEW AGENTS Ketolides: A New Class of Macrolide Antibacterials-Structural Characteristics and Biological Properties of RU 004 Constantin Agouridas, X Benedetti, A. Bonnefoy, P. Collette, A. Denis, P. Mauvais, G. Labbe, and Jean-Francois Chantot Isolation of an Antifungal Macrolide from Soil Sample Nocardioides Strain: Production and Structure Elucidation K Loppinet, L. Hilali, N. Youssef, R. Bonaly, and C. Finance

JI. LEGIONELLA, SPIROCHETES Azithromycin in the Treatment of Community-Acquired Legionnaires’ Disease I. Kuzman, S. Schonwald, and . l &.dig Temperature Dependence of MIC and MBC of Roxithromycin Against Borrelia burgdo$eri In Vitro I. Wendelin, R. Gasser, and E. C. Reisinger Tolerability of Treatment with g of Azithromycin Given in 5 Days Franc Strle, Vera Maraspin, Stanka Lotrit-Furlan, and JoZe Cimperman Azithromycin in the Treatment of Erythema Migrans J. GoriSek and J. Rogl

Contents Follow-up Study of Patients with Syphilis Treated with Azithromycin A. L. Mashkilleyson and M. A. Gomberg

m. MYCOPLASMA, CHLAMYDIA A Comparison of the In Vitro Sensitivity of Chlamydia pneumoniae to Macrolides and a New Benzoxazinorifamycin, KRM-1648 C.-C. Kuo, J Thomas Grayston, T. Hidaka, and L. M. Rose Susceptibilities to Azithromycin of Isolates of Chlamydia pneumoniae from Patients with Community-Acquired Pneumonia l? M. Roblin, N. Sokolovskaya, and M. R. Hammerschlag Azithromycin in Control of Trachoma Julius Schachter Microbiologic Efficacy of Azithromycin forthe Treatment of Community-Acquired Pneumonia Due to Chlamydia pneumoniae in Children M. R. Hammerschlag, P. M. Roblin, Michael Campbell, Antonia Kolokathis, M. Powell, and the Pediatric Pneumonia Study Group Community-Acquired Pneumonia in Children: Underestimation of Mycoplasma Infection Efficacy and of Macrolides Dominique Gendrel, Josette Raymond, Florence Moulin, JeanLuc Iniguez, Sophie Ravilly, Pierre Lebon, and Gabriel Kalija

Ureaplasma urealyticumIsolations from Young Children with Respiratory Sharon A. Poulin, Ruth B. Kuna'sin, and Rita D. DeLollis

Asthma? Clarithromycin PreventCould Rita D. DeLollis, Ruth B. Kuna'sin, and Sharon A. Poulin Azithromycin in the Treatment of Chlamydia trachomatis Infection Dajek Single-Dose Azithromycin inthe Treatment of Genital Chlamydial V: Ferianec, K. Holoman, J. Chmurny, V: Grba, and F. Stano

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Contents Single-Dose Azithromycin Treatment: A Solution for Uncomplicated Lower Genital Tract Chlamydial Infection B. Kobal Report of a Multicenter Clinical Evaluationof Azithromycin in the Treatment of Nongonococcal Urethritis Men in M. Urban, J. Flek, V. Herman, M. Chaloupka, B. Krhny, J. Klamo, D. Pactk, and P. Tomdtik Efficacy of Roxithromycin in Urogenital Infection I. I. Derevianko

IV. STAPHYLOCOCCI,

STREPTOCOCCI, PNEUMOCOCCI

Dissociated Macrolide and Lincosamide Resistance in Streptococcus pneumoniae and Variation of Susceptibility Testing Detecting Clindamycin Resistance Methods in E. L. Fasola, S. Bajaksouzian, P. C. Appelbaum, and M. R. Jacobs Prevalence of Antibiotic Resistance inStreptococcus pyogenes: Results of a National MulticenterSurvey Conducted in Italy During E. A. Debbia, P. Cipriani, G. l? Gesu, G. Ortisi, M. G. Menozzi, E. Nani, V . Nicolosi, R. Rigoli, R. Serra, M. Toni, K E. Vigand, and G. C. Schito In Vitro Activityof Some Macrolides Against S. pyogenes G. Tempera, P. M . Fumeri, V. Nicolosi, and G. Nicoletti Antipneumococcal Activity of RP (an Injectable Streptogramin), Erythromycin, and Sparlloxacinby MIC and Rapid G. A. Pankuch, M. R. Jacobs, and l? C. Appelbaum Effect of Macrolide Resistance on the Activity of the Oral Streptogramin RPR and Its Components Against Streptococcus M. R. Jacobs, S. K . Spangler, and P. C. Appelbaum

In Vitro Antibacterial Activity of RU 004, a New Ketolide piratory ns Against Active Constantin Agouridas, A. Bonnefoy, and Jean-Fraqois Chantot

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Antistaphylococcal Activity of QuinupristidDalfopristin, a WellDefined Mixture of Chemically Modified Streptogramins Christof von Eiff and Georg Peters E-Test for Susceptibility Testingof Streptococcus pyogenes to Azithromycin, Clarithromycin, Erythromycin, and Roxithromycin Gerard J. van Asselt and Jacobus H. Sloos Efficacy of Clarithromycin Against Experimental Pulmonary Infection Caused by Streptococcus pneumoniae Strains with a Novel Macrolide Resistance Mechanism J. A. Meulbroek, M. J. Mitten, A. Oleksijew, K D. Shortridge, S. K. Tanaka, and J. D. Alder Macro- and Microautoradiographic Studies on Penetrationof Azithromycin in Bacterially Infected Mice Issei Nakayama, Emiko Yamaji, Kaoru Shimada, Shuichi Yokoyama, Kazumi Miura, Hideya Muto, Kazunori Enogaki, Masatoshi Ogawa, and Kino Shimooka In Vivo Antibacterial Activity of RU 0 0 4 , a New Ketolide Active Against Respiratory Pathogens Constantin Agouridas, A. Bonnefoy, and Jean-Franco& Chantot Open Noncomparative Study of the Efficacy and Safety of Azithromycin in the Treatment of Adult Tonsilitis (Epidemiological Study of the Responsible Bacteria) Desaulty Open Study of Clarithromycin in the Treatment of Pneumonia Due to Streptococcus pneumoniae Murat Hayran, Mustafa Erman, Deniz Giir, Murat Akova, and Serhat Una1 Clinical Study of Rokitamycin on Pneumococcal Upper Respiratory Tract Infectionsin Pediatrics Yoshikiyo Toyonaga

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421

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V. PHARMACOKINETICS, PHARMACODYNAMICS, DRUG INTERACTIONS

Comparison of the Bronchopulmonary Pharmacokinetics of Clarithromycin and Azithromycin Kalpana B. Patel, Dawei Xuan, Charles H. Nightingale, Pamela R. Tessier, John H . Russomanno, and Richard Quintiliani

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Kinetics of Dirithromycin: Concentrations in Tonsils, Bronchial Secretions. Multiple-Dose Mucosa, and Studies C. Muller-Serieys, E. Bergogne-Berezin, E Lemaitre, M. Derriennic, A. Ohman, P. Gehanno, C. Le Royer, and J. Clavier

447

A Human Model of Local Abscess Using Skin Chambers: The Penetration of Azithromycin and the Chemiluminescence Response of Neutrophils (PMN) K. Takahashi, V: Duchateau, M. Husson, A. M. Bourguignon, and F. Crokaert

455

Separation of Presystemic and Post-Absorptive Influenceson the Bioavailability of Azithromycin Cynomolgus in Monkeys G. Foulds, A. G. Connolly, J. H. Former, and A. M. Fletcher

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Clinical Pharmacologyof Azithromycin Given at Various Sites Along the Gastrointestinal Healthy Tract in Subjects David R. Luke, G. Foulds, Joseph Scavone, Hylar L. Friedman, and William J. Curatolo Effect of Food and Formulationon Bioavailability of Azithromycin G. Foul&, David R. Luke, S. A. Willavize, William J. Curatolo, Hylar L. Friedman, M. J. Gardner, R. A. Hansen, R. Teng, and .l Vincent

ubjects Healthy Azithromycin inRectal David R. Luke, G. Foulds, G. Melnik, Paden C. Going, and Valerie Lawrence Comparative Pharmacodynamics of Clarithromycin and Azithromycin Against Respiratory Tract Pathogens Bauernfeind, R. Jungwirth, and E. Eberlein

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478

VI. HELJCOBACTER PYLORI, CAMPYLOBACTER Comparison of Clarithromycin Efficacy in Ferrets and Humans for Treatment of Helicobacter-Induced Gastritis P. Ewing, J. D. Alder, M. J. Mitten, A. Conway, A. Oleksijew, K. Jarvis, L. Paige, and S. K. Tanaka Azithromyciflanitidine Combined Treatmentof Helicobacter pylori in Patients with Duodenal Ulcer and Chronic Gastritis: A Pilot B. Desnica, V: Burek, and N. Makek

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Azithromycin Promising as a Part of Helicocidal Regimens 0.Shonovd, P. Petr, and 0. Hausner

498

ANAEROBES, ENTEROCOCCI, HAEMOPHILUS INFLUENZAE

Comparative In Vitro Activity of Five Macrolides Against Gram-positive Cocci, Campylobacter Species, and Anaerobes M. Marina, K. Ivanova, N. Hadjieva, and A. Urumov

505

Prophylactic Effect of Azithromycin on Experimentally Induced Infection Intraabdominal in Rats N. Panovski, P. MiloSevski, and N. Labatevski

511

Are Macrolides Active Against Haemophilus infiuenzae? Are In Vitro E Crokaert, M. Aoun, K Duchateau, H. Goossens, P. Grenier, A. Vandennies, and J. Klastersky

516

Macrolide Susceptibility of Isolates of Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis from Patients with Community-Acquired Lower Respiratory Tract Infection-Results of an International Multicenter Study(the 523 Project), 1992-1994 Alexander D. Felmingham, J. Linares, and the Alexander Project Group Resistance of Common Respiratory Pathogens to Erythromycin and Azithromycin Milan &.&an, Ana Zlata Drag&, Katja Seme, Andreja Orafem, and Metka Paragi Efficacy of Clarithromycin and Azithromycinat Human Pharmacological Dosage Against ExperimentalHaemophilus Infection infruenzae Pulmonary J. D. Alder, M. J. Mitten, A. Conway, A. Oleksijew, K . Jarvis, L. Paige, and S. K. Tanaka

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VIII. MYCOBACTERIA AND OTHER INFECTIONS IN HIV-INFECTED PERSONS

Clarithromycin 500 mg BID as Prophylaxis for MAC DiseaseA Follow-up John T. Sinnott, Douglas A. Holt, Sally H. Houston, Gary Bergen, Pamela Sakalosky, Julie A. Larkin, and Richard Oehler

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Clarithromycin Prophylaxis for Disseminated Mycobacterium avium Infection i? M. File, Jr., D. C. Claypoole, S. . I Longstreth, D. J. Signs, W. H. Ruby, and A. S. Indorf Azithromycin + Pentamidine + Pyrimethamine in the Prophylaxis of Opportunistic Infections in HIV-Positive Patients with CD4 Count Less Than G. Barbarini, G. Garavelli, G. Barbaro, B. Grisorio, A. Lucchini, S. Lopez, G. Del Buono, S. Edo, and R Alcini

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549

M. SPECIAL PATHOGENS Human and Animal Bite Wounds: Bacteriology, Macrolide Susceptibility, and Therapeutic Potential Ellie J. C. Goldstein and Diane M. Citron

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Azithromycinin the Treatment of PertussisinChildren: A Pilot Study A. Bade, N. Kuzmanovid, and i? Zmid

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The Use of New Macrolides in Experimental Brucella melitensis Infection R. Lang, D. Torten, B. Shasha, and Ethan Rubinstein

563

X. NONANTIBACTERIAL EFFECTS In Vivo Effect of Azithromycin Subinhibitory Concentrations on the Mortality of Experimental Pseudomonas aeruginosa Sepsis M. N. Marangos, M. E. Klepser, D. l? Nicolau, Charles H . Nightingale, and Richard Quintiliani

571

Clarithromycin Reduces Cl-Dependent Transepithelial Potential Difference in Tracheal Mucosa of Anesthetized Rabbits J. Tamaoki, H . Takemura, E. Tagaya, Y. Takeda,and K . Konno

576

Inhibition of Adherence of Klebsiella pneumoniae Strains to Intestine-407 Cell Linesby Roxithromycin S. Favre-Bonte, C. Forestier, A. Darfeuille-Michaud, C. Rich, J. Sirot, and B. Joly Clinical and Immunological Study of Roxithromycin on Infectious Diseasesin Obstetrics and Gynecology K . Izumi, H . Mikamo, K. Kawazoe, and T Tamaya

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XI. CLINICAL STUDIES OTITIS MEDIA Five-Day Treatment of Acute Otitis Media in Children with Clarithromycin Dimitris A. Kafetzis, Theodore Bairamis, Dimitra Dinopoulou, Stamatina Vlachou, and Nicholas Apostolopoulos Azithromycin Versus Amoxicillin for Acute Otitis Media Prophylaxis Nicola Principi, Paola Marchisio, Emanuela Sala, Luisa Lanzoni, and Stefania Sorella

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597

XII. CLINICAL STUDIES: BRONCHITIS Azithromycin for the Treatment of Community-Acquired Bronchitis: A Large Multicenter Efficacy-Tolerance Trial E Rafi Safety and Efficacy of Clarithromycin Compared with Cefpodoxime Proxetil in the Treatment of Acute Exacerbation ofObstructive Chronic Pulmonary Disease R Ltophonte and J. P. Chauvin

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609

m.CLINICAL STUDIES: PNEUMONIA Randomized Comparative Trial of the Safety, Efficacy,and Cost of Intravenous Cefuroxime Plus Intravenous Erythromycin Versus Intravenous Cefuroxime PlusOral Clarithromycin in the Therapy of Community-Acquired Pneumonia D. Skupien, A. Margulis, N. Kaczander, S. Jaworski, A. Ekleberry, J. Pypkowski, and M. J. Zervos

Azithromycin in the Treatment of Community-Acquired Pneumonia K. Golec, M. Rzeszutko-Grabowska, and D. BukowskaNierojewska

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Azithromycin: 3-Day Versus 5-Day Dosage Regimen for Community-Acquired Children Pneumonia in B. Ficnar, N. Huzjak, I. Klinar, and M. Matrapazovski

629

Single-Dose Azithromycin inthe Treatment of Atypical Pneumonia: S. Schonwald, I. Kuzman, V Car, J. tulig, and K. Oreskovid

634

Azithromycin in the Treatment of Patients with Upper Respiratory Tract Infections, Comparisonof 3-Day and 5-Day Regimens P. Dole2a1, M. Kro#l&k,and J. KlaZanskj Clinical and Bacteriological Evaluation of Clarithromycin (50-mg Tablets) in Children with Lower Respiratory Tract Infection Yoshikiyo Toyonaga Experience with Improved Complianceof Clarithromycin Granules in Children Kei3uke Sunakawa, Hironobu Akita, Satoshi Zwata, Yoshitake Satoh, Tatsuo Aoyama, and Ryochi Fujii Efficacy and Tolerability of Azithromycin Versus Josamycin in the Treatment of Children with Lower Respiratory Tract Infections Stt?p&nKutflek, Jozef Hoza, Daniela Markov&,and Milan Bayer Azithromycin in the Treatment of Respiratory Tract Infections in Children K. Galova, I. Marinova, J. Hractova, Kukova, S. Krifan, S. SuJliarska, H. Cintalanova, and A. Nogeova Efficacy of Roxithromycin in Elderly and Middle-Aged Patients with Respiratory Tract Infections S. Yakovlev

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645

649

656

660

664

OTHER CLINICAL STUDIES Experience with Azithromycin as Prophylaxis in Transrectal Biopsy of the Prostate M . Kvarantan and C. Dohoczky

671

Author Index Subject Index

675 681

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Contributors to Plenary Sessions and Workshops

JacquesF.Acar

Fondation Hdpital Saint-Joseph, Pans, France

ConstantinAgouridas Research Center, Roussel-Uclaf, Romainville, France Vincent T. Andriole Yale University School of Connecticut

Medicine, New Haven,

Daniel H.Bouanchaud* Rhdne-Poulenc Rorer, Vitry-sur-Seine, France Andr6Bryskier

Research Center, Roussel-Uclaf, Romainville, France

Jean-Paul Butzler University Hospitals, St. Pierre, Brugmann, and Queen Fabiola, Brussels, Belgium Sharon L. Cerwinka Rhdne-Poulenc Rorer, Collegeville, Pennsylvania Jean-FranqoisChantot Research Center, Roussel-Uclaf, Romainville, France Elisabeth Chaslus-Dancla Institut National de la Recherche Agronomique, Nouzilly, France C. Cocito University of Louvain Medical School, Brussels, Belgium

J Carl Craft Abbott Laboratories, Abbott Park, Illinois WilliamA.Craig

University of Wisconsin, Madison, Wisconsin

*Currentaffiliation:MEDICOM, Paris, France

Contributors

Michael W. Dunne Pfizer Central Research, Groton, Connecticut Roger G. Finch The City Hospital and University of .Nottingham, Nottingham, United Kingdom M. Di Giambattista University of Louvain Medical School, Brussels, Belgium J. Thomas Grayston University of Washington, Seattle, Washington I. M.Hoepelman

University Hospital, Utrecht, The Netherlands

Pentti Huovinen National Public Health Institute, lkrku, Finland INSERM U294, CHU X. Bichat, Paris, France

Marie-Th6rrbeLabro

Jean-Pierre Lafont Institut National de la Recherche Agronomique, Nouzilly, France Jean-Louis Martel Centre National d’Etudes Veterinaires et Alimentaires-LYON, Lyon, France KennethH.Mayer Rhode Island

Memorial Hospital of Rhode Island, Pawtucket,

George H. McCracken, Jr. University of Texas Southwestern Medical Center at Dallas, Dallas, Texas J. Allen McCutchan University of California School of Medicine, San Diego, California John E. McGowan,Jr.

Emory University, Atlanta, Georgia

FrancisMt5graud Hopital Pellegrin et Universite de Bordeaux 2, Bordeaux, France Jod Melo-Cristino University of Lisbon, Lisbon, Portugal

Robert C. Moellering, Jr. Boston, Massachusetts

Beth Israel Deaconess Medical Center,

Daniel M. Musher Baylor College of Medicine and Veterans’ Affairs Medical Center, Houston, Texas CharlesH.Nightingale

Hartford Hospital, Hartford, Connecticut

E. Nyssen University of Louvain Medical School, Brussels, Belgium Ian Phillips United Medical and Dental Schools of Guy’s and St. Thomas’s Hospitals, London, United Kingdom Jack Remington Stanford University School of Medicine, Stanford, and Palo Alto Medical Foundation, Palo Alto, California

Contributors

mi

Ethan Rubinstein Sheba Medical Center, Tel-Aviv University, School of Medicine, Tel-Hashomer, Israel JoelRuskin Kaiser Permanente Medical Center and Medicine, Los Angeles, California

UCLA School of

Urs B. Schaad University of Basel, Basel, Switzerland .

ShlomoSegev ShebaMedical Center, Tel-AvivUniversity,Schoolof Medicine, Tel-Hashomer, Israel Fritz Sorgel Institute for Biomedical and Pharmaceutical Research, Nurnberg-Heroldsberg, Germany

Charles W. Stratton Vanderbilt University School of Medicine, Nashville, Tennessee

M. J. Tarlow Birmingham Heartlands Hospital, Birmingham, United Kingdom P. Vannuffel University of Louvain Medical School, Brussels, Belgium

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PLENARY SESSIONS

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Overview of the Clinical Use of Macrolides and Streptogramins Roger G. Finch The City Hospital and University Nottingham Nottingham, United Kingdom

INTRODUCTION Among the macrolides, erythromycin has been extensively used for approximately 45 years. the prototype drug, it has defined the place of this class of antibiotic in therapeutics. It provides an effective alternative to thepenicillins in the treatment of many common community infections of the skin and respiratory tract, and to a lesser extent in the treatment of various sexually transmitted infections. It has been the mainstay for treating atypical infections such as those causedby Mycoplasma pneumoniae, the ureaplasma, Legionella spp., Coxiella burnetii, and, more recently, Chlamydia pneumoniae. It is used in surgical prophylaxis in some countries in relation to colonsurgeryandhasoccasionalusesin the control of pertussisanddiphtheria.However,itslimitationshaveincluded low biovailability, poor gastrointestinal intolerance, and the emergence of resistance among common target pathogens such as staphylococciandstreptococci,as wellasexhibitingunreliableactivityagainst Haemophilus influenzae.

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DEVELOPMENT OF STREPTOGRAMINS AND MACROLIDES The streptogramins belong to the peptide group of antibiotics and include the mikamycins,ostreomycins,virginiamycins,andpristinamycins. The latter naturally occurring antibiotics derived from Streptomyces pristinaespiru. Pristinamycin and virginiamycin have been used in continental Europe as oral agents for the treatment of staphylococcal and streptococcal infections. The pastfewyearshaveseenmajordevelopmentsinboth the macrolide and peptide classesof compounds,' most notably withthe availability of azithromycin,clarithromycin,androxithromycinamong the macrolides, and inthe case of the streptogramins, the development RP 59500 (quinupristiddalfopristin) from the original pristinamycins. The importance of thesedevelopmentscan be viewed fromseveral perspectives.First,theyprovideanexpansion of the macrolidelincosamide-streptogramin (MLS) group antibiotics at a time whenthe development of new anti-infective agents, and in particular new classes of drug, has shown a considerable slowing in comparison with former years. Second, we are currently faced with a number of new infectious challenges such as the emergence of multidrug-resistant enterococci and mycobacteria, and serious staphylococcal infections, particularly those caused by multidrug-resistant Staphylococcus aweus and coagulase-negative staphylococci. Furthermore, new diseases have been identified such as Lyme disease and HIV disease,withits attendant microbialcomplications,and Helicobacter pylori-associated gastroduodenal disease. Likewise, the reemergence of epidemic diphtheria in the countries of the former Soviet Union emphasizes the continuing need for notonlyeffectiveimmunoprophylaxis but also judicious chemoprophylaxis.

Structure Activity Issues The macrolides, lincosamides, and streptogramins are structurally diverse but are conventionally grouped together because of their common ability to bind to bacterial ribosome to inhibit protein synthesis and thereby bacterial multiplication. The macrolides form a large groupof related antibiotics, largelyderived from Streptomycetes. Structurally, they consist of a macrocyclic lactam ring to which various aminosugars are attached. The macrolides are classified into 14 and 16 carbon-containing rings. Among the C-l4 compounds are erythromycin, oleandomycin, and, more recently, clarithromycin,roxithromycin,troleandomycin,flurithromycin,anddirithromycin,

MacrolideslStreptogramim-Overview

5

which is a pro-drug of erythromycylamine. Among the C-l6 compounds are josamycin, midecamycin, rosaramycin, and spiramycin, as well as the semisynthetic miocamycin, characterizedby a single aminosugar. Azithromycin is a 15-membered compound with a methyl-substituted nitrogen at position which is therefore strictly an azalide, although for all practical purposes it can be considered as a macrolide. The lincosamides,lincomycinandclindamycin, are derivativesof Streptomyces lincolnis. Although there is some overlap in the areas of clinical use with those ofthe macrolides and streptogramins, theyare sufficiently distinct and will not be discussed further in this chapter. The streptogramingroup of antibioticsincludes the mikamycins, pristinamycins, oestreomycins, and virginiamycins which have been produced as secondary metabolites from a variety of natural Streptomycetes. They have a complex and often unstablestructure but lend themselves to modification without major loss of antimicrobial activity. Recent interest has focused on pristinamycin, which has two macrolactone componentspristinamycin I, and pristinamycin 11,. RP (quinupristin) and RP (dalfopristin) are the respective chemically derived water soluble components which when combined are bactericidal and synergistic against avariety RP [quinupristiddalfopristin target,pathogens. (Synercid it is undergoing clinical trials. The mode action of RP is to bind to the bacterial ribosome to form a stable dalfopristinribosome-quinupristin complex which irreversibly inhibits protein synthesis resulting in bacterial cell death. It would appear that this mechanism of action is unique and, therefore, distinguishes RP from other antibiotic classes. The focus of this chapter will be largely on the recently available macrolides, namely azithromycin, clarithromycin, and roxithromycin, and also quinupristiddalfopristinwith emphasis on their clinical use.The constraints of space do not permit a review of the safety aspects of these drugs. In order to appreciate their therapeutic potential, an understanding Of their in vitro performance and pharmacokinetic behavior is a necessary prerequisite.

In Vitro Activity The spectrum of activity of the recently available macrolides andstreptogramins is sufficiently distinctive among the major classesof anti-infectives, perhaps withthe exception tetracyclines, inthat it encompasses not only many common bacterial pathogens but also includes atypical pathogens, Mycobacteria spp., spirochaetes, and selective protozoa. Furthermore, the observed high tissue and intracellular concentrations broaden their clinical

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utility. The growing importance of drug resistance to other classes of antibiotic further emphasizes their growing importance. These resistance problems include penicillin-resistant pneumococci, multiresistant enterococci, and, in particular, vanocmycin-resistant strains, methicillin-resistant staphylococci, and multiresistant coagulase-negative staphylococci, as well as drug-resistant Mycobacteria spp. Macrolides

The in vitro activity of erythromycin encompasses most streptococci, including Streptococcus pneumoniae, Streptococcus pyogenes , and the viridans streptococci, but excludes the enterococci. Staphylococcus aureus is also susceptible with the exception of methicillin-resistant strains (MRSA) which are often multiresistant. Other sensitive gram-positive pathogens include corynebacterium and Listeria monocytogenes. Susceptible gram-negative pathogens include Neisseria gonorrhoeae, Moraxella catarrhalis, Legionella pneumophila, and Bordetella pertussis. Activity against Haemophilus influenzae is equivocal. Gram-positive anaerobic bacteria are often sensitive. The Enterobacteriaceae are not considered susceptible. Other susceptible pathogens include Mycoplasma pneumoniae, Chlamydia trachomatis, Chlamydia pneumoniae, Treponema pallidum, Borrelia burgdorferi, and Mycobacterium spp. The most striking in vitro differences for the most recently available macrolides is their enhanced activity against many common pathogens (Table 1). This is particularly so for clarithromycin, which is approximately twice as active as erythromycin against staphylococci and streptococci, and fourfold to eightfold more active than azithromycin. It should be emphasized that methicillin-resistant isolates of S . aureus are uniformly resistant to these new macrolides, as are the enterococci. The activity of roxithromycin most closely resembles that of the parent compound, erythromycin. Whereas the activity of erythromycin against H . influenzae has been unpredictable, azithromycin is twofold to eightfold more active and roxithromycin remains of comparable activity. The 14-hydroxy metabolite of clarithromycin has biological activity, which is twice as active as clarithromycin against H . influenzae. Bordetella pertussis also remains sensitive to these new macrolides. Some of the most striking differences in activity are seen against atypical respiratory pathogens; clarithromycin is the most active agent against C. pneumoniae, L. pneumophila, and M . pneumoniae. Erythromycin is an alternative agent for the treatment of gonococcal infections and also provides useful activity against nongonococcal genital tract pathogens such as Chlamydia. trachomatis and Ureaplasma urealyticum. Clarithromycin again demonstrates the greatest instrinsic activity and

MacrolideslStreptogramins- 0vervie w

7

Tuble I Macrolide Activity (MIC,,, mg/L) Against Common Bacterial Pathogens

Organism

Erythromycin

S . areus

Coagulase-negative staphylococcus S . pneumoniae S . pyogenes S . agalactiae E . faecalis E . faecium N . gonorrhoeae L . monocytogenes H . injluenzae B . pertussis H . pylori M . catarrhalis C . jejuni

Anaerobic streptococci B . fragi1i.s C. perfringens C . trachomatis U.urealyticum H . ducreyi

Azithromycin

Clarithromycin

0.5 0.5

0.12 0.25

0.25 0.25

0.02-0.06 0.03 0.03 >64 >64 2.0 0.5 4.0-8.0 0.03 0.25 0.25 2.0 4.0 2.0 1 .o 0.125 2.0 0.06

0.12 0.12 0.06 >64 >64 0.25 2 1 .o 0.06 0.25 0.06 0.5 2.0 2.0 0.25 0.25 2.0 0.003

0.02 0.06 0.06 >64 >64 0.5 0.25 8.0 0.03 0.03 0.25 2.0 4.0-8.0 2.0 0.5 0.016 0.2 0.015

Roxithromycin 1.o 0.25 0.03 0.06 0.25 >64 >64 1.0 1.0 8.0 0.03 0.5 1.o 4.0 32 32 2.0 0.125 2.0 0.06

~

Source: Data based on Refs. 3, 29, 51, and 62.

includes Haemophilus ducreyi. Azithromycin is the most active agent against N. gonorrhoeae whereas roxithromycin offers no microbiological advantage over erythromycin against this group of pathogens. Although the macrolides are inactive against common gram-negative pathogens, Cumpylobucter jejuni is susceptible. Furthermore, Helicobacter pylori is also susceptible, with clarithromycin showing the greatest activity (1). Of considerable interest is the activity of the new macrolides against Mycobucteria spp. M. tuberculosis is relatively insensitive to clarithromycin and azithromycin, for which the MIC,s are >10 mg/L and 32 mgIL, respectively. Roxithromycin inhibits most strains of M . tuberculosis at 4 mg/L, which with the addition of rifampicin or isoniazid is further reduced to 0.25 mg/L (2). Of greater interest is the activity of these compounds against atypical mycobacteria, in particular the Mycobucterium avium complex (MAC).

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Azithromycin and roxithromycinare relatively inactivein vitro withM I G s of 32-62m&. However,clarithromycindemonstrates greater activity with M I G s of 2-8 m& (3). The activity of clarithromycin against MAC is pH and media dependent. The MI& lies between2 and 8 m&, whereas the minimum bactericidal concentrationvanes between S6 and m@ (4) which emphasizes the importance of intracellular concentrations of drug in relation to any clinical utility (5). This activity has been supported by in vivo studies in beige mice, where bacterial counts the spleen in are reduced by clarithromycin and potentiated by clofazimine and rifabutin (6). The performance of azithromycin againstMAC, although less striking in vitro, has also reduced bacterial counts in beige mice (7), whereas macrophages infected with MAC of azithromycin inthe presence of interferonhave shown increased uptake gamma or tumor necrosis factor-alpha (8). Against other Mycobacterium spp., roxithromycin hasshown'potentially useful activity with MIC values against M. malmoenese, M. scrofulaceum, M. szulgai, M. xenopi, and M.kansasii but excluding M.marinum (9). Clarithromycin has demonstrated potential clinically useful activity against M. marinum resistant to other antituberculous agents-". kansasii (MIC90 0.25m&) (lo), M.chelonei spp., and M. forfuitum spp. (11). Other Organisms. Azithromycin, in keeping with other macrolides, has activity against amoebas and other protozoans. For example, the growth of Entamoeba histolytica trophozoites is inhibitedby concentrations of 40 m&, which is comparable to erythromycin (12). Giardia spp. appear to be insensitive. However,of greater interestis the susceptibility ofToxoplasma gondii to the macrolides. In the case of azithromycin, 50% inhibition of nucleotide synthesis has been demonstrated in macrophage grown cells at concentrations of 140 m& compared with a range of 54-246 mgL for clarithromycin, roxithromycin, and spiramycin. (13). Streptogramins

As-noted earlier, pristinamycin is composed of two components, PIA and PII,. Recently,thesetwofermentationproductshavebeenchemically modified to produce the water-soluble injectable derivatives quinupristin (RP 57669), derived from PI,, and dalfopristin (RP54476), derived from PIIA. When formulated a 30 : 70 ratio, they are synergistic and have been developed as RP 59500 (SynercidB). Quinupristiddalfopristin individually exhibit weak bacteriostatic activity against gram-positive bacteria. In combination, they are rapidly bactericidal against S. pneumoniae,Enterococcusfuecium, methicillin-sensitive(MSSA) and resistant (MRSA) S. aureus, and coagulase-negative staphylococci.

MacrolideslStreptogramins-Overview

9

The MIC,s are usually 1 m& and include strains resistant to erythromycin, ciprofloxacin, gentamicin, and rifampicin. The activity against staphylococci is comparable to vancomycin, erythromycin,and ciprofloxacin but less than ampicillin and erythromycin against streptococci. The in vitro activityof RP 59500 is summarized in Table 2. Considerable interest has focused on the activity of RP 59500 against enterococci.Althoughmany are susceptible,MICconcentrationshave ranged from 0.25-32 m@. E . faecium is usually more susceptiblethan E . faecalis. Likewise,vancomycin-resistant E. faecium are often sensitive. Those sensitive to vancomycin are often twofold or more susceptibleto RP 59500. Multiresistant strainsof E. faecium remain susceptibleto RP59500 with an MIC90 of1.0 m@. Gram-negative and intracellular pathogens such as-N. meningitidis, N. gonorrhoeae, Legionella spp., M . pneumoniae, and Chlamydia spp. are also susceptible with MIC, 1.0 m& (Table 2). H.influenzae and M . catarrhalis are less susceptiblethan other pathogens withMIC, of 4 m&. Table 2 Susceptibility of Pathogens toQuinupristinDalfopristin(m59500)

Dositive Gram ~~

MIC,
N.meningitidis N.gonorrhoeae M.pneumoniae

Clostridium spp.

L. pneumophila M . catarrhalis

Streptococci (groupsA, B) S. pneumonia@ S. bovis

Viridans streptococci L. monocytogenes

MIC,>lto<4pgL E. faecium (VSNR)d MIC,>4to<8mgL E. faecalis (ESER)

H . infruenzae

B. fiagilis

Nonfragilis Bacteroides PrevotellalPorphyromonas 'MSIMR = methicillin sensitive/methicillin resistant. bES/ER = erythromycin sensitive/erythromycin resistant. CIncludes penicillin sensitive, intermediate,and resistant strains. W S N R = vancomycin sensitive/vancomycin resistant. Source: Data based on Refs. 73-75.

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The Enterobacteriaeceae, Pseudomonas aeruginosa, and Acinetobacter spp. are not susceptible. Selected anaerobes such Clostridium as pefiingens, Bacteroides fragilis, and other Bacteroides spp., peptostreptococci, Fusobacterium spp., and prevotelldporphyromonasare sensitive withMIGs varying between2 and 4 m&. Synergistic activity withother agents is variable. However, a positive effect has been shown for vancomycin-resistant and multidrug-resistant E. faecium as well as MRSA with the combination of vancomycin and RP 59500. However, when combined with oxacillin or gentamicin, antagonism has been observed against S. aureus and againstE . faecah when combined with ampicillin. Quinupristiddalfopristinoften exhibits a prolonged postantibiotic effect (PAE) against gram-positive pathogens(14). For example, aPAE of > 2 h has been shown for S. aureus following exposure to 2 m&, which has increased to 8 h following exposure to 2.5 m&. Likewise, a prolonged PAE has been observed against coagulase-negative staphylococci (PAE h), S. pneumoniae (7.5-9.5 h), and S. pyogenes (>l8 h). The clinical relevance of these observations in relation to dosage.frequency and efficacy has yetto be confirmed.

Pharmacokinetic Properties Macrolides

Erythromycin baseis relatively poorly absorbed and results in fairly modest plasma concentrations made lowerif taken simultaneously with food.The development of various prodrugs includingsalts, esters, and saltsof esters (Table have hadthe aim of improvingthe bioavailability of erythromycin base. Table 3 Pro-drugs of Erythromycin ~~

Salts

Esters Propionyl Ethylsuccinate Salt Stearate

~

~

of an ester Propionyl-mercaptosuccinate

Acistrate Acetylcysteinate Troleandomycin Triacetyloleandomycin

MacrolideslStreptogramins-Overview

l1

Among the newer macrolides roxithromycin has improved absorption. Following a 150-mg oral dose, the C,, ranges from 6.61to 7.9 m@, rising to 9.1-11.02 mg/L following a 300-mg dose (15). This C,, after a single 150-mg dose is3.3 times greater than that following a single 250-mg dose of erythromycin. Furthermore, the area under the plasma concentration versus time curve (AUC) is 16-fold greater than that produced by erythromycin. Roxithromycin is widely distributed in tissues and body fluids, with concentrations generally higher than MIC, values for mosttarget pathogens (16). Protein bindingofisthe order of 96% and, in keeping with other macrolides, is largely bound to alpha-l-acid glycoprotein,rather than albumin. Clarithromycin is well absorbed with a bioavailability of greater than 50% despite substantial first-pass metabolism. C,, concentrations of 0.620.84 rise to 1.77-1.89 m@ following single doses of 250 and 500 mg, respectively, with AUCs ranging from 4 to 11 mg/L h for similar doses in volunteers (17). The rapid biotransformation of clarithromycin to 14-OH clarithromycin results in C, for this bioactive metabolite of 0.4 and 0.8 mg/L following and 500-mg doses, respectively, Food does not affect these pharmacokinetic parameters (18). Steady-state plasma concentrations for clarithromycinrangefrom2.4 to m@, and for the 14metabolite, 0.7-0.8 m@, following 500-mg dosing. In comparison with erythromycin, clarithromycin achieves a threefoldgreater C, and a fivefold greater AUC. The mean elimination half-life(Tin) ranges from 2.6to 4.4 h in volunteers given 250 or 500 mg, which is approximately twicethat for erythromycin. Protein binding is of the order of 42-50%, again to alpha-l-acid glycoprotein. Clarithromycin is also widely distributed, achieving high concentrations in various tissues and organs.The volume of distribution is also high, ranging from 226to 266 L. Peak concentration in tissues such as lung, nasal mucosa, skin, and tonsil exceeds plasma concentrations twofold to sixfold. In contrast, concentrations of the 14-hydroxy metaboliteare somewhat less than the parent drug in these tissues (19). Urinary excretionofisthe order of 32%, which includes the parent compound, the lbhydroxy metabolite, and other metabolites. Azithromycin presents a somewhat different profile. The pharmacokinetic parameters reflect rapid and extensive uptake into the intracellular compartments followedby slow release. Mean peak plasmaconcentrations are of the order of 0.4 m@ followinga 500-mg oral doseoccumng approximately 2.5 h after ingestion with a biovailability of 37% (20). The AUCis lower than that for erythromycin, roxithromycin,or clarithromycin. Cellular uptake includes polymorphonuclear leukocytes, monocytes, alveolar macrophages, and fibroblasts, as well other as tissues, and these concentra-

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tions considerably exceed serum concentrations. For example, concentrations in bronchoalveolar macrophages, bronchial epithelial lining fluid, and bronchial mucosa and sputum range from 0.56 to 26.59 m a , reflecting a tissue or fluid : serum concentration ratio of 52-1150 : 1 (21). In general, most tissue concentrationsare 2 2 m aProtein . binding varies from 37% to 50% and is again predominantly to alpha-l-acid glycoprotein. Serum elimination in man follows a polyphasic pattern, reflecting initial rapid distribution to tissues and subsequent slow release. The mean terminal elimination half-life following a 500-mg dose ranges 11 from to 14 h (from 8 to 24 h after oral administration). Following a 5-day regimen of 500 mg bid on day1and then500 mg daily for 4 additional days, the TI, varies between 11and 14 h afterthe last dose but increases to 48 h when calculatedfor the period 1-3 days after completion of the regimen and 57 h when calculated for the period 1-6 days after completion of the dosage regimen (20).

QuinupristinlDdfopristin The pharmacokinetic behaviorof RP 59500 has been evaluated in healthy controls and various patient populations in the ratio of30 : 70 for the components quinupristin and dalfopristin, respectively. Following a dose regimen ranging from 1.4 to 29.4 mgkg infused over 1 h the C, range from 0.95 ? 0.22 to 24.20 ? 8.82 mg/L with an elimination half-life of 1.27-1.53h. RP 12536is the activemetabolite of dalfopristin.When administered in a dose of 5 mgkg quinupristin, dalfopristin andRP 59500 produce C, values of 1.20,4.55, and 0.94 respectively withT,,’s of 0.87, 0.40, and 0.91. Blood concentrations following infusion of doses in excess of4.6 mgkg exceeded the MIC and MBC for moststreptococciand staphylococci (22).

Clinical Use

Azithromycin, clarithromycin, and roxithromycin have been widely evaluated in the treatment of upper and lower respiratory tract infections, including pneumonia, as well as skin and soft tissue infections and sexually transmitted diseases. Additional experience has been gained treating H . pylori-associated gastroduodenal disease and mycobacterial infections, especially those caused by MAC. Emerging areas of experience includethe treatment of toxoplasmosis, giardiasis, malaria, leprosy, rickettsioses, Lyme disease, and pneumocystosis. For the licensed indications, comparative clinical studies have been performed against a wideofvariety comparator agents including the aminopenicillins and oral cephalosporins, as well as competito macrolides. A selection of the published studies will permit an assessment of the current and future clinical utility of these macrolides.

MacrolideslStreptogramins-Overview Azithromycin

The antimicrobialspectrumanddistinctivepharmacokineticprofile of azithromycin have led to its evaluation in more abbreviated treatment courses of 5 days compared with 7-14 days for conventional agents. More recently, 3-day regimens have been studied in the treatment of a wide variety of common infections. Single-dose therapy has been evaluated in the treatment of chlamydial and gonococcal urethritis.

Upper Respiratory Tract Infections. Azithromycin has been used successfully to treat middle ear infections in both children and adults. In studies using dose regimens of mgkg once daily for the first day, and 5 mgkg daily for a further 4daysinchildren,incomparisonwith days amoxycillin-clavulanic acid, curerates of >95% have been achieved which compare favorably with the comparator (23). Likewise, acute maxillary sinusitis in adults has responded favorably with a dosage regimen of 500 mg once daily for the first day, followed by a further 4 days of 250 mg once daily (24). Comparators have included amoxycillin and erythromycin. Successful clinical response was again recorded in excess of 95% of those treated. Streptococcal pharyngitis has also responded favorably in comparisonwithpenicillin V andclarithromycin(25).Inchildren,comparable efficacy has been demonstrated in treating pharyngitis and/or tonsillitis compared with erythromycinor penicillin V; it has been shownto be superior to amoxicillin-clavulanate in acute otitis media in a large, multicenter study (26). Lower Respiratory Tract Infections. Azithromycin has been evaluated in a varietyof lower respiratorytract infections including hospitalized patients with acute exacerbationsof chronic bronchitis, acute community-acquired pneumonia, and acute bronchitis. Dosage regimens have varied from to 5 days. Some studies have included patients with atypical pneumonia caused by Chlamydia spp., Coxiella burnetii, and Mycoplasma pneumoniae (27). Bacteriologicaleradicationrateshavevariedbetween52%and 73%. Eradication rates of 82-100% have been recorded for S. pneumoniae (28) and 7595% for H. influenzae (29). These response rates are reassuring in view of the problem of increasing antibiotic resistance of these pathogens and the concerns that the low plasma concentration of azithromycin might not be effectiveagainst thesetarget pathogens. Experience with bacteremic pneumococcal pneumonia remains limited. Efficacy rates have been comparable to those achieved with amoxicillin, amoxicillin-clavulanic acid, cefaclor, as well as erythromycin;one study demonstrated superior efficacy against H.influenzae infections when compared with cefaclor (30).

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More recently, a 3-day regimenof azithromycin has been compared with 10 days of amoxicillin-clavulanic acid in adults with a variety of lower respiratory tract infection (LRTI). When treating acute infective exacerbations of chronic bronchitis, clinical response rates of 86% and92%, respectively, have been observed (31).In a larger studyof 369 adults with acute LRTI, the response rate for 3 days treatment with azithromycin was 95% compared with 96% for amoxycillin-clavulanic acid; S. pneumoniae and H.influenme predominated and were universally eradicated by azithromycin (32).A further study of azithromycin (a 3-day regimen) in 530 adults in the treatment of acute otitis media, streptococcal pharyngitis/tonsillitis or sinusitus showed comparable clinical efficacy to cefaclor administered in a dose of 250 mg tid for days (33). In children, a 3-day regimen has been compared with cefaclor for 10 days in an open, multicenter study of patients with acute otitis media; overall cure rates of 98% and 97%, respec-' tively, were recorded, although azithromycin had a statistically significant higher cure rate at 4 weeks posttreatment (34). Finally, two studies have compared 3 days ofazithromycinwith10daysofroxithromycinin the treatment of acute upper respiratory and lower respiratorytract infection in adults; clinical cure rates of 94% or higher were recordedfor both agents in those suffering from acute upper respiratory infections (35), whereas for lower respiratory infections,91.9% responded to azithromycin in comparison with 87.2% for roxithromycin, with bacteriological eradication rates of 92% and 81.1%, respectively (36).

Skin and Soft Tissue Infections. Skin and soft tissue infections in adults and children using a 5-day regimen of azithromycin in comparison with cephalexin (37), cloxacillin, and erythromycin (38) for 7-10 days have been evaluated; more recently a 3-day regimen has also been studied in children (39). Clinical cure rates have exceeded 94% in those assessable with bacteriological cure rates greater than 93%. Target pathogens have largely included S. aureus, S. pyogenes, and S. agalactiae.

Sexually Transmitted Infections. The susceptibility of Chlamydia trachomatis and Neisseria gonorrhoeaeto azithromycin has ledto its evaluationin genital infections using a variety of dosage regimens, including a single l-g dose (40), 500 mgs twice daily for two doses, and500 mgs on day 1,with.two subsequent daily doses of 250 mgin comparison with a standard 7-day course of doxycycline 100mg bid. Curerates for uncomplicated gonococcal urethritis have been 95% following a single l-g dose. Clinical cure and bacteriological eradicationrates for C. trachomatis,N . gonorrhoeae, and U. urealyticum have ranged between 56%, and 100%at 28 days (29). For chlamydial urethritiskervicitis, a singlel-g dose is comparable to 7 daysof doxycycline treatment. Superiority to ciprofloxacin inthe treatment of chla-

MacrolideslStreptogramim-Overview

15

mydial urethritis or cervicitis has also been confirmed Cure rates of for chancroid have been noted for a single l-g dose regimen

Other Infections. Azithromycin has activity against a variety of less common or unusual bacteria including intracellular pathogens, whereas a few protozoal infections have demonstrated susceptibility. Some of these areas of investigational useare as follows. Lyme Disease. The in vitro activityof azithromycin againstBorrelia burgdorferi has been translated into clinical studies of erythema chronicum migrans. Azithromycin, mg on day followed by mg per day for days,hasbeencomparedwithbothdoxycyclineandamoxicillinwith probenecid for days; the clinical resolution was greater than A further study comparingthe same regimen of azithromycin with penicillin V or doxycycline for days demonstrated comparable resolution of the cutaenous lesions of Lyme disease, although systemic and/or local symptoms resolved more rapidlyin those treated with azithromycin (44). Helicobacter pylori. Azithromycinhasprovedlessstrikingthan clarithromycin in the treatment of Helicobacter pylori associated gastritis/ duodenal ulcer with a tendency to relapse and develop drug resistanceat the end of treatment Toxoplasmic encephalitis. Toxoplasmic encephalitis complicating HIV disease presents a major therapeutic challenge. Anecdotal support for azithromycin as rescue therapy led to the comparison of azithromycin and mg daily in combination with pyrimethamine mg/day 1; mg/day thereafter) for weeks, after whichthe dose of azithromycin was maintained at mg daily. Using historical comparison of radiological response rates and drug toxicities, azithromycin demonstrated less observed toxicity,butprogressionrateswerehigheramong randomized and evaluable patients Study data on file: Pfizer Laboratories). Mycobacterium avium ComplexInfections. Mycobacterium avium complex (MAC) infections in HIV-infected and non-HIV-infected persons has been subjectto considerable attention. Azithromycin has been shown to be effective in those with AIDS, administered in a once daily of dose mg for periods ranging from 10to 30 days. A reduction in bacteremia has been demonstrated using sequential culture techniques. Clinical improvement, with reduction in fever and sweats, has also been recorded In doses of and mg/day, azithromycin produced a marked clinical improvement in and of patients, respectively. Multidrug regimens of azithromycin, ethambutol, rifabutin, and streptomycin are also under evaluation. Further information concerning the risk of drug resistance isrequired.

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Other areas of interest includethe treatment of acute pertussis where bacterial eradicationrates appear to be more efficient than for erythromycin. Bartonella infections such as B. henselae, which is responsible for cat scratch disease,are susceptible; clinical experience is awaited with interest for this challenging problem. Early responses of bacillary angiomatosis havealsobeen noted. The recognizedactivityofazithromycinagainst chlamydiae has stimulated investigation of its potentialtheinmanagement of trachoma. Likewise, the high and prolonged concentrations of drug achievable within the prostate suggest this should be investigated for the treatment of bacterial prostatitis. Cryptosporidiosis and Plasmodium falciparum infections present major therapeutic challenges where preliminary data suggest azithromycin deservesfurther clinical evaluation. Clurithromycin The therapeutic indications of clarithromycin again emphasizethe importance of infections of the upper and lower respiratory tracts, including those caused by atypical pathogens. It has also been evaluated in skin and soft tissue infections, urogenital infections, andtheinmanagement of atypical mycobacterial infections inthe HIV and non-HIV infected. Its role in managing H . pylori gastroduodenal diseaseis also achieving prominence.

Upper Respiratory Tract Infection. A large number of comparative studies in both children and adults using a variety of clinical trial designs were evaluated.Instreptococcalpharyngitis,clinicalandbacteriological response rates of >95% and 88%, respectively (47,48), have been published, including comparability with erythromycin and penicillin V. Acute mmillary sinusitis has also responded with satisfactory clinical responses of up to 92%, matched by similar bacteriological eradication rates (49); comparators have included amoxicillin-clavulanic acid. Acute tonsillitis and otitis media have shown similar cure rates of up to 87% and99%, respectively, in comparison to treatment with josamycin (50). The target pathogens have been representative of these infections, most notably S. pneumoniae, S. pyogenes, and S. aureus. Lower Respiratory Tract Infections. A variety of comparative and noncomparative studies have evaluated clarithromycin in such clinical syndromes as acute bronchitis, infective exacerbationsof chronic bronchitis, and community-acquired pneumonia. Clarithromycin250 or 500 mg twice daily for 1-2 weeks has proved effective in all these conditions. For example, clinical cure rates of up to 83% and bacteriological eradication rates of up to 100% have been recorded in community-acquired pneumonia (51), whereas in patients with acute exacerbations of chronic bronchitis, these figures have been of the order of96% and 100%, respectively. In the

Macrolides/Streptogramins-Overview

17

treatment of acute bronchitisor acute exacerbationsof chronic bronchitis, comparators have included josamycin, roxithromycin, erythromycin, ampicillin, cefuroxime axetil, and cefixime. With regard to atypical pneumonia, the results have been encouraging, with satisfactory responsesto infections causedby Chlamydia pneumoniae, Mycoplasma pneumoniae, and Legionella pneumophila. In the latter, a nonblinded multicenter study demonstrated clinicalcure in patients in doses of mg twice dailyfor periods of days With regard to H.infruenzae infection in patients withacute exacerbations of chronic bronchitis, clarithromycin was equivalent to cefaclor, cefuroxime axetil, or cefixime this emphasizesthe importance of the hydroxy metabolite against this pathogen.

Skinand Soft TissueInfections. A number of comparative multicenter studies have demonstrated the comparability of clarithromycin to agents such as erythromycin and cefadroxil with clinical successrates of or greater, and bacteriological curerates of Target pathogens have largely includedStaphylococcus aureus. Urogenital Infections. A number of studieshaveassessed the performance of clarithromycin in patients with nongonococcal and gonococcal urethritis/cervicitis. Response rates of up to have been recorded in patients with chlamydial urethritis and in persons with predominantly ureaplasma infections In the case of mixed gonococcaland chlamydial infections, response rates have been lower.

MycobacterialInfections. Atypical mycobacterial infections and, in particular, those caused by MAC complicating HIV disease have been a particular focusof assessment. Pilot studies have included randomized, some placebo controlled, double-blind crossover studies with clarithromycin dosages of g twice daily, either alone or in combination with clofazamine mgdaily, ethambutol 20 mglkgdaily,isoniazid mglkgdaily,and rifampicin mglkg daily, administered for up to 6 weeks in comparison with placebo These studies have monitored blood for colony-forming units of MAC. Bacteremia was eliminated in all recipients of clarithromycin and the four other antimycobacterial agents. In an additional small study of patients withAIDS, clarithromycin and clofazamine, and ethambutol and isoniazidfor 6 weeks, followed bymaintenance with clarithromycin and rifabutinfor weeks, followed by clarithromycin alone indicated that those who received clarithromycin responded clinically and developed negative blood cultures(56). Tho important studies have beenreported from Canada and France. The former prospective, randomized study compared ciprofloxacin mg

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18

bid, ethambutol mgkg qid, rifampin 600 mg qid, and clofazimine 100 mg qid with clarithromycin g bid, rifabutin 600 mg qid, and ethambutol mgkg qid in evaluable HIV-infected patients with MAC bacteremia followed to death. The clarithromycin-containingregimen was significantly better with regard to clearance of bacteremia, 67/97 (69%), and median survival months) In the French study, clarithromycin2 g/day g/day after months) and clofazimine mg/day mg/day after months) was compared with a similar clarithromycin regimen, combined with rifabutin mg qid and ethambutol mg qid.End points included decrease in fever and negative blood cultures and survival. No statistical significant differences were observed at 2 and 6 months terms in of the end points, although clarithromycin resistancewas more frequent (p < in the clofazime/clarithromycinregimen Clarithromycin has also shown some promise with early responses in immunocompromized patients infected withM.chelonae Helicobacter pylori. Clarithromycinhasshownconsiderablepromisein the treatment of H . pylori gastroduodenal disease.It is the most activeof the new macrolides against this pathogen (MIC, 0.03 m&). The objectives of therapy are not only to heal the associated H . pylori gastritis or duodenalulcerbutalso to prevent recurrence and alter the long-term natural history of the disease. monotherapy, eradication rates of have been observed 4-6 weeks posttreatment (60).Various strategies involving clarithromycin are under investigation or comparative clinical trials; the combinations are with acid-controlling agents suchas omeprazole, ranitidine-bismuth citrate, or bismuth.

Toxoplasmosis. A small number of patients with AIDS and complicating toxoplasma encephalitis have been treated with pyrimethamine and clarithromycin g twice daily for up to 6 weeks. Clinical and radiological response rates have been of the order of (61). Roxithromycin

Roxithromycin has antibacterial activitythat closely resembles erythromycin but with improved plasma, tissue, and body fluid concentrations and a much longer half-life.It has been evaluated for a variety of clinical indications and, in particular, infections of the upper and lower respiratorytracts skin and soft tissues, and the urogenital tract. selection of the published studies are reviewed.

Upper Respiratory Tract Infections. Pharyngitis, tonsillitis, sinusitis,and otitis media have been studied using 300 mg once or mg twice daily regimens with clinical response rates that have exceeded 92% for these

MacrolideslStreptogramins-Overview

19

conditions. Likewise, bacteriological response rates have been high, rangingfrom 9 0% to Incomparativestudies,comparability to clarithromycin-and amoxicillin-clavulanic acid has been demonstrated.

Lower Respiratory Tract Infections. Several trials have investigated the performance of roxithromycin in the treatment of acute bronchitis, acute exacerbations of chronic bronchitis, and pneumonia. These have been comparative against agents such as azithromycin, doxycycline, and amoxicillin. In the case of acute bronchitis, clinical efficacy rates of and have been reported for a regimen of mgbid. In treating acute exacerbations of chronic bronchitis, roxithromycin has been equivalent to amoxicillin; doxycycline, and azithromycin. Several small studies treating community-acquired pneumonia have also demonstrated efficacy rates of approximately withcomparability to erythromycin,azithromycin, clarithromycin, and amoxicillin. difference has been demonstrated between mg once daily and mg twice daily when treating atypical pneumonias; these have included infections causedby chlamydiae, mycoplasma, Legionella spp., and Coxiella burnetii. Responseshavebeen prompt, with efficacy rates comparable to erythromycin Despite modest in vitro activity against H . infiuenzae, analysis of the performance against this pathogen in various studies has demonstrated an response rate in treated patients. Intention to treat analysis also remains satisfactory at by meta-analysis There have been relatively few studies in children, although conditions such as tonsillitis, pharyngitis, impetigo, pneumococcal pneumonia, and pyoderma have responded satisfactorily, with response rates exceeding and bacteriological eradication rates ranging from to

Helicobacterpylon' Infections. Helicobacter pylori is susceptibleto roxithromycin with an MIC, of mg/L at pH H . pylori-associated duodenal ulcers have been treated with roxithromycin in combination with omeprazole and bismuth subnitrate. Eradication rates of approximately have been reported in small numbers of patients (66). Additional studies have confirmed healing month posttreatment using a regimenof roxithromycin forthe first weeks combined withthe addition of metronidazole forthe first days and lansoprazole for total a of weeks.

Skinand Soft Tissue Infections. Althoughprospective,comparative, double-blind studies have been few, roxithromycin has demonstrated clinicalefficacyratesvaryingbetween and in noncomparative studies In the treatment of erysipelas, roxithromycin mg bid has been as effective as penicillin with overall efficacy rates of and respectively.

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UrogenitalInfections. Roxithromycinhasbeenassessedinavarietyof gynecological and venereal infections. These have included nongonococcal urethritiskervicitis,genitalchlamydialinfections,andgonococcalcervicitis. Roxithromycin300 mg per day as a single dose or in divided dosesfor days has been shownto be effective inthe treatment of all these infections (67). Response rates have varied between 74% and 100%. Comparators have included doxycycline, minocycline, and erythromycin. OtherInfections. Roxithromycin150 mgsbd hasbeencomparedwith penicillin V in patients with uncomplicated erythema chronicum migrans; one study was stopped prematurely becauseof a less favorable response in the roxithromycin recipients (68). Further studies have indicated a more favorable response (69). The use of roxithromycin asthe primary preventionfor Pneumocystis curinii pneumonia (in combination with dapsone) and cerebral toxoplasmosis has been evaluated in a small number of patients with HIV infection (CD4 countsof <200/mm3). The initial results have been encouraging, but further experience is required (70). Likewise, early experience in treating cryptosporidiosis has produced encouraging preliminary information.

RP 59500 (QuinupristinlDalfopristin) Clinical experience with RP 59500 is limited to those studies included the in clinical trials program. Some indication of its likely performance in humans can be gained from animal studies. Animal Models. RP 59500 has been evaluated in a varietyof in vitro and animal models. In the rabbit model of endocarditis, it proved effective in treating both MRSA and MSSA infections, although strains with MICsof 0.5 m@ respondedlesswell(71).In the neutropenic mouse thigh model, RP 59500iseffectiveagainst S. uureus and S. pneumoniue and erythromycin-sensitive and erythromycin-resistant viridans streptococci. In addition, in amousesepticemicmodel,bactericidalactivitywasrapid against S. uureus and S. pneumoniue at 1 h postinfusion and persistingfor up to 8 h posttreatment, which is in contrast to the delayed bactericidal effect observed with vancomycin (72). Clinical Use. Quinupristiddalphopristin is currently undergoing trials in humans. Several hundred patients and healthy volunteers have received the drug in Phase I and Phase II/III studies. Dose-ranging studies on gramsensitive pneumonia and erysipelas have been conducted. In addition, comparative Phase I1 dose-ranging studies (at a dose of 5 or 7.5 m a g ) for community-acquired pneumonia and central venous catheter-related bacteremia are in progress. A Phase I11 comparative study in communityacquired pneumonia is scheduled.

MacrolideslStreptogramins-Overview

21

With the growing importance of vancomycin-resistant enterococcal infections, it is of interest to note that a noncomparative compassionate use study for the treatment of vancomycin-resistant E. faecium infections is in progress and includes patients with infective endocarditis and other challenging infections.

CONCLUSION The macrolides, azalides, and streptogramins are an expanding group of therapeutically important agents. Erythromycin, as the prototype macrolide, established the importance of these agents. The recognition of new infectious challengesof the respiratory tract added to its rolein managing atypical infections. However, it has significant limitations based largely on modest antimicrobial activity and side effects such as gastrointestinal intolerance. The new macrolides (including the azalides) have extended the indications of this classof drug into new therapeutic areas and have clearly demonstrated an improved safety profile. The streptogramins are a wellestablished group of agents whose clinical utility is just beginning to be explored through the availability of the parenteral synergistic agent RP 59500. This is timely, as clinical medicine is now increasingly confronted with a variety serious infections resistant to many conventional agents. The clinical results the in vitro promise ofRP 59500 in the management of such infections is eagerly awaited.

ACKNOWLEDGMENT The excellent secretarial assistance of Mrs M Thompson is gratefully acknowledged inthe preparation of this chapter.

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5. Rastogi N, Labrousse V. Extracellular and intracellular activitiesof clarithromycin used alone and in association with ethambutol and rifampin against Mycobacterium avium complex.Antimicrob.Agents Chemother 1991;35: 462-470. 6.KlemensSP, DeStefano MS,Cynamon "I. Activity of clarithromycin against Mycobacterium avium complex infection in beige mice. Antimicrob. Agents Chemother 1990;36:2413-2417. 7. Inderlied C. In vitro and in vivo activity of azithromycin against the Mycobacterium avium complex. J Infect Dis 1989;159:994-997. 8. Bermudez LE, Inderlied C, Young LS. Stimulation with cytokines enhances penetration of azithromycin into human macrophages. Antimicrob Agents Chemother 1991;35:2625-2629. 9. Rastogi N, Goh KS, Bryskier A. In vitro activity of roxithromycin against 16 species of atypical mycobacteria and effect of pH on its radiometic MICs. Antimicrob Agents Chemother 1993;37:1560-1562. 10. Wallace RJ, Brown BA, Jr, Onyi GO. Activity of clarithromycin (CL) against slow-growing nontuberculous mycobacteriausing a broth microdilution MIC system. 1stInternational Conference on the Macrolides, halides and Streptogramins. Santa Fe, NM, 1992. 11. Brown BA, Wallace RJ, Jr., Onyi GO, De Rosas G, Wallace RJ, 111. Activities of four macrolides, including clarithromycin, against Mycobacteriumfortuitum, Mycobacterium chelonae, and M . chelonae-like organisms. Antimicrob Agents Chemother 1992;36:180-184. 12. Ravdin JI, Skilogiannis J. In vitro susceptibilitiesof Entamoeba histolytica to azithromycin, CP-63956, erythromycin and metronidazole. Antimicrob Agents Chemother 1989;33:960-962. 13. Chang HR, Pechere J-CF. In vitro effects of four macrolides (roxithromycin, spiramycin, azithromycin, (CP-62,993) and A-56268) on Toxoplasma gondii. Antimicrob. Agents Chemother 1988;32:524-529, 14. Nougayrede A, Berthaud N, Bouanchaud DH. Post-antibiotic effects of RP 59500 with Staphylococcus aureus.J. Antimicrob. Chemother 1992; 30 (suppl A):101-106. 15. Pun SK, Lassman HB. Roliithromycin: a pharmacokinetic reviewof a macrolide. J AntimicrobChemother 1987;20 (suppl B):89-100. 16.Lassman HB, PuriSK, Ho I, Sabo R, Mezzino MJ. Pharmacokinetics of roxithromycin (RU 965). J Clin Pharmacol1988;28:141-52. 17. Chu SY, Deaton R, Cavanaugh J. Absolute bioavailability of clarithromycin after oral administration in humans. Antimicrob.Agents Chemother 1992;36: 1147-1150. 18. Chu SY, Park Y, Locke C, Wilson DS, Cavanaugh JC. Drug-food interaction potential of clarithromycin, anew macrolide antimicrobial. J Clin Pharmacol 1992;32:32-36. 19. Scaglione F, Fraschini F. Distribution of clarithromycinand its metabolite (14OH) in therapeutically relevant respiratory tract tissues and fluids. 1st International Conferenceon the Macrolides, halides and Streptogramins, Santa Fe, NM, 1992.

MacrolideslStreptogramim-Overview 20. Foulds G, Shepard RM, Johnson RB. The pharmacokinetics of azithromycin in human serum and tissues. J Antimicrob Chemother 1990; 25 (suppl A): 73-82. 21. Baldwin DR, Ashby JP, Andrews JM, Wise R, Honeybourne D. Pulmonary 500 mg oral dose. Thorax 1990; disposition of azithromycin following a single 45:324P. 22. Etienne SD, Montay G, Le Liboux A, Frydman A, Garaud JJ. A phase I, double-blind, placebo-controlled study of the tolerance and pharmacokinetic behaviour of RP 59500. J Antimicrob Chemother 1992;30 (suppl A):123-131. 23. Daniel R, AzithromycinStudy Group. Azithromycin,erythromycinand cloxacillin in the treatment of infections of skin and associated soft tissues. J Intern Med Res 1991;19:433-445. 24. Felstead SJ, Daniel R, European Azithromycin Study Group. Short-course treatment of sinusitis andother upper respiratory tract infections with azithromycin: a comparison with erythromycin and amoxycillin. J Intern Med Res 1991;19:363-372. 25. Hooton TA. A comparison of azithromycin and penicillinV for the treatment of streptococcal pharyngitis. J Med 1991;91:23S-26S. 26. Daniel R, AzithromycinStudy Group. Comparison of azithromycinand amoxicillinklavulanic acid in the treatment of otitis media in children. (Abstract) Proceedings of the 6th International Congress of Infectious Diseases, Montreal, 1990. 27. Schonwald S, Skerk V, Petricevic I, Car V, Majerus-Misic Lj, Gunjaco M. Comparison of three-day and five-day course of azithromycin inthe treatment of atypical pneumonia. Eur J Clin Microbiol Infect Dis 1991;10:877-880. Mertens JCC, vanBarneveldPWC,Asin HRG, Ligtvoet E, Visser MT, Branger T, Hoepelman AI. Double-blind, randomized study comparing the efficacies and safeties of a short (3-day) course of azithromycin and a 5-day course of amoxicillin in patients with acute exacerbationsof chronic bronchitis. Antimicrob Agents Chemother 1992;36:1446-1459. 29. Peters DH, Friedel HA, McTavish D. Azithromycin. A review of its antimicrobialactivity,pharmacokineticpropertiesandclinicalefficacy.Drugs 1992;44(5):750-799. 30. Dark D. Multicenter evaluation of azithromycin and cefaclor in acute lower respiratory tract infections. Am J Med 1991;91 (suppl3A):31S-35S. 31. Gris P. Once-daily,3-dayazithromycinversusathree-times-daily,10-day course of co-amoxiclav inthe treatment of adults with lower respiratorytract infections: results of a randomized, double-blind comparative study. J Antimicrob Chemother 1996;37(SupplC):93-101. 32. Zachariah J. A randomized, comparative study to evaluate the efficacy and tolerability of a 3-day course of azithromycin versus a 10-day course of coamoxiclav for the treatment of adult patients with lower respiratory tract infections. J Antimicrob Chemother 1996;37(SupplC):103-113. of azithromycin versus cefaclor in 33. O’Doherty B. An open comparative study the treatmentof patients with upper respiratory tract infections. J Antimicrob Chemother 1996;37(SupplC):71-81.

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34. Rodriguez AF. An open study to compare azithromycin with cefaclor in the treatment of paediatric patients with acute otitis media. J Antimicrob Chemother 1996;37(Suppl C):63-69. 35. Muller 0. open comparative studyof azithromycin and roxithromycin in the treatment of acuteupperrespiratorytractinfections. J Antimicrob Chemother 1996;37(Suppl C):83-92. 36. Laurent K. Efficacy, safety and tolerationof azithromycin versus roxithromycin in the treatment of acute lower respiratory tract infections. J Antimicrob Chemother 1996;37(SupplC):115-124. 37. Herbert A. Azithromycin in the treatment of skin and skin structure infections: a multicenter double-blind, double-dummy trial employing cephalexin as a comparative agent. (Abstract) Clin Res 1990;38929A. 38. Daniel R, European Azithromycin Study Group. Azithromycin, erythromycin and cloxacillin in the treatment of infections of skin and associated soft tissues. J Intern Med Res 1991;19:433-445. 39. Montero L. A comparative study of the efficacy, safety and toleration of azithromycin and cefaclor in the treatment of paediatric patients with acute skin and/orsoft tissue infections. J Antimicrob Chemother 1996. 40. WaughM. Open study of the safety and efficacy of a single oral dose of azithromycinfor the treatment of uncomplicatedgonorrhoeainmenand women. (Abstract) Proceedings of the Mediterranean Congressof Chemotherapy, Athens, 1992. 41. Stamm W. Azithromycin in the treatment of uncomplicated genital chlamydial infections. Am J Med 1991;91 (suppl3A):19-22. 42. Issoire C, Casin I, Perenet F, Brunat N, Janier M. Pilotstudy of azithromycin in the treatment of chancroid caused by Haemophilus ducreyi. (Abstract) Proceedings of the 6th International Congress of Infectious Diseases, 1990. Johnson, RC, 43. Massarotti EM, LugerSW, Rahn DW, Messner RP, Wong Steere AC. Treatment of early Lyme disease. Am Med 1992;92:396-403. 44. Strle F, Stanek G, Ruzic E, Susec-Michieli M. Erythema migrans: comparison of treatment with azithromycin, doxycycline and phenoxymethylpeniciln. (Abstract) Proceedings of the 6th International Congress of Infectious Diseases, 1990. 45. Glupczynski Y, Burette A. Failure of azithromycin to eradicate Campylobacter pylon from the stomach because of acquired resistance duringtreatment. Am J Gastroenterot 1990;85:98-99. 46. Young U,Wiviott L, Wu M, Kolonoski P,Bolan R, Inderlied C. Azithromycin for treatment of Mycobacterium avium-intracellulare complex infection in patients with AIDS. Lancet 1991;338:1107-1109. 47. Levenstein J. Clarithromycin versus penicillinin the treatment of streptococcal pharyngitis, J Antimicrob Chemother 1991;27 (suppl A):67-74. the treat48. Bachand R. A comparative studyof clarithromycin and penicillin in ment of outpatients with streptococcal pharyngitis. J Antimicrob Chemother 27 (Suppl A):75-82.

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49. Karma P, Pukander J, Penttila M, YlikoskiJ, Savolainen S, Oltn L, MelCn I, Lijth S. The comparative efficacy and safetyof clarithromycin and amoxycillin in the treatment of outpatients with acute maxillary sinusitis. J Antimicrob Chemother 1990;27 (suppl A):83-90. 50. Kawamura S , TakasakaT,Kobayashi T, Ikeda K, Endo S. Double-blind comparative clinical studyon TE-031 and josamycin in treatment of lacunar tonsilitis. Jibito Rinsho 1989;35:134-151. 51. Peters DH, Clissold SP. Clarithromycin. A review of its antimicrobial activity, pharmacokinetic properties and therapeutic potential. Drugs 1992;44(1): 117-164. 52. Hamedani P, Ali J, Hafeez S, Buchand R, Jr., Dawood G, Quereshi S, Raza R, Yab Z. The safety and efficacyof clarithromycin in patients with Legionella pneumonia. Chest1991;100:1503-1506. 53. Craft JC. Clarithromycin vs cephalosporin therapy for the treatment of H. influenrue bronchitis. 1st International Conference on the Macrolides, Azalides and Streptogramins,Sante Fe, NM, 1992. 54. Gupta S, Siepman H. Comparative safety andefficacy of clarithromycin versus stand'ard agents in the treatment of mild to moderate bacterial skin or skin structure infections. 1stInternational Conference on the Macrolides, Azalides and Streptogramins,Santa Fe, NM, 1992. 55. Dautzenberg B, Truffot C, Legris S , Meyohas M-C, Berlie HC, Mercat A, Chevret S , Grosset J. Activity of clarithromycin againstMycobacterium uvium infection inpatients with the acquired immune deficiency syndrome. Am Rev Resp Dis 1991;144:564-569. 56. Ruf B, Schurmann D, Mauch H, Jautzke G, Fehrenback FJ, Pohle HD. Effectiveness of the macrolideclarithromycinin the treatment of Mycobacterium avium complex infection in HIV-infectedpatients. Infection 1992; 20~267-272. 57. Shafran SD, Singer J, PhillipsP, the Canadian MAC StudyGroup. The Canadian Randomized Open-Label Trial of Combination Therapy for MAC Bacteremia: final results. 35thICAAC, San Francisco,1995. .58. May T, Brel F, Beucart C, Vincent V, Perronne C, Saint-Marc T, B. Dautzenberg B, Grosset J, CuraviumGroup. A French RandomizedOpen Trial of 2 Clarithromycin Combination Therapies for MAC Bacteraemia: first results. 35th ICAAC, San Francisco, 1995. 59. Wallace RJ, Jr, Tanner D, Brennan PJ, Brown BA, Craft JC. Preliminary results of an open noncomparative t i a l of clarithromycin (CL) in the therapy of (disseminated) infection due to Mycobacterium chelonae subsp. chelonae (Mcc). 1stInternational Conference on the Macrolides, Azalides andStreptogramins, Santa Fe, NM, 1992. 60. Graham DY, Opekun AR, Klein PD, Drnec J. Clarithromycinfor the eradication of H.pylori. 1st International Conference on the Macrolides, Azalides and Streptogramins, Santa Fe, NM, 1992. 61. Fernandes-Martin J, Leport C, Morlat P, Meyohas MC, Chauvin JP, Vide JL. Pyrimethamine-clarithromycin combination for therapy of acute toxoplasma

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65.

66.

67. 68.

69. 70. 71. 72.

73. 74. 75.

encephalitis in patients with AIDS. Antimicrob Agents Chemother1991;35: 2049-2052. Markham A, Faulds D. Roxithromycin. An update of its antimicrobial activity, pharmacokinetic properties and therapeutic use. Drugs 1994;48(2):297326. Schonwald S , Car V, Kuzman I, Mihaljevic F. Comparison of roxithromycin and erythromycin in the treatment of atypical pneumonias. (Abstract) 16th International Congress of Chemotherapy, Jerusalem, 1989. Cooper BC, Mullins PR, Jones MR. Clinical efficacyof roxithromycin in the treatment of adults with upper and lower respiratory tract infections due to Haemophilus influenzae. A meta-analysis of 12 clinical studies. Drug Invest 1994;7:229-314. BCgu6 P, Astruc J. The overall safety of oral roxithromycin in paediatric clinical studies. Infection 1995;23 (suppl 1):25-27. Okamoto S, Haruma K, Kawaguchi H, Inoue K, SumiiK,Kajiyama G, Vemura N, Sanuki E. Study on eradication of Helicobacter pylori by the combination useof roxithromycin, bismuth subnitrate, and omeprazole. 18th International Congress of Chemotherapy Stockholm, 1993; abstr. 960. Bircher Gelzer D, Rufli T. Roxithromycinin the treatment of nongonococcal urethritis.A double blind comparisonof two treatment regimens. (Abstract) 16th International Congress of Chemotherapy, Jerusalem, 1989. Hansen K, Hovmark A, Lebech AM, Lebech K, Ollsson I, Sorensen L. Roxithromycin in Lyme borreliosis: discrepant results of an in-vitro and invivo animal susceptibility study and a clinical trial inpatients with erythema migrans. Acta Derm Venerol1992;72:297-300. Gasser R, Wendelin I, Reisinger E, Bergloff J, FeiglB, Schafhalter I, Eber B, GrisoldM,Klein W. Roxithromycinin the treatment ofLymediseaseupdate and perspective. Infection 1995;23 (suppl 1):39-43. Durant J, Hazime F, Carles M, Pechere J-C, Dellamonica P. Prevention of Pneumocystis cariniipneumonia andof cerebral toxoplasmosisby roxithromycin in HIV-infected patients. Infection 1995;23 (suppl 1):33-38. Chambers HF. Studies of RP 59500 in vitro and in a rabbit model of aortic valve endocarditis caused by methicillin-resistant Staphylococcus aureus, J Antimicrob Chemother 1992;30 (suppl A):117-122. Berthaud N, Conard B, MontayG, Huet Y ,Bourges A, Bussiere JC, Santede M, Selingue M, Desnottes JF. RP 59500, in vitro bactericidal activity in a model of S. aureus mouse infection: influence of varying dosage and the number of treatments. Proceedings of the 32nd Interscience Conference of Antimicrobial Agents and Chemotherapy, Anaheim, 1992, pp. 69-71. Finch R. Antibacterial activityof quinupristiddalfopristin.Rationalefor clinical use. Drugs 1996; Sl(Suppl1):31-37. Goto S, Miyazaki S , Kaneko Y. The in-vitro activity of RP 59500 against gram-positive J Antimicrob Chemother1994;30 (suppl A):25-28. Verbist L, Verhaegen J. Comparative in-vitro activity of RP 59500, J. Antimicrob Chemother 1994;30 (suppl A):39-44.

2 Postantibiotic Effects and the Dosingof Macrolides, halides, and Streptogramins William A. Craig University Wkconsin Madison, Wisconsin

INTRODUCTION The optimal dosingof antibiotics is dependenton both the pharmacokinetics and pharmacodynamics of the drug. Pharmacodynamics, of course, is concerned with .the relationshipbetweendrugconcentrationsand the antimicrobial effect. The minimal inhibitory and bactericidal concentrations (MIC and MBC) have been the major parameters used over the years to describe the antimicrobial effect. Althoughthe MIC and MBC are good predictors of the potency of antibiotics, they do not describe the time course of antimicrobial activity.The effect of increasing concentrationson the rate of bactericidal activity andthe duration of the postantibiotic effect ( P M ) are much better predictors of the time courseof antimicrobial activity (1). Because increasing drug concentrations do not enhance bacterial killing by macrolides, azalides, and streptogramins, the duration of postantibiotic effectswith these drugs becomes an important determinant of their overallpharmacodynamicprofile.Thischapterwillsummarize current knowledge on the postantibiotic effects withthe various macrolides, azalides, and streptogramins. The impact of the postantibiotic effect on the appropriate dosing regimen for these antimicrobials will also be discussed. 27

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Craig

IN

POSTANTIBIOTIC EFFECTS

Persistent suppressionof growthafter intermittent drug exposureappears to be afeature of all macrolides, azalides, and streptogramins and has been observed with a varietyof respiratory tract pathogens In general, the duration of the in vitro postantibiotic effect has varied between and 9 h. Much of the variation is likely related to differences in media, environmental conditions, and bacterial strains. However, higher concentrations and longer exposure times produced more prolonged in vitro postantibiotic effects. The in vitro PAEs observedmyatlaboratory for erythromycin, clarithromycin, azithromycin, and the streptogramins (quinupristiddalfopristin and RP against multiple strains of Staphylococcus aureus, Streptococcuspneumoniae, and Haemophilus infruenzaeare shown in Table The duration of the in vitroPAEsweresimilar for erythromycin, clarithromycin, and azithromycin against the two gram-positive organisms. The streptogramins producedmuch longer in vitro PAEs against theseorganisms, especially following drug exposures of 1 h duration. Against H. injluenzae, clarithromycin and azithromycin exhibited longer in vitro PAEs than observed with erythromycin.

POSTANTIBIOTIC SUB-MIC EFFECTS Because drug levels in vivo do not fall abruptlyto barely detectable values as in the standard methodology for measuring the in vitro PAE, some investigators have examinedthe effects of sub-MIC concentrationson the duration of the in vitro postantibiotic effect The effect of sub-MIC concentrations of roxithromycin, clarithromycin, and azithromycinat onetenth and three-tenths of the MIC on the duration of the in vitro PAE observed with S. pyogenes and S. pneumoniae are shown in Table The duration of the in vitro postantibiotic was prolonged about when organisms were reexposed at one-tenth of the MIC and essentially doubled when exposed atthree-tenths of the MIC. The enhanced durationof the in vitro postantibiotic effect produced by sub-MIC concentrations largely explains the long durations of the postantibiotic effect exhibited in vivo in animal models

IN VIVO POSTANTIBIOTIC EFFECTS The neutropenic mouse thigh modelthe is only modelthat has been usedto evaluate the invivopostantibioticeffects of macrolides,azalides,and streptogramins An example of a typical in vivo postantibiotic effect study in this animal model is illustrated in Fig. Exposure of a

ug

Postantibiotic Effects and

the Dosing

29

MAS

Table I In Vitro Postantibiotic Effects with Erythromycin, Clarithromycin, Azithromycin, and Streptogramins AgainstStaphylococcus aureus ATCC and Streptococcus pneumoniae ATCC

Times MIC for Organism S. aureus

S. pneumoniae

H . influenzae

8 Times MIC for

lh Erythromycin Clarithromycin Azithromycin Streptogramins Erythromycin Clarithromycin Azithromycin Streptogramins Erythromycin Clarithromycin Azithromycin

-

-

-

-

-

-

Source: Data from Refs. 12-15 and based on 1-5 isolates for each organism.

standard strain of S. pneumoniae to 8 mgkg of azithromycin produced levels abovethe MIC for about 4.8 h. However,the organisms didnot start to resume logarithmic growthfor 11more hours. The duration of the in vivo PAEs obtained %vith erythromycin, clarithromycin, azithromycin, and quinupristiddalfopristinwith S. aureus and S. pneumoniae are shown in Table The values observed for all drugs are longer than the corresponding invitro PAEs listed in Table 1.Although the macrolides and azalides producedvitro in MICs of similar duration with Table 2 Impact of sub-MIC Concentrationson the Duration of the Postantibiotic Effect with S. meumoniae and H . influenzae

PAE (h) S. pneumoniae

H.influenzae

Roxithromycin Clarithromycin Azithromycin Roxithromycin Clarithromycin Azithromycin

Source: Data from Ref. 9.

.Increase inPAE (h) 0.1 MIC

0.3 MIC

30

Craig

r

1

lo

TzMlC

0

4

8

12

24

16

Time (h)

Figure l Time course of antimicrobial activity of m a g of azithromycin against S. pneumoniae ATCC in the thighs of neutropenic mice. Each point and error thighs. The solid bar representsthe duration bar represents the mean 2 S.D. of of time serum levels exceeded the MIC.

these two organisms, the durations of the in PAE were much longer for azithromycin than for clarithromycin and erythromycin. This is probably related to a longer persistence of subinhibitory concentrations with azithromycin than withthe other macrolides. The half-life of azithromycin in mice is at least fourfold longer than erythromycin and 1.6-fold longer than clarithromycin (12,13). Tabce In Vivo Postantibiotic Effects andthe Time Serum Levels Exceedthe MIC (T > MIC) for Various Dosesof Erythromycin, Clarithromycin, Azithromycin, and QuinupristidDalfopristinAgainst Staphylococcus aureus ATCC and Streptococcus pneumoniae ATCC Drug Organism S. aureus

S. pneumoniae

'

Erythromycin Azithromycin Quinupristinl dalfopristin Erythromycin Clarithromycin Azithromycin Quinupristinl dalfopristin

Source: Data from Refs. 12,13,15, and 16.

Dose Time (mag)

> MIC (h)

In vivo PAE (h)

Postantibiotic Eflects and the Dosingof MAS

31

lo

8 -

4 0

4

8

12

1624

20

Time (h)

Figure 2 Timecourse antimicrobialactivityof 100 mgkg quinupristinl dalfopristin againstS. aureus ATCC 25923 in the thighs neutropenic mice. Each S.D. 4-8 thighs.The solid bar pointanderror bar representsthemean

represents the duration time serum levels exceeded the MIC.

The regrowth of S. aurew and S. pneumoniae was also very prolonged following exposureto quinupristiddalfopriistin.The time courseof antimicrobial activity for 100 mg/kg of this drug combination against S. aurew is shown in Fig. 2. Because of the rapid elimination of quinupristid dalfopristin, serum levelsof this drug combination exceeded the MIC for only 1.8 h. Nevertheless, the in vivo PAE lasted h.

IMPACT OF DOSING INTERVAL EFFICACY

IN VIVO

The primary impact of the postantibiotic effect is on the dosing interval with intermittent drug administration. A prolonged PAE would prevent regrowth and maintain therapeutic efficacy even though serum and tissue levels of the drug fall below the MIC for considerable periods of time. The effects of varying dosing intervals on the in vivo efficacy of erythromycin, clarithromycin, azithromycin, andquinupristiddalfopristinagainst the same strain of S. pneumoniae used in the in vivo PAE studies are shown in Table 4. In vivo efficacy was measured bydetermining the cumulative 24-h dose (static dose) required to produce a net bacteriostatic effect over 24 h of therapy (17). With erythromycin and clarithromycin, increasing the dosing interval from 6toh12 and24 h resulted in a progressive rise theinstatic dose. With 24-h dosing, a bacteriostatic effect could notbe obtained even at doses erythromycin and clarithromycin that were 12-fold to 6.5-fold higher, respectively, than the cumulative dose requiredfor a bacteriostatic

.

32

Craig

Table 4 Impact of Dosing Interval on Static Dose for Erythromycin, Clarithromycin, Azithromycin, andQuinupristin/Dalfopristin with Streptococcus pneumoniue ATCC 10813 inthe Thighs of Neutropenic Mice

Drug Erythromycin Clarithromycin Azithromycin Quinupristid dalfopristin Source: Data from Refs.

MIC (mgn)

Half-life (min)

0.06 0.03 0.06 0.25

73 112 25

Static dose (mg/kg/U h) q 6 h q24 q 12h 12 11 13 10574

101 38 14 104

h >l44 > 72 12

and 15.

effect with 6-h dosing. Fernandeset al. (18) also observed lower mortality in a mouse peritonitis model infected with S. aurezu as the dosing frequency was increased from once to twice daily and from twice to thrice daily. On the other hand, longer dosing intervals had minimal or no effects on the cumulative dose requiredfor a bacteriostatic effect with azithromycin and quinupristiddalfopristin. The static doses for azithromycin were the same at all three dosing intervals. The cumulative 24-h dose required for efficacy of quinupristiddalfopristin increased only twofold as the dosing interval widened from 6 to 24 h. The prolonged in vivo postantibiotic effects produced by azithromycin and quinupristiddalfopristin likely account for the minimal impact of the dosing interval onthe in vivo efficacy of these drugs.

IMPACT OF NEUTROPHILS

IN VIVO EFFICACY

Neutrophils can enhance the in vivo activity of macrolides, azalides, and streptogramins by at least two mechanisms. McDonald and Pruul(l9) have demonstrated that organisms exposed to these drugsare more susceptible to the antimicrobial activity of neutrophils. With streptococci, pretreatment with erythromycin significantly enhanced phagocytosis. Macrolides, azalides, and streptogramins accumulate in cells and produce high intracellular concentrations (20-22). Lysisof neutrophils at sites of infection could release intracellular drug and enhance drug localconcentrations (21). Table 5 illustrates the activity of azithromycin, clarithromycin, erythromycin, and quinupristiddalfopristinagainst a standard strain of Streptu-

Postuntibiotic Efsects andthe Dosing of MAS Table S Impact of Neutrophils on Static Dose for Macrolides,halides, and Streptograminswith S. pneumoniae

Dosing

Static dose( m o d 2 4 h)

d-difference ormal tropenic (h)interval Drug Azithromycin 14.7

Clarithromycin Erythromycin Quinupristid dalfopristin

6 12 6

6 48.8 6

14.2-15.6 18.1 13.2 78.0

4.0-4.1 11.4 7.8

3.6-3.8 3.4 1.8 1.7 1.6

Source: Data from unpublished observationsby Craig.

coccus pneumoniue in the thighs of both normal and neutropenic mice (Craig, unpublished observations). The cumulative amount of drug (static dose) requiredto produce a net bacteriostatic effect over 24 h was 3.4-fold to 3.6-fold greater for azithromycin and 1.7-fold to 1.8-fold greater for clarithromycin, erythromycin, andquinupristiddalfopristinin neutropenic mice than in normal mice. The greater effect with azithromycin in the presence of neutrophils may be relatedto the very high accumulation of the drug in neutrophils andother phagocytes (21).

PHARMACOKINETIC PARAMETERS CORRELATING WITH IN VIVO EFFICACY There are several pharmacokinetidpharmacodynamicparameters, such as the area under the serum concentration versus time curve (AUC) to MIC ratio (AUCMIC), the peak level to MIC ratio, and the duration of time that serum levels exceed the MIC or MBC, that could be important in determining the in vivo efficacy of macrolides, azalides, and streptogramins. By evaluating the in vivo antimicrobial activity of several different cumulative drug doses administered at various dosing intervals, one can reduce the interdependence among these parameters and determine which parameter is of major importancein predicting efficacy (23). Studies with erythromycin have demonstrated that the duration of time serum levels exceedthe MIC isthe major parameter correlating with efficacyofthis drug against S. pneumoniue (23). Similar findings were obtained for clarithromycin with another strain of S. pneumoniue (12). The three panels in Fig. 3 demonstrate for clarithromycin against S. pneumoniue the relationship between the 24-h AUCMIC ratio (panel A), the

34

Craig

:

8

1 l 10 1

t

t """""_"

8

,!

" " " "

0 " "

g"--,

f3

0

Q 10

100

30

300

24-H AUCiMIC Ratio 11

I d (U

x i 9

0

1

3

0

10

30

100

(B)

Ratio PeaWMIC

(C)

Time Above MIC (percent)

300

Figure 3 Relationship of 24-h AUC/MIC ratio (panel A), peaWMIC ratio (panel B), and time above MIC (panel C) and the CFU of S. pneumoniae ATCC 10813 remaining in the thigh of neutropenic mice after 24 h of treatment with varying dosing regimensof clarithromycin. The dotted line represents the number of bacteria at the start of therapy.

Postantibiotic Effects and the Dosing of MAS

35

peak/MIC ratio (panel B), and the duration of time that serumlevels exceeded the MIC (panel C) and the efficacy of the drug in the thighs of neutropenic mice. In this study two to three mice were treated with 20 dosing regimensthat varied the total dose as well asthe dosing frequency. The number of bacteria remaining in the thigh after 24 h of therapy was then compared with each of three pharmacokinetidpharmacodynamic parameters. There was a poor correlation between bacterial numbers after 24 h of therapy andthe A U C M C and peak/MIC ratios. However,there was a highly significant correlation of bacterial numbers with the duration of time that serum concentrations exceededthe MIC. Serum concentrations needed to exceed the MIC for about 60% of the dosing intervalto produce a bacteriostatic effect after24 h of therapy. A similar study has been performed with azithromycin, but with markedly different results(13). The three panels in Fig.4 demonstrate for azithromycin againstthe same strainof S. pneumoniae the relationship between the three pharmacokinetic/pharmacodynamicparameters and the efficacy of the drug in the neutropenic murine thigh model. With drug, this the best correlation with efficacy was obtained with the AUC/MIC ratio followed by the peak/MIC ratio. In marked contrast to the results with clarithromycin, there was a poor correlation between bacterial numbers the andduration of time serum levels exceeded the MIC. Thus, with azithromycin,the amount of drug rather than the dosing regimen isthe major determinantof in vivo efficacy. Similar findings have been obtained with quinupristiddalfopriistin (15). As mentioned previously, the long in vivo postantibiotic effects observed with azithromycin and quinupristiddalfopristin most likely account for the different pharmacokinetidpharmacodynamic parameter correlating with efficacy for these drugs thanfor other macrolides.

CONCLUSIONS Macrolides, azalides, and streptogramins all produce postantibiotic effects with a variety of bacterial pathogens. The durationof in vitro PAEs is not always predictive of the magnitude in vivo PAEs. Azithromycin and quinupristiddalfopristinappear to produce much longer in vivo postantibiotic effects than observed with other macrolides. These long PAEs will maintain in vivo antimicrobial efficacy even with prolonged dosing intervals. The amount drug, rather than the dosing interval, is the major pharmacokinetidpharmacodynamicparameter determining the efficacy of azithromycin and quinupristiddalfopriistin. The postantibiotic effects with the macrolides are not long enough to prevent the lost of in vivo efficacy withwidelyspaceddosingregimens. The goal of dosingregimens for erythromycin and clarithromycin is to produce levels above the MIC for at

c

.$

"i

@ """_

4

" " " " " " " " " " " "

7

100

10

1000

24-H Ratio AUC/MIC 10

l

100

10

1

Ratio PeaklMlC lo

(C)

l

0

20

40

60

I

I

80

100

Time Above MIC (percent)

Figure 4 Relationship of 24-h A U W C ratio (panelA), p e a M C ratio (panelB),

and time above MIC (panel C) and the CFU of S.pnewnoniue ATCC 10813 remaining in the thigh of neutropenic mice after 24 h of treatment with varying dosing regimens of azithromycin. The dotted line represents the number of bacteria at the starttherapy.

36

Postantibiotic Effects theand

Dosing of MAS

37

least 50-60% of the dosing interval. The presence of neutrophils can enhance the in activity of these drugs, especially azithromycin, possibly by carrying the drugto sites infection.

REFERENCES 1. Craig WA,Ebert SC. Killingand regrowthof bacteria in vitro: areview. Scand J Infect Dis 1991; 74(suppl):63. 2. McDonald PJ, CraigWA, Kunin CP. Persistent effectof antibiotics on Staphylococcus aureus after exposure for limited periodsof time. J Infect Dis1977; 135:217. 3. Gerber AU, Craig WA. Growth kinetics of respiratory pathogens after short exposures to ampicillin and erythromycin in vitro. J Antimicrob Chemother 1981;8 (suppl):81. 4. Kuenzi B, SegessenmannC, Gerber AU. Postantibiotic effectof roxithromycin, erythromycin, and clindamycin against selected Gram-positive bacteria and Haemophilus influenzae.J. Antimicrob. Chemother 1987;20 (Suppl B):39. 5. Hardy DJ, Swanson RN, Rode RA, Marsh K, Shipkowitz NL, Clement JJ. Enhancement of the in vitro and in vivo activities of clarithromycin against Haernophilus influenzae by 14-hydroxy-clarithromycin,its major metabolite in humans. Antimicrob AgentsChemother 1990;34:1407. 6. Watanabe T, Kanno M, Tejima E, Orikasa Y. Effects of macrolides on ultrastructure of Staphylococcus aureus during postantibiotic phase. Drugs Expl. Clin Res 1992;18:81. 7. Scaglione F, Dugnani S , Demartini G, Saudelli M, Galmozzi G, Fraschini F. The postantibiotic effect of clarithromycin and its major human metabolite, 14-hydroxy clarithromycin. J Antimicrob Chemother 1993;32:507. Odenholt-Tornqvist I, Lowdin E, Cars 0. Postantibiotic sub-MIC effects of vancomycin, roxithromycin, spariloxacin, and amikacin. Antimicrob Agents Chemother 1992;36:1852. 9. Odenholt-TornqvistI, Lowdin E, Cars 0.Postantibiotic effects and postantibiotic sub-MIC effects of roxithromycin, clarithromycin, and azithromycin on respiratory tract pathogens. Antimicrob AgentsChemother 1995;39:221. 10. Craig WA. Post-antibiotic effect of macrolides. In: Macrolides Bryskier A, Butzler JP, Neu HC, and Tblkens PM, eds. Paris: Arnette Blackwell, 1993: p 205. 11. Nougayrede A, Berthaud N, Bouanchaud DH. Post-antibiotic effects of RP 59500 with Staphylococcus aureus. J Antimicrob Chemother 1992; 30 (suppl A):101. 12. Ebert S, Rikardsdottir S, Craig WA. Pharmacodynamic comparisonof clarithromycin vs erythromycin. Program and Abstracts of the Thirty-first Interscience Conferenceon Antimicrobial Agents and Chemotherapy, Washington, DC, 1991; abstr 509. 13. Craig W, Rikardsdottir S, Watanabe Y. In-vivo and in-vitro postantibiotic effects (PAEs) of azithromycin. Program and Abstracts of the Thirty-second

38

14.

15.

16. 17. 18. 19. 20.

21.

22.

23.

Craig Interscience Conferenceon Antimicrobial Agents and Chemotherapy,Washington, DC, 1992; abstr 45. Craig W, Watanabe Y. In-vivo and in-vitro postantibiotic effects (PAEs) of RP 74501/74502. Program and Abstracts of the Thirty-second InterscienceConference on Antimicrobial Agents and Chemotherapy, Washington, DC, 1992; abstr 1304. Craig W, Ebert S. Pharmacodynamic activities of RP 59500 in an animal infection model. Program and Abstracts of the Thirty-third Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 1993; abstr 470. Vogelman B, Gudmundsson S, 'hmidge J, Leggett JE, Craig WA. The in vivo postantibiotic effect in a thigh infection in neutropenic mice. J Infect Dis 1988;157:287. Fantin B, Leggett J, Ebert S, Craig WA. Correlation between in vitro and in vivo activity of antimicrobial agents against gram-negative bacilli in a murine infection model. Antimicrob Agents Chemother 1991;35:1413. Femandes PB, Swanson RN, Hardy DJ, McDonald EJ, Ramer N. Effect of dosing intervals on efficacy of clarithromycin and erythromycin in mouse infection models. Drugs ExpClin Res 1988;14:441. McDonald PJ, Pruul H. Macrolides and the immune system. Scand J Infect Dis 1992;83:34. Anderson R, Joone G, van Rensburg CE. Anin evaluation of the cellular uptake and intraphagocytic bioactivity of clarithromycin (A-56268, TE-031),anewmacrolideantimicrobialagent. J. Antimicrob. Chemother 1988;22:923. Gladue RP, Bright GM, Isaacson RE, Newborg MF. In vitro and in vivo uptake of azithromycin (CP-62,993) by phagocytic cells: possible mechanism of delivery and release at sites of infection. Antimicrob Agents Chemother 1989;33:277. Desnottes JF, Diallo N. Cellular uptake and intracellular bactericidal activity of RP 59500 inmurine macrophages. J AntimicrobChemother 1992;30 (suppl A):107. Vogelman B, Gudmundsson S, Leggett J, 'hmidge J, Ebert S. E, Craig WA. Correlation of antimicrobial pharmacokineticparameters with efficacy in an animal model. J Infect Dis1988;158:831.

Ketolides: New Semisynthetic 14-Membered-Ring Macrolides Andrc! Bryskier, Constantin Agouridas, and Jean-Franqois Chantot Roussel-Uclaf Romainville, France

Erythromycin A wasdiscovered at a timewhen a new drug active against penicillinase-producingS. uurew strains was needed. Penicillinaseproducing resistant strains appeared in London hospitals very shortly after the first clinical use of penicillin G, and soon spread worldwide (1). This led to parallel research projectsby the pharmaceutical industry and generated semisynthetic methicillin and isoxazolylpenicillins (oxacillin, cloxacillin, and flucloxacillin), as well the as first cephalosporinsfor parenteral use such as cephalothin and cephalorodine. Other antibiotic families have since been discovered, notably with the discovery of several natural macrolides produced from fermentationby Streptomyces species. Erythromycin A was the most active compound in vitro, but its use was rapidly limited bythe development of other drugs active on penicillinresistant S. uurew isolates and the fact that it shows only a bacteriostatic activity. In addition, erythromycin pharmacokinetic behavior iserratic, as the drug is unstable in acid conditions (2), despite attempts to circumvent this problem by means of physicochemical modifications such as esterification of the hydroxyl group in the 2’ position of the D-desosamine sugar, stochiometric admixture with the stearate, and the mixture of a salt and 39

40

Bryskier et al.

T-ester such as erythromycin A estolate (lauryl sulfate and erythromycin 2“propionate) (3). All these drawbacks led to a stagnationof clinical use of erythromycin A and other macrolides, with few exceptions. Semisyntheticmacrolidesbelonging to the secondwave,suchas roxithromycin, clarithromycin, azithromycin, and dirithromycin, were developed in response to the discoveries of Legionella pneumophila (4) and the majorroleplayed by Chlamydia trachomatis inhumanpathology. These new compounds were designedto have better activity than erythromycin A on Legionella spp., Chlamydia spp., and Mycoplasma spp., good stability in acid conditions, and good gastrointestinal absorption. Their in vitro activity is comparable to that of erythromycin A against common pathogens. Globally, these goals were achieved (5,6). The pharmacokinetic problems were overcomeby these new derivatives. W Okind of compounds could be described, those which show a high concentrations in plasma and tissues such as roxithromycin and clarithromyand thosewhose plasma levels are low (azithromycin and dirithromycin), and for whichthe term “tissue-directed kinetics”was coined (7). A new era opened with the use of these drugs, but it coincided with the post-penicillin era characterized by the emergence of strains resistantto mainy antibiotics, including macrolides. The new macrolide derivatives are intended to deal with the epidemiological environments, including the emergence of strains resistant to erythromycin A (gram-positive cocci, Campylobacter spp., H . pylon, Mycobacterium aviumcomplex). Other medical needs appeared with the emergence of S. pneumoniae isolates resistant to penicillinG , some of which are multidrug resistant. Several drugs belonging to different chemical classes will be necessaryto deal with these newpredators. Research has focused on new compounds with the pharmacokinetic advantages of existing drugs andthe same antibacterial activityon atypical organisms, but also active on strains resistant to erythromycin A and other antibacterials. No attempt was made to extend activity to Enterobacteriaceae. The first fruitsof this researchare the ketolides. Ketolides are semisynthetic 14-membered-ring macrolides (Fig. 1). W Ocategories of compounds have been synthesized on the basis of experience gained with previous macrolides: blockadeof the weak point in position C-9 of the erythronolide A ring, or blockade of the anchor points in C-l1 and C-6. During the development of new therapeutic agents, attention was focusedon blockade of the C-6 hydroxylgroup by a methyl group of the erythronolide A ring thereby preventing hemiketalization (Fig. 2). It had been shown that the presence of a carbamate in C-11-C-l2 increased the antibacterial activity of erythromycin A. A series of compounds with different side chains fixedto the carbamate in C-11-C-l2 was synthesized, and a numberof compounds were selectedfor preclinical evaluation.

Ketolides: New Semisynthetic Macrolides

Figure I

41

Ketolide, a semisynthetic 14-membered-ring macrolide.

The main structural innovation is the lack of the neutral sugar: Lcladinose. The 1Cmembered-ring macrolidesare considered bacteriologically inactive in the absence of an L-cladinose in position 3. This was shown with roxithromycin descladinose, clarithromycin descladinose, and azithromycindescladinose. The derivativesthusobtainedpossessa3hydroxyl (3-OH) group on the erythronolide A ring. However, oxidation of the 3-OH group gave riseto a new chemical subclass of 1Cmemberedring macrolides-ketolides, with a 3-keto instead of a 3-OH (Fig. 3). The 3-keto macrolides far produced differ by the side chain attached to the C-ll-C-12 carbamate or carbazate Ketolidespossess two new propertiesrelative to previoussemisynthetic macrolides: Strongacidstability Higher in vitro activity against gram-positive cocci isolates susceptible or resistant to erythromycin A The ketolides are far more stable in acid conditions than clarithromycin, roxithromycin, and azithromycin. It was shown that the latter compounds were degraded in acid conditions by transformation into descladinose derivatives, with a 3-OH group on the lactone ring (10,ll).

42

Bryskier et al.

Interaction (internal ketailization)

Stable derivative Figure 2 Blockade of the C-6 hydroxyl group by a methyl group of the erythronolide A ring.

These entities are stable in acid conditions but do not inhibit bacterial growth. The ketolides, which are 3-keto derivatives,are highly acid stable. Their in vitro activity remains at very lowpH for more than 6 h. On thecontrary, at pH1.2, clarithromycin or azithromycin is devoidof any in vitro activity after a l-h contact. The second novelty isthe lack of cross-resistance with strains resistantto erythromycin A by an inducible mechanism. The ketolides are active against S. aureus, S. pneumoniae, and S. pyogenes isolates resistant to erythromycin A. The antibacterial activityof ketolides against isolatesshowing a decreased susceptibility to erythromycin A, remains high evenif minimal inhibitory concentration(MIC) values are slightly higher than against wild strains. The ketolides have the same

Erythromycin A

Descladinose erythromycin A

3-keto Derivative

Figure 3 Oxidation of the 3-OH group, giving a ketolide.

Ketolides: NewMacrolides Semisynthetic

43

antibacterial spectrum as other macrolides,coveringgram-positiveand gram-negative cocci, gram-positive and gram-negative bacteria, including H . infZuenzae and M . catarrhalis, aswellas anaerobic bacteria and intracellular pathogens such as Chlamydia spp., Legionella spp., andRickettsia spp. Other atypical microorganisms are included such as Mycoplasma spp., Ureaplasma urealyticum, Campylobacter spp. , and H. pylori. Although ketolides sharethe activity of other macrolides on erythromycin A-susceptible strains, they differ in terms of their activity on strains resistant to erythromycin A. They also show good activityon S. pneumoniae isolates resistant to erythromycin A (inducible or constitutive) and those resistant to penicillin G . They are active against S. aureus strains resistant to erythromycin A by an inducible mechanism but not constitutively resistant to erythromycin A. Their L-cladinose counterparts are not active againstS. aureus isolates resistantto erythromycin A by an inducible mechanism. They possess better in vitro activity against clarithromycinsusceptible or clarithromycin-resistant isolates, as shown with H. pylori and Mycobacterium avium complex. Another highly interesting aspect of these 14-membered-ring compounds is their activity on bacterial species usually insensitiveto this typeof compound, such asMycoplasma hominis. The ketolides are also active on anaerobes. Overall, the mechanism of action is fairly classical(i.e., inhibition of protein synthesis). However, it no doubt differs from that of erythromycin A at the molecular level.

RU One compound, RU 64004, has been selected further for preclinical investigations. It is a ketolide derivative which bears a cyclic-hydrazano carbamate at theC-11-C-l2 positions of the erythronolide A ring on which a quinoline moiety is linked by an alkyl side chain (Fig.RU 4). 64004 isone of the most active macrolides against gram-positive cocci, anaerobes, intracellular organisms, and atypical bacteria. It is more active than clarithromycin against susceptible S. aureus isolates (MIC, 0.01m&). It isactiveagainst inducible erythromycin-resistantS. aureus isolates (MIC, 0.04 m&) but inactive against constitutively erythromycin A-resistant strains (Fig. 5 ) . It is highly active against enterococci susceptible to erythromycin A but also against inducible erythromycin resistant isolates (MIC, 0.01 m&), and vancomycin-resistant strains (MIC,, : 0.06 m&); lower in vitro activity is noted against constitutively erythromycin A-resistant isolates (MIC,,: 2.5 m&). Clarithromycin is inactive on such strains. RU 64004 is the most active macrolide against Streptococcus spp. (MIC, : 0.001 mgL). It exhibits goodactivityagainsterythromycin-

-

-

-

Bryskier et al.

44

\ Figure 4 The ketolide derivative RU

\ \

1

Figure

Ketolide RU

staphylococci.

Ketolides: New Semisynthetic Macrolides

Figure 6 Ketolide RU

45

S. pneumoniae.

resistant isolates (MIC,, : 0.01 m&) compared to clarithromycin. It also showshighactivityagainst the viridansgroup of streptococci (MIC,, -0.001m@).Against Streptococcus pneumoniae isolatessusceptible (MIC, : 0.016 m@) or resistant to erythromycin A (MIC,, : 0.06 m@), RU 64004 displays good activity. It exerts a strong and identical in vitro activity againstS. pneumoniue isolates susceptibleor intermediately susceptible to penicillin G (MIC, : 0.016 m&), but slightly lower in activity has been noted against penicillin G-resistant strains (MIC, : 0.06 m a ) ; clarithromycin can be considered inactive (MIC,, : 2 m@) (Fig. 6) (12,13). The available macrolidesare poorly active or inactive againstanaerobes as awhole(14).Compared to metronidazole, RU 64004shows enhanced activity against Bacteroides fragilis (MIC,, 0.5 m@). It is four times more active than metronidazole against B . thetaiotumicron (MIC, : 0.15 m@). It isrespectivelytwoandeighttimesmoreactive than clarithromycin and azithromycin against Fusobacteria spp. (MIC, : 0.06 m a ) .

-

Bryskier et al.

46

c

Figure 7 Ketolide RU

Mycoplasma spp.

It is highly active against Clostridium spp. andPeptostreptococcus spp. (15). It is more active than clarithromycin against H.influenzae (MIC, 1m&) and shows identical activity against Moraxella catarrhalis (MIC,, : 0.12 mgl L) (16).Unusualamong the macrolidesis the strongactivityagainst Neisseria meningitidis (MIC, : 0.007 m&) (17). Preliminary results show that RU 64004 iseffective againstChlamydia spp. (18) and Legionella spp. (19). One interesting characteristicis the good in vitro activity of RU 64004 against Mycoplasma hominis (Fig. 7) (MIC -0.2m a ) and M. fermentum (MIC m&) which are usually resistantto 14-membered-ring macrolides. It is effective againstM.pneumoniae (MIC : 0.01 m&) and respectively 5 times and 100 times more active againstUreaplasmu ureulyticum, whatever the resistance to tetracycline (MIC: 0.01 m&) (20). The in vivo antibacterial activityof RU 64004 wasexplored comparatively to that of erythromycin, clarithromycin, azithromycin,and pristinamycin. In murine septicemia due to S. aureus erythromycin-susceptible,

-

-

Ketolides: New Semisynthetic Macrolides

47

RU shows comparable activityto clarithromycin(PD, : mgkgMIC value0.01 m@), but it shows good in vivo activity againstS. aurew erythromycin-resistant byan induciblemechanism(PD, : mgkg), whereas clarithromycinwas totally inactive (PD50 < mgkg). It is also mgkg) than clarithromycin (PD50 : mg/kg) moreactive (PD, : against S. aurew resistant to erythromycin byan induciblemechanism and oxacillin. Against S. pneumoniae whatever the susceptibility of the isolate to erythromycin, RU showsgoodactivity. PD,s range between mgkg (erythromycin-susceptible strain) and mgkg (erythromycinresistant isolate-constitutive mechanism). Against erythromycin-resistant isolates whatever the mechanism of resistance, other tested macrolides were inactive(PD, < 50 mg/kg). RU displays a good in vivo activity against S. pyogenes (PD,, : mgkg) and S. agalactiae (PD, : mgkg). It is more active than pristinamycin against enterococci,PD, withbetween mgkg ( E . faeciurn erythromycin-R and Van-R)and mgkg ( E . faecium erythromycin-R). In experimental murine septicemia induced by H.influenzae, (Figure RU shows an in vivo activity closed to that of azithromycin. PD,g ranged between mgkg (ampicillin-S) and mgkg (ampicillin-R, lactamase neg) Experimental pneumoniawas induced inSwiss mice, byintratracheal inoculation of virulent S. pneumoniae isolates either susceptible to erythromycin A (S. pneumoniue or constitutively erythromycin-resistant(S. pneumoniae The drug administration started at h and h after challenge. live regimens have been given intraperitoneally : 50 and mgkg. MIC values for RU were and 0.5 mgL for erythromycinA susceptible and erythromycin-resistant isolates, respectively. Whenadministered every h or h, survival rates were and respectively. Comparatively,there was no survival with erythromycin. C5+ BV6 mice were intratracheally infected with H.influenzae type b (108 CFU). Bacterial lung clearancewas assayed after a singleoral dose of mgkg of RU or 50 mgkg of azithromycin, administered h after challenge. Both compounds exhibit identical activities with a log CFU reduction in burden compared to control. Initial clearance (at h) was more efficient withRU than with azithromycin

CONCLUSION These compounds will provide a therapeutic alternativeto currently available macrolides, and other oral antibiotics. They hold great promise for resolving problemsthat are likely to become acute inthe near future.

48

Bryskier et al.

Ketolides: New Semkynthetic Macrolides

49

REFERENCES 1. Bryskier A, Labro M-T. Macrolides nouvelles perspectives thtrapeutiques. La Presse Med 1994;23:1762-1766. 2. Bryskier A, Agouridas C, Chantot J-F. Acid Stability of new macrolides. J Chernofher 1993;5 (suppl1):158-159. 3. Bryskier A, Chantot J-F, Labro M-T, Gasc J-C. Evolution et tendance moderne de l'antibiothtrapie. InZribi A, Bryskier A, eds. L'antibiothtrapie d'aujourd'hui et dedemain Paris: Amette-Blackwell, 1989:ll-29. F. Legionnaires' disease: pathological and 4. Blackman J, Hicklin MD, Chandler historical aspect of a new disease. Arch Pathol Lab Med 1978;102:337-343. 5. Bryskier A. Newermacrolidesand their potential target organisms. Curr Opinion Infect Dis1992;5:764-772. 6. Bryskier A, Agouridas C, Chantot J-F. NewInsights into the structure-activity relationship of macrolides and azalides. In:Neu HC, Young LS, Zinner SH, Acar JF, eds. New Macrolides, Azalides and Streptogramins in Clinical Practice. Marcel Dekker Inc, New York: 19953-30. J Med 1991;92 7. Schentag J, Ballow CH. Tissue-directed pharmacokinetics. Am (Suppl3A):5S-llS. 8. Agouridas C, Benedetti Y,Denis A, Fromentin C, Gouin D'ambriBre S, Le Martret 0, Chantot J-F. Ketolides, a new distinct semi-synthetic class of macrolides. In: Program and Abstractsof the Thirty-fourth InterscienceConference on AntimicrobialAgentsandChemotherapy, Orlando, FL, 1994. Washington DC: American Societyfor Microbiology, 1994;abstr F-164. 9. Agouridas C, Bonnefoy A, Chantot J-F. In vitro antibacterial activity of RU 004, a novel ketolide highly active against respiratory pathogens. In :Program and Abstracts of the Thirty-fifth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 1995. Washington DC:Amencan Societyfor Microbiology, 1995; abstr F-158. 10. Fiese EP, Steffen SH. Comparison of the acid stability of azithromycin and erythromycin A. J Antimicrob Chemother 1990;25 (suppl A):39-47. 11. Pun SK, Lassman HB. Roxithromycin: pharmacokinetic review of a macrolide. J Antimicrob Chemother1987;20 (suppl B):89-100. 12. Ednie L, Jacobs MR, Appelbaum PC. Activityof the ketolide RU 004 compared to 15 other agents against 112 erythromycin-susceptible and resistant pneumococci. In: Program and Abstractsof the Thirty-fifth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 1995. WashingtonDC: American Societyfor Microbiology, 1995;abstr F-159. 13. Frtmeaux A, Sissia G, Chantot J-F, Geslin P. Antibacterial activity of the ketolide RU 004 against multi-resistant pneumococci. In: Program and Abstracts of the Thirty-fifth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 1995.WashingtonDC:American Society for Microbiology, 1995;abstr F-160. 14. Dubreuil, L. Activity of macrolides against anaerobes in vitro. In: Bryskier AJ, Butzler J-P, Neu HC, Tulkens PM, eds. Macrolides, Chemistry, Pharmacology and Clinical Uses. Paris: Amette-Blackwell, 1993:183-196.

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15. Ednie L, Spangler S, Jacobs M, Appelbaum P. Anti-anaerobic activity ofthe ketolide RU 004 compared to 4 macrolides, 5 p-lactams, clindamycin and metronidazole. In: Program and Abstracts of the Thirty-fifthInterscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 1995. WashingtonDC: American Societyfor Microbiology, 1995;abstr F-162. 16. Dabernat H, SeguyM, DelmasC.Invitroactivity of RU 004 against Haemophilus injluenzaeand Moraxella catarrhalis.In: Program and Abstracts of the Thirty-fifth Interscience Conferenceon Antimicrobial Agentsand Chemotherapy, San Francisco,CA, 1995. Washington DC: American Societyfor Microbiology, 1995;abstr F-161. 17. Fabre R, Cavallo JD, Chapalan JC, Meyran M. Comparative invitro activity of RU 004 against Neisseria gonorrhoeae and Neisseria meningitidis. In: ProgramandAbstracts of the Thirty-Fifth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco,CA, 1995. Washington, DC: American Society forMicrobiology, 1995; abstr F-164. 18. Haider F, Eb F, Orfila J. Ketolides and Chlamydia: in vitro evaluation of RU 004. In: Program and Abstractsof the Thirty-fifth InterscienceConference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 1995. Washington DC: American Societyfor Microbiology, 1995; abstr F-165. on 19. Bornstein N, Behr H, Brun Y, Fleurette J. In vitro activity of RU Legionella species. In Program and Abstractsof the Thirty-fifth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 1995. Washington DC: American Society for Microbiology, 1995; abstr F-166. 20. Renaudin H, Aydin MD, Btbtar C. Ketolides and Mycoplasma: in vitro evaluation of RU 004. In: ProgramandAbstracts of the Thirty-fifth Interscience Conferenceon Antimicrobial Agents and Chemotherapy, FranSan cisco, CA, 1995. Washington DC: American Society for Microbiology, 1995; abstr F-168. 21. Agouridas C, Bonnefoy A, Chantot J-F. In vivo antibacterial activity of RU 004, a novel ketolide highly active against respiratory pathogens.In: Program and Abstracts of the Thirty-fifth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 1995. Washington DC: American Societyfor Microbiology, 1995; abstr F-171. 22. Rajagopalan-Levasseur P, Vallte E, Agouridas C, Chantot J-F, Pocidalo J-J. RU 004: activity against erythromycin-resistant pneumococci and Haemophilus injluenzae in murine pneumonia model.In: Program andAbstracts of the Thirty-fifth Interscience Conference on AntimicrobialAgents and Chemotherapy, San Francisco, CA, 1995. Washington DC: American Society for Microbiology, 1995; abstr F-173.

4 Streptogramins: From Parenteral to Oral Daniel H.Bouanchaud* RhSne-Poulenc Rorer Vitry-sur-Seine, France

SUMMARY Streptogramins are complex mixtures of macrolactones, synthesized by Streptomyces spp. Until recently, the complex structure of streptogramins made it difficultto study their mechanismof action; it was not possible to prepare semisynthetic derivatives with improved pharmacokineticproperties nor to precisely determine the P W D parameters of these drugs in humans. Novel streptogramins consistof two well-defined components, which act synergicallyat the ribosomal level.RP 59500 (quinupristiddalfopristin) is a new semisynthetic injectable streptogramin. RPR 106972 (RPR 13919/ RPR 160950) isa cocrystalline association, derived from natural a streptogramin; this drug is well absorbedby the oral route and might be considered as an oral partner for RP 59500. Both drugs have similar effectiveness (MIC, 2 m&) against multiresistant S. aureus, S. epidennidis, Sa. pneumoniae, many other streptococci, E . faecium, Legionella spp., M. catarrhalis, and Mycoplasma spp. This activity has been confirmed with animal models of severe infections

*Current affiliation:MEDICOM, Paris,France

51

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Bouanchaud

and has also been observed in human infections (Phase I11 and I1 respectively for quinupristiddalfopristin and RPR

INTRODUCTION The streptogramins are antibiotics of natural origin. They have been isolated from Streptomyces, aswellasfrom several other genera (Actinoplanes, Actinomadura, and Micromonospora). They consist of a natural association of two types of molecules, groupA (or type 11) streptogramins and groupB (or type I) streptogramins. Of interest from a chemical standpoint is that the group A and B components are structurally dissimilar (group A compounds are olefinic macrolactones; groupB compounds ate peptidic macrolactones) and, from a microbiologicalstandpoint, that these two types of components are synergistic in terms of their antibacterial activity. Their antibacterial spectrum is fairly uncommon; it includes most gram-positive cocci and several respiratory pathogens (1). The streptogramins exert their activity by inhibiting protein synthesis (2). The two groups of components bindto the subunit of the bacterial ribosome. Group A streptogramins interfere with the function of peptidyl transferaseandalsotriggeraconformationalchangein the ribosome, which greatly increasesthe affinity of the group B streptogramins for these organelles. The group B streptogramins halt peptide chain elongation. Finally, the steric effects of the streptogramins may also destabilize the ribosome (3). The final result is that the two types of compound, which separately have only bacteriostatic activity, often have bactericidal activity when combined. Of the streptogramins, it is the pristinamycins that have been studied most extensively. Isolation of the pristinamycins from Streptomyces pristinaespiralis yields a number of molecules, of which the principal ones are pristinamycin I, and pristinamycin 11, (see Fig. 1)(1). The streptogramins found in nature are not water soluble; therefore, it is not possible to use them asparenteral drugs. However,by chemically modifying pristinamycin I, andpristinamycin 11, water-soluble derivatives have been obtained. These compounds, quinupristin (RP are dalfopristin (RP combined in a 30 : w/w ratio, constitute quinupristiddalfopristin (RP the first injectable streptogramin (4). This new semisynthetic antibiotic is currently undergoing Phase I11 clinical trials. The synthesis of quinupristin ((56R)- [(3S)-quinuclidinyl] thiomethyl pristinamycin],) involved the formation of a methylene ketone at the 56 position of the 4-oxopipecolinic acid residue of pristinamycin I,, followed by a stereoselective Michael-type addition of (3S)-quinuclidinyl thiol (Fig. The synthesis of dalfopristin ((264-diethylaminoethylsulphonylpristina-

53

Streptogramins: From Parenteral to Oral

Plistinamydn IA

Figure I

Pdsmmyckr

Major natural pristinamycin components.

mycin 11,) involved a stereoselective Michael-type addition on the dehydroproline ringof 2-diethylaminoethanethiol, followed bythe selective dation of the resulting26-thioether (1) (Fig.2).Thesemodifications yielded compounds as active as the natural pristinamycins(4). Reviewed inthe following are the in vitro and experimental vivo in activities of quinupristiddalfopristin (RP 59500) reported at the 35th Interscience Conferenceon Antimicrobial Agents and Chemotherapy (ICAAC) 1995 and at the Third International Conference on the Macrolides, Azalides and Streptogramins (ICMAS) 1996 for several gram-positive pathogens that cause severe disease andare increasingly implicated as agents of nosocomial infection. A streptogramin for oral use, RPR 106972, is at an early stage of development (Phase I1 clinical trials).The preliminary data obtained with this new compound will be discussed the second in part of this chapter. I

Figure 2 Structure of RP 59500.

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QUINUPRISTIN/DALFOPRISTIN(RP 59500) Activity of Quinupristinhlfopristin (RP 59500) Versus Methicillin-Resistant Staphylococcus aureus and other Staphylococci The results of testing the susceptibility of staphylococci to RP 59500 are shown in Table1. In a studyof 247 clinical isolatesof staphylococci belonging to 10 species, RP 59500 displayed MICs (minimal inhibitory concentrations) generally lower than those of ciprofloxacin (6). The range of MICs was 0.25-1 pg/ml andthe MIC, was 1 pg/ml for all strains tested, except for certain species of coagulase-negative staphylococci (CNS), for which the range was 0.25-2 pg/ml and the MIC, was 2 pg/ml. The susceptibility of Staphylococcus aureus to RP 59500 was not affectedby methicillin resistance. Shonekanet al. reportedthat the MIC rangeof RP 59500 was 0.1250.5 for oxacillin-sensitive strainsof staphylococci and0.125-2 pg/ml for oxacillin-resistant strains(100 strains studied)(5). Table I

In Vitro Activity

(Pm0 Phenotype, species (# strains)

RP 59500 Against Staphylococci Bacteriostatic activity Bactericidal activity (Pm!) MIC,

Methicillin-SaS. uureus (40) 0.5 Methicillin-R S. uureus Methicillin-S 0.5 0.5 S . epidermidis, S . huemolyticus (44) Methicillin-R 0.5 S . epidermidis, S. huemolyticus Other species of CNSb 0.5 Oxacillin-S S. aurezu Oxacillin-R S. uurelrs Oxacillin-S CNS CNS Oxacillin-R Erythromycin-S S. epidermidis Erythromycin-R S. epidermidis (4)

MIC,

MICrange

MBCrange Ref.

0.5

0.5

0.5

5

8

~~

.S: susceptible;R: resistant. -S: Coagulase-negative staphylococci.

MBC,

Parenteral Streptogramins: From

to Oral

55

The activity of RP 59500 against staphylococci adherent to a solid support was studied with two models designed to predict the capacity of the antibiotic to inhibit or killinfectiousbacteria that proliferate on biomaterial, such as catheters or prostheses. On the basis of experiments in which a nylon membranewas coated with fibronectin and then soaked ina bacterial culture, Berthaud et al. reported that RP 59500 was bactericidal against adherent Staphylococcusaureus (one strain tested) under these conditions. At a blood concentration of6.25 pg/ml, the activity of RP 59500 wasmore rapid thanthat of vancomycin [decrease in colony-forming units ( C m ) orders of magnitude within3 h versus 6 h for vancomycin] (7). In another study, silicone rubber disks were incubated withStaphylococcus epidennidis cultures (six strains tested) and the effect of RP 59500 on the resulting biofilm was compared to that of ciprofloxacin. RP 59500 was found to be bactericidal within 24 h, irrespective of slime productionor the erythromycin susceptibility status of the strain. It was concluded that the streptogramin was effective and could be useful for the treatment of infections of in-dwelling lines(8). A number of S. aurezu strains that have been testedfor susceptibility to RP 59500 exhibit an minimal bactericidal concentration (MBC) considerably higher than the MIC. It was suggested by Boswell et al. that such strains may have a phenotypic tolerance to the antibiotic (i.e., decreased susceptibility under certain conditionsof growth) (9).

Activity of QuinupristidDalfopristin (RP 59500) Versus Streptococcus pneumoniue That RP 59500 is an effective agent against Streptococcus pneumoniae, having not only bacteriostatic but also bactericidal activity, was confirmed by recently presented results (Table 2). Shonekan et al. reported low MICs (0.125-0.5 pg/ml) andMBCs equivalent or close to the MICs for the 20 S. pneumoniae strains that were studied (5). Reinert et al., in a study of German clinical isolates, foundthat the MIC, or RP 59500 was 0.5 pg/ml for 63 erythromycin-susceptible strains and 1 pg/ml for 30 erythromycinresistant strains; the MIC ranges were 0.25-1 pglml for the former group of strains and 0.125-2&m1 for the latter (10). In a study reported by Pankuch et al., the MIC, for 87 strains with various resistance profiles was 1pg/ml and the MIC range was 0.25-1 pg/ml. Resistanceto erythromycin or to penicillin did not affect the activity. These authors also tested RP 59500 for cidal activity against a subset of 50 strains and found it to be rapid. Approximately 40% of the strains tested were killed (decrease of three orders of magnitude) within 2 h of exposure to concentrations of greater than or equal to the MIC. At 50% MIC, cidal activity was also

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Bouunchuud

Table 2 In Vitro Activity of RP

Phenotype (#strains) of

Against Streptococcus pneumoniae

Bacteriostatic activity Bactericidal activity (Pg/ml)

MIC,,

MIC,

MIC range

MBC,

(Pdml)

MBC range

Not specified Penicillin-Sa, Erythromycin-S Erythromycin-R Penicillin-I, Erythromycin-S Penicillin-S Penicillin-I Penicillin-R Penicillin-S Penicillin-I (89) Penicillin-R

Ref. 5

b

1 0.5

OS:susceptible; I: intermediate; R: resistant. bRapid killing 2 h) observed at concentrations MIC.

observed for some of the strains (11). In a study by Tarasi and Tomasz of 217 isolates, most of which had intermediate or high-level resistance to penicillin, the MIC, of RP 59500 was 0.5 pg/ml and the MIC range was 0.03-1 pg/ml (12). Rapid in vitro killing was also reported for the two multidrug-resistant strains tested inanother study 30 log,, decrease in CFU/ml within 1 h of adding RP 59500 (13). In a rabbit model of meningitis,twodoses of RP delivered2h apart clearedbacteriafrom the cerebrospinal fluid (13).

Activity of Quinupristinklalfopristin (RP59500) Versus Enterococcus faecium

The recent disseminationof resistance to vancomycin and gentamicin among clinical isolates of Enterococcus fuecium has resulted in a more frequent occurrence of infections that do not respondto any available antimicrobial therapy. RP 59500 has been tested extensively for activity against this org ism in vitro and the results reported far are promising (Table Grimm reported that, of 118 E. fuecium strains isolated in Germany, all but 1 were inhibited by < 2 pg/ml of RP 59500 (14). Thirty-seven E. fuecium strains resistantto both vancomycin and gentamicin were reported by Hill et al. to have RP 59500 MICs ranging from to 1pg/ml (15). In a study by Williamset al., the MIC range for92 E.fuecium strains was 0.12-

Parenteral Streptogramins: From Table

In Vitro Activity of RP

Phenotype (# of strains) Vancomycin-Sa Vancomycin-R, Gentamicin-R Vancomycin-S Vancomycin-S Vancomycin-R Vancomycin-R, Erythromycin-I Vancomycin-R, Erythromycin-R Vancomycin-S Vancomycin-R

57

to Oral Against Enterococcusfaecium

Bacteriostatic activity (Pdm1) MC,,

MC, MIC range

Bactericidal activity (Pdml) MBC,

MBC range Ref.

5 0.5

b

0.5

.S: susceptible; I:intermediate; R resistant. bJSilling observed at 24 h for cells in exponential phase. CNO bactericidal activityobserved. dKilling observed at four timesthe MIC (three strains tested).

pg/ml. When tested against a subset of these strains, RP displayedbactericidalactivity(MBCrangingfrom to pg/ml for vancomycin-susceptible strains anda log,, reduction of viable countafter 4 h exposure to RP at 4 MIC forone vancomycin-resistant strain) Shonekan et al., however,reported that RP didnothave significant bactericidal activity againstE . faecium: The MBC, was 8pg/ml for strains, ofwhich were resistant to vancomycin. These strains however, were effectively inhibited by RP (MIC range pg/ml) In a study of vancomycin-resistant E. faecium strains, Caron et al. reported that the RP MIC, of the strains highly resistant to erythromycin (MIC> pg/ml) was slightly higher(RP MIC, = ml) than that of the strains with intermediate erythromycin resistance(RP MIC, = pg/ml). For all strains tested, the MIC range was &m1 also reported by Boswellet al. for S. aureus the susceptibility to RP was found in this study to be greater for E . faecium growing exponentially thanfor bacteria in stationary phase It was also reported that activity against E. faecium was increased if RP were combined with vancomycin, gentamicin, or teicoplanin, even if the strains being tested were resistant to one or more of these antibiotics For example, Hill et al. reported that the combina-

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tion of RP plus 8 pglml of teicoplanin reduced MICs to 0.06 pglml for 31 of vancomycin-resistant and gentamicin-resistant strains In a study intracellular activity, Herrera-Insuaet al. foundthat RP was more effective against polymorphoneclear-ingested vancomycin-sensitive E. faecium than against the ingested vancomycin-resistant strains.At four times the MIC, RP killed the former strains but only inhibited the latter. It was suggested that intracellular concentration and intracellular activity may not be directly related for this antibiotic (20).

Resistance to QuinupristidDalfopristin:Biochemical Mechanisms and Detection The rapidity with which resistance genes are disseminated through a bacterial population, especially in the hospital setting, is unpredictable. Both the frequency at which genes encoding proteins involved in resistance appear and the hardiness and transfer capacity of the genetic element carrying such genesare involved. The gene erm encodes a methylasethat modifies a base in 23s ribosomal RNA, reducing the affinity of the ribosome for macrolides,lincosamides,andgroup B streptogramins, such that these antibiotics cannot bind to their target (the MLS, resistance phenotype) (21). Other genes, frequently carriedby plasmids,. have been identified in staphylococci; these encode enzymes that can inactivate streptogramin components (22), aswell as an efflux mechanism. An inactivating enzyme has also been found inEnterococcus (23). For the streptogramins, it might be supposed that a prerequisite of resistance would be the appearance, in the same bacterial population, of genes that encode resistance to both components and, consequently, that resistance to RP would occur less frequently than it does for single agents. Resistanceto RP is apparently rare, but the low frequency of resistance observed far does notsimply reflectthe putative requirement for two coexisting mechanisms. It has been found that bacterial strains harboring genes that encode resistance to the group B streptogramins are susceptible to RP whereas those harboring genes encoding resistance to the group A streptogramins are resistant (24). In one in vitro study, itwas reported that, upon repeated exposure to concentrations of two or four times the MIC of RP E. faecium strains having MICs increased 16-foldor more could be derived from susceptible strains. Exposure to an initial drug concentration of at least 16 times the expected MIC prevented the emergence such resistant strains The implications of this study, however, are not entirely clear with respect to the clinical setting, wherethe genesis of resistance by mutation (presumably point mutation) may play a role of lesser importancethan the

Parenteral Streptogramins: From

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59

dispersal of genetic elements, such as transposons or plasmids, that encode complex resistance mechanisms. Susceptibility breakpoints forRP 59500 are now being determined.It has been suggested,on the basis of a study in which disks were loaded with and a disk zone diameter of 15 pg of RP 59500, that an MIC 2 1 18 mm could serve as provisional susceptibility breakpoints for S. pneumoniae, S. aureus, and Enterococcus spp. (26).

IN VITRO ACTIVITY OF RPR 106972, A NEW ORAL STREPTOGRAMIN, DERIVED (AS QUINUPRISTIN/ DALFOPRISTINE) FROM NATURAL PRISTINAMYCIN RPR 106972 is a cocrystalline associationof two natural molecules (in a4/2 molar ratio, or 45/55 in weight): type I pristinamycin (RPR 112808) containing PI, (RP 13919, > 95%)as the major component, and type I1 pristinamycin (RPR 106950) containing PIIB (RP 13920, > 95%) as the major component.

In Vitro Bacteriostatic Activity* Staphylococci

Minimal inhibitory concentration determination demonstrated that RPR 112808 inhibited the growth of sensitive and inducibly resistant S. aureus strains, with MICs ranging from 4 to 32m& but was inactive againstMI&constitutivelyresistant S. aureus strains. It inhibited S. epidermidis strains, with MICs ranging fromto264 m&, except the MLS, constitutively resistant strains (MIC> 128 m&). RPR 106950 inhibited all the S. aureus strains tested, with MICs ranging from 4 to 16 m&,and the S. epidermidis strains, withMICs ranging from 1 to 16 m a , except strains Mau., N 52, N 100, and N 122 (MIC > 128 m&). RPR 106972 (RPR 112808RPR 106950) was uniformly active against MLS,-sensitive and M L S , inducibly resistantS. aureus strains (MIC range: 0.12-1 m&), MLS, constitutively resistantS. aureus (MIC range:1-1 mg/ L),and S. epidermidis strains (MIC range:0.12-0.50 m&).

M L S ,

Streptococci The MIC determination demonstratedthat, against streptococci (groups B, C , G ) ,RPR 112808 was active, with MICs ranging from 0.50 to 8 m&, *Data on File, RhBne-Poulenc Rorer.

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except against the strains resistant to erythromycin (MIC 128 m&). RPR 106950inhibited the tested strains, with MICs ranging from 0.50 to m&. RPR 106972 demonstrated a potent activity against all the strains tested (MIC range: 0.03-0.12 m&), comparable to that of erythromycin (MIC range: 0.03-0.50 m&). Against Str. pneumoniae, RPR 112808 wasactive, with MICs ranging from 1to m& against the strains sensitiveto erythromycin. Against the strains resistant to erythromycin, it was less active (MIC range: > 128 m&). RPR 106950 did not demonstrate significant activity against anyof the Str. pneumoniae strains tested (MIC range: m&), but the RPR 112808/RPR 106950 association was strongly synergistic:RPR 106972 was uniformly and potently active against all the strains studied whatevertheir resistant profile (MIC range:0.06-0.50 m&). Against strains sensitive to the reference compounds, its activity was comparable to that of erythromycin (MIC range: 0.12-0.25 m a ) , about to four times inferiorto that of oxacillin (MIC range: 0.015-0.25 m&), and two to eight timessuperior to that of ciprofloxacin (MIC range: 0.50-1 m&).

Enterococci RPR 112808 inhibited the strains tested, with MICs ranging from 1 to 16 m&. RPR 106950 was slightly active against E . faecium, E. durans, and E . hirae (MIC range: 2- > 128 m&) but was inactive against E . faecalis (MIC range: 128- > 128 m&). RPR 106972 demonstrated potent activity against E . faecium, E durans, and E . hirae (MIC range: 0.06-0.50 m&). It was less active against E . faecalis than against the other enterococci (MIC range:0.50-2 m&).

Neisseria

Pasteurella species

Against Neisseria species, RPR 112808 was weakly active (MIC range: > 128 m&), whereas RPR 106950 demonstrated potent activity (MIC range: < 0.12-1 m&). RPR 106972 demonstrated strong activity (MIC range: 0.015-0.12 m&). Against P . multocida, RPR 112808 was inactive (MIC < 128 m&). RPR 106950 andRPR 106972 were poorly active (MIC ranges: 16-128 and 4-16 mg/L, respectively).

Moraxella catarrhali RPR 112808 did not demonstrate bacteriostatic activity against this species (MIC range: 32-128 m&). RPR 106950 inhibitedthe strains tested, with MICs ranging from 0.50to 2 m&. RPR 106972 demonstrated an activity (MICrange:0.12-0.25m&) tofourtimessuperior to that of erythromycin (MIC range: 0.12-1 m a ) .

Streptogramins: From Parenteral to Oral

61

Haemophilus influenzae RPR 112808 did not demonstrate activity ( M C range: > 128 m&). RPR 106950 was moderately active (MIC range: 4-16 m&). RPR 106972 (MIC range: 0.50-2 m&) was 2 to 16 times more active than erythromycin (MIC range: 4-16 m&). Enterobacteria and Pseudomonas aeruginosa Neither RPR 112808 (MIC > 128 m&), RPR 106950 (MIC > 128 m&), nor RPR 106972 (MIC range: 128- > 128 m&) demonstrated any activity against enterobacteriaor Pseudomonas aeruginosa. Anaerobes RPR 112808 and RPR 106950 were active against gram-positive anaerobes with MICs ranging from 0.50 to 64 m& and 1to 128 m&, respectively. They were generally inactive against gram-negative anaerobes (MIC ranges: 32- > 128 m& and > 128 m&, respectively.

Mycoplasma

Ureaplasma species

Against the M. pneumoniae strains tested, RPR 106972showed potent bacteriostatic activity (MIC = 0.12 m&). Against the M. hominis strain tested, RPR 106972 had an MIC of 1 m&. Against the U. urealyticum strain tested, RPR 106972 (MIC = 2 m&) demonstrated activity.

Legionella species RPR 112808and RPR 106950 demonstratedpooractivityagainst the strains tested (MIC ranges: 4-128 m& and 8-32 m&, respectively), and RPR 106972 (MIC range: 0.12-1 m&) was two to four times less active than erythromycin (MIC range: 0.06-0.25 m&).

Mycobacteria species PR 106972 was inactive (MIC range: 16> 128 m&) against the 15 strains tested [M.intracellulare (1 strain), M. avium (6 strains), M.fortuitum (1 strain), M. scrofulaceum (1 strain), M. simiae (1 strain), M. kansasii (2 strains), M.malmoense (1 strain), M.szulgai (1 strain), and M.xenopi (1 strain) (expt XWOl 079).

Synergism of the RPR 112808

RPR 106950 Combination

The RPR 112808 and RPR 106950 combination showed synergism for all bacterial strains tested.The FIC index obtained with the lowest concentration of eachcompoundrangedfrom0.034 to 0.13 for Staphylococcus aureus (seven m,-sensitive and MLS,-resistant strains), from 0.008 to

Bouanchaud

62

0.12 for Streptococcuspneumoniae (four strains including one eythromycinresistant strain and one penicillin-intermediate strain), and from 0.017 to for Enterococcus spp.(sevenstrainsincluding one multiresistant strain of Enterococcusfaecium).

Influence of the Ratio of RPR 112808 to RPR 106950 The RPR112808 andRPR 106950 combination was found to be synergistic over a wide range of ratios against all the strains tested (9040 to 80/20).

In Vitro Bactericidal Activity Staphylococcus aureus Against MU,-sensitive andMLS,-inducibly resistant strains (two strains of each), RPR 106972 was bactericidal at concentrations of 0.25-2 m& [two to four times the MIC accordingto the strain tested]in a 6-24-h period. A dose-dependent effect was found at concentrations corresponding to two and four times the MIC, but no difference in the effectwasobserved between the concentrations of four and eight times the MIC. AgainstM L S , constitutively resistant strains,RPR 106972 at the concentration of 8 m@ was bactericidal within 24 h (starting from of 6 contact) h againstone strain (8 m& = four times the MIC) and decreased viable counts by 2 log,, CFU/ m1 for the other (8 m& = eight timesthe MIC).

Streptococcus pneumoniae Against erythromycin-resistant strains (two strains), RPR 106972 wasbactericidal at the concentrations of 0.50 m& (two to four times the MIC according to the strain tested) within the first3h of contact. Against penicillin-intermediate strains (two strains), RPR 106972 was bactericidal at concentrations of 0.25-0.50 m& (two to four timesthe MIC) in a 6-8-h period. Against the two strains with a low levelof resistance to ciprofloxacin, RPR 106972 was bactericidal at the concentration of 0.25 m& (one to two times the MIC)ina3-6-h period.Againststrainsresistant to erythromycin, ciprofloxacin, and/or penicillin (three strains), RPR 106972 decreased viable countsby 2 log,, CFU/ml at the concentration of 2 m& (four to eight timesthe MIC) in a 6-8-h period.

Enterococcus species Against E . faecalis (two strains tested), RPR 106972 did not demonstrate any bactericidal activity. However, atthe concentration of eight hours the MIC (4-16 m&) according to the strain tested), it decreased viable counts by 2 log,, CFU/ml.

Streptogramins: From Parenteral Oral to

63

Against E . faecium (four strainstested), RPR was bactericidal period (one strain, at theconcentration of eight timesthe MIC in 24-48-h a MIC = m&) and decreased viable countsby to log,, CFU/ml (three strains, MICs = m&). Haemophilus influenzae .

Against the strains tested (including strains producing p-lactamases), RPR was bactericidal at the concentrations of or 4 m& or 4 the MIC accordingto the strain tested) in a to 6-h period strains) or in a to 24-h period strain). Conclusion

RPR has potent in vitro bacteriostatic activity against aerobic grampositive cocci, including staphylococci resistant to oxacillin and macrolideslincosamides-streptogramin, (MU,), gram-positive anaerobes, certain gram-negative bacteria responsible for respiratory tract infections (Neisseria spp., Moraxella catarrhalis, Haemophilus injluenzae), and fastidious due to the bacteria asLegionella spp. andMycoplasma spp. This activity is synergism of its components, RPR (containing PI, asthe major component) and RPR (containing PII, as the major component). This synergistic activity occurred over a wide range of RPR to RPR ratios and was affected only slightly by variations in culture conditions. RPR demonstrated in vitro bactericidal activity at concentrations ranging fromto to four times the MIC againstStaphylococcus aureus sensitive or resistant to oxacillin andM L S , (five ofsix strains tested), Streptococcus pneumoniae (six of nine strains tested, including erythromycinresistant penicillin-intermediate, ciprofloxacin-resistant,and multiresistant strains) and Haemophilusinfluenzae (six strains tested including strains producingp-lactamases).Againstenterococci (six strains tested), RPR was notbactericidalexceptagainstone strain of Enterococcus faecium; nevertheless it decreased viable counts by log,, CFU/mL at concentrations of eight timesthe MIC. RPR demonstrates promising in vitro activity against most of the bacteria responsible for respiratory tract, cutaneous and genital community-acquired infections. It may also be considered for oral follow on therapy for quinupristiddalfopristin.

ACKNOWLEDGMENTS I thank K. Pepper for helpful comments, and J.F. Desnottes and N. Berthaud for providing newdata from their laboratory.

Bouanchaud

64

REFERENCES 1. Bamere JC, Bouanchaud DH, Desnottes JF, Paris JM. Streptogramin analogues. Exp Opin Invest Drugs1994; 3(2):115-131. 2. Aumercier M, BouhallabS, Capmau ML, Le Goffic F. RP 59500: a proposed mechanism for its bactericidal activity. J Antimicrob Chemother 1992;30 (Suppl A):9-14. 3. Cocito C, Di Giambattista M,Vannufel P. The mechanism of action of streptogramins. 3rd International Conference on the Macrolides, halides and Streptogramins, Lisbon, 1996; abstr 12. 4. Barriere JC, Bouanchaud DH, Paris JM, Rolin 0, Hams Smith C. Antimicrobial activity againstStaphylococcus aureus of semisynthetic injectable streptogramins: RP 59500 and related compounds. J Antimicrob Chemother 1992; 30 (suppl A):1-8. 5. Shonekan D, Handwerger S, Mildvan D. Comparative in vitro activities of RP 59500 (quinupristiddalfopristin), CL 329,998, CL 331,002, CP-99,219, clinafloxacin,teicoplaninand vancomycinagainstgram-positive bacteria. 35th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, 1995; abstr E-124. 6. von Eiff C, Peters G. The in vitro activity of RP 59500, a new semisynthetic injectable pristinamycin, against staphylococci. 3rd International Conference on the Macrolides, Azalides and Streptogramins, Lisbon, 1996; abstr 3.25. 7. Berthaud N, Charles Y,Gouin AM, Houdebine C, Rousseau J, Desnottes JF. In vitro bactericidal activity of RP 59500 (quinupristiddalfopristin) against adherent Staphylococcus aureus. 35th Interscience Conferenceon Antimicrobial Agents and Chemotherapy, San Francisco,1995; abstr E-122. 8. Hamilton-Miller JMT, ShahS. Killing ofStaphylococcus epidermidisin biofilm by RP 59500 (quinupristiddalfopristin)and other antibiotics. 35th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, 1995; abstr A-104. 9. Boswell,FJ, Sunderland J, Andrews JM, Wise R. Time kill kinetics of RP 59500 (quinupristiddalfopristin)on Staphylococcus aureuswith and without a raised MBC evaluated using two methods. 35th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco,1995; abstr E-123. 10. Reinert R, Kresken M, Lemperle M, Lutticken R.In vitro antibacterial activity of RP 59500 (quinupristiddalfopristin),a semisyntheticstreptogramincombination, against Streptococcus pneumoniae. 3rd International Conference on the Macrolides, halides and Streptogramins, Lisbon,1996; abstr 3.15. 11. Pankuch GA, Jacobs MR, Appelbaum PC. Antipneumococcal activityof RP 59500 (an injectable streptogramin), erythromycin and sparfloxacin by MIC and rapid time-kill. 35th Interscience Conference on Antimicriobial Agents and Chemotherapy, San Francisco,1995; E-117. resistant clones of 12. Tarasi A, Tomasz A. Activity of RP59500 against multidrug Streptococcus pneumoniae. 35th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco,1995; abstr E-116.

Streptogramins: From Parenteral to Oral

65

13. Dever L, Tarasi A, Tomasz A. Bactericidal activityof RP 59500 (RP)against Streptococcuspneumoniaein the rabbit modelof experimental meningitis. 3rd International Conference on the Macrolides, Azalides and Streptogramins, Lisbon, 1996; abstr 3.31. 14. Grimm H. In vitro activity of quinupristiddalfopristin(RP 59500) and 8other antibiotics againstEnterococcus faecium. 3rd International Conference on the Macrolides, Azalides and Streptogramins, Lisbon, 1996; abstr 6.18. 15. Hill RLR, Smith C, Casewell MW. Bactericidal and inhibitory activityof RP 59500 (quinupristiddalfopristin)and its components against vancomycin- and gentamicin-resistant Enterococcus faecium. 35th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, 1995; abstr E-118. 16. Williams JD, Maskell JP, Whiley AC, Sefton A M . Comparative in vitro activity of RP 59500 (quinupristiddalfopristin)against Enterococcus spp. 3rdInternational Conference on the Macrolides, Azalides and Streptogramins, Lisbon, 1996; abstr 6.19. 17. Caron F, Gold HS, WennerstenCB, Moellering RC, Jr., Eliopoulos GM.Role of growth phase and of susceptibility to erythromycin on the bactericidal effect of RP 59500, a combinationof quinupristin and dalfopristin, against vancomycin-resistant Enterococcusfaecium strains. 3rdInternational Conference on the Macrolides, Azalides and Streptogramins, Lisbon, 1996;abstr 6.17. 18. Kang SL, Rybak MJ. In vitro bactericidal activity of RP 59500 (quinupristid dalfopristin) alone and in various combinations against resistant strains of Enterococcus species and Staphylococcus aureus. 35th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, 1995; abstr E-121. 19. Lorian V, Fernandes F. Synergistic activity of injectable streptogramin RP 59500 (quinupristidda1fopristin)-vancomycin combination. 35th Interscience Conference on AntimicrobialAgentsandChemotherapy,SanFrancisco, 1995; abstr E-126. 20. Herrera-InsuaI, Jacques-Palm K, Murray BE, Rakita RM. Intracellular activityof RP 59500 (quinupristiddalfopristin), aninjectablestreptogramin, against Enterococcus faecium. 35th Interscience Conferenceon Antimicrobial Agents and Chemotherapy, San Francisco, 1995; abstr E-119. 21. Weisblum B. Inducible resistance to macrolides, lincosamides and streptogramin type B antibiotics: the resistance phenotype, its biological diversity and structural elements that regulate expression-areview. J Antimicrob Chemother 1985; 16 (supplA):63-90. 22. Allignet J, Loncle V, El Solh N: Sequence of a staphylococcal gene, vat, encoding an acetyltransferase inactivating streptogramin A and related antibiotics (RP 54476). 33rd Interscience Conferenceon Antimicrobial Agents and Chemotherapy, New Orleans, 1993; abstr 218. 23. Leclercq R, Nantas L, Soussy CJ, Duval J. Activity of RP 59500, a new parenteral semisynthetic streptogramin, against staphylococci with various mechanisms of resistance to macrolide-lincosamide-streptogramin antibiotics. J Antimicrob Chemother 1992; 30 (suppl A):67-75.

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Bouanchaud

24. El Sohn N, Loncle V, Aubert A, Casetta A, Allignet J. Analysis of staphylococcal elements conferring resistance to streptogramin A (RP 54476). 33rd Interscience Conference on Antimicrobial Agents and Chemotherapy, New Orleans, 1993; abstr 217. 25. Millichap J, Ristow Noskin T, G, Peterson L.Selectionof Enterococcus fuecium strains with stable and unstable resistance to RP 59500 (quinupristiddalfopristin) using stepwise exposure in vitro. 35th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco,1995; abstr E-120. 26. Tenover FC, Baker CN. Development of provisional disk diffusion breakpoints for testing RP 59500 (quinupristiddalfopristin). 35th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, 1995; abstr D-30. 27. Neu HC, Chin N, Gu J. The in vitro activity of new streptogramins, RP 59500, RP 57669 and RP 54476, alone and in combination. J AntimicrobChemother 1992; 30 (Suppl A):83-94. 28. Dubois J, Joly JR. In vitro activity of RP 59500, a new synergic antibacterial agent, against Legionellu spp. J Antimicrob Chemother 1992; 30 (suppl A): 77-81. 29. Renaudin H, Boussens B, Bebear C. In vitro activity of RP 59500 against Mycoplasma. 31st Interscience Conferenceon Antimicrobial Agentsand Chemotherapy, Chicago,1991; abstr 897. 30. Nougayrede A, Berthaud N, Bouanchaud DH. Post-antibiotic effects of RP 59500 with Staphylococcus uureus. J Antimicrob Chemother 1992; 30 (suppl A):101-106. 31. Craig W, Ebert S. Pharmacodynamic activities of RP 59500 in an animal infection model. 33rd Interscience Conferenceon Antimicrobial Agents and Chemotherapy, New Orleans, 1995; abstr 470.

Azalides: Basicand Clinical Research Michael W. Dunne Pfirer Central Research Groton, Connecticut

The field of research inthe area of macrolides has beenquite productive in the last 10 years, as evidencedby the clinical developmentof a number of novel compounds. Among these agentsare included the azalides. The azalides as a class are defined by the inclusion of a nitrogen into the macrolidenucleus.Thisaminosubstitutionaddsanadditional positive charge to the compound and can be added to a 14membered, 15-membered, or, theoretically, a 16-membered ring, with the most productive substitutions occurring the at 8 and 9 position. An interest in altering the macrolide nucleus of erythromycin arose because of the acid instabilityof this compound. In fact, 10% of erythromycin is degraded in an acidic environment in approximately S, whereas substitution at the 9 position into the 15-membered ring, the structure of azithromycin, increases the time to acid degradation to about 20 min (1).

MICROBIOLOGIC ACTIVITY The azalides as a classhave the potential for improved gram-negative activity (Table 1). The minimal inhibitory concentrations (MICs) for the Enterobacteriaceae dropped into a range now potentially achievable with in vivo concentrations of the drug. Forthe compounds displayedin Table 1, 67

68

Dunne

Table l

MIc (cLg/ml)B halide Microorganism Enterococcus faecalis 5407 Staphylococcus aures MB 2865 S. haemolyticus MB 5412 Streptococcus pyogenes MB 2874 S. pyogenes MB 5403 (r. wn)b S. pyogenes MB 5406 (r. indy Enterobacter cloacaeCL 4298 Escherichia coli MB 2884 Klebsiella pneumoniae MB 4005 0.25-1 Haemophilus influenzae MB 5363 Pseudomonas stutzeri MB 1231

Erythromycin

Azithromycin analog

1-2

2-4

4

0.25

0.5-1

2-4

0.125 0.015

0.25 0.03

0.5 0.125

>l28 32 32-64 32-64 32-64

>l28 16 0.5-1 1-2 2

2-4 0.25-1.0

>l28 16 4-8 4-8 8

2-8 0.03-0.06

0.25

*Determinedby a standard broth microdilution assay. con: constitutive macrolide resistance. ind: inducible macrolide resistance. Source: From Ref. 2.

this enhancementof gram-negative activity did not depend on thering size, as both 14- and 15-member azalide rings were active (2). Although these azalides maintain activity against gram-positive organisms as well, there is a concomitant 1-2 tube dilution increase in MIC's over erythromycin.For example, the MIC of erythromycin to S. pneumoniae is pg/ml, whereasthat for azithromycin is 0.12 pg/ml. This typeof trade-off between gram-negative and gram-positive activity has been seen with other macrolide and azalide analogs. The mechanism for enhancement of gram-negative activity is not known; however, there is speculationthat the negative charge inthe outer surface of gram-negative bacilli attractsthe positively charged azalide nucleus. It does not appear, however, that the azalides are actively pumped into the gram-negative organism Although there is a slight enhancement of binding to gram-negative ribosomes,there is not enough improvement to explain all but a small degree of potency. It is important to note

Azalides: Basic and Clinical Research

69

that nitrogensubstitutionhasnotovercome the M L S , phenotype of macrolide resistancedue to methylation of ribosomal RNA.

PHARMACOKINETICS The nitrogen substitution within the azalide nucleus has also had a significant impact on thepharmacokinetic propertiesof the class. While in extracellular environments, the azalides have a relatively neutral pH, allowing for passive diffusion through the lipophilic cell membrane. Once inside an acidified vacuole,however,both of the aminoconstituents of the ringbecome charged, preventing diffusion back through the membranes, a phenomenon referred to as the ion-trapping mechanism(4). The azalides are sequestered intracellularly and slowly released back into the circulation (5). Consequently, a large reservoir of drug, free from degradation, is available to provide prolonged tissue exposure while extending the effective dosing interval. A second consequence of this sequestrationof drug involves its concentrationwithin the neutrophil. These cells concentrate the drug and subsequently accumulateat the site of infection, providing a targeted delivery to infected tissues (6). Third, the accumulation of antibiotic within the cells extends the microbiologic spectrumof activity to cover a variety ofintracellular pathogens includingMycobacterium avium, legionella, chlamydia, and rickettsia. Activity against these organisms is achieved because of a combination of the high intravacuolar levels as well as the prolonged retention in that space, maximizing exposureof the bacterium to the drug. This prolonged exposure distinguishes the azalides from other macrolides and quinolones which reach elevated peaks but diffuse more rapidlythe from cell Table 2 displays the murine pharmacokinetic properties of three azalide analogs and the preferential accumulation of drug within tissues(7). Table 2 Murine Pharmacokinetic Propertiesof Three halide Analogs and Accumulation of Drug in Tissues

AUcu-24 (PLg Wml)

Antibiotic

L-701,677 L735,659 L-708,365 azithromycin) G689,108 Source: From Ref. 7.

138 320

6 5

7

1038 2423 2768

271

70

Dunne

METABOLISM The metabolism of azalides is quite different from other macrolides. In vitro studies have demonstrated a lack of degradation of azithromycin within the cytochrome P450 system(8). In addition, numerous drug interaction studies have shown no impact of azithromycinon the metabolism of theophylline, terfenadine, estrogen, carbamazepine, rifabutin, and zidovudine (9-11). With regardto azithromycin, mostof the drug is not metabolized at all, with two-thirds being excreted unchanged the in bile. Metabolismof the remaining drug occurs via demethylation of the nitrogen groups.

CLINICAL STUDIES

The azalides have distinguished themselves with improved acid stability, enhanced gram-negative activity, and a pharmacokinetic profile allowing for prolonged exposure with less frequent dosing. The prototype compound in this class is azithromycin and its early clinical development was focused on the treatment of respiratory tract disease, buildingon the enhancedactivityagainst Haemophilusinfluenzae,Moraxellacatarrhalis, Chlamydia pneumoniae, andlegionella,whileshortening the course of therapy from 10-14 days, typical with erythromycin,to 5 and even 3 days. Its activity against sexually transmitted pathogens, both intracellular, such as Chlamydia trachomatis, and gram-negative, such as Neisseria gonorrhoeae and Hemophilus ducreyi, was developed with single-dose strategies, important in this area where lack of compliance remains a significant public health issue. Further development has expanded on the pharmacokinetic advantages of this drug by exploring single-dose therapiesfor other indications, by taking advantageof the potential for intermittent dosingwell as as by intervening to prevent new infections, all while broadening the scope of potential pathogens to include parasites and enteric gram-negative organisms. Single-dose therapies for treatment of chlamydia, gonorrhea, and chancroid have been studied and published, as thehas activity of azithromycin in the eradication of trachoma (12-15). Trialsare underway looking at singledose treatments of otitis media and shigellosis. Based on pharmacokinetic modeling, there is reason to believe that in addition to higher peak levels, the delivery of drug in shorter courses may provide a greater area under the plasma concentration versus time curve as well, possibly providing enhan activity comparedto similar total doses given over longer periods of time. These shorter courses of therapy must be studied carefully, however,the as inflammatory componentof manyinfectious diseases and the symptoms that come with them may continue beyond the first 24 h of therapy.

71

Azalides: Research Clinical Basic and Table

Pathogens in Intestinal Tract and Their MICs ~~

Azithromycin

Pathogen

Erythromycin

4.0 4.0

Salmonella typhi Salmonella enteritidh Shigella Shigella jejuni Campylobacter Vibrio cholerae ticus Vibrio Yersinia enterocolitica Escherichia coli

1.o 0.5

8.0

4.0

Enterotoxigenic Enteroinvasive

4.0

Clostridium difficile Aeromonas spp. Source: Data from Refs.

and 27.

Intermittent therapies may be of some advantage where prolonged therapy is required or direct observed therapy is considered necessary. This possibility is being explored in the treatment of M . avium disease with three times per week dosing (16). Azithromycinhasdemonstratedactivityin the prevention ofdisease caused by a variety of pathogens. A 1200-mg once weekly regimen has been shown to prevent the development of disseminated Mycobacterium avium infectioninpersonsinfectedwith AIDS. Prevention of bothsinusitisandpneumoniawasalsofoundinthesetrials(17,18). Efficacyin the prevention of malaria due to P. falciparum hasbeen demonstrated both in a controlled Phase I1 study in the United States (19)andafieldtrialinWesternKenya(20).Duringthis latter study, efficacy over placebo or doxycycline was also demonstrated in the prevention of shigellosis (21). Another area of early clinical research involves the treatment of bacterial infections of the gastrointestinal tract. Table 3 lists a variety these pathogens and the MI& are all generally < 4 Most of these are actually intracellular pathogens and consequently are located where azithromycin concentrationsare the highest. Activity has been seen the in treatment of Campylobacter enteritis (22), typhoid fever (23), and shigellosis (25). There are no data yet available concerningE . coli enteritis; however, levels of azithromycin in ileostomy fluidare in the range of 550-1000

72

Dunne

ml, as a consequence of both unabsorbed drug anda transintestinal route of elimination (24). In conclusion, the azalides have provided the clinician with an improvement over therapy with erythromycin. Azithromycin has shown activity in the treatmentof a wide variety of diseases while offering advantages in dosing that improve theease of administration.

REFERENCES 1. Fiese EF, Steffen SH. Comparison of the acid stability of azithromycin and erythromycin, A J Antimicrob Chemother1990; 25 (suppl A):39-47. 2. Jones AB, Herbert CM. J. Antibiot 1992;45:1785-1791. 3. Capobianco JO, Goldman RC. Erythromycin and azithromycintransport into Haemophilus injluenzae ATCC 19418 under conditions of depressed proton motive force. Antimicrob. Agents Chemother 1990;34:1787-1791. 4. Tulkens PM. Intracellular distribution and activity of antibiotics. Eur J Clin Microbiol Infect Dis 1991;lO:lOO-106. 5 . Gladue RP, Snider ME. Intracellular accumulation of azithromycin by cultured human fibroblasts. Antimicrob. Agents Chemother 1990;34:1056-1060. 6. Gladue RP, BrightGM,Isaacson RI, NewborgMF. In vitroandinvivo uptake of azithromycin (CP-62,993) by phagocytic cells: possible mechanisms of delivery and release at sites of infection. Antimicrob Agents Chemother 1989;33:277-282. 7. Pelak BA, Cerckens LS, Kropp H. Tissue distribution and pharmacokinetics of novel h a l i d e 9-Deoxo-8a-Aza-8a-Homoerythromycin derivatives. 33rdInNew Orterscience Conference on Antimicrobial Agents and Chemotherapy, leans, 1993. Amacher DE, Schomaker SJ, Retsema JA, etal. Comparison of the effects of the new azalide antibiotic, azithromycin, and erythromycin estolate on rat liver cytochrome P-450. Antimicrob Agents Chemother 1991;35:1188-1190. 9. Gardner M, Coates P, Hilligoss D, Henry E.Lack of effect of azithromycin on the pharmacokinetics of theophylline in man. 9th Mediterranean Congress on Chemotherapy, Athens, 1992. 10. Honig P, Wortham D, Zamani K, Conner D, Cantilena L. Effect of erythromycin, clarithromycin, and azithromycinon the pharmacokinetics of terfenadines. Clin PharmacolTher 1993;53:161. 11. Chave JP, Munafo A, Chatton J Y , Dayer P, Glauser M, Biollaz J. Once-aon weekazithromycinin AIDS patients:tolerability,kineticsandeffects zidovudine disposition. Antimicrob Agents Chemother 1992;36:1013-1018. 12. Martin DH, Mroczkowski TF, Dalu ZA, etal. A controlled trial of single dose azithromycin for the treatment of chlamydial urethritis and cervicitis. N. Engl J Med 1992;327:921-925. 13. Martin DH, et al. Comparison of azithromycin and ceftriaxonefor the treatment of chancroid. Clin Infect Dis 21:409-414.

esearch Clinical Azalides: and Basic

73

14. Handsfield HH, Dalu ZA, Martin DH, Douglas JM, McCarty JM, Jr, Schlossberg D, Azithromycin Gonorrhea Study Group. Multicenter trial of single-dose azithromycin vs. ceftriaxone in the treatment of uncomplicated gonorrhea. Sex. Transm Dis 1994;21(2):107-111. 15. Bailey RJ, Arullendran P, Whittle HC, Mabey DCW. Randomized controlled trial of single-dose azithromycin in treatment of trachoma. Lancet 1993;342: 453-456. 16. Wallace RJ, Jr, Griffith DE, Brown BA, et al. Initial results of three times weekly azithromycin(AZM) in treatment regimens for Mycobacterium aviumintracellulare (MAI) lung diseasein non-AIDS. 3rd International Conference on the Macrolides, Azalides and Streptogramins, Lisbon, 1996. 17. Havlir DV, Dube MP, Sattler FR, Forthal DN, KemperCA, Dunne MW, et al. Mycobacterium qavium complex with weekly Prophylaxis against disseminated agithromycin, daily rifabutin, or both. N. Engl. J. Med. 1996;335:392-398. 18. Oldfield EC, Dickinson G, Chung R, etal. Once weekly azithromycinfor the prevention of Mycobacterium avium complex (MAC) Infection in AIDS patients, 3rd Conferenceof Retroviruses and Opportunistic Infections, Washington, DC, 1996. 19. Andersen SL, Berman J,Kuschner R, et al.Prophylaxis of Plasmodium falciparum malaria with azithromycin administeredto volunteers. Ann Intern Med 1995;123:771-773. 20. Andersen SL, 0100 AJ, Gordon DM, et al. A double blinded, placebo controlled trial of azithromycin comparedto doxycycline for malaria prophylaxis in Western Kenya (submitted). 21. Shanks GD, Ragama OB, Aleman GM, Andersen SL, Gordon DM. Azithromycin prophylaxis prevents epidemic dysentery, Trans Roy SOC Trop Med Hygiene: 1996;90:316. 22. Kuschner RA, Trofa AF, et al. Use of azithromycin for the treatment of Campylobacter enteritis in travelers to Thailand, an area where ciprofloxacin resistance is prevalent. Clin Infect Dis 1995;21:536-541. 23. Tribble D, Girgis N, Habib N, Butler T. Efficacy of azithromycin for typhoid fever. Clin Infect Dis 1995;21:1045-1046. 24. Luke DR, Foulds G, Going PC, Connolly A. Oral absorption profile and disposition of azithromycin (AZM) in ileostomy subjects. 3rd International Conference on the Macrolides, Azalides and Streptogramins, Lisbon, 1996. (AZR) is 25. Khan WA, Seas C, Dhar U, Salam MA, Bennish ML, Azithromycin equivalent to ciprofloxacin (CIP) in the treatment of shigellosis: Results of a randomized, blinded, clinical trial. Abstracts of the 36th InterscienceConference on Antimicrobial Agents and Chemotherapy.1996. 26. Kitzis MD, Goldstein F W , MiCgi M, and AcarJF. In-vitro activity of azithromycin against various Gram-negative bacilli and anaerobic bacteria. J Antimicrob chemother 1990; 25, supplA: 15-18. 27. Jones K, Felmingham D, and Ridgway G. In vitro activity of azithromycin (CP-62, 993), a novel macrolide againstenteric pathogens. Drugs Exptl Clin Res 1988;14:613-615.

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Basic and Clinical Research on Macrolides J Carl Craft Abbott Laboratories

Abbott Park, Illinois

INTRODUCTION In 1952, a soil sample from the Philippineswas foundto contain astrain of Succhuropolysporu erythrueu(formally Streptomyces erythreus) which produced an antibacterialeffect. The discoveryof erythromycin, the first macrolide, set a high standard from which all subsequent macrolides have been judged. Erythromycin is very safe and useful in treating both outpatient and inpatient infections. Although it was initially developed for the treatment of staphylococcal infections in patients allergicto penicillin, it has been found to have much broader unanticipated uses and has in vitro activity against many organisms (Table 1).

ERYTHROMYCIN the full potential of erythromycin was defined by use, were several major disadvantages. These disadvantages include mainly variable pharmacokinetics, prokinetic activity, and unpredictable activity against HuemophiZ u s influenme. Pharmacokinetic studies have shown erythromycin to have variable absorption fromthe gastrointestinal tract dependent on formulation. Bioavailability ranges from 18% to 45%, with peak serum concentrations of 0.5-1.9 pg/ml after the administration 250-500-mg doses every 75

76 Table I

Craft Microbiology of Erythromycin

Streptococcus pyogenes (group A beta-hemolytic streptococcus) Alpha-hemolytic streptococcus (viridans group) Staphylococcusaureus Streptococcus pneumoniae Mycoplasma pneumoniae Chlamydia trachomatis Chlamydia pneumoniae Corynebacteriumdiphtheriae Corynebacteriumminutissimum Campylobacterjejuni Entamoeba histolytica Plasmodium species Listeria monocytogenes Bordetella pertussis Legionella pneumophilia Neisseria gonorrhoeae Treponema pallidum

6 h(1,2). The most frequent side effectsof oral erythromycinpreparations are gastrointestinal and are dose related. They include nausea, vomiting, abdominal pain, diarrhea, and anorexia. These are related to erythromycin’s activity as a motilin receptor agonist. The overall resultof this motilinlike activity isthat erythromycin may not be toleratedby up to one-third of patients Erythromycin has activity against Huernophifus influenzae with a minimal inhibitory concentration (MI%) of pg/ml (5). Due to the relatively high MICs of some isolates and variable pharmacokinetics, erythromycin does not always successfully treat H.influenzae infections. Macrolide research has focused for many years on developing macrolides which would overcome these problems while maintaining the many advantages of erythromycin.

CLARITHROMYCIN Clarithromycin, the 6-0-methyl semisynthetic derivative erythromycin, was discovered by Taisho Pharmaceutical Co., Ltd and developed worldwide byAbbott Laboratories. This relatively minor modification of the molecule provides substantial improvements over erythromycin. Clarithromycin has improved pharmacokinetics when comparedto erythromycin while maintaining the advantages. Orally administered clarithromycin is rapidly absorbed from the gastrointestinal tract and is not affected by food (6-8).

Basic and Clinical Research on Macrolides

77

Absorption is characterized by a brief lag time with maximum plasma concentrations usually occurring 2 h after dosing (6-8). Substantial amountsof a pharmacologically active metabolite, 14(R)-hydroxyclarithromycin, are formed followingthe oral administrationof clarithromycin(6-8). The absolute bioavailability of clarithromycin is 55% for the parent compound (g), but this does not take into account the first-pass metabolismof clarithromycin to 14(R)-hydroxyclarithromycin.Because boththe parent and metabolite are active, the true bioavailability is closerto 80-90% (5). Mean peak plasma concentrationsof clarithromycin are 1.0-2.8 pg/ml for the 250-mg bid dose and the 500-mgbid dose,respectively(5-8). The mean peak concentrations of 14(R)-hydroxyclarithromycinare 0.6-0.9 pg/ml for the 250-mg biddose and500-mg biddose, respectively (5-8). The pharmacokinetics of clarithromycinare nonlinear andare characterized by increases in half-life and greater than dose-proportional increases in area under the plasma concentration versus time curve (AUC) with increasing dose size (6-8). The nonlinearity of clarithromycin pharmacokineticsatisleast partially due to the capacity-limited formationof 14(R)-hydroxyclarithromycin (6-8). and AUC values forthe metabolite show lessthan proportional increases with increasing dose size, whereas the apparent elimination halflife increases with increasing dose. Despite the nonlinearity of the pharmacokinetics of clarithromycin,the steady state appears to beachieved by the fourth day of either once or twice daily dosing (6-8). Clarithromycin has a higher affinitythan erythromycin for penetration into cells and tissue. The excellent penetration into cells combined with 80-90% bioavailability and high serum concentrations results in even greater intracellular and tissue concentrations(10) (Table 2)Thus, clarithromycin has balanced pharmacokinetics achieving high intracellular concentrations needed for intracellular infections such as Chlamydia pneumoniae and disseminated Mycobacterium avium, withoutsacrificingserumand interstitial concentrations neededfor Streptococcus pneumoniae and Haemophilus infruenzae infections. Macrolides such as erythromycin and oleandomycin have been known to induce strong muscular contraction inthe gastrointestinal tract of dogs and humans. Intravenous erythromycin given at a O.OZmg/kg dose to dogs induced a pattern of migrating contractions in the gastrointestinal tract similar to the effect of motilin, a 22aminoacid peptide hormone, on gastrointestinal contractile activity (11). When given intravenously at a dose of 7 mg/kg, an immediate increase in contractile activity in the whole length of the intestinal tract was observed (12). The prokinetic activityof 14-membered macrolides and 15-membered azalides can also be related to their ability to bind to motilin receptors inthe gastrointestinal tract. These findings in dogs relate well to the frequent side effects of erythromycin

Craft

78

Tub& 2 Mean Bronchopulmonary Pharmacokinetics (pg/ml) for Clarithromycin, 14(R)-Hydroxyclarithromycin,and Azithromycin

Plasma Clarithromycin 14-Hydroxyclarithromycin Azithromycin Epithelial lining fluid Clarithromycin Azithromycin Alveolar cells Clarithromycin 14-Hydroxyclarithromycin Azithromycin ~~

4h

8h

a

a

h

a

24h

a

~~

.Below limit of assay (0.01 Source: From Ref.

which include nausea, vomiting, abdominal pain, diarrhea, and anorexia. Clarithromycin activity in the dog model is only 60% of that for erythromycin. Clarithromycin has a decreased affinity for the motilin receptors when compared to erythromycin and azithromycin (13) (Table 3). This decreased affinitywas shown to have clinical significance in two clinical studies comparing clarithromycin to erythromycin forthe treatment of pneumonia. In pneumonia studies conducted in adults comparing clarithromycin to erythromycin base or erythromycin stearate, there were fewer adverse events involving the digestive system in clarithromycin-treated patients compared to erythromycin-treated patients (13% versus 32%;~ < .01) (3,4). llventy percent the erythromycin-treated patients discontinued therapydue to adverseeventscompared to 4%of clarithromycin-treatedpatients. In other studies, clarithromycin was found to have gastrointestinal tolerance comparable to oral cephalosporins such as cefaclor (14,15). Clarithromycin is approximately equal to or one tube dilution less active than erythromycin againstH . influenzae and H . paruinfluenzae (1619). However, the 14-hydroxy metabolite has activity equal to orone tube dilutionmoreactivityagainst H . injruenzue aserythromycin, with an MIC, of 2-4 pg/ml (20). The combination of clarithromycin and 14(R)hydroxyclarithromycin produces additive and sometimes synergistic effects against H . influenzae both in vitro (21,22) and in vivo in a gerbil model of otitis media and a mouse model of pneumonia (23). To explain the ergistic effect between clarithromycin and 14(R)-hydroxyclarithromycin, their interactionwith gram-negative and gram-positive bacterial ribosomes

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on Macrolides

Tab& 3 Motilin Activity of Antibiotic Macrolides ~~

~

Clarithromycin Erythromycin Azithromycin

Smooth muscle (PED,,)

Dog (force/freq) stomach

5.85

1.00

Motilin binding

jejunum

PIC50

1.09

*p < .05 relative to azithromycin. Source: From Ref. 13.

was studied. It wasfound that for H. influenzae (1879) strain), 14(R)hydroxyclarithromycin bindsto two different sites. Similar to erythromycin and clarithromycin,it binds to the subunit of the ribosome. The 14(R)hydroxyclarithromycin molecule also bindsto the 70s ribosomal initiation complex which may provide the potential for a synergistic effect in some organisms (24). This increased activity against a full range of respiratory pathogens has allowed clarithromycin to obtain more extensive indications than erythromycin as shown below.

Clarithromycin Indications and Usage (3) Clarithromycin tablets and oral suspension are indicated for the treatment ofmild to moderate infections caused by susceptible strains of microorganisms in the conditions listed below: Adults: PharyngitislTonsillitis due to Streptococcus pyogenes (The usual drug of choice in the treatment and prevention of streptococcal infection and prophylaxisof rheumatic feveris penicillin administered by either the intramuscular or the oral route. Clarithromycin is generally effective in the eradication of S. pyogenes from the nasopharynx; however, data establishing the efficacy of clarithromycinin the subsequent prevention of rheumatic fever are not available at present.) Acute maxillarysinusitis due to Haemophilus influenzae, Moraxella catarrhalis, or Streptococcuspneumoniae. Acute bacterial exacerbationof chronic bronchitis due to Haemophilus influenzae, Moraxella catarrhalis,or Streptococcuspneumoniae. Pneumonia due to Mycoplasma pneumoniae or Streptococcus pneumoniae.

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Uncomplicated skin and skin structure infectionsdue to Staphylococcus aureusor Streptococcus pyogenes. Disseminated mycobacterial infections due to Mycobacterium avium or Mycobacterium intracellulare. Children:

PharyngitislTonsillitis due to Streptococcus pyogenes. Acute maxillarysinusitis due to Haemophilus infiuenzae, Moraxella catarrhalis,or Streptococcus pneumoniae. Acute otitis media due to Haemophilus infiuenzae, Moraxella catarrhalis, or Streptococcuspneumoniae. Uncomplicated skin and skin structure infectionsdue to Staphylococcus aureus or Streptococcus pyogenes. Disseminated mycobacterial infections due toMycobacterium avium, or Mycobacterium intracellulare. Prophylaxis: Clarithromycin tablets and oral suspension are indicated for the prevention of disseminated Mycobacterium aviumcomplex (MAC) disease in patients with advanced HIV infection.

Helicobacterpylori Clarithromycin in combination with ompeprazole are indicated for the treatment of patients with an active duodenal ulcer associated with H. pylori infection. The eradication of H. pylori has been demonstrated to reduce the risk of duodenal ulcer recurrence. Clarithromycin also has activity in vitro and in vivo activity against several other new pathogens of clinical importance. These pathogens include many of the environmental mycobacteria, which have become a major problem because of the AIDS epidemic and Helicobacterpylori which is associated with peptic ulcer disease. Unlike erythromycin, clarithromycin has activity against mycobacteria. The MICsof clarithromycin for M. chelonae, M. kansasii, and M. xenopi are generally 1 pg/ml(25-27). The MI&, for M. avium complex using pH-adjusted media in Bactec is 0.5 pg/ml (28). Clarithromycin has been shownto have activity againstM. leprae in the mouse foot pad model (29). This activity has been very useful in the treatment and prophylaxis against these previouslyuntreatable infections inAIDS patients. Clarithromycin, however, does not have sufficient activity against M. tuberculosis to be of clinical benefit.

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81

Macrolides for Peptic Ulcer Disease Peptic ulcer disease results in a high degree of morbidity and attendant economic cost. Early in this century,the pathogenesis of this disorder was believed to be related to diet complicated by stress.Palliative therapy focused on bed rest and a diet of bland foods. Subsequently, it was suspected that the gastric secretion of hydrochloric acid producedthe tissue injury associated with peptic ulcer disease, and antacids became'a standard therapy. In the early H,-receptor antagonists replaced antacids as the mainstay of therapy. More recently, inhibitorsof the proton pump gastric parietal cells, for example, omeprazole and lansoprazole, have also been shown to be effective in accelerating ulcer healingand, when continued, reduced the rateof recurrence. Even after complete ulcer healing has been obtained using thesepotent therapeutic agents, peptic ulcer disease recurs. The natural history of ulcer had been described as "once an ulcer, always an ulcer." Since the early research into gastritis andpeptic ulcer disease has been dominated by Helicobacter pylori. H . pylori is a small, gramnegative, spiral-shaped, urease-producing bacterium that inhabits the mucus layer coveringthe gastric mucosa.The association between this organism and human diseasewas first reported by Marshall and Warren in Initially, identification of the bacterium included it with the genus Campylobacter, but further research demonstrated a difference and the name Helicobacter pylori was adopted. Infection ofthe human stomachby H . pylori occurs worldwideand is virtually ubiquitous in many developing countries. Infection seems to be acquired early in life and is generally inversely proportional to social economic status. Means of transmission is unknown but is believed to be by way of the fecal-oral route, and infection, although acquired in childhood, generally persists unlesstreated.

Clarithromycin Treatment of Peptic Ulcer Disease Clarithromycin is very active against H . pylori, with aMIC, of pg/ml Clarithromycin and 14(R)-hydroxyclarithromycinare bound tightly to H . pylori ribosomes, having aKd value of 2 M which isthe tightest binding interaction observed for a macrolide-ribosome complex A comparison of kill kinetics and MICs demonstratedthat clarithromycin (with early bactericidal activityof at least a log reduction by h) was more active than azithromycin and erythromycin against H . pylori When tested againstH . pylori at pHvalues from5.5 to 8.0, clarithromycin was found to be the most activeof the macrolides tested (Table

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82 Table 4 Comparative Activityof Macrolides Against H . pylon

M C (pdml) Antimicrobial pH

pH 7.3 pH 5.5 6.5 pH

Clarithromycin 0.03 14-OH-clarithromycin Azithromycin 0.12 Roxithromycin 0.12 Erythromycin 0.25 0.25 Josamycin 1.00 Dirithromycin

8.0

< 0.015 0.06 0.06

0.25 1.oo

0.12

1.00 1.00 2.00 4.00 8.00

0.03

0.12 0.12 0.25

< 0.015 0.03

0.03 0.03 0.03

0.12 0.03

Source: From Ref.

Clarithromycin has been studied extensivelythefor treatment of peptic ulcer disease associated with H.pylori infection. Early pilot studies demonstrated clarithromycin to be the most active agent when used as monotherapy for the eradication of H . pylori. Clarithromycin monotherapy demonstrated eradication rates from15% to 54%, dependent on both dosage and frequency (34,35). Morefrequent dosing showing the highest eradication rates (Table 5). eradication rates of even 50% did not appear to be adequate for the treatment of peptic ulcer diseases associated with H.pylori, other agents were addedto clarithromycin. The addition of omeprazole to clarithromycin has improvedthe efficacy of clarithromycin (36,37) (Table6 ) . This is probably related to theincrease inthe mean pH of the stomach to 5.7, an effect of omeprazole which improvesthe activity of clarithromycinand by increasingthe concentration of clarithromycin at the site of the infection (38). Omeprazole also has limited activity againstH . Table

Results of Clarithromycin Monotherapy Studies Eradication

Study number MW-484” M91-602‘

rate dosage Clarithromycin (2 weeks)

Eradl evala

42mg QID 250 15 mg BID 500 1000mg BID 500 mg QID

5/12 U13 4111 7/13

.Eradicated/evaluable at 4 weeks after last dose. bData from Ref. CDatafrom Ref. 35.

36 54

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83

Table 6 Treatment of H.pylon Associated Duodenal Ulcer: Clarithromycin 500 mg tid and Omeprazole40 mg qid for2 Weeks

Studies United States8 Europeb

94% 99%

(60/64) (1481149)

72% 78%

(41157) (114/146)

29% 8%

(16155) (10/127)

'Data from Ref. bData from Ref.

pylori with MIC valuesof approximately 25 pg/ml. Clarithromycin in combination with ranitidine bismuthcitrate has also been shown to improve the activity of clarithromycin (Table 7). Smaller increasesin pH caused by ranitidine are augmented by the anti-H. pylori activity of bismuth citrate and probably account for the synergy seen with ranitidine bismuth citrate. Clarithromycin has become the cornerstone H . pylori eradication therapy. The best regimen using clarithromycin is yet to be determined, but many studies are under way.

PROKINETIC DRUGS Motility of the gastrointestinal tract is modulatedby the gastric pacemaker and spread distally by the myentericplexus. After feeding and during digestion, the plexus initiates the grinding of solids into chyme. Emptying of chyme from the stomach and progression through the gastrointestinal tract is proceeded by waves of electrical activity grouped in Phases I to IV, and known as the migrating myoelectric complex (MMC). Controlof gastrointestinal motilityis associated with cholinergic, adrenergic, and motilin receptors (Fig. 1). Stimulation of cholinergic nervesby acetylcholine leads to increase in motor activity, contractions fromthe esophagus to the small

Tab& 7 Treatment of H.pylon Associated Duodenal Ulcer Clarithromycin500 mg tid or250 mg qid and RBC400 mg bid for2 weeks

United Statesa Clarithromycin 500 mg tid Europeb Clarithromycin 250 mg qid 'Data from Ref. bData from Ref. 40.

Healing

Eradication

72%

82%

89%

94%

84

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Muscle

Figure l

Mode of action of prokinetic agents.

intestine, and maintenance of general muscle tone. This activity is countered by stimulation of dopamine receptors,which leads to gastrointestinal atony, lossof antroduodenal coordination, and reduced peristalsis (Fig. 1). Hence, exogenous cholinergic receptor agonists or dopamine antagonists lead to increased gastrointestinal motor activity and the induction of Phase I11 of the MMC (41), stimulation of coordinated antroduodenal contractions, and gastric emptying. Motilin is 100-fold more active than acetylcholine in stimulationof the gastrointestinal tract (42). Motilin is believedto be the major physiological factor inthe initiation and coordinationof the interdigestive muscle contractions which propel gastric contents through the gastrointestinal tract (Fig. 1). Exogenous administration of motilin results in an increase in lower esophageal sphincter pressure(44,45) and increased motility of the lower esophagus (46). In addition, induction of coordinated contractions resembling PhaseI11 the MMC leads tohccelerated gastric emptying(47). Erythromycin mimics the activity exogenous motilin when administered intravenouslyto rabbit, dog, and man (11). In humans, the intravenousadministration of erythromycininducesPhase I11 activity of the MMC (48) and coordinates contractile activities inthe esophagus, gastric antrum, and the intestine. Erythromycin specifically inhibits the binding of {lZI}-motilin to rabbit duodenal muscle strips (49) and displaces bound motilinfromisolatedrabbitcolonmonocytes (50). Erythromycinalso demonstrated clinical effectiveness in diabetic gastroparesis 32). (51 The prokinetic activityof the macrolide molecule is chemically unrelated to the antibacterial activityof the class of compounds. Thus, modifica-

85

Basic and Clinical Research on Macrolides Table 8 Ribosomal Binding Studies

Relative binding Compound Erythromycin ABT-229

1.5

10-7

1.5

10 M

M

lo00 lo00

Source: From Ref. 42.

tion of erythromycin led to compounds with improved prokinetic activity and reduced antibacterial activity, as demonstrated by reduced ribosomal binding (5334) (Table 8). ABT-229 is an excellent candidatefor use in humans withabnormal gastrointestinalmotility. In vitro data indicate that ABT-229displaces bound {'~I}-motilinfrom receptors in isolated rabbit antral smooth-muscle tissues; this displacement was logs greater than for erythromycin. Binding of ABT-229 is within an order of magnitude of that of motilin, indicating that ABT-229isa potent synthetic motilin agonist. In addition, tissue assays using rabbit duodenal smooth muscle indicate that ABT-229 is several orders of magnitude more potent than erythromycin as a prokinetic agent. ABT-229 has minimal affinity for bacterial ribosomes, which are the binding sitesof the antimicrobial activityof macrolides, withan affinity 1000-4000-fold lower than erythromycin. (Table 8). In addition, in vitro data against standard laboratory strainsof bacteria indicate that the mean MIC is 50 to > lOOpg/ml for ABT-229, comparedto erythromycin, which had a mean MICof 0.04-0.5 &m1 for susceptible organisms.Thus, there is minimal riskof altering the normal gut floraby long-term administration of ABT-229. Potency in animals has been demonstrated in the opossum by placement of strain gauge transducers inthe lower esophageal sphincter. ABT229 and erythromycin were each administered as1.0-mgkg a dose intravenously. Both drugs induce motility in the lower esophageal sphincter,but ABT-229 produces higher amplitude contractions and a more prolonged effect than erythromycin. In addition, ABT-229 also increasedthe toneof the esophageal smooth muscle. Thus, ABT-229 would be expected to be more effectivethan erythromycin for improving esophageal peristalsis and sphincter tone in man. The anesthetized dog model has been usedto evaluate the effect of ABT-229 and erythromycin on gastrointestinal motility. A single dose of eachcompound (0.4 mgkg) wasgiven intravenously.Bothdrugs had qualitatively similar activity, but ABT-229 induced more potent and sus-

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86

tained smooth-muscle contractions in the stomach, duodenum, and jejunumthanerythromycin. The response to ABT-229wasalso compared with that of cisapride in the same model; ABT-229 produced a higher motility index in the stomach, duodenum, and jejunum of the anesthetized dog. The motilin activitiesof erythromycin was always considered to be an unwanted side effect, but more recently erythromycin has been used successfully to treat diabetic gastroparesis. Thus, prokinetic macrolides have a potential to be usedto treat disorders of gastrointestinal motility in humans such as diabetic gastroparesis and gastroesophageal reflux.

THE FUTURE OF MACROLIDES The macrolides, after more than 40 years of use as antibiotics, are now on the verge a new age. New macrolides with improved activity against the resistant Streptococcus pneurnoniae will continue the antibiotic uses this class of drugs. Macrolides have become widely used as immunomodulators in the treatment of rejection during organ transplantation. Their usefulness in the treatment of inflammation associated with diseases such as asthma and Crohn's diseaseare just being defined andthe possibilities of treating some forms of cancer with combination therapy including macrolides has been reported. The prokinetic macrolidesare extending the nonantibiotics uses of the macrolides; this suggests that the potential may be unlimitedfor these extremely versatile molecules.

REFERENCES 1. Periti P, Mazzei T, Mini E, Novelli A. Clinical pharmacokineticproperties of the macrolide antibiotics, effectsof age and various pathophysiological states (Part I). Clin Pharmacokine 1989; 16:193-214. 2. Piscitelli SC, Danziger LH, Rodvold KA. Clarithromycin and azithromycin: new macrolide antibiotics. Clin. Pharm1992; 11:137. 3. BIAXIN", Abbott Laboratories, Physicians' Desk Reference@,Montvale NJ: Medical Economics Company, 1996: 405-410. P, Siepman N, ChanCK. Treatment of community4. ChienSM,Pichotta acquired pneumonia.A multicenter, double-blind, randomized study comparing clarithromycin with erythromycin. Canada-Sweden ClarithromycinPneumonia Study Group. Chest1993; 103(3):697-701. 5. Zuckerman JM, Kaye KM. The newer macrolides, azithromycin and clarithromycin. Infec. Dis Clin North 1995; 9(3):731-745. 6 . Chu S, Sennello L, Bunnell S, Varga L, Wilson D, Sonders R. Pharmacokineticsof clarithromycin,a new macrolide, after singleascending oral dose. Antimicrob. Agents Chemother 1992; 36:2447-2453.

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7. Chu S , Wilson DS, Park Y, Locke C, Cavanaugh JC. Drug-food interaction potential of clarithromycin, anew macrolide antimicrobial. J Clin Pharmacol 1992; 32(1):32-36. Chu S, Wilson DS, Deaton RL, Mackenthun AV, Eason CN, CavanaughJC. Single- and multiple-dose pharmacokineticsof clarithromycin, a new macrolide antimicrobial. JClin Pharmacoll993; 33(8):719-726. 9. Chu S , Deaton R, Cavanaugh JC. Absolute bioavailability of clarithromycin after oral administration in humans. Antimicrob. Agents Chemother 1992; 36(5):1147-1150. 10. Pate1 KB, Xuan D, Nightingale CH, Tessier PR, Russomanno JH, Quintiliani R. A comparisonof the bronchopulmonary pharmacokineticsof clarithromycin and azithromycin. 3rdInternational Conference on the Macrolides, Azalides, and Streptogramins, Lisbon,1996; abstr 4.06. 11. Itoh Z, Nakaya M, Suzuki T,Hiral H, Wakabayashi K. Erythromycin mimics exogenous motilin in gastrointestinal contractile activity in the dog. Am J PhysiollO:G688-G694. 12. Zara G, Thompson H, Pilot M, RitchieH. Effects of erythromycin on gastrointestinal tract motility. J AntimicrobChemother 1986; 16:175-179. 13. Nellans HN, Peterson AC, PeetersTL. Gastrointestinal side effects: clarithromycin superior to azithromycin in reduced smooth muscle contraction and binding. 31st Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago,1991; abstr 518. 14. Gooch W M , Gan V N , Corder W, Khurana CM, AndrewsW P Jr. Clarithromycin and cefaclor suspensions inthe treatment of acute otitis media in children. Pediatr Infect Dis J1993; 12(S3):S128-S133. 15. Guay DR, Patterson DR, Siepman N, Craft JC. Overview of the tolerability profile of clarithromycin in preclinical and clinical trials. Drug Safety 1993; 8(5):350-364. 16. Benson C, Segreti S , Beaudette F, Hines D, Goodman L, Kaplan R, Trenholme G. In vitro activity of A-56268 (TE-031), a new macrolide, compared with that of erythromycin and clindamycin against selected gram-positive and gram-negative organisms. Antimicrob Agents Chemother 1987; 31:328-330. 17. Barry A, Fernandes P, Jorgensen J,Thornsberry C, Hardy D, Jones R. Variability of clarithromycin and erythromycin susceptibility tests with Haemophilus injluenzae in four different broth media and correlation with the standard disk diffusion test. J Clin Microbioll988; 26:2415-2420. , 18. Fernandes P, Hardy D, Bailer R, McDonald E, Pintar J, Ramer N, Swanson R, Gade E. Susceptibility testing of macrolide antibiotics againstHaemophilus influenzae and correlation ofin vitro results with in vivo efficacy in a mouse septicemia model. Antimicrob Agents Chemother 1987; 31:1243-1250. 19. Liebers D, Baltch A, Smith R, Hammer M, Conroy J, ShayeganiM. Comparative in vitro activities ofA-56268 (E-031) and erythromycin against 306 clinical isolates. J Antimicrob Chemother 1988; 21565-570. 20. Hardy D, Hensey D, Beyer J, Vojtko C, McDonald E, Fernandes P. Comparative in vitro activities ofnew 14-, 15- and 16-membered macrolides. Antimicrob Agents Chemother 1988; 32:1710-1719.

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21. Hardy D, Swanson R, Rode R,Marsh K, Shipkowitz N, Clement J. Enhancement of the in vitro and in vivo activity of clarithromycin againstHaemophilus injluenrae by 14-hydroxyclarithromycin its major metabolite in humans. Antimicrob Agents Chemother 1990; 3431407-1413. 22. Jorgensen J, MahgerL, Howell A. Activityof clarithromycin and its principle human metabolite against Haemophilus influenzae. Antimicrob Agents Chemother 1991; 351524-1526. 23. Vallee E, Azouly-Dupis E, Swanson R, Bergogne-Berezin E, Pocidalo J. Individual and combined activities of clarithromycin and 14-hydroxymetabolite inmurinemodel of Haemophilusinfluenzae infection.JAntimicrob Chemother 1991; 27(supplA):ll-18. 24. ScaglioneF, Goldman C.Interaction of erythromycin, clarithromycin,and the 14hydroxyclarithromycin with gram-negative and -positive bacterial cells and ribosomes. 2nd International Conference on the Macrolides, halides and Streptogramins, Venice, 1994; abstr 113. 25. Biehle J, Cavalieri S. In vitro susceptibility of Mycobacterium kansasii to clarithromycin. Antimicrob Agents Chemother1992; 36:2039-2041. 26. Berlin 0, Young L, Floyd-Reising S, Bruckner D. Comparative in vitro activity of new macrolide A-56268 against mycobacteria.Eur J Clin MicrobInfect Dis 1987; 6:486-487. 27. Brown B, Wallace R, Onyl G, DeRosas V,Wallace R. Activities of four Mycobacterium fortuitum, Mycomacrolides including clarithromycin, against bacterium chelonae, and M. chelonae-likeorganisms,Antimicrob. Agents Chemother. 1992; 36:180-184. 28. Moinard D, Bourried Y, Bener-Melchior P, Raffi F. Activity of clarithromycin against Mycobacterium avium complex determined with Bactec: pH effect. 2nd International Conference on the Macrolides, halides and Streptogramins, Venice, 1994; abstr 156. 29. Ji B, Perani EG, Petinon C, Grosset JH. Bactericidal activities of single or multipledoses of variouscombinations ofnew antileprosydrugs and/or rifampin against M. leprae in mice. Int J Lepr Other Mycobact Dis 1992; 60(4):556-561. 30. Marshall BJ, Royce H, Annear DI, Warren JR. Original isolation of Campylobacter pyloridis from human gastric mucosa. Microbiosci Lett 1984; 25: 31. Hardy D, Hanson C, Hensey D, Beyer J, Fernandes P. Susceptibility of Campylobacterpylori to macrolides and fluoroquinolones.JAntimicrob Chemother 1988; 22:632-636. 32. Goldman R, Zukula D, Capobianco J. Tightbinding of clarithromycin, 14-hydroxyclarithromycinand erythromycinto Helicobacterpylori ribosomes. Antimicrob Agents Chemother1994; 38(7):1496-1500. 33. Flamm R, Beyer J, TanakaS, Clement J. Kill kinetics and activityof antibiotics against Helicobacter pylori. 2nd International Conference on the Macrolides, halides and Streptogramins, Venice,1994; abstr 139. 34. Graham DY, Opekun AR, Klein PD. Clarithromycin for the eradication of Helicobacter pylori. J Clin Gastroenteroll993; 16(4):292-294.

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35. Peterson WL, Graham DY, Blaser MJ, Genta R M , Klein PD, Stratton CW, Drnec J, Prokocimer P, Siepman N. Clarithromycin as monotherapy for eradication of Helicobacterpylon: a randomized, double-blind trial. Am GastroJ enteroll993; 88(11):1860-1864. 36. Hunt R, Schwartz H, Fitch D, Fedorak R, A1 KawasF, Vakil N. Dual therapy of clarithromycin and omeprazole for treatment of patients with duodenal H.pylori infection. 3rdInternational Conference on the ulcers associated with Macrolides, halides, and Streptogramins, Lisbon,1996; abstr 5.18. 37. O'Morain C, Logan RPH, the Clarithromycin European H . pylori Study Group. Clarithromycin in combination with omeprazole for healing of duodenal ulcers, prevention of duodenal ulcer recurrence, and eradication of H . pylori in two European studies. 3rd International Conference on the Macrolides, Azalides, and Streptogramins, Lisbon,1996; abstr 5.25. 38. Gustavson LE, Kaiser JF, Edmonds AL, Locke CS, DeBartolo ML, Schneck DW. Effect of omeprazoleon concentrations of clarithromycin in plasma and gastric tissue at steady state. Antimicrob Agents Chemother 1995;39(9): 2078-2083. 39. Peterson WL, Sontag SJ, Ciociola M, Sykes DJ, McSorleyDD, Webb DD, H. pylonUlcer Group. Ranitidine bismuth citrate plus clarithromycin is effective inthe eradication of Helicobacter pylori and prevention of duodenal ulcer relapse.3rd International Conference on the Macrolides, halides, and Streptogramins, Lisbon, 1996; abstr 5.32. 40. Bardhan K D , Dallaire C, Eisold H, Duggan AE. The treatment of duodenal ulcer with GR122311X (ranitidine bismuth citrate) and clarithromycin. 3rd International Conference on the Macrolides, halides, and Streptogramins, Lisbon, 1996; abstr 5.33. In: Physiology, Diagno41. Champion, M. Treatment of gastric motility disorders. sis andTherapy in G.I. Motility Disorders. Toronto:MES Medical Education Services, 1987:13-14. 42. Fox JET, Daniel EE, Jury J, Fox AE, Collin SM. Site and mechanisms of action of neuropeptides on canine gastric motility differin vivo and in vitro. Life Sci 1993; 33:819-825. 43. Lee K, Chey WY, Tail HH, Yajima H. Radioimmunoassay of motilin. Validation and studies on relationship between plasma motilin and interdigestive myoelectric activityof the duodenum of dog. Am J Dig Dis 1978; 23:789-795. 44. Lux G, Rosch W, Domsche S, Domsche W, Wunsch E. Intravenous 13-Nlemotilin increases the human lower esophageal sphincter pressure. Scand J Gastroenterol 1976; 11 (suppl39):75-79. 45. Jennewein HM, Bauer R, Hummelt H, Lepsin G, Siewert R. Motilin effects on gastrointestinal motility and lower esophageal sphincter (LES) pressure in dogs. Scand JGastroenteroll976; 11(suppl39):63-65. 46. Jennewein HM, Hummelt H, Siewert R, Waldeck F. The uniform effect of natural motilin inthe lower esophageal sphincter, fundus, antrum, and duodenum in dogs. Digestion 1975;13:246-250. In: Itoh Z, ed. 47. Yamagishi T,Debas HT. Biological activity in gastric emptying. Motilin. San Diego: Academic Press, (1990):122-132.

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48. Tomomasa T, Kuroume T, Arai H, Wakabayashi K, Itoh Z, Erythromycin induces migrating complexin human gastrointestinaltract. Dig Dis Sci 1986; 31:157-161. 49. Kondo Y, Toni K, Amura S , Itoh Z, Erythromycin and its derivatives with motilin-like biological activities inhibit the specific binding of 1-125 motilin to duodenal muscle. Biochem Biophys Res Commun 1988; 150:877-882. 50. Hasler W, Heldsinger A, Chung OY. Erythromycin contracts rabbit colon monocytes via occupationof motilin receptors. Am Physioll992; J 262:G50G55.

51. Urbain JL, Vantrappen G , Janssens J, VanCutsem E, Peeters TL, De Roo M. Intravenous erythromycin dramatically accelerated gastric emptying in gastroparesis diabeticorum and normals and abolishesthe emptying discrimination between solids and liquids. Nucl J Med 1990; 31:1490-1493. 52. Janssens J, Peeters TL, Vantrappen G, Tack J, Urbain JL, De Roo M, Mulus E, Bouillon R. Improvement of gastric emptying indiabetic gastroparesis by erythromycin. Preliminary studies.N Engl J Med 1990; 322:1028-1031. 53. Omura S, Tsuzuki K, SunazukaT, ToyodaH, Takahahsi I, Itoh Z. Gastrointestinal motor-stimulating activity of macrolide antibiotics and the structureactivity relationship. JAntibiot (Tokyo) 1985; 38:1631-1632. 54. Omura S, Tzuzuki K, Sunazuki T, Marui S, Toyoda H, Inatomi N, Itoh Macrolides with gastrointestinal motor stimulating activity. J Med Chem 1987; 30:1941-1943.

7 Nonantibiotic Effects of Macrolide Antibiotics: Suppression of the Bacterial Glycocalyx of Pseudomonas aeruginosa Isolates from Patients with Cystic Fibrosis Charles W. Stratton Vanderbilt University Schoolof Medicine Nashville, Tennessee

INTRODUCTION Macrolide antibiotics such as erythromycin, clarithromycin, and azithromycin, as well as clindamycin have been notedto have the ability to decrease sputum production in patients with chronic respiratory infections (1-3). Macrolide antibiotics also have beenreported to decrease the in vitro expression of mucoid exopolysaccharide (MEP), also called alginate, slime, and glycocalyx, produced by Pseudomonas aeriginosa (4,s). Although the decrease of sputum production in patients with chronic respiratory infections has beenattributed to a direct effect on the production of sputum it may, instead, be due to inhibition of MEP production by respiratory pathogens. Pseudomonas aeruginosa is a common cause of chronic pulmonary tract infections in patients with cystic fibrosis (CF) (6).Indeed, the predominant in vivo phenotype of P . aeruginosa that invariably emerges in chronic pulmonary infectionsof CF patients is this mucoid strain which is character91

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92

bed by the copious production of MEP (7,8). Emergence of this strain is associated with progressive respiratory distress and a poor prognosis (9). Suppression of MEP, therefore, may prove importantfor the therapy of CF patients with chronic pulmonary infections caused by this pathogen. This study usedthe E-strip agar diffusion method (10) to evaluate the suppressiveeffects of erythromycin,clarithromycin,azithromycin,and clindamycin on the in vitro productionof pseudomonal MEP using mucoid. strains of P . aeruginosa that werefreshlyisolatedfrom CF patients. Macrolide-containing E-strips were placed on the surface of inoculated agar plates and periodically assessed during 24 h of incubation. Other antimicrobial agents that inhibit protein synthesis (tetracycline and chloramphenicol)werealsoassessed.Inaddition,selectedcombinations of macrolides and antipseudomonal agents were evaluated using a doubledisk/E-strip agar diffusion technique.

MATERIALS AND METHODS Isolates

lbenty-five fresh mucoid strainsof P.aeruginosa were obtained and evaluated after initial isolation from cystic fibrosis patients. Because mucoid strains of P . aeruginosa often become nonmucoid after being subcultured in the clinical microbiology laboratory (11,12), only freshly isolated mucoid strains that had not been subcultured were used. The ATCC strain of P . aeruginosa (ATCC 27853) and other well-characterized strains [PsSOSAI', PsSOSAI-, and PsSOSAICONisogenic strains(13),kindlysupplied by Doctor David M. Livermore] were included as nonmucoid controls.

Antimicrobial Agents

Antimicrobial agents used in this study included erythromycin, clarithrom cin, azithromycin, and clindamycin, as well as tetracycline, chloramphenicol, andthe commonly used antipseudomonal agents gentamicin, ciprofloxacin, and piperacillin. Each agent was obtained as a graded carrier strip containing a predefined antibiotic gradient (AB Biodisk, Solna, Sweden) or on a susceptibility disk containing a fixed concentrationthe antibiotic (Difco) .

Media The agar media selected for this assessment included media limited in nutrients which promotes the growthofmucoidsessileformsof P. aeruginosa (1 1,12); these were cation-supplemented Mueller-Hinton agar

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(CSMHA, BBL) and Batco minimal agar Davis (BMAD, Difco). In addition, Bactec nutrient agar (BNA, Difco) was used; this medium nutrientis rich and promotesthe growth of nonmucoid planktonic forms(llJ2).

Assessment of Suppression The methodology selectedfor measuring the suppressive effectsof macrolides on MEP production of P . aemginosa was agar-based because agar promotes the microbial growthof mucoid sessile forms by providing attachment sitesfor these sessile forms as opposed to growth inbroth, which does not provide such attachment sites, thus promoting the growth of nonmucoid planktonic forms(14). The E Test (AB Biodisk) agar diffusion method (10) was used,as it providedlowandhighconcentrations of the selected macrolide and allowedthe determination of MIC (minimal inhibitory concentration) values. After inoculation with mucoid strains and placement of macrolide-containing E-strips, agar plates were incubated in ambient atair 35°C and evaluated after6, 8, 10, 12,24, and 48 h of incubation. Selected combinations of macrolides and antipseudomonal agents were evaluated cm) using a double-disk/E-strip agar diffusion. This technique used(10small plates of CA-MHB. These plates were inoculated with a mucoid stain by triple cross-streaking with a cotton swab asper the NCCLS recommendations forthe disk diffusion test(15). An E-strip containing the macrolide to be tested is placed on the agar surface and a disk containingthe antipseudomonal agent is placed at each end of the E-strip on opposite sides. The zone sizesof these two disksare assessed at 6,8,10,12,24, and 48 h of incubation. This allowed assessment ofthe effect of high and lowconcentrations of each macrolide on the inhibitory effect of the antipseudomonal agent.

RESULTS The results of this studyare summarized as follows:

1. Initial clinical isolates ofmucoid strains of P . aeruginosa from CF patients exhibited a characteristic mucoid appearance (wet, smooth, confluent colony growth) on CSMHA and BMAD; in contrast, these initial isolates exhibited a nonmucoid appearance (dry, rough, nonconfluent colony growth) when grownon BNA. 2. Each macrolide studied initially was noted to suppress the production of MEP at 6-12 hours of growth on CSMHA as evidenced by an eliptical zone aroundthe E-strip in which discrete nonmucoid colonies were seen. Nonmucoid colonies withinthe inhibitory zonesof the macrolides when suspended in an India inkhaline solution and viewed by

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Tabk Z Evaluation of the Potential for Tetracyclineand Chloramphenicol to Suppress Alginate Production in25 Mucoid Isolates of Pseudomonas aeruginosa. Results of Potential Glycocalyx Suppressionon the Antimicrobial Activity for Selected Antipseudomonal Agents

tested

Zone size (mm): top; bottom; -t standard

Antipseudomonal Suppressive Difference (mean agent Gentamicin Piperacillin Imipenem Pipltazo Ciprofloxacin Gentamicin Piperacillin Imipenem Pipltazo Ciprofloxacin

4.

5. 6.

7.

Tetracycline Tetracycline Tetracycline Tetracycline Tetracycline Chloramphenicol Chloramphenicol Chloramphenicol Chloramphenicol Chloramphenicol

'

19 (2 1.2); 18 (2 1.1); l ( * 0.8) 19 (2 1.3); 20 (2 1.2); l ( + 1.0) 26 (+ 1.5); 25 (2 1.6); none 27 (2 1.4); 28 (2 1.7); l ( + 1.5) 20 (+ 1.2); 19 (+ 1.4); 1 (2) 0.9) 19 (2 1.2); 20 ( 2 1.3); 1(+ 1.1) 20 (+ 1.4); 18 ( 2 1.7); 2 (+ 1.6) 25 (2 1.5); 25 (+ 1.7); none 27 (2 1.6); 27 (+ 1.5); none 20 (2 1.4); 19 (2 1.5); 1(-t 1.3)

phase-contrast microscopy were found to exhibit a marked reduction in capsular size in comparison with mucoidsurroundcolonies ing the inhibitory zones. These suppressive effectsof the macrolides studied wereranked as clarithromycin = azithromycin > erythromycin > clindamycin. At 24 h, this suppressive effectwas almost nonexistant withthe exception of azithromycin. Tetracycline and chloramphenicol did not suppress the production of mucoid exopolysaccharide. Instead, tetracyclinewas noted to inhibit the growth of these mucoid strains of P. aeruginosa at concentrations of 16-32 @m1 for up to 24 h after incubation on all three types of agar. Chloramphenicol was inhibitory at 24 h with pg/ml on BNA only. The inhibitory effectsof tetracycline werealsonotedfornonmucoidstrains,including the ATCC strain. In contrast, the inhibitory effectsof chloramphenicol were not seenfor any nonmucoid strains. (See Table 1.) Slimy strains of Pseudomonas often were found to besusceptible to gentamicin, ciprofloxacin, and piperacillidtazobactam at 24 h when tested on CS-MHA and BMAD but were resistant when tested on BNA. Nonmucoid strains were invariably resistant to gentamicinwhentested on BNA,yetweresusceptiblewhen tested on CS-MHA and BMAD. (See Table 2.)

etested

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Table 2 Evaluation of the Suppressive Effectof Macrolides on MEP Production in 25 Mucoid Isolatesof Pseudomonas aeruginosaand Their Effecton the Inhibitory Activity of Selected Antipseudomonal Agents

Zone size (mm); top; bottom; 2 standard

Antipseudomonal Suppressive difference (mean agent Gentamicin Piperacillin Imipenem Pipttazo Ciprofloxacin

Erythromycin Erythromycin Erythromycin Erythromycin Erythromycin

19 (2 1.2); 19 ( a 1.3); l ( + 1.1) 18 1.1); 17 (2 1.2); 1 (+ 0.8) 26 ( a 1.4); 27 (2 1.5); 1 (+ 0.9) 28 (2 1.5); 28 ( 2 1.6); none 23 ( a 1.4); 19 ( a 1.2); 4 (2 1.0)

Gentamicin Piperacillin Imipenem Pipltazo Ciprofloxacin

Clarithromycin Clarithromycin Clarithromycin Clarithromycin Clarithromycin

19 (2 1.3); 21 (+ 1.6); 2 (2 1.5) 20 (+ 1.4); 20 (2 1.5); none 27 (+ 1.3); 25 (2 1.9); 2 (2 1.7) 28 (2 1.1); 27 ( a 1.4); 1 (+ 1.2) 26 (+ 1.5); 19 (2 1.3); 7 (+ 1.4)

Gentamicin Piperacillin Imipenem Pipltazo Ciprofloxacin

Azithromycin Azithromycin Azithromycin Azithromycin Azithromycin

18 (+ 1.4); 19 (+ 1.5); 1(2 0.8) 20 (2 1.5); 19 (2 1.3); 1 (2 1.4 26 (a 1.4); 26 (+ 1.8); none 29 (+ 1.2); 28 (2 1.2); 1 (+ 1.1) 26 (2 1.6); 19 (2 1.3); 7 (2 1.4)

Gentamicin Piperacillin Imipenem Pipltazo Ciprofloxacin

Clindamycin Clindamycin Clindamycin Clindamycin Clindamycin

20 (2 1.7); 19 (+ 1.8); 1(2 1.7) 19 (a 1.6); 19 (2 1.4); none 26 (+ 1.4); 27 (2 1.8); l ( + 1.6) 28 (2 1.6); 28 (2 1.4); none 20 (a 1.3); 19 (+ 1.2); 1 (2 0.9)

8. Double-diskE-strip tests revealed that macrolides enhanced the inhibitory effects of ciprofloxacin on CS-MHA and, to a lesser

degree, on BMAD. In contrast, these macrolides were noted to be antagonistic for gentamicinon CS-MHA and BMAD. appreciable effects were seen for the p-lactam agents tested. 9. Hypersusceptible strains recovered from a number of CF patients and were characterized as follows: Mucoid appearance Lack of pigments Increasedsusceptibility to p-lactamagents(meanMICs to carbenicillin = 4 pg/ml) with the exception of imipenem to which susceptibilities remainedthe same.

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DISCUSSION Macrolide exopolysaccharides produced by P. aeruginosa is essentially an anionicpolymericdiffusionbarrierandcanbethoughtofasanionexchangeresin of almostinfinitesurfacearea(16).In addition, MEP protects Pseudomonas from heavy-metal toxicity (17) from most bacteriophages, from phagocytic white blood cells (18,19), from antibodies and or complement (20), and from an inhospitable milieu such as osmotic, pH, or enzymatic dangers (17). P . aeruginosa is able to excrete into its MEP and the surrounding medium several different classes of molecules, includingexopolysaccharides (the buildingblocks of MEP),pigmentswhich function as siderophores, protein enzymes, and toxins (21). Pseudomonal MEP also appears to serve as a repository for defensive substances such as p-lactamase (22). Finally, electron microscopy has revealed that there is an interaction of pseudomonal MEP with the mucus in the airways of the human lung (23). This allows mucoid phenotypes of P. aeruginosa to more readily and avidly attach to lower airways than nonmucoid strains. The synthesis of MEP by P. aeruginosa thus appears to be an important virulance factor (7-9). Exposure of planktonic cellsof Pseudomonas aeruginosa to a biofilm surface produced by cells of the same species triggersthe expression of at least genes, aZgC and aZgG (24,25), which inducethe synthesis of MEP. Nutrient limitation in isolates of P . aeruginosa also has been shown to result in increased synthesisof MEP, whereas availability of nutrients results in decreased MEP (11,12). Of interest is that several porin proteins (D-l and D-2 OMPs) of P . aeruginosa are induced in minimal media and repressed in nutrient media. The mucoid phenotypeof P. aeruginosa has been shownto be due to the production of copious amounts of MEP which is composed of one to four linked moietiesof D-mannuronate and L-glucuronate (26,27). MEP is well known for its abilityto form highly viscous aqueous solutions. Indeed, physicalpropertiescharacterizingpseudomonal MEP include(1)water retention, (2) ion binding, and (3) ability to form gels. The water retention and gel volume are at a minimum when the proportion of glucuronic acid residues is approximately 30%. MEP from mucoid P. aeruginosa always lacks poly-G blocks which have buckled polysaccharide chains and form so-called “egg-carton structures.” Instead, pseudomonal MEP has poly-M

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blocks which have extended “ribbon-like” polysaccharide. Poly” blocks are elastic and have high water-retention capability which is thought to be related to an increased amountof side-chain sugars The presence of 0 side-chain sugarson P . aeruginosa lipopolysaccharide (LPS) imparts hydrophilic properties on this LPS which, in turn, results inthe LPS being a virulence factor which can impart resistance to human serum Among other changes that occurunderstarvationconditions are those associated withthe stringent response The stringent responseis an adaptation to conditions amino acid starvation This response includes induction of specific enzymes such as guanosine tetraphosphate (Gp4) in the initial stagewhich then increases the transcription of both inducible and repressible enzyme operons involved the in stringent response Examples of the stringent response include phenotypic changes seen in marine bacterial cells These changesare characterized by rapid multiple divisions of starved cells leading to the formation of “ultramicropm in diameter) which are also called “dwarf” forms. bacteria” (< Dwarf forms ofP . aeruginosa have been described in microcolonies within the lung tissueof patients with CF Rapid formationof multiple copies is presumed to improve the chances of individual genomes surviving. These cell are dormant forms and are quite resistant to many antimicrobial agents and to osmotic stress. Of interest is that whereas long-term-starved cells are resistant to cellwall active agents as well as agents that inhibit DNA synthesis, they remain somewhat susceptibleto agents that inhibit protein synthesis. This may be due to the fact that maintenance of MEP involves synthesisof the precursors within the cytoplasm, translocation of these precursors to the outer portions of the cell walVmembrane, and final assembly of the biofilm matrix. Studies have shown that MEP maintenanceis dependent on a carbohydrate source, an energy source, certain enzymes, and a functioning efflux pump. There is an increasing body of evidence that macrolides interfere with the synthesis and repair of pseudomonal MEP (43) as well as the elaboration of exotoxins due to their codon-anticodon interactions in which translation of mRNA for inducible enzymes is inhibited. The results of this study confirm earlier reports and, more importantly, demonstratethat the suppressive effectof macrolides on pseudomonal MEP productionof is seen in the phenotypic forms typicallyfound in chronicpulmonaryinfections.Thisstudyalsoillustrates the mediumdependent variability of susceptibility test resultsthat has long been recognized asone of the major problems inherent with in vitro testing. However, if the medium can be selected to promote the growth of those microbial phenotypes actually found in infected patients, the results will be clinically relevant. The suppression of pseudomonal MEP production bymucoid

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strains bymacrolidesoffersafeasibleapproachfor CF patients with chronic pulmonary infections now that newer macrolides with better absorption andfew side effectsare available. Moreover, nonantibiotic effects such as suppressionof the MEP clearly will be clinically importantfor the therapy of other chronicinfectionsinvolvingglycocalyx-encasedsessile forms.

REFERENCES 1. Sawaki M, Mikami R, Mikasa K, Kunimatsu M, Ito S, Narita N. The longterm chemotherapy with erythromycin in chronic lower respiratory tract infections-second report: including cases with Pseudomonas infections. J Japan. Assoc Infect Dis1986; 60:45-50. 2. Goswami SK, Kivity S, Marom Erythromycin inhibits respiratory glycoconjugate secretion from human airways in vitro. Am rev Respir Dis 1990; 141:72-78. 3. Tamaoki J, Takeyama K, Tagaya E, Konno K. Effect of clarithromycin on sputum production and its rheologicalproperties in chronic respiratory tract infections. Antimicrob Agents Chemother1995; 39:1688-1690. 4. Yasuda H, Ajiki Y, Koga T, Kawada H, Yokata T. Interaction between biofilms formed by Pseudomonas aeruginosa and clarithromycin. Antimicrob. Agents Chemother 1993; 37:1749-1755. 5. Ichimiya T, Yamasaki T, Nasu M,In-vitro effects of antimicrobial agents on Pseudomonas aeruginosa biofilm production. J AntimicrobChemother 1994; 34~331-341. 6. Hoiby N, Olling S. Pseudomonas aeruginosa infection in cystic fibrosis.Acta Pathol Microbiol Scand C 1977; 85:197-114. 7. Govan JRW,Hams GS.Pseudomonas aeruginosa and cystic fibrosis: unusual bacterial adaptation and pathogenesis. MicrobiolSci 1986; 3:302-308. 8. Lam JS, Chan R, Lam K, Costerton JW. The production of mucoidmicrocolonies by Pseudomonas aeruginosa within infected lungs in cystic fibrosis. Infect Immunoll980: 28546-546-556. 9. Koch C, Hoiby N. Pathogenesis of cyctic fibrosis. Lancet 1993; 341:1065-1069. 10. Brown DFJ, BrownL. Evaluation of the E test, a novel methodof quantifying antimicrobial activity. J Antimicrob Chemother 1991;27:185-190. 11. Terry JM, Pina SE, Mattingly SJ. Environmental conditions which influence mucoid conversion in Pseudomonas aeruginosa PAOl. Infect Immunol 59 1991; 59:471-477. 12. Govan JRW. Mucoid strains of Pseudomonas aeruginosa. The influence of culture medium on the stability of mucus production. J Med Microbiol 1975; 8513-522. 13. Livermore DM. Penicillin-bindingproteins, porins and outer membrane permeability of carbenicillin-resistant and-susceptible strains of Pseudomonas aeruginosa. J Med Microbioll984; 18:261-270.

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14. Lorian V. In vitro simulation of in vivo conditions: physicalstate of the culture medium. J Clin Microbioll989; 27:2403-2406. 15. National Committee for Laboratory Standards. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically, 3rd ed; Approved Standard M7-A3. Villanova, PA: NCCLS, 1993. 16. Costerton JW, Lewandowski Z, DeBeer D, Caldwell D, Korber D, James G. Biofilms, the customized microniche. JBacterioll994; 176:2137-2141. 17. Magnusson KF. Physiochemical properties of bacterial surfaces. Biochem SOC Trans 1989; 454-458. 18. Costerton JW, LamK, Chan R.The role of the microcolony inthe pathogenesis of Pseudomonas aeruginosa. Rev Infect Dis1983; 5 (suppl):S867-S873. 19. Simpson JA, Smith SF, Dean RT. Alginate inhibition of the uptake of Pseudomonas aeruginosa by macrophages. J Gen Microbioll988; 34:29-36. 20. Meluleni GJ,Grout M, EvansDJ, Pier GB. Mucoid Pseudomonas aeruginosa growing in a biofilm in vitro are killed by opsonic antibodies to the mucoid exopolysaccharide capsule but not by antibodies produced during chronic lung infection in cystic fibrosis patients. J Immunol 1985; 155:2029-2038. 21. Doring G, Goldstein A, Roll A, Schiotz PO, Hoiby N, Botzenhart K. Role of Pseudomonas aeruginosa exoenzyme in lung infectionsof patients with cystic fibrosis. Infect Immunoll985; 4937-562. 22. Giwercman B, Jensen ET, Haoiby N, Kharazmi A, Costerton JW. Induction of p-lactamase production in Pseudomonas aeruginosa biofilm. Antimicrob Agents Chemother 1991; 35:1008-1010. 23. Vishwanath S, Ramphal R. Adherence of Pseudomonas aeruginosa to human tracheobronchial mucin,Infect. Zmmun. 45:197-202 (1984). 24. Davies DG, Chakrabarty AM, Geesey GG. Exopolysaccharideproduction in biofilms: substratum activation of alginate gene expression by Pseudomonas aeruginosa. Appl Environ Microbiol1993; 59:1181-1186. 25. Davies DG, Geesey GG. Regulation of the alginate biosynthesis gene algC in Pseudomonas aeruginosa during biofilm development in continuous culture. Appl EnvironMicrobioll995; 61:860-867. 26. Evans LR, Linker A. Production and characterization of the slime polysaccharide of Pseudomonas aeruginosa. J Bacterioll973; 116:915-924. 27. Sherbrook-Cox V, Russell NJ, Graceas P. The purification and chemicalcharacteristics of the alginate presentin extracellular material produced by mucoid strains of Pseudomonas aeruginosa. Carbohydr Res 1984; 135:147-154. Robertson JA, Trulear MG, Characklis WG. Cellular reproduction and extracellular polymer formationby Pseudomonas aeruginosa in continuous culture. Biotechnol Bioeng1984; 26:1409-1417. structure of lipopolysaccharides from Pseudo29. Wilkinson SG. Composition and monas aeruginosa. Rev Infect Dis1983; 5 (suppl5):S941-S947. 30. Cryz SJ, Pitt TL, Furer E, et al. Role of lipopolysaccharide in virulence of Pseudomonas aeruginosa. Infect Immunoll996; 44508-513. 31. Day DF, Marceau-Day ML. Lipopolysaccharide variability inPseudomonas aeruginosa. Curr Microbioll982; 7:93-98.

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Stratton Gilbert P, Collier PJ, Brown MRW. Influence of growth rate on susceptibility to antimicrobial agents:biofilms, cell cycle, dormancy and stringent response. Antimicrob AgentsChemother Roszak DB, Colwell RR. Survival strategiesof bacteria in the natural environment. Microb Rev Moyer CL, Morita RY. Effect of growth rate and starvation-survival on the viabilityandstability of a psychrophilicmarinebacterium. Appl Environ Microbioll989; 55: WrangstadhM,Conway PL, Kjelleberg S. The role of an extracellular polysaccharide producedby the marine Pseudomonas sp. in cellulardetachment duringstarvation. Can J Microbio Kita E, Sawaki M, Nishikawa F, et al. Suppression of virulence factors of Pseudomonas aeruginosa by erythromycin. J Antimicrob Chemother Molinari G, Guzmhn A, Pesce A, Schito GC. Inhibition of Pseudomonas aeruginosa virulence factors by subinhibitory concentrations of azithromycin and other macrolide antibiotics. J Antimicrob Chemother Mizukane R, Hirakata Y, Kaku M, et al. Comparative in vitro exoenzymesuppressing activitiesof azithromycin andother macrolides antibiotics against Pseudomonas aeruginosa. Antimicrob Agents Chemother Mizukane R, Hirakata Y, Kaku M, et al. Comparative in vitro exoenzymesuppressing activitiesof azithromycin andother macrolides antibiotics against Pseudomonas aeruginosa. Antimicrob Agents Chemother Haight "H, Finland M. Observations on mode of action of erythromycin. Proc SOC Exp Biol Med

8 Effects of Macrolides on Leukocytes and Inflammation Marie-ThCr&seLabro INSERM U294 CHU X.Bichat Park, France

INTRODUCTION Macrolide antibiotics, azalides (a subgroup of macrolides), and streptogramins (MAS) are all avidly concentrated by host cells, a property crucial to their intracellular bioactivity. Another consequence of this cell traping is the possibility to modify host cell functions and transport to the site of infection or inflammation by mobile cells (the basis for the concept of tissue-targeted pharmacokinetics). This chapter will review an important aspect of MAS interaction with host cell functions: their modulatory effects on the inflammatory response.As no data are yet availableon streptogramins in this context, only macrolides will be considered here. After a schematic overviewof the inflammatory response (beneficial anddetrimentalaspects)andrecognizedanti-inflammatoryactivities of various antibacterial drugs, the anti-inflammatory activity of macrolides will be approached on the basis of in vivo models (therapeutic efficacy in humans and animals) andthe underlying mechanisms identified in ex vivo and in vitro studies (e.g., cellular uptake mechanisms, effects on phagocyte functions, cytokine production and other interference with the immune system). A prospective conclusion will be drawn by examining the place of 101

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macrolides in the treatment of inflammatory diseases and looking atthese molecules in the extended continuum of macrocyclic structures which express dual effectson host cells and microorganisms.

INFLAMMATION: FROM THE GREEKS TO THECYTOKINES FromCelsus(30 B.C.-A.D.38), Rubor, Dolor, Calor, Tumor the four cardinalsigns ofinflammation-towhich Galen (or Wirchow?)added Functiolaesa-havestood the test of time.But the fundamentalapproach to the humoraYcellular factors involved in this phenomenon only began in the 19th century with the work of so many illustrious scientists as Cohnheim, Metchnikoff, Ehrlich, Von Behring, and Landsteiner. day, our visionof the scenario of eventswhichoccurseachtimehost integrity is altered by exogenous (mechanical, chemical, biological) factors or endogenous (tumoral, immune) disturbances has reached an extreme degree of complexity,parallel to our better understanding of pathophysiology at the level of cell-to-cell and intracellular communication pathways. It is outside of the scope of this chapter to analyze the successive (sometimes intricate) cascadesof humoral mediators and cellular activities involved in the inflammatory response, which may otherwise be found in various excellent books and papers(1-7). Here, I only will summarizethe cascade of events that may be possible targets for the anti-inflammatory activity of macrolides andother antimicrobials. When microorganisms have succeeded in overcoming the natural host barriers, a localized and beneficial inflammatory response is initiated to prevent tissue damage, destroy the infective pathogen, and activate repair processes. This well-orchestrated sequence of events is known the as acute-phase response. It involves the coagulation system in case of vascular breeches,the complement system (either activated by antigen-antibody complexes,or, in the alternative pathway, by the lipopolysaccharides present on bacterial surfaces) which leads to the formation of various chemoattractantsfor blood cells and mast cell activators (e.g., C3a, C5a) and opsonins (C3b,theiC3b); vasogenic response (increased dilatation and vascular permeability) mediated by histamine released from mast cells andthe kinin cascade; and the cytokine cascade initiated by resident macrophages activated by microbial or altered-tissueby-products. The early“alarm”cytokinestumornecrosis factor-a (TNF-a) and interleukin-l (IL-1) act both locally and distally by initiating “second wave” cytokines suchI Las8 and other chemokines that are highly chemotactic for blood phagocytes, and induce endothelial cell changes such as expression of adhesion receptors.

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All these changes rapidly promote (within minutes) the firm adhesion of neutrophils to the endothelium, followedby diapedesis and migration toward the infected site, where they perform their crucial function in infection (phagocytosis and bacterial killingby highly destructive oxidants, enzymes and antibacterial proteins, and peptides). Also, leukocytes synthesize and release their set of cytokines withinthe target tissue, aswell asmetabolites of arachidonic acid (prostaglandins, leukotrienes, and thromboxan M ) responsible for vasoconstrictionand bronchoconstrictioddilatation and secondary activationof leukocytes. In parallel, systemic inflammation mediated mainly by cytokines (IL-1, IL-6, TNF) alters the temperature setpoint in the hypothalamus (febrile response) and that of metabolism and gene regulation in the liver with production of acute-phase response proteins (a,-acid glycoprotein, C-reactive protein, complement component C3, etc.) and increased leukocytosis (mainly neutrophil leukocytosis). The involvement of blood monocytes and monocyte-derived tissue macrophages in bacterial eradication occurs later. However, these cells are potent producers of cytokine and other inflammatory mediators, which perpetuate the inflammatoryresponse.Exaggeratedinflammatoryreactions seen in the first stage of infection (when the excess of bacterial and cell products upregulate the host response) involve excessive production of reactive oxygen species targeting host cells and tissues, and cytokines which lead to septic shock, the systemic inflammatory response syndrome, and multiorgan failure.At a later stage, phagocytes initiate the second phaseof the immune response by digesting microbial antigens and transmittingthe messages to T cells, which become involved both in the specific immune response and the inflammatory reaction. Cell-mediated immunity is a major mechanism to terminate infection. In summary, the multifactorial nature of the inflammatory process offers an enormous potential range of therapeutic manipulation for its control. Based on the ultimate fundamental knowledge of the transduction pathways involved in the production, activity, and regulation of the various humorallcellular effectors, research is ongoingto widen our antiinflammatory armamentarium.

ANTIBACTERIAL AGENTS AND INF'LAMMATION It is now recognized that antibacterial agents, whose main target is the microorganism, may interfere with host cell functions (8). The many studies which have been performedvitro, in ex vivo, and in vivo have raised the possibility that some antibacterial drugs may indeed alter the inflammatory response. It is well documented that ampicillin, penicillin G and various aminothiazolyl-cephalosporinspossess oxidant-scavengingproper-

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tiesinvitro(9-11),althoughinvivop-lactam treatment may, on the contrary,enhance the inflammatoryresponseinmeningitis or lead to inflammatory sequelae, particularly in otitis. Recently, we demonstrated that OH-iminocephems directly inhibit myeloperoxidase in vitro (12,13), but no data areavailable on the in vivo effects of these drugs in inflammatory settings. Dapsone, clofazimine, and isoniazid also inhibit the myeloperoxidase system in vitro (14-15), and several authors have suggested is linked to that part of their therapeutic activity in mycobacterial diseases their anti-inflammatorypotential.Finally, the ansamycins and the cyclines have proved beneficial in various inflammatory settings not directly linked to infections. For both kinds of drugs, the hypothesis underlying clinical trials was their cellular uptake property and bioactivity on several microorganisms suggested to play a role in the pathogenesis of rheumatoid arthritis. This hypothesis has not been substantiated, but clinical trials have shown that intraarticular rifamycin SV was beneficial in rheumatoid arthritis, ankylosing spondylitis, and juvenile rheumatoid arthritis (16-18). Similarly, cyclines (particularly minocycline) have shown therapeutic benefit in reactive arthritis and mild to moderate rheumatoid arthritis (19-21). It must be noted that outside of their antibacterial activity these drugs have widely acknowledged effectson various inflammation mediators/effectors suchas inhibition of neutrophil functions and oxidant-scavenging properties (ansamycins, cyclines) and inhibition of metalloproteinases and prostaglandin synthesis (cyclines).The question as to whether macrolides could attenuate inflammatoryresponseswereraisedabout 25 yearsago (22-24) (e.g., erythromycin and troleandomycin in asthma, chronic bronchitis, and other inflammatory settings).In the last 5 years, an explosion of clinical trials and fundamental research in vitro and in animal models has again put this new potential for macrolideson center stage.

MACROLIDES AS ANTI-INFLAMMATORY AGENTS? In Vivo Studies Low-dose, long-term erythromycin treatment has beenreported effective in patients with chronic lower respiratorytract disease including diffuse panbronchiolitis (25-27). Diffuse panbronchiolitis is characterizedby chronic inflammation of the respiratory bronchioles and infiltrationof chronic inflammatory cells, accompanied by repeated episodes of respiratory infections, especially due to Pseudomonas aeruginosa, which finally result in respiratory failure. The clinical effectivenessof erythromycin in such pa-

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tients has been suggested to rely not on its antibacterial but on its antiinflammatory properties. In particular, in those patients receiving erythromycin mg/dayfor months), the number of neutrophils and the neutrophil-derived elastolytic-like activity in broncho alveolar lavage (BAL) fluid decreased andthe number of alveolar macrophages increased significantly compared tothe values obtained beforetreatment. Ampicillin (1 g/day) did not modify these parameters (28). A decreased number of neutrophils in BAL fluid of erythromycin-treated patients with diffuse panbronchiolitis was also observed another in study The authors found that neutrophil chemotactic activity of BAL fluidwasalsosignificantly decreased after a 4-week treatment with erythromycin. In addition, the intrapulmonary influx of neutrophils triggeredby intratracheal injection of IL-8 or lipopolysaccharide (LPS)was suppressed in mice given erythromycin mg/animal) h beforethe intratracheal challenge. Recently, Shirai et al. have reported that roxithromycin or mg) and clarithromycin or mg) given daily for at least months are as effective as erythromycin or mg) in diffuse panbronchiolitis (efficacy in and respectively) and in bronchiectasis (efficacy about Furthermore, chemotaxis of blood neutrophils from patients with diffuse panbroncholitis was significantly reduced after roxithromycin therapy. It has also been shownthat erythromycin and troleandomycin favorably affect the clinical status of patients with severe steroid-dependent asthma An effect of macrolides on theophylline or corticosteroid clearance has been postulated to contribute to their beneficial actions. Macrolide interactionswith various processess involved the in pathogenesis of asthma [e.g., bronchial inflammation and bronchial hyperreactivity is an area of active investigation (see below). The anti-inflammatory activity of macrolides has also been investigated in animal models. Erythromycin proved beneficial in various acute mouse ear inflammation models Similarly, inrats, roxithromycin (one dose of or m a g ) wasaseffective as indomethacin (5 m a g ) in reducing hind-paw edema induced by poly-L-arginine, but slightly less effective in-the L-carrageenan hind-paw edema model and in ear edema induced by croton oil in contrast, this macrolide mg/kg/day for days) was not active in a chronic inflammation model (polyester sponge implantation) Other animalmodelshavebeenused to study the protection afforded by erythromycin and roxithromycin mg/kg/day for days) in a model of endotoxin-induced vascular leakage and neutrophil accumulation inthe rat trachea The mechanism(s) involved in these anti-inflammatory activities have not been elucidated. According to Agen et al. the anti-inflammatory

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activity of roxithromycincouldbedue to a preferential action on the vasogenic component ofthe inflammatory response. However, Tamaokiet al. (37) suggested an effect of macrolides on neutrophil chemotaxis and/or activation. Thiswas also proposed in the model of zymosan-induced (aseptic) peritonitisof mice, where long-term administration of erythromycin (10 mgkg for 28 days) was as effective as dexamethasone (40pg for2 days) in reducing intraperitoneal extravasation and leukocyte (mainly neutrophil) accumulation, as well as prostaglandin E, (PGE,) production (38). That macrolides may modify immuneparameters relatedto inflammation was also suggested by various animalhuman models in which their administration resultedin the ability of immune cells to synthesize inflammatory/anti-inflammatory mediators, particularly cytokines, ex vivo. The mononuclear cells of volunteers receiving azithromycin 500 mg/day for 3 days released an increased amount of soluble IL-2 receptor after phylohemagglutinin (PHA) and phorbol myristateacetate (PMA) costimulation (39). The blood mononuclear cells of volunteers receiving erythromycin (600 mgfor 5 days) had an enhanced capacity to produce IL-la and TNF-a after stimulation with PMAor interferon-y (IFN-y), whereas IFN-yproduction was reduced (40). Ex vivo modification of cytokine production has also been reported in mice. Erythromycin stearate (10 mgkg/day for 28 days) increasedthe production of IL-1 by peritoneal macrophages and IL-2 by spleen cells (41). Similarly, in mice receiving roxithromycin mgkg for 28 days), IL-1and TNF-a production by LPS-stimulatedperitoneal exudate cellswasincreased, aswas that of IL-2, IL-4, andIFN-y by mitogen-stimulated spleen cells (42). Konnoet al. (43,44) observedthat LPS-stimulated resident peritoneal macrophages of mice receiving roxithromycin (5 mg/day) produced more IL-1 than controls on days 7-14 and 28, but on day 42, the production was decreased. Similarly IL-2 production by Con A-stimulated spleen cells or mesenteric lymph node cells was significantly increasedon day 14 and reduced on day 42. IL-5 production was significantly reduced throughout the test period (days 7 to 28). Finally Hirakata et al. (45) observed that peritoneal macrophages from mice receiving erythromycin, mgkg 250 for 7 days, produced significantly greater amounts of thymocyte-activating factors, possibly IL-1 and IL-6. Another immune parametermodified ex vivo by macrolide treatment is the motility of human neutrophils,which was increased after erythromycin treatment in healthy volunteers and in patients with defective chemotaxis (46-47). On the other hand, we have observedthat roxithromycin (300-mg single dose) increased various functions of human neutrophils90 min after ingestion (48), but multiple administrations (300 mg/day for 7 days) resul in a decreased oxidant production in fiveout of six neutrophil samples (49).

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In Vitro Studies Although some data indicate that certain macrolides may interfere with various pathophysiologic processes [for instance, inhibition of cholinergic neuroeffector transmission inthe human airway smooth muscle (50)], I will focus on the numerous aspects of macrolide interference with phagocytes which are corner stones inthe cascade of events associated with beneficial and detrimental aspects of inflammation. Because mostof the macrolideinduced modifications are thought to rely on their phagocyticuptake, this important property will be discussed first in relation with new findings made inmy laboratory. In Vitro Uptake of Macrolides All macrolides display intracellular (phagocytic) accumulation, with cellular to extracellular ratios greater than 20 (51). The precise mechanism underlying this cellular uptake is poorly understood. Trapping by protonation of these weakly basic molecules may explain their intragranular accumulation, particularly in the case of dibasic compounds (azithromycin, dirithromycin). We have recently comparedthe uptake of erythromycin A derivatives for their accumulation in human neutrophils and proposed a preliminary classificationof these compounds basedon their cellular kinetics, efflux from loaded cells, intracellular location, and activation energy (52) (Table 1).We also demonstratedthat extracellular Ca” channel regulating intracellular calcium homeostasis(53). This exchanger is unlikely to Table l Preliminary Classification of Erythromycin A Derivatives Accordingto Uptake by Polymorphonuclear Neutrophils (PMN) Groupkompounds

I:

Azithromycin Dirithromycin Erythromycylamine

11: Erythromycin A

Roxithromycin Clarithromycin

Characte Dibasic drugs Nonsaturable uptake Preferential intragranular location8 AG > kJ/mole Moderate efflux from loadedcells Monobasic drugs Rapid uptake + plateau Bimodal (granulekytoplasm) location AG 60-70 kJ/mole Rapid efflux

-

Erythromycylamine is located in cytoplasm and granules (poor liposolubility hinders membrane permeability).

a

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be the macrolide channel. Preliminary data from our laboratory suggest that PKA-mediated phosphorylation is critical for neutrophil uptake of erythromycin A-derived macrolides (54). Mod$c&n

of Phagocyte Functions by Macrolidees

The possibility that macrolidesdirectlymodifyphagocytefunctions,as suggested byinvivoandexvivo studies, has been widely investigated (reviewed in Ref. 55). In particular, many data suggest that erythromycin A derivativesimpairoxidantproduction by phagocytes(e.g., neutrophils) in a time-dependent and concentration-dependent manner and that thiseffectmaybeobtainedattherapeuticallyrelevantconcentrations (56-58). The mechanism has not been elucidated. Although Mitsuyama et al. (58) suggested that erythromycin-induced inhibition of the neutrophiloxidativeburstismediatedthroughPKAactivation,theydidnot check the importance of PKA for macrolideuptake.Perry et al.(59) have clearly shown the importance of the phospholipase D and phosphatidate-phosphohydrolase transduction pathway in roxithromycin-induced inhibition of neutrophil NADPH oxidase, the crucial enzyme in oxidant production by phagocytes. Parallel to their inhibitory effect onthe oxidative burst, erythromycin A derivatives have a stimulating effecton neutrophil exocytosis (60-62). This activity may serveto explain, partlyat least, the intracellular bioactivity of macrolides on some microorganisms which inhibit phagolysosome fusion; it does not ruleout potential anti-inflammatory activity, as various anti-inflammatory drugs (chloroquint, amodiaquine, staurosporine, etc.) impair oxidant generation while inducing neutrophil exocytosis. It must be noted that 16-membered-ring macrolides do not display these two neutrophil-modulating activities; onthe contrary, josamycin has been reported to enhance oxidant production by phagocytes, an activity suggested to underly its inhibitory effect on antibody production (63). The in vitro effect of macrolides on cytokine production has been also demonstrated. Spiramycin at the high concentrations of10-50 mgL enhanced IG6 production by LPS-stimulated human blood mononuclear cells (64) and josamycin > midecamycin > clarithromycin decreased IL-2 production in these cells (65). This effect was additive withthat of FK 506 and cyclosporin A. Clarithromycin > erythromycin decreased IL-1 production by LPS-stimulated peritoneal macrophages of mice (66), and roxithromycin (2-10 m&) decreased that of TFN-a bymouse spleencells and macrophages (42). This latter effect was suggested to involve increased IL-4 production. Recently, Takizawa et al. (67) demonstrated that erythromycin was also able to suppress IL-6 expression by human bronchial epithelial cells.

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Other interactions of macrolideswithvariousparameters of the inflammatory response have been described. Briefly, erythromycin (1-10 m&) has been reported to shorten neutrophil survival by accelerating apoptosis (68), to promote monocyte to macrophage differentiation and to inhibit respiratory glycoconjugate secretion from human airways (70),whereasroxithromycinhasbeenshown to inhibitcell cycle progressionin HL cells (71) and T-lymphocyte transformation induced by mitogenstimulation(72).Furthermoreithasbeenshown that erythromycin may also alter various virulence factors of Pseudomonas aeruginosa, apathogenofteninvolvedininfectionsinpatientswithpanbronchiolitis (45).

CONCLUDING REMARKS Whatever the in vivo mechanisms(effectson phagocyte numbers and functions, cytokine production, or other inflammatory parameters) there is a worldwide consensusthat in additionto their antibacterialproperties, some macrolide antibiotics are also endowed with anti-inflammatory activity. The host target in most studies isthe phagocyte, which is a cornerstone in host defenses and inflammation, whereasthere is no concern for a beneficial anti-inflammatory activityof macrolides in the case of acute infection with exaggerated destructive inflammatory responses. What wouldbe the consequences of long-term use of these drugs in inflammatory diseases (asthma or panbronchiolitis)? The specter of increasing microbial resistance is a major limitation. Second, anti-inflammatory drugsare also potentially immunosuppressive. What might be the consequences on host natural defences themselvesof uncontrolled useof macrolides in inflammatory diseases? Indeed, there are experimental data showing that erythromycin may induce the suppression of pulmonary antibacterial defences, a potential mechanism of superadded infection (73).It is not easyto answer these questions, but I advocate careful management of clinical trials and extensive cooperation between clinicians and fundamentalists to elaborate new prospects. In particular, elucidation of the structures and mechanism underlying macrolide anti-inflammatory activity will be crucial to develop new therapeutic drugs. The extended classificationof macrolides fromthe classical definition of Woodward (Fig. 1) presents a vast continuum of macrocyclic lactonic structures in which some molecules are mainly antibacterial (true macrolides), whereasothers possess mainly immunosuppressant activity (FK 506, rapamycin) or even antifungal activity with host cell inhibitoryproperties (bafilomycin, concanamycins).

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Macrolides True .

Lankacidins (17)

FK 506 (23)

Rapamycin (311

No Sugars Immunosuppressants

Antibiotics Antifungals

(Antifungals Immunosuppressants) Antibiotics

+

(MIP-producing organisms) Inflammation

Figure I “True” macrolidesare characterized by a 12-to 16-membered ring macrocyclic lactone nucleus with few (if any) double bonds and no nitrogen atom, substituted by several aminoand/or neutral sugars. Recently, azalides were proposed as a new subgroup of macrolides andare characterized by the presence of a nitrogen in the lactonic ring. All these molecules display a homogeneous antimicrobial spectrum. Another subgroup in the macrolide family corresponds to the bafilomycins and concanamycins, which are not therapeutically used, owing to their strong immunodemessant potential related to the property of inhibitingmammalian V-typeH+ATPases, which are crucialin regulating intracellular pH. An extended definition of macrolides will include lankacidins (which are active against microorganisms but do not possess glucidic substituents) and the macrocycliccompounds, FK 506, rapamycin,and their derivatives (which are mainly immunosuppressants). New therapeutic potentials are looked for throughout this vast continuum of macrocyclic compounds with the development of new antimicrobial drugs in the group of rapamycin derivatives and that of the antiinflammatory properties of the macrolide derivatices. The number atoms the lactonic ring given inparentheses. Abbreviations:MIP: Macrophage infectivity potentiating factor (immunophih);organisms which produce these factorsinclude Neisseria, Chlamydia, and Legionella.

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Understandingthe mechanisms that confer on these chemically related molecules different degrees of immunosuppressantlantimicrobial activity will help to define the optimal structures for both therapeutic properties.

Z like the dreams of the f i t w e better than the history of the past. Thomas Jefferson

(letter to J. Adams).

ACKNOWLEDGMENT The author thanks Miss Fr. Breton for her expert secretarial assistance.

REFERENCES 1. Spector WG, Willoughby DA. The Pharmacology of Inflammation. London: English Universities Press, 1968. 2. Armstrong W. Recent Trends in Researchon Inflammation and Treatment of Inflammatory Diseases.SCRIP, PJB PublicationsLtd., 1991. 3. Hellewell PG, Williams TJ eds. Immunopharmacology of Neutrophils. London: Academic Press/Harcourt Brace and CO,1994. 4. Bauman H, Bauldie J. The acutephaseresponse.Immunol Today1994; 15(2):74-80. 5. Gaur S , Kesarwala H, Gavai M, Gupta M, Whitley-WilliamsP, Frenkel LD. Clinical immunology and infectious diseases. Pediatr Clin North Am 1994; 41(4):745-782. 6. Bellanti JA, Kadlec J V , Escobar-Gutierrez A. Cytokines and the immune response. Pediatr Clin North Am 1994; 41(4):597-621. 7. Luger TA,Schwarz T. The role of cytokines andneuroendocrine hormones in cutaneous immunity and inflammation. Allergy 1995; 50:292-302. 8. Labro MT. Immunomodulation by antibacterial agents. Is it clinically relevant? Drugs 1993; 45:319-328. 9. Ottonello L, Dallegri F, Dapino P, Pastorino G , Sacchetti C. Cytoprotection against neutrophil-delivered oxidantattack by antibiotics. Biochem Pharmac01 1991; 42~2317-2321. 10. Gunther MR, Mao J, Cohen MS. Oxidant-scavenging activities of ampicillin and sulbactam and their effects on neutrophil function. Antimicrob Agents Chemother 1993; 37:950-956. 11. Lapenna D, Cellini L,De Gioia S, Mezzetti A, Ciofani G, Festi D, Cuccurullo F. Cephalosporins are scavengers of hypochlorous acid. Biochem Pharmacoll995; 49:1249-1254. 12. Labro MT, El Benna J, Charlier N, Hakim J. Cefdinir (CI-983), a new oral amino-2-thiazolyl cephalosporin inhibits human neutrophil myeloperoxidase in the extracellular medium but not the phagolysosome. J Immunol 1994; 152~2447-2455.

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13. Labro MT, AbdelghaffarH, Cooper R, Munroe J, Ternansky R, Hornback W, Skelton J. 1993. Structure-activity relationships of new carbacephems with antioxidant properties. 33d ICAAC, 1993; abstr 925. 14. Van Zyl JM, Basson K, Kriegler A, Van der Walt BJ. Mechanisms by which clofazimine and dapsone inhibit the myeloperoxidase system. A possible correlation with theiwnti-inflammatory properties. Biochem Pharmacol 1991; 42: 599-608. 15. Van Zyl JM, Basson K, Uebel RA, Van der WaltBJ. Isoniazid-mediated irreversible inhibition of the myeloperoxidase antimicrobial system of the human neutrophil and the effects of thyronins. Biochem Pharmacol 1989; 38:2363-2373. 16. Caruso I, Boccassini L, Cazzola M, et al. Multilocal intra-articular treatment of rheumatoid arthritis: a randomized prospective study comparing rifamycin SV with pefloxacin. JIntern Med Res 1992; 20:27-39. 17. Caruso I, Santandrea S , Sarzi Puttini P, et al. Prevention of appearance of radiologic lesions in early rheumatoid arthritis: a randomized single-blind study comparing intra-articular rifamycin with auranofin in116 patients followed for 60 months. J intern Med Res 1992; 20:61-77. 18. Caruso I, CazzolaM, Santandrea S. Clinicalimprovementinankylosis spondylitis with rifamycin SV infiltrations of peripheral joints. J Intern Med Res 1992; 20:171-181. 19. Lauhio A, Leirisalo-Rep0 M, LahdevirtaJ, Saikku P, Rep0 H. Double-blind, placebo-controlled studyof three-month treatment with lymecycline in reactive arthritis with special reference to Chlamydia arthritis. Arthritis Rheum 1991; 34~6-14. 20. Tilley BC, Alarcon GS, Heyse SP, Trentham DE, Neuner N, et al. Minocycline in rheumatoid arthritis. A 48-week, double-blind, placeboantrolled trial. Ann Intern Med 1995; 122:81-89. 21. Sanchez I. Tetracycline treatment in rheumatoidarthritis and other rheumatic diseases. BrasilMed 1968; 82:22-31. 22. Itkin IH, Menzel ML. The use of macrolide antibiotic substance inthe treatment of asthma. J Allergy1970; 45:146-162. Spector SL, Katz FH, Farr RS.Troleandomycin:effectivenessin steroiddependent asthma and bronchitis. J Allergy Clin Immunoll974; 54:367-379. 24. Plewig G , Schopf E. Anti-inflammatoryeffects of antimicrobialagents. Drugs 1976; 11:472-473. Kudoh S, Uetake T, Hagiwara K, Hirayama M, Hus LH, Kimura H, Sugiyama Y. Clinical effect of low-dose long-term erythromycin chemotherapyon diffuse panbronchiolitis. JapanThorac Dis 1987; 25:632-643. 26. Yamamoto M, Kondo A, Tamura A, Izumi T, Ina Y, Noda M. Long-term therapeutic effects of erythromycin andnew quinolone antibacterial agents on diffuse panbronchiolitis. Japan Thorac J Dis 1990; 28:1305-1313. 27. Nagai H, Shishido H, Yoneda R, Yamaguchi E, Tamura A, Kurashima A. Long-term low-dose administration of erythromycin to patients with diffuse panbronchiolitis. Respiration 1991; 58:145-149.

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28. Ichikawa Y, Ninomiya H, Koga H, Tanaka M, KinoshitaM, Tokunga N, Yano

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T, Oizumi K. Erythromycin reduces neutrophils and neutrophil-derived elastolytic-like activityin the lower respiratorytract of bronchiolitis patients. Am Rev Respir Dis 1992; 146:196-203. Kadota JI, Sakito 0, Kohno S, Sawa H, Mukae H, Oda H, Kawakami K, Fukushima K, Hiratani K, Hara K. Amechanism of erythromycin treatment in patients withdiffusepanbronchiolitis.AmRev Respir Dis 1993; 147~153-159. Shirai T, Sato A, Chida K. Effect of 1Cmembered ring macrolidetherapy on chronic respiratorytract infections and polymorphonuclear leukocyte activity. Intern Med 1995; 34:469-474. Zeiger RS, Schatz M, Sperling W, Simon RA, Stevenson DD. Efficacy of troleandomycin in outpatients with severe steroid-dependent asthma. Allergy Clin Immunoll980; 54:367-379. Kamada AK, Hill MP, IklC DN, Brenner M, SzeflerSJ. Efficacy and safety of low-dose troleandomycin therapy in children with severe, steroid- requiring asthma. J Allergy Clin Immunol 1993; 91:873-882. Miyatake H, Taki F, Taniguchi H, Suzuki R, Takagi K, Satake T. Erythromycin reduce the severity of bronchial hyperresponsiveness in asthma. Chest 1991; 97:670-673. Poulter LW, Janossy G, Power GC, Sreenan S, Burke C. Immunological/ physiologicalrelationshipsinasthma: potential regulation by lungmacrophages. Immunol Today 1994; 15:258-261. Tarayre JP, Aliaga M, Barbara M, Villanova G, Ballester R, The-Versailles J, Couzinier JP. Cutaneously applied erythromycin base reduces various types of inflammatory reactions in mouse ear. Int. J Tissue React 1987; 9:77-85. Agen C, Danesi R, Blandizzi C, Costa M, StacchiniB, Favini P, Del 'hcca M. Macrolide antibiotics as anti-inflammatory agents: roxithromycin in an unexpected role. Agents Action 1993; 30:85-90. Tamaoki J, SakaiN, Tagaya E, Konno K. Macrolide antibioticsprotect against endotoxin-induced vascular leakage and neutrophil accumulation in rat trachea. Antimicrob Agents Chemother1994; 38:1641-1643. Mikasa K, Kita E, Sawaki M, KunimatsuM, Hamada K, Konishi M, Kashiba S, Narita N. The anti-inflammatoryeffect of erythromycin in zymosaninduced peritonitisof mice. J AntimicrobChemother 1992; 30:339-348. Tomazic J, Kotnic V, Wraber B. In vivo administration of azithromycin affects lymphocyte activities in vitro. Antimicrob Agents Chemother 1993; 37: 1786-1789. Katahira J, Haruki K,Shibata Y, Kibuchi K, Hasegawa H, Totuska K, Shimizu H, Kawada H, Takizawa T. Stimulation of interleukin-l and tumor necrosisfactorproduction by oral erythromycin.Chemotherapy(Tokyo) 1991; 39:320-328. Kita E, Sawaki M, Nishikawa F, Mikasa K, Yagyu Y, 'hkeuchi S, Yasui K, Narita N, Kashiba S. Enhanced interleukin productionafter long-term administration of erythromycin stearate. Pharmacology 1990; 47:177-183.

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42. Kita E, Sawaki M, Mikasa H, Hamada H, 'Pdkeuchi S, Maeda K, Narita N. Alterations of host response by a long-term treatment of roxithromycin. J Antimicrob Chemother 1993; 32:285-294. 43. Konno S, Adachi M, Asano K, KawazoeT, Okamoto K, Takahashi T. Influence of roxithromycin on cell-mediated immune responses. Life Sci1992; 51:PL107-PL112. 44. Konno S, Adachi K, Asano K, Okamoto F, Takahashi T. Antiallergic activity of roxithromycin: inhibition of interleukin-5 production from mouse T-lymphocytes. Life Sci 1993; 52:PL25-PWO. 45. Hirakata Y, Kaku M, MizukaneR, Ishida K, Furuya N, Matsumoto T, Tateda K, Yamaguchi K. Potential effects of erythromycin on host defence systems and virulence of Pseudomonas aeruginosa. Antimicrob Agents Chemother 1992; 36:1922-1927. 46. Anderson R, Fernandes AC, Eftychis HE. Studies on the effects of ingestion of a single500 mg oral dose of erythromycinstearate on leucocyte motilityand transformation and on release in vitro of prostaglandin E2 by stimulated leucocytes. J Antimicrob Chemother 1984; 14:41-50. 47. Ras GJ, Anderson R. An in-vitro study of oral therapeutic doses of cotrimoxazole and erythromycinstearate on abnormal polymorphonuclear leucocyte migration. J Antimicrob Chemother 1986; 17:185-193. 48.' Labro MT, Bryskier A, Babin-Chevaye C, Hakim J. Interaction de la roxithromycine avecle polynuclkaire neutrophile humain in vitro et ex vivo. Pathol Bioll988; 36:711-714. 49. Labro MT, El BennaJ, Barre J. Effects of roxithromycin and erythromycinon human neutrophil functionsex-vivo. 1st ICMAS 1992;abstr 175. 50. Tamaoki J, Tagaya E, Sakai M, KonnoK. Effects of macrolide antibioticson neurally mediated contraction of human isolated bronchus. J Allergy Clin 'Immunol1995; 95:853-859. 51. Labro MT. Intraphagocytic uptake of macrolide antibiotics. In: Bryskier A, Butzler JP, Neu HC, Tulkens PM, eds. Macrolides: Chemistry, Pharmacology and Clinical Uses. Paris: Amette-Blackwell, 1993:379-388. 52. Abdelghaffar H. Mkcanisme de modulation des fonctions du polynuclkaire neutrophile humain par les macrolides. Thesis in Science, Paris VI1 University, 1995. 53. Mtairag EM, Abdelghaffar H, Douhet C, Labro MT. Role of extracellular calcium in vitro uptake and intraphagocytic location of macrolides. Antimicrob Agents Chemother 1995;39: 1676-1682. 54. Abdelghaffar H, Vazifeh D, Bryskier A, Labro MT. Investigation of the mechanism underlyingthe stimulation of neutrophil exocytosis by roxithromycin. IIId ICMAS, 1996; abstr 902. 55. Labro MT. Effects of macrolides on host natural defenses. In: Bryskier A, Butzler JP, Neu HC, lblkens PM, eds. Pans: Amette-Blackwell, 1993:389408. 56. Labro MT, El Bema J, Babin-Chevaye C. Comparison of the in-vitro effectof several macrolideson the oxidative burstof human neutrophils. JAntimicrob Chemother 1989; 24561-572.

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57. Anderson R. Erythromycin and roxithromycin potentiate human neutrophil locomotion in vitroby inhibition of leukoattractant-activatedsuperoxide generation and auto-oxidation.J Infect Dis1989; 159:966-973. 58. Mitsuyama T, Tanaka T, Hidaka K, Abe M, Hara N. Inhibition by erythromycin of superoxide anion production by human polymorphonuclear leukocytes through the action ofcyclic AMP-dependentproteinkinase. Respiration 1995; 62:269-273. 59. Peny DK, Hand WL, Edmonson DE, Lambeth JD. Role of phospholipase D derived diradyl glycerolin the activation of the human neutrophil respiratory burst oxidase.J Immunoll992; 149:2749-2758. 60. Abdelghaffar H, Mtairag EM, Labro MT. Effect of dirithromycinand erythromycylamine on human neutrophil degranulation. Antimicrob Agents Chemother 1994; 39:1548-1554. 61. Labro MT, Abdelghaffar H, BryskierA.Effect of macrolides on human neutrophil degranulation. 33d ICAAC, 1993; abstr 309. 62. Labro MT, Abdelghaffar H, Douhet C, Bryskier A. Investigation of the mechanism underlyingthe stimulation of neutrophil exocytosisby macrolides. 35th ICAAC, 1995; abstr G39. 63. Villa ML, ValentiF, Mantovani M, ScaglioneF, Clerici E. Macrolidic antibiotics: effects on primary in vitro antibody responses.Int. J. Immunopharmacol. 1988; 10~919-924. Bailly S, Pocidalo JJ, Fay M, Gougerot-Pocidalo MA. Differential modulation of cytokineproduction by macrolide:interleukin-6productionisincreased by spiramycinanderythromycin.Antimicrob Agents Chemother 1991; 35~2016-2019. 65. Morikawa K, Oseko F, Mirikawa S, Iwamoto K. Immunomodulatory effects of three macrolides, midecamycin acetate, josamycin and clarithromycin on human T-lymphocyte function in vitro. AntimicrobAgents Chemother 1994; 38~2643-2647. 66. Takeshita K, Yamagishi I, Harada M, Otomo S, Nakagawa T, Mizushima Y. Immunological and anti-inflammatory effectsof clarithromycin: inhibitionof interleukin 1 production of murine peritoneal macrophages. Drugs Exp Clin Res 1989; 15527-533. 67. Takizawa H, Desaki M, OhtoshiT, Kikutani T, Okazaki H, Sat0 M, Akiyama N, Shoji S, Hiramatsu K, Ito K. Erythromycin suppressesinterleukin-6expression by human bronchial epithelial cells: a potential mechanism of its antiinflammatory action. Biochem BiophysRes Commun 1995; 210:781-786. 68. Aoshiba K, Nagai A, Konno K. Erythromycin shortens neutrophil survival by accelerating apoptosis. Antimicrob Agents Chemother 1995; 392372477. 69. Keicho N, Kudosh S, Yotsumoto H, Akagawa KS. Erythromycin promotes monocyte to macrophage differentiation. Antibiot 1994; 47:80-89. Erythromycin inhibits respiratory glyco70. Goswami SK, Kivity S , Marom conjugate secretion from human airways in vitro.Am Rev Respir Dis 1990; 141:72-78. 71. Nagai M, YamadaH, Nakada S, Ochi K, NemotoT, Takahara S, Hoshima S, Horiguchi-Yamada J. Amacrolideantibiotic,roxithromycininhibits the

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growth of human myeloid leukemia HL 60 cells by producing multinucleate cells. Mol Cell Biochem 1995; 144:191-195. 72. Konno S, Adachi M, Asano K, Okamoto K, Takahashi T. Inhibition of human T-lymphocyte activation by a macrolide antibiotic, roxithromycin. Life Sci 1992; 51~231-236. 73. Nelson S , Summer W, Terry PB, Warr GA, Jakab GJ. Erythromycin-induced suppression of pulmonary antibacterial defenses. Am Rev Respir Dis 1987; 136~1207-1212.

Animal Usesof Macrolides and Related Antibiotics Jean-Pierre Lafont

Elisabeth Chaslus-Dancla

Institut Nationalde la RechercheAgronomique Nourilly ,France

Jean-Louis Martel Centre National d'Etudes Vktkrinaires Alimentaires-LYON et Lyon, France

ANIMAL USES OF MACROLIDES AND RELATED ANTIBIOTICS Macrolides and related antibiotics (MRAs) are widely used in the various animal sectors, fortwo main purposes: therapy and growth promotion.

Growth Promotion This particular useof antibiotics originates in experiments dating from the early fifties (1) demonstrating that the addition of very low doses in feed has stimulating effects on animal growth and, more generally, on animal production (e.g., eggs,milk).Thiseffectwasshown to result from increased feed efficiency,due to modifications of bacterial metabolism in the presence of subinhibitory concentrationsof antimicrobials inthe intestinal tract. Despite a sizable amount of research done by animal nutritionists, the mechanisms of this growth promotion have not been clearly elucidated

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and the bacterial targetsare still unidentified.The best documented mechanisms are reduction of the microbialproduction of growthdepressing metabolites, in the microbial destruction of essential nutrients such as amino acids in the intestinal tract, enhanced efficiency of absorption of nutrients due to a thinner intestinal wall,and sparing of nutrients for the host because of a lower turnover of the intestinal epithelium in treated animals (2). It should be noted in this respect that germ-free animals frequently exceed conventional controls in growth rate This nontherapeutic use of antibiotics in animal husbandry became widespread and controversial (4). Public health concerns led the agreement commissions of the European Union (EU) to restrict the list of substances authorized in this respectto specific families not (e.g., flavophospholipol) or scarcely (e.g., bacitracin) employed in animal or human therapy and unlikely to select for transferable drug resistance of medical importance. Tetracyclines and penicillins, for instance, were banned from this use in the EU, whereas certain MRAsare among the few exceptions to this rule. In France, spiramycin, tylosin, and virginiamycin are therefore used as growth- (and production-) promoting feed additives in birds, pigs, and ruminants. The doses (usually5-50 grams per ton of feed) are well below therapeutic doses. The selective effectof such low doses of MRAs hasnot been conclusivelydemonstrated, but this question remains open.Another undesirable consequence of long-term growth promoting supplementation of feed with MRAs could be the impairment of the “barrier effects” (“resistance to colonization,” “competitive exclusion”) exerted by the normal intestinal microflora of animals, especially poultry, againstSalmonella (5). The most dependable experimentson this topic suggestthat virginiamycin does not increase Salmonella shedding in animals, whereasan increase in shedding canbe occasionally observed with tylosin (6-9).

Therapy Macrolides and related antibioticsare invaluable therapeutic agents in animals. This results from their activity against prominent bacterial pathogens of animals, chiefly Mycoplasma (e.g., M . gallisepticum, M . iowae, M . meleagridis, and M. synoviae in birds, M. hyosynoviae and M. hyorhinis in pigs, M . agalactiae, M. capricolum, M . dispar, and M . mycoides in ruminants, etc.)but also Pasteurella, the trepanematous agentSerpulina hyodysenteriae, gram-positive cocci, and forth. The relative absenceof toxicity or adverse effectsof most MRA drugs in animals [except in horsesand in rabbits (lo)] and their low prices, at least for the older molecules of the family,which are the most extensively used (erythromycin, spiramycin, tylosin), have also fostered their use in veterinary medicine. Macrolides

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and related antibiotics are thus used under veterinary prescriptionto treat respiratory, intestinal, systemic, genital, and skin infections in most animal species (11). The molecules used in France and in European countries belong to the macrolide group with both 14-membered ring (erythromycin, oleandomycin)and16-memberedring(spiramycin,tylosin,tilmicosin, josamicin) molecules, to the lincosamides (lincomycin, and clindamycin in dogs andcats), and to the pleuromutilin group (tiamulin). This last group is specific to veterinary medicine andis tentatively presentedhere along with the MRAs because. its therapeutic uses are similar, targeted at the same infectious microorganisms(Actinobacillus, Mycoplasma, Pasteurella, Serpulino), but its structure with a cyclopentacyclooctene ring differs noticeably from that of the other MRAs. Streptogramins are no longer intherapeutic useandazalides are absentfrom the veterinarymarket.Tilmicosin, a semisynthetic derivative of demyracosyltylosin with an improved activity against Pasteurella species responsible for pneumonia cattle in and pigs, has been recently introduced asa new veterinary antibiotic (12). Depending on the drug, administration is by the oral andor parenteral routes with, in addition, pharmaceutical preparations proper to veterinary practice: intramammary suspensions used to treat or prevent mastitis, and medicated feed madeby mixing concentrated “premixes”to the other feed components. Medicated feed is distributed for curative purposes but also for prevention in large populations of intensively reared farm animals exposed to a risk of infection (e.g., at the onset of the first symptoms ina few individuals);the concentrations are in the therapeutic range (e.g., 400 grams/ton or ppm). Some preparations associate a MRA with another antibiotic substance; this takes into consideration the frequent involvement of severalpathogens inmanybacterialdiseasesofanimalsofwhich colisepticaemiaandchronicrespiratorydisease of poultry,associating Mycoplasma gallisepticum and pathogenic strains of Escherichia coli, are typical examples. Antibiotic use in animals is ruled by a vast array of national and European regulations (13). In particular, these regulations impose withdrawal periods before marketing, to avoid the presence of residuesin human food products coming fromtreated animals. The administration to laying hens of drugs liable to be excreted in eggs (14) is banned.

MICROBIAL RESISTANCE Evolution of Susceptibility in Bacterial Pathogens The evolution of susceptibility to MRAs has not been the subject of extensive work in veterinary medicine. Therapeutic failures are not commonly

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reported by fieldpractitioners, andthe major speciesof Mycoplasma which cause diseases frequently requiring MRAs treatments appear as remaining clinically susceptibleto the drugs Resistant mutantsof M . mycoides can be obtained in several steps in vitro However, although minimal inhibitory concentrations (MICs) of the MRAs can be higher for recent avian isolates than for reference strains isolated a long the time microorago, (18). ganisms still can often be considered as susceptible This situation concerning the MRAs is in contrast with an increase in resistance to the tetracyclines reported in MRA-susceptible bovineMycoplasma species The existence of resistant isolates has been reported in many other bacterial pathogensof animals such as Actinobacillus (formerly Haemophilus) pleuropneumoniae Actinomyces (formerly Corynebacterium) pyogenes Campylobacter coli Clostridium per,fringens various coagulase-negativeStaphylococcus species Staphylococaureus Staphylococcus epidermidis Staphylococcus hyicus Staphylococcus intermedius Streptococcus suis Streptococagalactiae, S . dysgalactiae, S. uberis and Rhodococcus .equi but not in Corynebacteriumpseudotuberculosis Serpulina hyodysenteriae appears as intrinsically resistant to macrolides and lincosamides but susceptible to tiamulin and Haemophilus somnus and Renibacterium salmoninarum appear as generally susceptible species. If the existence of resistant strains is well established for many species of pathogenic microorganisms of animals, the evolution of this resistance has not been systematically surveyed. Few specific studies have been devoted to this topic, and they have yielded somewhat discrepant results which need further clarification by more systematic investigations. Mostof these studies deal with a limited number of isolates, and the samples of strains have not been collected in a systematicway over the years to avoid biases in comparing these samples and in concluding about the evolution of the bacterial species considered. Although resistant strains do exist, gram-positive cocci involved in bovine (and small ruminant) mastitis remain generally susceptible whereas resistance is frequently observedStaphylococcus in hyicus or Streptococcus suis frompigs A possible evolution toward increased resistance has been reported for Campylobacter, by comparing pig and human isolatesof C . coli It should be notedhere that MRAs are not commonly used to treat animal Campylobacter infections. In Pasteurella, a 4-year survey of antimicrobial susceptibilitytrends of bovine isolates from the United States and Canada ledto the conclusion that the proportion of resistant strains had increased, but wide variation in the year-to-year results was observed By contrast, results obtained in France by the

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network monitoring antibiotic resistance in bovine pathogens (40,41) does not point to such atrend, with samplesof strains of similar size. The selective effect of treatments in animals can be easily expected and has been reported in field conditions (42). However, experimental studies in controlled conditions are generally lacking and it is difficult to appreciate the actual contributionof selective pressure to the evolution of resistance to the MRAs. Thus, it is presently impossible to establish whether resistance to these drugs in animals constitutes a public health problem. One has to recall in this respect that resistance to tylosin, a macrolide widely used in animal production, is very rare in human isolatesof Staphylococcus aureus, Streptococcus pneumoniae, and Campylobacter coli in many countries (43). A staphylococcal isolateof possible human origin was isolated fromthe udder skinof cows byPereira and Siqueira-Junior(38), and Saikiaet al. (44)reported the isolation of macrolide-resistantandlincosamide-resistantstaphylococci from cowsreared in a closed flock (taking no replacement other fromfarms) in whichthere was no useof any antibiotic, in a country (India) where none of the MRAs is usedfor treatment or growth promotion. Exchange of resistant strains between man and animals is, thus, also highly probable, but its significance for veterinary medicine is still unknown.

Genetic Supportof Resistance Few studies have been conducted on the genetic support of MRA resistance in veterinary medicine.Such studies are required for a better understanding of the public (and veterinary) health significance of this resistance: among pathogenic microorganisms causing diseases treated by MRAs in animals, none is a prominent zoonotic agent. Species of gram-positive cocci are generallyhost-specificand the critical :risk here is the selection of resistance determinants liable to be exchanged between animal and human pathogenic bacteria. Although MRAs are not first-choice antibiotics for the treatment of human listeriosis, the appearance of macrolide-resistant strains of Listeria monocytogenes may be of particular concern in this respect. These strainsare multiresistant and their multiresistance is encoded by plasmids bearing the ermB gene in addition to resistance genes for tetracycline/minocycline, streptomycin,andchloramphenicol (45). The presence of similar plasmids, showing extensive homology with plasmids from Streptococcus agalactiae, is established in L. monocytogenes strains from different countries (46). Although such strains are infrequently encountered (47), it is not known whether they were selected in animals, and particular attention should be paid to resistance in veterinary isolates of Listeria: resistance plasmids can be acquired readily from Enterococcus or

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Streptococcus donors in this species (47,48). Resistance to the macrolidelincosamide-streptogramin ( M U ) in gram-positive cocci from animals is generally constitutive and can be plasmid mediated (29,49,50). Some of theseplasmidsfrom Staphylococcus hyicus are close to plasmidsfrom strains of human origin (49). However, interspecific conjugal transfer of the resistance is often not obtained from staphylococcal strains isolated from bovine mastitis (51). Medical concerns arise here mostly from results suggesting that resistant Enterococci from animal sources could colonize humans (52,53) and thereby exchange transferable determinants of resistance in this occasional host.

Mechanisms and Determinants of Resistance Several mechanisms of resistance to the MRAs are known in various species of bacteria (54-56). All have been initially described from medical microorganisms of human origin. Whereas intrinsic resistance due to outer membrane impermeability is a general characteristic of gram-negative bacteria, three main mechanisms of acquired resistance have been reported: posttranscriptionalmethylation of. ribosomalRNA(target protection), modification inactivatingthe drug(s), and active.efflux. All have been documented in bacteriaof animal origin,by hybridization with geneor oligonucleotideprobes,obtention of PCRproducts withspecificprimers, or biochemical identificationof the mechanism. Here again, one can deplore the absence of systematic studies in veterinary medicine.The distribution of r-RNA methylase genes in bacteria causing disease in animals has received the attention of severalgroups:mostdeterminantsidentifiedin gram-positive cocci correspond to genes belonging to hybridization classes ermB and ermC (30,31,57,58). In contrast to human medicine, genes belonging to hybridization class..ermAappear as very rare in animal strains being far reported from one Staphylococcus strain only(S. intermedius), isolated from a dog (the samestrainalsohybridizedwith ermB) (58). Sequences homologous to Tn917, a well-characterized transposon from a human Enterococcusfaecalis strain, are present on plasmids of Enterococisolated from healthy chickens and pigs, which suggests that ermBrelated genes can spread by transposition in fecal streptococci of animals (59). Homologies with Tn916, the prototype of which does not bear MLS resistance, have also been recorded in macrolide-resistant plasmids from veterinary gram-positive cocci (50).The ermP determinant ( e m A M class) has been detected in animal erythromycin-resistant isolatesof the toxinogenic anaerobic speciesClostridium perfringens (60). The linA gene encoding 4-lincosamide-O-nucleotidyltransferase has been identified inStreptococcus uberis strains from dairy cows (61), andthe

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msrA gene encoding an active efflux system has been detected Staphyloin coccus strains from dogs and pigs (58). It should be stressed that these studies rest on a limited number of strains and can be considered only as occasional and welcome forays intothe question. As yet unidentified erm determinants encoding potentiallynew methylases are probably present in animal strains (27,28,58).

SPECIFIC VETERINARY PROBLEMS In veterinary aswell as in human medicine, the interpretation antibioticsusceptibility testing follows rules edicted by specific ad hoc scientific committees such asthe CA-SFM in France orthe NCCLS in the United States. This interpretation, categorizing strains as susceptible, intermediate, or resistant, is based on bacteriological, pharmacological, and clinical considerations. Veterinary laboratories carryout antibiograms and in vitro methods of susceptibility testing according to these rules and are subjected to qualitycontrolswithreference strains, usually E. coli and S. aureus (40,41). However, some specific veterinary pathogens such as Pasteurella or Actinomyces pyogenes (21) require the addition to culture media of growth factorswhich mayinterfere with diffusion.International agreement about well-characterized reference strains of these particular species would be needed for more accurate interpretation susceptibility testing. The pharmacological data used for interpretation should be specific to the target animal species for new molecules proper to the veterinary market such tilmicosin (62). But for old antibiotics nowin common use and no longer covered by patents, no pharmaceutical firm is willing to embark on costly pharmacological studies in several animal species, and the data used, as recommended by the official committees, are frequently those obtained in man. Thesedata may not be appropriate for veterinary medicine, which may explain some discrepancies between the results of susceptibility testing inthe laboratory and clinical results inthe field. The use breakpoints recommended by the official committees may not be relevant to veterinary situations, even withdrugsforwhichveterinary pharmacological data are available. This is especiallytrue with macrolides such as spiramycin for which, asfor azalides the use of blood levels as breakpoints for susceptibilitywould appear to be inappropriate: levels of drugs at the tissue site of infection could be a better guide to predicting efficacy. In all the animal species studied, including man, spiramycin concentrations reachvery high levels, far in excess of serum levels, in particular organs such as the lung (64,65). In bovine medicine, most Pasteurella strains should be considered as resistant according to the recommended breakpoints. However, lung levels in calves are very high, exceeding for

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long periods of time the MICs of most strains, and good clinical results are obtained with this antibiotic in treating infections the lower respiratory Better tissue penetration could also tract of calves due to Pasteurella be responsible for better in vivo activity in the female genital tract (67). This is moreor less true of other macrolide antibiotics, and the definition of specific veterinary breakpoints should be considered here

CONCLUSION AND PROSPECTS In veterinary medicine, MRAs have probably not received the attention they deserve, especially as far as bacterial resistance is concerned. Thisis probably due to the generally satisfactory results reported by field therapists with the use of these antibiotics in most animal species. Although resistant strains are known, the investigation of resistance has not been given a high priority by the small number of veterinary microbiologists involved in this field of research. MRAs were considered as raising no particular problem concerning public health: no prominent “life-saving” molecule usedin hospitals belongedto this class, and most target veterinary microorganisms did not represent a direct threat to human health. Their rational use could thus simply derive from the general recommendations and codes for good practices about antimicrobial use in animals expressed by registration expert groups(68). The situation may change with the new molecules this class introduced in human medicine, suchthe asazalides, requiringmore attention aboutveterinaryuseanditspotentialconsequences. In this respect, several questions would have to be answered: 1. Does the prolongeduse oflow doses of MRAs asgrowthpromoting feed additives contribute to the selection of resistant strains? Even if they are not the mostcontroversialgroupof antibiotics in this respect, MRAs require specific experimental studies here. 2. Is there a definite trendof certain veterinary pathogens toward increased resistanceto the MRAs? Veterinary surveillance networks (40) could be focused on this question, and pharmaceutical firms should work on this problem, especially regarding post marketing surveillance of molecules which have recently come the intoveterinary market.It would be of specific veterinaryinterest to consider the situation in major Mycoplasma species, and Campylobacter should be specifically surveyed with particular attention (69). Are intestinal gram-positive cocciof animal origin liable to carry transferable resistance determinants, ableto colonize the intestinal tract of humans, and transfer their resistance to indigenous

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strains or to cause human infection directly? Both experimental and genetic studies using the all modem tools of genomic analysis are needed to establish this most important point. 4. Forparticularclinicalsituations(e.g.,therapy of bovinepasteurellosis), should we use levels of drugs at the tissue site of infection as guidesto predicting efficacy rather than breakpoints for susceptibility deducedfrom blood levels?

ACKNOWLEDGMENTS The authors thank R. Leclercqforcriticalreading of the manuscript. Thanks are also due to G . Menou and P. Maillard for their help in the search for the relevant literature, and to F. Tardo-Din0 for typing.

REFERENCES 1. Stockstad ELR, Jukes TH. Growth-promoting effectsof aureomycin on turkey poults. PoultSci 1950; 29:611-612. 2. Visek WJ. The mode of growth promotionby antibiotics. J AnimalSci 1978; 46~1447-1469. Hamson GF, Coates ME. Dietary aureomycin 3. Freeman BM, Manning ACC, and the response of the fowl to stressors. Br PoultSci 1975; 16:395-404. 4. Levy SB. Antibiotic usefor growth promotion in animals: ecologic and public health consequences. J FoodProtect 1987; 50:616-620. Nurmi E,Rantala M. New aspects of Salmonella infection in broiler production. Nature 1973; 241:210-211. 6. Abou-Youssef MH, Di Cuolo (3,Free SM, Scott GC.The influence of a feed additive level of virginiamycin on the course of an experimentally induced Salmonella typhimurium infection in broilers. Poult Sci 1982; 62:30-37. 7. Jones FT, Langlois BE, Cromwell GM, Hays GW. Effect of feeding chlortetracycline or virginiamycin on shedding of Salmonellae from experimentally induced Salmonella typhimurim infectioninbroilers.JAnimal Sci 1983; 57:279-285. 8. Williams-Smith H, Tucker JF.The effect of feeding diets containing permitted antibiotics on the fecal excretion of Salmonella typhimurium by experimentally infected chickens. Hyg J Camb 1975; 75:293-301. 9. Williams-SmithH, 'hcker JF. The effect of antimicrobialfeed additives on the colonization of the alimentary tract of chickens by Salmonella typhimurium. J Hyg Camb 1978; 80:217-231. 10. Licois D. Risques associCs B l'utilisation des antibiotiques chezle lapin. World Rabbit Sci. (in press). 11. Lafont JP, Martel JL, Mourot D, Guittet M, Kobisch M, Colin P, Kempf I, eds.AntimicrobialsinIntensiveAnimalProduction.Ploufragan,France: ISPAIA-Zoopole-DCveloppement, 1994; 1-306.

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12. Kirst HA. New development in macrolides. Prog Med Chem 1994; 31:265-295. 13. Pinault L, Milhaud G. Ugislation en pharmacie v6tCrinaire. In: Meissonnier E. Devisme P, Join-Lambert P, eds. Dictionnaire des MCdicamentsvCtCrinaires. Paris: Editionsdu Point VCtCrinaire, 1995; Gl-G41. 14. Roudaut B, Moretain JP. Residues of macrolide antibioticsin eggs following medication of laying hens. Br. Poult Sci 1990; 31: 661-675. 15. Inamoto T, Takahashi H, Yamamoto K, Nahai Y, Ogimoto K. Antibiotic susceptibility of Mycoplasma hyopneumoniae isolated from swine.J Vet Med Sci 1994; 56:393-394. 16. Poumarat F, Martel JL. Antibiosensibilite in vitro des souches fransaises de Mycoplasma bovis. Ann Rech V& 1989; 20:145-152. 17. Lee DH, Miles RJ, Inal JR. Antibiotic sensibility and mutation rates to antibiotic resistancein Mycoplasmamycoides spp. mycoides. EpidemiolInfect 1987; 98~361-368. 18. Lin MY, In vitro comparison of the activity of various antibiotics and drugs against newTaYwan isolates andstandard strains of avian Mycoplasma. Avian Dis 1987; 31:705-712. 19. Ter Laak EA, Noordergraaf Verschure MH. Susceptibilities of Mycoplasma bovis, Mycoplasma dispar and Ureaplasma diversum strains to antimicrobial agents in vitro. Antimicrob Agents Chemother 1993; 37:317-321. 20. Gutierrez CB, Piriz S, Vadillo S, Rodriguez-Feni EF. In vitro susceptibilityof Actinobacillus pleuropneumoniaestrains to 42 antimicrobial agents. Am Vet J Res 1993; 54546-550. 21. Gutrin-FaublCe V, Flandrois JP, Broye E, ThpinF,Richard Y. Actinomyces pyogenes: susceptibility of 103clinicalanimalisolates to 22 antimicrobial agents. Vet. Res 1993; 24:251-259. 22. Cabrita J, Rodrigues J, Braganca F, Morgado C, Pires I, Penha-Goncalves A. Prevalence, biotype, plasmid profile and antimicrobial resistance of Campylobacter isolated from wild and domestic animals from northeast Portugal. J Appl Bacterioll992; 73:279-285. 23. Elharrif MCgraud F, Marchand AM. Susceptibilityof Campylobacterjejuni and Campylobacter coli to macrolide and related compounds. Antimicrob Agents Chemother 1985; 28:695-697. 24. Rood1 JI, Buddle JR, Wales Sidhu R. The occurrence of antibiotic resistance inClostridium perfringem from pigs. Aust Vet J 1985; 62:276-279. 25. Myllys V. Staphylococci in heifer mastitis before and after parturition. J Dairy Res 1995; 6251-60. 26. Buragohain J, Dutta GN. A new type of resistance in staphylococci from bovine subclinical mastitis.Res Vet Sci 1990; 49:248-249. 27. Schwan S , Blobel H. Isolation of aplasmidfromcanine Staphylococcus epidermidis mediating constitutive resistanceto macrolides and lincosamides. Comp Immunol Microbiol Infect Dis 1990; 13:209-216. Wegener HC, Schwan S. Antibiotic-resistance and plasmids in Staphylococcus hyicusisolated frompigs with exudative dermatitis and from healthypigs. Vet Microbiol 1993; 34~363-372.

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in 29. Greene RT, Schwarz S. Small antibiotic resistance plasmids Staphylococcus intermedius. Zbl Bakt 1992; 276:280-289. 30. Wasteson Y, Hoie S, Roberts MC. Characterization of antibiotic resistance in Streptococcus suis. Vet Microbioll994; 41:41-49. 31. Roberts MC, Brown M B . Macrolide-lincosamide resistance determinants in streptococcal species isolated from the bovine mammary gland.Vet Microbiol 1994; 40~253-261. 32. Kenney DG, Robbins SC, Prescott Kaushik A, Baird JD. Development of reactive arthritis and resistanceto erythromycin and rifampinin a foal during treatment for Rhodococcus equi pneumonia. Equine Vet J 1994; 26:246-248. 33. Judson R, Songer JG. Corynebacteriumpseudotuberculosis: in vitro susceptibility to 39 antimicrobial agents.Vet Microbiol 1991; 27:145-150. 34. Buller NB, Hampson DJ. Antimicrobial susceptibility testing of Serpulina hyodysenteriae. Aust Vet J 1994; 71:211-214. 35. Watts JL, Yancey RJ, Salmon SA, Case CA. A 4-year survey of antimicrobial susceptibility trends for isolates fromcattle with bovine respiratory disease in North America. Antimicrob AgentsChemother 1994; 34:725-731. 36. Bandin I, Santos Y, Toranzo EA, Barja JL. MICs and MBCs of chemotherapeutic agents against Renibacterium salmoninarum.Antimicrob Agents Chemother 1991; 35:lOll-1013. 37. Brown MB, Scasserra AE. Antimicrobial resistance in streptococcal species isolated from bovine mammary glands. Am J Vet Res 1990; 51:2015-2018. 38. Pereira MSV, Siqueira-Junior JP. Antimicrobial drug resistance in Staphylococcus aureus isolated from cattle in Brazil. Lett. Appl. Microbiol 1995; 2 0 391-395. AA, Gomwalk NE. Antibiogram of Staphylococcus 39. UmohVJ,Adesiyun strains isolated frommilk and milk products. Zent Vet Med 1990; 37:701-706. 40. Martel JL, Chaslus-Dancla E, Coudert M, Poumarat F, Lafont JP. Survey of antimicrobial resistance in bacterial isolates from diseased cattle in France. Microb Drug Res 1995; 1:273-283. 41. Martel JL, Coudert M. Bacterial resistance monitoringin animals: the french national experience of surveillance schemes.'VetMicrobioll993; 35321-338. 42. Noble WC, Allaker RP. Staphylococci on the skin of pigs: isolates from two farms with different antibiotic policies.Vet Rec 1992; 130466-468. bacteria. Vet Rec 43. Lacey RW.Rarity of tylosin resistance in human pathogenic 1988; 122:438-439. 44. Saikia PK,Dutta GN, KalitaCC. Naturally occumng macrolide-lincosamide resistance in staphylococci from bovine mastitis. J Antimicrob Chemother 1986; 17~685-686. 45. Poyart-SalmeronC, Carlier C, Trieu-Cuot P, Courtieu AL, Courvalin P. Transferable plasmid-mediated antibiotic resistance Listeria in monocytogenes.Lancet 1990; 335:1422-1426. 46. Hadorn K, Hachler H, Schaffner A, Kayser FH. Genetic characterization of plasmid-encoded multiple antibiotic resistance in a strain of Listeria monocytogenes causing endocarditis.Eur J Clin Microbioll993; 12:928-937.

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47. Charpentier E, Gerbaud G, Jacquet C, Rocourt J, Courvalin P. Incidence of antibiotic resistance inListeria species, JInfect Dis 1995; 172:277-281. 48. Vicente MF, Baquero F, Perez-Diaz JC. Conjugative acquisition and expression of antibiotic resistancedeterminants in Lkteria spp. J Antimicrob Chemother 1988; 21:309-318. 49. Noble WC, Rahman M, Lloyd DH. Plasmids inStaphylococcus hyicus. J Appl Bacterioll988; 64:145-149. 50. Stuart JG, Zimmerer EJ, MadduxRL. Conjugation of antibiotic resistance in Streptococcus suis. Vet Microbioll992; 30:213-222. 51.Muhammad G , Hoblet KM, JackwoodDJ,Bech-Nielsen S, Smith KL. Interspecific conjugal transfer of antibiotic resistance among staphylococci isolated from the bovine mammary gland. Am J Vet Res 1993; 54:1432-1440. 52. Bates J, Jordens JZ, Griffiths DT. Farm animals as a putative reservoir for vancomycin-resistant enterococcal infection in man. J Antimicrob Chemother 1994; 34507-514. 53. Le Blanc DJ, Inamine JM, Lee LN. Broad geographical distribution of homologouserythromycin,kanamycin,andstreptomycinresistance determinants among group D streptococci of human and animal origin. Antimicrob Agents Chemother 1986; 29549-555. 54. Leclercq R, Courvalin P. Bacterial resistance to macrolide, lincosamide and streptogramin antibiotics by target modification. Antimicrob Agents Chemother 1991; 35:1267-1272. 55. Leclercq R, Courvalin P. Intrinsicandunusualresistance to macrolide, lincosamide, and streptogramin antibiotics in bacteria. Antimicrob Agents Chemother 1991; 35:1273-1276. 56.Leclercq R, Courvalin P. Resistance to macrolides,azalidesand streptogramins. In: Neu HC, Young LS, Zinner SH, Acar J, eds. New Macrolides, Azalides and Streptograminsin Clinical Practice.New York: Marcel Dekker, Inc., 1995;31-42. 57. Buu-Hoi A, Le Bouguenec C, Horaud T. Genetic basis of antibioticresistance in Aerococcus viridans.Antimicrob Agents Chemother1989; 33529-534. 58. Eady EA, Ross JI, Tipper JI, Walters CE, Cove JH, and Noble WC.Distribution of genes encoding erythromycin ribosomal methylases and an erythromycin efflux pumpinepidemiologicallydistinctgroups of staphylococci. J AntimicrobChemother 1993; 31:211-217. 59. Rollins LD, Lee LN, Le Blanc DJ. Evidence for a disseminated erythromycin resistance determinant mediated by Tn917-like sequences among group D streptococci isolated from pigs, chickens and humans. Antimicrob Agents Chemother 1985; 27:439-444. 60. Berryman DI, Rood JI. Cloning and hybridization analysisof ermP, a macrolide-lincosamide-streptogramin B resistance determinant from Clostridium perfringens. Antimicrob Agents Chemother1989; 33:1346-1353. 61. Arthur M, Brisson-NoelA, Courvalin P. Origin and evolutionof genes specifying resistance to macrolide, lincosamide and streptogramin antibiotics: data and hypotheses. J AntimicrobChemother 1987; 20:783-802.

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62. Gourlay RN, Thomas LH, Wyld SG. Effect of a new macrolide antibiotic (tilmicosin) on pneumonia experimentally induced in calvesby Mycoplasma bovis and Pasteurella haemolytica. Res Vet Sci 1989; 47:84-89. 63. Neu HC. Clinical microbiology of azithromycin. Am J Med 1991; 91 (suppl A):12S-l8S. Osono T, Umezawa H. Pharmacokinetics of macrolide$, lincosamides and streptogramins. J Antimicrob Chemother1985; 16 (supplA):151-166. 65. Rolin 0, Bouanchaud DH. ActivitB antipneumococcique de l’drythromycine et de la spiramycine dans deuxmodtles expdrimentaux chez la souris. Pathol Biol 1987; 35742-745. B, Van Goo1 F,Bayle R, Libersa M, Espinasse 66. Alzieu JP, Bichet HJ, Levrier Efficacy and long-lasting activity of spiramycin in young beef cattle with infections enzootic broncho-pneumonia. Bovine Practitioner 1989; 24:38-41. 67. Cester CC, Laurentie MP, Garcia-Villar P, Toutain PL. Spiramycinconcentrations in plasma and genital-tract secretions after intravenous administration in the ewe. J Vet Pharmacol Therap 1990; 13:7-14. 68. Espinasse J. Responsible useof antimicrobialsin veterinary medicine: perspectives in France.Vet Microbioll993; 35:289-301. 69. Kerr R, Moore JE. Potential for erythromycin resistancein porcine Campylobacter species. Trends Microbiol 1995; 3:440.

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l0 The New Macrolidesfor Treatment of Pediatric Infections: Roundtable Discussion George H.McCracken, Jr. University of Texas SouthwesternMedical Center at Dallas Dallas, Texas

Urs B. Schaad University of Basel Basel, Switzerland

M.J. Tarlow Birmingham HeartlandsHospital Birmingham, United Kingdom

Dr. McCracken: In this discussion we will present various aspects of the use of the macrolides in pediatrics. At the outset, wewill not mention every study of every drug, although some reference will be madeto the studies presented inthe poster sessions. As a general dictum,what applies to one macrolide appliesto the others as well, althoughthere are very specific differencesthat distinguish them. I would liketo provide a brief introduction on these drugs, reviewing their in vitro susceptibilities as they apply to pediatrics and, then, their pharmacokinetics in infants and children. 131

I32 Table l

McCracken et al. Potential Uses of New Macrolides in Pediatrics

1. Alternative when erythromycin is not tolerated 2. RespiratoryInfections Otitis media (shortened regimens) Sinusitis Bronchitis (pertussis in pediatrics) Pneumonia, especially in children and adolescents 3. Nontuberculous mycobacterialand helicobacter infections

The macrolides have enjoyed tremendous success in pediatrics, and pediatricians worldwide are very familiar and very confident inthe use of these drugs (Table 1).They have been used for a number of indications from microbiological standpoint, although several of these agents represent new developments for the use the new macrolides and azalides such as Bordetella pertussis, but they do not yet have an indication for this organism. My ownbias with‘regard to the new macrolides in pediatricsthat is they will find their greatest usefulnessin the therapy of respiratory infectionspharyngitis, acute otitis media, acute sinusitis, bronchitis (which really not is a disease in children, except for pertussis), and pneumonia. The drugs shown in Table2, erythromycin, clarithromycin, roxithromycin, and azithromycin, are active against the common organisms of the respiratory tract in pediatric patients. ForStreptococcus pneumoniae, they all are active, although azithromycin may be a little less active than the others. It’s very difficult to make any conclusion about relative activity, especially in light of Dr. Craig’s discussion on how to correlate in vitro Table 2 In Vitro Activity of Newer Macrolides

Erythromycin Clarithromycin Roxithromycin S. pneumoniae Staph. aureus H.infruenzae M.catarrhalis Legionella M . pneumoniae C. pneumoniae C. trachomatis MAC a+

(least) ++

++ + ++ + ++++ ++++ ++ 0

++ + (most) activity.

++++ ++ ++ ++ ++ ++++ ++++ ++++ +++

Azithromycin

+++ + ++ ++ ++ ++++ ++++ +++ ++

++ + +++ +++ + ++++ ++ ++ +

New Macrolides Pediatric and Infections: Discussion Table 3 Susceptibilities pneumoniae

133

Penicillin-Resistant Isolates of Streptococcus Intermediate 1.0 pg/ml)

MIGO

Macrolide Erythromycina Clarithromycina Azithromycina of isolates had MIC

High (> Pg/ml)

0.03 0.016

8 2 4

MGO

MIGO

> 16 > 16 > 16

1 pglml (data fromTrudy Murphy).

Source: Antimicrob Agents Chemother 1995; 39:533.

activity with in vivo success, when dealing with the pharmacokinetics and intracellular concentrationsof these drugs. The other two respiratory pathogens of importance are Chlamydia pneumoniae and Mycoplasma pneumoniae and allof these agents appear to be active for these organisms. The technique usedto test MIC valuesfor S. pneumoniae is critical as Michael Jacobs has pointed out. Not only is it important whether they are penicillin resistant (i.e., intermediate versus high resistant) but also whether they are erythromycin resistant, as crossresistance isthe rule among macrolides. The MIC, and MIC, values for the intermediately penicillin-resistant and highly resistant strainsare presented in Table When we compare the MIC, values, especially for thosethat are highly penicillin resistant,these agents have minimal effect, although in some instances they may be clinically effective, depending on thesite andthe type of infection. The footnote in the table presents Dr. Murphy’sdata on strains isolated in Dallaswhich show that 90% of the isolates tested from patients in Dallas County had MIC values for clarithromycinandazithromycinless than 1 pg/ml by Mueller-Hinton broth dilution testing. One of the issues of concern is middle ear infection caused by Streptococcus pneumoniae. Table 4 presents the representative peak concentrations of erythromycin estolate, clarithromycin, and azithromycin in the middle ear fluid versus those of amoxicillin givento pediatric patients.The dosages are given in milligrams per kilogram. The achievable concentrations in middle ear fluid exceedthe MIC, value for intermediate penicillinresistant organisms in most instances, and these values be should successful for management. The MIC, values are not necessarily representative of usual values encountered in practice. Whether you determine bacteriologic response by peak concentration over MICor time over MIC is an issue that hasn’t been resolved, although

lrg)

McCracken et al.

134 Table 4 Macrolide Therapy for Acute Otitis Media

MC, values

MEP

Drug 8

4-5 10-12 8-10 4-6

Erythromycin (est.) (15) Clarithromycin (7.5) Azithromycin (10) Amoxicillin (15)

2 4 0.5

>l6 >l6 >l6 2-8

'Middle ear fluid. Concentration inpg/ml. bIPR Intermediate penicillin resistant;HPR highly penicillin resistant.

Bill Craig has shown in the March 1996 issue of The Pediatric Infectious Diseuse Journal that bacteriologicsuccesscan be predicted on either pharmacodynamic marker (concentration over MIC or time over MIC) for most of the commonly used antimicrobial agents. Finally, Table 5 reviews some of the pharmacokinetic profilesof the macrolides in pediatric patients. Weknow that witherythromycin one expects peak serum concentrations the in range of 4-6 pglml; with clarithromycin 3-5 pglml; and for azithromycin, less than1 pglml. The half-life values in childrenare shown inthe table. Clarithromycinand azithromycin have very long half-life values, particularly azithromycin. The dosage schedule is based principally on the half-life values.The shorter the half-life, the more frequentlythe agent mustbe administered. Also, we might addressthe issue of the effect of food on theabsorption or bioavailability of thesedrugs. There appears to be no adverse effect; in fact, there may even be an enhancing effectof coadministration of food with the macrolides to pediatric patients. It has been known for years that erythromycin suspensions are better absorbed if taken with food, and it appears that there is no interference or diminished bioavailability with the ingestion of food withthe other macrolide suspensionsas well. In Table 5 Pharmacokinetic Profile of Macrolide Antibiotics

mycin rithromycin Erythromycin Indices Serum conc. (pg/ml) Half-life (h) Vol. Dist. (L) Urin. Exc. (%) Dosage (mgflrg)

4-5

1.5 50 5

10.5 q &12h

3-5 5 250 35 7.5 q12h

~0.4 12 2100 15 5-10 q24h

New MacrolidesPediatric and Infections: Table

Discussion

135

General Aspects of Respiratory Infections

Respiratory tract infections (RTI)-most common infectious disease worldwide Majority of RTIs-viral Principal challenge-promptly diagnose bacterial RTIs

fact, this could be an advantage because children sometimes don’t like antibiotic suspensions, but this is not a problem because they can be disguised in a food substance. Dr. Schaad: Mr. Chairman, ladies and gentlemen. For the next couple of minutes, I will include some general remarks with regard to respiratory tract infections in children, then some specific comments to the group A streptococcal tonsillopharyngitis, and finally, some pharmacokinetic and pharmacodynamic considerations andthe results of clinical studies with the macrolides forthat indication. Respiratory tract infections in childrenare the most common infectiousdiseasesworldwide(Table 6). This isalso true for adults. A big problem is that the majority of these respiratory tract infectionsare viral; therefore, the principal challenge for all of us to promptly diagnose the bacterial infections because neither the new oral cephalosporins nor the new macrolides have antiviral activity. We also know the bacterial respiratory tract infections can be either primary or secondary (Table 7). Secondary refers to superinfections to initially viral or even allergic disease. One more fact we all know is that antibiotics are effective for bacterial respiratory tract infections. The diagnosis is often difficult because it is principally based on clinical findings (Table 8) and, especially in pediatric patients, on only a few additional investigations (Table9). The therapeutic problems with groupA streptococcal tonsillopharyngitis are quite remarkable. The goalsare more or less clear:We would like to cure the sick patient of the sore throat; we would liketo stopcontagion, and then, of course, prevent bacterial and nonbacterial complications. Tabk 7 GeneralAspects

ndary orPrimary RTIs: Bacterial Antibiotics are effective Clinical findings Diagnosis on: based Few investigations (microbiological, radiological, laboratory)

al.

136

McCracken et

Table 8 Group A StreptococcalTonsillopharyngitis-Diagnostic Problems

Incidence of group A streptococcal (GAS) pharyngitis: (age, season) “Typical” clinics of GAS pharyngitis +headache, fever +sore throat, t tonsils + pharyngeal erythema and exudation + tender and t cervical nodes + absence of rhinorrhea, cough, and hoarseness The choice of drug is also a hotly debated issue. Is there any reasonto delay treatment in order to allow the patient to mount a protective immune response, or is this not necessary? Also, is the appropriate length of treatment really days or is it 5 days? Another big problem isthe frequency of recurrence that we all see. It is hardto decide if this is reinfection, which is probably much more common thantrue relapse. The recommendations for the treatment of group A streptococcal tonsillopharyngitis are presented in Table10. I think that even for this very simple disease, it is essentialto have a proper diagnostic workup.Another point is that the 40-year-old dominance of penicillin is clearly being challenged. It is not that penicillin is no longer a good drug, but there are conditions or situations whereother therapeutic modalitiesare indicated. I believe, and we hope to confirm this in our upcoming new study, that a delay of approximately 48 h after the start of symptoms is a reasonable approach to allow the patient to mount a protective immune response.Of course, there are indications where individualized management is necessary to adequately control recurrence epidemicsor outbreaks of rheumatic fever. The clinical efficacy of treatment can be predicted either by pharmacokinetic factors or, as is more fashionable, by pharmacodynamic factors. Table 9 Group A StreptococcalTonsillopharyngitis-Diagnostic Problems ~~~

~

~

Throat culture= gold standard +false

+:

+false -:

Carriers of group A streptococcal (GAS) swabbing Faulty Inadequate bacteriology Occult antibioticRx

Rapid antigen detection tests +false +: +false -:

Carriers of GAS Validated by culture

Microbiologic search for GAS requires proper clinical indication

New Macrolides and Pediatric Infections: Discussion

137

Table l 0 Recommendations for GAS Tonsillopharyngitis

Proper diagnostic workup >40-year-old dominance of penicillin is clearly challenged (oral cephalosporins: 5d; azithromycin: Rx delay of 48 hoften reasonable Individual management of recurrence, epidemics, and outbreaks ofARF

Especially for the macrolides, the time above the minimal inhibitory concentration or the MBC is most important. These drugs also produce a clinically relevant postantibiotic effect. In Table 11, the ratios between drug concentrations achieved in the extracellular space (serumor interstitial fluid) comparedto intracellular or tissue concentrationsare presented for the macrolides and penicillins.The first column represents the peak concentrations achievable with normal dosing dividedby the MIC, value for groupA streptococci. The drugs that accumulate the most will have a significantly higher value. It is not clear where the relevant multiplicationof the group A streptococci takes place in this simple disease. Based on the analysis of histopathological findings, I believe that both intracellular and extracellular organismsare important. The results of clinical studies with group A streptococcal tonsillopharyngitis are summarized in Table 12. The clinical responses with penicillin, erythromycin, roxithromycin, clarithromycin, and azithromycin are usually very satisfactory and there is no clear difference. The microbiologic responses in these older and newer studies appears to be slightly less with penicillin than the macrolides; however, there is some indication that in some studies,the bacteriologic response to azithromycin is somewhat lower. In arecentlycompleted Swissstudy, 170 pediatricpatientswith proven groupA streptococcal tonsillopharyngitis were treated with azithromycin for days, and a similar sized cohortwas randomized to penicillin Table I l

Drug ConcentratiodMIC, for Group A Beta-Hemolytic Streptococci Serum, interstitial, intracellular Tissue, extracellular ~

Erythromycin Roxithromycin Clarithromycin Azithromycin Penicillin

=

= =

0.410.03 = =

50

~~

4-8

138 Table 12

McCracken et al. Results of Clinical Studies in Streptococcal Tonsillopharyngitis Clinical response Bacteriologic response

Penicillin Erythromycin Roxithromycin Clarithromycin Azithromycin

90%

twice daily for 10 days. The clinical response was 93% and respectively.However,microbiologicresponseswerelower for azithromycin (65%) than with penicillin(82%). What is the explanation for this relatively low group A streptococcal eradication in our study? Although wewere quite strict withinclusion criteria that included really ill patients,we may have had an unusual number of asymptomatic carriers.There were only very few cases of reinfection with discordant strains. Mostof the strains isolated atthe end of treatment in these children were the same as the initial isolate, according to serotyping. We did not find resistance, andwe monitored compliance withthe measurement of antibacterial activity in the urine, but we cannot exclude some reinfections with identical strains. Both drugs were well tolerated, and at 6-month follow-up examinations, there were no cases of rheumatic fever.There was only one case of post streptococcal glomerulonephritisin the penicillin group, andrecurrent episodes of documentedgroup A streptococcaltonsillopharyngitisoccurred at a similar frequency in both groups (17% with azithromycin and 20% with penicillin). Even though it is a very simple disease, streptococcal pharyngitis remains a quite attractive issue. Thank you.

Dr. McCracken: Dr. Tarlow will now talk on acute otitis media and the study withthe macrolides.

Dr.Tarlow:

Thank you very much. In the next few minutes,I will discuss the bacterial etiology of acute otitis media in children and the specific pharmacokinetics of the new macrolides as they particularly affect middle ear infections in children. I will outline in general terms the results of comparative studies of the new macrolides in acute otitis media in childhood and consider overalltreatment regimens. The first table (Table 13) shows the typical frequency of bacterial isolates by tympanocentesis in acute otitis media. A caveat of these data is that tympanocentesis in otitis media is not practiced in all countries: it is

New Macrolides and Pediatric Infections:Discussion

139

Table 13 Acute Otitis Media-Etiology Streptococcus pneumoniae 4 0 % Haemophilus influenzae catawhalis Moraxella 10% Others-e.g., Strep. pyogenes,Staph, aureus, etc. of minor importance

not performed in Britain and Switzerland. These data are largely but not entirely from the United States and I am not sure whether they will have general geographic relevance. About 40% of the cases are associated with Streptococcus pneumoniae; the proportion of penicillin-resistant, intermediate, andsensitive strains will vary with the geographical area. About 25% will be associated with Haemophilus influenzae, and it is importantto mention that these are largely nontypeable strains;therefore, immunization against withthe new HIB vaccine is not likely to have a significant impact. About 10% are associated with Moraxella catarrhalis. Also, a few percent may be associated with chlamydial infection. Other bacteria, except inthe newborn and the immunocompromised, are really of minor importance in contributing to the overall pattern. In about 30% of cases, no bacterial etiology can be determined, and these caseswe think are viral. Table 14 summarizes the approximate MICsof azithromycinand clarithromycin against these organisms has been mentioned repeatedly, azithromycin is rather more active against hemophilus and clarithromycin may be slightly more active againstthe pneumococcus. The concentrations of clarithromycin and its active metabolite, 14 hydroxyclarithromycin, inthe middle ear fluid and in plasmaare shown in Table 15. Plasma peaks at around 2-4 h after the dose, whereas these antibiotics will continue increasing in middle ear effusion fluid in acute otitis media upto about 12 h when concentrations approaching8 pg/g can be found. Table 14 Approximate MICs of Principal Pathogens ~~~

~~

Azithromycin Clarithromycin (Pdd

Pneumococci Haemophilus Moraxella Source: Khurana, 1995.

(Pdd

ErythromycidSulfisox

140

McCrucken et al.

Table IS Antibiotic Concentrations in Middle Ear Fluid-Clarithromycin

Multiple doses (7.5 mgkg bid) After dose 6: Plasma conc.: 2.9 pglg after 2 h; 0.7 pglg after 12 h MEEa conc.: 3.0 pg/g after 2 h;7.4 pglg after 12 h 14-OH C 2.5 pglg after 2 h; 3.8 pglg after 12 h MEE conc. WEE = Middle ear effusion. Source: Cam, 1996.

The high levels foundare probably associated withthe accumulation of both clarithromycin and its metabolites in polymorphs which migrate into middle ear fluidwith the inflammation.Similarly,azithromycinis taken up and is concentrated to an even greater extent in the middle ear fluid (Table 16). After a single 10-mg dose, azithromycin is still present in the middle ear fluid in a concentrationof almost pg/g after 24 h and still has a significant concentration at 48 h. After multiple doses of azithromycin given daily, around 8.61 pg/g are present 24 h after the dose. After the end of a courseof azithromycin, effective tissue concentrations are still present for several days. These concentrations are effective against the majority of bacterial pathogens in acute otitis media. Specifically, theyare effective againstthe vast majority of penicillin-sensitive and penicillin intermediately resistant strain of S. pneumoniue, although there is a significant minorityof strains which are resistant to macrolides, including azithromycin and clarithromycin. Some of the reasons for in vitro and in vivo efficacy differ, and we cannot just extrapolate from these in vitro studies to assume that an antibiotic will or will not have an appreciable clinical effect.We need to look at Table 16 Antibiotic Concentrations in Middle Ear Fluid-Azithromycin

Single dose (10 mgkg) 3.97 pg/g after 24 h;

1.42 pglg after 48 h

Multiple doses 8.61 pg/g after 24 h;

9.43 pglg after 48 h

Middle ear effusion (MEE) conc. lOOX greater than serumconc. Source: Pukander, 1994.

New MacrolidesPediatn'c and Infections: Discussion

141

clinical trialsto see how these newer macrolides compare with established treatment in the management of otitis media in childhood. First, clarithromycin. Urs Schaad has presented inthe past an overview of seven trials of clarithromycin in which days of clarithromycin were compared with amoxicillin or with amoxicillin and clavulanic acid, with really equivalent success in both groups but rather fewer adverse effects inthe groups given clarithromycin than in the others. More recently, a multicenter blinded trial has been published by McCarty showing, again, that clarithromycin isas good as amoxicillin clavulanate withrather fewer side effects inthe clarithromycin-treated group.Another trial comparingit with cefaclor showedthat 10 days of clarithromycin wereas effective as days of cefaclor with moreor less equal incidence side effects. As far as azithromycin is concerned-I'm just picking out heresome of the clarithromycin and azithromycin studies-there are three comparative UnitedStates studies published by McMin last year covering over 1200 children in which 5 days of azithromycin was compared with days of amoxicillidclavulanate. Again, equivalent efficacy was found in the two groups with fewer side effects in those treated with azithromycin. Because of the long action of azithromycinafter the last dose has been given and because even very short courses have been shown to be effective, Urs Schaad presented a study where only days of azithromycin were compared with 10 days of amoxicillidclavulanate, again showingsuccess with azithromycin and fewer side effects than the amoxicillidclavulanate in group. A group in Belgium has recently reduced the course of clarithromycin in a slightly smaller study in which only 5 daysof clarithromycin were compared with azithromycin; both appeared successful, with very similar levels of adverse effects. what can we conclude from thisand where do we go from here? First all, both azithromycin and clarithromycinare useful drugs in the management of childhood otitis media. Both have a more effective spectrum of antibacterial activity than erythromycin. They can be given less frequently than many conventional antibiotics, clarithromycin twice daily and azithromycin once daily, and they are both well accepted and tolerated. Most, but not all, studies show a significantly lower incidence of side effects in children with otitis media given these drugs compared with traditional antibiotictreatment. In most studies, clarithromycin has been given for 10 days, but it seems that 5 days may possiblybe sufficient. Azithromycin is given 5 days in the United States, but outside the United States, the course has been cutto days and it still seemsto very effective. Couldthe course of azithromycin in otitis media be cut any further? Maybe 2 days or even 1 day of treatment might be possible. I think I will stop there and pass overto you again, George.

142

et

McCracken

al.

Dr. McCracken: Actually, I can add one interesting sidelight. There are only twocenters in the world that are doing two tympanocenteses to demonstrate bacteriologic cure: the one in Galveston which has done it for years with Dr. Howie and colleagues and the other with Ron Dagan in Israel. These are studies that are very hardto accomplish but probably provide the most enlightening information about bacteriologic responses. In the last moments we’re goingto talk about pneumonia, which is a very large subject, but fortunately there is only one published study that I think addresses this best. This is by Stan Block and a numberofcollaborators in a multicenter study published in the Pediatric Infections Disease Journal last year.There are several studies here presentedon azithromycin by Gail Cassel, Maggie Hammerschlaag, and others that look almost identical in termsof bacteriologic and clinical responses. In ambulatory pneumonia in the age groupof 3-12 years, the etiology was detected in 50% by culture, PCR, serology, or a combinationof these. Interestingly, M . pneumoniae was detected in 27% of the individuals by culture or PCR; with positive cultureor PCR had serologic response. This represents an interesting question in that, although found by culture, only half had a serologic response, and this makes it difficult to determine whether this, the isolated organism, is a true pathogen or a colonizer. C . pneumoniae infection was detected in 28%,essentially the same percentage of children hadeither M. pneumoniae or C. pneumoniae. l7venty-three percent of these with positive cultures had serologic responses. Was therapy started early enough to interfere with this response or are our serologic techniques not refined or sensitive enoughto demonstrate a rise intiter? Coinfection was seen in 37patients, 14% of all patients and 22% of those with etiology defined. In some instances, bothC . pneumoniae and M.pneumoniae were recovered. Coinfection doesn’t necessarily mean that something went wrong because we’ve known for yearsthat viral infections predispose to bacterial disease of the lower respiratory tract. It is possible that either M . pneumoniae or C . pneumoniae infection maydo the same thingor predispose one to the other. The data of Stan Block and colleagues on the bacteriologic response in the children with pneumonia and a documented etiologic agent show that for C . pneumoniae and M . pneumoniae, the microbiological response to clarithromycin was similar to that of erythromycin, as was the clinical response. Clarithromycin appeared to be comparable to erythromycin in this study. They are both well tolerated. In studies that are presented in posters ofICMAS 111, azithromycinappeared to be comparable to its comparator, and both clarithromycin and azithromycin appear to be effective for ambulatory infections in children of 3-12 years of age, perhaps younger. One of the interesting aspects of Dr. Cassels’ study is that M.

New Macrolides and Pediatric Infections: Discussion

143

pneumoniue was found in a certain percentageof children youngerthan 5 years, which isagainsttraditionalthinking. We don’tusuallythink of mycoplasma as a pathogen in the 2-3 or 4-year-old’ but in her study this was the case. Well, we’ve gonethrough agreat deal of information. I think wecan summarize by sayingthat the new macrolidesare interesting and important to pediatrics. Hopefully they will be used carefully. I have some concern that the overuse of these agents, as has been shown in Finland and Japan with erythromycin, can lead to resistance. I hope that this will not be the case. I do not think for otitis media, for example, that the macrolides represent the drugs of choice. In the United States, we stillprefer amoxicillin andthen use the macrolidesor otheragents for those failuresor when amoxicillin should notbe given. They are well tolerated and appear to be very safe. Thank you.

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l1 The Molecular Mechanismof Action of Streptogramins and Related Antibiotics C. Cocito, M. Di Giambattista, E. Nyssen, and P. Vannuffel University of Louvain Medical School Brussels, Belgium

INTRODUCTION Proteins are the main polymers endowed with catalytic function and constitute the basic structural and superstructural components of the cell. Inhibition of protein synthesis.is, thus, a prominent intervention in the chemotherapy of infectiousdiseases, for bacterialmultiplicationisprevented when formation of these essential polymers is halted. Protein structure is highly specific. It is the amino acid sequence of single proteins that provides this specificity, which is precisely recognized by the immune system and by the immunoglobulins. Instructionsfor the synthesis of the specific protein structure, which is encoded in the cell genome, is transmitted to the translating machinery of the cell (the ribosome), by vector molecules [messengerRNA (mRNA)]. Fidelityof translation of the genetic message is providedby a complex mechanism, wherein ribosomes faithfully hang amino acids in a sequence collinear with that of the corresponding gene. Such a ribosome-catalyzed template-dependent polymerization is a multistep process, to which a series of cytoplasmic protein factors participates. 145

Cocito et al.

I46

Antibiotics inhibit protein synthesis by interfering withthe metabolic pathway leadingto the assembly of these polymers. Essentially, each step of this path is blocked by a specific inhibitor and, in fact, antibiotics have been invaluable reagents for dissecting this pathway and identifyingits components. Only a limited number of the in vitro active compounds, however, has found clinical applications in chemotherapy of infectious diseases. We will briefly sketch the scheme of protein synthesis in bacteria before introducing the antibiotics that interfere with single steps of this pathway and analyzing the molecular bases. Special emphasis willbe cast on streptogramins and related antibiotics of the macrolide-lincosamidestreptogramin. (MM)group.

THE METABOLIC PATHWAY FOR BACTERIAL PROTEIN SYNTHESIS

Specificity of protein synthesis relieson the order in which some 20 amino acids are hooked together, a specific order encoded in the nucleotide sequence of DNA. Ribosomes ensurethe fidelity oftranslation of the genetic message. Bacterial ribosomesare ribonucleoprotein particleswith a sedimentation coefficientof 70S,a mass of 2.5 lo6Da and a size of about 200 A (1,2). There are about 2 lo4ribosomes per bacterial cell, representing a substantial fractionof total cell proteins (10%) and RNA (80%). Some ribosomes are free in the cytoplasm, whereas others are attached to the membrane. Ribosomes (70s) contain two subunits (Fig. 1):a large subunit including 2rRNA molecules (5s and 23s) and some 31 rProteins, and a small subunit made of 16s rRNA and 21 rProteins (1,2) (Table 1). During the course of protein synthesis,the two subunits separate after completion of the polypeptidechainandjoinagainwhenaminoacidconcatenation starts (ribosomal cycle). A series of cytoplasmic protein factors promote some steps of this process by transiently binding to the ribosome in an active form. From a chemical pointof view, protein synthesis consists in establishing a covalent link between the COOH group of one amino acid and the cr-NH2 group of the next. In reality, the activated forms of amino acids undergo the polymerization reaction on linkage with a specific aminoacyl transfer RNA (AA-tRNA) whose anticodon triplet recognized the codon triplet of mRNA. Initiation takes place on the subunit, which binds mRNA and the first AA-tRNA containingformylmethionine (Met-tRNA).The three initiation factors ( E l , IF2, IF3) which promote the binding reaction recycle when the large subunit joins to form the initiation complex (Met-tRNA.70S.mRNA).

Molecular Mechanism of Action of Streptogramins

-,

,-Head

147

Head

(3 ‘len

Plateform

d

,-Central protuberance

a

I s e

I ,- Centralprotuberance

Figure Z Consensus models of ribosomes and subunits. Projections in dimensions the (A, B) and SOS (C, D) ribosomal subunits, and of ribosomes (E, F) of E. coli: front (A, C, E) and side (B, D, F) views of the particles.

Cocito et al.

148 Table l

Properties of Bacterial Ribosomes and Subunits Ribosome Small

Sedimentation coefficient Mass (kDa) Number of rRNA bases Mass of rRNA (kDa) Proportion (%) Number of polypeptides Total mass (kDa) Proportion (%) Dimensions (nm)

subunit

Large subunit

60

Elongation consists of three step that are sequentially repeated for each amino acid joining. The accepted model for this process is based on the presence on the subunit of two sites (A and P), to which two tRNA molecules bind. Peptide bond (-CO-NH-) formation occurs between the COOH group of the growing peptide chain (pep-tRNA) attached to the peptidyl P site(donorsite)and the AA-tRNA at the aminoacyl A site (acceptor site) (Fig. 2). Thiscrucialeventinprotein synthesis occurs inthree steps: (a) binding of AA-tRNA to the A site, (b) peptidyltransfer reaction from the P site to the A site, and (c) translocation

peptidyl transferase

center PTC

Figure 2 Peptide bond synthesis. Peptide bond synthesis, promoted by the catalytic PTC center of SOS subunits, consists in the transfer of a peptidyl strand (n

aminoacids) at the P site to the AA-tRNA at the A site; a one-unit longer peptide chain (n+l aminoacids) is thus formed.

Molecular Mechanism

Action

Streptogramins

149

reaction whereby the polypeptide chain is transposed from A to P, thus allowing the process to be repeated. These steps are catalyzedby the elongation factors EF'Ib and EFTS (step a) and EFG (step c), respectively. Peptide bond formation (stepb) is promoted by the catalytic locusof the subunit (peptidyltransferase centeror PTC). When the polypeptide chain is completed, it detaches from the ribosome togetherwith tRNA and mRNA, a reaction promoted by PTC as well as by the three termination factors(RF1, FW2, RF3). The overall pathway is depictedin Fig. including the interwound ribosomal cycle.

Figure 3 Metabolic pathway for protein synthesis and ribosomal cycle. I: initiation complex;11: initiation complex;111: binding (elongationstep I), IV:peptidization (elongation step 11); V: translocation (elongation step 111). The asterisk (*) repetition of cycle I11 to V.

150

Cocito et al. ,ANTIBIOTIC INHIBITORS OF PROTEIN SYNTHESIS

Numerous independently discovered substances, mostly produced by streptomycetes, were foundto block bacterial proliferation by inhibiting protein synthesis. According to their inhibitory action, antibiotics are classed in three groups: bacteriostatic (transient inhibition of growth limited to the presence of the inhibitor), bactericidal (irreversible bacterial inactivation), and bacteriolytic (lysisof the cell). Most inhibitors of protein synthesis are bacteriostatic; those interfering with nucleic acid metabolism are lethal, and those halting cell wall formation (which confers resistance to the bacterial envelope) have lytic properties. Protein synthesis inhibitors may act either at the ribosome level or on cytoplasmic factors. Antibiotics can interfere with particle functions by binding either to the small or to the large subunit(3-6). Because the unique functionof 30s particles isto start the first step of protein synthesis, antibiotics binding tothe small subunits primarilyinterfere with initiation. However,30s particles are also involved in elongation, being responsible forthe correct codon-anticodon pairingthat ensures the proper selection of the AA-tRNA that corresponds to a given mRNA triplet. Consequently, the aminoglycosides (a large antibiotic group including widely used therapeutic agents such as gentamicin, kanamycin, kasugamycin, neomycin, paramomycin, spectinomycin, streptomycin, and tobramycin) produce two kinds of effects. They interfere with initiation, thus halting protein synthesis completely, and they induce the formation of erroneous proteins (incorporation of amino acids differing in one of the anticodon nucleotides, a lesion known as misreading). W Oother antibiotic families, edeines and tetracyclines, interfere not only with initiation but also with elongation (the growing peptide chain is, in fact, located at the interface betweenthe two subunits). The main function of the large subunit is to promote peptide bond formation. The activecenter of the enzymaticactivityresponsible for amino acid polymerization (peptidyltransferase) is located at the base of the central protuberance. This enzyme is specifically inhibited by chloramphenicol, the firstbroad-spectrumantibioticproducedsynthetically andusedtherapeutically.Affinity-labeling of ribosomeswithchloramphenicol analogs has led to identification of proteins L2, L6, L16, L24, and L27 at the binding site of this antibiotic, henceat the catalytic center of 50s (6). Three other antibioticfamilies(macrolides,lincosamides,and streptogramins) are clustered within the M U group because of similarities in their mode of action and their common resistance patterns. The MLS are also peptidyltransferase inhibitors, thoughtheir binding sites do

Molecular Mechanism of Action of Streptogramins

151

not overlap that of chloramphenicol. From a functional viewpoint, the catalytic center of PTC is to bedistinguishedfrom the two substrate binding sites of the enzyme, which correspond to the A and P sites of 50s subunits. Macrolides include several subgroups differing in the number of atoms in the macrocyclic lactone rings: M12 (methymycin), M14 (erythromycin, oleandomycin), and M16 (carbomycin, lincomycin, spiramycin, and tylosin). Proteins U,L15, and L22, which were found to be altered in some MLS-resistant mutants, are located at the binding sites of these antibiotics 7-9). Puromycin is an antibiotic endowed with a peculiar action on 50s particles. Owing to its structure mimicking the OH-terminal end of PhetRNA, this antibiotic bindsto the ribosomal A site and triggers a peptidyltransfer reaction. The peptidyl-puromycin, which formsunder these conditions, is released from ribosome, thus causingthe premature interruption of peptide chains. This antibiotic, with no therapeutic application, has thus become an irreplaceable reagent for the assay of the peptidyltransfer reaction and its inhibitors (the aforementioned antibiotics, for instance). Proteins L11 and L23 have been located at the puromycin binding site of 50S, which corresponds to the A site of the particle (10). A last group of antibiotics interfere with the function protein factors. Neglecting the inhibitors acting onIF and RF factors, we restrict our interest to those that interfere with EFTu and EFG, which promote the AA-tRNA bindingreactionandtranslocation,respectively.Kirromycin attaches to the complex AA-tRNA.GTP.EFlb, which binds to the ribosomal A site; elongation is stopped at this level (11).W Oantibiotics, fusidic acid and thiostrepton, which bind to ribosomes in the proximity of the A site, inhibit the EFG-mediated translocation (driven by the energy of hydrolysisof the ribosome-bound EFG.GTP complex). Protein L11 has been located at the thiostrepton binding site (12).

BIOLOGICAL EFFECTS OF STRJWTOGRAMINS The unique property of streptogramins (synonyms: mikamycins, pristinamycins, synergimycins, synergistins, and virginiamycins) is that they contain two groups of components A and B), producing a synergistic inhibition of bacterial growth.W Oaspects of such a synergism should be mentioned. One is quantitative: the inhibitory power of B components undergoes a hundredfold increasein the presence of A components. The other is qualitative: single components, A or B, are bacteriostatic, whereastheir mixture is bactericidal. Consequently, protein synthesis is halted transiently by A or B, and permanentlyby A + B (13-17) (Table 2).

Cocito et al.

152

Table 2 In Vivo Synergistic Actions of A and B Streptogramin Components Growth proteinviability inhibition (MIC)a Single components(A or B) Mixture of components (A+B)

100 1

cell

Inhibition of synthesis

Unchanged Lowered

Reversible Irreversible

*Minimalinhibitory concentration (pglml). bColony forming units.

Virginiamycin S

I h

I

/ \2

!

I l l

Thr

AmBut

Pm

PhGly

Pipec

Ma&/

/

\$' I

0

N-

{C

\

/

Virginiamycin

M

Figure 4 Chemical structure of streptogramins: virginiamycin M nent) and virginiamycinS (type B component).

A compo-

Molecular Mechanism of Action of Streptogramins

153

The two streptogramin components are produced together by some strains of streptomycetes. Although chemically unrelated, both of them are macrocyclic lactone rings. Type B components are cyclic hexadepsipeptides of about 800 Da, whereas A compounds can be considered as highly modifiedcyclopeptides of about 500 Da, with multiple,conjugateddouble bonds (16) (Fig.4). Multiplication of most gram-positive bacteria is rapidly stopped upon incubationwith single streptogramin components. Proliferation is resumed, however, when cellsare transferred to antibiotic-free media, although growth resumption of cells treated with type A compounds occurs after a prolonged lag (bacteriopause). When bacteria are incubated with a mixture of A and B components, a viability drop of several logs within a single generation time can be witnessed (17). Most gram-negative bacteria are insensitive to streptogramins, owing to the impermeability barrier of LPS-containingenvelops.However,permeabilitymutants of gram-negative bacteria have been described which proved to be sensitiveto streptogramins and related antibiotics. Ribosomal RNA is metabolically stable under normal growth conditions,becauseassoonassynthesized, it becomesassociatedwith rProteins within newly formed ribosomes. Nonetheless, when protein synthesis is halted by streptogramins, the newly synthesized rRNA becomes metabolically unstable and undergoes turnover when protein formation resumes (16). Density gradient ultracentrifugation of cytoplasm samples from bacteria growing under physiological conditions invariably shows peaks of 70S, 50S, and S particles. An additional 60s peak appears under certain experimental conditions in bacteria treated with A streptogramins (Fig,5 ) . Such an unusual peak isdue to the occurrence of pressure-labile particles, which split at critical centrifugal speed and Mg concentration. No such alteration (whose molecular mechanism will be further discussed) appears in cells treated with type B components or other MLS antibiotics (17).

MECHANISM OF ACTION OF TYPE A STREPTOGRAMINS These antibiotics halt protein synthesis in intact bacteria as wellasin cell-free systems: They are powerful inhibitors of the poly(U)-directed poly(Phe) synthesis (Nirenberg system) (18). Type A compounds bind to free ribosomes in a monomolecular reaction, whose kinetic constants have not yet been determined with precision (19,20). It has been shownthat in the presence of type A streptogramins such as virginiamycinM (W),initiation complexesare assembled in anapparently normal fashion butare functionally inactive (Fig.6), thus pointingto

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154 60s 705

SOS

1.37 1.36 1.35 1.37

-

1.36 1.35

z_

L

fr 1.37 1.36 1.35 1 1

1.35

1.34 10

20

30

FRACTIONS

Figure 5 Roduction of pressure-labileribosomesin the presence type A synergimycins. [3H]uracil-labeled bacteriaare incubated witheither type A (B), or type B (C) components and their mixture (D) prior to ribosome fractionation by density gradient centrifugation. Sample A is the control (no antibiotic) and sample E is glutaraldehyde-fixed sample B. Wherever cells are incubated with type A components, pressure-labile ribosomesappear unless they are fixed.

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Figure 6 Assembly and reactivityof initiation complexes in the presence of type A synergimycins. To initiationcomplexes (30s ribosomalsubunits,MS,-RNA, [3H]fMet-tRNA, and IF-1, IF-2, IF-3), 50s subunits wereadded, in the presence of either GTP [(A) and (B)] or its nonhydrolyzable analog G-PPCP(C); only sample (B) contained typeA synergimycin. Samples were splitinto two parts, one of which was incubated with puromycin (o----o = no puromycin) and fractioned by density gradient centrifugation. Radioactivity tracing shows that initiation complexes were formed in all cases, but those assembled inthe presence of the antibiotic (B) and those with G-PPCP (C) held unreactive Met-tRNA.

an inhibition of the elongation phase (21). Moreover, of the three elongation steps (AA-tRNA binding to the A site, peptidyltransfer from site P, and translocation from the A to the P site), the first two proved to be inhibited by type A compounds. The AA-tRNA binding reaction assayed at equilibrium was indeed found to be blocked by V M (18). However, this step can be further dissected by the use of an additional inhibitor, kirromycin, which binds to

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EJ3b. The overall reaction sequence is as follows: (a) formation of the AAtRNA.EFTh.GTP complex, (b) binding of the complex to the ribosomal A site, (c) release of ElTu.GDP, and (d) irreversible fixation AA-tRNA to the substrate acceptor site of peptidyltransferase (11).Kirromycin prevents reaction (c), irrespective of the presence V M , whose interference isthus restricted to step (d) (12). Indeed, in the presence of GTP and E m u , labeled AA-tRNA firmly binds to ribosomes, from which it cannot be released by an excess unlabeled AA-tRNA; this exchange reaction occurs whenV M is present (22,23) (Fig. 7A). In addition, the puromycin reaction with peptidyl-tRNA linked to the ribosomal P site was found to be inhibited bytype A streptogramins (18,21) (Fig. 6). Here again, the P-site-bound substrates (labeled acAA-tRNAfor

Time (mint

0

10 20 Time ( m i n )

Figure 7 (A)TheVM-inducedexchangebetweenfreeandA-sitebound

aminoacyl-tRNA. Control (R)-ribosomes and those transiently incubatedVM with (R*), both carrying (14C)Phe-tRNA at the A site, were incubated with unlabeled Phe-tRNA, GTP, and EF-Tb in the presence or absence ofVM; at various times, ribosome-bound radioactivity was measured. R ribosomes: no VM (o),plus VM (A); R* ribosomes:no VM plus VM (A). (B) Translocational ejection of AA-tRNA from Psite of ribosomes incubated with type A synergimycins. Poly(U).ribosomes complexes containing Ac(14C)Phe-tRNA at the P site were incubated with tRNA (A site filling) and then with EF-G and GTP (to promote translocation), in the presence (filled symbols) and in the absence (open symbols) of V M . Ribosomebound radioactivity was measured. Samples: complete system (A,A), minus tRNA (0, minus EF-G+GTP(o, Translocational ejection occurred in the presence of

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instance) cannotbe chased out by the corresponding unlabeled derivatives, a sign of irreversible linkage with ribosomes. However, this exchange does take place inthe presence of VM, which prevents a stable interaction of peptRNA with the donor site of peptidyltransferase (Fig. 7B). The overall conclusion is that type A streptogramins are specific inhibitors of both substrate binding sites of the enzyme. How can such an interference be reconciled with the well-known fact that only one VM molecule can bind to each 50s particle? The clue has been furnished by a new model, whereby the acceptor and donor substrate binding sites of peptidyltransferase exchange position, conformation, and function at each elongation step in a jigsaw-like motion (Fig. 8). 'Qpe A streptogramins can bind to both subunits and free 70s particles but notto ribosomes engaged in protein synthesis or topolysomes. In fact, translating particles carrying peptidyl-tRNA or AA-tRNA are proIn agreement withthe proposed model tected against these antibiotics of interchangeable peptidyltransferase sites, particlesare protected by Asite bound AA-tRNA, as well as by P-site-bound pep-tRNA, or by AAtRNA derivatives translocated from the A to the P site. These observations imply that type A components can only bindto the free arms of peptidyltransferase, and thusto particles at the termination-initiation phase (runoff ribosomes) As previously mentioned, in cells treated with type A streptogramins, pressure-sensitive ribosomes accumulate, yielding unusual ultracentrifugal patterns (Fig. 5). It has been shown that particles carrying uncharged tRNA are unstable in centrifugation at low Mg. This contrasts with the stability of ribosomes containingAA-tRNA and pep-tRNA linkedat the A and P sites, respectively. When the latter two types of complexes are assembled in the presence of type A compounds, they become pressure sensitive It is the stable interaction of the aminoacyl and peptidyl moietiesof tRNA derivatives with the substrate binding sites of peptidyltransferase that renders particles pressure-resistant.

THE MECHANISM OF ACTION OF TYPE B STREPTOGRAMINS Unliketype A components,type B components are ineffectivein the Nirenberg system This observation has promptedthe development of other cell-free systems with copolymers as messengers. In these models, polypeptides with diverging propertiesare synthesized: charged and hydrophilic, those primed by adenine polymers; and neutral and hydrophobic, those directed by uracil-containing messengers.Indeed, the system containing poly(A,C) copolymers as templatesare highly sensitiveto inhibition by

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translocation

recycle

I

I

aa-tRNA binding peptidebond formation

translocation

translocation aa-tRNA binding peptidebond formation

recycle

translocation

{tRNA

peptidyl-RNA

Figure 8 Models of peptidyltransferase function. Iko schemes for the events occumng at the peptidyltransferase centerof the SOS subunit during translocationare compared. According to the conventional model (A), the donor and acceptor sites of the enzyme have fixed positions, and translocation involves the detachment of peptidyl-tRNA from the acceptor site. The proposed model (B) postulates an exchange of position, conformation, and function of the two sitesof the enzyme at each elongation round, with no detachment of peptidyl-tRNA in translocation.

type B components which, in addition, produce a premature release of short polypeptides (28) (Fig. 9). The followingconclusionswere then drawn: (a) Streptogramins B inhibit the in vitro peptide bond synthesis; (b) the inhibition level is templatedependent; (c) these antibiotics also induce a detachmentof incomplete protein chains; and (d) the overall inhibition is proportional to the peptide length. This indicates that in the presence of these antibiotics, the elongation process becomes increasingly difficult as the protein chain extends, by a steric hindrance effectthat slows the poly-

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lime (min)

Figure 9 Inhibition of polypeptidesynthesisandprematurechainreleaseproduced byVS and Ery and in acell-free system. Poly(A, C)-directed protein synthesis was carried out in a cell-free system of Escherichia coli containing [4C]lysine,

[“Clproline, and unlabeled threonine, histidine, glutamine, and asparagine, in the absence (0)and in the presence of VS (A, type B synergimycin) and Ery (0,14membered macrolide). Amino acid incorporation into ribosome-bound peptides total peptides(B), and high-molecular-weight peptides (C) was measured.

merization process and promotes its interruption at the level of basic amino acids (points of physiological instabilityof the elongation complexes). Indeed, peptides isolated from cell-free systems incubated with B compotype nents werefar shorter than controls and bore basic amino acids in carboxylterminal position (29). Unlike type A streptogramins that bind exclusively to naked particles, type B compounds can link also ribosomes engaged in protein synthesisand polysomes. Note that comparable inhibition of poly(A, C) and poly(U, C) systems was observed in the presence ofvirginiamycin S (type B streptogramin) and erythromycin (M14 macrolide) (28,29)(Fig.9). The same mechanism of action canbe proposed for the two antibiotics: inhibitionof peptide bond synthesis and interruption of protein elongation (26). Although unfinished peptides were not previously detected in wild-type bacteria incubated with these inhibitors, an accumulation of peptidyl-tRNA was observed in a hydrolase-negative mutant incubated with erythromycin (this precursor being rapidly degraded by the enzymes of wild-type cells) (30). As type B streptogramins are peptidyltransferase inhibitors, identification of their binding site would give a hint of the, PTC location. This information was search by the useof the biophysical technique of non-

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radiant energy transfer between fluorophores.The 50s subunit holds two L7L12 dimers: a strong site located at the base of the central protuberance, and a weak site composing the stalk. Each dimerwas removed selectively, labeled by the addition of a fluorescent coumaryl group, and replaced in its original location. Energy transfer measurements between each coumaryl-17Ll2 dimer and virginiamycin S (a type B streptogramin endowed with intrinsic fluorescence) on the same particle yielded the distances between the fluorophore couples. the result of this triangulation process, the VS binding site corresponding to the PTC was located at the base of the central protuberance (31) (Fig. 10). The proteins at the binding site of type B streptogramins were identified by an affinity-labeling approach. This relies on the synthesis of an antibiotic derivative that carries a reactive arm susceptible to forming a covalent linkage withthe receptor protein under certain experimental con-

Figure Localization of the binding site for type B synergimycins on the large ribosomal subunits.The distances between ribosome-boundVS (a type A synergimycin withinherent fluorescence) and coumaryl fluorophores located on the strong and weak L7L12 sites and on the L10 site were measured by nonradiant energy transfer. The topology of the VS binding site (hatched surface)was made by triangulation with respectto the known position of L7L12 dimers.

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Figure ZZ Topological model of the binding site for MLS antibiotics on the 50s ribosomalsubunit.Ribosomalproteins (LE,L18, L22,andL27),whichwere affinity-labeled by different MLS antibioticsandlocated by immuneelectronmicroscopy, and bases in domains I1 and V of rRNA, which were altered by M L S mutations (A) are depicted. The dottedarea in the insert indicates the consensus topological situation of the PTC domain, whichis overlapped by the MLS binding site.

ditions. The hydroxysuccinimide ester of a VS oximation derivativewas the chosen reagent; it was able to reversibly bind to particles at and to attach irreversibly at 30°C. After dissociation of the affinity-labeled particles and protein fractionation by two-dimensional electrophoresis, proteins L18 and. L22 wereidentified (32). These rProteins, in addition to the previously recognized L15 and L27, components of PTC (Fig. 11). The presence at the base of the central protuberance, of the four aforementioned proteins, and of L2 and L16 (a conformation protein) has been confirmed by the immune electron microscopy approach(33-35).

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THE MOLECULAR BASIS OF THE SYNERGISM BETWEEN A AND B STREPTOGRAMINS The synergistic inhibitory action exerted by the two streptogramin components, a biological phenomenon unique innature, was reproduced in vitro inabiophysicalmodelmeasuring the antibiotic-ribosomecomplex at equilibrium. Q p e B streptogramins,suchasvirginiamycin S (VS), are endowed with intrinsic fluorescencedue to thepicolinyl component at the ribosome binding portion of the molecule. When these antibiotics bind to the 50s subunits, there isanincrease of fluorescenceintensity that is proportional to the concentration of the formed complex; such an increase provides a valuation of the affinity of the particle for the bound inhibitor (36). In the presence of type A components,such as virginiamycin M (VM), the fluorescence intensityof the ribosome.VS complex undergoes afurther increase. Indeed, the association constantof this complex formation is about tenfold higher in the presence of VM (K,= 2.5 lo6 M" without W, and 15 lo6 M" with VM) (37) (Fig. 12).

B

of synergimycinsAand B to ribosomes. (A) Scatchard plot of the binding of type A component to ribosomes, in the presence (A----A) and inthe absence (o----o)of type B components. (B) Scatchard plotof the binding of a type B component to ribosomes in the presence (o----o) and in the absence of type Acomponents. The two binding reactions were monomolecular; although bindingwas not modified by their partners, the K , of the binding reaction of components Bwas strongly increased inthe presence of synergimycin A.

Figure 12 Bindingreactions

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163

0 Z 3

g

50

0.2 0.4 0.6 08 100

0

I

0

0.4

ERYI50 S

Figure 13 Competitive displacement of type B components by macrolides in the presence of synergimycin A. 50s ribosomal subunits were incubated with synergimycin B (VS), in the presence (e----*) or in the absence (o----o) of type A synergimycin (W).Increasing amounts of erythromycin were added, and ribosomebound VS was measured. Erythromycin completely displaced bound VS but was ineffective in the presenceof W.

Fast kinetics measurements (stopped-flow techniques) have provided evidence for a decrease of the dissociation rate constant of the VS.ribosome complex, which is inducedby VM Further structural details were gathered with fluorescence attenuationmeasurements.Accordingly, the VSbinding site on the ribosome surface was viewed as having the shape of an open well, undergoing a transannularconstrictionin the presence of VM The abovementioned spectrofluorometric model and fast kinetics techniques have also allowed the delineation of the synergistic and antagonistic interactions existing among ribosome-binding antibiotics. Thus, there is antagonism between erythromycin (ERY, M14 macrolide) and virginiamycin S (VS, type B streptogramin) for bindingto Because these antibioticshavesimilarmechanisms of action and overlapping binding sites, their ribosome fixation is mutually exclusive. The association constant of ERY (K,= 2 lo7 M-'),being higher than that of VS (2.5 lo6 " l ) , ERY addition to VS.ribosome complexes induces VS detachment and its replacement by ERY. Such an exchange does not occur, however, in the presence of type A streptogramins (Fig. 13). The following succession of events has been identified by stopped-flow studies.Attachment of V M to ERY.ribosomes complexes induces a conformational change reducing the ribosome affinity for ERY (which is thus released) (Fig. 14) and

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0

5 TIME, (min)

10

Figure 14 Displacement of ribosome-bound erythromycin by virginiamycin M. A ribosome suspension was incubated with [14C]erythromycin (Ery,a 14-membered macrolide) and splitinto three parts which were incubated (zero time) respectively with V M (a type A synergimycin) (a), unlabeled Ery (O), none (A); ribosomebound radioactivity was measured.

Tabk Kinetic Parameters forthe Binding Reactionsof Some Antibiotics .to Ribosomes

Rate constants Antibiotics Type A synergimycins 1.4 (virginiamycin M) Type B synergimycins (virginiamycin S) 14-Membered macrolides 3.2 (erythromycin) 16-membered macrolides (leucomycin A3) Lincosamides 4.5 (lincomycin)

k+ (M-' s-l)

Dissociation constant

k- (s-l)

X

104

-

X X X

l6 l6 16

0.042 0.008 4.0 0.0039

1.5 x

lo4

0.00032 1.5

R 2.8 2.1 R*

x l@ 0.25

Ribosomes nonincubated (R) or incubated (R') with type

Kd(M)

3.1 X

(2.0 X

1.8 X 107

x

lo8

1.4.x X 1O"l

3 X 10-~ synergimycins.

.

< .

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of Action

165

Streptogramins

Antibiotic

Peptidyl-transferase domains

I

II

I

111

vs

Type B streptogramins

ERY 14-membered macrolides

Type A streptogramins Lincosamides

I

Figure 15 Topological model of the PTC domain on the large ribosomal subunit. The binding sites five antibiotic families inhibiting peptidyltransferase have been located according to competitive (overlapping) and noncompetitive (nonoverlapping) interactions with ribosomes.

increasing the affinity for VS (which binds with a tenfold higherstrength) (26,37). This approach has allowed the kinetic constantsof all the members of the MLS group of antibiotics to be determined (40) (Table 3). These constants define the behavior of these inhibitors, their affinity for the particles, as well as synergistic and antagonistic interactions withthe other antibiotics; By these works, a topographic mapof PTC has been sketched, which contains three sections with partly overlapping antibiotic binding sites. One section comprises the sites of type A streptogramins andM16 macrolides, another those of lincomycin plus M14 and M16 macrolides, and a third the sites of B streptogramins, M14 and M16 macrolides (26,40) (Fig. 15).The first section might correspondto the catalytic center of PTC, whose function could also be altered by antibiotics binding to the nearby sections I1 and 111. In conclusion, typeA streptogramins induce a conformational change of 50S, leading to increased affinity for typeB components and decreased affinity for macrolides. Additional confirmationof such aninterference has beengathered byanalysisof the patterns of protectionafforded by ribosome-bound streptogramins against chemical reagents altering rRNA structure.

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THE PEPTIDYLTRANSFERASE CENTER: THE TARGET OF MOST 50s SUBUNIT INHIBITORS Ribosomalsubunits are notsimplesuperpositionsof protein shells on RNA nuclei. Although singlestranded, rRNA molecules acquire complex tridimensional configurations due to the occurrence of double-stranded regions (base pairing of RNA segments with complementary sequences) (41).WhereasrProteins adhere tospecificpartsof the folded rRNA structures, portions of the latter molecules protrude on the particle surface, hence their interaction with cytoplasmic nucleic acids and proteins (42). In fact, incubation of ribosomes with RNAse causes a splitting ofthe particles and the separation of their components. A series of chemical reagents specifically attacking nucleotide bases proved to be able to alter discrete portionsof rRNA within intact particles,the corresponding RNA alterations beingrevealed by sequencing(43). One of the approaches used to identify the ribosomal receptors for antibiotics is based onthe use of these nucleic acids reagents: Ribosome-bound antibiotics would protect against chemical attack the RNA segment present at the corresponding binding site As the result of these studies,two regions of the 23s rRNA molecule lying at the PTC locus have been identified:the central loop of domain V with adjacent sequences, and a stem of domain 11. Within these regions, type A streptogramins protected basesA2037, A2042, G2049, and C2050 (stem adjacent to the central loop of domain V) (49, whereas type B streptogramins protected basesA2062 and G2505 (central loop of domain V) plus base A752 (stemof domain 11, presumably folding over domain V) (44,45) (Fig. 16). Note that macrolides and chloramphenicol also protect bases within this region. In fact, A2058, A2059, and G2505 are protected by erythromycinandcarbomycin, the latter alsoshieldingA2062and A2451, and chloramphenicol segment2497-2507 This approachalsohasprovidedevidence for the conformational ribosomal changes inducedby type A streptogramins, which is responsible for synergism between compounds of type A and B. Indeed, base A2062, which is protectedby ribosome-bound VS, becomes unshielded whenVM is addedto the system (46). Moreover, typeA streptogramins increasethe reactivity of C2073, U2086, and U2092 toward RNA reagents, other indications of conformational particle changes (47). Notethat although protection could be due to different alternative effects (direct shielding, steric hindrance, or conformational change),the increased accessibility of a base (A2062 in our case) is only compatible with a conformational change. An-additional observation inthiscontextis the lasting ribosomal alteration produced by the attachment of type A streptogramins. Ribosomes isolated from VM-treated cells or incubated in vitro with this antibi-

Molecular Mechanism of Action of Streptogramins

167

.

A U

mo~

2

m

2460

m

I

I

o^ t

d

0

V V A

h

tl$

: :: l Figure 16 The protection patterns of rRNA bases inducedby V M , other antibiotics, andtRNA derivatives. In this illustrationof the central loop of domain V of 23s rRNA and related stems, the bases displaying analtered reactivity toward chemical reagents, in the presence of ribosome binding substances (tRNA derivatives and antibiotics) are indicated as follows: (a) bases protected by VM (D),(b) bases protected by other antibiotics are encircled; (c) basedprotected by aminoacyl-(A) and peptidyl-(P)tRNA; and (d) base A2062, while protected by VS alone, became accessible inthe presence of VM+VS (filled arrow).

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otic, after purification by sedimentation or bycolumn chromatography (procedures supposedly removing all the bound inhibitor), were found to (48). Analysis be inactive in directing protein synthesis in cell-free systems of these particles has shownthe presence of noncovalently bound residual VM, removable by anti-VM immunoglobulins. However, VM-free particles, although able to catalyze peptide-bound formation, did still bindVS with increased affinity(49). The overall conclusions are as follows: (a) type A streptogramins bind to ribosomes with an affinity level higher than previously reported (Kd = 3.1 (b) residual V M rendersparticlesunsuitable to catalyze peptide bond formation; (c) after complete V M removal, ribosomes still hold a lasting conformational change, conferning higher affinity for type B streptogramins; and (d) the latter alteration can only be removed by particles dissociation and reassociation(50).

CONCLUSIONS AND SUMMARY Bacterial protein synthesis is a multistep process catalyzed by ribosomes and cytoplasmic protein factors; initiation, elongation, and termination are the phases of this path. In parallel, ribosomes undergo an associationdissociation cycle, whereby 30s and subunits separate at termination and rejoin at initiation, started by 30s. Most antibiotics transiently inhibit protein synthesis (bacteriostatic action) by binding either to ribosomes or to cytoplasmic factors. Only aminoglycosides (pleiotropic inhibitors of and streptogramins have a bactericidal (i.e., permanent action). The unique trait of streptogramins isthe presence of A and B components, which are separately bacteriostatic and jointly bactericidal. Both components bind to SOS subunits with monomolecular reactions,the affinity of ribosomes for B components being tenfold increased by their partners. The two-site model for protein synthesis is basedon the presence at the SOS surface of two binding sites,P and A, for two moleculesof precursors AA-tRNA, which are also linkedto mRNA by codon-anticodon pairing. Elongation occurs in three steps: AA-tRNA binding to the A site (promoted by E m ) , peptidization between pep-tRNA at the P site and AAtRNA at the A site (promoted by the PTC of subunits), and translocation of pep-tRNA from P to A site (promoted by EFG). Most of the best known therapeutically active antibiotics that act on elongation interfere with PTC function. Within the PTC domain, a catalytic center (inhibited by chloramphenicol) is to be distinguished from the substrate donor and acceptor sites (corresponding to the P and A sites of ribosomes), to which the aminoacyl or peptidyl portions of the two reacting substrates are bound. Type A streptogramins specifically inhibit the substrate interac-

Molecular Mechanism

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I69

tion with the corresponding PTC sites: A release of precursors and a halt of polypeptide synthesis followthe attachment 'of these inhibitors to the 50s subunits. 'T)lpe B streptogramins and macrolidesslow the elongation process and cause a premature halt at the level of basic amino acids, hencethe release of incomplete polypeptide threads. Thus, type A compounds can only bind to naked particles (i.e., to the free substrate binding arms of peptidyltransferase), whereas type B streptogramins can attach to polysomes in a pointof the 50s surface situated at a certain distance from the catalytic center,of PTC (and yet withinthe domain of the enzyme). A streptogramins cause a conformational change of 50s subunits, which is also responsible for the synergistic actionof A and B components. Several proofs for suchachangehavebeenprovided: (a) after contact with A streptogramins, ribosome affinity for type B components undergoes a tenfold increase,high a affinitystate that lasts after removal of A components; (b) a decreased ribosome affinityfor erythromycin is also producedundertheseconditions;(c) the accessibilityoffluorescence quenchers to the ribosomal bindingsite of B streptogramins is decreased in the presence of type A compounds; and (d) protection of rRNA bases against nucleic acids reagents, which is provided by type B components, is modified by the binding of type A streptogramins to particles. In conclusion, antibiotics may interfere in three ways with ribosome functions and protein synthesis: (a) direct functional block (the case of chloramphenicol which binds to the catalytic sector of PTC); (b) steric hindrance (typeB streptogramins and macrolides which interfere with elongation, by binding at adistancefromcatalytic center); and (c) conformational changeof ribosome triggered by antibiotics which bind in the proximity of the catalytic center (typeA streptogramins). Conformational PTC changes are responsible for the synergistic and antagonistic interactions among different antibiotic families and also for the synergism between A and B streptogramins.

REFERENCES 1. Wittman HG. Components of bacterial ribosomes. Annu RevMicrobioll982; 51:155-183. 2. Wittman HG. Architecture of prokarioticribosomes.Annu Rev Microbiol 1983; 52~35-65. 3. Pestka S. Inhibitors of ribosome functions. AnnuRev Biochem 1971; 40:697710. 4. Vasquez D. Inhibitors of proteins synthesis.FEBS Lett 1974; 40:S63-S84. 5. Pestka S. Insightsintoproteinbiosynthesis and ribosomefunctionthrough inhibitors. Prog NucleicAcid Res Mol Bioll976; 17:217-245.

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6. Vhzquez D. Inhibitors of Protein Synthesis. Berlin: Springer-Verlag,1979. 7. Teraoka H, Nierhaus KH. Proteins from Escherichia coli ribosomes involved in the binding of erythromycin. JMol Bioll978; 126:185-193. 8. Tejedor F, Ballesta JPG. Reaction of some macrolide antibiotics with the ribosome. Labeling of the binding site components. Biochemistry 1986; 25: 7725-7731. 9. ArCvalo MA, Tejefor F, Polo F, Ballesta JPG. Protein components of the erythromycin binding site in bacterial ribosomes. J Biol Chem 1988;263: 58-63. 10. Olsen HM, NicholsonAW, Cooperman BS, Glitz DG. Localization of sites of photoaffinity labelingof the large subunitof E . coli ribosomes by an arylazide derivative of puromycin. J Biol Chem 1985; 260:10326-10331. 11. Parmeggiani A, Swart GWM. Mechanism of action of kirromycin-likeantibiotics. Annu Rev Microbiol1985;3937-577. 12. Thomson Y, Cundliffe E, Stark M. Binding of thiostrepton to a complex of 23s rRNA with ribosomal protein L11. Eur J Biochem 1979; 98:261-265. 13. VAsquez D. The streptogramin family of antibiotics. In: Gottlieb D, Shaw PD, eds. Antibiotics, Vol. I. Berlin: Springer-Verlag, 1967:387-403. 14. Vhzques D. The streptogramin family of antibiotics. In: Corcoran J W , Hahn FE, eds. Antibiotics, Vol 111. Berlin: Springer-Verlag 1975:521-534. 15. Tanaka N.Mikamycin. In: Corcoran JW, Hahn FE, eds. Berlin: SpringerVerlag, 1975521-534. 16. Cocito C. Antibiotics of the virginiamycin family, inhibitors which contain synergistic components. Microbiol Rev 1979; 43:145-198. 17. Cocito C. Properties of virginiamycin-like antibiotics (synergimycins), inhibitors containingsynergisticcomponents.In:CorcoranJW, Hahn FE, eds. Antibiotics, Vol. IV. Berlin: Springer-Verlag, 296-332. 18. Cocito C, Kaji A. Virginiamycin M-A specific inhibitor of the acceptor site of ribosomes. Biochimie 1971;53:763-770. . 19. Cocito C, Di Giambattista M. The in vitro binding of virginiamycin M to bacterial ribosomesandribosomalsubunits. Mol Gen Genet 1978; 166: 53-59. 20. Aumercier M, BouhalladS, Capmau ML, Le Goffic F. Irreversible binding of pristinamycin IIA (streptogramin A) to ribosomes explains its “lasting damage” effect.J Antibiot 1986; 39:1322-1328. 21. Cocito C, Voorma H, Bosch L. Interference of virginiamycin M with the initiation and the elongation of peptide chains in cell-free systems. Biochim Biophys Acta 1974; 340:285-298. 22. Chinali G. Moureau Ph, Cocito C.The mechanism of action of virginiamycin M on the binding of aminoacyl-tRNA to ribosomes directed by elongation factor Tu. Eur J Biochem 1981; 118577-583. 23. Cocito C, Chinali G. Molecular mechanism of action of virginiamycin-like antibiotics (synergimycins)on protein synthesis in bacterial cell-free systems. J Antimicrob Chemother 1985; 16 (supplA):35-52. 24. Chinali G, Moureau Ph, Cocito C. The action of virginiamycin M on the

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acceptor, donor and catalytic sitesof peptidyltransferase. J BiolChem,

25.

Chinali G, Di Giambattista M, Cocito C. Ribosome protection by tRNA derivatives against inactivation by virginiamycin M. Evidence for types of interaction of tRNA with the donor site of the peptidyltransferase. Biochemistry Di Giambattista M., Chinali G, Cocito C. The molecular basis of the inhibitory activities of type A and type B synergimycins and related antibiotics on ribosomes J. Antimicrob Chemother Chinali G, Vanlinden F, Cocito C. Actionof virginiamycin Mon the stability of differentribosomalcomplexes to ultracentrifugation.BiochimBiophys Acta Chinali G, Nyssen E, Di Giambattista M, Cocito C. Inhibitionof polypeptide synthesis in cell-free systems by virginiamycin S and erythromycin. Evidence for a common mode of action of type B synergimycins and 14-membered macrolides. Biochim Biophys Acta Chinali G, Nyssen E, Di Giambattista M, Cocito C. Action of erythromycin and virginiamycin S on polypeptides synthesis in cell-free systems. Biochim Biophys Acta Menniger JR, Otto DP. Erythromycin, carbomycin, and spiramycin inhibit protein synthesis by stimulating the dissociation of peptidyl-tRNA from ribosomes. Antimicrob AgentsChemother Di Giambattista M, Thielen A, Maassen J, Moller W, Cocito C. Localisation of virginiamycin S binding site on bacterial ribosome by fluorescence energy transfer. Biochemistry Di Giambattista M, Nyssen E, Pecher A, Cocito C. Affinity labeling of the virginiamycin S binding site on bacterial ribosome. Biochemistry Stoffler G, and Stoffler-Meilicke M. Immuno electron microscopy of ribosomes. Annu Rev Biophys Bioeng Stoffler G, and Stoffler-Meilicke M. Immuno electron microscopyon Escherichia coli ribosomes In: Hardesty B, KramerG, eds. Structure, function and genetics of ribosomes. New York: Springer-Verlag Lake JA. Evolving ribosome structure: domains in archaebacteria, eubacteria, eocytes and eukaryotes.Annu Rev Biochem Parfait R, de BCthuneMP, Cocito C. Aspectrofluorimetric study of the interaction between virginiamycin S and bacterial ribosomes.Mol Gen Genet Moureau Ph, Engelborghs Y, Di Giambattista M, Cocito C. Fluorescence stopped flowanalysisof the interaction of virginiamycin components and erythromycin with bacterial ribosomes. J Biol Chem Di Giambattista M, Ide G, Engelborghs Y , Cocito C. Analysis of fluorescence quenchingof ribosome-bound virginiamycinS. J Biol Chem Cocito C, Di Giambattista M, Nyssen E, Vannuffel P. Inhibition of protein

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synthesis by streptogramins and related antibiotics. J Antimicrob Chemother suppl A, in press. Di Giambattista M, Nyssen E, Engelborghs Y, Cocito C. Kinetics of,binding of macrolides, lincosamides and synergimycins to ribosomes. J Biol-Chem Noller HF. Structure of ribosomalRNA.AnnuRevMicrobiol Noller HF. Ribosomal RNA and translation. Annu Rev Biochem

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Stem S , Moazed D, Noller HF. In: Noller HF, Moldave K. eds. Methods in Enzymology NewYork: AcademicPress, . .. 44. Moazed D, Noller Chloramphenicol, erythromycin, carbomycinand vernamycin B protect overlapping sites inthe peptidyl transferase region of . rRNA. Biochimie Vannuffel P, Di Giambattista M, Cocito C. Chemical probing of virginiamycin M-promoted conformational changeof the peptidyltransferase domain. Nucl 22 Ac. Res. 46. Vannuffel P, Di Giambattista M, Cocito C. The role of rRNA bases in the interaction of peptidyltransferase inhibitors with bacterial ribosomes. J Biol Chem Vannuffel P. L'interactiondesinhibiteurs de la peptidyltransferase, avec YARN Le r61e des bases du domainV. PhD thesis. Faculty of Sciences, UniversitB catholique de Louvain, pp. 48. Parfait R, CocitoC.Lastingdamage to bacterialribosomes by reversiblybound virginiamycinM. Proc Natl Acad SciUSA Nyssen E, Di Giambattista M, Cocito C. Analysisof the reversible bindingof virginiamycin Mto ribosome and particle functionsafter removal of the antibiotic. Biochim Biophys Acta 50. Moureau Ph, Di Giambattista M, Cocito C. The lasting ribosome alteration produced by virginiamycin M disappears upon removal of certain ribosomal proteins. Biochim Biophys Acta

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12 Early Clinical Results with Quinupristid Dalfopristin for the Therapy of Bacteremia Due to Resistant Gram-Positive Bacteria C. Moellering, Jr. Beth Israel Deaconess Medical Center Boston, Massachusetts

Sharon L. Cerwinka RhSne-Poulenc Rorer Collegeville, Pennsylvania

Quinupristiddalfopristin (Synercide) is a novel streptogramin antibiotic with potent activity against gram-positive organisms, including methicillinresistantstaphylococciandvancomycin-resistant Enterococcus fuecium (VREF) Even thoughthe drug has yetto be released for clinical use, it has attracted widespread attention inthe infectious diseases community, as it is often the only or one of the few agents with useful activity against multiresistant staphylococci and enterococci. Because of this, an emergency use program was initiated in June and as of December patients had been enrolled in this program.Of the patients treated with the drug thus far, 608 have been infected with vancomycin-resistant Enterococcus fuecium, with other vancomycin-resistantenterococci, with staphylococci, and with streptococci or Corynebacteria. Criteria for enrollment in this study include the,following:

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1. Positive culture for a pathogen presumed susceptible to quinupristiddalfopristin 2. Signs and symptoms of infection 3. Infecting organism resistant to all clinically appropriate antibiotics or patient was a documentedtreatment failure for all clinically appropriate antibiotics or patient was intolerant of all available clinically appropriate antibiotics The majority of patients enrolled in the study thus far (613 patients) have come from the United States. Includedin the 234 patients for whom completed data are currently available are 126 patients with bacteremia due to VREF or staphylococci who form the basis for this interim report: 115 patients (111 adults and 4 children) with Enterococcusfueciurn bacteremia and 11 with staphylococcal bacteremia (one or more positive blood cultures within 7 days prior to quinupristiddalfopristin). Of the 115 patients with Enterococcus fueciurn bacteremia treated with the study drug, 76% were immunosuppressed, 22% were leukopenic (total white blood cell count <500), 19% were on hemodialysis, and48% had had prior surgery. Included among the early enrollees were 19 liver transplant, 5 renal transplant, and 5 multiorgan transplant patients. Thus, the patients enrolled in this study represent a group of patients with particularly severe underlying disease. The doses of quinupristiddalfopristin given to these patients varied from 5 mg/kg q12h to 7.5 mg/kg q8h, and duration of therapy lasted a mean of 13 days (range 1-72 days). In keeping withthe severe underlying illness in these patients,the subjects had received an average of 4.6 antibiotics per patient prior to initiation of quinupristiddalfopristin therapy. Only 8 patients received monotherapy withquinupristiddalfopristinand 71 received a noneffectiveconcomitant antibiotic.The remaining patients received concomitant therapy with a variety of agents, including chloramphenicol(19), rifampin (lo), tetracycline or doxycycline ( l l ) , or teicoplanin (1). Sixtynine of the bacteremias occurredin patients withoutan identifiable source. In 26 of the patients, an intraabdominal sourcewas thought to have been the primary site of infection, followedby 12 with catheter-related bacteremias and 5 with endocarditis. Skin and skin structures (4), urinary tract infections (2), vascular sites(2), and mediastinitis(1) represented the other sites of bacteremic infection.Of these patients,70% had 2 2 positive blood cultures within 7 days prior to quinupristiddalfopristintherapy, and 42% had positive blood cultures within 24 h of initiation of treatment. All of the organisms were susceptibleto quinupristiddalfopristin[minimal inhibitory concentration (MI%) = 1, range = 0.03-4 pg/ml], and 100% were resistant to vancomycin. Ninety-five percent were resistantto gentamicin, 91% to ampicillin, and 8% to chloramphenicol.

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Clinical cureor improvement was seen in 4165ofpatients (63%) who had 2 2 positive blood cultures and were treated for at least 5 days. The bacteremia was cleared in 46 of 65 (71%) of the patients, and in 4% the bacteriologicoutcome was indeterminatebecause of failure to obtain follow-upcultures. There wasessentiallynodifferenceinoutcome for those patients receiving quinupristiddalfopristinmonotherapy (or concomitant noneffective treatment) and those who received concomitant antibioticswith potential activity against VREF. The mortality rate in patients treated for less than5 days was 92%, and in those treated for more than 5 days, 58%. VREF was thought to be a direct cause of death in 4% of the patients treated for more than 5 days and felt to be contributory (by the clinical investigators)to death with other causes in60% of the cases. Overall, 50% of the patients diedwhile receivingquinupristiddalfopristin,confirming the serious nature of the underlying disease in these patients.This is consistent with what has been seen in a number of other studies of bacteremia due to VREF treated with a variety of other agents (2). Overall, the 11 patients with staphylococcal bacteremia appeared to have somewhat less severe underlying disease, and evaluation of these patients was thus more straightforward. These patients had received a significant number of antibiotics (averaging 3.1 per patient) prior to receiving quinupristiddalfopristin therapy. Although only 1 received monotherapy with quinupristiddalfopristin,9 additional patients receiveda noneffective concomitant antibioticto which the organism either was resistant or which had not been effective against the Staphylococcus in prior therapy. Sevenof 11patients had12 positive blood cultures for Staph. aureus within 7 days of quinupristiddalfopriistin therapy, and 6 of 11 had positive blood cultures within 24h of the start of treatment. All of the organisms were susceptible to quinupristiddalfopristinand to vancomycin. Nine patients were treated for more than 5 days, and bacteremiawas cleared in all nine ofthese patients. Despite this,44% of the patients died, but staphylococci were not felt to be the direct causeof death in any of these patients. Adverse effects were rare. Arthralgia (3), abnormal liver function tests venous intolerance(2), and myalgia (2), aswell as vomiting,rash, hyponatremia, rigors, seizure, hemolysis, and thrombocytopenia (seen in (6%) one patient each) led to discontinuation of therapy in17of234 evaluable cases inthe overall study. Noneof these stood out as significant problems, particularly given the severe underlying disease of the patients treated. On balance, quinupristiddalfopristin appears to be very effective in the therapy of staphylococcalbacteremiaand appears to havedefinite utility in patients with vancomycin-resistantEnterococcus faecium bacteremia as well. The exact efficacy of quinupristin/dalfopristin in the latter

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setting, however, is virtually impossible to ascertain from this uncontrolled series because so many patients had severe terminal underlying disease and/or received concomitant antibiotic therapy. This phenomenon is not unique to therapy of VREF infections withquinupristiddalfopristin (3,4).

REFERENCES Collins LA, Malanoski GJ, Eliopoulos GM, Wennersten CB, Ferraro MJ, Moellering RC, Jr. In vitro activity of RP an injectable streptogramin antibiotic against vancomycin-resistant gram-positive organisms. Antimicrob Agents Chemother a semisynthetic injectable pristinamyFass RJ. In vitro activity of RP cin, against staphylococci, streptococci and enterococci. Antimicrob Agents Chemother Feldman RJ, Paul S, Cody R, Noveck H, Silber L, Weinstein MP. Analysis of treatment of patients with vancomycin-resistant enterococcal bacteremia (VREB). 34th Intersci. Conf. Antimicrob. Agents Chemother., Orlando, FL, abstr 5/54, Noms AH,Reilly JP, Edelstein PH, Brennan PJ, Schuster MG. Chloramphenicol for the treatment of vancomycin-resistant enterococcal infections. Clin Infect Dis

13 Drug Interactions of Macrolides and halides Ethan Rubinsteinand Shlomo Segev Sheba Medical Center Tel-Aviv Universiv, School ofMedicine Tel-Hashomer. Israel

The macrolides and azalidesare antibiotics in common use in a large number of patients. Due to the fact that these antibiotics are frequently prescribed to patients with respiratory tract infections who receive many other agents such as bronchodilatators, mucolytic agents, cardiac medications, diuretics, and forth, drug interactions of this group of agents are of particular importance. In addition, at the present time, many subjects in the population take a large numberof agents on a habitual basis,either as primary or secondary prophylaxis for various degenerative diseases or conditions such as atherosclerosis, weight reduction, inhibiting organ rejection, cardiac conditions, and metabolic support. At present, the number of subjects on immunomodulating agents is the largest in history, and the number of immunosuppressed patients has never been greater. All these background factors place any new agent through rigorous testing for the possible interaction with other medicines before being launched. This is particularly true if one considers the large numbers with various compromised organ functions, with advanced age, and with a variety of underlying diseases and other confounding conditions. With this background in mind and with the recent experience with unforeseen adverse events and drug interactions produced by some of the new fluoroquinolones, it is rather 177

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surprising that the macrolides and azalides have had few adverse events and drug interactions described despitethe large number of patients who have benefited from these antibiotics.

MECHANISM OF DRUG INTERACTION Over the last 25 years there have been many reports indicating that the macrolides and azalides may interfere with the metabolism ofother drugs. Erythromycin and triacetyloleandomycin have hadthe longest experience. The most studied and controversial interaction has been betweenerythromycin and theophylline. The background for understanding these drug interactions lies inthe understanding of the interaction ofthe macrolide or its metabolite withthe cytochrome P-450 enzyme complex. Critical evaluation of this interaction requires considerationof several factors, including (a) dose, formulation,route, serum concentration, timeof dose relativeto meals, and duration of therapy with the antibiotic: (relative to possible saturable elimination) of the other drug; (b) characteristics of the patients or study subjects (e.g., age, gender, smoking history and status, pathophysiology of the underlying condition, and environmental factors); and (c) study design (1). With the appearance of many semisynthetitic macrolidesand azalides, drug interactions between these agents and other drugs can be classified into three different groups, all basedon the primary processof the binding between the macrolide and the cytochrome P-450. The first group consists of the macrolides proneto forming nitrosoalkanes and the consequent formationof inactive cytochrome P-450-metabolite complexes; troleandodomycin and the erythromycins belong to this group. The second group also forms such complexes, but to a lesser extent, and rarely produces drug interactions; josamycin, flurithromycin, roxithromycin, clarithromycin, miocamycin, and midecamycin belong to this group. The third group does not inactivate the cytochrome P-450 nor forms complexes and therefore is to unable modify the pharmacokinetics and metabolismof other drugs; spiramycin, rokitamycin, dirithromycin, and azithromycin belong to this group. It appears that two structural factors are important for a macrolide antibiotic to lead to theinduction of cytochrome P-450-iron-nitrosoalkane metabolite complex: the presence of a nonhindered readily accessible Ndimethylamino group and the hydrophobic characterof the macrolide (2).

.BIOCHEMICALSTUDIES Most drug interactions involving macrolides result fromthe inhibition of hepatic drug-metabolizing enzymes. Some hepatic cytochrome P-450 en-

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zymes are inactivated by a suicidal process during the oxidative metabolism of certain macrolides, andas a result, the metabolism of other drugs normally catalyzed by these enzymes is inhibited. This leadsto accumulation of the second drug that may have clinical relevance, as is the case with ergotamine derivativesthat may cause ergotism when coadministered with the macrolides, with theophylline that may cause seizures, and with anticoagulants that may cause bleeding The cytochromeP-450 are alargefamily of monooxygenase enzymes which convert hydrophobic drugs into more hydrophilic metabolites, thus facilitating their elimination. Cytochrome P-450 enzymes are present in almost all organs but are found in the liver in their highest concentrations.Cytochrome P-450have acommon site, an iron (111)protoporphyrin IX, bound to the protein by an iron-cysteinate bond (4). Monooxygenation of theirsubstratesinvolvesacommonmechanism whereby 0, binds to the free axial coordination position of the iron is activated, with the formation of a high-valence iron-oxo-active species after a two-electron reduction. Many compounds act as inhibitors of cytochrome P-450. One category acts as reversible inhibitors by binding to the active site or to the iron; a second category acts as irreversible inhibitors by binding covalently to the iron, the porphyrin, or the protein. The formation of strong iron-nitrogen bonds between cytochrome P-450 and compounds containing basic and accessible nitrogen atoms is often implicated in inhibition of the cytochromeP-450. On the other hand, someagentscontaining imidazole, triazole, or pyridine groups form stable iron-nitrogen complexes. Some compounds containing an amine such as propoxyphene and amphetamines are oxidized into nitrosoalkanemetaboliteswhichbind Porphyrin-Fe(I1)-nitrosoalkane strongly to cytochromeP-45O-Fe(II). complexeshaveastrongiron N(0)R bondwhen the iron isin the ferrous state but not in the ferric state. Treatment of these complexes with ferricyanide regenerates cytochrome P-450 and releases the nitrosoalkaneligand by converting the iron to itsbivalence state. Troleandromycin and erythromycin estolate are metabolized to the formation of Fe(I1)-nitrososalkane complexes which are stable. Troleandomycin and erythromycin estolate are metabolized in the liver to form P-450-Fe(II)nitrosoalkane stable complexes(43).

ANIMAL STUDIES In rats, triactyloleandomycin administered IP 1mmolkg daily for 4 days induced its own demethylation,the demethylated metabolite possibly a nitrosoalkane formed a stable complex with iron (Fe2+)of reduced cytochrome

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P-450. Hexabarbitone sleeping timewas prolonged, and the in vivo disappearance of the hexabarbitone from the liver was prolonged. The same dose of troleandomycin increasedthe canalicular bile acid-independent bile flow but decreased the activity of cholesterol 7-alpha hydroxylase andthe synthesis pool size and biliary secretion rate of bile acids. Similar observations were made for oleandomycin and for erythromycin (2 mmolkg administered orally for 4 days.) Single doses of triacetyloleandomycin (1 mmol/ kg), oleandomycin, and erythromycin (4 mmol/kg) caused glutathione depletion in rats. Glutathione prevented the in vivo formation of the cytochrome P-450-triacetyloleandomycin complex. However, glutathione did not inhibitthe demethylation of triacetyloleandomycin and did not destroy the complex once itwas formed.

HUMAN STUDIES Treatment of patients withtriacetyloleandomycin 2 g for 6daysincreased NADPH-cytochrome-C reductase activity by 48% as well as the total concentration of cytochrome P-450; but one-third of the total cytochrome was complexed by the triacetyloleandomycin metabolite. In patients treated with triacetyloleandomycin for 7 days, antipyrine clearance was decreased by 45%,whereasasingledosehadnoeffect.Similar results were also obtained with erythromycin propionate (2g orally daily for 7days)whichincreasedNADPH-cytochrome C reductase activity and increased cytochrome-C concentrations, but again part of the cytochrome wascomplexedbyanerythromycin metabolite. The activity of hexabarbitone was unchanged, and clearance of antipyrine was also unchanged under these conditions.

EFFECT OF MACROLIDES ON THE PHARMACOKINETICS OF OTHER DRUGS Theophylline (Table The first clinicalreport of theophylline-erythromycin interaction appeared in 1977. In thisreport, the blood levels of theophylline increased 37% and 46% after the addition of4.5-5.0mg/kg/q6hof erythromycin A in two children. In healthyvolunteers,aftera 10-day course erythromycin stearate (250 mg tid orally), the half-life of serum theophylline increased from 4.79 to 7.53 h, whereas drug clearance decreased from 91.6 to 54.8 ml/h/kg. Theophylline metabolites 3-methylxanthine and 1.3 dimethyl uric acid also decreased (7). Other investigators demonstrated prolongation of theophylline T, in asthmatics but not in patients with bronchitis and most investigators failedto demonstrate changesin theophylline clearance.

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Other investigators failed to demonstrate changes in theophylline pharmacokinetics and metabolism both in volunteers and inpatients with a variety of respiratory conditions (bronchitis, asthma, chronic bronchoectasis, etc.). Someof these controversiesmay be due to differences amongthe studied individuals, some may be due to differences in assay methods of theophylline and its metabolites, and others may be due to different brands of erythromycin used. The state of the art at the present time is that an interaction betweenerythromycinandtheophyllinecan be expectedwhen the daily dose of erythromycin exceeds1000 mg/day, whenit has been administered for several days,and when the serum level of theophylline is inthe upper limits of the therapeutic range. Because one cannot predict which patient will suffer from this interaction, close monitoring of such patients is mandatory (8). Theophylline probably affectsthe disposition of erythromycin little if at all.Althoughearlierstudieshaveshowedareductionin erythromycin area under the plasmaconcentrationversustime (AUC) following its oral administration, the kinetics of IV administered erythromycinwas unchanged. The only possible effect may be a reduction in tubular reabsorptionof erythromycin (or enhanced secretion)by theophyllinemetabolites (9). Triacetyloleandomycin (=troleandomycin)significantly reduces the elimination theophylline to about 50%, with a 30% increasein T,, anda100%increaseinterminal T,, andsignificantly increases serum theophylline levels. Clinical signs of theophylline toxicity were noted (epileptic seizures) in a patient taking both agents together (10).Roxithromycincausesaslightdecline of theophyllineclearance versus 0.292 U); however, no increases in serum theophylline levels were observed (11).

Carbamazepine (Table 2)

Carbamazepine is almost entirely metabolized throughthe hepatic monooxygenase system inthe cytochrome-C systemto epoxide. The remaining 2% is eliminated unchanged. This metabolic pathwayto epoxide is accentuated during chronic carbamazepine administration due to autoinduction of this pathway. Triacetyloleandomycin-induced carbamazepine intoxication occurred within1 or 2 days after administration, whereas after discontinuation of this macrolide, carbamazepine levels soon decreasedto nontoxic levels (12). Few reports have been published describing clinical case of carbamazepine intoxication in patients who received erythromycin. In a kineticstudy,asignificantdecreaseincarbamazepineclearance (5%41%) was demonstrated with the coadministration of erythromycin without anysubstantialdelay(13).Troleandomycinsignificantlyincreased

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carbamazepine serum levelsby twofoldto fourfold in epilepticpatients and was responsible for carbamazepine intoxication

Cyclosporine (Table 3) Erythromycin increases cyclosporine levels fivefold to the toxic range, giving rise to clinical manifestations, both in transplant patients and in children with diabetes mellitus.The association is independentof the dose and formulation of erythromycin and occurs between days 2 and of erythromycin therapy The mechanisms responsible are either inhibition of oxidative transformationof cyclosporine inthe liver duringthe hepatic first pass or due to increased absorptionin the intestinal tract probably secondary to the effect of the macrolide on the gastrointestinal flora In clinical practice, close monitoringof cyclosporine serum concentrations is mandatory to prevent intoxication if erythromycin is coadministered.

Digoxin Several patients have been reported who have suffered increase to toxic levels of digoxin when erythromycin therapy was added. This phenomenon occurs in the minority of patients who excreteunusuallyhighdigoxin amounts into their intestinal tract. By its ability to alter the intestinal flora, erythromycin eliminates the bacteria that metabolize digoxin; thus, excreted and unmetabolized digoxin is reabsorbed from the intestine andmay cause toxicity

Warfarin Several patients have been described to have elevated prothrombin times when warfarin was coadministered with erythromycin A kinetic study in volunteers demonstrated an average decrease of of warfarin clearance, in particularin subjects who already have a slow warfarin clearance The nature of this interactionis not yet completely understood.

Bromocriptine Bromocriptine is an ergot derivative used in the therapy of Parkinson’s disease and as an antiprolactin. Erythromycin estolate mg qid) increased the AUC of bromocriptine by > with a maximal plasma concentration four to six times higher than inthe control period. Erythromycin increases bromocriptine bioavailability through alterations in its absorption or hepatic first pass.In patients receiving erythromycin,the dose of bromocriptine shouldbe reduced to a thirdor a quarter (23).

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Methylprednisolone On the basis of a favorable response of asthma patients to erythromycin therapy a kinetic study was done on the interaction of erythromycin with methylprednisolone. It was found that erythromycin reducedthe clearance of methylprednisolone by and increasedthe half-life from to h Troleandomycin also impairs significantly the elimination of methylprednisolone, reducing clearanceby Reduced tissue binding or increased plasma protein binding of methylprednisolone by troleandomycin have been reported to increase the steady state of the hormone. Similarly, the metabolism of estrogen- and progesterone containing contraceptives were inhibitedby troleandomycin giving riseto cholestatic jaundice in female patients who received troleandomycin.

Ergot Alkaloids Ergotism described in a patient who took concomitantly ergotamine tartarate andtroleandomycin was the firstdescribedinteractionbetween macrolide and another agent The phenomenon has been repeatedly described not only with troleandomycin but also with erythromycin and is presumably due to inhibition of the ergot compound’s metabolism in the liver. The occurrence of ergotism is unpredictable; therefore, this drug combination should not be used(8).

Alfentanil Alfentanil is a synthetic opioid usedin anesthesia; inhibition of alfentanil elimination by erythromycin was studied in volunteers.The average clearance decreased from to ml/h/kg and T,, increased from to min following erythromycin mg bid orally for days Triazolam elimination was reduced by troleandomycin threefold (from to giving riseto psychomotor disturbances

Disopyramide Disopyramide is an antiarrhythmic agent that undergoes oxidative metabolism inthe liver. Concomitant administration of this agentwith erythromycin caused prolongationof the QT interval on electrocardiogram and intraventricular conduction disturbances as a signof increased plasma level of disopyramide Amiodarone, another antiarrythmicagenthasalso caused increasedQT intervals in patients who receivedit with erythromycin concomitantly.

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Terfenidine Erythromycin alters the metabolism of terfenidine an antihistaminic agent which resulted in altered cardiac repolarization disturbances(29). In summary, the newer macrolides seem to offer a far greater degree of safety from drug-drug interactions, mainly because of lower degree of interaction with the hepatic cyctochrome P-450 enzyme complex. At the presenttime,azithromycin,roxythromycin,anddirithromycinhave no clinically recognizable significant interactions.

REFERENCES 1. Ludden TM. Pharmacokinetic interactionsof the macrolide antibiotics. Clin. Pharmacokineti 1985; 10:63. 2. Periti P, Mazzei T,Mini E, Novelli A. Pharmacokinetic drug interactions of macrolidesstudies. Clin. Pharmacokineti1993; 24:70. 3. Pessayre D, Larrey D, Funck-Brentano C, Benhamou JP. Drug interactions and hepatitis producedby some macrolide antibiotics. J Antimicrob Chemother 1985; 16 (suppl A):181. 4. Ortiz de Montellano PR. Cytochrome P-450: Structure, Mechanism and Biochemistry. 1986. New York: Plenum Press 5. Delaforge M, JaouenM, Mansuy D. Dual effects of macrolide antibioticson rat liver cytochromeP450 induction and formationofmetabolite complexes. A structure activity relationship. BiochemPharmacoll983; 32:2309. 6. Cummins D, Kozak PP, Jr. Gillman SA. Erythromycin effecton theophylline blood levels. Pediatrics 1977; 59:144. 7. Renton KW, Gray JD, Hung OR. Depression of theophylline eliminationby erythromycin. Clin Pharm Ther 1981; 30:422. Used. 8. Ludwig M. In:Macrolides,Chemistry,PharmacologyandClinical Bryskier AJ, Butzler J- P,Neu HC, "kens PM,eds.Oxford: AmetteBlackwell, 1993:495. 9. Hildebrandt R, Moller H, Gundert-Remy U. Influence of theophylline on renal clearance of erythromycin. Int J Clin Pharmacol Toxicol Therap 1987; 25:601. 10. Weinberger M, Hudgel D, Spector S, Chidsey C. Inhibition of theophylline clearance by troleandomycin. J AllergyClin Immunoll977; 59:228. 11. Saint-Salvi B, TremblayD, Surjis A, Lefebvre M. A study of the interaction J Antimicrob Chemoof roxithromycin with theophylline and carbamazepine. ther 1987; 20 (suppl B):121. 12. Mesdjian E, Dravet C, Cenraud B, Roger E. Carbamazepine intoxicationdue to triacetyl-oleandomycin administration in epilepticpatients. Epilepsia 1980; 21:489. 13. Wong W, Ludden TM, Bell RD. Effect of erythromycin on carbamazepine kinetics. Clin Pharmacol Ther 1983; 33:460-464.

Rubinstein and Segev 14. Dravet C, Mesdjian E, Cenraud B, Roger J. Interaction between carbamazepine and triacetyloleandomycin (Correspondence). Lancet1977; 1:810. 15. Ben-Ari J, Eistenstein B, Davidovits M, Shmueli D, Shapira Effect of erythromycin on blood cyclosporin concentrations in kidney transplant patients. Pediatrics 1988; 112:992 16. Martell R, Heinrichs D, Stiller CR, Jenner M, Keown PA, Dupre J. The effects of erythromycin in patients treated with erythromycin. Ann. Intern Med 1986; 104:660. 17. Freeman DJ, Martell R, Carruthers SG, Heinrichs D, Keown PA, Stiller Cr. Cyclosporin-erythromycin interaction in normal subjects.Br J Clin Pharmacol 1987; 23:776. 18. Gupta SK, Bakran A, Johnson RWG, Rowland M, Cyclosporin-erythromycin interaction in renal transplant patients. Br J Clin Phrmacol 1989; 27:475. 19. Lindenbaum, Rund DG, Butler W, Tse-Eng D, Saha JR. Inactivation of digoxin by the gut flora: reversalby antibiotic therapy. New Engl JMed 1981; 305789. 20. Maxwell DL,Gilmour-White SK, HallMR. Digoxin toxicity due to interaction of digoxin with erythromycin. Br Med J 1989; 198572. 21. Husserl FE. Erythromycin-warfarin interaction (Correspondence). Arch Intern Med 1983; 143:183. 22. Bachmann K, Schwartz JI, Fornet R, Frogameni A, Jaregui LE. The effect of erythromycin on the dispositionkinetics of warfarin.Pharmacology1984; 28: 171. 23. Nelson MV, Berchou RC, Kareti D, LeWitt PA. Pharmacokinetic evaluation of erythromycin and caffeine administered with bromocriptine. Clin Pharmacol Ther 1990; 47:694. 24. LaForce CF, SzeflerSJ,Miller MF, Ebling W, Brenner M. Inhibition of methyl-prednisolone elimination in the presence of erythromycin therapy. Allergy Clin Immunoll983; 72:34. Heyton A. Precipitation of acute ergotism by triacetyloleandomycin (Correspondence). Med 1969; 69:42. 26. Bartkowski RR, McDonnell "E. Inhibition of alfentanilmetabolism by erythromycin. Clin Pharmacol Ther 1989; 86:465. 27. Warot D, Bergougnan L, Lamiable D, Berlin D, Bensimon G, Danjou P, Puech N.Troleandomycin-trialam interaction in healthy volunteers:pharmacokinetic and psychometric evaluation. Eur J Clin Pharmacoll987; 32:389. 28. Ragosta M, Weihl AC, Rosenfield LE. Potentially fatal interaction between erythromycin and disopyramide.Am J Med 1989; 86:465. 29. Honig PK, Zamani K, WoosleyRL, Conner DP, Cantilena LR, Jr. Erythromycin changes terfenidine pharmacokinetics and electrocardiographic pharmacodynamics. Clin PharmacolTher 1992; 51:156. 30. Szefler SJ, Brenner M, Jusko WJ, Spector SL, Flesher KA, Ellis EF. Doseand time-related effectof troleandomycin on methylprednisolone elimination Clin Pharmacol Ther 1982; 32:166.

Macrolides, halides, and Streptogramins in Treatment of Opportunistic Infections in Immunocompromised Patients Jack S . Remington Stanford UniversitySchool of Medicine, Stanford, and Palo Alto Medical Foundation, Palo Alto, California

INTRODUCTION The purpose of thismeetingwas to reviewnewerinformation on the macrolides, azalides, and streptogramins. For this reason, we shall attempt to confine our remarks to certain newer observationson these drugs as they relate to the treatment and prevention of infections in immunocompromised individuals. Much of the available data presented at the meeting were incomplete;many of the studies are still in progress.The usefulness of the newer macrolide and azalide antibiotics has only begun to be appreciated (Table 1). Thisisreminiscent of the many antibiotics licensed by governmental drug regulatory bodies that initially were consideredto have a relatively narrow spectrumof activity. Withfurther study andneed, a far broader spectrum was often discovered that turned out to be lifesaving. Here, we refer to such antimicrobial agents as trimethopridsulfamethoxazole (a “urinary tract antibiotic”) with its extended spectrum against Pneumocystis carinii and Toxoplasmagondii, the extended useof clindamycin (a “gram-positive antibiotic”) to include not only anaerobes but also 189

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Advantages compared with erythromycin Improved bioavailability Fewer adverse effects Fewer drug interactions Improved antibacterial spectrum Activity against MAC Disadvantages compared with erythromycin No parenteral form available in the United States Cost

Toxoplasma gondii and Pneumocystis carinii. It is likely that the full antimicrobial spectrum of the newer macrolides, azalides, and streptogramins and their usefulness for treatment of opportunistic infectionsin immunocompromised patients will be evengreater than now appreciated.

LEGIONELLA Many of the newer macrolides and the azalide azithromycin have significant advantages when compared with erythromycin (see Table 1). These include improved bioavailability, fewer adverse effects, fewer drug interactions,andimprovedantibacterialspectrum. One disadvantagein the United States is that parenteral forms of clarithromycin and azithromycin are not available. Depending on the geographical locale, there may be a significant cost differential between the use of erythromycin andthe newer macrolides that must be evaluated in light ofthe features mentioned above. Because of the appreciable untoward side effects of high-dose oral and intravenous erythromycin, many physicians who treat Legionnaire’s disease frequentlynow use clarithromycinor azithromycin in preferenceto erythromycin. Although each of these drugs is active against a variety of strains of legionella, meaningful differences in clinicalor bacteriologic response and outcomein humans have not been demonstrated. number of studies have been performed to assess the in vitro activity of different macrolides on intracellular Legionella. Considerable differences in activity were obtained by different investigators, likely reflectingthe lack of standardization of the procedure. Thus, greater or lesser activity of a given macrolide or group of macrolides against intracellular Legionella should not necessarily be interpreted as a reflection of their in vivo activity or extrapolated to what might be their potential usefulness in humans. Any significant differences inthe clinical efficacy of these drugs against Legion-

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naires’ disease will only come from appropriately designed clinicalTotrials. date, almost all such trials have been retrospective. One recently published trial by Kuzman et al. (1) was a retrospective, noncomparative study in 16 patients in whom the diagnosis was serologically confirmed. Azithromycin days. was administered orallyfor a total dose of 1.5 g over a periodof All of the patients were stated to have been cured. Stuart and Yu have recently stated that because of gastrointestinal intolerance, the requirement for large fluid volume administration andthe toxicity the 4-g dose of erythromycin, the newer macrolides will likely displace erythromycin as the drugs of choice for the treatment of legionellosis (2).

PNEUMOCYSTIS CARINII The AIDS epidemic in the United States was heralded by Pneumocystis carinii pneumonia (PCP). Inthe early yearsof the epidemic, diseasedue to this organism accounted for more than 60% of AIDS-defining illnessesand occurred in80% of persons with AIDS. Prophylacticregimens,primarily trimethoprim/sulfamethoxazole (TMP/SMZ) or pentamidine have dramatically reduced the incidence of this opportunistic disease. Unfortunately, many patients suffer significant untoward effects when given TMPMSZ propylaxis, and pentamidine is less effective. Thus, there is a needfor effective and less toxic drugs for prophylaxis of thisinfectionin AIDS patients.Whether the macrolidesand azalides or streptogramins will play any role in such preventive measures remains to be shown. The activity of clarithromycin alone and in combination with sulfamethoxazole inrata model would suggestthat studies will be conducted in humansto clarify this. An interesting observationin this regard was presentedat this meeting by Crampton and colleagues In a study similar to that described below for giardiasis, these authors assessed whether clarithromycin as administered in the multinational clarithromycin prophylaxis trial described abovehadanyeffecton the occurrence ofPCP andconcluded that clarithromycin prophylaxis formycobacterium aviumcomplex (MAC) provided additional prophylaxis for PCP. It should be notedthat these patients were allowedto continue on whatever prophylaxisfor PCP their physicians considered appropriate. Whether there were differences in prophylaxis, duration of prophylaxis, dosing, and forth, during the trial was not stated. Thus,whether the remarkableandstatisticallysignificantresult between the clarithromycin recipients and placebo group (p = .021) was indeed due to clarithromycin or to some other factor(s) will hopefully be clarified whenthe results are analyzed andthe data published.

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GIARDIASIS

Giardia lamblia, the most common parasitic cause of traveler’s diarrhea (4), has been described in HIV-infected patients with diarrhea and was classically associated with the “gay bowel syndrome..” In other patients classified as immunocompromised,it is those with dysgammaglobulinemia in whom giardiasis is most closely associated with an immune defect (5). Whether diseasedue to this parasite is more severe in the immunologically impaired patient is unclear. Craft and his colleagues presented some intrigu ing data on the effect of clarithromycin on giardiasis inAIDS patients (6). In the large prospective randomized multicenter, multinational study of clarithromycin versus placebo for preventionof MAC described above, Giardia lambia wasfoundin10(2.9%) of the patients who had been randomized to the placebogroup,whereas it wasidentifiedinonly (0.9%) of patients randomized to receive clarithromycin (p = .048). Of interest isthe observation that less diarrhea was experienced bypatients in the clarithromycin group than inthe placebo group. Althoughthe author’s conclusion of their data was that clarithromycin prophylaxisfor MAC infections also prevents development of giardiasis in patients with AIDS who have CD4 cell countsof < 100 cells/pl, there was insufficient information in the abstract to allow one to determine whetherother differences, including other therapy and/or disease entities, influenced the presence or absence of giardiasis and whether all patients in the study were screened for this pathogen if they developed diarrhea.

TOXOPLASMOSIS Toxoplasma gondii, a cause of life-threatening infectionsin immunocompromised patients including those with hematologic malignancies, organ transplants (especially bone marrow and heart), and AIDS (7-g), is susceptible to the newer macrolides-roxithromycin, clarithromycin, and azithromycin. The latter twohavebeenstudiedinclinicaltrials. In 1991, Fernandez-Martin and colleagues (10) reported the first resultsof a trial in which clarithromycin was usedfor the treatment of toxoplasmic encephalitis. These investigators obtained promising results in their pilot study performed in11AIDS patients with toxoplasmic encephalitistreated with the combination pyrimethamine (75 mg/day) and clarithromycin (1 g q12h). The protocol was completed in eight patients and discontinued in five patients (2 = voluntary withdrawal,2 = deterioration of neurological condition and thrombocytopenia,1 = suspicion of liver toxicity). Sixty-twopercent of these patients had a complete and23% a partial clinical response; 15% diedby week of therapy. Of the surviving patients,27% had clarith-

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romycin discontinued because of drug toxicitythat resulted in thrombocytopenia and/or liver enzyme abnormalities. Clinical and CT scan responses at 6 weeks of treatment were 80% and 50%, respectively. l b o patients died, one of toxoplasmic encephalitis and the other of cerebral bleeding considered to becausedbypyrimethamine-inducedthrombocytopenia. lbenty-four percent of the patients experienced significant increases in liver enzymes and hearing loss was noted in 15%; 31% experienced severe hematologic abnormalities. The authors concluded that their results with clarithromycidpyrimethaminewere comparable to the results of previous separate studies inwhich pyrimethamine/sulfathiazine or pyrimethamineklindamycin were used for therapy of acute toxoplasmic encephalitis in patients with AIDS. They alsostated that the optimal doseof clarithromycin is yet to be defined. Further studies examining the efficacy of clarithromycinisyet to bedefined. Further studiesexamining the efficacy of clarithromycin have not been reported and, to the best of my knowledge, none are in progress. In another report in which onlya single case is described, there was a dramatic responseto azithromycin. The patient had toxoplasmic encephalitis, was allergic to sulfonamide and pyrimethamine, and had beentreated unsuccessfully with clindamycin and doxycycline (11). In a recent trial (ACTG 156; data and information provided by Dr. Benjamin Luft) designed to characterize the clinicalandradiologicresponse rates in the treatment of toxoplasmic encephalitis by cohort, patients were treated with azithromycin in combination with pyrimethamine as shown in Table 2. The numbers of patients in each cohort were relatively small, making analysisof the data more difficult. The data presented here are froman initial analysis. Classificationof the 32 evaluable patients was as follows: 10 (31%) responders; 3 (9%) potential responders; 7 (22%) relapse failures; 9 (28%) induction failures; 3 (9%) indeterminate. There was no significant difference in theserates by cohort. When the figures for induction and relapse failures were combined, they yielded an overall fail-

Table 2 AzithromycinPyrimethamine for Treatment of Toxoplasmic Encephalitis inAIDS (ACTG Trial 156) Drug regimen

Pyrimethamine Cohort

I I1

111

Azithromycin mg qd mg qd mg qd

25-50 mg qd mg qd mg qd

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ure rate of 73% (8/11), 43% (6/14), and50% (2/4) respectivelyfor cohorts I, 11, and 111, without a significant difference between cohort groups. The percentage of patients respondingby week 6 was 64% (7/11), 79% (11/14), and 50% (2/4) respectively for cohortsI, 11, and I11 and, again, no significant differences betweenthe cohorts were noted. Untilthe data analysis is completed, a final conclusion about the effectiveness of the azithromycin/ pyrimethamine regimen cannot be made. What is evident from this study i that azithromycin is effective (when used in combination with pyrimethamine) for treatment of some cases of toxoplasmic encephalitis in AIDS patients.

CRYPTOSPORIDIUM

Cryptosporidia, a protozoan parasite, may cause severe and persistent diarrhea in immunologically impaired patients including the neutropenic patient, bonemarrowandrenaltransplantrecipients,thosewith certain immunoglobulin deficiencystates, and those with AIDS (12-14). Severe, unremitting infectionof the gall bladder and gastrointestinal tract occurs in such settings. Unfortunately, no single therapeutic regimen has been described that dependably eradicated cryptosporidia fromthe intestine and gall bladder in sufficient numbers of patients to allow for afirm recommendation regarding optimal specifictreatment for the parasite. In a trialof roxithromycin, mg orally bidfor 4 consecutive weeks, for therapy of AIDS-related diarrhea caused by cryptosporidium, Sprinz reported his initial results in 21 patients in Port0 Alegre, Brazil (15). In 11 (52.4%), remission in symptoms was complete and in 6 (28.6%), the response was partial with a decrease in numbers of diarrheic evacuationsfor 1 month of follow-up;4(19%)failed to respond to the treatment. This investigator concluded that roxithromycin treatment resulted in an overall favorable outcomein more than 75%of their cases andthat the antibiotic was relatively safe and well tolerated. Their study is ongoing. In a similar study from Sao Paulo, Brazil, Ulp and his colleagues (16) studied 22 adults with AIDS who were infected with cryptosporidia; 17 had significant diarrhea. The treatment regimenwas the same as described above forthe study by Sprinz. In this study,the investigators stated that good results [absence of cryptosporidium on fecal examination or colonoscopy after the end of treatment (50%), or improvement defined as persistence of cryptosporidium inthe feces but an increase in weight of the patient and/or a decrease in number of evacuations/day (27%)] with roxithromycin were obtained in 72.7% of their patients and that the drug was well tolerated. Amsden and Bessette from Worcester, Massachusetts described their results with azithromycin in the treatment of an HIV-negativepatient with

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severe diarrhea caused by cryptosporidium (17). This case,in an otherwise normal individual, andthe therapy used appear relevant to theproblem of cryptosporidiosis in immunocompromised patients. The patient was a 42year-old male whose diarrhea continued despite treatment with loperamide and by the time of hospitalization for severe diarrhea and dehydration; he had lost 24 pounds. He was placed on azithromycin 1250 mg/day by mouth and within 48h he had resolution ofhis diarrhea and subsequently was asymptomatic. He was treated for 14 days and his stools became negative for cryptosporidia.The investigators feltthat they had ruledout spontaneousresolutionas a cause of the excellentresults. Further studies of azithromycin seem warranted for this disease inAIDS patients and other immunocompromised patients as well as in otherwise normal individuals. One wonders why further studies as follow-up to the promising results of Soave and her colleagues (18) have not been published to clarify where this drug belongs inour armamenarium fortreatment of this disease.A trial of intravenous azithromycin is presently ongoing and hopefully will answer the question as to whether this antimicrobial willeradicate cryptosporidia from extraintestinal sites such as the biliarytractandtherebyprevent recurrence. MYCOBACTERIUM AVIUMCOMPLEX The mycobacterium avium complex (MAC) is the most common cause of systemic bacterial infection inAIDS patients in the United States, where the lifetime incidenceof this disease ranges from18% to 43%. Individuals at greatest risk for disseminated disease are those with CD4+ cell counts of less than75 cellslpl andwho have hada prior AIDS-defining opportunistic infection. The portals of entry appear to be both the gastrointestinal and respiratory tracts. Disseminated MAC predisposes to earlier death than occurs in AIDS patients without this disseminated infection (19). Because of its frequency andthe severe debility it causes, it has been a prime focus for drug discovery for both prevention and treatment. Optimally, it would be ideal were a single drug, or even two drugs, identified that would prevent multiple opportunistic infections in AIDS patients. Becauseof their broad antimicrobial spectrum,that includes both bacterial and protozoal pathogens, the newer macrolide/azalide drugs have the potential for preventing a number of such infections. Inthe past several years, giant steps have been made in both prevention and treatment of MAC infections in AIDS patients. These advances have extended to and benefit non-AIDS patients who acquire this infection and for whom we previously had littleto offer inthe way of effective treatment. A number of interesting studies and observations were presented at this meeting.

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Prophylaxis Chrithromycin In a recent prospective, randomized, double-blind, multicenter, multinational,placebo-controlledtrial(M91-561; Abbott) in682 patients of clarithromycin for prophylaxis of disseminated MAC infection in AIDS patients with CD4 countsof < 100 cells/mm3, this macrolide was foundto provide effective and safe prophylaxis andwas associated with longer survival than observed in untreated patients (data provided by Dr. J Carl Craft, Abbott Laboratories). There were 341 patients in each arm of the study. Clarithromycin was given at a dose of 500 mg PO bid. The overall beneficial effectswereclassifiedas either bacteriologic or clinical. The primary bacteriologic benefit was a 69% reduction inofrisk developing MAC infection. The primary clinical benefit was improved survival. A significantly higher proportionof clarithromycin patients than placebo patients reported one or more adverse events; these were attributable to higher rates of digestive events andtaste perversion. Patients inthe clarithromycin group had a statistically significant reduction in mortality of 28%, compared to the plaof MACprophycebo group(p C .OS). This is the first well-controlled study laxis to reveal such a significant survival benefit. Of interest, patients in the clarithromycin arm experienced a statistically significant(p < .05) reduced riskof hospitalization and of developing symptoms associated with MAC bacteremia. Risk of hospitalization was reduced by 23% and the risk of bacteremia accompanied by weight loss, pyrexia, night sweats, and anemia were each reduced by more than 80% when comparedto the placebo group. Greater than 50% of MAC strains isolated from the clarithromycin recipients with breakthrough bacteremia were resistantto clarithromycin, whereas noneof the MAC isolates fromthe placebo recipients were resistant to clarithromycin might have been due to a previously existing (prior to clarithromycin prophylaxis) active (without bacteremia) MAC infection in these patients. In regard to breakthrough bacteremias, it is interestingto note that when such breakthroughs have occurredin studies in which patients were receiving rifabutin, the breakthrough usually involved rifabutin-susceptible strains,whereasinthosereceivingclarithromycin it usuallywaswith clarithromycin-resistantstrains. Azithromycin Following the report of promising results with azithromycin treatment of MAC infection in patients with AIDS by Young and his colleagues from San Francisco, California (20),there has been increased interest infurther

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evaluation of this drug for both prevention andtreatment of this infection in AIDS patients. In two recent randomized, double-blind comparative trials, azithromycin, 1200 mg once weekly, was evaluated to determine its role in prevention of disseminated MAC inAIDS patients withCD4 counts of < 100 cells/mm3 (see Ref. 21 and data provided by Dr. Michael Dunne, Pfizer Central Research). Azithromycin alone was compared with rifabutin alone or with azithromycin in combination with rifabutin.The mean duration of therapy was 11months. The incidence of disseminated MAC inthe 3 groupsof patients in an intent-to-treat analysis was: azithromycin alone, 13.9%; rifabutin alone, 23.3%; azithromycin plus rifabutin, 8.3%. The difference betweenthe azithromycin and rifabutin groups was significant (p = 0.11). The percentages of side effects in the three groups of patients at any time were azithromycin 78.1% rifabutin 59.7%; and azithromycin plus rifabutin 83.5%. Side effects were primarily (approximately 60%) diarrhea, mostly mild to moderate. In approximately 5% of patients, the diarrhea was stated to have been severe. The percentage of discontinuations were azithromycin 13.5%, rifabutin 15.9%, and azithromycin plus rifabutin 22.7%. In the second study, azithromycin was evaluated against placebo in 180 HIV-infected patients with CD-4 counts of < 100 cells/mm3.The mean duration of therapy was 1year. The incidence of disseminated MAC inthe two groups of patients was azithromycin 15.3% and placebo 30.3% (p = The percentage of side effects were azithromycin 79.8% and placebo 31.9%; of discontinuations, azithromycin 8.2% and placebo 2.3%.

Treatment Chrithromycin

Chaisson and his colleagues (22) presented data obtained in a controlled trial of clarithromycin plus ethambutol with or without clofazamine for treatment of MAC bacteremia in AIDS patients. The doses given were clarithromycin 500 mg bid orally and ethambutol 15 mg/kg/day with randomization to receive either clofazimine 100 mg/day or no clofazimine.The median time to obtaining negative cultures was 58 days for the two-drug regimen and 63 days the for three-drug regimen.The proportion of patients that became culture negativewas in the two-drug group and 54% in the three-drug group. Fever and night sweats improvedin approximately the same number of patients in each group, whereas the proportion of patients that had to be withdrawn from therapy was higher in the threedrug group (22% versus 13% in the two-drug group). The authors concluded that clarithromycin is effective in treating MAC bacteremia, that it prevents emergenceof resistance, andthat the addition of clofazimine does

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not contribute to the clinical response [it was actually associated with a higher mortality (p = .03)]. In another study, Craft and his colleagues (23) reported that the addition of ethambutol or ethambutol plus clofazimine to clarithromycin significantly reduced developmentof clarithromycin-resistantMAC in patientsbeing treated for disseminatedMACinfection. The dosages of clarithromycin, ethambutol, and clofazimine were essentially the same as in the study reported by Chaisson et al. In another study by Craft and his colleagues (24), clarithromycin-resistant MAC was noted during prophylaxis only in patients with CD4 counts of 25 cells/mm3and CD8 counts of 780 cells/mm3. The investigators stated that their results suggest that clarithromycin-resistant MAC reflect treatment failures in patients who had disseminated nonbacteremic MAC infections at the start of prophylaxis. The latter seems likely. Of potential importance to the non-AIDS immunocompromised patient with MAC isthe report at this meeting by Wallaceand his colleagues from the University of Texas Health Center in Qler, Texas (25). These investigators employed clarithromycin in treatment regimens for pulmonary infections caused by MAC. Non-AIDS patients were treated until culture negative for 1 year with clarithromycin, 500 mg bid (as initial monotherapy in some of them), ethambutol, rifabutin or rifampin and, initially, with streptomycin.In 6 of 39 patients, the organism became resistant to clarithromycin, most likely related to thefact that they had received clarithromycin as monotherapy. None of the 25 patients who completed their therapy relapsed during a 19-month period of follow-up. Despite reduced levels of clarithromycin serum levels caused by their rifampin, of 13 patients that received these two drugs together in their treatment regimens were treated successfully. The authors concluded that the clarithromycin-containingtreatment regimen is superior to those in which clarithromycinwas not included. patients were randomized In a studyby Dube and his colleagues to receive clarithromycin1gm bid plusclofazamine withor without ethambutol inan attempt to prevent microbiologic relapse in AIDS patients with MAC bacteremia.The two groupsof patients were generally comparable. The response rate was similar in both groups. The risk of relapse was remarkably and significantly greater in those patients who were receiving the two-drug regimen. Thus, the three-drug regimen of clarithromycin/ clofazamine/ethambutol wasmoreeffective than the two-drugregimen clarithromycin/clofazamine in preventing microbiologic relapse in AIDS patients with MAC bacteremia. Pierce et al. demonstrated in a prospective trial that MAC was a predictor of mortality. In the multicenter prophylaxis trial mentioned above, the data support their hypothesis that

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Table 3 Manifestations of Bartonella (Rochalimaea) Infection in Immunocompromised Patients

Peliosis hepatitis Bacillary angiomatosis Endocarditis Chronic lymphadenopathy Meningitis

the favorable effect of clarithromycin on survival in advanced AIDS was due primarily to prevention of MAC.

BARTONELLA Bartonella species are fastidious gram-negative bacilli that recently have been recognized asthe cause of significant and often life-threatening infections inimmunocompromisedpatients.Certainmanifestations of these infections are shown in Table 3. Macrolide antibiotics, includingerythromycin,clarithromycin,azithromycin,roxithromycin,anddirithromycin, have been shown to have excellent in vitro activity against B. visionii, B. hemelae, and B. quinfana (Table 4) (28,29). A number of antibiotics have been used successfully to treat various manifestationsof bartonellosis, including erythromycin and doxycycline, with resulting resolution of skin lesions and improvementin constitutional symptoms(30). Recently, Guerra and colleagues reported a case of AIDS-related bacillary angiomatosis ina 28-year-old male witha CD4 count of 100/mm3 who presented with fever, hepatomegaly, weight loss, and multiple polypoid, angiomatous lesionson his facethat were found on biopsyto be due to bacillary angiomatosis.He was treated with oral azithromycin, 1g daily as a single dose, and experienced rapid resolutionof skin lesions. After 1 Table 4 MICs (pg/ml) for 14 Strains of Bartonella

Erythromycin Clarithromycin Azithromycin

0.06-0.12 0.003-0.03 0.006-0.03

Note: Similar M 0 I for B. visionii, B. henselae, B. quintana. Source: Maurin et al. (1995).

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week of therapy, there was adiminution in size of his liverand spleen noted on computed tomography with resolution of the attenuation lesions in these organs. At 10-month follow-up, there was no trace of skin lesions suggestive of bacillary angiomatosis and neither liver nor spleen were pal ble. Blood cultures remained negative. At this meeting, Paucar and his colleagues reported a case inwhich there was coexistence of bacillary angiomatosis and Kaposi’s sarcoma in a black African woman who was treated successfully forher Bartonella infection with azithromycin, mg/ day for days and, thereafter, clarithromycin (500 mg/day) was administered for months. The in vitro results along with recent clinical experienc in limited numbers of cases suggestthat these new macrolides hold promise for treatment of this infectionin immunocompromised patients.

COMMUNITY-ACQUIRED PNEUMONIA

As is true for immunologically competent patients, those who are immunologically compromisedare susceptibleto organismsthat arecommon causes of community-acquired pneumonia. Of particular interest in this regard is that the macrolides and azalides are active against five of the common pathogens (Table Thus, althoughthese organisms do not always have a particular predilection for the immunocompromisedpatient, those individuals whoare not severely immunocompromised ill will likely do well when treated with the oral preparations of these antimicrobials. The more severely ill immunocompromised patients with community-acquired pneumonia will likely require hospitalization and intravenous antibiotic therapy. Recently completed is a multicenter trial of erythromycin plus cefuroxime versus azithromycin alone (500mg IV for days followed by 500 mg PO for days for a total duration of azithromycin of days) in community-acquired pneumonia. This five-center trial in the United States has foundthat azithromycinwas as efficaciousas erythromycidcefuroxime. Furthermore, azithromycin was better tolerated than erythromycin. (Personal communication from Dr. Victor Yu, Table MacrolidedAzalides are Active Against Five Common Pathogensof Community-Acquired Pneumonia

S. pneumoniae H . infiuenzae M . pneumoniae C. pneumoniae L. pneumophila

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VANCOMYCIN-RESISTANT E . FAECZUM The streptogramin, quinupristiddalfopriistin is being used in multiple trials for treatment of gram-positive infections, including those caused by vancomycin-resistant E. faecium. Undoubtedly, this latter organism will prove a significant problem in immunocompromised patients. W Osuch cases of life-threatening infectionsin the setting of severe immunosuppression, one in a patient with chronic renal failureon peritoneal dialysis who developed vancomycin-resistant E. faecium peritonitis and the other in a severely neutropenic patient with lymphoma who was receiving chemotherapy were seenat the consulting service at Stanford. The latter patient was a 44-year-old female with large cell lymphoma and recentCHAD therapy. On November 6,1995, she developed fever in a setting of severe neutropenia. Blood culturesgrew E. faecium sensitive to chloramphenicol, without high-level resistance to streptomycin but with resistance to penicillidampicillin, vancomycin, gentamicin, ciprofloxacin, tetracycline, andtrimethopridsulfamethoxazole.It had intermediate sensitivity to rifampin. On November 10, chloramphenicol was begun for the vancomycin-resistantE. faecium isolated from multiple blood cultures taken fromNovember6 to November10. On November 11, quinupristid dalfopristin 7.5 mgq8hwas begun. All subsequent blood cultures were negative. It cannot be stated with assurance that quinupristiddalfopriistin resulted in the negativebloodculturessinceshehadbeenplaced on chloramphenicol the daybefore. Quinupristiddalfopristin hadbeenrequested on a compassionate plea basis because the patient’s infection was life-threatening and in a situation in which chloramphenicol has not been proven in sufficient numbers of cases to be effective when usedalone.

ACKNOWLEDGMENT This work was supported by National Institutes of Public Health Services Grants A130230 and AI04717.

REFERENCES 1. Kuzman I, Soldo I, Schonwald S, Culig J. Azithromycin for treatment of community acquired pneumonia cased by Legionella pneumophila: A retrospective study. Scand J Infect Dis 1995; 27503-505. 2. Stuart JE, Yu, VL. Current concepts: Legionellosis. N Engl J Med 1996; Cramptom S, Craft JC, Notario G , Henry D. Prevention of Pneumocytstis carinii pneumonia in A I D S patients by clarithromycin prophylaxisfor Mycobacterium avium complex (MAC).The Third International Conference on the Macrolides, Azalides and Streptogramins, Lisbon,1996; abstr. 7.09.

Remington 4. Connolly GM, Forbes A, Gazzard BG. Investigation of seeminglypathogennegative diarrhoea in patients infected with HIV-1. Gut 1990; 31:886-889. Ament ME, Ochs HD, Davis SD. Structure and function of gastrointestinal tract in primary immunodeficiency syndrome: A study of 39 patients. Medicine 1973; 52:227-248. 6. Craft JC, Henry D. Giardia lamblia prevention during MAC prophylaxis with clarithromycin 500 mg BID in AIDS patients with CD4 counts < 100 cells/ mm3. The Third International Conference on the Macrolides, halides and Streptogramins, Lisbon, 1996: abstr 7.28. 7. Israelski DM, Remington JS. Toxoplasmosis in the non-AIDS immunocompromised host. In: Current Clinical Topics in Infectious Diseases, Vol.13. Remington JS and SwartzMN, eds. Boston: Blackwell Scientific Publications, 1993~322-256. 8. Israelski DM, Remington JS. Toxoplasmosisin patients with cancer. Clin Infect Dis 1993; 17:S423-S435. 9. Wong SY, Remington JS.Toxoplasmosis in the setting of AIDS In: Broder, S, Merigan TC, Bolognesi D, eds.Textbook of AIDS Medicine. Baltimore: Williams and Wilkins,1994:223-257. 10. Fernandez-Martin J, Leport C, Morlat P, Meyohas MC, Chauvin JP, Vilde JL. Pyrimethamine-clarithromycin combination for therapy of acute Toxoplasma encephalitisin patients with AIDS.AntimicrobAgents Chemother 1991; 35~2049-2052. 11. Farthing C, Rendel M, Cume B, SeidlinM,Azithromycin for cerebral toxoplasmosis. Lancet 1992; 339:437-438. 12. Current WL, Reese NC, Ernst J V , et al. Human cryptosporidiosis in irnmunocompetent and immunodeficient persons: Studiesof an outbreak and experimental transmission. N Engl J Med 1983; 308:1252-1257. 13. DuPont HL. Cryptosporidiosis and the healthy host. N Engl J Med1985; 312:1312-1320. 14. Weisburger W R , Hutcheon DF, Yardley JH, et al. Cryptosporidiosis in an immunosuppressed renal-transplant recipientwith IgA deficiency. J Clin Patholl979; 72:473-478. 15. Sprinz E, AIDS-related cryptosporidiumdiarrhea: A pilot study with roxithromycin. The Third International Conference on the Macrolides, Azalides and Streptogramins, Lisbon, 1996; abstr 7.26. 16. Ulp DE, Lima, ALLM Amato, VS, Neto, VA. The use of roxithromycin in the diarrhea caused by cryptosporidium sp associated with A I D S . The Third International Conference on the Macrolides, Azalides and Streptogramins, Lisbon, 1996; abstr 7.27. 17. Amsden GW, Bessette RE, Treatment of non-HIV cryptosporidial diarrhea with azithromycin. The Third International Conference on the Macrolides, Azalides and Streptogramins, Lisbon,1996; abstr 8.12. 18. Soave R, Havlir D, Lancaster D, Joseph P, Leedom J, Clough W, Geisler Dunne M. Azithromycin (U) therapy of AIDS-related cryptosporidial diarrhea (CD): A multi-center, placebo-controlled, double blind study. Program

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and Abstracts of the 33rd Interscience Conferenceon Antimicrobial Agents and Chemotherapy, American Societyfor Microbiology, New Orleans, abstr Benson C. Disseminated Mycobacterium avium complex disease in patients with AIDS. AIDS Res Hum Retroviruses Young LS, Wiviott L, Wu M, KolonoskiP, Bolan R, Inderlied CB. Azithromycin for treatment of Mycobacterium aviumintracellulare complex infection in patients with AIDS. Lancet 1991; 338:1107-1109. Havlir DV, McCutchan JA, Bozzette SA, Dunne M, CCTG/MOPPS Study Investigators. University of California, San Diego, and Pfizer Center Research, Groton, CT. A double-blind randomized studyof weekly azithromycin, daily rifabutin, and combination azithromycin and rifabutinfor the prevention of Mycobacterim aviumcomplex (MAC) inAIDS patients. The Third International Conference on the Macrolides, Azalides and Streptogramins, Lisbon, abstr Chaisson R E , Keiser P, Pierce M, Fessel WJ, Ruskin J, Lahart C, Meek K. Controlled trial of clarithromycidethambutolwith or without clofaziminefor the treatment of mycobacterium avium complex bacteremia Ain I D S patients. The Third International Conference on the Macrolides, Azalidesand Streptogramins, Lisbon, abstr Craft JC, Siepman N, Notario G, Gupta S. The addition of ethambutol with or without clofazimineto clarithromycin for disseminated Mycobacterium avium complex (MAC) treatment. The Third International Conference on the Macrolides, Azalides and Streptogramins, Lisbon abstr Craft JC, Henry D, Olson CA, Notario G, Hom R, Crampton S, Pierce M. Prevention of resistance (R) to clarithromycin (C) during prophylaxis for disseminated Mycobacterium aviumcomplex (MAC).The Third International Conference on the Macrolides, Azalides and Streptogramins, Lisbon, abstr 25. Wallace RJ Jr, Brown BA, Griffith DE, Girard W, Murphy D. Clarithromycin (CLARI) regimens for pulmonary Mycobacteriumavium-intracellulare (MAI) in non-AIDS: longterm followup of the first 50 patients. The Third International Conference on the Macrolides, Azalides and Streptogramins, Lisbon, abstr Dub6 M, Sattler F, Tomani F, See D, Havlir D, Kemper C, Dezfuli M, Bozzette S , Bartok A, Leedom J, TillesJ, McCutchan J. Clarithromycin plus clofazimine withor without ethambutol for the prevention of relapse of MAC bacteremia inAIDS. The Third International Conference on the Macrolides, Azalides and Streptogramins, Lisbon, abstr Pierce M, CramptonS , Henry D, Craft C, Notario G. The effect of MAC and its prevention on survival inpatients with advanced HIV infection. The Third International Conference on the Macrolides, Azalides and Streptogramins, Lisbon, abstr Maurin M, Raoult D. Antimicrobial susceptibilityof Rochalimaea quintana, Rochalimaea vinsonii, and the newly recognized Rochalimaea henselae. Br SOC Antimicrobi Chemother

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29.Ives TJ, Regenery R L , ManzewitschKebedeM. In vitro evaluation of macrolide antibiotics against bartonela (rochafimaea)hemselae, B. quintana and B. elizabethae via immunofluorescent antibody testing. The Third International Conference on the Macrolides, halides and Streptogramins, Lisbon, 1996; abstr 30. Guerra LG, Neira C Y , Boman D, H0 H, Casner PR, Zuckerman M, Verghese A. Rapid response of AIDS-related bacillary angiomatosis to azithromycin. Clin Infect Dis 1993; 17:264-266. 31. Paucar D, Kabeya K, HermansP, Van Laethem Y,Clumeck N. Coexistence of bacilaryangiomatosis (BA) and kaposi's sarcoma (KS) in black African woman: First description and treatment with macrolides. The Third International Conference on the Macrolides, halides and Streptogramins, Lisbon, 1996; abstr 7.25.

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15 Legionella and Spirochetes I. M. Hoepelman University Hospital Utrecht, The Netherlands

Daniel M. Musher Baylor College of Medicine and Veterans’ Affairs Medical Center Houston, Texas

LEGIONELLA Depending on the seasonandlocation, about 2-10% of communityacquired pneumonias (CAPS)are caused by Legionella spp. Most cases of Legionnaires’ diseaseare caused by Legionella pneumophila, although infections by other species are well documented. Legionella spp. belong to the group of supposedly “atypical” organisms, together with Chlamydia pneumoniae, Coxiella burnetti, and Mycoplasma pneumoniae. The formerly sharp distinction between typical pneumonia (usually caused by S. pneumoniae and said to be characterized by an abrupt onset, high fever, chest pain, tachycardia, and consolidation, together with the production of purulent sputum) and atypical pneumonia, caused by a different set of microorganisms and causing a different clinical picture (vide infra) is probably of historical interest. Recognitionof substantial clinical overlap,better diagnostic tools,the adoption of broad-spectrum regimensfor empiric cov207

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erage of CAP, and the introduction of newer antimicrobial agents that are equally effective against typical and atypical pathogens (1) have all ledto the blurring of this distinction.

Clinical Picture The clinical syndromeof Legionnaires’ diseaseis characterized by a nonproductive cough, pulse-temperature dissociation, abnormal liver function tests, rise in serum creatinine, diarrhea, hyponatremia, hypophosphatemia,myalgia,confusion,andrespiratoryfailure(2,3).However,ithas been shown that none of these signs, symptoms,or laboratory abnormalities is characteristic for legionellosis (2). In several prospective andretrospective comparative studies, chest roentgenographic findings have been found to benonspecific(3).Moreover,oneshouldnotforget that extrapulmonary forms (without initial radiology abnormalities) also and exist may present as peritonitis, pericarditis, and endocarditis.

Laboratory Diagnosis Laboratory diagnosis of Legionnaires’ disease may be done by antibody detection with a sensitivity of 75% and specificity of 95-99%, provided samples are taken during the acute phase and 6-9 weeks later (3). However, this is not usually helpful in caring for patients. Moreover, seroconversion typically occurs with type1L. pneumophila. Detection of L.pneumophila antigenuria is a convenient and sensitive method of diagnosing the disease, although at present this test only detects disease caused by serogroup I (which causes 70-90% of all cases). Direct fluorescent antibody (DFA) testing is an excellent method for the detection of L. pneumophila in sputum and tissue fluids, but the laboratory should have experience with this technique andthe monoclonal antibodies have a short shelf life. DNA probes and polymerase chain reaction (PCR) are currently in their developmental stages. Culture remainsthe gold standard; special media and selective techniquesare needed for optimal yields.

Therapy

Currently, the only FDA-approved therapy for Legionnaires’ disease is erythromycin 500mg-1 gqid, with or without rifampin 600 mg orally or intravenously bid, for 2-3 weeks. However, one should realize that’noprospective, controlled clinical trials of Legionnaires’ disease exist, and all available treatment information is based on retrospective analyses and clinical anecdotes (4). On average, the fatality rates associatedwith erythromycintreated or tetracycline-treatedLegionnaires’disease are about twofold

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lower than those associated with nonerythromycin-treated Legionnaires’ disease (4). In a recent study(5) of severe community-acquired Legionella pneumonia, patients (30%) who received erythromycin died compared to 5 of (33%) who received rifampin in addition. Fourpatients received no antibiotics and survived. This study highlights the difficulty in treating severe Legionella pneumonia in which mortality rates can approach 3342% for patients who require intensive supportive therapy (4), as well as difficulty in evaluating efficacyof new therapies. Antimicrobial therapy for infections caused by Legionella should be dictated by what is known from retrospective clinicaldata, in vitro susceptibility testing (taking into account the facultative intracellularnature of this agent), and animal studies (using intratracheal challenge). Extracellular susceptibility testing of L. pneumophila shows that macrolides, azalides, streptogramins, fluoroquinolones, and p-lactam antibiotics are all highly effective (6). However, neither direct minimal inhibitory concentrations (MICs)nortime-killcurveassaysaccuratelypredictintracellular L. pneumophilu susceptibility In vitro methodsusing intracellular models show that the newfluoroquinolonesandrifampin are highly effective, generally somewhat more active than the new macrolides and azalides and more activethan erythromycin (6-8). Some data suggest that azithromycin andciprofloxacin are bactericidalintracellularly,whereaserythromycin and clarithromycin are bacteriostatic (3’7). Regarding the newer macrolides and azalides, animal studies on experimental Legionella pneumonia show that clarithromycin and azithromycinare consistently more active than erythromycin (9). Josamycin, tested in animal models, is almost as active as erythromycin(8). already stated, the only drug currently approved for Legionnaires’ disease is erythromycin. In patients using immunosuppressive drugs, in other patients with impaired host defenses(HIV), and patients developing ARDS, because of mortality ratesthat approach 50% even withtreatment, it is advised to add rifampicin 600 mg bid. Animal studies show that the addition of rifampicin reduces lung bacterial cell counts and diminishes lung damage (9). In one preliminary report, spiramycin was studied in patients who required intensive care admission and the mortality was 30%. The compares favorably to data from patients with severe Legionnaires’ disease treated with erythromycin(4).

Role of the Newer Macrolides and halides Clinical data on clarithromycin as therapyfor Legionella pneumonia have been presented in four studies [Parolaet al. (lo)] in a total of 79 patients (Table 1). Overall cure rates were excellent (approaching loo%), indicative

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; m

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Legionella Table 2 Clinical Studies with Azithromycin

Author

criteria Entry

Myburgh et al. Kuzman et al.

CAP Retrospective

Dorrell et al.

CAP -failure Erythromycin/ rifampin CAP

Rizzato et al.

Dose Outcome study mg days mg days (5) mg 5d mg (iv) mg5d 500 mg

3d

Total no. in

66

-

818

4/4

of the fact that probably only mild cases of Legionnaires’ disease were treated. However,given the fact that in one of these studies, (U), 15 patients had failed previous therapy, this suggeststhat clarithromycin can be safely prescribed in patients with Legionnaires’ disease. The recommended therapy is250-500 mg bid for at least days. Moreover, Abbott has data on file on an additional 50 patients (84% previously treated with other antibiotics) in whom a 100% clinical cure rate was seen. (J.C. Craft, personal communication). An even smaller numberof patients have beentreated with azithromycin (12) with similarly good results. The duration of therapy ranged from to 5 days (12-15). It is known that this drug has an extremely long half-life and accumulates intracellularly in the phagocyte, where L . pneumophila is located (16). In acute exacerbations of chronic bronchitis and community-acquired pneumonia, days of therapy with this azalide has given excellent results, comparableto p-lactam antibiotics (16). Currently, neither azithromycin nor clarithromycin is available for intravenous administration. Based on intracellular susceptibility testing, animal models, and clinical studies, we believe that either of these drugs can safely replace erythromycin orally, which must be administered four times daily and isnot well tolerated. Clarithromycin shouldbe given for at least days, 250-500 mg twice daily (the highest dose we personally favor). Azithromycin, when prescribed at 500 mg daily for 10 days, is certainly as effective as clarithromycin, and therapy forshorter a period is likelyto be effective. Azithromycin showsno capacity to induce or inhibit cytochrome P-450 enzymes. This may be an advantage in immunocompromised patients treated in cyclosporineor methylprednisolone and in patients also receiving theophylline and digoxin. A compassionate use program for the IV formulation existsfor both clarithromycin and azithromycin.

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At ICMAS 111, six posters were presented on Legionella spp. Segreti et al. (abstract 1.01) showed that in their fibroblast MBC model clarithromycin was more effective than azithromycin or erythromycin. Martin et al. (abstract 1.02) showed in their extracellular MIC model that 14-hydroxy clarithromycin was additive to its mother compound; however, we have already discussedthe value of extracellular susceptibility testing. Pendland et al. (abstract 1.03) showedthat in the vitro activity of the streptogramin RPR 106972 againstLegionella spp. is similar to that of erythromycin and ciprofloxacin, and the same authors (abstract 1.04) showed in their extracellularmodel that RPR 106973ismorerapidlybactericidal than erythromycin at 2 m MIC. The study of Kuzman (abstract 1.05) was recently published (14). Additionaldata on the efficacy of clarithromycin in Legionnaires’ disease came from Parola et a1 (abstract 1.06), who treated 20 patients with proven Legionnaires’ disease(out of 200 with CAP) with this drug 500 mg bid for IV days and continuedtreatment orally for 10-15 days; all patients were cured (12). Every investigator who treats patients with community-acquired pneumonia with azithromycin and finds casesof Legionnaires’ disease is urged to report these in the literature, especially if failures occur, because this can guide expertson the dose and durationof optimal therapy.In severe cases, combination with rifampin remains advisable. Fluoroquinolones may become the drugs of choice for these severe cases, as it has been shown that they are very active in animal models and in patients, some of whom were . severely ill or immunocompromised. Exceptionally high cure rates with this class of drugs have been reported.

DISEASES CAUSED BY SPIROCHETES Erythema Chronicum Migrans Erythema chronicum migrans (ECM)the is most important skin manifestation ofLyme disease. This tick-borne disease (the most important one, accounting for over 90% of such diseasesin the United States) is caused by the spirochete Borrelia burgdorferi. Lyme disease is a systemic infection which particularly targets the skin,. joints, heart, eyes, and the nervous system. In 1994, over 13,000 Lyme cases werereported to theCenters for Disease Control, although the diagnosis may bequestioned in someproportion of these. As with syphilis, Lyme borreliosis presents in stages, all requiring different treatment regimens. The primary lesion occurs at the site of the tick bite, 3-14 days after the feed. It begins as a macule or papule that expands to become an annular lesion witha raised, red border and central

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clearing called ECM. It slowly expands, the center may become necrotic and new adjacent rings may form. Inthe untreated patient, the skin lesion usually disappears over a period of weeks. Thereafter, a period of spirochetemia associated with constitutional symptoms occurs before the viable spirochetes settle invariousorgansystems,leading to immunologically mediated damage. The second stage consists of neurologic and cardiac abnormalities, andarthritis marks the third stage. Treatment of ECM is aimed at abolishing early disease and preventing late-stage disease. Therefore, therapeutic studies should always use the development of late-stage disease as part of the evaluation criteria.There is no current consensus on the optimal antibiotic or duration of treatment, and theissue of whetherB. burgdorferi causes intracellular infection in vivo is unresolved. In general, oral doxycycline or amoxicillin have been used for 2-3 weeks to treat ECM (Table 3), whereas intravenous ceftriaxone is recommended for neurological,arthritic, and cardiac disease. The in vitro activityof the new macrolides, azithromycin, clarithromycin, and roxithromycin was compared in different studies. Tests using at least 10 clinical isolates yield M I G s of 0.06 pg/ml for erythromycin,0.015 pg/ml for azithromycin and clarithromycin and0.03 pg/ml for roxithromycin (17), comparing favorably with MIC90s for ceftriaxone 0.06 pg/ml, tetracycline 0.5 pg/ml, and ampicillin 0.25 pg/ml(l8). Although azithromycin wasmore potent than other macrolides in some experimental infections (19), other studies in mice, using an arthritis model, show that p-lactam antibiotics effectively eliminate the disease, whereas macrolides do not (19). Studies in humans show conflicting results. Disappointing results have been seenwith roxithromycin (20) and azithromycin (21) in comparison to penicillin. However,Strle et al. (22) treated ECM with either azithromycin (3 gin 5 days) or doxycycline 100 mg bid for 14 days, and a study by Weber et al. comparing with penicillin V (23) yielded favorable results with azithromycin. A comparative studyby the group of Steere also found comparaTable 3 Therapy of Lyme Borreliosis ~~

Clinical (stage) Early (stage ECM

1)

Tetracycline

10-21 qid PO mg 250 10-21 bid PO mg 100 500 10-21 tid PO mg Amoxicillin plus Probenicid 10-21 tid PO mg mg 250 PO10-21 qid Erythromycin Cefuroxime 21 bid PO 500 mg

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ble results among azithromycin (1.5 g), amoxicillidprobenicid, and doxycycline for the treatment of early Lyme disease (24). However, patients with Lyme arthritis treated with oral antibiotics (doxycyclineor amoxicillin plus probenicid)may still develop neuroborreliosis (25). At the third ICMAS, five posters were presented on Lyme disease. One poster (abstract 1.07) from Bryskieret al. showed that roxithromycin and cyclines (e.g., tetracycline) displayed synergistic activity. Another study from Gasser (abstract 1.08) showed that MICs for Borrelia spp. increase when the temperature declines from38°C to 30°C, a finding which may be important in ECM. Strle et al. (abstract 1.09) presented follow-up data from their previousstudycomparingazithromycin (AZI) 3gin 5dayswith doxycycline (DOX) 100 mg bidfor 14 days (22). During the follow-up of 12 months, one patient in each group developed majorlate manifestations of Lymeborreliosis. In 10patients(16.4%) treated with AZI and in 11 (23.4%) receiving DOX, minor manifestations were observed. GoriSeket al. (abstract 1.11) treated 30 adults with ECM withAZI 1g on day 1and mg on days 2-5. Of 25patients studied,only two were documented late complications. deMarco (abstract 1.12) studied a cohort of 89patients in a retrospective fashion to determine the effectiveness of clarithromycin, in late Lyme disease.lbenty-eight were given CL 250 mg bid for 8 weeks, 37 were given CL 500 mg for 8 weeks, and 24 were given CL 250 mg or qd500 mg in combination with a cephalosporin. In the lower-dose group 71% (20/28) improved, comparedto 78% inthe high-dose group (29/37), whereas the combination group displayed slightly less improvement. However, these alldata should be considered premature, because in our opinion more long-term follow-up data are needed to judge efficacy of a regimen in Lyme (ECM).

Syphilis It isespeciallydifficult to interpret the efficacy of therapy for syphilis because of the interaction between host defenses andthe infecting organism, on the one hand, and the natural history that includes late recurrence of tertiary disease, on the other (26). In the preantibiotic era, in the absence of therapy, host immunity brought active syphilis under control, but tertiary disease still followed in about one-thirdof untreated cases. Treatment with arsphenamine eventually succeeded in producing a clinical cure of the early lesions of syphilis in all cases, but a late relapse to tertiary disease occurred in5-770ofcases. The later in the natural course of disease that therapy was given,the lower wasthe likelihood of late disease, further illustrating the lifetime importanceof host immunityafter the initial beneficial effect of therapy.

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Penicillin represented a spectacular advance in therapy. Active disease was eliminated with vastly shorter courses of treatment. Standard regimens were developed during careful prospective study of thousands of patients under a wide variety of dosage schedules. Initially it could not have been known, butnow it appears clear that accepted courses of penicillin also prevent late syphilis. Some authorities point out that careful prospective studies have not been completed, but, as reviewed elsewhere (26), virtually all anecdotal retrospectivedata suggest that penicillin is effective in producing a lifetime clinical cure ofsyphilisin the absence of HIV infection. Several linesof evidence suggest that Treponema pallidum may persist after therapy, indicating that ongoing host immunity plays some role in preventing a recrudescenceof infection (26). Alternative drugs such as erythromycin have been used to treat relatively tiny numbers of cases. Courses of erythromycin therapy totaling20 g were associated with > 20% early failure rate and, although one study showed uniform cureof patients using g of erythromycin, others have reported failures in 5-10%ofcases at that dosage (27,28). It is simply unknown whether erythromycin abolishes the risk of tertiary syphilis, although it greatly reduces it. Similar problems exist with availabledata on tetracycline or, more recently, ceftriaxone (29), although it seems reasonable to believe that ceftriaxone will provide cure rates identicalto those of penicillin. Evaluation of any newantibiotic regimen must be carried out with the understanding that penicillin remains a clear regimen of choice because, by now, the high rate of clinical cure throughout life is certain. Roxithromycin, azithromycin, and/or clarithromycin have been shown to be effective in vitro against Treponema pallidum and in animals experimentally infected with this organism A prospective study of 16 patients who had early syphilis using500 mg azithromycin dailyfor days appeared to yield good results, although not entirely free of relapseheinfection The work of Professor Mashkilleyson, Dr. Gomberg, and their colleagues in Moscow, as reportedat this conference, isof great importance This group has treated 100 patients who have primary or secondary syphilis using azithromycin 0.5g daily for 10 days. At the third ICMAS, they report a uniform clinical cure during observation for up to years. Serologic response was seen in 90% of cases. In the context of the very small numberof patients previouslyreported after treatment with erythromycin, this experience with azithromycin is large and important. Based on available in vitro and in vivo (animal) data, as well as available clinical observations, azithromycin might be preferredto erythromycin. Compliance will almost certainly better, be and the Russian experience

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suggests an excellent short-term clinical response. However, very strong preference should continue to be given to penicillin, for which adequate data are available on long-term outcome. Alternative therapy should be given only to those patients who have a convincing history of penicillin allergy.Such patients should be followed closely for short-term clinical response and be advised that a long-term cure is not assured. Failure to prevent congenital syphilis by treating pregnantwomen is welldocumented not onlyfor macrolides (36) but also for penicillin Finally, the efficacy of macrolides in treating syphilis inthe presence of HIV infection has not been reported.

REFERENCES 1. Niederman MS, Bass Campbell GD, Fein AM, Grossman R, Mandell LA, Marrie TJ, SarosiGA, Torres A, Yu VL. Guidelines forthe initial management of adults with community-acquired pneumonia. Am Rev Respir Dis 1993; 148~1418-1426. 2. Mane TJ. Coxiella burnem’ (Q fever) pneumonia. Clin Infect Dis 1995; 21 (SUppl3):253-264. 3. Edelstein PH. Legionnaires’ disease. Clin Infect Dis 1993; 16741-749. 4. Edelstein PH. Antimicrobial chemotherapy for Legionnaires’ disease: a review. Clin Infect Dis 1995;5 (suppl3):265-276. 5. Hubbard RB, Mathur RM, MacFarlane JT. Severe community-acquired legionellapneumonia: treatment, complicationandoutcome. Quart JMed 1993; 86:327-332. 6. Havlichek D, Saravolatz L, Pohlod D. Effect of quinolones and other antimicrobialagents on cell-associated Legionella pneumophila. Antimicrob Agents Chemother 1987;31:1529-1534. 7. Edelstein PH, Edelstein MAC. In vitro activity of azithromycin against clinical isolates of Legionella species. Antimicrob Agents Chemother 1991; 35: 180-181. H, Tomonaga A, 8. Saito A, Sawatari K, Fukuda Y, NagasawaM,Koga Nakazato H, Fujita K, Shigeno Y, Ama YS, Yamaguchi K, Izumikawa K, Hara K. Susceptibility of Legionella pneumophila to ofloxacin in vitro and in experimental Legionella pneumonia in Guinea pigs. Antimicrob Agents Chemother 1985; 28:15-20. 9. Fitzgeorge RB, Lever S , Baskerville A. A comparison of the efficacyof azithromycin and clarithromycin in oral therapy of experimental airborne legionnaires’ disease. J Antimicrob Chemother 1993; 3l(suppl E):171-176. 10. Parola D, De Maio F, Minniti R, Tronci M. Clarithromycin in Legionnaires’ disease. ICMAS 3,1996; abstr 1.06. 11. Hamedani P, Ali J, HafeezS , Bachand R Jr, Dawood G, Quereshi S, Raza R, Yab S. The safety and efficacy of clarithromycin in patients with Legionella pneumonia. Chest 1991; 100:1503-1506.

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Rizzato G, MontemurroL,Fraioli P, Montanan G, Fanti D, Pozzoli R, Magliano E. Efficacy of a three day course of azithromycin in moderately severe community-acquired pneumonia.Eur Respir J Myburgh J, Nagel GJ, Petschel E. The efficacy and tolerance of a three-day course of azithromycin in the treatment of community-acquired pneumonia. J Antimicrob Chemother (suppl Kuzman I, Soldo I, Schonwald S, Culig J. Azithromycin for treatment of community acquired pneumonia caused by Legionella pneumophila-a retrospective study. Scand J Infect Dis Dorrell L, Fulton B, Ong ELC. Intravenous azithromycin as salvage therapy in patient with Legionnaires' disease. Thorax HoepelmanIM,Schneider "E. Azithromycin: the first of the tissueselective azalides. Int J Antimicrob Agents Preac-Mursic V, WilskeB,Schierz, SUB E, GroBB.Comparative antimicrobial activity of the new macrolides against Borrelia burgdorferi. Eur J Clin Microbiol Infect Dis E. In vitro and 18. Preac-Mursic V, Wilske B, Schierz G, Holnburger M, in vivo susceptibility of Borrelia burgdorferi. Eur J Clin Microbiol Moody K D , Adams RL, Barthold SW. Effectiveness of antimicrobial treatmentagainst Borrelia burgdorferi infectioninmice.AntmicrobAgents Chemother Hansen K, HovmarkA, Lebech A-M, LebechK,Olsson I, Halkier-S0rensen L, Olsson E, Asbrink E. Roxithromycin in lyme borreliosis: discrepant results of an in vitro and in vivo animal susceptibility study and a clinical trial in patients with erythema migrans. Acta DermVenereoll992; Luft BJ, Dattwyler R, et al. Azithromycin compared with amoxicillin in the treatment of erythema migrans. A double-blind randomized, controlled trial. Ann Intern Med, Strle F,Preac-Mursic V, CimpermanJ,Ruzic E, Maraspin V, Jereb M. Azithromycin versus doxycycline for treatmentof erythema migrans: clinical and microbiological findings. Infection Weber K,Wilske B, Preac-Mursic V et al. Azithromycin versus penicillin V for the treatment of early lyme borrellosis. Infection Massarotti EM, Luger SW, Rahn DW, Messner RP, Wong JB, Johnson RC, Steere AC. Treatment of early Lyme disease. AM J Med Steere AC, Levin RE, Molloy PJ, Kalish RA, Abraham JH, Liu N Y , Schmid CH. Treatment of Lyme arthritis. Arthritis Rheumatism Musher DM, Baughn RE, Hamill RJ. Effect of human immunodeficiency (HIV) infection on the course of syphilis and on the response to treatment. Ann Intern Med Willcox RR. Treatment of early venereal syphilis with antibiotics. Br J Vener Dis Lucas JB, Price EV. Co-operative evaluation of treatment for early syphilis. Br J Vener Dis

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29. Dowel1 ME, Ross PG, Musher DM, Cate TR, Baughn RE. Response of latent syphilis or neurosyphilis to ceftriaxone therapyin persons infected with human immunodeficiency virus. Am J Med1991; 93:481-488. 30. Stamm LV, PamshEA. In-vitroactivity of azithromycinand CP-63,956 against Treponema pallidum. J Antimicrob Chemother1990; 25:ll-14. 31. Lukehart SA,Baker-Zander SA. Roxithromycin(RU 965): effective therapy for experimental syphilis infection in rabbits. Antimicrob AgentsChemother 1987; 31:187-190. 32. Lukehart SA,Fohn MJ, Baker-Zander SA. Efficacy of azithromycin for therapy of active syphilis in the rabbit model. J Antimicrob Chemother 1990; 25~91-99. 33. Alder J, Jarvis K, Mitten M, Shipkowitz NL, Gupta P, Clement J. Clarithromycin therapy of experimental Treponema pallidum infections in hamsters. Antimicrob Agents Chemother1993; 37:864-867. 34. Verdon MS, Handsfield HH, Johnson FU3.Pilot studyof azithromycin for treatment of primary and secondary syphilis. Clin Infect Dis 1994; 19:486-488. 35. Mashkilleyson AL, Gomberg MA, Mashkilleyson N, Kutin SA. Treatment of syphilis with azithromycin.Int J STD AIDS 1996; 7:13-15. 36. El Tabbakh GH, Elejalde Br, Broekhuizen FE. Primary syphilis and nonimmune fetal hydrops in a penicillin-allergic woman. A case report. J Reproduct Med 1994; 39:412-414. 37. McFarlin BL, Bottoms SF,Dock BS, Isada NB. Epidemic syphilis: maternal factorsassociatedwithcongenitalinfection. AM J Obstet Gynecol 1994; 170535-540.

16 Mycoplasma and Chlamydia J. Thomas Grayston University of Washington Seattle, Washington

Pentti Huovinen National Public Health Institute Thrku, Finland

Data on the incidence of total pneumonia and pneumonia due to Mycoplasmapneumoniae and Chlamydiapneumoniaewas presented. Pneumonia occurs most often in the youngest and oldest persons the in population. In a 12-year study in a SeattleHMO, the annual incidenceof pneumonia averaged 1in 80. Similar rates have beenreported from other studies, including a nationwide survey of practitioners in the United States. Similar data also have been reported from Europe. These data suggesting that, on average, everyone has one episode of pneumonia during his lifetime is undoubtedly an underestimationdue to the failure of diagnosis in milder cases. In clinical and microbiologically controlled studies, it has been shown that clinical findings do not correlatewell withthe organism specific diagnosis of pneumonia. Although this sessionwas primarily concerned with atypical pneumonia, it was pointed out that the classification of pneumonias bythe typical and atypical criteria is difficult, because of the overlapping of symptoms and the rarity of typical lobar pneumococcal pneumonia. Both M. pneumoniae and C. pneumoniae are periodic. Efforts to define a cycle of a regular period of years for both these organisms have 219

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failed. Whereas countrywide epidemics have seen beenin the Nordic countries of Europe, fairly consistent endemic rates of 1620% for the two organisms have been seen Europe in and the United States in recent years. Although C. pneumoniae is far more apt to be associated with hospitalized pneumonia than M.pneumoniae, it is unclear how often the more severe cases of hospitalized pneumonia associated with TWAR infection are due to that organism alone. Dual infections with bacteria and viruses are found frequently. There was general agreementthat the macrolides werethe antibiotics of choice for treatment of atypical pneumonia. Data were presented from recent studies showingthat azithromycin was highly effective against pneumonia caused by M. pneumoniae and by C. pneumoniae. Some of these studies were compared with the effect of erythromycin which was also highly effective.A discussant stated that roxithromycin had been shownto be highly effective in atypical pneumonia. Another discussant told of the study showingthat clarithromycin.was equally effective as erythromycin in treatment of pneumonia due to M. pneumoniae and C. pneumoniae. It appears that the newer macrolides or azalides are at least as effective as erythromycin intreatment of C. pneumoniae and M.pneumoniae and that they have fewer side effects. A question was raised about whether any treatment was necessaryfor M.pneumoniae infection and perhaps evenC. pneumoniae infection, even when a clinical syndrome of pneumonia was present. The suggestion was that these patients gotwell spontaneously andthe cure rate with antibiotics was falsely inflated. There was little support for this position, although it was acknowledgedthat asymptomatic and mildly symptomatic pneumonitis due to bothorganismswaswelldocumented. The fact that prolonged moderately debilitating cough illnesses often followed untreated or inadequately treated C. pneumoniae lower respiratorytract infection argued for appropriate treatment of this infection. In a discussion of antibiotic resistance, no one in the audience had an isolate of either M.pneumoniae or any Chlamydia species that was resistant to macrolides or tetracycline.Duringtherapy, the MICs(minimal inhibitory concentrations) of thesemicroorganismsusuallystays at the same level or changes onlyone step up or down. There have been reports of cases where the MIC of C. pneumoniae jumped by dilutions after receivingazithromycin. There havebeenclinicalcaseswhereseveral courses of macrolides were neededfor clinical cure, although the MIC of C. pneumoniae isolated remained unchanged. Studies of isolates of C. trachomatis from patientswith clinical failure have been tested and always found to be susceptibleto macrolides and tetracyclines.It is probable that a 1 or 2 dilution difference in MIC susceptibility is an insignificant variation

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because of the cell culture technique used for measurement. The only known antimicrobial agent to which resistance has developed in these organisms is rifampin. There is a report that the MIC of chlamydia can be pushed up to the MBC (minimal bactericidal concentration) level by several passages. However, when concentrations of antibiotic at the MBC level have been reached, the organisms always die. Someof the problems of measuring the MIC of these organisms was discussed. For example, control of the pH of the media is important when testingM. pneumoniae. wide varietyof different cell typesare now usedfor measuring antibiotic susceptibility. There is a need for standardizationof the susceptibility testing methodsfor mycoplasmas and chlamydias. In the past decade, abouthalf of M. hominis isolates have provento be resistant to tetracycline, carrying tetM-resistantdeterminant. The same determinant can be seen in Ureaplasma urealyticum. In the preceding decade, isolates of these organisms were susceptible to tetracycline. This phenomenon is similar to that that has been seenfor Nekseria gonorrhoea. Several attempts in the laboratory to put the tetM-resistant determinant into M . pneumoniae have failed. Among the mycoplasma, there is concern about M. hominis, which can cause chronic systemic infectionsof the joints. The best antimicrobial has been the tetracycline group, but there is now resistance to these antimicrobials. It remains to be seen what effect some of the newer quinolines, such as spadoxacin will have on this organism, as it is known that temafloxacin and ciprofloxacinare not effective inM. hominis infections. There was a discussion on whatthe future holds in the way of antibiotic resistance if the newer macrolides are increasingly used for treatment of mycoplasmas and chlamydias.The concern expressed wasnot that resistance would develop in these organisms against these antibiotics, butthat the wideruseofmacrolidescould further promote resistance in other organisms such asStreptococcus pneumoniae. There was considerable discussion about the use of macrolides as a first-line drug inthe treatment of outpatient pneumonia. One of the issues was that because classical pneumococcal pneumonia is rarely seen, pneumococcal pneumonia often cannot be differentiated from pneumonia due to M . pneumoniae and C. pneumoniae. It was stated that persons who die of pneumococcal pneumonia duringthe first 24 h will die despite therapy and that the figures on such deaths have not changed in 70 years. It was suggested that if this type of infection is to be successfully treated, the target may be the cytokine response. The best predictorof mortality and serious complicationsthe is severity of the pneumonia illness at presentation. The nature of the patient has to be taken into account when planning antimicrobial treatment. When the

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patient has risk factors (e.g., age, smoking habits, diabetes), it is necessary to plan treatment according to these risk factors.The macrolides in uncomplicated outpatient pneumonia should be safe and effective. Future improvement in fatal and severe disease with pneumonia will probably be best effected by efforts to develop active vaccination programs andto develop new vaccinesfor young children. The next major topicfor discussion was specific diagnosis of clinical cases of atypical pneumonia. It is recognized that even specialists do not carry out laboratory tests for specific diagnosisof atypical pneumonia unless theyare involved in a clinical trial. This behavior is strongly influenced by the availability of effective rapid laboratory diagnostic methods. For M. pneumoniae, there are many antigen-detection tests and DNA/RNA probes available. However,the specificity of these tests islow. A PCR (polymerase chain reaction) test now is available, but how effective it will be inthe clinical setting is not yet known. Diagnosis of Chlamydia pneumoniae infection outside the research laboratory is difficult. Cell culture techniques for isolation of the organisms have yielded widely variable results. For the most part, isolation of the organism has been difficult. This is duepart in to the fact that the infection isin the lung and the specimens are taken from the throat. Obtaining specimens from a lung is rarely justified. There is general but not unanimous agreement that the MIF (microimmunofluorescent) serologic test is the most sensitive for diagnosingC. pneumoniae infections. However, because of the requirement for a convalescent serum specimen, this method is usually not practical for immediate clinical decisionsabout therapy, but it may be useful in cases with prolonged illnessesthat are not responding to treatment. Several PCR testshavebeendevelopedandmaymeet the criteria for more rapid diagnosisin the future. Even with the development of more rapid and sensitive tests to identify the organisms, there will remain decisions to be made clinically about the significance of the identified organism. Some asymptomatic camage of both organisms has been demonstrated. There was discussion of a need to diagnose M. pneumoniae or C. pneumoniae infection when pneumonia was not present. In the case of bronchitis caused by these organisms, there is general agreementthat specific therapy is useful. Although it is true that children and some adults may have mild infections with these organisms and recover fairly rapidly without treatment, some patients are debilitated for months and benefit from treatment. The question of treating asymptomatic infection was also discussed. Whereas asymptomatic female patients with genital C. trachomatis are always treated, there is no information to show whether patients with asymptomatic respiratory infections needto be treated.

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Finally, there was a discussion of the use of the new macrolides to treat M. hominis genital tract infection. M . hominis in the lower genital tract is usually a commensal. There are reports that M. hominis can cause pelvic inflammatory disease, but these appear to be rare cases. Infectionsin newborns and immunocompromised patients are different and treatment should be considered.

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Staphylococci, Streptococci, and Pneumococci Jacques F. Acar Fondation HSpital Saint-Joseph Paris, France

J& Melo-Crlstino University of Lisbon Lisbon, Portugal

The authors of abstracts and discussants developed an overview of S. aurew, S. pneumoniae, and S. pyogenes reporting on susceptibility and susceptibility testing; epidemiology of resistant strains and insights on therapy of infections. The susceptibility and susceptibility testing of S. uurew to the macrolides and macrolide-related antibiotics should be considered regarding the different classesof compounds: C,, macrolides, azalides,C,, macrolides, lincosamides, and streptogramins and B. Table 1summarizes the morefrequent resistance characters and the corresponding resistance phenotypes. Inactivation mechanisms with different enzymes are seldom reported. The E m genes . . . the major resistance determinants found in clinical isolate. Because the new macrolides are in the C,, group, this gene mediates the resistance to the whole group of compounds (including the azalides) inthe inducible state as well as in the constitutive state. In the consti-

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tutive form, E m mediates resistance to C,, macrolides, lincosamides, and streptograminB. The prevalence of macrolide resistance (usually in the constitutive form) in methicillin-resistant Staphylococcus aureus (MRSA) ranges from 40% to 95% of isolates. On the contrary, methicillin-susceptibleStaphylococcus aureus (MSSA) often remain susceptible with a prevalence of resistance only from 5% to 15%. Coagulase-negative staphylococci share the same resistance phenotype with Staphylococcus aureus. However, efflux mechanisms were recognized in coagulase-negative staphylococci. Pneumococcal resistanceto the macrolides tendsto increase inEuropean countries.The penicillin resistanceof pneumococci has beenreported more frequently associatedwith resistance to other antibiotics, particularly the macrolides. However, the prevalence of pneumococci resistant to the macrolides varies widely from country to country: 10-20% reported from France,whereas the prevalencein the Netherlands is only 1% and in Germany 1.5%. The occurrence of penicillin resistance in S. pneumoniae strains was variable, asreported at this meeting.A Spanish studyby Garcia de Lomas et al. with 296isolates demonstratedthat the resistance to penicillin (intermediate and high levels) in healthy carriers was higher in strains recovered from children than adults (70% in children and 42.5% in adults). The authors found the coexistence of susceptible and resistant strains in 17 specimens from children and 8 from adults. Resistance rates to macrolides (erythromycin and clarithromycin) were 5.6% among children and 6.3% among adults. Paris et al. studied the incidence of disease and antibiotic susceptibility of S. pneumoniae from a population-based study in Dallas County in 19921993; 732 S. pneumoniae isolates were identified. Penicillin-intermediate strains accounted for 14% and resistant strains 3%. Theoverall susceptibility to the macrolides, clarithromycin and azithromycin (MIC d 1pg/ml) was estimated at 93%. Visalli et al. determined the activity of erythromycin, azithromycin, and clarithromycin against 120 pneumococci isolated from the United States. Results ofMICandtime-killstudiesshowed that clarithromycin was the most active macrolide. The authors suggested that clarithromycin has a potential for use against infectionsbycaused penicillinsusceptible and -intermediate and -resistant pneumococci in the United States, provided that the strains are susceptible to macrolides. From Germany, Knothe et al. studied 710 S. pneumoniue isolates from non-hospitalized children. Only 21 strains showed decreased susceptibility to penicillin (18 intermediate and 3 resistant). Roxithromycin was tested as representative of the macrolides and only11(1.5%) of the strains were resistant (MIC 4 pg/ml). These results are in agreement with the favourable situationin Germany regarding antibiotic susceptibility of pneu-

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mococci. S. pneumoniae strains isolated from acute otitis media in children were less resistant to the macrolides, azalides, and streptogramins (MAS) than to other antibiotics commonly used to treat otitis media (p-lactams andco-trimoxazole)in the reports ofMcLinnwithazithromycinand clarithromycin, and Blocket al. with clarithromycin and clindamycin. The effect of CO, incubation in antimicrobial testing of pneumococci was discussed in two studies. Moore et al. and Fasola et al. found differences in two incubation atmospheres (CO2 and air). The latter authors made the point that the dissociationinvitro(diskmethod)between macrolide and lincosamide susceptibility inS. pneumoniae is artificial due to the better expression of resistance to the macrolides. They made recommendations for macrolide and lincosamide testing of pneumococci, which include the following:minimuminhibitoryconcentration(MIC)breakpoints for erythromycin 0.5 pg/ml,susceptible; lpg/ml, resistant; clindamycin 0.25pg/ml, susceptible; OSpg/ml resistant; no intermediate category should be used. Testing of these agents can be reliably performed by agar dilution, E-test and disk diffusion with incubation in 510% CO,. Testing of these agents by microdilution should be performed under 5-10% CO, or aerobic incubation prolongedto 48 h. P. Appelbaum stressed the cross- resistance of pneumococci to macrolides, azalides, and lincosamides and the susceptibility of such strains to the ketolides and the streptogramins. Streptococcus pyogenes and pharyngitis/tonsilitis werethe subject of a few posters. The efficacyofazithromycinwas stressed by several authors. O’Dohertyet al., from Ireland, demonstrated in a doubleblind prospective studyof 489 patients, that azithromycin at a doseof 20 or mg/kg/day for 3 days was as safe and effective as penicillin V in the treatment of pediatric patientswith acute pharyngitis/tonsillitis. The MAS resistance in S. pyogenes was presented by Katala et al. from Finland, a country where an increasein the erythromycin resistance was found in the beginning of this decade. A novel resistance phenotypein which clindamycin resistance could not be induced by erythromycin has increased and accounted for70% of the resistant strains isolated in1994. Ninety-five percent of these strains belonged to the T4 serotype (DNA analysis showed genetic heterogenicity among T4 serotypes), whereas 91% of the isolates showingthe inducible typeof resistance belongedto theT28 serotype. The authors concludedthat erythromycin resistance in Finland is due to the spread of a few serotypes of S. pyogenes. In Italy,anationalmulticentersurveyconductedduring1995included 2739 strains and revealed that resistance in S. pyogenes was 8.2% for clindamycin, 14.7% for tetracycline, and 24.7% for erythromycin. Disk diffusion was the prevailing technique adopted. The authors stressed that these findings raise serious concerns given the fact that severe S. pyogenes-

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associated diseasesare increasing worldwide and that in patients allergic to p-lactams, resistance to alternative agents may cause serious therapeutic problems. In contrast to the two previous studies, Van Asselt et al. from the Netherlands, presented a study of 180 Xpyogenes strains, none of which was resistant to macrolides, which confirms the low rate of erythromycin resistance in this country. Three methods (E-test, microbroth MIC, and diskdiffusion)wereused to study the susceptibility of S.pyogenes to azithromycin, clarithromycin, erythromycin, and roxithromycin. Clarithromycin and azithromycin were compared with erythromycin, vancomycin, and amoxicillin for prophylaxis of viridans streptococcal experimental endocarditisby Rouse et al. from United States. Azithromycin and clarithromycin were as effective as amoxicillin, and erythromycin was less effective (p < than amoxicillin. A report from Shibl et al., from Saudi Arabia evaluated roxithromycin postantibiotic effect (PAE)on several Streptococcus species. The data indicate that PAE could not only be considered as prolonged suppression of bacterial growth but also could include an inducedstate of decreased microbial virulence through impaired adherence, diminished tissue invasion, and modulation of bacterial susceptibility to phagocytosis. Three new compounds were evaluated in several studies and discussions: RP 59500 (Quinupristiddalfopristin),Synercida, a new injectable streptogramin developedby RhBne-Poulenc-Rorer, was testedby Pankuch et al. using time-killing curves of penicillin-susceptible, -intermediate, and resistant pneumococci. They found that MICs, for all groups were 1pg/ml. Only RP 59500 killed pneumococci on initial exposure and within 1 h of exposure and it also was the most active at 2 h.RP 59500 yieldedthe most rapid killingof all drugs tested and was equally active against erythromycinsusceptible and -resistant strains. Reinert et al. from Germany also found a goodactivityagainstallpneumococcitested(penicillin-resistantstrains were not tested), including erythromycin-resistant isolates, and suggested that RP 59500 might be useful for the treatment of pneumococcal infections. Tarasiet al. fromthe United States tested 217 pneumococcal isolates that included 200 penicillin-resistant strains intermediate (89 and 111highly resistant) and4 isolates with specific resistanceto third-generation cephalosporins. The isolates included representatives of five major multiresistant pneumococcal clones as defined with pulsed-field gel electrophoretic patterns. More thanhalf of the highly penicillin-resistant isolates were representatives of the 23F capsular type. RP 59500 was active against all these isolates, with MIC, = 0.25 andMIC, = 0.5 pg/ml, includingthe 49 strains that were resistantto erythromycin. Using a rabbit modelof experimental meningitis, Denver et al. of the United States suggested that there is suffi-

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cient penetration through the inflamed meninges of RP 59500 to cause extensivecidaleffectinpneumococcalmeningitis. Further studies are needed to confirm this finding. Berthaud et al. from France studied in vitro bactericidal activity of the compound against S.aurew and compared it with vancomycin. Both drugs, at concentrations achieved in blood, demonstrated potent a bactericidal activity, but the bactericidal effect of RP 59500 was more rapid than that of vancomycin. Tenoveret al. fromthe United States developed provisional disk diffusion breakpointsfor testing RP 59500. Using a susceptibility breakpoint of < 2 pg/ml, the best correlation zone sizes and MICs was achieved with 5/10(quinupristiddalfopristin) ratio disk and a zone diameter of > 18 mm. Clinical correlation data are needed to choose the optimal disk concentration for testing. Studies with RPR 106972, a new oral streptogramin developed by RhBne-Poulenc Rorer, was presented. Berthaud et al. from France verified that in vitro, the drug showed promising antibacterial activity against gram-positive cocci (staphylococci, streptococci, and enterococci) and certaingram-negativebacteriaresponsibleforrespiratory tract infections (Neisseria, Moraxella, H . influenzae) and mycoplasmas. In vivo, in experimentalgram-positiveinfectionsinmice,theydemonstrated that RPR 106972 was active in the treatment of infections caused by sensitive and macrolide-lincosamide-streptogramin B (MLS,)-resistant S. aurew and erythromycin-sensitiveand-resistant S. pneumoniae. The antipneumococcal activity of RPR 106972 was also studied by Spangler et al. from the UnitedStates.These authors compared its activitywith9 oral lactams against 75 penicillin-susceptible, 55 penicillin-intermediate,and 73 penicillin-resistant pneumococci and verified that RPR 106972 hadthe lowest MICs (< 0.5 pg/ml) of the compounds tested. Macrolide resistance did not interfere with the results because MICs distributions were similar for the different subgroups of susceptible and resistant strains. RU 004 is representative of a new class of 1Cmembered macrolide antibacterials, the ketolides, generatedby Hoechst Marion Roussel. Ketolides are characterized by a keto function in position of the macrolactone ring, which replaces the cladinose moiety. Agouridas et al. from France evaluated in vitro and in vivo, the antibacterial activity of the compound against respiratory pathogens in experimental murine septicemia and pne monia models. They showed that this compound demonstrates outstanding activity against macrolide-susceptible, macrolide-inducibleMU,-resistant staphylococci, constitutively MLS,-resistant cocci(e.g., pneumococci), except staphylococci.The compound showed activity similar to azithromycin against H.influenzae and also was very active against bacteria such as Legionella, Mycoplasma, and Chlamydia spp. The in vivo studies confirmed

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the potential usefulness of RU 004 in respiratory infections causedby H. influenzae and pneumococci resistant to macrolides, p-lactams, and other drugs. Edine et al. fromthe United States studied the activity of RU 004and compared it with15 other agents against228 erythromycin-susceptible and -resistant pneumococci and found that the compound had excellent activity against penicillin- and erythromycin-sensitive and -resistant strains,showing, in all cases, MICs, values much lower than those of erythromycin, azithromycin, and clindamycin.

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l8 Pharmacokinetics and Pharmacodynamics Charles H.Nightingale Hartford Hospital Hartford, Connecticut

Fritz Sorgel Institute for Biomedical and Pharmaceutical Research Nurnberg-Heroldsberg, Germany

This workshop discussed several important pharmacodynamic and pharmacokinetic issues.The most important issue involved understanding how the current pharmacodynamic model appliesto the macrolides. Currently, it is widely believedthat for concentration-dependent killing antibiotics (aminoglycosides, quinolones), the pharmacodynamic parameter that correlates with bacterial eradication is the area under the plasma concentration versus time curve/minimal inhibitory concentration(AUCMIC) ratio and, under certain conditions, the C#IC ratio. For concentration-independent killing drugs such as p-lactams, the important pharmacodynamicparameter is the time that the serum concentrations remain abovethe MIC (t > MIC). In both cases, the serum concentrationis indexed to the organism’s MIC, and for antibiotics to eradicate organisms, the serum or, by extension, the interstitial fluid concentrations needto exceed the MIC in someway. For macrolides, it can be demonstrated that these models hold for most organisms (e.g., S. pneumonaie), but for other organisms such asH. influenme, these models predict drug failure. Clinical studies indicated that macrolides are quite effective in the treatment of diseases where H . influ-

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mzae is a major pathogen. This disconnect between the model and reality is an indication that the existing model cannot completely describe the pharmacodynamics of the macrolides. Upon further discussion, it was felt that the current model assumesthat the liquid surroundingthe microorganism contains a homogenous concentration of drug. This is probably not correct for drugs such as macrolides that are capable of accumulating in mammalian tissue. As drug leaves these tissue repositories, a concentration gradient is established with the highest drug concentrations just on the outer membranes of the mammalian cell and decreasing the further one travels away fromthat site. Measurable interstitial fluid levels represent an average drug concentration as do measurable serum or blood concentrations. For drugswith little tissue uptake (e.g., p-lactams), serum and interstitial fluid levelsare sufficiently high, even on average,to eradicate target pathogens; the current model predicts their activity very well. For drugs like macrolides, the same situation occurs (i.e., for very sensitive organisms, the average blood and interstitial fluid levels are sufficientlyhigh to eradicate the organism), however, when the organism’,is moderately susceptible, (i.e., has a higher MIC),the current modelis incapableof predicting organism eradication. important issue isto determine wherethe organism resides inthe body in relationship to where the drug is at high concentration. It is believed that pathogens reside attached to surfaces (i.e., the outside membranes of cells). It is further believed that due to the concentration gradient of macrolides, the bacteria reside in just the place where interstitial fluid concentrations are highest. Additionally, macrolides accumulate in macrophages. Macrophages migrate to infection sites to engulf bacteria. Upon doing they can release drug, thus increasing drug concentrations in bacteria andmay also changethe permeability of drug intothe bacteria, in effect reducing the in vivo MIC.It was generally feltthat the in vitroMIC is higher than the in vivo MIC and this may explain, in part, the lack of concordance between clinical studies and pharmacodynamic predictions for macrolides. It is believed that such a modified model is capable of describing the clinical observations associated with macrolides. Regarding pharmacodynamic parameters in general, it is felt there,is on a sigmoidal dose-response curve. For concena gradient of effects based tration-dependent killing drugs, the dose-response curve is verysteep and the drug is usually cidal using therapeutic doses. For p-lactams, the curve is less steep; at lower concentrations, the drugs’ actions may be bacteriostatic, and at higher concentrations, bactericidal. Macrolides may have a dose-response curvethat is less steep than that of p-lactams. Regarding the postantibiotic effect, it could not be decided whether this was generally goodor problematic. It was thought to be good because

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it allows infrequent dosing; however, there was some indication that prolonged exposureto ineffective concentrationsmay result in mutations and selection of resistant organisms. To clarify this requires differentiation between postantibiotic effect and subtherapeutic concentrations. The workshop also focused on tissue distribution issues, noting that lactams are distributed extracellularly, aminoglycosides are similar, whereas quinolones have some intracellular distribution and macrolides have the most intracellular distribution. Not all macrolides are alike, however, because roxithromycin shows a distribution pattern similar to p-lactams. It was also pointedout that erythromycin pro-drugs may cause erythromycin to behave more like azithromycin than clarithromycin. It was postulated that the pro-drug can penetrate rapidly into tissue, be hydrolyzed, and egress out of the tissue, resulting in prolonged but low serum erythromycin concentrations. There was some discussion about azithromycin absorption probably due to loss by first-pass effects in the liver. Also, bioavailability of the suspensiondosageformdidnotchangewithage of the patients. Clarithromycin suspension dosage forms was stated to have AUC/24 hthat were similar to the adult dosage form, including similar Cm,’s and Cmin’s. The reporting of tissue concentration data was discussed. The point was made that tissue/serum ratios provide little useful information. It is possible to have a high ratio if the numerator is high, and it is possible to have a high ratio if the denominator islow. Antibiotics kill bacteria based on drug concentrationsat theplace wherethe bacteria reside. Actual tissue concentrations should be compared, not the penetration ratios. Penetration ratios have marketing value but little scientific merit and its use should be discouraged. Finally, the difficulties of conducting tissue uptake studies were discussed. It is realized that there is a wide variety oftechniques usedfor this purpose withlittle standardization. It was suggestedthat standardized methodologies be established as a guide in conducting these studies that the results canbe more easily compared from different study centers.

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19 Campylobacter and Helicobmter pylori Jean-Paul Butzler University Hospitals, St. Pierre, Brugmann, and Queen Fabiola Bmsels, Belgium

Francis M6graud Hopital Pellegrin et Universitkde Bordeaux 2 Bordeaux, France

MACROLIDES IN CAMPYLOBACTER ENTERITIS

In the last 10 years,Cumpylobucterjejuni has emergedas the most common cause of bacterial gastroenteritis in humans. The symptoms C. jejuni infection are usually mild but systemic and postinfectious manifestations such as the Guillain-Barr6 syndrome may occur (1,2). In general, Campylobacter enteritis is a self-limiting disease and the isolation of campylobacters from stools does not warrant chemotherapy. In the absence of chemotherapy, the feces of patients remain positive for about 2-7 weeks after the illness. Antimicrobialtreatment, when started within 4 days of onset, has a clinical benefit and shortensthe fecal excretionof C. jejuni. Salazar-Lindo et al. evaluated the efficiencyof erythromycin, starting immediatelyon presentation. They found that early administration of erythromycin significantly reduced the duration of both diarrhea and fecal excretion the organism in infants and children with acute C. jejuni dysentery. In children, roxithromycin treatment initiated within 4 days the onset of C. jejuni diarrhea appeared to reduce the camage of the

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and

Butzler

pathogen (4). It was also effective in reducing boththe severity and duration of the diarrhea and seemed to accelerate the disappearance of abdominal pain. Antimicrobial treatment is indicated in prolonged disease with severe symptoms, in high fevers or with bloody stools, in relapses, in pregnancy,andinimmunocompromisedpersons. The clinicalcourseof C. jejuni diarrhea in AIDS patients very often requires long-term treatment. Campylobacterjejuni shows rather unusual antimicrobial sensitivities. The macrolides, furazolidone andthe aminoglycosides are the most active compounds; all strains are resistant to benzylpenicillin, cephalothin, colistin, vancomycin, rifampicin, and trimethoprim. Up to 20% of strains are resistant to tetracycline. Susceptibility to ampicillin, metronidazole, and trimethoprim sulphamethoxazole is variable.C. jejuni is susceptible to the newer macrolides such as roxithromycin, clarithromycin, and azithromycin (Table 1). In industrialized countries, macrolides continue to be the drugs of choice for the treatment of Campylobacter infections because the preva(2-5%) and stable lence of erythromycin-resistantstrainsremainslow Several studies showthat macrolides like clarithromycin and azithromycin have invitro activities similar to that of erythromycin and superiorto that of roxithromycin (6). The situation for quinolones is totally different. Several authors have showna remarkable increasein the level of resistance since the introduction of norfloxacin and ciprofloxacin andthe inclusion of enrofloxacin in veterinary practices (5,7,8). Sanchez et al. ( 5 ) studied the evolution of antimicrobial susceptibilities of 275 clinical Campylobacter strains isolated in their institution duringa 5-year period and Tabk l

Susceptibility of Campylobacter jejuni to Various Antimicrobia'l Agents MIC(mgn) Range

Erythromycin Clarithromycin Roxithromycin Erythromycylamine Dirithromycin Flurithromycin Azithromycin Josamycin

90%

4.0

0.50

0.50

Spiramycin

Miokamycin Rokitamycin

50%

0.5->4

4.0

Campylobacter andpylori Helicobacter found a stable macrolide activity with a rapid development of quinolone resistance. The evolution of resistance (percent resistance in 1988 versus percent resistance in 1992) was as follows: erythromycin, 2.6 versus 3.1; clarithromycin, 2.6 versus 3.1; azithromycin, 2.6 versus 3.1; ciprofloxacin, versus 49.5; norfloxacin, 2.6 versus 55.5; ofloxacin, 0 versus 45.6. A number of studies (5-7) have confirmed that the species C. coli is much more resistant to erythromycin and the other macrolides than C. jejuni. Several investigators demonstratedthat the strains of C. coli isolated in pigs were those withthe highest resistance rate, close to 70% (9). This phenomenon may be dueto the indiscriminate useof the macrolide antibiotic tylosin (analogousto erythromycin) for fattening these animals, which should leadto the appearance of strains with cross-resistanceto erythromycin, which would subsequently be ingested by humans (7,9). Endtz et (8) determinedtheminimalinhibitoryconcentrations (MICs) of erythromycin and three new macrolide antibiotics for 36 quinolonesusceptible and 106 quinolone-resistantC. jejuni strains. The MIC, values of azithromycin,clarithromycin,roxithromycin,anderythromycinwere 0.5, 4, 16, and 4 m&,respectively. difference was found between macrolide activity against the quinolone-susceptible and the quinoloneresistant strains. Taylor and Chang (6) studied the MICs of azithromycin and erythromycin for 20 Campylobacter coli and 20 Campylobacter jejuni strains. The results demonstratedthat for Campylobacter species, all highlevel erythromycin-resistant strains were also resistant to azithromycin and that-azithromycin did not exhibit increased potency in comparison with that of erythromycin. Yan and Taylor (10) investigatedthe mechanism of resistance to erythromycin. Erythromycin resistance (MICs, greater than 1024 pglml) in three clinical isolates of Campylobacter jejuni and one C. coli isolate was determined to be constitutive and chromosomally mediated. If patients are caught in the early stages of the disease or if they are distressed, appropriate chemotherapy is justifiable. Erythromycin has been advocated as the agent of choice for the treatment of Campylobacter enteritis. It hasexcellentinvitroactivity,lowtoxicity,afairlynarrow antibacterial spectrum, and relatively low cost. Resistant strains are, on the whole, rare and almost confinedto C. coli. It probably matters little what preparation of erythromycinisgiven (other thanenteric-coatedpills). There are theoretical reasons for favoringthe stearate, at least for adults. Apart from being acid resistant andstable, it is incompletelyabsorbed, there is a chance of a contact action in the bowel lumen as well as a systemic action in the blood. A dosage of 500 mg twice daily for 5 days has proved satisfactory in practice; higher doses of the stearate are liable to cause acute abdominal pain. Erythromycin ethylsuccinate at 40 mg/kg/day in divided doses is recommended for children.

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Although the newer macrolides (roxithromycin, clarithromycin, and azithromycin) may have a future because of their pharmacologic advantages, there is not sufficient evidence to prove that they are superior to erythromycin, but theymay be better tolerated. Funke et al. (11) reported the development of resistance to clarithromycin in vivo during treatment of an invasive Cumpylobucterjejuni infection in anAIDS patient. Because of the chemical relatednessof clarithromycin and erythromycin, resistanceto clarithromycin may be due to the same mechanism, suggested by the fact that the isolatesbecameresistanttobothdrugs at the sametime. As previouslymentioned,TaylorandChang (6) found that erythromycinresistant C. jejuni and C. coli strains always showed cross-resistance to azithromycin. The clinical course of C. jejuni diarrhea in AIDS patients very often requires long-term treatment that may not be successful. with other long-term antimicrobial therapies, clinicians should be aware of in vivo development of macrolide resistance in immunocompromized as well as in immunocompetent patientstreated for C. jejuni infection. In the industrialized countries, dehydration caused by C. jejuni is infrequent, but fluid and electrolyte replacementare sometimes necessary in infected infants.The best treatment forC. jejuni infections in developing countries could well prove to be different. Factors such as low socioeconomic status and malnutrition may determine the severity of a C. jejuni infection and its great prevalence in very young children. Vomiting and watery diarrhea are frequent, and sometimes, oral rehydration is required in children. Antibiotics should be reserved for very severecases. It is certain that education andbetter hygiene havefar greater roles in reducing infections than do antibiotics. In conclusion, the current consensus isthat antibiotic therapy is indicated in patients with Campylobacter infection who are acutely ill with enteritis, have persistent fever, bloody diarrhea, more than eight bowel movements per dayor significant volume loss,or more than a 7-day history of diarrhea. HIV-infected individualsor immunocompromised persons and pregnant women should receive antibiotic treatment. When antimicrobial therapy is indicated, erythromycin is the drug of choice, given its efficacy, low toxicity, and low cost. There is not sufficient evidence to prove that the newer macrolides, roxithromycin, clarithromycin, and azithromycin, are superior to erythromycin, but theymay be better tolerated. HELICOBACTER PYLORIAND MACROLIDES The discovery of Helicobacterpylorihas beenone of the most important in the field of medical bacteriology.The diseases of the stomach, in particular peptic ulcer for which numerous causes had been advocated in the past

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241

including diet, stress, and forth, are now recognized as infectious diseases (12). Furthermore, for the first time, a bacterial infection is considered to be at the origin of a carcinogenic process as wasthe case for viral infections (e.g., hepatitis B) and parasitic infections. for anyother bacterial infection, H.pylori infection must betreated with antibiotics. WhenMICs were determined on this bacterium, most antibiotics were found to be effective, but clinical experience selected only a few compounds or groups of compounds: a p-lactam: amoxicillin, nitroimidazoles, and macrolides.

MICs of Macrolides on H.pylon When MICs were determined on strains of H.pylori, a small group of resistant strains was identified (e.g., 10% in France) (14). The MICs of susceptible strainsto clarithromycin at neutral pH range from to 0.06 m&. This compound isthe most effective macrolide with aMIC, of m& (i.e., almostaslowasamoxicillin). Other macrolideswithgood activity are azithromycin and roxithromycin When MICs are determined at a lowerpH, their values increase,but, again, clarithromycin isthe least affected compound (Table2). A question which was debated during ICMASI11 was the optimal way of testing strain susceptibilityto antibiotics and especially to macrolides. It was agreed that for macrolides, the method was not critical because all the methods can discriminate between susceptible and resistant strains (i.e., E-test, and even disk diffusion can be used); the agar diffusion method is not mandatory. However, it was stressed that a heavy inoculum should be plated.The reason is that a mixed population of susceptible and resistant strains may be involved and it may be missed if one uses a light inoculum or a single colony. Table 2 Susceptibility of Helicobacterpylon Strains to Various Antimicrobial Agents at Three Different pHs

pH 7.5 Erythromycin Roxithromycin Dirithromycin Clarithromycin Azithromycin Spiramycin Mdecamycin

0.2 0.25

pH 6.5 2

pH 5.5 16 4

1

1 4

0.03 0.12

0.06 1

1 1

8

32

2

4

16 0.25 8

242

and

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Mbgraud

However, there are asmallnumber of strains with MICs slightly higher than normal (i.e., 0.5-1 m&), the question remains: Where should the breakpoint be set? Based onU.S.and European studies (16,17), a breakpoint of 0.5 m& was proposed but was not accepted by the U.S. Food and Drug Administration. In fact, noone knows what to do with the isolates which have an MIC of 0.5-1 m& and which are seldom found. It would be interesting to know if they possess the same genetic mutationas the high-level resistant strains. More data on clinico-bacteriological correlations are needed before concluding onthe breakpoint values.

Pharmacokinetics of Macrolides in the Stomach It is well known that macrolides have the ability to concentrate in tissue. This phenomenon also seemsto apply in the gastric mucosa. A study was performed with azithromycin given to patients before stomach resection. Azithromycin concentration was determined at different time intervals. Concentrations of azithromycin of 2.27 mg/g were still present in gastric tissue h or more after oral administrationof 500 mg of azithromycin. The concentration in gastricmucus was much lower mg/g, standard deviation: 0-1.6) but still of potential interest (18). More recently, clarithromycin concentrations have been determined in gastric.mucosaof patients receiving this antibiotic alone or in association with omeprazole. Whereas in two instances clarithromycin concentration did not differ significantly the at antrum and fundus level, its concentration in the mucus increased by a factor of 10 when omeprazole was given also. Information is lackingfor the other macrolides.

H.pylori Resistance to Macrolides Recent studiesreported during this meeting by Versalovicet al. showedthe 2058 molecular basisof this resistance which is a point mutation in position or 2059 on the 23s ribosomal RNA genes. It has been shown for Mycobacteria, a genus with a singly copy of ribosomal RNA genes, that a point mutation is responsible for resistance. The best evidence now is that H. pylori has copies of ribosomal RNA genes and, despite this, resistance can occur. One mutation seems sufficientto cause macrolide resistance in H. pylori; this is contradictoryto the dogma that when multiple copies of ribosomal RNA genes are present, point mutations do not lead to resistance. Versalovicet al. reportedthe case ofa heterozygotestrain with a one point mutationon one gene andthe wild type onthe other gene which was resistant. Furthermore, the mutation seemsto be present whenthe MIC of the strain is 2 mgL(l9).

Campylobacter andpylori Helicobacter

243

0.5% 5 - 10%

10

4 I

-

Resistance of H. pylori to clarithromycinin European countries in1994.

Although the mechanism is most likelyto be the same for all strains, only 10 strains fromthe United States have been tested, it is important to study this problem further. The frequency of this mutation has been studied in the past and is foundto be in the range of lo-’ to which is lowerthan for nitroimidazoles (20). Based on this mechanism, it is easy to understand that macrolide resistance is definitive and not reversible, aswas wrongly proposed inthe past. There are different situations with regard to the level of this resistance in different parts of the world. The 1994 data presented during the meetings in Edinburgh and Berlin in 1995 are summarized in Fig.1. Three countrieshavealevel of resistance of approximately 10%: Belgium, France, and Spain. A level of 7% was reported in Hungary, but in other countries likethe United Kingdom,the resistance level is almost nil. These data most likely reflectthe common use of macrolides for respiratory infections in these different countries inthe past. It was noted in Belgium, for example, that the resistance rate was 1.7% before 1992 and it has risento 10.5%sincethen(21). A dramaticincreasewasobservedinSpainby Lopez-Brea (22). Baquero also reported at this meeting an increase from to 12% between 1987 and 1995 in Madrid. We have evaluated the resistance of all our strains isolated in Bordeaux since 1985 and were surprised to note that, during thisperiod, resis-

Butzler and Mdgraud

244

tancehasbeen in the range 8-11%; thisresistance rate waspossibly reached between 1982, the date of the recommendation to use macrolides for respiratory infections, and 1985 (14). The audience concluded that the resistance problem should be kept in mindbut that it does not seem to be a menacing problemat this stage.

Antibiotic Regimens to Eradicate H. pylori

The history of H.pylori treatment is short but already rich. In 1990, the firsteffective triple therapyincludingbismuthcompoundswasrecommended Because of side effects and poor compliance, alternatives were then proposed, especially bitherapies associating a proton pump inhibitor with an antibiotic. The best result was obtained with clarithromycin but the eradication rate did not surpass 75% and, therefore, was considered insufficient. Accordingly, triple therapiesare now favored, especially combinations ofproton pump inhibitorswith clarithromycin and amoxicillin or clarithromycin and metronidazole. These regimens allow a cure of the infection in more than90% of patients (Fig.2). However, a question debated during the session wasthe comparative advantages of bitherapy versus tritherapy. Bitherapies using clarithromycin (1.5 g/day) with aproton pump inhibitor during2 weeks leadto an eradica100

80

70 60 50 40

30 20 10 0

0-A 0 ”- C - A

Figure 2 Meta-analysis of the results obtained with different eradication therapies (based on data from patients). (FromRef. 24.)

Cumpylobacter and Helicobacter pylori

245

% eradication

"l

Figure 3 Eradication rates obtained in studies using clarithromycin as the only antibiotic during 14 days in ulcer patients. American trial (Ref. 25):(1) Clari = clarithromycin 500 mg tidyOme = omeprazole 40 mg;(2) Clari = clarithromycin 500 mg tid;(3) OME = omeprazole 40 mg. European trial(Ref. 26):(4) Clan = clarithromycine 500 mg tid, Ome = omeprazole 40 mg; (5) Ome = omeprazole 40 mg.

tion rate of 80% at best (Fig. whereas clarithromycin at a lower dose administered with amoxicillin and a proton pump inhibitor during only 1 week leads consistentlyto eradication rates higher than 90%. There was a consensus that the best way to prevent the development of resistance isto have the highest possible eradicationrate. With regardto bitherapies, different populations seem to have different response rates. For example, northernEuropeans tend to have higher eradication rates than southern Europeans. Because clarithromycin can inhibit omeprazole metabolism andtherefore improve the treatment efficacy, one hypothesis is that there is some variability in this interaction between populations. An interesting report is that clarithromycin given alone, despite an unsatisfactory eradication rate, was able to heal ulcers (27) and even perform better than antisecretory drugswith regard to pain relief. Obviously, suchdata need to be confirmed, butif they are, it would be additional proof of the infective nature of peptic ulcer disease.

Butzler and Mbgraud

246

At this stageit is stillthe rule to use an antisecretory drug, butwhich one is the best? Omeprazole and lansoprazole seem to be equally effective. The new compound, ranitidine bismuth citrate, which brings forth ranitidine activity has been tested with clarithromycin, but there has not yet been a head-to-head comparison between omeprazole and this compound. It is also difficultto compare the results of these studiesto others because only the rates of eradication in healed ulcers were considered. With .regard to in vitro data, some results concerning the association between roxithromycinandlansoprazolewerepresentedandshowed that there was an additive effect (28). Most of the invivo data have been obtained with clarithromycin; however, some studies have been performed with other macrolides. There were two multicenter studies using roxithromycin aspart of a triple therapy regime. Dammann et al. in Germany associated roxithromycin (300 mg bid) with metronidazole andthe H, antagonist roxatidine during 7-10 days and obtained eradication rates ranging from 78% to 90% (31). The same dose was used by Lamouliatte et al. in association with amoxicillin and lansoprazole during 2 weeks the andresult was a promising 85% eradication rate (32). Azithromycinwasusedin two studies in Eastern Europe. In the Czech Republic, Shonovaet al. used 250 mg/day of azithromycin during a week with omeprazole (85% eradication rate)(23), and in Croatia 1g/day during 6 days with ranitidine (68% eradication rate) (34). There was also one report on the use of spiramycin in association with metronidazole and lansoprazole during 2 weeks in children with a 72% successrate (35). The cost-effectiveness of this new triple-therapy regimen must be considered. All the studies performed have shown that eradication therapy is considerably cheaper than the traditional antisecretory drug therapy. Short-term triple therapies which were not considered the in study by Vakil et al. (36) are also cost-effective comparedto dual therapies andstandard triple therapiesor quadruple therapiesif the rate of eradication observedis as high asthe 90% reported. In conclusion, macrolidesand, in particular, clarithromycin have confirmed their important role in therapeutic regimens designed to eradicate H . pylori. At this stage, tritherapies seem the most efficient. They canbe used on a short-term basis, but there are still questions remaining, especially with regard to the possibility of expansion of antimicrobial resistance.

REFERENCES 1. Butzler JP, Glupczynski Y, Goossens H. Campylobacter and helicobacter infections. Curr Opin InfectDis 1992; 5:80-87.

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2. Mishu Allos B, Blaser M. Campylobacterjejuni and the expanding spectrum of related infections. Clin Infect Dis1995; 20:1092-1101. 3. Salazar-Lindo E, Sack RB, Chea-Woo E, Kay BA, Piscoya Early treatment with erythromycin ofCampylobacterjejuni-associated dysentery in children. J Pediatr 1986; 109:355-360. 4. Butzler JP, Levy J, Goossens H, Glupnynski Y ,De Mol P. Macrolides in campylobacter enteritis. In Macrolides, Chemistry, Pharmacology, and Clinical Uses. Bryskier A, Butzler J-P, Neu HC, lklkensPM, eds. Arnette-Blackwell, Paris:1993. 5. Sanchez R, Fernandez-Baca V, Diaz MD, Munoz P, Rodriguez-Creixems M, Bouza E. Evolution of susceptibilities of Campylobacter spp. to quinolones and macrolides. Antimicrob Agents Chemother 1994; 38(9):1879-1882. 6. Taylor DE, Chang N. In vitro susceptibilities of Campylobacter jejuni and Campylobacter coli to azithromycin and erythromycin. Antimicrob Agents Chemother 1991; 35(9):1917-1918. 7. Reina J, RosMJ, Serra A. Susceptibilitiesto 10 antimicrobialagents of 1,220 Campylobacter strains isolated from 1987 to 1993 from feces of pediatric patients. Antimicrob Agents Chemother1994; 38(12):2917-2920. 8. Endtz H P , Broeren M, Mouton RP. In vitro susceptibility of quinoloneresistant Campylobacterjejuni to new macrolide antibiotics.Eur J Microbiol Infect Dis 1993; 12(1):48-50. 9. Wang WL, Reller LB, Blaser UT. Comparison of antimicrobial susceptibility patterns of Campylobacterjejuni and Campylobacter coli.Antimicrob Agents Chemother 1984; 26(3):351-353. 10. Yan W, Taylor DE. Characterization of erythromycin resistance inCampylobacter jejuni and Campylobacter coli. Antimicrob Agents Chemother 1991; 35(10):1989-1996. 11. Funke G, Baumann R, Penner JL, Altwegg M. Eur J Clin Microbiol Infect Dis 1994; 13(7):612-615. 12. Mtgraud F,Lamouliatte H. Helicobacterpylori and doudenal ulcer. Evidence suggesting causation. Dig Dis Sci 1992; 37:769-772. 13. Schistosomes, Liver Flukes and Helicobacterpylori;IARC, Lyon, 1994. 14. Camou C, Ancelle J, Lamouliatte H, Mtgraud F. Evolution of the resistance of Helicobacter pylon to macrolides, The Third International Conference on the Macrolides. Azalides and Streptogramins, Lisbon, 1996;abstr 5-12. 15. Darmaillac V, Bouchard S, Lamouliatte H, MBgraud F. Effects of pH on the susceptibility of Helicobacter pylori to macrolides. The Third International Conference on the Macrolides, Azalides and Streptogramins, Lisbon 19%; abstr 5-13. 16. Ghoneim AT, Langdale P, Edmonds A, Green R, Reitmayer R, Flamm RK, Tanaka SK. Interpretive criteria for in vitro susceptibility testingof clarithromycin against Helicobacter pylori from a multicenter European clinical trial. The Third International Conference on the Macrolides, Azalidesand Streptogramins, Lisbon, 1996; abstr 5-05. 17. Graham DY, VersalovicJ, Flamm RK, Clamdge JE, Evans DG, Edmonds A, Cox S, Tanaka SK. Interpretive criteria for in vitro susceptibility testing of

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18. 19.

20. 21. 22.

23. 24.

25.

26.

27.

29.

Butzler and Mdgraud clarithromycin againstHelicobacterpylori from a multicenterUS clinical trial. The Third International Conference on the Macrolides, Azalidesand Streptogramins, Lisbon, 1996; abstr 5-03. Harrison JD, Jones JA, Morris DL. Azithromycin levels in plasma and gastric tissue, juice and mucus, Eur J Clin Microbiol Infect Dis 1991; 10:862-864. Versalovic J, ShortridgeD, Kibler K, GriffyW ,Beyer J, FlammRK, Tanaka SK, Graham DY, Go ME Mutations in 23s rRNA are associated with clarithromycin resistance in Helicobacter pylori. Antimicrob Agents Chemother 1996; 40. Haas CE, Nix DE, and SchentagJJ. In vitro selection of resistant Helicobacter pylori, Antimicrob AgentsChemother 1996; 34:1637-1641. Glupczynski Y, Goutier S, Van den Borre C, Butzler JP, Burette A. Surveillance of Helicobacterpylori resistance to antimicrobial agents in Belgium from 1989 to 1994, Gut 1995; 37(suppll):A56. Lopez-BreaM,Martinez MJ, Doming0 D, SanchezTomero I, Sanz JC, Alarcon T. Evolution of the resistance to several antibiotics in H.pylori over a 4 year period. Gut 1995; 37 (suppl 1):A97. Helicobacter pylori: causal agent in peptic ulcer disease? A working team report. Gastroenterol Hepatoll991; 6:103-140. Chiba N, Wilkinson JM, Hunt RH. Clarithromycin (C) or amoxicillin (A) dual and triple therapies in H. pylori (Hp) eradication: a meta-analysis.The Third International Conference on the Macrolides, Azalides and Streptogramins, Lisbon, 1996; abstr 5-26. Hunt R, Schwartz H, Fitch D, Fedorak R, AI Kawas F, Vakil N. Dual therapy of clarithromycin (CL) and omeprazole (OM) for treatment of patients with duodenal ulcers (DU) associated with H. pyloninfection. The Third International Conference on the Macrolides, Azalides and Streptogramins, Lisbon, 1996; abstr 5-18. O'Morain C, Logan RPH. the Clarithromycin European H. pylon Study Group. Clarithromycin (CL)in combination with omeprazole (OM) for healing of duodenal ulcers (DU), prevention of DU recurrence, and eradication of H.pylon (HP) in European studies. The Third International Conference on the Macrolides, Azalides and Streptogramins, Lisbon, 1996; abstr 5-25. Cave D, Vakil N, Hunt R, Graham DY, the clarithromycin H. pylori Study Group, Therole of clarithromycin without anti-secretorytherapy in the treatment of H. pylon and prevention of duodenal ulcer recurrence. The Third International Conference on the Macrolides, Azalides and Streptogramins, Lisbon, 1996; abstr 5-29. MBgraud F, Briigmann D, Darmaillac V. Roxithromycin-"ICs and bactericidal effecton Helicobacterpylori. The Third International Conference on the Macrolides, Azalides and Streptogramins, Lisbon, 1996; abstr 5-01. Bardhan KD, Dallaire C, Eisold H, Duggan AE. The treatment of duodenal ulcer with GR122311X (ranitidine bismuth citrate) and clarithromycin. The Third International Conference on the Macrolides, Azalides and Streptogramins, Lisbon, 1996; abstr 5-33.

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Peterson WL, Sontag SJ, Ciociola AA, Sykes CL, McSorley DJ, Webb DD. Ranitidine bismuth citrate (RBC) plus clarithromycin (CLAR) is effective in the eradication of Helicobacter pylori and prevention of duodenal ulcer relapse. The Third International Conference on the Macrolides, Azalides and Streptogramins, Lisbon, abstr Dammann HG, Walter TA. Roxithromycine, metronidazole and roxatidine acetate triple therapy andH. pylori eradication rates.The ThirdInternational Conference on the Macrolides, halides and Streptogramins, Lisbon, abstr Lamouliatte H, Coumer A,Mion F, MBgraud F, Rio Y,Reverdy ME, FlBjou JF, Topeza M. Roxithromycin mg bid in association with amoxicillin and lansoprazole on eradication of Helicobacter pylori: results of an open noncomparative multicentre study. The Third International Conference on the Macrolides, halides and Streptogramins, Lisbon, abstr Shonova Petr P, Hausner 0. hithromycin as a promising part of helicocidal regimens. The Third International Conference on the Macrolides, halides and Streptogramins, Lisbon, abstr Desnica B, Burek V, Makek N, Azithromycidranitidine combined treatment of H. pylori in patients with duodenal ulcer and chronic gastritis-a pilot study. The Third International Conference on the Macrolides, halides and Streptogramins, Lisbon, abstr Raymond J, Ralach N, Bergeret M, Benhamou P, Barbet P, Briet F, Flourie R, Senouci I, Gendrel D, Dupont C. A controlled studyof efficacy of lansoprazole in combinationwithtwodifferentdualantibioticassociationsduring Helicobacterpylori gastric infection in children.The Third International Conference on the Macrolides, halides and Streptogramins, Lisbon, abstr Vakil N, Fennerty B, Wi U. Economic modeling of medical therapy for H. pylori related peptic ulcer disease. The Third International Conference on the Macrolides, halides and Streptogramins, Lisbon, abstr

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20 Haemophilus influenme,Enterococci, and Anaerobes Vincent T. Andriole Yale University Schoolof Medicine New Haven, Connecticut

Ian Phillips United Medical and Dental Schools of Guy’s and St. Thomas’s Hospitals London, United Kingdom

The difficulties of in vitro susceptibility testing in relation to the MAS group of antibiotics in general, and this otherwise heterogeneous group of microorganisms in particular, were repeatedly stressed throughout the session. Wide discrepancies in results obtained byanyof the conventional susceptibility-testing methods may be related to differences in the media used, including subtle changes in the composition of what purports to be the same medium as well more obvious differences between media such as Haemophilus Test Medium, Mueller-Hinton medium with additives such as L-cysteine, hemin, horse or sheep blood, and on. Differences in pH, even within the NCCLS-recommended range, usually associated with incubation in atmospheres containing carbon dioxide which may be necessary for the adequate growth some these bacteria can have profound effects, particularly on macrolides and azalides. Finally, the sizeof the bacterial inoculum may affect results, evenfor otherwise robusttests such as the E-test (which is also affected by carbon dioxide). 251

252

.Phillips

Andriole and

HAEMOPHILUS INFLUENZAE

After considerable discussion of the technical problems associated with susceptibility testingof this organism, includingthe additional factor ofthe osmolarity of the medium for an organismthat readily produces L-forms, it wasgenerallyagreed that forerythromycin and, to alesser extent, clarithromycin (even allowing for potentiation by the 14-OH metabolite) minimal inhibitory concentrations (MICs) cluster around the generally recognized breakpoints. The same may be true for dalfopristidquinupristin when breakpoints are established. On the other hand, azithromycin and, on the basis of the preliminary results, the ketolide RU 64004 appear to have rather greater anti-gram-negative activity, and thusdo not pose quite the samedifficulty. It isindicative of the problems that Barry,having proposed higher breakpoints for clarithromycin, still felt that infections caused by strains identified as having intermediate sensitivity might be well treatable as easily as those caused by fully sensitive strains. It was generally agreed that high-level MAS resistance has not yet been convincingly demonstrated for H. influenzae, but there was concern that somestrainsmightbegenuinelylesssensitive to macrolidesand azalides,some of thembeingampicillin-resistantP-lactamase-negative strains, which are rare but do occasionally cause infection. It also appears that these strainsof border-line resistancemay not share withStrep. pneumoniae and Strep. pyogenes, the property of susceptibility to streptogramins. Several contributors feltthat although properly controlled susceptibility testing is essential for epidemiological purposes, it contributes little at present to the management of the individual patient. However, when MAS treatment of haemophilus infection fails, it is advisable to carry out full invitro susceptibility teststo identify resistance whenit occurs. For this purpose, it was suggested that the E-test might be useful. Results of the use of MAS inexperimental septicemia and pneumonia added the important dimension of differential pharmacokinetics to the discussion, and the importance of area under the plasma concentration versus time curve (AUC)/MIC as opposedto @la/MIC for some of these antibiotics was mentioned. Considerable amountsof clinical data were availablefor both clarithromycin and azithromycin, demonstrating their reliable microbiological and clinical efficacy in the treatment of patients with acute otitis media which (in antibiotic concentrations in middle-ear fluid do appear to be adequate), acute sinusitis, bronchitis, and lower respiratory tract infection in children. Clearly, H. influenzae is notthe only or even the most common pathogenin these conditions, but the overall good results imply that these macrolides are generally effective for this organism as well asthe others. However, we

Haemophilis influenzae,

Enterococci, and Anaerobes

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were reminded that at least a proportionof the patients with eachof these infections recovers without chemotherapy, although this may not be a practicable optionin the face of patients’ and parents’ expectations.

ENTEROCOCCI This session was introduced with an historical perspective of the emergence of multidrug-resistant enterococci. The following reflections were emphasized. Although the field of antimicrobial therapy has experienced many changes since the discovery of the sulfonamides more than 60 years ago, advances inour knowledge of molecular bacteriology have led to the development ofnewer and better antibacterial agents. New agents were designed to inhibit and kill bacterial pathogens by interfering with factors essential for bacterial survival and multiplication. Despite this progress in antimicrobialtherapy,bacterialpathogenshavebeenable to develop mechanismsof resistance,even to the newestclassesof antibacterial agents. One of our major problems today is the increasing incidence of serious infections caused by gram-positive bacteria, especially those that are now resistant to previously effective antibiotics. These gram-positive bacteria include strains of Staphylococcus aureus and coagulase-negative staphylococci resistant to methicillin, Streptococcus pneumoniae resistant to penicillin and macrolides, and enterococci, particularly, Enterococcus fuecium, resistant to vancomycin, gentamicin, and other antibiotics. Thus, there is an urgent need to develop new antimicrobial agents which will be effective inthe treatment of these life-threatening infections. The development of the streptogramins represents a new and unique class antibiotics, which inhibit protein synthesis at the ribosomal level and which are active against many gram-positive and gram-negative bacteria. Quinupristiddalfopristin (Q/D) currentlyis the mostdeveloped streptogramin. There wasconsiderablemeaningfuldiscussion about recent data, both in vitro and in vivo, which evaluated the potential efficacy of Q/D against enterococci, particularly against vancomycin-resistant Enterococcus faecium. Three issues were emphasized. The firstissueinvolved the role of erythromycinresistance of vancomycin-resistant Enterococcus faecium (VREF) as a predictorof Q/D susceptibility of these VREF strains. Although some data suggested that erythromycin-resistant strains of VREF were not killed in vitro by Q/D, other data suggested that erythromycin resistance had no impact on susceptibility of VREF strains to Q/D. This dichotomy was resolved favorably because two different definitions of erythromycin resistance were used in

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these studies. Specifically, high MICs to erythromycin correlated with loss of bactericidalactivity to Q/DagainstVREF,whereaslowerMICs to erythromycin (even though these MICs were relatively high) had no predi tive value of VREF resistance to Q/D. The second issue involved the clinical efficacy of Q/D onthe outcome of VREF bacteremia in surgical patients with comorbidities ofshock, dialysis, mechanical ventilation, liver failure, and immunocompromised state. Mortality was high in boththe Q/D treated patients andthe control group. However, the QD-treated cohort was less likely to die as a direct consequence of VREF infection, which suggested a beneficial effect of Q/D. However, larger clinical trialsare needed to determine the true efficacy of Q/D treatment. In this context, much discussion centered on the proper identification of VREF bacteremia. Specifically, blood cultures obtained from contaminated intravascular lines and defined as serious VREF bacteremia was considered an inappropriate definition for true VREFbacteremia because it would interfere with proper efficacy studies, not only of Q/D treatment but also for any new antimicrobial agent. We all agreed that proper technique was to obtain blood cultures from a site peripheral to an intravascular line. The third issue involved considerable discussion about the potential value of combination antibiotic therapy in the treatment of VREF infections because,in vitro, the combination of a new oral streptogramin, RPR with vancomycin or ampicillin enhanced the in vitro activity compared to either agent used alone. Additional benefit was also seen when RPR wascombinedin triplecombinations ofvancomycinplus gentamicin, or ampicillinplusgentamicin.Clearly,clinicalstudies are needed to assess the in vivo efficacy of combination therapy. All agreed that we need innovative approaches to hopefully treat serious VREF infections successfully.

ANAEROBES Again there was considerable discussion of the technical problems of susceptibility testing of anaerobes for these antibiotics.It was suggested that the rather high MICsobtained in a multicenterEuropean study might have been the result of the inactivating effect of Gcysteine, whereas those of other studies might have been adversely affected by carbon dioxide andthe consequent loweringof pH. It was agreed that MAS antibiotics are generally more active against gram-positive than gram-negative anaerobic species, although azithromycin is less imbalanced than the macrolides in this respect. Givenappropriate in vitro conditions,the ketolide RU and streptogramin combina-

Haemophilis influenzae,

Enterococci, and

Anaerobes

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tions such asdalfopristidquinupristinhave potentially usefulantianaerobe activity, as indeed do erythromycin and clarithromycin. Even the gramnegative anaerobes such asBacteroides fragilis are probably within therapeutic range. The intrinsically macrolide-resistant fusobacteria such as F. varium are resistant to RU but strainswith acquired resistancemay not be More information is needed on cross-resistance of anaerobic bacteria among MAS antibiotics. The report of bactericidal activity of the streptogramin antibioticRU for the single strainof B. fragilis tested as well as the considerable postantibiotic effect were noted. Perhaps paradoxically in view of in vitro results, azithromycin appears to be the only one of the new MAS agents to have been tested against experimental E. colilB. fragilis infections. Treated animals fared significantly better in terms of early mortality andlate abscess formationthan did untreated controls. We were reminded that erythromycin in combination with an aminoglycoside has a long and apparently successful history as a bowel “sterilizer’’ before major largebowel surgery forthe prevention of postoperative infection. Although this does not confirm erythromycin asantianaerobe an drug, as mostof these infections involve anaerobes as well aerobes, as it is an interesting pieceof collateral evidence.The best evidence proferred of the clinical efficacy of MAS antibiotics against anaerobic infections again paradoxically relatedto azithromycin. In a placebo-controlled trial, azithromycin was shownto be useful adjunctto standard dental maneuvers inthe treatment of periodontal disease, both in terms of the depth of pockets and in the reduction of counts of Porphyromonas gingivalis and spirochetes. Clearly,beforenew MAS antibiotics are accepted as good agents for the treatment of anaerobicinfections,moreexperimentalworkis required-and justified.

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21 The Newer Macrolides,halides, and Streptogramins for the Management of HIV-Associated Opportunistic Infections Kenneth H.Mayer Memorial Hospital of Rhode Island Pawtucket, RhodeIsland

J. Allen McCutchan University of California Schoolof Medicine San Diego, California

The discussion of the uses of macrolides and azalides (MAS antibiotics) to HIV-associated infections during the workshop was dividedinto three parts: . the first two focused on the treatment and prophylaxis of Mycobacterium avium complex (MAC) infections. The third area of discussion focused on MAS antibiotics for prophylaxis and treatmentof other opportunistic infections:toxoplasmosis, Pneumocystitiscarinii, bartonellosis(rochalimeae) and cryptosporidium.

TREATMENT OF MAC The MAC treatment discussionwas introduced bydefining therapeutic goals in microbiologic and clinical terms. Microbiologically,the goal is the elimination of both MAC bacteremia and tissue reservoirs, which have 257

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Mayer and

recently been shown to be both anatomically and quantitatively highly variable among patients at autopsy Clinically, the goal is reliefof the symptoms of fever, weight loss, diarrhea, and fatigue. Although multidrug regimens had reduced levels of circulating mycobacteria and symptomsof MAC (3,4), the recent introductionof clarithromycin dramatically improved and simplified the therapy of MAC (5), and azithromycin has also shown promise (6). The major limitation of monotherapy with these drugs is recurrence after a few months of MAC bacteremia with organisms resistant to both macrolides. An expert panel recommended that macrolides be used in combination ethambutol, with rifabutin, clofazimine, or other drugs with demonstrated activity against MAC (7). The effectiveness of this approachin prolongingthe control of MAC by clarithromycin had not been demonstrated, however, at the time of those recommendations. This question has been subsequently addressed in a seriesof comparative studies utilizing clarithromycin in combination with two or three additional drugs to suppress resistance. As presented at this meeting (8), the California Collaborative TreatmentGroup (CCTG) demonstrated that the addition of ethambutol delayed emergence of resistance, but a significant proportion of patients (about half of the responders by around nine months) still broke through with MAC bacteremia. In a complementary study, clofazimine had no effect when added to ethambutol and clarithromycin (5). About two-thirds of patients responded initially, regardless of the number of typeof drugs addedto clarithromycin, suggestingthat clarithromycin drives the dramatic clearance of MAC bacteremia. Thus, ethambutol appears to delay emergenceof clarithromycin-resistantMAC, but clofazimine does not. The high rate of recurrent bacteremias with clarithromycin-resistant MAC documented by the CCTG study necessitates acontinuedsearch for amore potent seconddrug to augment clarithromycin intreatment of MAC bacteremia. During the discussion, Dube addressed several unsolved. technical problem in MAC treatment studies: the definitions of both response and relapse bymicrobiologicandclinical criteria. He showed that twomicrobiologic definitions of relapse utilized in the CCTG trial led to substantiallydifferingestimates of the rates of relapse by Kaplan-Meier survivalanalysis. The first(protocol-defined)methodwhich required sustained recurrence of bacteremia appeared to seridemonstration ously underestimate the rate of relapse (only 5% relapsed on clarithromycinplus ethambutol and clofazimine). A second(posthoc)method (i.e., any positive blood culture for MAC after any negative culture in patients who responded to treatment) appeared to be moreclinically relevant (about half of patients relapsed) but might not be capable of

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distinguishing relapses due to poor compliance from those due to failure of treatment. A third method based on the isolation of clarithromycin-resistant MAC from blood may be preferable, as such breakthroughs presumably reflect failure of sustained treatment, rather than noncompliance. In other words, only patients who fail during a sustained exposure to clarithromycin should relapse withclarithromycin-resistant MAC. Noncompliant patients would be more likelyto relapse withclarithromycin-sensitiveMAC. In fact, the third method agreed closely in the CCTG study with the second microbiologic definitionof relapse: a positive blood culture following a negative culture. Thus, recurrent macrolide-resistant MAC bacteremia may be the best measureof true treatment failure, and recurrent macrolide-sensitive MAC may measure noncompliance withtreatment. Another methodological problem has been to identify clearlythe clinical manifestationsof relapse in the face of multiple other potential causesof fever, wasting, and diarrhea in advanced AIDS patients. Future studies will also need to evaluate the role of other new drugs to add either to clarithromycin or azithromycin for improving the duration of treatment responses centered on the aminoglycosides (e.g., amikacin), quinolones (e.g., ciprofloxacin and sparfloxacin), newer rifamycins (e.g., rifabutin and KR”1648), and immune modulators (such as gamma interferon andGM-CSF). ACTGstudy is currently examining rifabutin, ethambutol, and both drugs added to clarithromycin for MAC treatment.

PROPHYLAXIS OF MAC Issues of prophylaxis of MAC were introduced by a comparison of the regimens for which substantial evidence of efficacy has been developed. The first, rifabutin mg, was the original drug approvedfor this indication (9). The three other regimens were discussed in abstracts of this conference (8, 10-18). Clarithromycin is licensed inthe United States for treatment for MAC and is widely used for prophylaxis (500 mg) based on a large,placebo-controlledstudy (13). Inaddition,twotrialscomparing azithromycin 1.2 g weekly either to placebo (19) or to rifabutin alone and to the combination of rifabutin and azithromycin(16) have establishedthe value of weekly azithromycin for MAC prophylaxis. Daily clarithromycin and weekly azithromycin appearto have similar efficacy(about 70%) and are superior to daily rifabutin (50% efficacy). In order to compare the four regimens, the annual costs of prophylaxis for 100 patients was divided by the estimated number of cases pre-

Mayer and McCutchan

260 Table I

Agent

Candidate Regimens for MAC Prophylaxis in AIDS Other Costlcase Annual Cases mstb prevented8 Efficacy

benefits prevented ~

Rifabutin 300 mg qd Clarithromycin 500 2O1,94gc21 Azithromycin 70% 1200 Azithromycin 85 PCP 1200 qw, and rifabutin 300 mg

50%

%

15

297,367

19,842

21

355,741

16,940 respir. PCP infect. ? Giardia 9,617 Respir. infect.,

25.5

Respir. 19,580 499,315

?

infect.,

.Annually per 100 patients assuming per annum incidence in target population (< 100 CD4 cells). bEstimated pharmacy dispensingcost (“Red Book” wholesale cost of drug + 30% + $8.50 dispensing fee every 2 months) for 100 patients. CBased on price of 2 600-mg tablets approved for prophylaxis.

vented in 100 patients to generate the cost per case prevented (Table 1). The number of cases prevented per 100 patients is calculated by multiplying the annual incidence of MAC in patients with CD4 < 100 (approximately 30 cases/100 patientdyear) times the prophylacticefficacy(e.g., 50% for rifabutin, 70% for either azithromycin or clarithromycin, and 85% for azithromycin plus rifabutin). The costs for each drug were calculated using wholesale acquisition costs fromthe ‘Red Book‘ to which pharmacy dispensing costsare added as outlinedin footnote b of Table 1. The cost of azithromycin was based on the 2 600 mg, lactose-free capsules used in the Pfizer/CCTG study. Costsper case of MACprevented for daily clarithromycin($16,940) and for daily rifabutin ($19,842) are higher than for weekly azithromycin ($9,617). The most effective regimen, the combination of azithromycin weekly and rifabutin, is more costly ($19,580 per case prevented) and requires more than four times as many capsules per week as azithromycin given weekly for monoprophylaxis. In addition to the issues of cost, both rifabutin and clarithromycin have potential drug interactions with multiple drugs frequently taken by advanced AIDS patients. For example, fluconazole increases rifabutin conce trations which can result in anterior uveitis (20). Clarithromycin increases levels of theophylline, carbamazepine, and terfenadine. Thus, the use of macrolides for prophylaxisof MAC inAIDS appears well established, and initial estimatesof costs and benefits favors weekly azithromycin over other currently licensed drugs. More sophisticated pharmacoeconomic analysis

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taking into account benefitsof these drugsfor prophylaxis of other opportunistic infections andthe costs of drug interactions shouldbe undertaken to better estimate their total impact.

OTHER OPPORTUNISTIC INFECTIONS Although the combination of the sulfa drug plus a folic acid antagonist has been shown to be effective inthe treatment of HIV-infected patients who develop cerebral toxoplasmosis, relapses are not uncommon and intolerance to a one-drug regimen maybe particularly frequent in HIV-infected persons The dose-limitingtoxicity of pyrimethamine maybe leukopenia, whichmay or may not respond to treatment with colonystimulating factors, and many HIV-infected patients become sulfonamide intolerant over time-however, many will respondto desensitization protocols. The second-line treatment agents,suchasclindamycin,mayalso result in unacceptable toxicities, such as refractory diarrhea. Newer regimens may include drugs like atoviquone. However, the experience of using spiramycin in the treatment of nonimmunocompromised patients with a variety of toxoplasmosis syndromes has raised the question of whether the macrolides alone or in combination withother drugs may constitute effective therapy for HIV-infected patients with cerebral toxoplasmosis. Initial reports indicated that failures were seen in HIV-infected patients who received monotherapy witheither spiramycin or roxithromycin (24). Several patients have been reported to have developed toxoplasmosis whilereceivingclarithromycin Subsequent studies have suggested that pyrimethamine plus clarithromycinwas comparable to pyrimethamine plus clindamycin However, complete responses were noted in only about 50% of patients in both treatment groups, and patients who failed this regimenreported anemia, nausea, and increased liver function tests, as well as hypoacusis. At the current conference, Brun-Pascaud presented a poster which indicated that in a rat model, roxithromycin plus dapsone or in combination with a sulfonamide was active against both Pneumocystis carinii pneumonia(PCP)andtoxoplasmosis.Thisfindingraised the hope that the combination of a macrolide plus a sulfa, sulfone,or pyrimethamine could result in prophylasis against three very common opportunistic infections (i.e., PCP,toxoplasmosis,and M. avium-intracellulare infection). Craft, Crampton, and Henry presented several posters at the conference suggesting that among participantsin a placebo-controlled studyof clarithromycin 500 mg twiceper day to perventM. avium infection, there was significantly lessPCP versus 10% in the placebogroup,p-value = .021). In

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addition, there was less community-acquired pneumonia (7% versus 13%, p = .Ol), as well as less giardiasis(0.9% versus p = .048). However, the prevalenceoftoxoplasmosiswasnotshown to be prophylaxis of M. aviumsignificantlyreducedin the Craftstudy intracellulare infection with clarithromycin; but it is importantto note that the study was done in North American where the prevalence of toxoplasmosis antibodies is significantly lower than in Europe. It is possible that future prophylaxis studies conducted overseas might be ableto show some added benefit from the use of a macrolide for the prophylaxis or other opportunistic infections. However, atthe present time, the data are not sufficiently strong to suggest the use of a macrolide as first-line treatment for toxoplasmic encephalitis, or for the prophylaxis of patients who have positive toxoplasmosis antibody titers andlow CD4 counts. However,inthosepatientswho are notable to tolerate sulfonamides or pyrimethamine or who progress despite appropriate first-line therapy, the next choice wouldbe clindamycin. However, there is a substantial number people livingwith HIV whomay not be able to tolerate long-term clindamycin therapy because of diarrhea, and for those individuals, the useof amacrolideincombinationwith other activeantitoxoplasmosis agentsmaybeuseful. Another alternative wouldbe the use of atovaquone, and further studies have to be done on alternative salvage therapies for patients who do not respond to pyrimethamine/sulfadiazine. The macrolides have also been shown to have some efficacy against cryptosporidiosis in animal models (27). However, studies by Soave and colleagues have shown partial responses with monotherapy using azithromycin at high daily doses (up to 1.8 g per day), with frequent relapses (28). The useof intravenousmacrolidetherapy for thisopportunistic cause of diarrhea is also undergoing study. Another study by Blanshard and colleagues revealedthat azithromycin given 1 g as a loading dose and then 500 mg per day did not decrease the number of stools per day or oocyst burden (29). However, at the conference, roxithromycin given 300 mg twice per day for 4 weeks resulted in complete responses in two different studies from Brazil (Sprinzet al. and Uip et al.) and partial responses inabout an additional quarter of individuals. The authors indicated that in patients for whom there was not anappropriate clinical response,or when the response was onlypartial, they triedother macrolides such as azithromycin, occasionally with success, and alsoadded other active agents, such as paromomycin The consensus at the meeting was that more efficacious therapy of cryptosporidial diarrhea could possibly be achieved by the addition of a macrolideplusparomomycinand that thiscombinationwarrants more careful critical study.The discussion also notedthe high degree of variabil-

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ity with regardto the syndrome associated with cryptosporidial enteritis, in that in patients with higher CD4 counts who developed cryptosporidiosis, the disease may be self-limited or be more likely to respond to a short course of an oral macrolide; in other individuals with extremely lowCD4 counts, the likelihood that the macrolide could be adequately absorbed and efficacious would be less likely. Thus, the interpretation of nonrandomked, carefully controlled clinical trials is difficult, although the data presented the hope that the newer macrolides could play a role in the management of cryptosporidial diarrhea inAIDS patients. The workshop concluded with an increased awareness that the macrolides are highly efficacious for many diverse HIV-associated opportunistic infections. Thus, verysoon, virtually all patients with advanced HIV infection will likely receivea macrolide aspart of their prophylaxisor treatment regimen (31). This will raise many confusing management questions in the future if clinical illnesses such as M.avium-intracellulare, toxoplasmosis, or cryptosporidiosis occur while they are receiving an MAS antibiotic. By ICMAS IV, hopefully there will be new reports as to how macrolides may be used in future prophylaxis and therapeutic regimens. In addition, at this conference other novel uses of the newer macrolides were discussed, such as the treatment of bacillary angiomatosis (Paucaret al.) andother atypical non-MAC mycobacterial infections. Thus, the use of the newer macrolides can be expected to continue to increase in HIV-infected populations over the next few years, with precise indications being further refined as clinical trials are completed and clinical experience matures.

REFERENCES 1. Tbrriani FJ, McCutchan JA, Bozzette SA, Grafe M R , Havlir DV. Autopsy findings in AIDS patients with mycobacterium avium complex bacteremia. J InfectDis 1994; 170:1601-1605. 2. Tomani FJ, Behling CA, McCutchan JA, Haubrich RH, Havlir DV. Disseminated mycobacterium avium complex: correlation between blood and tissue burden. J Infect Dis1996. 3. Chiu J, Nussbaum J, Bozzette S, Tiles JG, Young LS, Leedom J, Heseltine NR, McCutchan JA, the California Collaborative Treatment Group. Treatment of disseminated mycobacterium aviumcomplex infectionin AIDS with amikacin, ethambutol, rifampin, and ciprofloxacin. Ann Intern Med 1990; 113~358-361. Bartok AE, Leedom 4. Kemper CA, Meng TC, Nussbaum J, Chiu J, Feigal DF, JM, Tilles JG, Deresinski SC, McCutchan JA, the California Collaborative Treatment Group. Treatment of mycobacterium avium complexbacteremia in AIDS with a four-drug oral regimen. Ann Intern Med 1992; 116:466-472.

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5. Chaisson RE, Benson CA, Michael DP, Leonid BH, Korvick JA, Elkin S, Smith T, CraftC, Sattler FR. Clarithromycin therapyfor bacteremic mycobacterium avium complex disease: a random, double-blind, dose-ranging study in patients with AIDS. AnnIntern Med 1994; 121:905-911. 6. Young LS, Wiviott L,Wu M, KolonoskiP, Bolan R, Inderlied CB. Azithromycin for treatment of mycobacterium avium-intracellulare complex infection in patients with AIDS. Lancet 1991;338:1107-1109. 7. Mazur H, etal. SpecialReport: recommendations on prophylaxis andtherapy for disseminated mycobacterium avium complex disease in patients infected with the human immunodeficiency virus.N Engl J Med 1993;898-904. 8. Dube M, Sattler F, Torriani F, See D, Havlir D, Kemper C, Dezfuli M, Bozzette S, Bartok A, Leedom J, Tiles J. McCutchan J. Clarithromycin plus clofazimine, withor without ethambutol for the prevention of relapse of MAC bacteremia in AIDS. In: Program and Abstracts of the Third International Conference on Macrolides, Azalides and Streptogramins, Lisbon, 1996;abstr 7.07. 9. Nightingale SD, Cameron W, Gordin FM, Sullam PM, Cohn DL, Chaisson RE, et al. lbo controlled trials of rifabutin prophylaxis against mycobacterium avium complex infection in AIDS. N Engl J Med 1993; 329:828-833. 10. Miller DA, Clarithromycin:safeandeffectiveprophylaxisagentagainst MAC. In: Program and Abstracts of the Third International Conference on Macrolides, Azalides and Streptogramins, Lisbon, 1996; abstr 7.01. 11. Sinnott JT, HoltDA, Houston H, Bergen G, Sakalosky P, Larkin J. Clarithromycin 500 mg BID as prophylaxis for MAC disease: a follow-up review. In: Program and Abstractsof the Third International Conference on Macrolides, Azalides and Streptogramins, Lisbon, 1996; abstr 7.02. 12. Grieger-Zanlungo P, Zev C. Clarithromycin 500 mg BID for dMAC prophylaxis. In: Program and Abstracts of the Third International Conference on Macrolides, Azalides and Streptogramins, Lisbon, 1996; abstr 7.03. 13. Pierce M, Crampton S, Henry D, Craft C, NotarioG. Theeffect of MAC and its prevention on survival in patients with advanced HIV infection. In: Program and Abstracts of the Third International Conference on Macrolides, Azalides and Streptogramins, Lisbon, 1996; abstr 7.04. WH, IndorfAS. 14. File TM, Klippel DC, LongstrethSJ,SignsDJ,Ruby Clarithromycin prophylaxis for disseminated mycobacterium avium (dMAC). In: Program and Abstracts of the Third International Conference on Macrolides, Azalides and Streptogramins, Lisbon, 1996; abstr 7.05. 15. Craft J, Henry D, Olson CA, Notario G, Hom R, Crampton S, Pierce M. Prevention of resistance to clarithromycin during prophylaxis for disseminated mycobacterium avium complex (MAC). In: Program and Abstracts of the Third Internationql Conferenceon Macrolides, Azalides and Streptogramins, Lisbon, 1996; abstr 7.06. 16. Havlir DV, Dube M P , Sattler FR, et al. Prophylaxis against disseminated Mycobacterium avium complex with weekly azithromycin, daily rifabutin or both. N Engl J Med 1996;335:392-398.

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17. Crampton S, Craft JC, Notario G, Henry D. Prevention of pneumocystis carinii pneumonia in AIDS patientsby clarithromycin prophylaxis for mycobacterium avium complex (MAC). In: Program and Abstracts of the Third International Conference on Macrolides, Azalides and Streptogramins, Lisbon, 1996;abstr 7.09. 18. Crampton S, Craft JC, Henry D. Clarithromycin prophylaxis for MAC in reduces the incidence of AIDS patients with CD4 counts < 100 ~elldmrn-~ community-acquiredpneumonia.In:ProgramandAbstracts of the Third International Conference on Macrolides, Azalides and Streptogramins, Lisbon, 1996;abstr 7.10. 19. Oldfield et al. Third National Conference on Retroviruses, Washington,DC, 1996. 20. Havlir D, Torriani F, Dube M. Uveitis associated with rifabutin prophylaxis. Ann Intern med 1994; 121510-512. 21. Richards FO, Kovacs JA, Luft BJ. Preventing toxoplasmic encephalitis in persons infected with human immunodeficiency virus. Clin Infect Dis 1995; 21(~~ppl):S49-S56. the acquired immuno22. Luft BJ,et al. Toxoplasmic encephalitis in patients with deficiency syndrome. N Engl J Med 1993; 329:995-10oO. 23. Porter SB, Sande MA. Toxoplasmosis of the central nervous system in the acquired immunodeficiency syndrome. N Engl J Med 1992; 327:1643-1648. 24. Saba J,et al. Pyrimethamine plus azithromycin treatment for of acute toxoplasmic encephalitis in patients with AIDS. Eur J Clin Microbiol Infect Dis 1993; 12~853-856. 25. Ruf B, Schurmann D, Pohle HD. Failure of clarithromycin in preventing toxoplasmic encephalitis in AIDS patients (letter). J Acquired Immune Defic Syndr 1992; 5530-531. 26. Leport C, et al.Combination of pyrimethamine-clarithromycin for acute therapy of toxoplasmic encephalitis. A pilot study in 13 AIDS patients. 30th ICAAC, 1990;abstr 1158. 27. Blanshard C, et al. Cryptosporidiosis in HIV-seropositive patients. Quart J Med 1992; 85307-308. 28. Soave R, et al. Oral diclazuril therapy for cryptosporidiosis. Abstract of the VI International Conference on AIDS, San Francisco, 1990. 29. Blanshard D, et al. Azithromycin, paromomycin and letrazuril in the treatment of cryptosporidiosis. Third European Conference on Clinical Aspects and Treatmentof HIV Infection, Pans, 1992;abstr P28. 30. White AC, et al. Paromomycin for cryptosporidiosis in AIDS: a prespective double-blind trial. J Infect Dis1994; 170:419-429. 31. Centers for Disease Control:USPHSLIDSA Guidelines for the prevention of opportunistic infections in persons infected with Human Immunodeficiency Virus. MMWR 1995; 44:l-24.

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22 Special Pathogens John E. McGowan, Jr. Emory University Atlanta, Georgia

Joel Ruskin Kaker Permanente Medical Center andUCLA School Los Angeles, California

Medicine

The development of new macrolides, azalides, and streptogramins has led to unique and unusual applications of these antimicrobial agents. There was extensive discussion in the seminar regarding how best to utilize the drugs withinthe clinical context ofcurrent therapeutic options, what advantages new regimens would offer in terms of increased efficacy, safety and tolerance, and whether the new data presented at the meeting might, in fact, alter the treatment of a varietyof infectious diseases.

HUMANAND ANIMAL BITE WOUND INFECTIONS Human and animal bites may cause a wide spectrum tissue damage, ranging from superficial breaks in the integument to cellulitis, septic arthritis, and osteomyelitis. In an emergency-room setting, the bite victim is often treated empirically with an oral antibiotic. The drug selected should possess activity against the polymicrobial flora most likely to cause such an infection. The culpable bacterial species include streptococci, staphylococci 267

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(usually S. aureus), Pasteurella multocida (especially important in cat bites), oral anaerobes (Prevotellu and Porphyromonas spp. are common, whereas Fusobacterium nucleatumis not), and Eikenellu corrodens. Amoxicillidclavulanate is generally consideredthe best choice to cover infection with these organisms. However, penicillin-allergic patients need alternative treatment. Goldstein and Citron (Santa Monica, CA) evaluated the in vitro efficacy of the newer macrolides azithromycin, clarithromycin, and roxithromycin against isolates commonly cultured from bite wounds. Azithromycin was foundto be the most active against many of the aerobes, including R multocida [minimalinhibitoryconcentration for 90% of isolates (MI(&,) < 2pg/ml] and E. corrodens, and was 2 to 4 dilutions more active thanerythromycinversusanaerobes.Overall,clarithromycinwasless activethanazithromycin,androxithromycinwasevenlessactive than erythromycin. Neither Goldstein nor others in the workshop had substantive clinical experience with the newer macrolides in bite wound management. Yet, in light of the above in vitro data, there was consensusthat at least azithromycin merited clinical evaluation in the penicillin-allergic patient. However, two caveats were stressed. First, in the emergent care of bite wounds, particularly clenched-fist injuries, copious irrigation followedby elevation of the affected extremity is essential for cure; no antimicrobial is likely to be of benefit without such intervention. Second,if the bite wound extends to bone and osteomyelitis ensues, the consequences may be devastating. In this scenario (which is not uncommon when the infection is due to P. multocida), it is probably bestto avoid therapy with any ofthe macrolides.

LISTERIA AND CORYNEBACTERIA

A concern of the 1990s is the growing microbialthreat posed byresistant croorganisms. Bacteria resistant to p-lactam drugsare now seen to be resistant as well to glycopeptides. Among these are the Corynebacteria, which have begunto play an increasingly important role as nosocomial pathogens. Corynebacterium jeikeiumis the most common organism of thisgroup and is often associated with intravascular infection. In studies of this bacterium, Holloway and associates (Wilmington, DE) determined its in vitro susceptibility to the streptogramin quinupristiddalfopristin (RP59500).Susceptibility to quinupristiddalfopristin could addto our therapeutic armamentarium an alternative drug for strains resistant to the current antibiotics of choice, vancomycin andthe quinolones. The early stage of these investigations demonstrated differencesin in vitro susceptibilityof the organism to quinupristiddalfopriistin when testing was performed in glass tubes asop-

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posed to plastic containers. Ultimately, the laboratory test system to be used must be defined by a clinically relevant gold standard. However, none is yet available, as few patients have beentreated with this drug. Listeriu monocytogenes, another organism of nosocomial importance, wasconsideredinpresentationsfrom the United States andGermany. Variable in vitro susceptibility to quinupristiddalfopristin was noted in different test media. The study from Hof and colleagues (Mannheim,Germany) suggested that this drug, as well as clindamycin and other macrolides, are much less active against Listeria when present inside cells with an efflux pump. The clinical impact of these fascinating observations remains to be determined.

BRUCELLOSIS Brucellosis, although uncommon in the United States, still exists worldwide, especially in the Middle East, the Indian subcontinent, and in parts of Mexico and Central and South America. Rubinstein and colleagues (Tel Aviv, Israel) reported findingsfromamurinemodelofinfectionwith Brucella melitensis. In Rubinstein’s view, the model mimics the subacute, disseminated granulomatous infectionin man, where brucellae localizein organs (viz. liver and spleen) rich in elements of the reticuloendothelial system. In the murine model and in man, combination antibiotic regimens have been shownto be the optimal therapyof Brucella infection. Excellent results have been achievedwith tetracycline plus streptomycin,or tetracycline plus rifampin. Single-drug treatment, notably with cotrimoxazole or the quinolones, has been unsuccessful. It was proposed that single-drugtherapywith one of the newer macrolides might yieldbetter results. To test this hypothesis in their murine model of the infection, Rubinstein and his colleagues administered azithromycin or clarithromycin for 7-14 days, beginning 7 days after intraperitoneal inoculation of B. melitensis. Azithromycingiven for either 1 or 2 weekswasnearly100%efficacious.Moreover,azithromycinprevented relapse for a 4-week follow-up period. other antimicrobial previously tested in this model produced such dramatic results. Rubinstein believes these data warrant a clinicaltrial of azithromycin therapy of human brucellosis. Infected infants and pregnant women, in whom the use of tetracyclines is precluded, would be desirable subjects. On the other hand, he was less sanguine about the use of azithromycin or macrolides at this time for the treatment of the osteoarticular complications of brucellosis. Unfortunately,the latter have been notoriously refractory to protracted courses of standard drug combinations, including those that incorporate parenteral streptomycin.

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PERTUSSIS Whooping cough continuesto be an important causeof infectious morbidity in New Zealand. Brett and co-workers at the Communicable Disease Center (Porirua, NZ) wereinterested, therefore, in assessingthe activity of roxithromycin andother drugs against recent isolates there. Roxithromycin was found to bebactericidal at easilyachievableconcentrations.They noted inthe discussion that clinical trialsof this drug, a more easily administered alternative to erythromycin, are planned. InZagreb, Croatia, Bace et al. used azithromycin in a clinical trial in children with pertussis. Bacteriologic eradication byday 7 wasseeninall15children treated, and no adverse effects were seen. They suggested to the group that the advantage of the drug in preference to erythromycin (shorter treatment course and thus better patient compliance) madefurther evaluation of azithromycin in whooping cough reasonable.

SALMONELLOSIS Multidrug-resistant salmonellae, includingS. typhi, are being isolated with increasing frequency throughout the world. Treatment regimens with chloramphenicol, ampicillin,or cotrimoxazole are no longer reliable. The quinolones (notably ciprofloxacin) have been utilized with some success, but concerns remain about their use in children and pregnancy. Azithromycin is known to achieve huge intracellular concentrations in intestinal lymphoid tissue, where salmonellae reside and multiply. Kelneric and co-workers from a veterinary institute (Zagreb, Croatia) studied the activity of azithromycin and erythromycin against animal isolates of S. enteriditis and S. typhimurium, as well as against isolates of S. virchow and S. typhi recovered from humans. More than 90% of the 67 strains tested were inhibited by azithromycin at concentrations between 2 and 4 &ml, whereas high-level resistance to erythromycin was demonstrated. Rubinstein and Ruskin noted that there is already-published salutary human experience withazithromycinin treatment of salmonellagastroenteritis as well as typhoid fever. In viewof these supportive in vitrodata from Croatia, azithromycin and possiblyother new macrolides merit testing in large-scaletreatment trials of clinical salmonellosis.

MALARIA Malaria remains a leading cause of morbidity and mortality in the developingworld; 100-300millioninfectionsand1-1.5million deaths occur yearly. The highly developed world is by no means immuneto this “tropi-

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cal” disease; in 1994, 1229 cases werereported in the United States. New and safe prophylactic agents effective against R falciparum are urgently needed. Currently, mefloquine is recommended for some settings and is about 90% effective. However, mefloquine-resistant strains already exist and are likely to spread withincreasinguseof the drug.Moreover, mefloquine has disadvantages: It is expensive and is not recommended in pregnancy, in infants weighing
BARTONELLOSIS Although cat-scratch disease (CSD) has been a well-known clinical syndrome for decades, bacillary angiomatosis (BA) is a relative newcomer. Commonly seen in HIV-infected patients, BA is characterized by cutaneous and subcutaneous vascular lesions that (like lymph nodes in CSD) contain silver-staining bacilli. More recently, BA lesions have been found in virtually every organ system, including lymph node, bone, brain, liver (peliosis hepatitis), and spleen. Through novel genotypic methodology, the etiologic organisms of CSD and BA have been shownto be identical. Bartonella (formerly Rochalimaea) henselae appears to cause CSD and BA, and sometimes bacteremia or endocarditis. B. quintana also causes BA, aswellasclassical “trenchfever,“bacteremia,andendocarditis. Another related organism, B. elizabethae, has been isolated from an immunosuppressedindividualwithendocarditis.Given that bacteria-like forms had been visualized in lesions of CSD and BA (before cultivation and speciation had been achieved), a host of antibacterial agents have

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beenemployedempirically to treat bothconditions.Aminoglycosides, chloramphenicol, quinolones, cotrimoxazole, and erythromycin have exhibited variable efficacy; p-lactams have generally been less active. Ives and colleagues (Chapel Hill, NC and Atlanta) determined the in vitro susceptibility of the three Bartonella species to erythromycin and newer macrolides (azithromycin, clarithromycin, dirithromycin, roxithromycin). Antibacterial activitywas measured in cell cultures, utilizing indirect immunofluorescent staining with Bartonella-specific fluorescent IGg antibody. Azithromycin had the lowest minimal inhibitory concentration (MIC,,) and azithromycin, clarithromycin, and roxithromycin the hadlargest area under the curve of plasmaconcentrationversustime (AUC)/ MIC,,. However, all the tested agents were deemed active in vitro against the three organisms. It was emphasized that, at least for erythromycin, these resultsare not necessarily indicativeof clinical efficacy. Mostpatients with CSD improve spontaneously, regardless of treatment, whereas immunoincompetent patientswith BA sufferfrequent relapses. It is thought that intracellular growth of Bartonella and the inhibitory rather than bactericidal activity of the drugs commonly used heretofore (e.g., tetracycline or erythromycin) may account in part for the clinical relapses and failures observed. It was speculated that protracted therapy withthe newer macrolides and azalides, drugsthat attain high intracellular concentrations,may yield superior clinical outcomes. Thus, studies with these agents in BA (and possibly CSD) seem warranted.

GONORRHEA Many countries today report a high prevalence of p-lactamase-producing strains of the gonococcus.Thishasled to useof fluoroquinolones or broad-spectrum cephalosporins as first-line drugsfor treatment. However, the increasing appearance of isolates of Neisseria gonorrhoeae resistant to the quinolone drugs, especially ciprofloxacin and ofloxacin, has led to a search for treatment alternatives. In this context, a clinical trialby Gruber et al. (Rijeka, Croatia) compared azithromycin to ciprofloxacin for therapyofgonococcalinfection.Frequency of cure and adverseeffects were similar for both drugs. Because the study was conducted in wartime in a busy port city with transient naval and merchant marine personnel, follow-up to ascertain treatment effectiveness was quite difficult. Nonetheless, in thissetting, the ability to use a single 1 g dose of azithromycin for treatment bothgonorrheaandchlamydialinfection was felt to be highly advantageous.

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CONCLUDING REMARKS The other workshopsweredesigned to summarize data about specific pathogen(s) and related infections. In contrast,our seminar dealt with an array of apparently disparate infectious diseases. Initially, it appeared that there would be little linkage between conditions as diverse as bite wounds and malaria, or whooping cough and bacillary angiomatosis. However, a common theme soon emerged. Each infection has become increasingly resistant to "traditional" therapeutic regimens. It is abundantly clear that we mustfindnew (or modify existing) antimicrobial agents in order to regain control over many of these endemic and nosocomial infections. In this regard,the newer macrolides and reformulated streptogramins are of great interest. Although the studies presented here are preliminary, and largely elucidate the in vitro efficacy of these compounds, their broad spectrum of activity is remarkable. There was consensus that several clinical trials with these agents should start as soon as possible. It is hoped that the results of such studies will be fruitful and availablefor presentation at the next ICMAS.It may turn out that we have only just begunto realize the full therapeutic potential of the macrolides and streptogramins.

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EXTENDED ABSTRACTS

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I NEW AGENTS

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Ketolides: A New Class of Macrolide Antibacterials-Structural Characteristics and Biological Propertiesof RU 004 Constantin Agouridas,Y. Benedetti, A. Bonnefoy, P. Collette, A. Denis, P. Mauvais, G. Labbe, and Jean-Francois Chantot Roussel-Uclaf Romainville, France

INTRODUCTION Macrolides are a well-known family of oral antibiotics. Their spectrum of activity covers allthe relevant strains responsiblefor URTI and LRTI. In contrast with the p-lactam family, they also display strong activity against atypical bacteria, an increasing causeof LRTI. The recent recognition of penicillin-resistant S. pneurnoniue and its epidemic spread in some areas (10-40% worldwide except in the United Kingdom and Scandinavia) has caused justifiable concern and focused attention on therapeutic alternatives. Clearly,botherythromycinand the newercompounds(roxithromycin, clarithromycin, azithromycin, and dirithromycin) are among the alternative agents to be considered. Unfortunately, penicillin-resistantS. pneurnoniae also may demonstratemultiple-drugresistance,includingresistance to macrolides. Furthermore, resistance to macrolides has increased among S. pneurnoniue in several areas of the world but levels as high as 60% have been reported in certain areas). 279

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280 ...............................................

RU

"P

RU 252

Top: induction experimentsin S. aureus. The inhibitory concentration of ERY was 50 pg/ml. The inducible subinhibitory concentrations were 0.06, and pg/ml for ERY, RU004,and RU respectively. Bottom: structures of RU 004 and RU 252.

Figure I

After the discovery of erythromycin and other natural compounds, including spiramycin, josamycin, and midecamycin, considerable energy has been devoted to the development of longer-acting erythromycin analogswithenhancedbioavailability.Also,opportunitiesformodifying available products to broaden the antimicrobial spectrum havebeen fully explored.However,despite attempts to quantitatively or qualitatively improve the spectrum of activity, the results havebeen unsuccessful.

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At Roussel Uclaf, we focused our efforts on finding new antibacterials active against multiresistant pneumococci, H. influenzae, streptococci, and atypical bacteria (Mycoplasma, Legionella, and Chlamydia sp.). Such compounds couldbe considered as excellent alternativesfor first-line therapy in respiratory tract infections. A breakthrough was recently made inthe field of macrolides withthe discovery of ketolides. These are characterized by a 3-keto function in place of the L-cladinose moiety, a sugar long thought to be essential for antibacterial activity.RU 004, a new member of this class, is active in vitro and in vivo against multiresistant pneumococci and displays well-balanced activity against other respiratory pathogens (e.g., H. influenzae, group A streptococci, and atypical bacteria.

STRUCTURE AND CONFORMATIONAL ANALYSIS OF RU 004 Previous conformational analysis of the basic ketolide skeleton clearly demonstrated that the ketolide desosamine ring had more conformational freedom than clarithromycin. In addition, superimpositionof both compounds revealed two changes that may be of importance for the ketolide mode of action: a shiftingof 3.6 of the Ddesosamine dimethyl-amino group and bondanglemodificationsin the 3-keto-4-methylarea:clarithromycin (CLA) : C3C4C5C6 = 98"; ketolide: C3C4C5C6 = 75". RU 004 (Fig. .l) presents the same characteristics as shownby x-ray analysis. Moreover, an interesting feature of RU 004 has been revealed by the x-ray data: The quinoline moiety overlapsthe macrolactone ring. This positionof the aromatic nucleus may allowthe U electrons or the nitrogen of the quinoline to interact with additional residuesof the ribosome (U-U interactions, hydrogen bonds, etc.). All these physical and structural features may account for a strengthening of the interactions betweenRU 004 and bacterial ribosomes.

INDUCTION EXPERIMENTS IN S. AUREUS In contrast to most other 16-membered-ring macrolides which show poor activity (e.g., josamycin), all inducibly erythromycin (ERY)-resistant strains are remarkably susceptibleto RU 004 (staphylococci, enterococci, pneumococci). Induction experimentsin liquid medium withRU 004 compared to its corresponding cladinose derivative (RU 252), clearly demonstratedthat RU 004 has no inducing properties (Fig. 1). The observations made here with ketolides will certainly revive interest the in role of L-cladinoseon the induction of resistance.

Agouridas et al.

STABILITY OF RU IN ACIDIC MEDIUM AND PHYSICOCHEMICAL PROPERTIES Cladinose sugar is an acid-labile group which can be hydrolyzed rapidly in gastricmedium.Thisis why the commonchemicalmetabolite of 14membered-ring macrolides is the corresponding descladinosyl derivative. Acid-stability experiments using RU 004 versus CLA and azithromycin (AZI), clearly demonstrate its high acid stability. Therefore, we do not expect chemical metabolism which would contribute to erratic absorption (Fig. 2). RU 004, as shown by Log P values ( 4 3 , is more lipophilic than ERY (3.5) and presents two pKa's above and below the physiological pH (pK, 1 = 5.3; pK, 2 = 8.6). Some interesting properties regarding tissue accumulation and cellpenetration may be anticipated from these physicochemical parameters.

EFFECT OF pH ON MINIMAL INHIBITORY CONCENTRATIONS Because of RU 004's physicochemical characteristics (pKa, log P ) , environmental conditionsappear to have little influence on its antimicrobial properties (Fig. 2). Regarding the high activityof RU 004 even at pH 5, in vivo and ex vivo experiments with intracellular bacteria or bacteria surviving in acidic conditionswould be ofinterest basically and clinically.

UPTAKJI BY HUMAN MONOCYTIC CELLS RU 004 accumulateshighlyinmonocytes (m1cellline, data not shown). This property, together withthe low influence of pH on antibiotic uptake and on antibacterial potency, couldensure high bioactivity against pathogens that survive in cellular acidic compartments. Efflux kinetics of RU 004 frommonocytes (at least for THPl lineage)suggest no late persistence and, consequently, complete eliminationof the drug (data not shown).

IN VITRO AND IN VIVO EFFECTS ON HEPATIC CYTOCHROME P-450 With RU 004, no nitrosoalkane complexes were detected in vitro in microsomes from dexamethasonepretreated rats. A negligible percentage of nitrosoalkane complexes were detected in vivo after an acute doseof RU 004 in dexamethasonepretreated rats (Fig. 3). nitrosoalkane complexes were detected in vivo in rats after repeated doses of RU 004. Only weak

Ketolides: RU 004-Structure and Biological Properties

70

283

-

60

50

AZI

40

30 20 10

lime (in hours)

0

3.5

b

0

3 0.5

1

2.5

Figure 2 Top: stability of RU MICs.

2

4

in acidic medium and (bottom) effectof pH on

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25

- 20 8

RU 004

15

roxithromycin

10

clarithromycln

5

0

formation of cytochrome Fe(l1)-metabolite complexes with microsomes from dexamethasone pretreated rats. RU 004 or macrolides (0.1 mM) were incubated with NADPH (1 mM) and hepatic microsomes from dexamethasone-treated rats. Results are means f S.E.M. for 5 experiments.

50 45

"

40 --

35 -S! 30 --

25 --

2 20

"

8 15 --

lo -. 5 0-

"

Effects of a single dose ofRU W4 and various macrolideson cytochrome P450 in rats pretreated with dexamethasone. Dexamethasone-pretreated rats received various macrolides (0.5 mmol.kg-l ) in methylcellulose and were sacrificed 1 hr later. Results are means f S.E.M. for 5 rats.

Figure 3 In vitro and in vivo effects on hepatic cytochrome P-450.

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285

induction was observed after repeated doses of RU 004 in rats (data not shown). Therefore, no drug-drug interaction is expected with RU

CONCLUSIONS One of the mostactiveketolides, RU 004 demonstratesgoodactivity against multiresistant pneumococci and well-balanced activity against respiratory pathogens (H.influenzae, group A streptococci, and atypical bacteria). Its physicochemical properties give RU 004 high stability in acidic medium, little pH influence on antibacterial activity, and high intracellular accumulation. In addition, no interference with P-450isozymes was observed, suggesting no drug-drug interaction. Therefore, RU 004 may be a goodcandidate for further investigationin the development of new antibacterials for the treatment of respiratory tract infections.

Isolation of an Antifungal Macrolide from Soil SampleNocardioiiles Strain: Production and Structure Elucidation V. Loppinet, L. Hilali, N. Youssef, C. Finance

Bonaly,

Universitt! Henri Poincart? Nancy, France

INTRODUCTION

The need for new and nontoxic antifungal for agents medical use led tous isolate actinomycete strainsthat produce nonpolyenic antifungals. Actinomycetes were described the as main sourceof antibiotics, which are considered as secondary metabolites. They show a wide rangeof chemical structures (aminoglycosides, anthracyclines, glycoproteins, p-lactamins, macrolides, nucleosides, polyenes, polyethers, and tetracyclines) are and used bylaboratories interestedin antimicrobial researchto diversify their sources for producing microorganisms, by using exotic samples, and by achieving new se tion methodsfor rare species. We have isolated from a soil sample collected in Lorraine (France) an actinomycetal strain identified Nocurdioi'des as a sp. (unpublished data) that produces an antifungal metabolite. Thispaper describes the production, isolation, purification, andstructure elucidation of the active secondary metabolite. 286

Isolation of Antifingal Macrolide from Nocardioides Strain

287

MATERIALS AND METHODS Fermentation and Medium Conditions Inocula were prepared (1) from the Nocardiofdes sp. strain on Olson's medium and cultured48 h at 27"C, under stirringin liquid medium, first in 20 of synthetic broth, then again for 48 h in m1 this medium. Finally, the culture was scaled upto 1.5 L in a fermentor and grown under stirring at 250 rpm and aeration of 30 Wmin. Samples were sterilely regularly collected to estimate the strain growth by variation of absorption at 620 nm andthe production of the antifungal molecule. Antifungal extracts were obtained from 25 m1 of this culture, after centrifugation at rpm, for 15 min. The supernatant was either used directly or submitted to extraction and purification assays.The mycelium was washed twice in sterile distilled water and treated with 20 m1 (2). The mixture was then centrifuged as above and the methanolic extract was harvested. All extracts were stored at - 20°C.

Biological Properties Antifungal activity against Candida albicans (ATCC 2091) and Candida tropicalis R2(IP 203) amphotericin B-nystatin resistant was estimated by diffusion methods in Sabouraud's agar medium (4% D-glucose, 1% peptone Diagnostic Pasteur, and 1.5% agar) usingeither double-layer activity test, wells, or disk techniques.

Purification by Chromatographic Methods Samples were treated by thick-layer chromatography to remove residual impurities, the active zone was scraped out of the gel, andthe molecule was solubilized in n-butanol-acetic acid-water (45 : 10 : 15). After concentration and solubilization in methanol, the relative molecular weight was estimated by membrane ultrafiltration.The active filtratewas further purified bysilicagel60columnchromatography(3)using the elution system nbutanol-acetic acid-water (45 : 5 : 25). Gel filtration on Sephadex LH20 was then carried out. Purity of the active fractionwas checked by thin-layer chromatography and spectrophotometry at 254 and 366 nm. Chromatograms were revealed chemically and microbiologically Further (4). analyticalstudy of the activemoleculewaseffectedbyKieselgelthick-layer chromatography and high-performance liquid chromatography. The methods described above were adapted for preparative chromatography.

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Bacterialfermentation

1 Cells extraction by methanol

I

Organic phase

Olson's medium (7 days,

centrifugation

rpm. 10 min)

Supernatant (+) e x W o n by

I

(+)

Organic phase

l I

methanolln-bu (+)

resuspcnsiodwatcc mechanol extracliodethyl acefate

Biologically active crude metabolite purification by

chromatographic me(h0dS

Antimicrobialmacrolide Figure I

Isolation procedure of an antifungal macrolide from a Nocardioi'des SP.

Isolation of Antifingal Macrolide from Nocardioides Strain

289

Structural Analysis Biochemical methods: Hydrolysis was realized on antifungal extracts in methanol. After evaporation of a 2-mL fraction, 2 mg of metabolite and 0.5 mL 6 N HC1 were added, the tubes were sealed and were incubated 3 h at 105°C. The hydrolysate was evaporated and extracted by CHC1,-H,O (1 : 1). The lipid fraction and the hydrosoluble phase were dried under vacuum and analyzed. Lipid analysis was carried out by G60 silicagel thinlayer chromatography. The solvent system was petroleum ether-diethyl ether-acetic acid, and detection was as described in Ref. 5. Sugar content was estimated after hydrolysis and identified by paper or gas chromatography. Proteins were analyzed as described in Ref. 6. Physico-chemical methods: Ultraviolet spectra of active extracts were recorded between220 and 440 nm. Massspectra was obtained on VG ZAB2SEQ equipment. 'H NMR spectra were performed on Briiker AC 200 (200 MHz) spectrometer and infrared spectra on Perkin Elmer-l310 apparatus.

RESULTS AND DISCUSSION Extraction of the antifungal agent was camed out on culture supernatant, as shown in Fig. 1. The active filtrate showed a molecular weight lower than 10,OOO. Extracts were analyzedby silica gel-thin-layer chromatography, and chromatograms revealed microbiologically showed only one active dot by a bioautographic method.Further analytical studyof the active molecule by Kieselgel thick-layer chromatography and HPLC showed a purity of more than90%. The results of the structural analysis realized as described in Material and Methods are listed in Table 1. The antifungal product is a macrolide characterized by a large 14-membered lactone ring, to which are attached a chain of two glucose molecules connected via glucoside linkage and an amino group. The macrocycle is substitutedby various groups (6-oxo, 10hydroxy, 8,13-dimethyl) and has an ethylenic bond between C,, and C12. This antifungal metabolite hasthe same molecular weight as Maridomycin I1 (C,H,&IO,,), a16-memberedmacrolideisolatedfrom Streptomyces hygroscopicus and Streptomyces platensis strains, but it differs from this antibacterial antibioticby its infrared 'H NMR spectrum.

CONCLUSION We have identified an antifungal macrolide (14 member) produced by a Nocardioides actinomycetales strain.

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Table l Physico-Chemical Characteristics of the Antifungal Metabolite Physico-chemical data Thermostability Solubility

W/MeOH (Arnm nm) IW(Nujo1) (v cm")

Product Stable at Loss of 100% activity after 15 min at Soluble in DMSO,methanol, n-butanol Less soluble in water Insoluble in hexane and petroleum ether 229 1650,1270,1110,1070,1030

FAB-MS (M+Na+) mlz (kelative intensity according matrix) a: glycerol ( M W : 92) b: thioglycerol ( M W : 108) c: thioglycerol+l% TFA (trifluoracetic acid)

867 (M: 844) 867 (a: 4.28%; b: 8.57%; c: 8.57%), 845 (a: 5.71%; b: 6.64%; c: 5.00%), 732 (a: 2.14%; b: 4.64%; c: 3.21%), 624 (a: 6.42%; b: 7.85%; c: 12.14%), 512 (a: 2.58%; b: 2.14%; c: 1.78%), 277 (c: loo%), 204 (a: 100%; b: 100%) Calcd for C&&NZOl9 Elemental analysis C% 53.01, H% 8.05, N% 2.77,0% 36.08 Calcd. 36.08 2.77 8.59 53.01 Found. 10.64 (lH, S , CHO, --HC-OH); 6.67-6.64 'H NMR' (200 MHz, DMSO-d,) (lH, d, CH--C); 5.43-5.39 (2H, m, NH, . lH, H ose); 5.00-4.85 (6H, m, H ose); 4.51-4.26 (5H,m, H ose, HC-CH--C, l H , H ethylenic); 3.81-2.92 (19H, m, C-CH); 2.64-2.22 (9H, m, CO-CH or NCH); 2.18-1.20 (15H, m, CH, cyclic or not, 8 1.19-1.16 (6H, S, S, CH3, CH(CH3)J; 0.87-0.83 (6H, S, S , CH,, Composed sugar(%) Composed amino acids(%) Composed lipid(%) TLCb R, valueC ~~

~~

% = singlet,

~

~~~~~

d = doublet, m = multiplet. bTLC = thin-layer chromatography. en-butanol-acetic acid-water (45 : 30 :25).

Glucose 39% Not present Not present 0.67

Isolation

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Until now such antifungal agents had been isolated primarily from Streptomyces strains. This original metabolite is active against pathogenic yeasts such asCandida albicans and Candida tropicalis which is resistantto. polyenic agents (amphotericin and nystatin). Further studies are now in progress in order to complete its chemical structure.

REFERENCES 1. Shirling EB, Gottlieb D. Methods for characterization of Streptomyces species. Int J Syst Bacterioll966; 16:313-340. 2. Lindenfelser LA, Shotwell OL, Bachler MJ, Shannon GM, Pridham TG. Antibiotics against plant disease. Screening for nonpolyenic antifungalantibiotics producedby Streptomycetes. Appl Microbiol 1964; 12508-512. 3. Issaq HJ, Barr EW, Wei T, Meyers C, Aszalos S. Thin-layer chromatographic classification of antibioticsexhibiting antitumor properties. J Chromatogr 1977; 183:291-302. 4. Bettina V, Bioautography in paper and thin-layer chromatography and its scope inthe antibiotic fields.J Chromatogr 1973; 78:41-51. 5. Higgins JA. Separation and analysisof membranes lipidcomponent. In: Biological Membranes, a Practical Approach. (Findlay JBC, Evans WH, eds.) Oxford, WA: I.R.L. Press, 1987:103-137. 6. Bradford MM. A rapid and sensitive method for quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Anal Biochem 1976; 72:248-254.

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II LEGIONELLA, SPIROCHETES

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Azithromycin in the Treatment of Community-Acquired Legionnaires’ Disease I. Kuznlan

S . Schiinwald

University Hospital of Infectious Diseases“Dr. Fran MihaljeviP Zagreb, Croatia

J. Cuiig Pliva d.d. Pharmaceuticals Division Zagreb, Croatia

INTRODUCTION Legionellapneumophila is a relativelyfrequent causative pathogen of pneumonia.According to data fromvarious countries, itcauses 5-10% of community-acquired pneumonias(1,2) and a significant amountof nosocomial pneumonias In one clinical trial conducted in Croatia, 8.1% of patients with community-acquired pneumonia had serological evidenceof Legionnaires’ disease (4). There are no reliable clinical, radiological, or analytical findings indicating that pneumonia is caused byLegionella spp. Therefore, the rational initial treatment of pneumonia, especially atypical pneumonia, should utilize antibiotics effective against legionellaswell as as other common pathogens (5). Azithromycin, a novel azalide antibiotic, is very active against L. pneumophila and it is superior to erythromycin invitro as well as in experi295

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mental legionellosis (6,7). Our early clinical experience with azithromycin in the treatment of Legionnaires’ disease has been published recently(8). A retrospective, noncomparative study was undertaken to assess the efficacy and tolerability of azithromycin in patients with community-acquired pneumonia causedby L. pneumophila.

PATIENTS AND METHODS A total of 21 patients with serologically confirmed diagnoses of communityacquired pneumonia caused by L. pneumophila were included. All patients werehospitalizedassporadiccasespresenting at the University Hospital of Infectious Diseases in Zagreb from 1990-1995. These patients did not receive prior or concomitant antibiotics effective against legionellas (macrolides, tetracyclines, rifampicin, quinolones, chloramphenicol, co-trimoxazole). L. pneumophila, serotype 1, wasconfirmedinall patients by the LegionellaIndirectFluorescentAntibody(IFA) test usinga standard method as described by Wilkinson (9). In two patients the diagnosis was confirmed by the presence of antigen in sputum using a direct immunofluorescent method (DFA) (10). Clinical diagnosis of pneumonia was confirmedbychestx-ray.Azithromycinwasadministeredorally,once daily, at a total dose of 1.5 g. Eleven patients weretreated for 5 days (500 mg on day 1 followed by 250 mg on days 2-5) and 10 patients for days (500 mg daily). Cure was defined as resolution of fever within 96 h after the start of treatment with improvement of other signs and symptoms of pneumoniaandregression of pneumonicinfiltrate on follow-upchest x-ray. Allpatientsweremales,aged 27-60 years.Nonehad severe chronic cardiacor pulmonary diseaseor signs of immunodeficiency. There were 15 smokers.

RESULTS All patients were successfully cured with azithomycin. Sixteen patients (76%) became afebrile within48 h, 2 within 72 h, and within 96 h after the initiation of treatment (Fig. 1). Azithromycin was very well tolerated and no clinical side-effects were observed. Laboratory abnormalities occurred in five patients. A transient increasein eosinophil count (11%) was registered in one patient and twofold to fourfold elevation of liver aminotransferases in four. All .abnormalities resolved spontaneously withinthe subsequent weeks. There were no differences in efficacy or tolerability between the or 5-day azithromycin dosage regimens.

Azithromycin

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48

72

96

Time after the initiationof treatment (hours)

Figure I The regression of fever in patients with Legionnaires’ disease with azithromycin.

treated

CONCLUSION The results indicate that azithromycin, given orally at a standarddose total of 1.5 g, is effective and well tolerated in the treatment of Legionnaires’ disease. REFERENCES 1. Marrie TD, Durant H, Yates L. Community-acquired pneumonia requiring hospitalization: 5-year prospective study. Rev Infect Dis 1989; 11586. 2. Fang, GD, Fine M, Orloff J. New and emerging etiologies for communityacquired pneumonia with implications for therapy. Medicine 1990; 69:307. NguyenMLT,Yu VL. Legionella infection. Clin. Chest Med 1991; 12:257. Kuzman I. Doctoral dissertation, Zagreb,Croatia, 1994. Roig J, Carreres A, Doming0 C. Treatment of legionnaires’ disease. Current recommendations. Drugs 1993; 46:63. 6. Edelstein PH, Edelstein MAC. In vitro activity of azithromycin against clinical isolates of Legionella species. Antimicrob Agents Chemother 1991; 35: 180.

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7. Fitzgeorge R B , Featherstone ASR, BaskenrilleA. Efficacy of azithromycin in the treatment of guinea pigs infected with Legionella pneumophila by aerosol. J Antimicrob Chemother 1990;25 (suppl A):101. 8. Kuzman I, Soldo I, Schonwald S, Culig J. Azithromycin for the treatment of community-acquired pneumonia caused by Legionella pneumophila: a retrospective study. ScandJ Infect Dis 1995;27: 503. 9.WilkinsonHW,Fikes BJ, Cruce DD. Indirectimmunofluorescencetest for serodiagnosis of legionnaires’disease:evidencefor serogroup diversity of legionnaires’ disease bacterial antigens and for multiple specificityof human antibodies. J Clin Microbioll979; 9:379. 10. Edelstein PH, Meyer RD, Finegold SM. Laboratory diagnosisof legionnaires’ disease. Am Rev Respir Dis 1980; 121:317.

Temperature Dependenceof MIC and MBC of Roxithromycin AgainstBorrelia burgdo~en'In Vitro I. Wendelin, R. Gasser,and E. C.Reisinger Karl Franzens University Graz ,Austria

INTRODUCTION Borreliaburgdorferi (Bb) is susceptible to roxithromycin in vitro (1,2). Clinical data show that erythema migrans is not affected by roxithromycin (3), whereas late Lyme disease (cardiovascular, neurological manifestations, etc.) does respond to roxithromycin (4). In vitro susceptibility testing usually has been performed at 35°C. In the human body, Bb is localized in organs with temperatures ranging from28°C (skin) to 37°C (core temperature). We investigated both growth and susceptibility of Bb to roxithromycin at temperatures of 30"C, 35"C, and 38°C in vitro.

MATERIALS AND METHODS Borrelia burgdorferistrains ATCC 53899(isolate from cerebrospinal fluid), ATCC 35210 (B31, tick isolate), and PKo (skin isolate) were cultured in modified Barbour-Stoenner-Kelly medium (5). Broth macrodilution susceptibility testing with roxithromycin was performed in 24-well (6) plates at 299

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300

30"C, 35"C, and 38°C in electronically controlled air incubators. After 4 days of incubation, growth of borreliae was determined by counting the cells with a Petroff-Hausser counting chamber and dark-field microscopy. The minimal inhibitory concentration (MIC) was defined as the lowest antimicrobial concentration with 5 X l@motile borrelidml (i.e., the original inoculum). Subcultures were performed (5% volhol) and incubated at 35°C for 3 weeks. The minimal bactericidal concentration (MBC) was defined as the lowest antimicrobial concentration with no spirochetes detectable by dark-field microscopy . The experiments were performedon three separate occasions, using duplicates each time.

RESULTS Borrelia1 Growth Growth of Lyme spirochetes in BSK I1 medium without roxithromycin was equal at temperatures of 30°C and 35°C and decreased at38°C. The numbers of motile Bb cells/ml on day 4 of incubation at differenttemperatures are shown in Fig. 1.

MIC and MBC The MICs of roxithromycin forthe Bb strains testedat 38°C were lower by 3-4log, dilutions than those obtained at 30°C. The respective MBCs at 38°C were lower by 1log, dilution than those at 30°C. MICs and MBCs of roxithromycin for Bb at different temperaturesare summarized in Table1.

ATCC 53899

30°C

lt h 3 a ~

Incubation temperature

Figure Z Growth of Borrelia burgdorferi strains ATCC 53899, B31, and PKo in modified BSK I1 medium at different temperatures after 4 days of incubation.

Temperature Dependence of MZC and MBC of Roxithromycin

301

Table l MICs and MBCs of Roxithromycin for Borrelia burgdofleri Strains ATCC 53899, B31, and PKoat Different Temperatures Modal MIC (pg/ml)a Modal MBC (pg/ml)a 30°C

Strain 38°C

35°C

ATCC 53899 B31 PKo

30°C 0.25 0.5

0.25

0.125 0.125 0.125

0.03 0.03 0.03

0.5 0.5 0.25

0.25 0.5 0.125

0.25 0.25 0.125

'Mean value of six determinations.

DISCUSSION AND CONCLUSIONS The activity of roxithromycin against Bb determined at was significantly lowerthan that observed at and B31 in BSKI1 medium withGrowth of the strains out roxithromycin was comparable when determined at and In the cultures, growthof the strains B31 and PKo was decreased compared with the cultures. The human body temperature ranges from abut (skin) to (core temperature). Temperature-sensitive strains might localize predominantly inthe skin, whereas strains less sensitive to temperature might establish also in organs with higher bodytemperature. The reduced activity of roxithromycin in erythema migrans could be explained by the lower temperature of the skin and needsfurther investigation.

REFERENCES Gasser R,Wendelin I, Reisinger EC, Bergloff J, Feigl B, Schafhalter I, Eber B, Grisold M, Klein W. Roxithromycin in the treatment of Lyme diseaseUpdate and perspectives. Infection 1995; 23:39-43. Preac-Mursic V, Wilske B, Schien G, SUS E, Gross B. Comparative microbial activity of the new macrolides against Borrelia burgdogeri. Eur J Clin Microbiol Infect Dis 1989; 8:651-653. HansenK,Hovmark A, LebechAM,LebechK,Olsson I, Halkier SorensenL,Olsson E, Asbrink E. Roxithromycin in Lymeborreliosis: Discrepant results of an in vitro and in vivo animal susceptibility study and a clinical trial in patients with erythema migrans. Acta Derm Venerol 1992; 72~297-303. Gasser R, Dusleag J. Oral treatment of late borreliosis with roxithromycin plus co-trimoxazole. Lancet 1990;336:1189-1190.

Wendelin et al. 5. Barbour AG. IsolationandcultivationofLymediseasespirochetes.Yale J Bioi Med 1994; 6. Reisinger EC, Wendelin I, Gasser R, Halwachs G, Truschnig M, Krejs GJ. Enhancement of antibacterial efficacy of penicillin and ceftriaxone by temperature. Scan J Infect Dis (in press).

Tolerability of Treatment with3 g of Azithromycin Given in 5 Days Franc Strle, Vera Maraspin, Stanka LotriE-Furlan, JoZe Cimperman University Medical Centre Ljubljana Ljubljana, Slovenia

INTRODUCTION The total recommended dose of azithromycin for the treatment of most susceptible infections is g, given over or 5 days It is well known that side effects of such therapy are relatively rare and usually mild Double total dosage is recommended in the management of erythema migrans (6,7). However, data on tolerability of g azithromycinare sparse (6,7). A large number of patients with erythema migrans treated with azithromycin gave us an opportunityto analyze the tolerability of azithromycin, given in atotal dose of g.

MATERIALS AND METHODS Data were analyzed on consecutive adult patients with early Lyme borreliosis (solitary erythema migrans) that were enrolled in prospective studies at University Medical Centre, Department of Infectious Diseases, Ljubljana, Slovenia in and that compared the efficacy and safety of azithromycin with severalother antibiotics. 303

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Table I Nature and Incidence of Side Effects in Patients Treated with g3 of Azithromycin

Side effect

No. of patients (%) 15 (4.6%) 10 (3.1%)

10

Abdominal pain Nausea Diarrhea

3

Dizziness Headache Sleepiness

7 (2.2%)' 5 (1.5%)8 (0.9%)

Total 'One patient had two side effects.

All the patients wereolder than 15 years and none received any other medication inthe period of 14 days prior and14 days after the institution of azithromycin treatment. Azithromycin was administered in atotal dose of g, 500 mg bid on the first day followed by 500 mg once daily for 4 days. All patients were seen by at least one of the authors before the institution of treatment as well as 14 days and 2 months later. Data on concomitantmedicationandsideeffects of therapywere obtained by means of questionnaire. Laboratory tests (erythrocyte sedimentation rate, blood cell count, and liver function tests) were performed prior to and 14 days after the institution of treatment. All findings out of the reference limits wereanalyzed. When abnormal findings were observed,the tests were repeated at later examinations.

RESULTS Forty-nine patients (15.1%) reported side effects, all within first 14 days after the institution of treatment; The nature and incidence of side effects are presented in Table1. Mild (27patients) to moderate (7 patients) gastrointestinal complaints were most common. Vomiting or allergic skin manifestations were not reported. As a rule, side effects appeared during treatment (usually on the first day of therapy) and disappeared 1-2 days after the cessation of treatment. No treatments were discontinued because of side effects. Laboratory tests were performed in 297patients. "bo weeks after the institution of azithromycintherapy,laboratoryabnormalitieswere observed in (28.3%) patients (Table 2). Mild abnormalities of liver func-

Tolerability

Azithromycinfor 5 Days

Treatment with

Table 2 Type and Incidence of Laboratory Abnormalities in Patients Treated with g of Azithromycin

normalityTest

No. of patients (%)

Increase tests function Liver se countEosinophil se countLeukocyte

unt

305

Increase Platelet Total

42 (14.1%) 17 (5.7%) 12 (4.0%) (3.4%) 3 (1.0%) (28.3%)

tion tests were most frequent (Table Mild aberration of previously normal liver function tests occurred 28 in(9.4%) patients andfurther slight deterioration was observed in 14 (4.7%) patients, whohad abnormal liver function testsat baseline. In all patients with normal baseline findings, the observed abnormalities were transient.

DISCUSSION All our patients were informed in advance about the course of their illness and potential side effectsof therapy. That might have induced someof the complaints. Gastrointestinalsymptoms are highly infrequentin the course of Lyme borreliosis(7) and patients’ reportson nausea, abdominal pain, and diarrhea probably represent side effects of azithromycin treatment. Such causal interpretation is more difficult for complaints such as headache, sleepiness,anddizziness whichmay be manifestations of earlyLyme borreliosis Table The Abnormalitiesof Liver Function Testsin Patients Treated with3 g of Azithromycin

normalityTest

No. of patients (%) ~~

Alkaline phosphatase Gamma-GT Serum bilirubin SGPT SGOT

Increase

28 (9.8%)

Increase Increase Decrease

20 (6.7%) (3.4%) 5 (1.7%) 2 (0.7%)

Total In some patients more than one test was abnormal.

42 (14.1%)

306

et

Strle

al.

The observed laboratory abnormalities were generally mild and transient, but their incidencewas higher than reported for the treatment with 1.5 g of azithromycin However, the assessment is delicate, especially for the abnormalities of liver function tests and eosinophil count, as the samefindingsmaybefoundduring the natural course of earlyLyme borreliosis(8-10). A similarincidence of laboratory abnormalities has been observed in our patients with early Lyme borreliosis treated with amoxicillin with even higher rates in patients treated with doxycycline(data not shown).

CONCLUSION Side effectsof azithromycin given at doublethe usual dosage are mild and do not differ substantially from findingsreported for treatment with 1.5 g of the agent given over 5 days. Mild transient abnormalities of liver function tests and eosinophilia were considerably more common than reported with 1.5 g of azithromycin; however, interpretation is uncertain, as these findings may also be a manifestation of the natural course of early Lyme borreliosis.

REFERENCES 1. Foulds G, Shepard R M , Johnson RB. The pharmacokineticsof azithromycin in human serum and tissues. J Antimicrob Chemother 1990; 25 (suppl A):73-82. 2. Lode H. The pharmacokinetics of azithromycin andtheir clinical significance. Eur J Clin Microbiol Infect Dis 1991; 10:807-812. 3. Hopkins S. Clinical toleration and safetyof azithromycin. J Med 1991; 12 (SUPPI3A):40S-45S. Drew RH, Gallis HA. Azithromycin-Spectrum of activity, pharmacokinetics, and clinical applications. Pharmacotherapy 1992;12:161-173. 5. Ballow CH, Amsden GW. Azithromycin: the first azalide antibiotic. Ann Pharmacother 1992; 26:1253-1261. 6. Strle F, Preac-Mursic V, CimpermanJ, RuZiC E, Maraspin V, Jereb M. Azithromycin versus doxycycline fortreatment of erythema migrans: clinical and microbiological findings. Infection 1993; 21:83-88. 7. Strle F, Maraspin V, LotriE-Furlan S , RuziSabljiC E, Cimperman J. Azithromycin and doxycycline for treatment of Borrelia culture positive erythema migrans. Infection 1996(in press). Steere AC. Lyme disease. N Engl J Med 1989;321586596. 9. Weber K, Neubert U. Clinical features of early erythemamigrans disease and related disorders. Zbl BaktHyg A 1986; 263:209-228. 10. Steere AC, Bartenhagen N H , Craft JE, Hutchinson GJ, Newman JH, Rahn D, Sigal, LU, Spieler PN, Stem KS, Malawista SE. The early clinical manifestations of Lyme disease. Ann Intern Med 1983;99:7682.

Azithromycin in the Treatment of Erythema Migrans J. GoriSek

J. Rogl

Maribor Teaching Hospital Maribor, Slovenia

INTRODUCTION Lyme disease is the most frequent tick-borne disease in Slovenia. It is caused by spirocheta Borrelia burgdorferi and it appears wherever ticks are found. The clinicalpresentation ofLymediseaseis variegated; it affects numerous organs and organ systems and its course is very unpredictable. Lyme disease is divided into early and late stages. Early infection may be localized or disseminated.Erythemamigrans(EM)is the cardinal symptom of the early stage occuring at the site of the tick bite. The appearance of EM is characteristic that clinical diagnosis is sufficient. EM may be accompanied by local complications at the site of the tick bite, by general symptoms, and, sometimes, bysignsof disease in individual organs or organ systems. Although EM resolves spontaneously, with adequate antibiotic treatment skin changes disappear more rapidly and the development of late stage of Lyme disease can be prevented with high probability. The aim of the present studywas to assess the efficacy of azithromycin,anazalideantibiotic,in the treatment of patients with erythema migrans.

307

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PATIENTS AND METHODS

.

This prospective study was conducted from March1994 to October 1995. Thirty adult patients of both sexes with EM who gavetheir informed consent were included. All patients came from the broad region of Maribor. Patients who received antibiotics before the beginning of treatment and those allergic to azithromycin were excluded. The diagnosis of EM was based on historical (tick bite, time of EM occurrence) and epidemiologic data (seasonal appearance, geographic region) as well as the characteristic clinical picture. The presence of specific IgM and IgG antibodies for B. burgdorferi was assessed by IFT and ELISA. Azithromycin was givenper for 5 days: 500 mg twice daily on the first day and 500 mg once daily for the following 4 days. Clinical examinations were performed14 days, 6 months, and 1 year after the termination of the treatment. Besides the appearance of EM, local (itching, burning, pain) and general (fatigue, malaise, headache, myalgia, arthralgia) clinical symptoms(CS)weremonitored.Follow-upserologicaltestswere .performed 2 months, 6 months, and 1year afterthe termination of treatment. Side effectsof treatment were recordedat the first follow-up visit. At each examination, clinical response was evaluated as successful (complete reso tion of EM and CS), improved (complete resolution of EM but notof CS), and unsuccessful (incomplete resolution of EM and CS or the appearance of signs of late Lyme disease).

RESULTS Baseline patient characteristics are summarized in Table1. The time elapsing between the tick bite and the appearance of EM was days to 4 months, 17 days on average. Seasonal appeararlce of and erythema migrans is shown in Fig. 1. The geographic regionwas identical for patients presenting with either of the findings. Clinical response to azithromycin treatment is presented in Table 2. Fourteen days after treatment, fatigue, malaise, and periodic headaches were present in seven patients, and in two patients, in addition to fatigue Table I

Baseline Patient Characteristics

Total no.of patients Sex (maledfemales)

30 lor20

Mean age (range)

41.9 (17-68)years 22 (73.3%)patients

No tick bite stated

8 (26.7%) patients

Tick bite stated

Azithromycin Treatment in

309

of Erythema Migram

.l . l

Figure l

Seasonal appearance of tick bites and erythema

and malaise, there were also visible moderateskin changes with local signs of itching. After 6 months, nine patientsreported general symptoms such as fatigue, malaise and headache. Fiveof these also had periodic myalgias and arthralgias.After 1year, 25 patients were symptom-free. Five patients (16.7%) reported fatigue, periodic headaches, malaise and temporary myalgias and arthralgias, without manifest signs of late Lyme disease. No patient had any side effectsof treatment.

DISCUSSION The analysis of epidemiologic and historical data revealed that the results were comparable to those of similar studies carried out in other parts of Table 2 The Results of Azithromycin Treatment in Patients with Erythema Migrans

Time after terminationof treatment

6 months

14 days response Clinical Successful Improved 6.7 Unsuccessful No. of patients

1 year

N

%

N

%

N

21 7 2

70.0

21

70.0

25

0

0

5 0

100

30

%

16.7 0

100

Gorsek and Rogl

310

Slovenia and neighboring Austria(1-4). In the diagnosis of EM, serologic investigations forthe confirmation of specific IgGand IgM antibodies were not considered because they were positive in less than50% of patients at the onset of disease. The treatment patients with EM can be evaluated as successful because by 14 days after the termination of azithromycin treatment there wascompleteregressionofskinlesionsin 28 patients and within 1 year, no patient showed any signs of late Lyme disease.

CONCLUSION The results of the study can be considered satisfactory, because in the course of 1 year, none of the 30 patients showed any objective signs of late Lyme disease. However, caution is required when evaluating the results, because subjectivedata were included andthe course of Lyme disease is very unpredictable. Therefore, follow-up of these patients is continuing. The advantages of azithromycin inthe treatment of early Lyme disease includes single daily dosage, good tolerability, and short duration of treatment.

REFERENCES 1. Weber K, Burgdorfer W. (eds.). Aspects of LymeBorreliosis,NewYork: Springer-Verlag, 1993. 2. Stanek G, Hofmann H. Krankdurch Zecken. FSME und Lyme-borreliose. Wein: Verlag Wilchelm Maudrich, 1994. 3. Strle F. Lymska borelioza, Zdrav Vest 1988; 57: 4. Herzer P.Lyme-Borreliose. Epidemiologie. Atiologie. Diagnostik. Klink und Therapie. Darmastadt: Steinkopff Verlag, 1989.

Follow-up Study of Patients with Syphilis Treated with Azithromycin A. L. Mashkilleyson and M. A. Gomberg Semaskho Moscow Medical and Dental School Moscow, Russia

INTRODUCTION Azithromycin is very effective against Treponema pallidum as shown in vitro and in experimental syphilis (1,2). The first results of its use in patients with early syphilis are very promising this disease requires a very long follow-up period to evaluate the overall effectiveness, allpatients must be evaluated clinically and serologically for at least2 years (43). The aimof this study was to evaluate the long-term results of azithromycin treatment of early syphilis.

PATIENTS AND METHODS As of this writing, more than

patients with early syphilis have been treated with azithromycin. Azithromycin was administered in a total dose of 5 g, 500 mg once daily for consecutive days.We were able to analyze follow-up data on patients. The duration of follow-up was years. There were 25 patients with primary seropositive syphilis,15 patients with secondary syphilis, and with recurring syphilis (Table

311

Mashkilleyson and Gomberg

312

Table I Baseline Stage of Syphilis and Duration of Follow-up After Azithromycin Treatment

Duration of follow-up Stage of syphilis

year

years

years

6

Primary syphilis Secondary syphilis Recumng syphilis

15

4 6 9

Total No. of patients

26

19

8

Total No. of patients 25

Clinical examination and serological tests were performed monthly during the firstyear of follow-up, and every 3 months thereafter. The monitoring of the Wassermann reaction (WaR) and the Venereal Disease Research Laboratory (VDRL) testwas started 1month after therapy; the monitoring of the T. pallidum immobilization (TPI) test as well as the fluorescent treponemal antibody-absorption (FTA-ABS)test was initiated 12 months aftertreatment. All patients have been examined by an internist, an ophthalmologist, a neurologist, and an otorhinolaryngologist.

RESULTS

In all patients signsofsyphilis disappeared within 1-2 weeks after the initiation of treatment. In 70 patients (91%), WaR and VDRL became negative within4 months after the treatment (Fig. 1). At the end of the firstyear of follow-upWaRand VDRL, were negative in 75patients (98.7%). In addition, inone male patient these tests became negative within 15 months afterthe treatment. In one female patient with recumng syphilis, the clinical and serologic relapse was seen 4 months after the disappearance of the clinical symptoms of syphilis, but reinfection could not be completely excluded. A second attempt to treat this patientwith azithromycin was ineffective. The complete normalizationof TPI and FTA-ABS tests was observed in 38 out of 77 patients (Table In 7 patients, the negative tests were observed within 1year, in 9 within 18 months, in10 within 2 years, andin 12 within years after treatment. There were no signs of neurosyphilis or visceral syphilisin any case. Tolerability of azithromycin was very good and there were no treatment discontinuations due to side effects. In some patients, mild gastrointestinal side effectsor transient elevation of liver enzymes wasobserved.

313

Azithromycin-Treated Syphilis Follow-up

--

"

treatment (months)

lime after

Figure I Resolution of WaR and VDRL test in patients with seropositive syphilis treated with azithromycin.

Table 2 Resolution of TPI and FTA-ABS Tests in Patients with Syphilis Treated with Azithromycin

Follow-up after azithromycin treatment years

3

years

2

Total No.of patients Patients with negative tests (%)

year

1 26 7(26.9)

32

19

19(59.3)

12(63.1)

314

Mashkilleyson and Gomberg

CONCLUSION The results of long-term follow-up confirm the efficacy of azithromycin in the treatment of early syphilis.. Azithromycinat a total dose of 5 g can be recommended in patients with hypersensitivity to penicillin and inpatients with concomitant chlamydial infection.

REFERENCES 1. Stamm LV, Parish EA. In vitro activityof azithromycin and CP-63,956 against Treponemapallidurn.J Antimicrob Chemother 1990;25 (suppl A):ll. 2. Lukehart SA,Fohn MJ, Baker-Zander SA. Efficacy of azithromycin for therapy of active syphilis in the rabbit model. J Antimicrob Chemother 1990;25 (suppl A):91. 3. Mashkilleyson AL, Gomberg MA. Azithromycin in patients with syphilis. The first International Conference on the Macrolides, Azalides and Streptogramins, Santa Fe, 1992:abstr 60. 4. Jaffe HW. Management of the reactive serology. eds. Holmes KK, MBrdh P-A, Sparling PF, WiesnerPJ.Sexually Transmitted Diseases, NewYork: McGraw-Hill, 1984:313. 5. WHO Recommendations for the Management of Sexually Transmitted Diseases. Geneva:WHO Advisory Group Meeting on STDs Treatments, 1993:29.

III MYCOPLASMA, CHLAMYDIA

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A Comparison of the In Vitro Sensitivityof Chlamydiapneumoniue to Macrolides and a New Benzoxazinorifamycin, KRM-1648 C.-C.

and J. Thomas Grayston University of Washington Seattle, Washington

T.Hidaka Kaneka Corporation Osaka, Japan

L. M.Rose PathoGenesis Corporation Seattle, Washington

INTRODUCTION Chlumydiupneumoniue(WAR) is a frequent causeof community-acquired pneumonia in adults, accounting for approximately 10% of cases (1). Current recommendations forthe treatment of C.pneumoniueare based primarily on in vitro sensitivity studies and analogies to the treatment of other chlamydial infections.It has been suggestedthat prolonged antibiotictherapy may be necessary to cure W A R infections because symptoms recur frequently after short or conventional coursesof appropriate antibiotics, and persistent infection has been documentedby culture after treatment. The 317

Kuo et al.

318

question of persisistent infection after treatment is of potential importance because C. pneumoniue has been associated with chronic diseases, such as atherosclerosis and coronaryheart disease in sero-epidemiological studies and by direct detectionof C. pneumoniue in atheromatous lesions. of TWAR pneumonitis in We have developed an experimental model mice which has been used to evaluate the effectiveness of new compounds invivo. We report here onpreliminaryresultswithanewsynthetic rifamycin,KRM-1648,which,like other drugs in the same class, is an inhibitor of microbial DNA-dependent RNA polymerase (2).In addition to the animal studies, we have initiated in vitro studies to evaluate the effectiveness of KRM-1648 and azithromycin in combination.

MATERIALS AND METHODS In Vitro Susceptibility of pneumoniue and to KR"1648

trachomatis

The prototype C. pneumoniue strain (ocular isolateTW-183) and a C. truchomutis strain (Blnv-5lOT) were grown in HL cells at inocula to yield two 400X in to five inclusions per field at a magnification ofcontrols. After centrifugation, the inoculum was removed and replaced by culture media contain ing KRM-1648at twofold dilutions.After incubation for 3 days, cells were stained and examined for inclusions under a fluorescence microscope. Unstained cells were harvested, passed, cultured without antibiotics for 3 day and examined. Minimal inhibitory concentrations (MICs) were defined as concentrations causingthe complete inhibitionof inclusion formation in the original inoculum; minimal bactericidal concentrations were defined as co centrations causing the complete inhibition of inclusion formation in the second passage (3). Experiments were performed in duplicate tubes per dilution.

In vitro Testing for SynergisdAntagonism by the Microdilution Checkerboard Method A two-dimensional microdilution checkerboardwas set up to evaluate the antichlamydial activity ofKRM-1648 in combination with azithromycin. Dilutions of the two antibiotics ranged from 2X to 0.125X the MIC for each antibiotic. Inoculaof C. pneumoniue (strain TW-183) were prepared as described above. In vitro interactions were interpreted as synergistic/ additive,indifferent, or antagonistic,depending on whether the antibacterial activityof the combination wasgreater than, equivalent to, or less than, respectively, the activities of the individual agents. Second passage evaluations of MBCs have not been completed.

Sensitivity of C.pneumoniae to MucrolidesVersus KRM-1648

31 9

Experimental Animal Model Four-week-old male Swiss Webster mice were inoculated intranasally with 0.5 108 inclusion-forming units (IFUs) of C. pneumoniue (respiratory isolate AR-39) under lightether anesthesia to induce hyperventilation(4). Antibiotic treatment was started 2 days after inoculation, when pneumonitis was most severe. Different groups of five animals were sacrificed at different time points after treatment to either assess viable counts of organisms in lung tissueor histology.

Antibiotic Treatment R o days after inoculation, animals were randomly chosento be injected intraperitoneally (IP) with either phosphate-buffered saline (PBS) once a day for 3 consecutive days, with KR"1648 at 10 mgkg of body weight once a day for 3 consecutive days, or with KRM-1648 at 1 mgkg of body weight for 3 consecutive days. KR"1648 was dissolved in DMSO (10% final concentration) andfurther diluted in PBS prior to dilution. Culture of Lung Tissue Lungswereweighed,minced,andhomogenized to make a 10% (w/v) suspension in cold SPG buffer. Tissue suspensions were centrifugedat 500 g for 10 min at 4°C to remove debris and frozenat -70°C until testing. Viable counts were assayedby titration of tissue homogenates inHL cells grown on a 12-mm-diameter coverslip in a flat-bottomed l-dram (4-ml) shell vial. Inoculated cells were incubated at 36°C for 3 days; infected cells Chlamydia genus-specific werefixedwithacetoneandstainedwitha monoclonal antibody (CF-2) conjugated to fluorescein isothiocyanate. Inclusionswerecountedunderafluorescencemicroscope.Viablecounts were expressed as log,,IFU per gramof lung tissue.

Histopathology Lungs were removed and placed in 10% formalin for histology. Formalinfixed tissues. wereembeddedinparaffin,sectioned,andstainedwith hematoxylin and eosinby standard methods.

RESULTS In vitro susceptibility testingwas conducted in cell cultures againstprotoa type C. pneumoniue strain (TW-183) and aC. .truchomutis strain (BrrW-5/ using HL cells. Boththe MICs andMBCs were determined. C.pneumoniue and C. fruchomutiswere equally sensitiveto KRM-1648 with MICsof

Kuo et al.

320

Table I In Vitro Susceptibilityof C. pneumoniae (TW-183)to KM-1648and Azithromycin in Combination KRM-1648

Test drug

noKRM

MIC

0.000125-0.00025 pg/mL andMBCs of 0.~125-0.OO1pg/mL. KR”1648 was tested in combination with azithromycin against C. pneumoniae and found to have an additiveeffectinvitro(Table 1). The MIC of azithromycinwas twofoldto fourfold lower when combined with sub-MICs Of KR”1648. A mouse model of chlamydial pneumonitis was applied for in vivo testing of KR”1648. Pneumonia was induced by intranasal inoculation of C. pneumoniae strain AR-39. All animals challenged withC.pneumoniae developed pneumonitis, as determined histologically postmortem. KRM1648 was administered by IP injection of 1 mgkg (Table 2) or 10 mg/kg qd

Table2 Isolation of C. pneumoniae from Lungs Following Treatmentof Experimental Pneumonitiswith KM-1648 mgkg days) Days after (deaths)b Untreated Treated (treatment) inoculation’

Number of positive animals total

m 515 015 015

2l4=

.Inoculum size: 1.0 108 IFU/mouse. bFive control died, one on day 2 and two each on days 7 and 9 postinoculation;isolations were not attempted in dead animals. CReisolation from the fifth mouse in the group was contaminated.

Sensitivity of C. pneumoniae to Macrolides Versus KRM-1648

321

Table Isolation of C. pneumoniae from Lungs Following Treatment of Experimental Pneumonitis withKR"1648 mglkg X days)

Days after Untreated inoculationa l (treatment)

Number of Positive animalstotal Treated

ND 015 015 018

for days (Table The efficacy of treatment was evaluated by isolation of C. pneumoniae from lungs. Five days aftertreatment, C. pneumoniae was not recoverable fromany animals treated with KRM-1648,whereas 100% of control animals were still infected (Table 2).

CONCLUSIONS Thesepreliminaryresultsindicate that KR"1648 has potent activity against C. pneumoniae, butcombinationswithazithromycin or other macrolides may be even more effective. In vivo combination studies are planned.

REFERENCES 1. Kuo C-C, Jackson LA, Campbell LA Grayston JT. Chlamydia pneumoniae (TWAR). Clin Microbiol Rev 8:451-461. 2. Saito H, Tomioka H, Sato K, Emori M, Yamane T, Yamashita K, Hosoe K, Hidaka T. In vitro antimycobacterial activities of a newly synthesized benzoxazinorifamycins. Antimicrob Agents Chemother Kuo C-C, Grayston JT. In vitro susceptibility of Chlamydia sp. strain TWAR. Antimicrob Agents Chemother Malinverni R, Kuo C-C, Campbell LA, GraystonJT. Effects of two antibiotic regimens on course and persistence of experimental Chlamydia pneumoniae W A R pneumonitis. Antimicrob Agents Chemother

Susceptibilities to Azithromycin of Isolates of Chlamydia pneumoniaefrom Patients with Community-Acquired Pneumonia P. M. Roblin, N. Sokolovskaya,

M.R. Hammerschlag SUNY Health ScienceCenter at Brooklyn Brooklyn, New York

INTRODUCTION Chlamydia pneumoniae is a frequent cause of community-acquired respiratory tract infection, including pneumonia and bronchitis There are limited data on the treatment of these infections; most studies have usedserologyonly,thusmicrobiologicefficacycould not be assessed. Azithromycinisactiveagainstawiderange of pathogens and has superior pharmacokinetics and tolerance comparedto erythromycin. Preliminarystudiesfrom our laboratoryhavedemonstrated that azithromycin hasinvitroactivityagainst C. pneumoniae similar to erythromycin part of a nationwide, multicenter study that evaluated a 5-day course of oral azithromycin for the treatment of community-acquired pneumonia and bronchitis in adults, we isolated C. pneumoniae from (18%) of the 168 patients enrolled. We performed in vitro susceptibilities to azithromycin on isolates of C. pneumoniae from patients with pneumonia. 322

Azithromycin: Susceptibility

of Isolates of pneumoniae C.

323

METHODS Patients Patients 12years of age or older presenting with community-acquired pneumonia or bronchitis were enrolled in the study. Patients were obtained as part of a multicenter study fromsix sites in five states and the District of Columbia (Georgia, New York, Wisconsin, Texas, Massachusetts).

Drug Azithromycin (Pfker) was supplied as powder and solubilized according to the instructions of the manufacturer. Culture of C. pneumonia Culture was performed at S U N Y Health ScienceCenter at Brooklyn utilizing cycloheximide-treated HE-2 cells grown in 96-well microtiter plates. After 72 h incubation, all specimens were passed once. Cultures were confirmed by fluorescent antibody staining with a C. pneumoniae-specific monoclonal antibody (Washington Research Foundation).

Isolates Patient isolates were passed five to six times in cell culture in antibiotic-free medium.

Susceptibility Testing Testing of C. pneumoniae was performed in cell culture using HEp-2 cells grown in 96-wellmicrotiter plates. Eachwell was inoculated with 0.2 m1 of the organism dilutedto yield 103inclusion-forming units(IFUs) per ml, and centrifuged at 2000 X for 1 h. The wells were then aspirated and overlayed with 0.2m1of medium containing1pg of cycloheximide per mL and serial twofold dilutionsof the test drug. After incubation at 35°C for 72 h, cultures were k e d and stained for inclusions with fluorescein-conjugated antibody to the lipopolysaccharide genus antigen (Pathfinder Chlamydia Culture Confirmation System, Kallestad Diagnostics, Chaska, MN). The minimal inhibitory concentration (MIC) wasthe lowest antibiotic concentration at which no inclusions were seen.The minimal chlamydiacidal concentration (MCC) was determinedby freezing the cultures at -7O"C, then thawing, passing the disrupted cell monolayersonto new cells, incubating for 72 h, then fixing and staining as above. The MCCwas the lowest antibiotic concentration which resulted in no inclusions after passage. All tests were run in triplicate.

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Table l Results of Azithromycin Multicenter TreatmentStudy of Patients with Pneumonia and Bronchitis C. pneumoniae culture-positive (%)

No. with pneumonia No.with bronchitis Total No.patients

RESULTS shown in Rble of of the patients with bronchitis and of patients with pneumonia were culture-positive for C. pneumoniae. Three of the pneumonia patients were culture-positive during and after treatment. We were ableto retrieve five additional isolates from thesethree patients. The MICs and MCCs of all isolates are shown in Table The MIC,, and MIC, were pg/ml (range pg/ml). The MCC,s were pg/ml. The in vitro susceptibilities of the isolates from the persistently infected patientsare shown in Table The MICs did not change during or after therapy except for patient The MIC and MCC of this isolate increased fourfold after therapy from pg/ml to pg/ml; Despite persistenceof C. pneumoniae, all patients improved clinically.

CONCLUSIONS The efficacy of azithromycin for eradication of C. pneumonia from the nasopharynx of patients with pneumonia was which iscomparable to our previous experience with clarithromycin and erythromycin for treatment of C. pneumoniae infection in children The MIC and MCC of one isolate increased fourfold after treatment, although it remained susceptible to azithromycin. It is not clear if this is an isolated event or suggestive of possible development of resistance. However, the isolates Table 2 In Vitro Susceptibilitiesto Azithromycin of Isolates of C. pneumoniae from Patients with Community-Acquired Pneumonia

Range

MIc (Pf5Wl) MCC (Pg/ml)

90%

Azithromycin: Susceptibility of Isolates of C. pneumoniae

325

Table In Vitro Susceptibilitiesto Azithromycin of Isolates of C. pneumoniae from Three Persistently Positive Patients with Pneumonia Patient

Date

MIC (/.%W

MCC (Pg/ml)

ARD ARD ARD CJA CJA WF WF WF

from the other two persistently infected patients did not change duringor after treatment with azithromycin. Inour previous experiencewith the use of clarithromycin and erythromycin,we did not find any change MIC in or MCC despite persistence of the organism in children with pneumonia(4). Further studies of treatment of C.pneumoniae infection, utilizing culture, are needed to both assess efficacy andto monitor forthe possible development of resistance.

REFERENCES Grayston JT, Kuo C-C, Wang SP,Altman J. A new Chlamydia psittaci strain, W A R , isolated in acute respiratory tract infections. N Engl J Med Grayston, JT, Campbell LA, Kuo C-C, MordhorstCH, Saikku P, Thom DH, WangSP. A new respiratory tract pathogen: Chlamydia pneumoniae strain W A R . J Infect Dis Hammerschlag MR, QumeiKK, Roblin PM. In vitro activities of azithromycin, clarithromycin, L-ofloxacin andother antibiotics againstChlamydiapneumoniae. Antimicrob Agents Chemother Roblin PM, Montalban G ,Hammerschlag MR. Susceptibilities to clarithromycin and erythromycinof isolates of Chlamydia pneumoniaefrom children with pneumonia. Antimicrob Agents Chemother

Azithromycin in Control of Trachoma Julius Schachter UCSF Chlamydia Research Laboratory, San Francisco GeneralHospital San Francisco, California

INTRODUCTION Trachoma, a chronicfollicular'keratoconjunctivitis, isconsidered the world's leading cause of preventable blindness.It occurs in many developing countries and affectsthe poorest members of societies livingunder the worst hygienic conditions. It is currently estimated that several hundred million people live in trachoma endemic areas andmillionshavebeen blinded as a result of this infection. In trachoma hyperendemic areas, virtually all individuals are infected very early in life (prior to 2 years of age). Active disease is a disease of children. The infection rates drop dramatically after age 7-10 years. With resolution, the conjunctivae are scarred. Thosethat have had severe inflammation are more likely to develop scarring,which causes distortionof the eyelids. The cornea is then abraded by inturned eyelashes. This causes blindness. In the hyperendemic areas, the risk of blindness can be as high as 25% or more in those survivingto age 60 years. Trachoma control is currently based on use of topical tetracyclinefor infected children and corrective lid surgery for adults with trichiasis. The causative organism, Chlamydia trachomah, is sensitive to a number of antibiotics, but formany years, tetracyclines were consideredthe drugs of choice. Recent introduction of azithromycin has dramatically changed our 326

Azithromycin in Control of Trachoma

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perspectivesontreatingchlamydialinfection.Azithromycinhasbeen shown to be effective in a single-dose therapy for genital chlamydial infection (C. trachomatis is a very common genital tract pathogen) (1). Thus, there is a potential for single-dose therapy of trachoma rather than the long-term treatment currently requiredwith antibiotics such as tetracycline or erythromycin. Single-dose azithromycin has been shown in Gambia and in Egyptto be as effectiveas the current long-termWHO-approvedregimen of ophthalmic ointments(2). The use these ointmentsis aimed at reducing the time of infection and thus reducingthe risk of complications. It is not considered curative. If single-dose azithromycin is curative, it offers the possibility for trachoma control, rather than simply mitigating its consequences in endemic areas.

MATERIALS AND METHODS In each country, two or more villages with high levels of endemic active trachoma were identified. These villages were randomized to receive 30 days of topical oxytetracyclineor three once-weekly doses of azithromycin. Oral doses of azithromycin were 1g for adults and 20 mgkg for those under10years of age. Womenof childbearingagewerescheduled to receive week-long courses of erythromycin 500 mg four times daily or amoxicillin 500 mg three times daily. Standard trachoma scoring systems were used in of each the countries (3). Infection rates were assessedby use of ligase chain reaction to detect chlamydial DNA in conjunctival and nasopharyngeal specimens. The project, as designed, involves follow-up for l-year period after treatment. Treatment was finished at the various sites between October 1994 and March 1995 and the l-year follow-up is scheduledto be completed in March 1996.

RESULTS Becausemost of the results are codedand the researchers have been blinded, we are not yet in a position to present any of the microbiological results or to discuss most of the clinical findings. Preliminary evaluation of the 3-month follow-up in Egypt can be discussed. Those results are shown in Table 1. There was a statistically significant reduction in active disease (p < loT5)in both the oral azithromycin andthe topical oxytetracycline groups. The reduction in the azithromycin group was seen from both severe and moderate disease,whereas the topicaloxytetracyclinegroup,although

Schachter

328

Tabk I Trachoma Therapy (Egypt)-Interim Results 1995: Clinical Response to Oral Azithromycin or Topical Oxytetracycline

Post" Pre-RX (N=180)

#l 131 days N=178

Azithromycin Severe Moderate Not active

Oxytetracycline Severe Moderate Not active

30%

4.4%

-

44.5% 51.1%

(N=166)

146 days (N=164)

-

14% 62% 24%

showing a diminution in overall activity, did not show significant reductions by level of clinical activity. It may be of interest to look at the different effects that were observed in this trial where there was communitywidetreatment as compared to a previous trial in which only children with active disease weretreated. In that clinical trial, with follow-upof approximately the same time period (4-5 months after treatment), 9/38 (24%) of the azithromycin group and 10/40 (25%) of the topical oxytetracycline group showedno active disease at the 4-5 month period. In contrast, whenvillagewide treatment was carried out, 9/178 (51%) of the azithromycin group was clinically inactive at the first follow-up, whereas again, only about aquarter (39/164) of the topical oxytetracycline group were inactive(p <

CONCLUSIONS Obviously, this study is underway and we have only the most preliminary results to discuss. What has been shownat all sites isthat short courses of oralazithromycin areat leastaseffectiveaslongcourses of topical oxytetracycline, suggesting from a program viewpoint that this may be useful therapy even if it is not an effective control measure. Preliminary results show that in Egypt, at least, there appears to be an increased benefit amongthe most highly infected group ofthe community (those children with active disease) when the entire village wastreated

Azithromycin in Control

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as compared to when just the children with active disease were treated. This was shown by an approximate twofold increase inthose who became clinically inactive atthe 4-5 month follow-up period. Because these were isolated treated villages in hyperendemic or endemic trachomaareas, it is predictablethat this communitywide intervention must fail asthe organisms will be reintroduced from nearby untreated villages and by migration patterns. However, the dramatic reduction in disease suggeststhat azithromycin may be usefulthe in control of trachoma. Such a conclusion must awaitfurther analysis of the results, together with the confirmation of clinical findings by community-based microbiologic testing to assess infection status.

REFERENCES Martin D, Mroczkowski T, Dalu AZ, McCartyJ,Jones RB, HopkinsSJ, Johnson RB, and the Azithromycin for Chlamydial Infections Study Group. A controlled trial of a single dose of azithromycin for the treatment of chlamydial urethritis and cervicitis.N Engl J Med Bailey R L , Arullendran P, Whittle HC, and Mabey DC. Randomized controlled trialof single-dose azithromycinin treatment of trachoma. Lancet Thylefors B, Dawson CR, Jones BR, West SK, andTaylor HR. A simple WHO system for the assessment of trachoma and its complications. Bull

Microbiologic Efficacy of Azithromycin for the Treatmentof Community-Acquired Pneumonia Due toChlamydia pneumoniae in Children M. Hammerschlag, P. M. Roblin, Michael Campbell, Antonia Kolokathis,M. Powell, and the Pediatric Pneumonia Study Group SUNY Health ScienceCenter at Brooklyn Brooklyn, New York Pfirer Central Research New York, New York

Chlamydia pneumoniaeis emerging as an important cause of pneumonia in adults and children. Initial serologic studies suggested that this organismis an infrequent cause of pneumonia in young children. However, a recent multicenter treatment study fromthe United States isolated C. pneumoniae from 14% of children, 3-12 years of age, withradiographicallydocumented pneumonia (1).Data on treatment of these infections are limited; most studies have relied on serologic diagnosis; thus, microbiologic efficacy couldnotbeassessed. The purpose of the present study was to compare azithromycin suspensionto erythromycin suspension and amoxicillinclavulanate for the treatment of pneumonia in children. 330

Microbiological EfJicucy of AzithromycinAgainst C.pneumoniae

331

METHODS Patients Children, 6 months through 16 yearsof age, presenting with communityacquired pneumonia were enrolled in the study. They had to have radiographic evidence of pneumonia, no history of allergy to macrolide antibiotics, or no serious underlying disease. Written informed consent was obtained from the parent or legal guardian. The children were randomized (2 : 1) to receive pediatric suspensionsof either azithromycin or the comparative agent (amoxicillin-clavulanate if <5 years; erythromycin if 2 5 years of age). The dose of azithromycin suspensionwas 10 mgkg once on day 1 (maximum 500 mg) followed by 5 mgkg qd (maximum 250 mg/ day) on days 2-5. Amoxicillin-clavulanate suspension was given at a dose of 40 mgkg per day, in three divided doses for 10 days and erythromycin estolate suspension was given at a dose of 40 mgkg per day, in three divided doses for 10 days.

Specimens Nasopharyngeal swab specimens were obtained for C. pneumoniue culture. A throat swab was alsoobtained for culture of Mycoplasma pneumoniue. Sera were obtainedfor antibody to both organisms.

Culture of

pneumoniue

Culture was performed at S U N Y Health ScienceCenter at Brooklyn utilizing cycloheximide-treated HEp-2 cells grown in 96-well microtiter plates. After 72 hincubation,allspecimenswerepassedonce.Cultureswere confirmed by fluorescent antibody staining with a C. pneumoniue-specific monoclonal antibody (Washington Research Foundation). pneumoniue Serology

C.pneumoniue antibody testingwas performed at MRL Reference Laboratory, Cypress, CA by the microimmunofluorescence (MIF) method with IgG and IgM conjugates. Susceptibility Testing of

pneumonim Isolates

Susceptibility testing of C. pneumoniue was performed in cell culture by usingHEp-2cells grown in96-wellmicrotiterplates.Eachwellwas inoculated with0.1mLof the testisolatediluted to yield103-104 inclusion-forming units (IFUs) per mL, centrifuged at 1700 X for 1 h.

332

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Hammerschlag

al.

Wells were then aspirated and overlaidwith mL medium containing &m1of cycloheximide and serial twofold dilutions of azithromycin or erythromycin. After h incubation, cultures were fixed and stained for inclusions,asdescribedabove. The minimalinhibitoryconcentration (MIC) was the lowestantibioticconcentration at which no inclusions were seen. The minimum chlamydicidal concentration (MCC) was determined as previously described. The MCC was the lowest antibiotic concentration which resulted in no inclusions after passage. All tests were run in triplicate.

M.pneumoniae Culture, PCR, and Serology Cultures, PCR, and serology by ELISA were performed at theUniversity of Alabama, Birmingham.

Patient Follow-up Patients were seen again at days, days, and 4-6 weeks after enrollment. Specimens for cultures and sera for antibody determinations were collected at baseline andthe visit.

RESULTS

A total of children with pneumonia were enrolled from sites in states. There were males and (44.5%) females. The mean age of the children was years; one-half were less thanyears of age. C. pneumoniae was isolated from of the children. The mean ageof the chlamydia-positive childrenwas years; were lessthan years of age. Only 6 of culture-positive children had serologic evidence of acute infection (IgM titer IgG 1 or fourfold rise). Table I Microbiologic Response to Treatment in Children withC. pneumoniae Infection

Drug

No. patients with cultures/total negative (%)

Azithromycin Amoxicillin-clavulanate Erythromycin Total

414 (loo) 7/7 (loo) 30/34 (88)

Microbiological Efficacy of Azithromycin Against C. pneumoniae

333

Table 2 In Vitro Susceptibilitiesof 38 Isolates of C.pneumoniae from 32 Children with Community-Acquired Pneumonia

MIc (Pg/ml) D m Azithromycin Erythromycin

MCC (Pdml)

Range

50%

90%

Range

90%

0.015-0.5 0.015-0.5

0.125 0.062

0.25

0.015-0.5 0.015-0.5

0.25

W Oculture-positive patients did not return for any follow-up visits. shown in Table 1, C. pneumoniae was eradicated after treatment from the NP of 19 of 23 (83%) evaluable patients who received azithromycin; 4 of 4, and 7 of 7 who received amoxicillin-clavulanate and erythromycin, respectively (p = The MICs and MCCs of 38 isolates of C. pneumoniae from 32 patients were performed against azithromycin and erythromycin.shown in Table 2, the MIC, for azithromycin and erythromycin was 0.5 pg/ml and 0.25 pg/ml, respectively.The MICs of the isolates fromthree or four persistently infected children did not change aftertreatment, one increased dilutions, 0.031-0.125 &m1 for both azithromycin and erythromycin but was still susceptible (Table 3). lbo (5.5%) of the children with culturepositive C. pneumoniae infection were coinfected with M. pneumoniae. Both children were treated with azithromycin and both organisms were eradicated. The MICs to azithromycin of all the M.pneumoniae isolates were < 0.008 pg/ml. Table MICs for Isolates for C.pneumoniue from Four Persistently Positive Patients

Treatment Patient

Date

hromycin015 7/26/94

Azithromycin 2/7/94 1321

hromycin356

hromycin032

12/21/94 U8194 3/22/94 U1194 U16194

Erythromycin Azithromycin 0.031 0.125 0.5 0.5 0.25 0.25 0.25 0.125

0.031 0.125 0.25 0.25 0.125 0.125 0.125 0.031

Hammerschlag et al.

334

DISCUSSION Azithromycin eradicated C. pneumoniae from the nasopharynx of 81% of the infected children with pneumonia in this study. These resultsare very similar to those reported by Block et al. (1) comparing clarithromycin to erythromycin, which had eradication rates forC. pneumoniae of 79% and 86%, respectively. the numbers of children with culture-positive C. pneumoniae infectionwhoreceived the comparativeagentswerevery small,wecannotmakeanyconclusionsabout the relative efficacy of erythromycin or amoxicillin-clavulanate. Persistence of C. pneumoniae infection after treatment with azithromycin was not associated with the development of antibiotic resistance, asthe MICs remained stable inthree of the patients. These findingsare similar to those we described for isolates of C. pneumoniae from persistently infected children in the clarithromycin study (2). The MICs to azithromycin and erythromycin of one strain did increase by two dilutions but remained susceptible.

REFERENCES 1. Block S, Hedrick J, Hammerschlag MR, Cassell GH, Craft JC. Mycoplasma pneumoniae and Chlamydia pneumoniae in pediatriccommunity-acquired pneumonia: comparative efficacy and safetyof clarithromycin vs. erythromycin ethylsuccinate. Pediatr InfectDis J 1995; 14:471-477. Roblin PM, MontalbanG, Hammerschlag MR. Susceptibilitiesto clarithromycin and erythromycinof isolates of Chlamydia pneumoniae from children with pneumonia. Antimicrob Agents Chemother 1994; 38:1588-1589.

Community-Acquired Pneumoniain Children: Underestimation of Mycoplasma Infection and Efficacy of Macrolides Dominique Gendrel, Josette Raymond, Florence Moulin, Jean-Luc Iniguez, Sophie Ravilly, Pierre Lebon, and Gabriel Kalifa Hopital Saint Vincent de Paul Paris, France

The initial antibiotic treatment in pneumonia of children remains largely empiric because the determination of etiologic agents is difficult in most patients (1-4). However, the frequency of various pathogens involved in childhood respiratory infections is changing. Penicillin-resistant Streptococcuspneumoniae is increasing rapidly Haemophilus and injluenzae b immunization has resultedin adecrease in invasive infections due to this organism. The aim of our study was to determine the etiology of community-acquired pneumonia in children overthe age of 18 months.

PATIENTS AND METHODS Between 1 June 1992 and 1 December 1994, 104 children, mean age 5.6 years (range: 18 months-l3 years) presenting with a community-acquired pneumonia to the outpatient clinic of Saint Vincent de Paul Hospital in Paris were enrolled in the study.Bloodsampleswere taken for blood culture, acutekonvalescent antibody assay for viruses and Mycoplasma

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Gendrel et al.

336

pneurnoniae (IgM and IgG). A nasopharyngeal aspirate was obtained at the first visitfor viral studies.

RESULTS Etiologic Agents

A potentialcausativepathogen was identifiedin 871104 of the children (Table 1).A viral originwas detected in patients (29%) (mean age: 4.1 years). Pneumonia was due to Streptococcus pneurnoniae (SP) in 12 patients (mean age: 4.5 years). Blood culture for SP was positive in eight patients, with all strains sensitiveto penicillin. In 41 patients (40%) (mean age: years), Mycoplasma pneumoniae was the cause of pneumonia. Specific IgM titers against Mycoplasma pneumoniae (MP) were positive and a threefold or more increase of specific IgG titers between two sera was observed. In two other cases, MP and SP infections occurred together. In eight children with a bacterial infection, serology showed an asso ated viral infection: parainfluenza with pneumococcal infection in two children, mycoplasma infection with respiratory syncytial virus (RSV) in three children, with parainfluenza in two and with Epstein-Barr virus (EBV) in two others. Table l Etiology of Community-Acquired Pneumonia in 104 French Children, 18 Months to 15 Years

Viruses RSV Parainfluenza 1or Influenza A or B Adenoviruses Other

30/104 10 6 4 4

6

Bacteria 571104 (55%) Staph. aureus 1 Chlamydia 1 Streptococcus pneumoniae Mycoplasma pneumoniae 41 Pneumococcus + Mycoplasma No agent identified

17/104

PneumoniainChildren:

Mycoplasma InfectionlMacrolideEfficacy

337

Clinical and Radiological Data There was a difference in baseline signs and symptoms and the seventy of illness by identified pathogens. Eleven (92%) children with pneumococcal infection were hospitalized emergently, and 7 of them were hypoxemic. Only 15/41 (36.6%) children with mycoplasma infection required hospitalization (2/41 were hypoxemic), and 18/30 (60%)of those with viral infection (nine with hypoxemia). Radiological findings were not specific for one etiology. Uniquelobar consolidation, considered characteristic of pneumococcal infection, was present in 50% of patients with pneumoniadue to S. pneumoniae, 12.2% in mycoplasma infections, and 10% in viral pneumonia.

Response to Antibiotic Therapy Children with pneumococcal pneumonia had not received antibiotics before hospitalization. Apyrexia was obtained with amoxicillin in 24 h inll/ 12 patients. Among the 41 patients with MP infection, 32 had initially received amoxicillin or penicillin V. In 30 patients, fever persistedfor 2-18 days, untiltreatment was switchedto a macrolide.In the nine patients with MPinfectioninitially treated withmacrolidesandin others given the macrolide treatment subsequently, fever decreased and definitive apyrexia was observed in 2-3 days. Macrolides given in the 41 patients when M P infection wasassessedincludedspiramycinin31,josamycinin 7 , and erythromycin in Eighteen children infectedby Mycoplasma pneumoniaewho failed lactam therapy were suspectedof infection with penicillin-resistant pneumococci and were referredto the hospital.

DISCUSSION Data were obtained from 104 children, 18 months to 13 years old, living in Pans or in nearby suburbs.A pathogen was detected by blood culture, viral isolation from the nasopharynx or serology in 85% of patients (89/104). Mycoplasma pneumoniae (40%), Streptococcus pneumoniae (11%) , and RSV (9%) were the most common causative agents isolated.The percentage of infections due to Chlamydia pneumoniae probably was underestimated because specific tests with high sensitivity for this pathogen were not performed. A pure viral infection was found in 30 patients and RSV was the predominant viral pathogen, as inother studies (1). Mycoplasmapneumoniae was the predominantpathogeninthis study, but the percentage of40% is higher than the 20-28% previously reported (1-4). The frequency ofMPin the etiology of community-

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acquired pneumonia is an important consideration the in choice of empiric antimicrobial treatment. Neither radiological findings nor CRP or WBC values are sufficient to distinguish pneumococcal from mycoplasmal infections. The best indicationof Mycoplasma pneumoniae infection ina patient with community-acquired pneumoniathe is failure of initial p-lactam antibiotic treatment. Because pneumococcal pneumonia remains the most lifethreateningillness in our experience, we consider that highdoses of amoxicillin is the best initial choice for antibiotic treatment in childhood pneumonia.When the p-lactam treatment failsclinically, it mustbe switched within48 h to a macrolide becauseMycoplasmapneumoniae infection is highly probable.

REFERENCES Claesson BA, Trollfors B, Brolin I, Granstrom M, Henrichsen J, Jodal U, Jut0 P, KallingsT,Kanclerski K, Lagergard T, et al. Etiology-of communityacquired pneumonia in children based on antibody responses to bacterial and viral antigens. Pediatr Infect Dis 1989; J 8:856-861. Ruuskanen Nohynek H, Ziegler T, CapedingR, Rikalianen H. Pneumonia in childhood; etiology and responseto antimicrobial therapy. Eur J Clin Microbiol Infect Dis 1992; 11:217-223. Block S , Hedrick J, Hammerschlag MR, Cassell GH, Craft JC. Mycoplasma pneumoniae and Chlamydiapneumoniae inpediatriccommunity-acquired pneumonia: comparativeefficacy and safety of clarithromycinvs. erythromycin ethylsuccinate. Pediatr Infect Dis J1995; 14:471-477. Nohynek H, Eskola J, KleemolaM, Jalonen E, Saikku P, Leinonen M. Bacterial antibody assays in the diagnosisof acute lower respiratory tract infection in children. Pediatr Infect Dis J. 1995; 14:478-484.

Ureaplasma urealyticumIsolations from Young Children with Respiratory Problems Sharon A. Poulin, Ruth B. Kundsin, and Rita D. Dehllis Brigham and Women’sHospital, The Children’sHospital Medical Center, Boston, Massachusetts

INTRODUCTION Meta-analysis of data from publications comprising 1092 subjects confirm that there is a significant association between Ureaplasma urealyticum colonization of the respiratorytractandsubsequentdevelopment of bronchopulmonarydysplasia,alsoknownaschroniclungdisease,in preterm infants (1). These studies all dealt with neonates. a logical sequel, this study was designed to look for U. urealyticum in older infants and young children with respiratory problems.It was also important to investigate whether preterm birth predisposed young children to both colonization and respiratory problems. Children, positive for U. urealyticum and older than6 months, would be treated with clarithromycin to establish whether eradicationof the microorganism would improve their clinical symptoms.

339

al. 340

et

Poulin

MATERIALS AND METHODS Eighty-eight children ranging in age from to months were seen in a pediatric practice for pulmonary symptoms including wheezing, bronchiolitis, cough, and respiratory distress. a comparison,throat cultures were also taken from twenty-two, well, asymptomatic children. The cultures were taken by one of the authors (RDD), a pediatrician with a practice devoted to high-risk young children, children who had been discharged from the intensive care nursery. Throat cultures were taken with a rayon swab with a plastic shaft which was immediately placed into Boston Broth The broth cultures were brought to the laboratory where the swab was streaked on A7 agar '' and replaced in the broth. Incubation of the plate was done anaerobically using the Fortner method Both plate and broth were observed daily. The change in pH of the broth to alkaline was an indicationthat a ureaseproducing organism was present and subcultures were madeto an A7agar plate. Cultures were considered positive when colonies were seen on the original agar plate or on the subculture. Some cultures positivefor ureaplasmas and mycoplasmas may have been missed because of overgrowth by other bacteria.

RESULTS Isolations from eighty-eight children with wheezing, bronchiolitis,or cough ranging in age from 2 to months are shown in Table1. Overall, of the had U. urealyticum isolated from the throat. Of infants, 12 months or younger at the time of culture, had U. urealyticum. Of children older than months but younger than months, 9 had U. urealyticum isolated. No ureaplasmas were isolated from seven children older than months. One child (1%) had Mycoplasma hominis as well as

l

%

32%

Tabik I Ureaplasma urealyticum from the Throats of 88 Symptomatic Young Children Versus Age

Age (Yearn)

U. urealyticum-positive childredtotal Percent done

0-1 >l-2 >2-3 >3-5

19/55

Total

28/88

positive

40% 116

On

17% 0

U.urealyticum Isolations and Respiratory Problems Table 2 Ureaplasma urealyticum from the Throats of Children VersusAge

341 Asymptomatic Young

U. urealyticum-positive childredtotal done

>

Percent positive

OD

5%

Total

U. urealyticum. Five children (6%) had Mycoplasma salivarium but not ureaplasmas. Children fromwhom U. urealyticum was cultured were ill significantly more frequently and more severely than children with negative cultures. Culture-positive children had an average of 46 days of wheezing inthe first year of life, comparedto 7 days of wheezingfor culture-negative children. As shown in Table 2, cultures of a control group of 22 well children resulted in one isolation of U. urealyticum (p < .Ol). The well children had had an averageof 2 daysof wheezing inthe first year of life. Ten (34%) of 29 children born preterm (less than 37.5 weeks) were found to be positive comparedto 31% (18/59) born at term (Table3). This finding is particularly interesting, because it suggests that colonization of a term infant may be associated with respiratory problems. All 28 children positive for U. urealyticum in the throat have been treated with 15mgkg clarithromycin for 14 days. In 16 children, symptoms improvedandsubsequentposttreatment throat cultures were negative. children were positive aftertreatment, and two other children didnot complete treatment. Eight children still need posttreatment follow-up cultures. In vitro antibiotic susceptibility testing of U. urealyticum -demonstrated that 100% of isolates were susceptibleto clarithromycin. Tabh 3 Ureaplasma urealyticum from the Throats of Children Versus GestationalAge U. urealyticum-positive Percent age

Gestational weeks 237.5 weeks

Symptomatic Young

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Poulin et al.

CONCLUSION This report documents the presence of U. urealyticum in the respiratory tract of symptomatic children upto the age of years. U. urealyticum may play a role in the etiology of severe respiratory symptomsin these young children. Because tetracyclines,the most effective antibioticsfor ureaplasmas, are contraindicated in children, these infections are not eliminated and may continue to cause pulmonary symptoms. Whether these microorgansims are causal is not clear. However, eradication of U. urealyticum from these young children withthe macrolide clarithromycin did result in improvement of their respiratory symptoms.

REFXRENCES 1. Wang EE, Ohlsson A, Kellner JD. Association of Ureaplasmaurealyticum colonization with chronic lung disease of prematurity: results of a metaanalysis. J Pediatr 1995; 127:640-644. 2. Leland DS, Lapworth MA, Jones RB, French ML. Comparative evaluation of media for isolation of Ureaplasma urealyticum and genital mycoplasma species. J Clin Microbioll982; 16:709-714. 3. Kundsin RB, Poulin SA. Laboratory diagnosis of genital mycoplasma infection. Ann New York Acad Sci 1988; 549:72-80.

Could Clarithromycin Prevent Asthma? Rita D. DeLollis, Ruth B. Kundsin, and Sharon A. Pouli Brigham and Women’sHospital, The Children’sHospital Medical Center and Harvard Medical School Boston, Massachusetts

BACKGROUND Asthma isthe most disablingof all chronic diseases in childhood, and one of the commonest. About 30% of individuals who are subsequently diagnosed, in middle childhood or later, as having asthma began with symptoms of recurrent wheezing, cough, and bronchospasm inthe first year of life. A longitudinal study of infants who wheezed in the first year has revealed that only a portion,about B%,who have had more severe symptoms and abnormal pulmonary function develop asthma in later life (1). Most severe childhood asthmatics become asthmatic adults. Prior studies have concentrated on family history and demographic factors such as low risk, and lower maternal age. Infection birth weight and prematurity, withrespiratorysyncytial virus (RSV) and chlamydiapneumoniae also have been implicated in some studies of asthma, possibly through hostmediated IgE responses Ureaplasma urealyticum is a common organism in the genitourinary tract of women, found in up to 70% at obstetric examination. Infected women may inoculate their infants at or before parturition. U. urealyticum has been linkedto premature birth (5,6) and implicated in pneumoniaand inchronicbronchopulmonarydysplasia of premature infants (6,7). U. urealyticurn had been found to persist in pulmonary secretions in these infants for several months, butour work has newly demonstrated the ongoing presenceof U. urealyticum in term andpreterm infants through years 343

DeLollis et al.

344

of age. No prior study has been carriedout to determine if U. urealyticum plays a pathogenic role in children born at term. This report explores the effects of colonization with Ureaplasma urealyticum in infants who present with bronchospasm inthe first 3 yearsof life, with attention to the possible role of U. urealyticum as a precursorto later asthma.

OBJECTIVE The objective of this study is to assess the clinical effects ofneonatal colonization with U. urealyticum with regard to later reactive airway disease.

PATIENTS AND METHODS Subjects were drawn from a neonatal high-risk follow-up program and from a primary care pediatric practice. Ninety children aged 2-36 months (mean: months) were seen for well or sick ambulatory care and were evaluated for respiratorysymptoms.Seventy-three of the children had wheezing and other symptoms of reactive airway disease, including cough, tachypnea, retractions, or hypoxia at the time of the visit, and were assigned to the trial group. Seventeen children without evidenceof respiratory disease were enrolledas controls. (See Table1.) Throat cultures (Table 2) were taken from the 90 children and placed in Boston Broth. They were forwarded to the same laboratory without indication of groupstatus.Serialcultureswere undertaken to isolate ureaplasmas and genital mycoplasmas. Not all samples were tested for classical mycoplasmas. Children who had positive cultures then weretreated with clarithromycin at 15 mgkg per day in bid dosage. The first 5 patients received a 10-day course, and the subsequent 21 patients hadtreatment for a 1Cday period. Treatment was followed by a repeat culture in 2-8 weeks (in progress). Evaluation of the clinical course was accomplished by review birth and ambulatory medical records and physical examination. All children were assessed clinically when cultured, before and after treatment. Clinical follow-up was carriedout for a periodof 1-24 months after culture (mean: 10.7 months).

RESULTS Ureaplasma urealyticum was cultured from 26 children: subjects (34%) and 1 control (5%) (p < .Ol). Mycoplasma salivarium was found in subjects and Mycoplasma pneumoniae and Mycoplasma hominis in one

Could Clarithromycin Prevent Asthma? Table I

345

DemographicData

Subjects

Controls

Total Age at culture months months months months Sex Female Male Gestational Age weeks weeks weeks Race White Black Hispanic

5 8

9

46 9

0 55

Other Birth weight (kg)

0

4

1

0

(mean)

child each. Urine was collected from 21 of the children who had positive throat cultures. Three (18%) grew urealyticum. Renty-one subjects positive for U. urealyticurn were treated with clarithromycin, 15mgkg per day. The initial five subjects weretreated for 10 days, andthe remainder for 14 days. Follow-up cultures wereobtained to date on 11children. lbo subjects, one each treated for 10 and 14 days, required a second course of 14 daysto eradicate the organism. Eight of the 11 eventually had negative follow-up cultures. Three children had inadequate courses medication. One (not cultured) had colitis and could not tolerate treatment, and compliance was an issuefor the other two who are twins (positiveon reculture). Follow-up examination was completed for all children treated, and for all but five of negative children who had later follow-up by telephone. A study of medical records revealed little difference between control children and subjectswho had a negative culture for U. urealyticum. Children who had negative cultures for U. urealyticurn, whether in control or in study groups, had a mean 7 days of wheezing in the first year of life. Children who had positive cultures spent an average of 46 days (p < .01) U.'

DeLollis et al.

I I I

do00

347

Could Clarithromycin Prevent Asthma? Table 3 ClinicalData

Age period 0-6 months 1.08 Controls N= 17

Days spent wheezing 6-12 months

0-12 months

16.48

46.12

2.50

Subjects 3.73 Negative

3.40

N=48 Positive

19.64

N=25

wheezing inthe first year. Positive infants presented with wheezing, usually diagnosed as bronchiolitis, earlier than did negative children, at a mean of 4 months versus 7.5 months for negative infants (p < .05); were clinically more ill; and spent more days in treatment with beta-adrenergic medication (usually albuterol). They did not have more prior courses of antibiotics. (See Table Culture for other agents was camed out on a clinical basis. Five subjectssent to the hospitalhadpositiverapidtesting for respiratory syncytial virus. One of these also had U. urealyticum. All of the children who have been treated adequately with clarithromycin showed clinical improvement. Those who did not complete treatmenthavecontinued to have intermittent or chronicbronchospasm. Treated children also subsequently had more wheezing episodes than negative children for the same age period, but fewer than untreated children. Children having positive cultures who entered the study after the first year were more likely to have evidence of invasive disease (e.g., presentation with pneumonia) and then continued episodes of bronchospasm after treatment than were infants treated in the first year.

DISCUSSION Host factors and environment are known to affect the later course of children who have asthma. Theseare not assessed in this study. However, the range of severity of symptoms among colonized children was great and probably due to these factors. It is not supposedthat infection with U. urealyticum is the only source for chronic asthma.Other agents, including respiratory syncytial virus and Chlamydiae, may be implicated in some patients.

DeLollis et al.

348

In this very preliminary study,the association between asthma symptoms andthe presence of U. urealyticurn in the throat, and the amelioration of symptoms followingtreatment suggest a causal relationship. Because the children who were positive werethe most severely affected inthe practice population in the first year, they are the most likely to continue to have reactive airway symptoms in later childhood and adulthood.Thus, eradication of this organism,if carried out early in life,may prevent some cases of adulthood asthma. Further study is required to confirm these hypotheses. It is notable that virtually allthe subjects had been previously treated with antibiotics. Many had fouror five courses beforeculture. Clearly, for the colonized infants, these prior courses had not been effective in eradica ing this organism.

CONCLUSIONS Clarithromycin may have a new indication for use in children. Wheezing children identified early and treated seem to have abetter clinical outcome regarding asthma thando children treated later, or left untreated.

REFERENCES 1. Martinez FD, Wright AL,, Taussig LM, HolbergC J , Halonen M, MorganWJ. Asthma and wheezing the in first six years of life. N Engl J Med 1995; 323:133. 2. Duff AL, Pomeranz ES, Gelber LE, Price GW,Fams H,Hayden FG, PlattsMills TA, Heymann PW. Risk factors for acute wheezing in infants and children: viruses, passive smoke, and IgE antibodies to inhalant allergens. Pediatrics 1993; 92535. 3. Emre U, Roblin PM, GellingM, Domornay W, Rao M, Hammerschlag MR, Schachter J. The association of Chlamydia pneumoniae infection and reactive airway disease in children. Arch Pediatr AdolesMed 1994; 148:727. 4. Emre U, Sokolovshaya N, Roblin PM, Schachter J, HammerschlagMR. Detection of anti-Chlamydia pneumoniae IgE in children with reactive airway disease. J Infect Dis 1995; 172:265. 5. Eschenbach DA. Ureaplasma and premature birth. Clin Infect Dis 1993; 17 (suppl1):S100. 6. Kundsin RB, Leviton A, AllredEN,PoulinSA.Ureaplasmaurealyticum infection of the placentainpregnancies that endedprematurely. Obstet Gynecoll996; 87:122. 7. Cassell GC, Waites KB, Watson HH, Crouse DT, Harasawa R. Ureaplmmu urealyticum intrauterine infection: role in prematurity and disease in newborns. Clin MicrobiolRev 1993; 6:69.

Azithromycin in the Treatment of Chlamydia trachomatis Infection Dajek Warsaw MedicalSchool, Institute of Venerology Warsaw, Poland

INTRODUCTION Single-dose therapy is an ideal in venerology. This is especially true in the treatment of infections causedby Chlamydia truchomatis because they are the most widespread sexually transmitted diseases; the symptoms of infectionin men are often mildandinfectioninwomen is veryoften asymptomatic. Moreover, chlamydial infections may be implicated as a casue of infertility in both sexes, pregnancy complications, and/or newborn diseases. Asymptomatic patients, especially female sex partners of men with chlamydial urethritis, or patients with mild symptoms tend to take prescribed antibiotics incompletely. Poor compliance results therapeutic in failures and complications and contributes to the spread of infection. Thus, availability of effective single-dose therapy of chlamydial infections with azithromycin is important.

MATERIALS AND METHODS This study was runf'rom 8 July 1994 to 7 June 1995 at the outpatient clinic of the Institute of Venerology in Warsaw. Adult males and females with non349

Dajek

350 Table I

Baseline Patient Data

Females

Males

No. of evaluable patients (years) Age Symptomatic patients Asymptomatic patients

73 18-51 (mean 30.7) 72 (98.6%) 1 (1.4%)

28 18-46 (mean 27.3) 13 (46.4%) 15

complicatedchlamydialurethritidcervicitiswereincluded in the study. Chlamydial infection was diagnosed by direct immunofluoresdent (DIF), test Microtrac (Spa) or Chlamyset (Orion). Patients with gonorrhea, trichomoniasis, syphilis,or epididymitis were excluded. A single dose of azithromycin (Surnamed, Pliva)was administered orally, at least1h beforeor 2 h after a meal. Clinical examination, microscopy of urethral or cervical swab, and DIF were repeated 8-14 days after treatment. All patients were askedto avoid sexual intercourse until the completion of follow-up examinations. In men, cure was defined as disappearance of symptoms of urethritis with negative DIF test at the follow-up visit. Becauseof a high proportion of asymptomatic cases, women patients with negative DIF test at follow-up were considered cured.

RESULTS total of 108 patients, 80 males and 28 females, aged 18-51 years, were enrolled inthe study. Men presented spontaneously, but most women were referred sexual partners. Seven males were excluded from evaluation because they missed the follow-up. Baseline data on 101 evaluablepatients are presented in Table 1. Postgonococcal urethritis was diagnosed in 10 males given had been previouslytreated with penicillin, spectinomycin, or Table 2 Presence of Chlamydial Infection and Azithromycin Treatment Among Examined Pairs of Sexual Partners (Couples)

male partnerMale couples No.of DIF+b 24 DIF+b 2

+b

12 1

epididymitis

DIF+E*b DIF+ DIF+b DIF-

.Dm+: positive directimmunofluorescenttest immunofluorescent test for C. trachomatis. bPatients treated with azithromycin.

DIFfor C. trachomafis;DIF-: negative direct

Azithromycin for C. trachomatis

351

Tab& 3 Efficacy of Single-Dose Azithromycin inthe Treatment of Genital Chlamydial Infections Females

Males

No. of treated patients No. of cured patients No. of therapeutic failures

(100%)

rosoxacin. Although 39 couples or sexual partner pairs were examined, only 24 couples were treated with azithromycin (Table 2). Three males were homosexual. Among those not treated with azithromycin were 3 males and up to 12 DIF-negative female partners of males with DIF-positive urethritis. Patients withchlamydialepididymitisreceived standard treatment with doxycycline. Prophylactic treatment with doxycycline also was givento sexual partners with negative DIF but were at highriskofdeveloping chlamydial cervicitis and urethritis. Seventy (96%) men and28 (100%)women were cured (Table 3).In most men, discharge and dysuria regressed within 48-96 h after the administration of azithromycin; eight patients regressed within 24 h. In nine men urethral leukocytosis was present at follow-up but resolved within the following 7 days. Therapeutic failure, confirmed by a positive DIF test, was observed in three men. Tho had been without discharge and with a negative DIF test at follow-up, but recurrence occurred at 6 and 10 days, respectively. The third patient had discharge and positive DIF test at the first follow-up; he had a disulfiram implant.His sexual partner was cured. The tolerance of azithromycin was very good. Side effects were noticed in only two patients (2%). One patient reported vomiting 1h after taking the drug, but shewas cured. In the other patient, candida1 vaginitis was observed.

CONCLUSIONS

A single l-g dose of azithromycin is very effective and well tolerated in the treatment of symptomaticandasymptomatic urethral andendocervical infections caused by C. trachomatis. Single-dose azithromycin isverywnvenient and can be particularly recommended the in treatment of asymptomatic and chlamydia test negative female sexual contacts of menwith chlamydial urethritis.

Single-Dose Azithromycin in the Treatmentof Genital Chlamydial Infections V. Ferianec, K. Holoman, and J. Chmurny Comenius University Bratislava, Slovak Republic

V. Grba and F. Pliva-Bratislava Bratislava, Slovak Republic

INTRODUCTION Chlamydial infection is the most frequently encountered sexually transmitted disease and causes serious sequelae. Azithromycin is an azalide antibioticwithaverylowminimalinhibitoryconcentration(MIC) for Chlamydia trachomatis andachieves high intracellularconcentration which may be beneficial in eradicating this obligate intracellular pathogen. Azithromycin has high tissue bioavailability and a tissue half-life of days. These characteristics enable single-doseadministration of azithromycin in the treatment of uncomplicated genital chlamydial infections The objective of the study was to state the therapeutic efficacyandtolerance of azithromycin in the treatment of female genital chlamydial infections. 352

Single-Dose Azithromycin for

Genital Chlamydial Infections

353

PATIENTS AND METHODS Thirty women with genital chlamydial infection were treated with a l-g single oral dose of azithromycin. The same treatment was prescribed for the patient’s sexual partners.The presence of C . trachomatis was detected by cervical culture which was performed before and 2 weeks after the administration of azithromycin. Cure was defined as an absence, and failure as a presence of C. trachomatis in the cervical culture performed 2 weeks after the administration of azithromycin. Toleranceof azithromycin was assessed byquestionnaires that identified the incidence of drug-related side effects.

RESULTS lbo weeks after the administration of a single l-g oral dose of azithromycin, cervical culturewas negative in 28 women The treatment was unsuccessful in two women (6.6%). One gave a historyof unprotected intercourse with an untreated partner and probably was reinfected. One patient had a mild, probably drug-related, gastrointestinal side effect (nausea). CONCLUSION Single-dose oral azithromycin therapy is an advance in the management and controlof genital chlamydial infections.

REFERENCES 1. Johnson RB, The role of azalide antibiotics in the treatment of chlamydia, J Obstet Gynecoll991; 164:1794. 2. Majeroni BA. Chlamydial cervicitis: complications and new treatment options. Fam Physician 1994; 49:1825. Weber JT, Johnson RE. New treatments for Chlamydia trachomatk genital infection. Clin Infect Dis1995; 20 (suppll):S66.

Single-Dose Azithromycin Treatment: A Solution for Uncomplicated Lower Genital Tract Chlamydial Infection B. Kobal University Medical Centre Ljubljana Ljubljana, Slovenia

INTRODUCTION

Uncomplicated chlamydial cervicitis is a mild disease, predominantly affe ing young women at the beginning of their reproductive life. Spread of chlamydial infectionto the upper genital tract may result in serious tubal damage, increasing the risk for ectopic pregnancy and tubal infertility. Appropriate treatment of uncomplicatedlowergenital tract infection (LGTI) should preventfuture reproductive problems in women. A 10-day antibiotic course for uncomplicated cervical chlamydial infection isoften poorly accepted by otherwise healthy young women. Single-dose therapy would represent a solutionto this problemif adequate bacterial eradication could be achieved. A prospective, open, comparative study has been undertaken to evaluate the efficacy and tolerabilityof a single-dose azithromycin treatment in uncomplicatedLGTI.

354

Single-Dose Azithromycin

for Uncomplicated LGTI

355

PATIENTS AND METHODS Patients of both sexes, 16 years or older, with signs of uncomplicated LGTI and C. trachomatis isolated fromurethra or cervix were eligible for study. Patients with gram-negative diplococci on Gram-stained smear were excluded. Females were examinedat Department of Gynecology, University Medical Centre Ljubljana, and males at Department of Dermatovenerology. Patients were randomized to receive azithromycin (single l-g dose) or doxycycline (100 mg bidfor 7 days). Clinical examination and microbiologic assessments were performed before treatment and 7 and 28 days after treatment. Chlamydial infection was documented by isolation of C. trachomatisinMcCoycellculture. Hematologicaltests,bloodbiochemistry,andurineanalysiswereperformed before treatment and on the first follow-up visit.

RESULTS Ibenty-eight patients (11 male and 17 female) were included in the study. Thirteen patients weretreated with azithromycin and 15 with doxycycline. Baseline clinical signs of LGTI are presented in Table 1. Bacteriological efficacy is presented in Table At the first follow-up (day 7) chlamydial cultures were negative in all patients.At day 28, cultures were still.negative inallazithromycin-treatedpatientsandinonly10 of 15 doxycyclinetreated patients; long-term eradication rate in this group was 67%. As presented in Fig. 1. Clinical improvement was more rapid in azithromycintreated patients. There were two peaks of clinical improvement: seven patients were free of symptoms on the second or third day; five responded on the fifth day. In the doxycycline group, 11 patients reported clinical improvement by the seventh day of treatment. Regardless of the time to clinical improvement, all C . trachomatis-negative patients in both groups

Table Z Clinical Signs of Lower Genital Tract Infection at Admission Doxycycline Total No. of patients Females No symptoms Vaginal discharge Cervical discharge Males Urethral discharge

Azithromycin 15

6 1 2 7

7

7

7 4 4 4

Kobal

356

Tabkit?2 Eradication of C. trachomatis in Patients with Lower Genital Tract Infection Treated with Azithromycin and Doxycycline

Doxycycline Day 7 Day 28

Azithromycin 13/13 13/13

15/15 10115

were clinically improved on the second control visit, 28 days after treatment. Both treatments were well tolerated. No serious side effects were registered. Seven patients in the doxycycline group reported nausea, and one patient in the azithromycin group reported vomiting on the second day after treatment. No laboratory abnormalities weredetected.

DISCUSSION

The appropriate treatment for uncomplicated chlamydialLGTI should be short (i.e., single dose), and the eradication of pathogen should be complete with few side effects and low cost. All patients were highly motivate for treatment, but noneof their sexual partnerswished to participate in the treatment under the study conditions. With the use of barrier contraceptive

.-2m

a

(c

Days after treatment initiation

I Theimprovementofclinicalsigns lowergenitaltractinfectionin patients treated with azithromycin and doxycycline.

Single-Dose Azithromycin for Uncomplicated LGTI

357

methods, the study participants were somewhat protected from reinfection. We have observed a high eradication rate of C.trachomatis at day 7 in both treatment groups, butit was much higher in azithromycin recipients at day 28. The relatively lowlate eradication rate in the doxycycline group may be partly the result of reinfection due to withdrawal of barrier contraceptives. In addition to bacteriologicalefficacy,improvementofsymptomswas achieved earlier in azithromycin-treated patients and, azithromycin was better tolerated than doxycycline.

CONCLUSION Because only one-third of the planned 100 patients have completed the study, the conclusionsinthispresentation are strictlypreliminary. The results suggest high bacteriological efficacy, rapid clinical effect, and good tolerance of single-dose azithromycin inthe treatment of lower genitaltract chlamydial infections. The main advantage of azithromycin isits ability to be given as a single dose the in treatment of a common sexually transmitted disease.

Report of a Multicenter Clinical Evaluation of Azithromycin in the Treatment of Nongonococcal Urethritis in Men M. Urban University Hospital Kr4lovsk6Vinohrady Prague, Czech Republic ,

J. Flek, V. Herman, M. Chaloupka, andB. KraSny Dr. MulaE's First Hospital Plzen, CzechRepublic

J. Klamo General University Hospital Prague, Czech Republic

D. Pacfk and P. TomaStik University Hospital Brno Brno-Bohunice, Czech Republic

INTRODUCTION Chlamydia trachomatis and Ureaplasma urealyticum are the main demonstrable causes of nongonococcal urethritis (NGU). C. trachomatis is known to be present in 30-50% of cases (1). It is a slow-growing intracellular 358

359

Azithromycin for Nongonococcal Urethritis Menin

organism, difficult to treat. To be effective, the antibiotic must penetrate intracellulary and be present in therapeutic concentrations for several days (i.e., over the whole chlamydial growing cycle). Azithromycin is a novel azalide antibiotic with unique pharmacokinetic properties that enable sustained high tissue levels after a single oral dose. The aim of the study was to assess the efficacy and safetyof a singlel-g oral dose of azithromycin inthe treatment of men with NGU caused by C. trachomatis.

PATIENTS AND METHODS Men with symptoms and/or signs of NGU,urethral leukocytosis (more than five polymorphonuclear leukocytesper high-power fieldin a Gram-stained urethral smear), and positive culture for C. trachomatis were included in this open and noncomparative study. All patients were treated with a single l-g oral dose of azithromycin (Surnamed, Pliva). Clinical examination was performed before treatment and 8 -t 2 and 14-21 days after treatment. Signsandsymptoms of urethritis (urethral discharge,itching,dysuria, meatal erythema, and tenderness) were graded from 1to Specimens for microscopy and screening cultures, including C. trachomatis cell culture, were obtained at each visit. Treatment outcome was assessed on days 1421. Clinical efficacy was evaluated ascure, improvement, or failure (based on subjective criteria), and microbiological efficacy was evaluated as eradication, persistence, or eradication with recurrence.

RESULTS A total of 105menaged16-68years (meanage:38.1years)with chlamydial urethritis were recruited intothe study. The clinical and bacteriological efficacyof azithromycin is presented in Table 1. Two weeks after the treatment, cure was achieved in 78 patients (74%) and improvement in 27 (26%); there were no treatment failures. The eradication of C. Pachomatis was observed in 100 patients (95%). Tolerance of azithromycin was very good. No drug-related side effects were recorded. Table Z Clinical and Bacteriological Efficacyof Single l-g Dose of Azithromycin in Men with Chlamydial Urethritis

Clinical efficacy

No. of patients Bacteriological efficacy

Cure Improvement Failure

78 (74%) 27 (26%) 0

Eradication Persistence Recurrence

No. of patients

loo (95%) 5 (5%) 0

Urban et al.

360 DISCUSSION

We have observed high bacteriological efficacy of azithromycin that is in agreement with resultsof a large multicenter U.S.study in whichthe eradication rate of C. truchomutis was 96% (2). The complete or partial disappearance of symptoms of NGU was achieved in all patients. The absence of side effects was surprising because in other published studiesthe incidence of side effects was 8-17%

CONCLUSION A single 1-g dose of azithromycin is highly effective and safe inthe treatment of uncomplicatedchlamydialurethritis inmen. The assurance of compliance, enabled by single-dose administration, is a significant advantage of azithromycin in clinical practice.

REFERENCES Judson FN.Epidemiology and control of nongonococcal urethritis and genital chlamydial infections: review. Sexually Transmit Dis 1981; 8:117. Martin DH, Mroczkowski TF, D a h ZA, McCarty J, Jones RB, Hopkins SJ, Johnson RB, the Azithromycin for Chlamydial Infections Study Group. A controlled trial of a single dose of azithromycin for the treatment of chlamydial urethritis and cervicitis. N Engl J Med 1992; 327921. Stamm WE. Azithromycin in the treatment of uncomplicated genital chlamydial infections. J Med 1991; 9l(suppl3A):19S.

.

Efficacy of Roxithromycin in Urogenital Infixtion I. I. Derevianko Institute of Urology Moscow, Russia

INTRODUCTION Regardless of the achievements of modem chemotherapy, the treatment of urogenital infections remains unresolved. This is mainly due to the long course of urological diseases with clear tendencyto chronicity because of complicatedpathogenesiswithobstructionusuallyan important factor. Treatment is further complicated by the increasing incidence of strains resistant to antibiotics in heavy use in routine clinical practice. In addition, several microorganisms are responsible for the development of inflammation. These microorganisms often require combined treatment. Urogenital infection is predominantly caused by gram-positive bacteria-Staphylococcus and Streptococcus;seldom by gram-negative microorganisms, unless acquired nosocomially. At thepresent time, when diagnostic methods are improving, “atypical” microorganisms such as, Chlamydia trachomatis, Mycoplasmahorninis, Ureaplasmaurealyticum, Gardnerella vaginalis, and forth are often responsible for urogenital infections. Atypical microorganisms usually are found inside cells and cannot be reached by many antibacterial preparations (in particular, p-lactams, whose ability to penetrate cell membranes ispoor).

362

Derevianko

Macrolides have been used in the treatment of urological infections for many years. Erythromycin has the disadvantages of instability at low pH, a relatively narrow spectrum of activity, disposition to the speedy selection of stable microorganisms, and a high incidence of gastrointestinal side effects. The new generation of macrolide antibiotics with improved chemical structure alsohaveimprovedbiologicalandpharmacokinetic properties. Roxithromycin (Rulide “Roussel”) is a semisynthetic macrolide antibiotic that differs from erythromycin by the presence of a 2-methoxyethoxy-methoxymino groupat the ninth position.The present report shows high efficacy of roxithromycin in urogenital infections treatment caused by trachoma^). gram-positive organisms and atypical bacteria (Chlamydia

MATERIALS AND METHODS This open randomized study was conducted with two groups of patients. The first group consisted of 68 patients with history of inflammatory diseases of the lower urinary tract and the male urogenital system causedby gram-positive bacteria. Staphylococcus plus gram-negative flora-E.coli and Pseudomonas aeruginosa caused infection in eight patients who underwent invasive measures including cystoscopy and bladdercatheterization. All included patients had no renal or liver insufficiency.The differentiation of patients by age, diagnosis, and bacterial agents is shown in Table 1. In the second group, 23 patients withhistoriesofinflammatorydiseases Table Z Characteristics of Male Patients (17-83 Years Old) with Gram-positive Bacteria (n=68)

No. of Datients ~~

Diagnosis: Prostatitis Epididymoorchitis Balanopostitis Nongonococcal Bacteriological pathogens: Staphylococcus epidermidis Staphylococcus aureus Streptococcus sp. Staphylococcus epidennidis Escherichia coli Staphylococcus epidermidis Pseudomonas aeruginosa

{

~~

‘22 32 6

8 18

13 29 5

3

Infection Roxithromycin Urogenital in

363

Tabk 2 Characteristics of Patients (19-48 Years Old) with Chlamydia trachomafis(n = 23; 18 Males, 5 Females)

Sex

No. of patients

Male

urethritis cystitis Chronic Female

Chronic prostatitis Chronic Chronic urethritis

+ epididymoorchitis

+ epididymoorchitis

Chronic cystitis+ cervicovaginitis

7 3 5

3 2

caused by Chlamydia trachomatis were included. Table2 shows the clinical symptoms of these patients. Diagnoses of infection was based on typical clinical signs (pain the in lower abdomen, dysuria, fever) confirmedby special urological examination, blood, urine, prostate secretion, ejaculate analysis, and bacteriological examinations (urine and ejaculate culture). Laboratory tests were performed before and48 h after treatment. Roxithromycin was given orallyat a doseof 150 mg bid. Patients were instructed to take the medicine before meals with a sufficient quantity of water. Duration of treatment ranged from 5-10 days to 21 days according to the severity of the infection, typeof pathogen and clinical symptoms. Clinical response was assessed as “excellent” (complete relief from symptoms within 5-7 days), “good” (relief of symptoms within7-10 days or satisfactory decrease of symptoms at the end of treatment), and “poor” (clinical signs remained or worsened). Patients with C. trachomatis infections weretreated for 21 days and results were assessed after the treatment was completed, dependingon the presence (or absence) of the pathogen. If the pathogen remained duringtreatment, the clinical response was considered “poor;” disappearance of signs and symptoms was assessed as “excellent.” Bacteriological efficacy was considered as “satisfactory” when the microorganism was eradicated and “unsatisfactory” when the organism was not eradicated or superinfection developed.

RESULTS Treatment results are represented in Table 3. Clinical efficacy in patients with gram-positive infectionwas estimated at 38%. Symptoms disappeared or decreased within 5-10 days after the treatment started. The duration of treatment in this group varied, from5 to 10 days. Durabilityof treatment

Derevianko

364

Table 3 Clinical and Bacteriological Efficacy of Roxithromycin in the Treatment of Urogenital Tract Infections Patients with gram-positive bacteria (n=68) Clinical efficacy: Excellent Good Poor rate success clinical Total Bacteriological response: Satisfactory Unsatisfactory Total bacteriological Success rate Adverse effects

Patients with Chlamydia trachomatk

4

60 55 4 55

-

depends on the severity of clinical signs of the inflammatory process and the specific type of infection. For example, 5-7 days of roxithromycin is effective in treatingbalanoposthitis or nongonococcal urethritis. But, epididymorrchitis and prostatitis needs longer treatment, usually from 810 days. “Poor” results were obtained by eight patients who had grampositivebacteriaassociatedwith E. coli and Pseudomonas aeruginosa. These patients were treated with other antibiotics. Bacteriological response in this group was rather high (80.9%). The pathogen was eradicated in 55 patients. The second group, patients with inflammatory processes caused by Chlamydia trachomatis, also achieved goodtreatment results with roxithromycin. Clinical and bacteriological responses were both 82.6%. The organism was eradicated in 19 patients. The duration of antibacterial therapy should beno less than 21 days. Roxithromycin was well tolerated. Onlytwo patients developed side effects (nausea and diarrhea, each in one patient). No laboratory abnormalities were detected.

DISCUSSION The results of our clinical study are in agreement with several other published trials (1-3). The clinical efficacy rate of roxithromycin in patients with nongonococcal urogenital infection approaches 86-100%. Bacterio-

Roxithromycin Urogenital in

Infection

365

logical efficacy is more than 80%. For chlamydia infection these rates are 74-92% and 72-84%, respectively. Roxithromycin at doses of 150 mg bid is sufficient because of the elimination of half-life, (Tsi) of 12.4 h. This allows roxithromycin to be administered once a day at a dose of mg (233).

feature of roxithromycin pharmacokinetics is that it accumulates in high concentrations in body fluids (semen, ejaculate) and intracellularly. This feature, which is seen with all new macrolides, enables treatment of inflammatory urogenital diseases causedby atypical microorganisms. Successful treatment can be achieved with new macrolides, quinolones and tetracyclines. However higher dosages of these latter drugs are needed and these agentsare more toxic.

CONCLUSIONS Roxithromycin, at a doseof 150 mg bid, is an effective, well-tolerated treatment for urogenital inflammatory diseases caused by gram-positive bacteria and atypical microorganisms (in particular,Chlamydia trachomatis).

REFERENCES 1. Markham A, Faulds D. Roxithromycin. update of its antimicrobial activity, pharmacokinetic properties and therapeutic use. Drugs 1994; 48(2):297-326. 2. Neu H. Roxithromycin-an overview. Br J Clin Pract 1988; 42 (suppl55):l-3. 3. Young R, Gonzalez T, Sorkin E. Roxithromycin. A review of its antibacterial activity, pharmacokinetic properties and clinical efficacy. Drugs 1989; 37:8-41.

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Dissociated Macrolide and Lincosamide Resistance inStreptococcus pneumoniue and Variationof Susceptibility Testing Methods in Detecting Clindamycin Resistance E. L. Fasola, S. Bajaksouzian, P. C. Appelbaum, and M. R. Jacobs Case Western Reserve University and UniversityHospitals of Cleveland Cleveland, Ohio Hershey Medical Center Hershey, Pennsylvania

INTRODUCTION Streptococcuspneumoniae is the mostcommoncausativeorganism of community-acquiredpneumonia,bronchitis,otitismedia,andsinusitis. Pathogens in these diseases are rarely identified due to lack of adequate clinical specimens, and antimicrobial therapy is usually chosen empirically. Erythromycin has been considered one of the antibiotics of choice for treating many of these community-acquired infections because it is active against the pathogens commonly associated with these diseases. Erythromycin is also the agent of choice for patients allergic to penicillin. However,

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al.

isolation of erythromycin-resistant strains of S. pneumoniae has been increasing in several countriesof Europe, Japan, Korea, South Africa, and, more recently, also inthe United States Erythromycin is a macrolide antibiotic, which inhibits protein synthesis by binding the ribosomal subunit Cross-resistance among macrolides, lincosamides, and streptogramin B-type antibiotics usually occurs (5). Therefore, erythromycin-resistant strains of in gram-positive organisms S. pneumoniae have been considered resistant to all macrolides and lincosamides, including clindamycin, clarithromycin, and azithromycin However, more recent reports have documented erythromycin-resistant strains in the United States that are susceptible to clindamycin (9,lO). Based on these facts, it is advisableto maintain routine surveillance of resistance inS. pneumoniae to erythromycin andother agents, and it now is necessary to test clindamycin as well. This study evaluated the following methods for susceptibility testing of S. pneumoniae to erythromycin and clindamycin: broth microdilution minimal inhibitory concentrations (MICs) with incubation in ambient air andin 5% CO,, agar dilution (MICs), disk diffusion, and E- test.

MATERIAL AND METHODS Strains One hundred twenty-four strains were tested. Ninety-two erythromycinresistant strains were chosen to represent as many countries, resistance patterns, and serotypes as possible. In addition, erythromycin-susceptible strains were chosen as controls.

Susceptibility Testing Broth Microdilution M K S

Microdilutiontrayswerepreparedin-houseusingcation-supplemented Mueller-Hinton broth supplemented with 5% lysed horse blood. Erythromycin and clindamycin were tested at doubling dilutions from to pg/ml. Trays were incubated aerobically, with duplicate trays incubated in a 5% CO, atmosphere. MICs were read at and hours. Agar Dilution MICs

Mueller-Hinton agar with 5% sheep blood was used. Erythromycin and clindamycin were tested at doubling dilutions from to pg/ml. Plates were tested in duplicate, incubated in air5% and CO,, and MICs read at and h.

S. pneumoniae

Macrolide and Lincosamide Resistance in E-Test MICs

Disk Difluswn

Mueller-Hinton agar with 5% sheep blood was used. Erythromycin and clindamycin E-tests and disks were placed on plates, which were incubated in 5% CO, and read at 24 and 48h. Criteriafor Interpretation of Results Interpretations used were those for S. pneumoniae published by NCCLS M100-S6 inDecember1995 (11). MIC interpretations were as follows: susceptible, 50.25 pg/ml; intermediate,0.5 pg/ml; and resistant,21 pg/ml for both erythromycin and clindamycin. The criteria for disk diffusion were as follows: susceptible 221 mm and resistant 515 mm for erythromycin, 221 mm and 516 mm for clarithromycin.

RESULTS Table 1 shows the comparison of the different susceptibility testing methods used. Broth microdilutionwithincubationinambientairshowedfalse susceptibilityto clindamycin for 25 resistant isolates at 24 h andfor 5 at 48 h. However, after incubation in CO,, only one of these isolates remained susceptible at 24 h and this strain became resistant at 48 h. For erythromycin, there was one false-susceptible isolate and three intermediate strains at 24hof incubation in air; these strains became resistant after incubation for 48 h. When the trays were incubated in CO,, there were no false-susceptible isolates. Tables 2 and 3 show the MICs for susceptible and resistant isolates by microdilution and agar dilution in ambient air and CO,. The MIC,, and MI& for erythromycin-resistant isolates in ambient andair CO, at 24 and 48 h were 232 &m1 by both methods. For clindamycin-resistant strains,the MIC,, was 1pg/ml bybroth microdilution with incubation in for air24 h, but increased to 232 &m1 at 48 h and with incubation CO,. in MIC,, and MIC, were 232 pg/ml by agar dilution usingeither incubation in airor CO,. Table l Number of Strains of Pneumococci Susceptible to Erythromycin and Clindamycin According to Method (n=124); Results at h

Test method

oth usion microdilution E-Test Antimicrobial dilution Erythromycin Clindamycin

Agar

91/71

Fasola et al.

.4 E G

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d 3

E

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Erythromycin-susceptible isolates had erythromycin MIC, and MIC, of pg/ml by both microdilution and agar dilution with incubation in air and 0.06 &m1 with CO, incubation. Clindamycin-susceptible isolates had clindamycin MIC,, of 50.03 &m1 and MIC, of 0.06 &m1 by both methods with incubation in air. After CO, incubation, the MIC,, was 0.06 pg/ml and the MIC, was 0.12 pg/ml by microdilution, as well as by agar dilution.

DISCUSSION As reported in the literature some erythromycin-resistant strains were fully susceptible to clindamycin by all methods when strains were incubated in CO,. However, many strains of erythromycin-resistant pneumococci appeared susceptible to clindamycin by microdilution when incubated aerobically but were resistant when tested with methodsthe in presence of CO, or by prolonging aerobic incubation to 48 h. Disk diffusion was the simplest and most convenient test method, with the same accuracy as the MIC methods. Although erythromycin-resistant pneumococci are cross-resistant to allmacrolidesandazalides,strainsmaybesusceptible or resistant to clindamycin. Disk diffusion wasthe simplest and most convenient method for susceptibility testingof pneumococci to erythromycin and clindamycin, whereas the NCCLS broth microdilution method often resulted in false susceptibility of strains to clindamycin and required modification to produce correct results.

REFERENCES 1. b n k s JR, Medeiros AA. High rate of erythromycin and clarithromycin resistance among Streptococcuspneumoniae isolates from blood cultures from Providence, R.I. Antimicrob Agents Chemother1993; 37:1742-1745. Strep2. Geslin P, Buu-Hoo A, Frimaux A, Acar JF.Antimicrobial resistance in tococcus pneumoniae: an epidemiological survey in France, 1970-1990. Clii Infect Dis 1992; 15:95-98. 3. Marton A, Gulyas M, Muqoz R, Tomasz A. Extremely high incidence of antibiotic resistance in clinical isolates of Streptococcus pneumoniae in Hungary. J Infect Dis 1991; 163542-548. 4. Weisblum B. Insights into erythromycin action from studies of its activity as inducer of resistance. Antimicrob Agents Chemother1995; 39:797-805. 5. Lonks JR, Medeiros AA. Emergence of erythromycin-resistant Streptococcus pneumoniae. Infect Med 1994; 11:415-418,423-424. 6. Jacobs MR, Appelbaum PC. Antibiotic-resistant pneumococci. Rev Med Microbiol 1995; 6:77-93.

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7. Klugman KP. Pneumococcalresistance to antibiotics. Clin MicrobiolRev 1990; 3~171-196. 8. AppelbaumPC.Antimicrobialresistancein Streptococcus pneumoniae: an overview. Clin Infect Dis 1992; 15:77-83. 9. Nelson C T , Mason EO, Kaplan SL. Activity of oral antibiotics in middle ear and sinus infections causedby penicillin-resistant Streptococcus pneumoniae: implications for treatment. Pediatr Infect DisJ 1994; 13585-589. 10. Block SL, Harrison Cl, Hedrick JA, et al. Penicillin-resistant Streptococcus pneumoniae in acute otitis media: risk factors, susceptibility patterns, and antimicrobial management. Pediatr Infect Dis J 1995; 14:751-759. 11. NationalCommittee for Clinical Laboratory Standards. Performance Standards for Antimicrobial Susceptibility Testing: Sixth Informational Supplement. Villanova PA: NCCLS, 1995; M100-S6.

Prevalence of Antibiotic Resistancein Streptococcuspyogenes: Results of a National Multicenter Survey Conducted in Italy During 1995 E. A. Debbia, P. Cipriani, G. P. G. Ortisi, M. G. Menozzi, E. Nani, V. Nicolosi, R. Rigoli, R. Serra, M. Toni, F. E. Viganb, and G . C. Schito Committeefor the Studyof Antimicrobial Drugs of the ItalianAssociation of Clinical Microbiologhts (AMCLI) Milan, Italy

INTRODUCTION Given the total absence of relevant data concerning the incidence of antimicrobial resistanceto drugs employed inthe therapy of upper respiratory tract infections causedby Streptococcuspyogenes, a nationwide survey was conducted during February-May1995in Italy. AMCLI members were requested to submit the results of erythromycin, tetracycline, and clindamycin susceptibility testing as routinely performed in their laboratories on S. pyogenes strains circulating in their regions. We report the data obtained in this study.

376

in Resistance Antibiotic

S. pyogenes

377

MATERIAL AND METHODS Information was received from widely dispersed centers northern, central, and southern Italy). A total of strains were analyzed. Most pathogens wererecoveredfrom outpatients and throat swabs Systemic infection isolates were represented by organisms (5.0%). Disk diffusion on Mueller-Hinton agar supplemented with sheep blood was the prevailing technique used for susceptibility testing of centers). Antibiotic contents and interpretative criteria were those suggested byNCCLS guidelines Microscan Sceptor (5.8%), VITEK and other methodologies also were employed.

RESULTS AND CONCLUSIONS Overall, resistance to the three antibiotics tested in S. pyogenes was for clindamycin, for tetracycline,and for erythromycin (Table The distribution of resistance to erythromycin appears to be homogeneous, as only out of the 52 centers reported rates lower than S. pyogenes isolated from outpatients showed an incidenceof resistance exceedingthat of strains recovered from inpatients. This feature was shared by all the antibiotics tested (Table2). Given the multicenter nature of the present survey,the heterogeneity of sampling and susceptibility testing methods, and the lack of quality controls, these data await validation from more accurate determinations conducted in a single reference laboratory performing minimal inhibitory concentration(MIC)analysesonnational S. pyogenesisolates.Nonetheless, the present findings raise serious concerns given that severe S. pyogenes-associated diseasesare increasing worldwide(2). In patients allergic to p-lactams or, in the case of penicillin failures, possiblydue to lack of compliance or p-lactamase productionby the normal microbial pharyngeal Table I Prevalence of Antibiotic Resistance in During

S. pyogenes Isolates in Italy

N ("/.l Antibiotic

R1

Erythromycin Tetracycline Clindamycin

.R = resistant; I = intermediate; S = susceptible.

I 102

S

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Debbia et al.

Table 2 Origin of S. pyogenes and Prevalenceof Antibiotic Resistance

N Outpatients Erythromycin Tetracycline Clindamycin Inpatients Erythromycin Tetracycline Clindamycin

Ra

I

610 (26.2) 311 (15.4) 193 (9.1)

134 (5.7) 89 (4.4) 118 (5.6)

1587 (68.1) 1623 (80.2) 1812 (85.3)

66 (16.2)

25 (6.1) 13 (3.6) 20 (5.1)

316 (77.6) 305 (85.4) 353 (90.5)

39 (10.9) 17 (4.4)

S

aR = resistant; I = intermediate; S = susceptible.

population the useof alternativedrugs(macrolides,lincosamides, tetracyclines) for the treatment of infections sustained by S. pyogenes is inevitable (4). Widespread resistance of this microorganism to the above agents might reproduce inour country the serious therapeutic threats that already have been faced other in countries (5-7). The present findings differ from thosereported in other studies with respect to important pathogens of lower respiratory tract infections (S. pneumoniae, H.influenzae, M.catarrhalis)that exhibit a surprising susceptibility to the major groupsof antibiotics currently used in this country (8). On the contrary, S. pyogenes shows an unexpected high rate of resistance to these agents, which are employed as alternatives t o p-lactam drugs. If the highlevelsofresistancedescribedhere are confirmed, their origin might be tracedto excessive selective pressure frominappropriate usage of these otherwise precious drugs.

REFERENCES National Committee Clinical for Laboratory Standards (1994). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically. Third Edition, Approved Standard M7-A3. Villanova, PA: NCCLS, 1994. Gunzenhauser JD, Longfield JN, BrundageJF,Kaplan EL, Miller RN, Brandt CA. Epidemic streptococcal disease among armytrainees, July 1989 through June 1991. J Infect Dis1995; 172:124-131. Brook I. The role of P-lactamase-producing bacteria in the persistence of streptococcal tonsillar infections. Rev Infect Dis 1984; 6501-607. Mandell GL, Douglas RG, Bennett JE. Principles and Practiceof Infectious diseases. New York:Churchill Livingstone, 1995.

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5. Phillips G, Pamatt D, Orange CV, Harper McEvan H, Young N. Erythromycin-resistant Streptococcus pyogenes. J Antimicrob Chemother 1990; 2 5 : 723-724. 6. Po-Ren Hsueh, Hung-Mo Chen, Ay-Huey Huang, Jiunn-Jong Wu. Decreased activity of erythromycin againstStreptococcus pyogenes in Taiwan. Antimirob Agents Chemother 1995; 39:2239-2242. 7. Seppala H, Nissinen A, Jarvinen H, Huovinen S, Henriksson T, Herva E, et al. Resistance to erythromycin in group A streptococci. N Engl J Med 1992; 326:292-297. 8. Marchese A, Debbia EA, Arvigo Pesce A, Schito GC. Susceptibility of Streptococcus pneumoniae strains isolated in Italy to penicillin and ten other antibiotics. J Antimicrob Chemother 1995; 36:833-837.

In Vitro Activity of Some Macrolides Against S. pyogenes G. Tempera, P. M. Fumeri, V. Nicolosi, and G . Nicoletti University of Cutuniu Cutuniu, ZtuZy

INTRODUCTION An increase in the incidence of severe S. pyogenes (GAS) infections has been observed in the last decade It follows that an aggressive management of streptococcal pharyngotonsillitis becomes a necessityto avoid the persistence of S. pyogenes in the throat. Penicillin is stillthe drug of choice for the treatment of streptococcal pharyngitis, whereas erythromycin is an alternative drug for individuals who cannottake p-lactam antibiotics. Recent clinical trials have suggested an increased incidence of clinical relapse and bacteriological failure following oral penicillintreatment due both to penicillin tolerance in streptococci and to the presence of p-lactamaseproducing copathogens(S. uurew, H.influenzue, oropharyngeal anaerobes, M . cuturrhulis) that inactivate penicillin at the site of GAS infection (4). Thus,erythromycin,awell-established,safeandeffectiveantimicrobial agent, has received renewed interest over the last decade. New macrolides, with expanded activity spectra, improved gastrointestinaltolerance, and better pharmacokinetics than erythromycin, are now available. Among these, azithromycin and clarithromycin are most commonly used in the treatment of streptococcal pharyngotonsillitis. Resistance of GAS to

380

In Vitro Activity

of Macrolides Against S. pyogenes

381

macrolides (mainly to erythromycin) has been evaluated worldwide and varies from countryto country and from yearto year. The purpose of this studyis to determine the minimalinhibitoryconcentration(MIC) of erythromycin, azithromycin, and clarithromycin for several freshly isolated GAS, in order to estimate the rate of macrolide-resistance ineastern Sicily.

MATERIALS AND METHODS Strains A total of GAS strains were obtained from cultures of throat swabs of school children clinically diagnosed with pharyngotonsillitis. Beta-hemolytic colonies were identified as group A streptococci by a latex agglutination test (Slidex Strepto Kit, bioMCrieux, Italy). The strains were maintained in glycerol brothat -70°C until all isolates were collected. Stocks of group A streptococci were subculturedtwice on defibrinated sheep blood 5% agar (Oxoid) priorto susceptibility testing.

Antibiotics Antibiotic preparations were obtained from their respective manufacturers: azithromycin from Pfizer Inc. (New York, USA); clarithromycin from Abbott Laboratories (Abbott Park, IL, USA), and erythromycin from Sigma Chemical Co. (Sigma Aldrich s.r.l., Milan, Italy). Disks of erythromycin azithromycin, and clarithromycin were supplied by BBL (BBL Sensi-Disc, Becton Dickinson Microbiology System, Cockeysville, MD, USA).

Susceptibility

Procedure

Broth Microdilution

The MICs of azithromycin, clarithromycin, and erythromycin for the isolates were determined by a broth microdilution method (5). For each assay,antibioticsweretestedfrom &m1 to &m1 for all macrolides. For resistant strains, the range was extended from256 pg/ml to 1pg/ml. The MICs, defined as the lowest concentration of each antibiotic that completely inhibited visible growth, were read visually after incubation at h at 37°C. Disk-DiffusionAssay The disk-diffusion methodwas performed on Mueller-Hinton agar supplemented with 5% sheep blood, according to the National Committee for Clinical Laboratory Standards (NCCLS) (5). The breakpoints used andthe

Tempera et al.

382 no. of strains l

Figure I

MICs (pg/ml) of er)rthromycin, azithromycin, and clarithromycin.

three-category classification scheme (susceptible, intermediate, and resistant) were those suggestedby the NCCLS-1994 (6).

RESULTS The in vitro activities (MICs) of the three macrolides against 112 GAS performed by broth microdilution are presented in Fig. 1, whereas the results of the disk-diffusion assay are shown in Fig. 2. MIC,$ of erythromycin, azithromycin, and clarithromycin were0.12,0.25, and 0.12 pg/ml, respectively. M I G s were 1, 4, and 4 pg/ml, respectively. We found 8% resistant strains; as expected, these strains showed cross-resistanceto the three macrolides tested. regards the disk-diffusion assay, excellent agreement in results between the two methods was found for susceptible strains, wliereas the differences inintermediate and resistant strains can be explained comparing the results, strain by strain. Actually,three of the four.strainswith intennediate MICs for erythromycin and clarithromycin were resistant when tested by disk-diffusion assay, and 1of the 99 susceptible strains became intermediate. A similar phenomenonwas observed for azithromycin.

CONCLUSION Considering the different breakpoints between erythromycin the andnewer macrolides, no differences in susceptibility were found among the three

In Vitro Activityof Macrolides Against S. pyogenes

383

Ciarithromycin and Azkhromycin

i:


>l8

Figure 2 In vitro activity by categories by means of disk-diffusion test for emhromycin, clarithromycin, and azithromycin.

macrolides tested, even if erythromycin showed the highest activity (82 strains with MIC
i

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Tempera etal.

REFERENCES 1. Kaplan EL. The resurgence of group A streptococcal infections and their sequelae. Eur Clin Microbiol Infect Dis 1991; 1055. 2. Bisno AL. Group A streptococcal infections and acute rheumatic fever. N Engl Med 1991; 325783. Kim KS, Kaplan EL. Association of penicillin tolerance with failureto eradicate group A streptococci from patients with pharyngitis. Pediatr 1985; 106:681. 4. Brook I. The role of beta-lactamase-producing bacteria in the persistence of streptococcal tonsillar infection. Rev Infect Dis 1984; 6501. 5 . National Committee for Clinical Laboratory Standards. Methods for Dilitution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically. Third Edition, Approved StandardM7-A3. Villanova, PA: NCCLS, 1993; Vol 13, No. 25. 6. National Committee for Clinical Laboratory Standards. Methods for Dilitution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically. Third Edition, Approved Standard M100-S5. Villanova, PA: NCCLS, 1994; Vol. 17, No. 16.

Antipneumococcal Activity of RP 59500 (an Injectable Streptogramin), Erythromycin, and Sparfloxacin byMIC and Rapid Time-Kill G. A. Pankuch, M.

Jacobs, and P. C. Appelbaum

Hershey Medical Center Hershey, Pennsylvania Case Western Reserve University Cleveland, Ohio

INTRODUCTION Previous studies demonstrated that RP was bactericidal at

6 h at concentrations of one-half to one-fourth the minimal inhibitory concentration (MIC) in7/10 strains, and bacteriostatic in 3/10 strains, with regrowth at 24 h. Additionally, at 10 min after inoculation, a (1-3)log1, unit reduction in the original inoculum wasseen for 6/10 strains with RP at concentrations'greater than or equal to the MIC. One penicillinresistant strain was examined by time-kill at 0,1,2, and h: RP at a concentration equalto the MIC, was bactericidal within 1h andat a concentration one-half the MIC was bactericidal within h. The above rapid bactericidal effect was not seen with the other compounds tested (erythromycin, sparfloxacin, ciprofloxacin, vancomycin) (1).

I

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Pankuch et al.

386

To confirm and extend the above findings, we examined (i) the in vitroactivity of RP its constituentcomponents RP (quinupristin), and RP (dalfopristin), erythromycin, and sparfloxacinagainst penicillinsusceptible, intermediateand resistant pneumococci using agar dilution MIC methodology; and (ii) the activity of these compounds using time-kill methodologyat and h.

MATERIALS AND METHODS Seventy-six clinicalisolatesof S..pneumoniue obtained from blood, cerebrospinalfluid,nasopharynx, or sputumweretested.Theseincluded penicillin-susceptible (MICCO.l pg/ml), intermediate (MIC ml), and resistant (MIC pg/ml) organisms. Of these strains, were erythromycin susceptible ( k c pg/ml) and were erythromycin resistant (MIC pg/ml). MICs were determined by the agar dilution method For time-kill experiments, the method described previously (1) was used. Viability counts (0 h) were performed immediately (within min) after inoculation. Viability countsof antibiotic-containing suspensions were performed at and h, respectively, by plating tenfold dilutionsof 0.1 m1 aliquots from each tube in sterile Mueller-Hinton broth on trypticase soy agar5% sheep blood agar plates (BBL). Recovery plates were incubated for at least h. Colony counts were performed on plates yeilding 30-300 colonies. Time-kill results were expressed as percentageof strains with Alog,, CFU (colony-forming units) per milliliter lower than the original inoculum at each of the time intervalstested.

RESULTS RP was active (MIC l.O.pg/ml) regardlessof the strain's penicillin or erythromycin susceptibility status. The components of RP were muchlessactive.Sparfloxacinwasactive(MI% 0.5 pg/ml) against all strains. Erythromycin was active at MICs pg/ml against of penicillin-susceptible strains; by' contrast, of penicillin-intermediate and 35% of penicillin-resistant strains were erythromycin resistant (MIC .O pg/ml) . Comparativetime-killactivities of RP sparfloxacin, and erythromycin against strains are shown in Table Only - R P inhibited pneumococci at 0 h at one to four times the MIC. RP also was the most active after 1and h. Penicillin and sparfloxacinat and 4 X MIC reduced the original inoculum by log at h in and of strains, respectively. RP RP and

Antipneumococcal Activity

RP 59500, Erythromycin, Sparjloxacin 387

m

c

+

W

I

m

I

M

n

m 4

Pankuch et al.

388

erythromycin were either inactive or bacteriostatic at 2 h. Results of RP time-killsfor erythromycin-susceptible and 20 erythromycinresistant strains were similar.

DISCUSSION The streptogramins are the only members ofthe macrolide-lincosamidestreptogramin group which are consistentlyactiveagainstmacrolideresistantpneumococci(1,3). In the current study,agardilution MIC confirmed the activity of RP against penicillin-and/or erythromycinresistant pneumococci (1,3). MICs all fell within a very narrow range of 0.25-1.0 pg/ml, even againststrains with penicillin MICs of 4.0 pg/ml and erythromycin MICs of .>128.0 pg/ml. The two constituent components alone were less active, but act synergistically. Spariloxacin was also found to be active against all strains, with MICs between and 2.0 pg/ml, regardless of their susceptibility to other compounds. Time-kill results inour study confirmthe rapid killingof most strains by RP Only RP inhibited pneumococci at 0 h and was the most active agent after 1and 2 h. After 2 h, penicillin G and spariloxacin showed increased killing, but the latter was still significantly less than RP At a concentration equal to the agar dilution MIC, RP reduced the original inoculum by 3 log,, killing) in 32% and of strains after 1 and 2 h,respectively.By contrast,penicillin G and spariloxacin, at concentrations equal to the agar MIC, failed to similarly reduce the original inoculum of all strains but one (penicillin G) after 2 h. Erythromycin was either inactive or bacteriostatic at 2 h. RP was equally effective against erythromycin-susceptible and -resistant strains, supporting agar dilution MIC results obtained in the current study and those reported previously by us (3). In summary, only RP was rapidly bactericidal killing) against most pneumococcal strains tested. This phenomenon was not seen with other compounds andmay be unique to the streptogramins. The clinical significance of this observation remainsto be determined and may affect the clinicalresponse to theseagentsinpatientsinfectedwith pneumococci.

REFERENCES 1. Pankuch GAYJacobs MR, Appelbaum PC. Study of comparative antipneumococcal activities of penicillin G, RP 59500, erythromycin, spartloxacin, ciprofloxacin and vancomycin by using time-kill methodology. Antimicrob Agents Chemother 1994;

Antipneumococcal Activity of RP 59500, Erythromycin, SparJIoxacin 389 2. Jacobs M R . Treatmentanddiagnosis of infections caused by drug-resistant Streptococcus pneumoniae. Clin Infect Dis 1992; 15119-127. Spangler SK, Jacobs MR, AppelbaumPC.Susceptibilities of penicillinsusceptible and -resistant strains of Streptococcus pneumoniue to RP 59500, Win vancomycin,erythromycin, PD 131628,sparfloxacin,temafloxacin, 57273,ofloxacin and ciprofloxacin.Antimicrob Agents Chemother1992; 36:856-859.

Effect of Macrolide Resistance on the Activity of the Oral StreptograminRPR 106972 and Its Components Against Streptococcus pneumonia? M. R. Jacobs Case WesternReserve University Cleveland, Ohio

S. K. Spangler and P. C. Appelbaum Hershey Medical Center Hershey, Pennsylvania

INTRODUCTION With the recent rapid increase in the prevalence of strains of Streptococcus pneumoniue resistant to p-lactams, macrolides, tetracyclines, and trimethoprim-sulfamethoxazole,the availability of oral agents that can be used to treat pneumococcal infections is severely limited (1-6). Currently available oral p-lactams that can be used to treat infections due to penicillinresistant strains are constrained by poor therapeutic indices ofmanyof these agents, with amoxicillin and cefuroxime axetil beingthe most active (6).Trimethoprim-sulfamethoxazoledoes not have much clinical potential, as most penicillin-resistant strains are also resistant to this combination (43). Quinolones have limited application for pneumococcal infectionsdue

390

Effect

Macrolides on RPR 106972 Against S. pneumoniae

391

to poor therapeutic indices of available agents as well as development of resistance and are not approved for pediatric use Tetracyclines have similar limitationsin pediatrics, andmany strains are resistant Of the macrolide-lincosamide-streptogramin (MLS) group, erythromycinhasmanyof the problems encountered with trimethoprimsulfamethoxazoleandtetracyclines,anderythromycin-resistant strains are cross-resistant to other approvedmacrolidesandazalidessuchas clarithromycin and azithromycin Clindamycin has some merit, as some erythromycin-resistant pneumococci are susceptible to this agent A parenteral streptogramin, RP (a combination of quinupristin and dalfopristin), is active at &m1 against all strains studied to date, regardless of erythromycin susceptibility A streptogramin preparation suitable for oral administration, RPR acombination of RPR and RPR also has been developed

MATERIALS AND METHODS Strains A total of 203 isolates of S. pneumoniue of varying penicillin susceptibility isolated from blood, cerebrospinal fluid, ear, nasopharynx, or sputum were examined.

Antimicrobial Agents RPR RPR and RPR were obtained from RhonePoulenc Rorer (Paris) erythromycin from Abbott Laboratories (Chicago, IL), and clindamycin fromthe Upjohn Co. (Kalamazoo,MI).

MIC Determination Minimal inhibitory concentrations (MICs) of RPR RPR RPR erythromycin, and clindamycin were determined by agar dilution on Mueller-Hinton agar supplemented with sheep blood Agents were tested at concentrationsof &m1 in twofold increments. Plates were incubated overnight in ambient air at All strains grew well without added Standard quality control strains were included in each run Strains were considered susceptible to erythromycin at MICs of 50.5 pg/ml, and to clindamycin at MICs of &m1 (5).

Statistical Analysis The MIC ranges and the MIC, and MIC, values were determined for each agent. Results also were analyzed according to the macrolide suscep-

Jacobs et al.

392

tibility of strains. Correlation of results of the three streptogramin compounds with each other was performed by regression analysis using the leastsquaresmethod,andregressionlinesandcorrelationcoefficients determined.

RESULTS Results of susceptibility testing are presented in Table 1. All strains were inhibited by RPR 106972 at a concentrationof 50.5 pg/ml, andthe activity of this agentwas independent of penicillin, erythromycin, and clindamycin susceptibility. MIC, and MIC, values of RPR 106972 for all strains were 0.12 and pg/ml, respectively.The two constituent componentsof RPR 106972 were considerably less active than the combination product, with

Table I MIC Ranges and MIC, and MIC, Values of Strains According to Erythromycin and Clindamycin Susceptibility; Penicillin Susceptibility of Strains Tested: Penicillin Susceptible,75; Penicillin Intermediate, 55; Penicillin Resistant, 73

RPR 106972 Ery-S (142') Ery-R, cli-S (25) Ery-R, cli-R(36) RPR 112808 Ery-S (142) Ery-R, cli-S (25) Ery-R, cli-R (36) RPR 106950 Ery-S (142) Ery-R, cli-S (25) Ery-R, cli-R(36) Erythromycin Ery-S (142) Ery-R, cli-S (25) Ery-R, cli-R (36) Clindamycin Ery-S (142) Ery-R, cli-S (25) Ery-R, cli-R(36) 'Number of strains.

0.25 0.12 0.5

50.015-0.5 0.03-0.25 50.015-0.5

0.12 0.12 0.12

50.015-8 0.03->l6 0.06-4

0.5 8 1

2 >l6 4

0.06-16 0.06->l6 0.06-16

1 8 2

8 >l6 16

0.03-0.12 1->64

0.03 4

0.06

50.03 50.03 16.

50.03 0.06

50.03 50.03-0.06 24-64

Effect of Macrolides on RPR 106972 Against S. pneumoniae

393

MIC, and MIC, values for all strains of 2 and 16&m1 for RPR 106950, and 0.5 and 4&m1 for RPR 112808. The MIC results of the constituents of RPR 106972 were analyzed according to the susceptibility of strains to erythromycin and clindamycin. MIC distributions, MIC,s and MIwere similar forstrains in two ofthe groups-thegroupsusceptible to macrolidesandlincosamidesand the group resistant to these MLS agents. However, the constituents of RPR 106972 hadthe least activity againstthe third group, resistant to erythromycin but susceptible to clindamycin, with M I G s of the components being >l6 pg/ml. Nevertheless, RF'R 106972 was equally active against allthree groups of organisms. Regression analysisof scatterplots of PRP 106972 and its components showed no relation between the activity of RPR 106972 and that of its components, with correlation coefficients(r2 values) of 0.34and 0.41. However, the components of RPR 106972 did show some relation to each other, with a correlation coefficientof 0.56.

DISCUSSION This study evaluated the relation between the activities of the new oral streptogramin combination, RPR106972, and its two constituent components, FWR 106950 and RPR 112808, as well as the effect of macrolide and lincosamide resistance onthe activity of these three compounds. The data presented in this study show that the components of RPR 106972 demonstrate true synergismwhencombined. The activity of the combination product, RPR 106972, was unaffected by the presence or absence of resistance to erythromycinand/orclindamycin. The components of RPR 106972, however, did show some correlationMLS withsusceptibility. Activity of the components was similar against strains fully susceptibleto MLS agents as well as strains resistant to both erythromycin and clindamycin. However, the constituents(particularly RPR 112808)werelessactive against erythromycin-resistant but clindamycin-susceptible strains. The reasons for this difference are unclear but may be associated withthe mechanism of resistance in these strains, whichthe inMLS group of agents results in selective macrolide resistance. Evaluation of the relationbetween the activities of macrolides, lincosamides, and streptogramins has shown the value of streptogramin combinations, the components of which are inactive alone, against a variety of macrolide-susceptible and -resistant pneumococci. The association of decreased activityof the streptogramin componentsfor strains resistantto macrolides but not lincosamides is of interest and hopefully will be explained when the resistance mechanismof these strains is determined.

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394

REFERENCES 1. Appelbaum PC. Antimicrobial resistance in Streptococcus pneumoniae: an overview. Clin Infect Dis 1992; 1577-83. 2. Fremaux A, Sissia G, Cohen R, Geslin P. In-vitro antibacterial activityof RP 59500, a semisynthetic streptogramin, against Streptococcus pneumoniae. J Antimicrob Chemother 1992;3O(suppl A):19-23. 3. Friedland IR, McCrackenJr. GH. Management of infectionscaused by antibiotic-resistant Streprococcus pneumoniue. N EnglJ Med1994;331: 377-382. 4. Hoffman J, Cetron MS, Farley MM, et al. The prevalence of drug-resistant Sfrepfococcuspneumoniuein Atlanta. N Engl J Med 1995; 333:481-486. 5. Jacobs MR. Treatment and diagnosis of infections caused by drug-resistant Sfreptococcuspneumoniue. Clin Infect Dis 1992;15:119-127. 6. Klugman KP. Pneumococcalresistance to antibiotics.ClinMicrobiolRev 1990; 3~171-196. 7. Nelson C T , Mason Jr. EO, Kaplan SL. Activity of oral antibiotics in middle ear and sinus infections causedby penicillin-resistant Streptococcus pneumoniue: implications for treatment. Pediatr Infect Dis J1994; 13585-589. National Committee for Clinical Laboratory Standards. Methods for Detection of Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically. Third Edition, Approved Standard M7-A3. Villanova, PA. NCCLS, 1993.

In Vitro Antibacterial Activity of RU 004, a New Ketolide Active Against Respiratory Pathogens Constantin Agouridas,A. Bonnefoy, andJean-FrangoisChantot ,

Roussel-Uclaf Romainville, France

INTRODUCTION Macrolides are a well-known family of oral antibiotics whose spectrum of activity includes most relevant species responsible for URTI and LRTI. However, all contemporary drugs, including clarithromycin and azithromycin, present several drawbacks such as cross-resistance against MU,(macrolides-lincosamides-streptogramin B) resistantpneumococciand borderline activity againstHuemophilus influenzue (with the exception of azithromycin). Indeed, the resistance of Streptococcuspneumoniue to macrolides has increased significantly recent.years in due to their extensive therapeutic use but levels as high as 60% have been reported in certain areas). Furthermore, there is a high percentage of cross-resistance to penicillin among these pneumococcal isolates, with epidemic spread of 10-40% in some areas, except in the United Kingdom and Scandinavia. Therefore, available macrolides cannot be used inthe treatment of infections causedby multiresistant S. pneumoniue. 395

Agouridas et al.

3%

In the search for new compounds likelyto overcome the threatening problem of pneumococcal resistance, a new class of 14-membered-ring macrolide antibacterials has been generated. Ketolides are characterized by a keto function in position of the macrolactone ring, which replaces the L-cladinose moiety, a sugar thought to be essential for antibacterial activity. RU 004 has beenshown to be one of the most active compounds in a novel seriesof 11,12 cyclo-disubstituted ketolides synthesized by Roussel Uclaf. In this report, we describe the in vitro antibacterialproperties of RU 004 in comparison with those of erythromycin A (ERY), clarithromycin (CLA), azithromycin (AZI), josamycin (JOS), pristinamycin (PRI), and ampicillin (AMP) inthe case of H.influenzae.

MATERIALS AND METHODS Antibiotics RU 004, clarithromycin, and azithromycin were synthesized by Roussel Uclaf (Romainville, France). Josamycin and pristinamycin were obtained from RhBne-Poulenc-Rorer (Vitry, France), rokitamycin from Toyo-Jozo (Japan), and ampicillin from Beecham (UK). Antibiotic stock solutions were prepared extemporaneously in Mueller-Hinton broth.

Bacterial Strains More than 500 strains of gram-positive and gram-negative bacteria were used in this study. Most were recent clinical isolates from various European hospitals. Special attention was paidto penicillin- and/orMU,-resistant strains of gram-positive cocci. Inducibly and constitutively, MU,-resistant strains of staphylococci were conveniently sorted out by using josamycin, a 16membered-ring macrolide which is only active against inducibly resistant staphylococci. With streptococci, pneumococci, and enterococci, rokitamycin, another 16-membered-ring macrolide, was used to distinguish between inducibly and constitutively resistant strains. Minimal inhibitory concentrations (MICs)of rokitamycin were 1mg/L and>4 mg/L for inducibly and constitutively MLS,-resistant strains, respectively, as shown by induction experiments carriedout in parallel.

Susceptibility Testing (1) In vitro susceptibility tests were performed by using a twofold agar dilution method. Mueller-Hinton agar medium (pH 7.4; Diagnostic Pasteur, France) was used throughout the study. The medium was appropriately supplemented to support the growth of some fastidious microorganisms

In Vitro Antibacterial Activity

R U 004

397

(4% globular extractfor Haemophilus injluenzae;7% horse bloodfor streptococci and pneumococci). Reference organisms were included for quality control: S. aureus ATCC 29213 and E. faecalis ATCC 29212. A standard inoculum of 104CFW (colony-forming units)/ spot was used throughout. All plates were incubated at 36°C for 20 h and the MIC was defined as the lowest concentration at whichnovisiblegrowthwas detected on agar plates.

Determination of Minimal Bactericidal Concentrations First, a broth microdilution test was used to determine MICs. Subcultures on Mueller-Hinton agar or other appropriate medium were made from those dilutionsin microdilution plates which failed to show visible growth. The agar plates were read after 24 h incubationat 36°C. The lowest dilution of the antibiotic yielding no bacterial growth on the agar subculture was taken as the minimal bactericidal concentration (MBC).

In Vitro Resistance Studies The development of resistance to RU was studied with several bacterial strains using a macrodilution method. An M C . of RU 004 was first determined in Mueller-Hinton broth against a test strain. Bacteria from the highest concentration of RU 004 showing growth were subculturedto tubes containing doubling concentrations of RU 004. Again, an MIC was determined andthe highest concentrationof RU 004 was subcultured once more in doubling concentrations the antibiotic. This subculture process was repeated several timesto study possible buildupof resistance.

RESULTS Results are shown in Table1 and Figs. 1and 2. Tuble I

In Vitro Antibacterial Activity of RU

(GM,m a ) ~~

Strains (No.)

Phen.

Staphylococci (65i)b Enterococci (13i+19c) Streptococci (27) Pneumococci (38i+34c)

ET-R Ery-R ET-R

H. influenza (80)

Ery-R -

RU 0.027 0.20

0.027 0.039 0.23

ERYa

CLA

AZI

PRI

>40 22.7 7.4 29.1 1.59

23 17.7 3.7 23.6 1.77

>40 33.6 15.1 >40 0.16

0.12 1.14 0.057 0.22 0.37

'ERY: erythromycin M I : azithromycin; C L A : clarithromycin; PRIS: pristinamycin. c: inducible, constitutiveM L S , resistance.

Agouridas et al.

398

Figure I

In Vitro activity of RU

against ERY-R gram-positive

DISCUSSION

In Vitro Activity of RU

Against Relevant Nonatypical Respiratory Pathogens Gram-PositiveBacteria

I

Against strains susceptible to macrolides, the activity of RU 004 is one (staphylococci, P-hemolytic streptococci, pneumococci) or two orders of magnitude (enterococci) higher than that of other available drugs [e.g., clarithromycin and erythromycin A (data not shown)]. Because RU 004 does not induce M L S , resistance, all clinical isolates inducibly resistantto macrolides are highly susceptibleto the ketolide at a level identicalor very close to that found with susceptible bacteria (staphylococci, enterococci, pneumococci). It isconsiderablymoreactivethanjosamycin,a 16membered-ring macrolide considered the as reference against strains exhibiting inducible macrolide resistance. With the exception of staphylococci, RU 004exhibits high activity against all other constitutively resistantMU, gram-positive cocci. In this respect, RU 004 displays potent activity against pneumococci resistant to macrolides andor penicillin G andor several

In Vitro Antibacterial Activity of RU 004

399

Figure 2 In VitroActivity of RU againstatypicalbacteria. (A) Antichlamydial activity (MLC) (after Haider F, et al. 35th Intersci Conf Antimicrob Agents Chemother, 1995; abstr. F-165).(B) Antibacterial activity against45 strains of Legionella (afterBorstein N, et al., 35thIntersciConfAntimicrob Agents Chemother, 1995; abstr. F-166).

Agouridas et al.

400

Gram-Negative Bacteria The activity of RU 004against H. influenzae is high, closeto that found for azithromycin. In addition, the new compound does not show dissociated activity against isolates susceptible or resistant to ampicillin. RU 004shares the same antibacterial potency the as 14-membered-ring macrolides against Moraxella catarrhalis. RU 004 is slightlymore activethan available macrolides against several species of the family Enterobacteriaceae, with the exception of Salmonella sp. against which its efficacy is slightly less than that of azithromycin (data not shown). Other Properties of RV 004 RU provides MBC values equivalent to the MICs for pneumococci and H. influenzae. The Potential selectionof mutants by RU 004 wasstudied by serial passaging in broth. In contrast to josamycin, the increase in MICs was very slow (data not shown). Thus, the emergence of resistance to the ketolide is expected to be slow for pneumococci and enterococci.

In Vitro Activity of RU Against Atypical Bacteria (Fig. 2) Mycophma The new ketolide RU was very active in vitro against all mycoplasma species tested, M. pneumoniae, M. genitalium, M. penetrans, U. urealyticum, and even M. hominis and M.fermentam. Its activity was better than the other available drugs (macrolides or azalides). Furthermore, this antibiotic was bactericidal against most mycoplasma species tested, except M. homink (data not shown). Chlamydia RU displays very high activity against the three species of Chlamydia tested far, a resultwhich confirmsits well-balanced spectrum.There is a clearbactericidaleffect of the drugwhichmay eradicate intracellular chlamydia. Legionelha RU 004 displays excellent activity against clinical and reference Legionella strains (MI% 0.03 and 0.03 m& compared with 0.5 and 0.12 m& for erythromycin). In comparison with macrolides,RU 004is the only drug whose MICs are never above 0.06 m&. Its activity against L. pneumophila is identical to that of the most active macrolide, clarithromycin.It is superior to all macrolides againstother species of Legionella.

In Vitro Antibacterial Activity of R U 004

401

Considering these results, as well asthe high intracellular accumulation inside phagocytes,RU 004 appears to be a promising new agent for the therapy of infections due to intracellular pathogens. In vivo experiments are now needed to assess the potential clinical value of the new ketolide.

CONCLUSIONS RU 004 isactiveagainstmultiresistantpneumococciincluding penicillin-resistant and macrolide-resistant strains. It possesseswell-balancedantibacterialactivityagainstrelevant pathogensinvolvedinRTI(gram-positivecocci, H . influenme, and M. catarrhah, regardless of resistance antibiophenotypes, as well as atypical bacteria). Unlike macrolides, it does not induce the so-called MU, resistance (staphylococci or pneumococci), thereby exhibiting potent activity against all macrolide inducibly-resistant strains and opening a fundamentaldebate on the role of cladinose inthe induction of resistance. These findings strongly suggest a mechanism action dissociated fromthat of macrolides. Its physicochemicalpropertiesand structure give RU 004 high stability in acidic medium, little influence of pH on antibacterial activity, and high intracellular accumulation, accompanied by adequate efflux kinetics whichensure total elimination of the drug. Conforming to its in vitro profile,RU 004 displays potent therapeutic efficacy in animals infected by all relevant strains of bacteria. RU 004thus appears as avery promising agent inthe treatment of infections causedby difficult-to-treat respiratory pathogens.

REFERENCE 1. Lorian V. Antibiotics in Laboratory Medicine, Baltimore,MD: Williams and Wilkins, 1993.

Antistaphylococcal Activity of Quinupristidlalfopristin, a Well-Defmed Mixture of Chemically Modified Streptogramins Christof von Eiff and Georg Peters Westjalische Wilhelms- Universitat Munster Munster, Germany

INTRODUCTION Pristinamycin is a mixtureof naturally occurring streptogramin antibiotics isolated fromStreptomyces pristinaespiralis. It consists of a complex oftwo main componentsof water-insoluble compounds, pristinamycin I, (a group B streptogramin) and pristinamycin 11, (a group A streptogramin), which have synergistic antibacterial activities. Pristinamycin I, and 11, have been chemically modified to provide a well-defined mixture : 70) of watersoluble semisynthetic derivates suitable for parenteral administration (1). In our study, the in vitro activity of this mixture, quinupristiddalfopristin (RP59500) was compared with thoseof other antimicrobial agents against methicillin-susceptible and -resistant S. aureus and speciated coagulasenegative staphylococci.

402

Antistaphylococcal Activity of QuinupristiniDalfopristin

MATERIAL AND METHODS A total of 247 staphylococcal strains freshly isolated from clinical material was tested. Only one isolate per patientwas included. In addition, because methicillin-resistant Staphylococcus aureus strains can cause epidemics, we used several criteriato avoid multiple copiesof the same strain: (i) strains were from different geographic locations (both within hospitals and from different hospitals), which would reduce the chance of obtaining a single clone, (ii) collecting strains over a period of years would also make a single clone unlikely, and (iii) when strains with similar antibiograms and phenotypes were obtained, we performed pulsed-field gel electrophoresis and selectedonly one clone. The S. aureus strainsincluded 29 penicillinsusceptible S. aureus (PSSA), 40 methicillin- susceptibleS.aurews (MSSA), and61methicillin-resistant S. aureus (MRSA). The coagulase-negative staphylococci (CONS) comprised24 methicillin-susceptible S. epidermidis (MSSE), 28 methicillin-resistant S. epidermidis (MRSE), 20 methicillinsusceptible S. haemolyticus (MSSH), 23 methicillin-resistant S. haemolyticus (MRSH), and 22 other coagulase-negative staphylococci belongingto novobiocin-susceptiblecoagulase-negativestaphylococci [S. hominis (7), S. warneri S. simulans (3), S. capitis (2), S. lugdunensis (l)] and to novobiocin-resistant coagulase-negative staphylococci[S. saprophyticus ( 9 , S. cohnii (l)]. The minimalinhibitoryconcentrations(MICs)were determined on Mueller-Hinton agar (Difco, Augsburg, Germany) using the agar dilution technique. A dilution of a fresh overnight culture was applied to agar platesby using a multipoint inoculator (MastLaboratories Ltd., Bootle,England),yieldingafinalinoculum of105 CFU (colonyforming units). Isolates were confirmed to be methicillin resistant by supplementation of the agar with 2% NaCl (48 h, The antibiotics were tested in 13 different concentrations ranging from 0.031 m& to 128 m&. The following substances were used and obtained from their respective manufacturers: RP 59500, vancomycin, and ciprofloxacin. The results were read after 18 h incubation at 36°C. Additionally, the following reference strains were included:S. aureus: ATCC 25923, ATCC 29213;Enterococcus faecalis: ATCC 29212; Escherichia coli: ATCC 25922, ATCC 35218;Pseudomonas aeruginosa: ATCC 27853.

RESULTS The ranges of MICs, MIC,$, and MIC,s for the 130 strains of S. aureus are showninTable 1 and the correspondingvalues for the 117 strains of coagulase-negative staphylococci tested are giveninTable2. The MIC

von Eiff and Peters

404 Table I

In vitro activity of RP 59500,Vancomycin, and Ciprofloxacin Against

130S.aureus Strains Range

S. awe@

nwa

mc50 ~~

RP 59500 Vancomycin Ciprofloxacin

PSSA MSSA

RP 59500

MRSA

Vancomycin Ciprofloxacin RP Vancomycin Ciprofloxacin

0.5 1-2 0.5-2 0.25-1 1-2 0.25-32 0.5-1 1-2 0.5->l28 64

0.5 1 0.5 0.5 1 0.5 0.5 1 16

0.5 2 1 0.5 1 1 1

2

‘Number of strains tested: PSSA, penicillin-susceptibleS. aureus (29) MSSA, methicillin-susceptibleS. aureus (40) MRSA, methicillin-resistantS. aureus (61)

Table 2 In Vitro Activity of RP 59500,Vancomycin, and Ciprofloxacin Against 117Strains of Coagulase-Negative Staphylococci ~~~~

~

~~~

Range

CONS= MSSE

0.25-0RP .5 59500 Vancomycin Ciprofloxacin

MRSE

1-2 0.25-64

0.25-1 RP 59500 Vancomycin Ciprofloxacin

RP

MSSH

Vancomycin Ciprofloxacin RP 59500 Vancomycin Ciprofloxacin RP Vancomycin Ciprofloxacin

MRSH CoNS (others)

MIGO

MI%

0.5 2

0.5 2 0.5 0.5 2 64

0.25

0.5

2 0.25-64 0.5-10.5 1-2 0.25->l28 0.5 1-4 64 0.125-64 0.25-2 0.5-4 0.063-32

~~

‘Number of strains tested: MSSE, methicillin-susceptibleS. epidemidis (24) MRSE, methicillin-resistantS. epidemidis (28) MSSH, methicillin-susceptibleS. huemolyticus MRSH, methicillin-resistantS. huemolyticus (23) CoNS, other coagulase-negative staphylococci(22)

2

1 0.5 2

2

0.25

2 0.5 2

0.5 2 16 0.5 2 0.25

2 2 1

Antistaphylococcal Activity of &uinupr&inlDalfoprktin

405

Table 3 In Vitro Activity of RP 59500, Vancomycin, and Ciprofloxacin Against

Reference Strains Reference strain

Vancomycin RP Ciprofloxacin 59500

S. aurew

25923 ATCC 29213 ATCC

2

1

1

1 0.5

E. faecah

29212 ATCC

4

8

E. coli

0.031

8 25922 ATCC 8 35218ATCC

0.031

P. aeruginosa

27853 ATCC

>128

>l28

values for the control reference strains were within the expected range throughout the testing andare given in Table

DISCUSSION Depending on local epidemiological conditions, a significant number of staphylococci are resistant to penicillin, methicillin, clindamycin, erythromycin,aminoglycosides,and/orquinolones(2).Additionally,emerging glycopeptide resistance in some staphylococcal species is a newpotential threat particularly in nosocomial infections. Occasionally, clinical CONS with elevated MICs for vancomycin have been isolated and transformagene has been tion of S. aurew strainswithavancomycin-resistant achieved under laboratory conditions(4). In this context,the ability of RP 59500 to inhibit staphylococcal strains resistantto methicillin is potentially of major clinical importance.In ourstudy, all staphylococci were inhibited by 2pg of RP 59500 per milliliter. The new streptogramin was equally active against both methicillin-susceptible and methicillin-resistant strains of S. aureus, the MI&, values being0.5 pg/ml and 1pg/ml, respectively.A similar pattern was seen for all other Staphylococcus spp. studied. The results concerningthe antimicrobial susceptibilities of our strains were generally in agreement with those reported in previous studies showing that RP 59500was as activeas or moreactive than vancomycinagainst methicillin-susceptible and -resistant staphylococci (1,5-8), even though we tested a larger number of methicillin-resistant strains and excluded multiple copies of the same strain. Additionally, we tested widely used reference strains and a major number of different species of coagulase-

von Eiff and Peters

406

negative staphylococci, separating the methicillin-susceptible and -resistant strains. In summary, our data indicate that RP 59500 exhibits wide-spectrum antistaphylococcal activity against both methicillin-susceptibleand -resistant strains, stimulating further in vitroand, particularly, in vivo evaluation of this promising new antimicrobial agentfor therapy of infections due to multiple-resistant staphylococcal microorganisms.

REFERENCES 1. Bamere JC, Bouanchaud DH, Paris JM, Rolin 0, Hams SmithC. Antimicrobial activity againstStaphylococcus aureus of semisynthetic injectable streptogramins: RP 59500 and related compounds. J Antimicrob Chemother 1992; 30 (supplA):l. 2. Voss A, Milatovic D, WallrauchSchwarz C,Rosdahl W, Braveny I. Methicillin-resistant Staphylococcus aureus in Europe. Eur J Clin Microbiol Infect Dis 1994; 1350. 3. Sanyal D, Johnson AP, George RC, Cookson BD, Williams AJ. Peritonitis due tovancomycin-resistant Staphylococcus epidermidis. Lancet 1991;33754. 4. Noble WC, Virani Z, Cree RG. Co-transfer of vancomycin and other resistancegenesfrom Enterococcusfaecalis NCTC12201 to Staphylococcus aureus. FEMS Microbiol Lett 1992; 72:195. 5. Brumfitt W, Hamilton Miller JM, ShahS. In-vitro activityof RP 59500, a new semisynthetic streptogramin antibiotic, against gram-positive bacteria. J Antimicrob Chemother 1992; 30 (suppl A):29. 6. vonEiff C, Peters G . In vitro activity of RP 59500, a new semisynthetic injectable pristinamycin against staphylococci. Zbl Bakt. 1966; 283:497. 7. Fass RJ. In vitro activity of RP 59500, a semisynthetic injectable pristinamycin, against staphylococci, streptococci, and enterococci. Antimicrob Agents Chemother 1991; 35553. 8. Leclercq R, Nantas L, Soussy Duval J. Activity of RP '59500, a new parenteral semisynthetic streptogramin, against staphylococci with various mechanisms of resistance to macrolide-lincosamide-streptograminantibiotics. J Antimicrob Chemother1992; 30 (suppl A):67.

E-Test for Susceptibility Testing. of Streptococcus pyogenes to Azithromycin, Clarithromycin, Erythromycin, and Roxithromycin Gerard J. van Asselt University Hospital Leiden, The Netherlands WesteindeHospital Den Haag, The Netherlands

Jacobus H. S l m University Hospital Leiden, The Netherlands

INTRODUCTION The macrolides are alternatives for treatment of group streptococcal infections after penicillin failure and are often used as empirical therapy for infections of the respiratory tract. Compared with erythromycin, the new macrolides azithromycin, clarithromycin, and roxithromycin have an expanded spectrum, reduced adverse effects such as dose-related epigastric distress, improved oral bioavailability, increased tissue penetration, and longer clearance times. Reports on the activity of the newmacrolides against Streptococcus pyogenes are scarce. 407

van Asselt and Sloos

408

STUDY OBJECTIVE The purpose of the study was to determine the in vitro activity of the macrolides azithromycin, clarithromycin, erythromycin, and roxithromycin against group A streptococci and to compare the results of the E-test method, the broth microdilution method, and disk-diffusion assay.

MATERIALS AND METHODS Group A streptococcal strains included 180 clinical isolates originating from 6 regions in The Netherlands. W Olow-level erythromycin-resistant group A streptococci from Leiden, a high-level erythromycin-resistant group A streptococcus from Finland, and the Enterococcus faeculis ATCC29212 strain were included as controls.

E-Test Method The inoculum was 0.5 McFarland at 5% sheep blood agar plates; the time of incubation was 18-24 h.

Broth Microdilution Method The inoculum was 106 CFU (colony-forming units)/ml in Mueller-Hinton broth; the time of incubation was 18-20 h. The concentration of macrolide ranged from 0.0039 to 4.0 pg/ml (extended to 128 pg/ml for resistant strains).

Minimal Bactericidal Concentration The inoculum was 1 p1 of the minimal inhibitory concentration (MC) dilutions, appliedto sheep blood agar platesby a multipoint inoculator;the time of incubation was 24 h.

Disk-Diffusion Assay The inoculum was a tenfold dilution of a 0.5 McFarland standard at 5% sheep blood agar plates;the time of incubation was 18-24 h. The disk load was 15 pg. All tests were repeatedon a separate occasion.

RESULTS AND DISCUSSION The results are summarized in Tables 1 and 2. The MICs and minimal bactericidal concentrations (MBCs) of erythromycin and clarithromycin

nge

E-Test for Susceptibility Testing Table I

MIC for

of S. pyogenes

409

Erythromycin-Susceptible Group A Streptococci

MIC(Pg/ml)MIC microbroth method by byE-test

(Pg/ml)

Macrolide

MC,

Azithromycin Clarithromycin Erythromycin Roxithromycin

0.094

Range

MIC,

.

were lower than those of azithromycin and roxithromycin. The MICs obtained by microbroth and E-test methods were reproducible: For 2 95% of strains, the outcome of the repeated experiments was within two dilution steps. No major discrepancies were found among the E-test method, the microbroth MIC method,and the disk-diffusionassay. The somewhat lower MIC values obtained with the E-test method compared to the microbroth method could be explainedby use of different media with different pH conditions (sheep blood agar versus Mueller-Hinton broth). The reproducibility of the MBCs was low: For 27%of strains, the values of the repeated experiments differed more than two dilution steps (possibly because of the inhomogeneous suspensionsof some groupA streptococci and the small volume of inoculum).

SUMMARY The E-test method was comparedto a microbrothM C method and a diskdiffusion assay for susceptibility testing of 180 clinical isolates of group A streptococci to the macrolide antibiotics azithromycin, clarithromycin, Table 2 Zone Diameters and MBC for Streptococci

diameters Zone (mm)

an Range Macrolide Azithromycin Clarithromycin Erythromycin Roxithromycin

Erythromycin-Susceptible GroupA (Pg/W MBc microbroth by

method MBGO

MJ3c,

410

Sloos

van Asselt and

erythromycin,androxithromycin. MBCs alsoweredetermined. The MIGs of azithromycin, clarithromycin, erythromycin, and roxithromycin for group A streptococci were &m1 (MI% obtained by the E-test was 0.250, 0.047, 0.094, and 0.250 pg/ml, respectively). The bactericidal pg/ml) were activities of clarithromycin and erythromycin (MBC,, reached at lower concentrations than those of azithromycin and roxithromycin (MBC,, pg/ml). Both the MIC and MBC data proved that clarithromycin and erythromycin have higher antistreptococcal activities than azithromycin and roxithromycin. MICs obtained with the E-test were one or two steps lower than those found with the microbroth method. Only minor discrepancies were observed among the three methods. The Etest method is applicable in a diagnostic laboratory for susceptibility screening of group A streptococci forthe macrolides tested in this study. None of the 180 strains was resistant to erythromycin and the other macrolides, confirming the low rate of erythromycin resistance (0.5%) in The Netherlands, as foundin our previous study.

REFERENCE 1. van Asselt GJ, Sloos Mouton RP, Van Boven CPA,van de Klundert JAM. Susceptibility of Streptococcus pyogenes to azithromycin,clarithromycin, erythromycin and roxithromycin in vitro. J Med Microbiol 1995; 43:386-391.

Efficacy of Clarithromycin Against Experimental Pulmonary Infection Caused by Streptococcus pneumoniaeStrains with a Novel Macrolide Resistance Mechanism J. A. Meulbroek, M. J. Mitten, A. Oleksiew, V. D. Shortridge,

S. K. Tanaka, and J. D. Alder Abbott Laboratories Abbott Park, Illinok

INTRODUCTION Macrolide resistance has been increasingly frequent in Streptococcus pneumoniae. Although onlyl-2% of all S. pneumoniae are resistant to erythromycin, >50% of penicillin-resistant S. pneumoniae are also erythromycin resistant (1). The most thoroughly described mechanism of macrolide resistance is a ribosomal methylase encoded by e m , a large family of related genes found in many different bacterial species. In Sfreptococci, methylase, production is encodedby e m A M (2). PCR analysis has recently shownthat the majority of S. pneumoniae strains with intermediate erythromycin resistance lacked DNA homologous to known e m sequences (data not shown). These strains have a novel macrolide resistance mechanism. In thisreport, the in vitro susceptibilities of S. pneumoniae strains bearing the novel resistance mechanism are de411

412

Meulbroek et al.

scribed.Additionally, the comparativeefficacies of clarithromycinand azithromycin against rat pulmonary infection caused by susceptible and novel resistant strainsof S. pneumoniae are reported.

MATERIALS AND METHODS All strains of S. pneumoniae were clinical isolates maintained the in clinical culture collection of Abbott Laboratories. Minimal inhibitory concentrations (MICs) were determinedby agar dilution testing using guidelinesset by the National Committee for Clinical Laboratory Standards. Twentyfour-hour cultures,grown in BHI broth with 5% sheep blood, were usedas inocula.Inoculawereprepared bymixing undilutedculturewith 2% molten agar in a1:3 ratio. Sprague-Dawley (male)rats, 7-10 weeks of age (Charles River Breeding Laboratories, Wilmington, MA), were inoculated intratracheally, per via a blunt-end feeding tube, delivering a volume of 0.2 m1 of the inoculum. Medications were given orally, beginning at 5 h postinoculation, then twice daily (7 A . M . /P.M.) ~ for 2 additional days. Untreated rats were determined not to be bacteremic at the termination of the efficacy trial. Lungs were harvested 12 h following the last medication. Homogenates wereplatedonColumbiaCNAAgar with 5% blood.Colonieswere counted 24 h later.

RESULTS The results are summarized in Tables1-3.

Tabh I

Comparison of MIC Values of Sensitive and Resistant S. pneumoniae MIc (CLg/ml)

Resistance

MIC

ery-Sensa

0.008 MIcso 0.015 MIC, 2 MIGO 4 MIC, 16 MGO MIC,>l28 >l28

ery-Novel ery-Em .

Clan = clarithromycin; = penicillin.

Claria

Azi

0.03 0.03 8 32

Clinda

0.06 0.12 0.12 16 >l28

Pen

0.03 0.06 1

4 2 4

= azithromycin; ery = erythromycin; Cliida = clindamycin; Pen

S. pneumoniae

MacrolideinResistance

413

Tabk 2 In Vitro Susceptibility of Streptococcus pneumoniae to Clarithromycin, Azithromycin, Clindamycin, and Penicillin

esistanceStrain

Claria

Pen

Clinda

Novel Novel Novel Susceptible Clan = clarithromycin;Azi = azithromycin; Clinda= clindamycin; Pen = penicillin.

DISCUSSION Streptococcus pneumoniae which bear the novel resistance mechanism to macrolides are a subset of penicillin-resistant pneumococci. These organisms are distinct in their resistancepattern from pneumococci bearinge m gene sequences: The novel resistant organisms have a moderate level of resistance to macrolides andare sensitive to clindamycin (Table 1). A rat pulmonary infection was utilizedto determine the comparative efficacies of clarithromycin and azithromycin against a number of strains Table Efficacies of Clarithromycin and Azithromycin Against Rat Lung Infection Caused by Novel Resistant S. pneumoniae

from lungs Log Cmr recovered Strains of S. pneumoniae

of

Untreated rats

Clarithromycintreatedb rats

Azithromycintreatedb rats

f

& f f f

f -t f f

2 f

f

% Reduction (compared to untreated rats) following Rx with

Clan

Azi

.With respect to macrolide susceptibility, strains 5649, 5654, and 5659 bear the novel resistance mechanism; strain 5739 is a susceptible strain (see Table Inoculum doses for the individual trials are strain 5649, log 6.15 CFU; strain 5654, log 6.69 C F U ; strain 5659, log 6.60 C F U ; strain 5739, log 6.78 CFU.( C F U = colony-forming units.) bFor individual involving strains 5649, 5654, and 5659, rats were treated with macrolide at 100 mglkg per day, twice per day, for days. For the trial involving strain 5739, rats were treated with macrolidesat 5 mg/kg per day, twice per day, for days.

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414

bearing the novel resistance mechanism (Table 2). As indicated in Table clarithromycin therapyat 100 mgkg per day resulted in greater a reduction in the bacterial burden recovered fromthe lungs of infected rats than did azithromycin fortwo of the novel resistant strains tested (strains and Against a third novel resistant strain (strain 5654), the efficacies of clarithromycin and azithromycin were comparable. Both macrolides were highlyefficaciousagainstasensitive strain (strain These results indicate a potential role for the use of clarithromycin against a subset of penicillin-resistant pneumococci.The novel resistance mechanism isunder investigation inour laboratories.

CONCLUSIONS Clarithromycin has efficacy superior to azithromycin for the treatment of pulmonary infection caused by novel resistant Streptococcus pneumoniae. Novel resistant S. pneumoniae make up alargeportion of penicillinresistantpneumococci and are still potentially treatable withclarithromycin. The use of this macrolide as a treatment option for penicillinresistant pneumococci needsto be more thoroughly investigated.

REFERENCES 1. Lonks JR,Medeiros AA. The growing threat of antibiotic-resistant Streptococcus pneumoniae. Med Clin N 1995; 79523-535.

2. Leclercq R,Courvalin P.Bacterial resistance to macrolide, lincosamide, and streptogramin antibioticsby target modification. Antimicrob Agents Chemother 1991; 35:1267-1272.

Macro- and Microautoradiographic Studies on Penetration of Azithromycin in Bacterially Infected Mice Issei Nakayama and Emiko Yamaji Nihon University Schoolof Medicine Tokyo, Japan

Kaoru Shimada Tokyo Senbai Hospital Tokyo, Japan

Shuichi Yokoyama, Kazumi Miura, Hideya Muto, Kazunori Enogaki, Masatoshi Ogawa, andKino Shimooka Pfizer Pharmaceuticals, Inc. Nagoya, Japan

INTRODUCTION Azithromycin is a new acid-stable 15-membered-ring macrolide developed at Pfizer Inc. inthe United States. Azithromycin well is absorbed following oral administration in mice, rats, dogs, and cynomolgus monkeys, exhibits a uniformly long elimination half-life, and is distributed exceptionally well into all tissues compared with erythromycin(1).In vivo efficacy has been observed in various infection models, which suggest that penetration into infectionsitesishigherthannoninfectedtissues (2-4). The ability of 415

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azithromycin to accumulate markedly in human phagocyte cells also has been demonstrated(5). According to studies on the intracellular accumulation of azithromycin, one potential for phagocyte transport to sites of infectionwassuggested.Inthisstudy,wedescribe the distribution of azithromycin in the infection site by autoradiography and the distribution of azithromycin in inflammatory cells by microautoradiography.

MATERIALS AND METHODS Radiolabeled Compounds The specific radioactivityof [W]azithromycin was 0.92 MBq/mg.(24.8pCi/ mg) and that of [3H]azithromycin was 13.2 MBq/mg (357 pCi/mg). The radiochemicalpurity of eachcompoundwas more than 98%, as determined by radio-thin-layer chromatography [Three types of solvents: chloroform/hexane/diethylamine/triethylamine(15 : 15 : 2 : 2), ethylacetate/hexane/diethylamine(15 : 15: 2), diethylether/methanoYammoniawater (85 : 15 : 2)]. 'Ib prepare the dosing solution, a radiolabeled compound was dissolved in0.05 M citrate buffer(pH 5.3) and unlabeled azithromycin was added to dilute the radioactivity.

Experimental Animals ICR mice (male, 4 weeks, SPF) were purchased from Nihon SLC Co. ('Ibkyo). Mice were given water and fed [CE-2, Nihon Clea (Tokyo)] ad libitum. Infection models were produced in mice as described byGirard et al. [4]. A localized infection was induced by implanting apaper disk (0.08 mm) impregnated with Staphylococcus aureus ATCC25923 (3 X CFU/ disk/mouse) on the mid-back of anesthetized mice.The incision was closed with wound stitchesto prevent removalof the suture.

Autoradiography of Infection Sitein Locally Infected Mice At 5 dayspostchallenge,micereceived 20 mgkg [Wlazithromycin by gavage. At 2,6,24, and 72 h after administration,mice were sacrificed by diethylether anaesthesia.The mice were rapidly frozen by immersion in an acetone-dry ice mixtureat -80°C, and the CMC block wasmounted to the sample holder of the cryotome. The frozen mice were sagittally sectioned (30 pm) and lyophilized. All cryosections were exposed for 3 days in an imaging plate(20x40 c m , FUJI film, Japan) before being scanned with the BioimagingAnalyserSystem(BAS2000 FUJI film, Japan). The autoradiographic optical densityof the film was measured and handled with 32 colors, with output expanding fourfold.

Autographic Studies

on Penetration of Azithromycin

41 7

Microautoradiography Mice were given 20mgkg [3H]azithromycin by mouthat 5 days after challenge. At 24 h after the dose, mice wereanesthetized with diethylether and the infection site wasexcisedandmountedusingO.C.T.compound (TISSUE-TEK, Miles Inc.). The tissue was sectioned into 4-pm sections withacryostat(CRYOCUT Leica) at -20°C.Each section was placed on slides precoated with emulsion for microautoradiography by the thaw mount method(6) and dried.The sections were exposedat -80°C for 2 weeks, developed with Dectol (Kodak), and fixed with Konik (Konica). Sections were then stained with hematoxylin-eosin for microscopic examination.Microautoradiogramsandphotomicrographswere prepared at x200 and ~ 1 0 0respectively. , In addition photomicrographs were madeat X400 to examine the cells.

RESULTS Infection Site Autoradiography Autoradiograms of the infection site following oral doses of [Wlazithromycin at 5 dayspostimplantation of a S. aureus-impregnateddisk are shown in Figs. 1 (whole-body autoradiogram) and 2 (autoradiograms of infected site). The distribution of radioactivity aroundthe implanted paper disk (infection site) was similar to that of noninfected sites 2 h after the dose. The level of radioactivityin the infectionsitewashigher than noninfected tissue siteat 6 h afterthe dose, and increased progressively by 24 h. In addition, the level in the infection site around the paper disk was

Z Whole-body autoradiogram of infected mouse showing the distribution of radioactivity after oral administration of [W]azithromycin (20 mglkg) 5 days postimplantation of a S. aureus-impregnated disk.

Nakayama et al.

2h

24h

72 h

Figure 2 Autoradiograms of infected sites showing the distribution of radioactivity after oral administration of [14C]azithromycin (20 mgkg) to mice, 5 days post implantation of a S. aureus-impregnated disk.

also higher than at noninfection site at 72 h and suggestedthat the tissue of infection site retained [14C]azithromycin.

Microautoradiography Microscopic examination of infection site showeda massive influx of polymorphonuclear leukocytes,a few macrophages and fibroblasts, and formation of large foci of S. aureus. Neutrophil accumulation and layered distribution were observed aroundthe implanted paper disk. The. silver grains indicating radiolabeled drug distributed around the paper disk and concentrated in the area of neutrophil accumulation. The distribution of the radiolabeled drug corresponded to the area of inflammatory cell aggregation.

Autographic Studies on Penetration

Azithromycin

419

DISCUSSION Autoradiograms of the infection site in mice following an oral dose of [Wlazithromycin showed that extremely high level of azithromycin accumulated at the local infection site where a large number of phagocytic cells infiltrated. In addition, microautoradiograms using [3H]azithromycin suggested that phagocytes retained high concentrations of azithromycin. Wildfeuer et al. reported that uptake of [Wlazithromycinby human neutrophil polymorphonuclear leukocytes and macrophages resulted in intracellular concentrations greater than the extracellular medium (5). Retsema et al. described the possibility of augmentation of azithromycin delivery to the infected sitewith the use of S. aureus thigh infection models in CD-l mice. Azithromycin concentrationsin the infected leg were 20-foldgreater than in saline-injected legsat 145 h (2). In other rodent infection models (e.g., S. aureus paper disk infection in rat and Salmonella models of acute systemic and tissue infection in rats), azithromycin was shownto be effective even though azithromycin plasma concentrations were belowthe minimal inhibitory concentration (MIC) at all times during and after the challenge In viewof the above two studiesand our results, highlevelsof azithromycin in the localized infection site may be a consequence of augmented drug distribution as a result of chemotaxis of azithromycin-loaded phagocyte cells and may help explain why azithromycin was effective even though plasma concentrations were below the MIC of key pathogens.

CONCLUSION Autoradiograms of the infectionsiteinmicefollowing oral doses of [“C]azithromycin showedthat an extremely high level of azithromycin accumulated in the localized infection site where large amounts of phagocytes infiltrated. Microautoradiograms of the infection site suggested that the phagocytes retained high concentrationsof azithromycin. High levels of azithromycin in the localized infection sites may be a consequence of augmented drug distribution as a result of chemotaxis of azithromycin-loaded phagocytic cells even though the azithromycin plasma concentrations were below the MIC of key pathogens.

REFERENCES 1. Girard AE, Girard D, English AR, Gootz TD, Cimochowski CR, Faiella JA, Haskell SL, Retsema JA. Pharmacokinetic andin vivo studies with azithromycin (CP-62,993), a new macrolide with an extended half-life and excellent tissue distribution. Antimicrob Agents Chemother 1987; 31:1948.

420 2.

3. 4. 5.

6.

Nakayama et al. Retsema JAYBergeron JM, Girard D, Milisen WB, Girard AE. Preferential concentration of azithromycininaninfectedmousethighmodel.JAntimicrob Chemother 1993; 31 (supplE):S. Girard AE, Girard D, Retsema JA. Correlation of the extravascular pharmacokinetics of azithromycin with in vivo efficacy in models of localized infection. J. Antimicrob Chemother 1990;25 (suppl A):61. Girard D, Bergeron JM, Milisen WB, Retsema JA. Comparison of azithromyroxithromycin, and cephalexin penetration kinetics in early and mature abscesses. J Antimicrob Chemother 1993;3l(suppl E):17. Wildfeuer A, Reisert I, Laufen H. Uptake and subcellular distribution of azithromycin in human phagocytic cells. Armeim.- Forsch/Drug Res 1993; 43(I), No. 4:484. Stumpf WE. Techniques for the autoradiography of diffusible compound. In: PrescottDM, ed. Methods in Cell Biology 13, New York: Academic Press, 1976:171.

In Vivo Antibacterial Activityof RU 004, a New Ketolide Active Against Respiratory Pathogens Constantin Agouridas, A. Bonnefoy, and Jean-Franqois Chantot Rowel-Uclaf Romainville, France

INTRODUCTION RU 004 is a new ketolide which displays excellent in vitro activity against all relevant pathogens involved in RTI , including erythromycin-resistant pneumococci, Huemophilus infruenzae (Hi) and atypical bacteria. We report here on the in vivo antibacterial activityof RU 004 in various murine experimental septicemia models and in two different murine models of pulmonary infections.

MATERIALS AND METHODS mouse septicemia modelwas usedto assess the protective efficacy of RU A (ERY), clarithromycin (CLA), azithromycin (AZI), josamycin (JOS), pristinamycin (PRI), and ampicillin inthe case of Hi. PD, (protective doses) were calculated according to the Reed and Munch method(1). In pneumonia models,the following bacterial strains were used:

004 in comparison with erythromycin

421

Agouridas et al.

422

Streptococcus pneumoniae (Sp)serotype01,originallyisolated fromabloodculture(strainSp6254) : erythromycin-resistant strain. Hi serotype b, originally isolated from cerebral spinal fluid (strain 87169); a P-lactamase-producing strain. Infections were induced in female Swiss (OF-l) mice, 6-7 weeks of age (Sp model) and in female C57BI/6 mice, 6 monthsof age (Hi model). Mice were infected intratracheally via the mouth with 105 CFU (colonyforming units)of Sp 6254; they developed bacteremic pneumonia and fatal diseasewithin 2-5 days.In the case of Hi, micewereinfected intratracheally via the mouth with 108 CFU; they developed inflammatory bronchopulmonary diseasethat was spontaneously cured. Therapeutic assays were conducted as follows;

-

-

Survival studies (Sp):Treatmentwas initiated 6 h (protective)or 18 h (curative) postinfection. Drugs were administered orally bidfor 3 days (n=10-15 mice per treatment group). Bacterialclearance(Hi):Kinetics of bacterialkillingwererecorded over a 24-h period following a singlePO administration of the drug 16 h postinfection(n=3 mice).

RESULTS Results are shown in Table1 and Figs. 1and 2. Table Z In Vivo Antibacterial Activityof RU 004 (PD,,, mgkg)

Strains S. pyogenes Ery-Sa 300 S. aureus Ery-S S. aureus Ery-Ri E.faecium Ery-R, Van-R S. pneumoniae Ery-S >50 S. pneumoniae Ery-Ri S. pneumoniae Ery-RC S. pneumoniae Ery-RC S. pneumoniae Ery-RC H. influenzae Amp-S H. influenzae Amp-R325

RU004 16 20 13 11 16 30 19 17 15 142 116

ERYa

>l00 >l00

>l00 >l00 >l00 >l00

CLA

AZI

PIU

AMP

-

16 13 >l00

16

-

>l00

-

>l00 >l00 >l00 >l00 346 110

18 >l00 >l00 >loo >l00 110 117

-

-

-

-

>l00

-

>IO0 >l00 >l00 67

-

-

-

5

aEry-Ri, Ery-RC: inducible, constitutive resistance to erythromycin A; ERY: erythromycin; C M : clarithromycin; M I : azithromycin;PRI:pristinamycin; A M P : ampicillin; VAN: vancomycin.

In Vivo Antibacterial Activity

423

RU 004

Experimentalmurinesepticemia.

Figure I

DISCUSSION In septicemia caused by gram-positive cocci susceptible to ERY, RU 004 shows in vivo activity similar to that of CLA but quite superior to that of ERY. In infectionscaused by ERY-R strains, RU displays a high antipneumoccocal efficacy, unlike available 14- or 16-membered-ring macrolides which are completely inactive. The protective doses fall withinthe range of values foundfor susceptible bacteria (15-42 mg/kg). PR1 showed measurable activity in only two out of five infections, with the PD,, at 67 and 76 mgkg. In Hi-induced septicemia, the ketolide was two to three

Q

1

2

3

4

6

e

7

e

o

l

Q

l

l

I

Z

Pmtecthre treatmant

Figure 2 Experimental murine pneumonia. (From Ref. 2.)

~

~

Curative treatment

~

B

~

Agouridas et al.

424

times more active than ERYor CLA. Conversely, PD,, were closeto those of M I . Moreover, RU 004 displays high efficacy inEnterococcus infections, whatever the phenotype of infecting strains (VAN-R and/or ERY-R) (Table l). In pneumonia induced by ERY-R Sp, RU 004 demonstrates the same level of efficacy as virginiamycin (Fig. 2). Despite a significant decrease inefficacy between protective and curativetreatments, the ketolide remains notably effective in severe conditionsof bacteremic experimental disease. In these experiments, no resistant mutants were isolated. In pneumonia induced by Hi, RU 004 exhibits an activity similarto that of AZI. The initial bacterial clearance was more efficient with the ketolide than with AZI.

CONCLUSIONS Unlike macrolides and streptogramins, RU 004 displays high therapeutic efficacy in mice infected by various common respiratory pathogens and gram-positive cocci, thus conforming to its well-balanced and potent in vitro activity. RU 004 appears to be very promising for the treatment of infections causedby difficult-to-treat respiratory pathogens.

REFERENCES 1. Lorian V. Antibiotics in Laboratory Medicine. Baltimore,MD: Williams and Wilkins, 1993. 2. Rajagopalan, Levasseur P, VallCe E, et al., 35th Intersci Conf Antimicrob Agents Chemother, 1993; abst F-173.

Open Noncomparative Studyof the Efficacy and Safetyof Azithromycin in the Treatment of Adult Tonsilitis (Epidemiological Studyof the Responsible Bacteria) A. Desaulty Centre Hospitalier, Rkgional Universitairede Lille Lille, France

INTRODUCTION Cases apparently commonplace bacterial tonsilitis occur frequently in patients of all ages. Such cases require treatment designed not only to relieve symptoms as rapidly as possible but also to avoid those complications which mightresult from infections due to group streptococci (rheumatic fever, glomerulonephritis, phlegmonous adenitis, peritonsillar abscess, etc.). Azithromcyin is a new azalide (nitrogen-containing macrolide) antibiotic that provides a useful alternative in the treatment such tonsilitis in view ofthe following: It has a suitable spectrum of activity; it shows excellent diffusioninto tonsillar tissue; and its pharmacokinetic parameters are such that it persists for a prolonged period in tonsillar tissue. The objective of the present trial was to evaluate the clinical efficacy and safety 425

Desaulty

426

of treatment with azithromycin (once-daily dose for 5 days) in adults with acute tonsilitis and also to define the bacterial epidemiologyof communityacquired acute tonsilitis.

METHODS Patients Patients included in the trial had to comply with the following criteria: age over 18; monitored as outpatients; presenting with acute erythematous or erythematous pustular tonsilitis accompanied bysignsof acute tonsilitis; treated with azithromycin when the reference therapy (penicillin)couldnot be used;havinggiveninformedconsentinwriting. A throat swabhad to be taken foreachpatientfrom the tonsillar area, purulent or cryptic areas, and/or the posterior wallof the pharynx for bacteriologic studies.

Treatment Patients received500 mg of azithromycin (2 X 250 mg capsules) on the first day and 250 mg of azithromycin dailyfor the following 4 days. Any other antibiotic therapy, digitalis glycosides, and ergot derivatives were prohibited throughout the trial.

FOllOW-up Clinical efficacy was assessed at day evaluation.

-t

2 during a complete physical

Assessment Criteria Success was defined as follows: Apyrexia (defined as a temperature equal to or below before the control evaluation at day 2 2 without the need for any other antibiotic; the disappearance of the following signs at day 10: pharyngeal pain (spontaneous or on swallowing), alteration in appearance of the tonsils and/or pharynx, and difficulty in swallowing. For the purposes of the analysis, all patients entered in the trial were included in the description of the population and evaluationof the safety of azithromycin. The efficacy of azithromycin wasevaluated in the population sample with streptococcal tonsilitis as documented by bacteriologic tests.

Azithromycin for Adult Tonsilitis

427

RESULTS Description of the Population Three hundred nine patients were included in the trial between December and July The population was balanced in terms of sex men, women) and had a large majorityof patients under years of age In compliance with protocol recommendations, all included patients exhibited analteration in the appearance of the tonsils and/or pharynx together with a bodytemperature equal to or greater than More than half the patients were suffering from severe pharyngeal pain and had great difficulty in swallowing or had already taken an antipyretic

Bacterial Epidemiology Bacteriologic tests in patients revealed bacterial tonsilitis in 55% of cases includingstreptococcaltonsilitisin 16% The other microorganisms most frequently detected were as follows: Staphylococcus aureus in cases; Haemophilus parainfluenzae in cases; Haemophilus influenzae in cases. Bacteriologic tests were negative in of the patients

Efficacy Evaluation The efficacy evaluation was carried out for a sample size of patients with streptococcal tonsilitis initially confirmed by bacteriologic tests. Treatment was successful in of cases Of the three cases regarded as failures, the test treatment had been discontinued and days after day 0 respectively as local and systemic signs either persisted or worsened, warranting the prescription of another antibiotic. No complications (particularly phlegmonous adenitisor peritonsillar abscess) werereported during the trial.

Safety Evaluation Ttventy-three out of patients reported at least 1 adverse event during the trial. Gastrointestinal symptoms were most frequently involved (six cases of diarrhea, four casesof abdominal pain, two casesof gastralgia, and two cases of nausea). patients had to discontinue treatment prematurely becauseof adverse reactions (diarrhea and exacerbation of hemorrhoids in one case, and nausea and erythemaof exposed parts in the other). None of the other adverse events mentioned was regarded as serious (i.e. leading to permanent sequelae, adversely affecting the patient’s prognosis for survival or to hospitalization).

Desaulty

428

DISCUSSION AND CONCLUSION The trial involved 309 adult patients suffering from acute erythematous or erythematopultaceous tonsilitis and provideddata concerning its bacterial epidemiology. The data may be compared with previously published relevant results (1): A high proportion of negative bacteriologic, results were obtained (45% of cases): the infection was most frequently diagnosed as streptococcal in origin during the trial (16% in the total sample size and 29% of the population with bacteriologically documented tonsilitis). However, this figure is lower that thanpresented in previously published results. The trial also confirmed the efficacy of once-daily azithromycinfor'5 days for the symptoms of acute streptococcal tonsilitis in adults.At day'l0, the success rate attained 91%; thisis similar to the results noted inother trials for the same indication (2,3). Safety analysis revealed that 7.4% of patients experienced at least one adverse event duringthe trial and confirmedthat none of the adverse events was serious.

REFERENCES 1. Gehanno P, Portier H, Longuet P. L e pointactuelsur1'6pid6miologie des angines aigueset dessyndromes post-streptococciques.La Revue du Praticien 1992; 42(3):284-287. 2. Hooton TM. A comparison of azithromycin and penicillinV for the treatment of streptococcal pharyngitis.Am J Med 1991; 9l(suppl3A):23S-26S. 3. Carbon C, etal. Economic analysis of 3 antibiotic regimens in acute pharyngia prospective naturalistic comparison of azithromycin versus roxithromycin. J Antimicrob Chemoth (in press).

Open Studyof Clarithromycin in the Treatment of Pneumonia Due to Streptococcus pneurnoniue Murat Hayran, MustafaErman, Deniz Gur, Murat Akova, and Serhat Una1 Hacettepe UniversitySchool of Medicine Ankara, %key

INTRODUCTION Clarithromycin is a novel macrolide antibiotic active against a variety of organisms responsible for community-acquired pneumonia. Streptococcus pneumoniue invariably has been the most commonly encountered pathogen and appears to be sensitive to clarithromycin in vitro. In this open, prospective, noncomparative study, 41 patients with community-acquired lobar pneumonia were evaluated to establish the efficacy and safety of clarithromycin.

PATIENTS AND METHODS Forty-one adult patients with community-acquired pneumococcal pneumonia were included.The diagnosis was established by the presence of symptoms which included cough, sputum production, dyspnea, pleuritic chest pain and rigors, x-ray findings, fever, and the demonstration of abundant 429

Hayran et al.

430

polymorphonuclear leukocytes( P m ) and gram-positive diplococci on microscopic examinationof sputum. Patientswith evidence oftuberculosis or malignancy, chronic liver or renal impairment, or receiving theophylline/ theophylline analogs, as wellas pregnant or lactating women were excluded. Patients with severe or complicated lower respiratory tract infections necessitating parenteral antibiotictreatment were not eligible. Complete physical examinationwas performed daily. Cough, sputum production, and dyspnea were recorded by using a grading scale as absent, mild, moderate, or severe. Pyrexia was considered as present when body temperature was greater than 37.8"C. Complete blood counts with differential, blood urea nitrogen, creatinine,total bilirubin, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were performed, and sputum samples for microscopic examination and culture were collected on the first day, during, andat the end of treatment, if available. Chest x-ray was obtained on the firstand tenth daysandrepeatedweeklyduring follow-up until pneumonia resolved. Blood cultures were taken before the initiation of treatment and daily as long as the patient remained febrile. Gram-stained sputum smears were examined and sputum cultures were performedon sheep blood and chocolate agar plates incubated at 37°C in an atmosphere containing 5-10% carbon dioxide daily as long as sputumsamplecould be obtained.Suspiciouscoloniesweretested for susceptibility to optochin. Susceptibility to penicillin and clarithromycin was determined by disk-diffusion method. n7vo tablets of clarithromycin (250 mg) were administered everyh for 10 days. Clinical curewas defined asthe resolution or improvement pretreatment symptoms and signs with defervescence of fever before study day

RESULTS Of the 41 patients, (54%) were males and 19 (46%) females.The mean age was 40.5 years (range:18-67). Four (9.8%) patients had underlying chronic obstructive pulmonary disease. Clinical characteristics of patients are summarized in Table1. Streptococcus pneurnoniae was isolated in the sputum cultures of 16 patients (39.0%). None of the patients was bacteremic. Susceptibility testing by the disk-diffusion method disclosed all isolates were sensitive to clarithromycin in vitro. Clinical cure with microbiological eradication was achievedin 37 patients(90.2%). Pretreatment signsandsymptoms resolved in days after initiation of clarithromycin treatment (median: days).In patients whodevelopedpleuraleffusion, treatment was switched to parenteral p-lactam antibiotics. Another two remained febrile

Clarithromycinfor S. pneumoniae Infection

431

Table Z Clinical Characteristicsof Patients Treated with Clarithromycin Characteristics Sex, FM (min, Mean Median (min, max) duration of infection (day) 2 (9.8) disease Underlying (COPD) (%) Abundant gram-positive diplococci in sputum smear (%) S. pneurnoniae culture sputum in (%) Cure (%) (90.2) eradication Microbiological (%) (95.1) Failure (%) (%) (7.3) Side Nausea (%) (2.4) transaminases Increase in (%) (2.4) discomfort Abdominal (%) (2.4)

19/22

40.5 (18-67) (1-5) 4/41 41 (100.0) 16 (39.0) 37/41 39/41 2/41 (4.9) 3/41 1/41 1/41 1/41

as long as the fifth day and were, therefore, assigned to have “indeterminate” response and the antibiotic regimen was modified. Clarithromycin was well tolerated by all but three patients who exhibited mild side effects of nausea, abdominal discomfort, and elevation of aspartate aminotransferase (ALT). However, none of these side effects required discontinuationof treatment.

CONCLUSION Clarithromycin is effective and welltolerated in the treatment of uncomplicated lower respiratory tract infections due to Streptococcus pneumoniae. The use of clarithromycin in severeor complicated pneumonia needsto be further evaluated.

REFERENCES 1. Fass RJ. Aetiologyand treatment of communityacquiredpneumoniain adults: an historic perspective. J Antimicrob Chemother 1993; 32 (suppl A): 17-27. 2. Uzun Hayran M, Akova M, Gur D, Akahn HE. Efficacy of a three day course of azithromycin inthe treatment of community acquired pneumococcal pneumonia: a preliminaryreport. J Chemother 1994; 653-57. 3. Chien SM, Pichotta P, Siepman N, ChanCK. Treatment of communityacquired pneumonia. A multicenter, double-blind, randomized study wmparing clarithromycinwith erythromycin. Chest1993; 103(3):697-701.

Clinical Studyof Rokitamycin on Pneumococcal Upper Respiratory Tract Infections in Pediatrics Yoshikiyo Toyonaga YamanashiRed Cross Hospital Yamanashi, Japan

INTRODUCTION Pediatricians most frequently encounter upper respiratory tract infections (URTI) in outpatient clinics. An especially high isolation rate of penicillininsusceptible (MIC 0.01-0.78 pg/ml)/resistant (MIC 2 1.56 pg/ml) Streppneumoniue (PISPPRSP) has become a recent problem (1) (MIC = minimal inhibitory concentration). To investigate the clinical efficacy of rokitamycin (RKM), a 16-membered ring macrolide antibiotic, on pediatric URTI caused by S. pneumoniue, we have reviewed the clinical performances of cases of pediatric URTI treated in the department and the results are reported below.

PATIENTS AND METHODS Rhinopharyngeal cultures were taken from pediatric who patients visitedthe department from February to October 1995. Patients with URTI purportedly due to S. pneumoniue were given RKM, cefditoren pivoxil (CDTR), or 432

Rokitamycin in Pneumococcal URTI

in Children

433

cefdinir (CFDN) at random, and the clinical efficacy and bacteriological effects were investigated. The dosages were mgkg, mgkg, and mgkg for RKM, CDTR and CFDN, respectively,three times daily.

RESULTS MIC Distribution Of the cases of URTI, significant organisms weredetected in cases. S. pneumoniae was isolated from52.2% of the cases cases), of which 80 cases were complicated infections with various other bacteria, such as Haemophilus influentae and Moraxella subgenus Branhamella catarrhalis. There were isolates of S. pneumoniae, of which isolates) were penicillin susceptible (MIC &ml) S. pneumoniae (PSSP) and isolates) were PISPPRSP. The MIC distributions of various antibacterial agentsfor isolated PSSP andPISPPRSP areshown in Figs. and 2. MIC distributionsof the penicillins (PCs) and the cephems (CEPs) for PISPPRSP were evidently inferior to those against PSSP. Among the macrolides (MLs), 14-membered-ring erythromycin (EM) and clarithromycin (CAM) showed marked increases inthe number of resistant strains

Figure l Antibacterial activity of various antibiotics against penicillin-susceptible S. pneumoniae.

Toyonaga

434

"0-

--

"e

--&

CFDN

-.d-.RKM

-e*---

Figure 2

Antibacterial activityof penicillin-insusceptiblelresistantS. pneumoniae.

for PISPPRSP; however, 16-membered-ring RKM showed similar MIC distributions forPSSP and PISPPRSP.

Clinical and Bacteriological Efficacy The clinical efficacyrates of RKM, CDTR, and CFDN were 89.5% (34/38), 87.1% (27/31), and 72.4% (21/29), respectively, for the PSSP infections, and 90.0% (18/20), 73.9%, (17123) and 71.4% (10/14), respectively,for the PISPPRSP infections, indicating no significant decrease in efficacy with these agents.On the other hand, the eradication rates of CDTR and CFDN for the PISPPRSP infections were evidently lowered compared with the PSSP infections, whereas a reduction in the eradication rate was not observed for RKM for the PISPPRSP infections (Table1).

DISCUSSION Streptococcus pneumoniae, in addition to H. influenzae and M. (B.) catarrhalis, is an important causal organism of pediatric upper respiratory tract infections. In recent years, PISPPRSP have been isolated at a high frequency, and insusceptibilityof the organism not only to PCs and CEPs but also to MIS (1) is a serious problem for pediatricians. Also in this study a marked insusceptibility of PISPPRSP toPCs and CEPs was found. This was reflected in bacteriological efficacy, and reduc-

Rokitamycin in Pneumococcal URTI

in Children

435

Table Z Bacteriological Effects Classified by Penicillin Sensitivity (Eradication Rate of S. pneumoniae)

Agent RKM

.

CDTR CFDN

'No. of strains eradicatemo. of strains isolated.

tions in eradicationrates of CDTR and CFDN were found inPISPPRSP infections, compared with PSSP infections. On the other hand, regarding MLs, PISPPRSP showed insusceptibilityto EM and CAM. However, the MIC distributionof RKM for PISPPRSP was similar to that for PSSP, and high eradication rates for bothPSSP and PISPPRSP were observed inthe clinical performance results. RKM is reported to have a MIC of 1.56-12.5 pglml for S. pneumoniae, for whichEM and other 16-membered-ring macrolideshaveMICs of notlessthan100 &m1 (2). The excellent antibacterial activityof RKM, even againstPISPPRSP, which are resistant to EM and CAM, suggests low cross-resistanceto other MLs. In addition, RKM enters polymorphonuclear leukocytes at higher concentrations than EM andCAM and may explainthe excellent eradicationrates achieved with RKM in this study.

CONCLUSIONS Rokitamycin showed similar MIC distributions for both PISPPRSP and PSSP and exhibited excellent clinical efficacy and bacteriological effects. With the recent problem of increasing PISPPRSP, RKM is considered useful for the treatment of URTI due to S. pneumoniae.

REFERENCES Working Group for Pc-resistant S. pneumoniae; organizer: Masatoshi K. An epidemiological study of penicillin-resistant Streptococcus pneumoniae in Japan. JJA Inf D Koichi D, et al. Study of susceptibilities of freshly isolated strains to macrolide antibiotics. Jpn J Antibiot Ishiguro M, Koga H, Kohno S, Hayashi T, Yamaguchi K, Hirota M. Penetration of macrolides into human polymorphonuclear leukocytes. J Antimicrob Chemother

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V PHARMACOKINETICS, PHARMACODYNAMICS, DRUG INTERACTIONS

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A Comparison of the Bronchopulmonary Pharmacokinetics of Clarithromycin and Azithromycin Kalpana B. Pate1 Arnold and Mane Schwartz College of Pharmacy, Long Island University Brooklyn, New York

Dawei Xuan, Charles H. Nightingale, Pamela R. Tessier, John H. Russomanno, and Richard Quintiliani Hartford Hospital Hartford, Connecticut

The purpose of this investigation wasto measure and comparethe concentrations of clarithromycin, 14-hydroxyclarithromycin, and azithromycin in plasma, epithelial lining fluid, and alveolar macrophage cells in healthy volunteers atthe end of a typical courseof therapy.

MATERIALS AND METHODS

Study Design This was a randomized, prospective, nonblinded trial in healthy adult volunteers. All potential subjects were required to be 18 years or older and within 10% of acceptable weight for their height according to the Metropolitan Life heightlweight tables(1). 439

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440

After giving informed consent, 42 volunteers underwent a complete medicalhistory,physicalexamination,andbaselinelaboratorytesting. lkenty-one (12 males, 9 females) were randomized to the clarithromycin group and 21 (11 males, 10 females) to the azithromycin group. The median age (range)of subjects in the clarithromycin and azithromycin were 28 (21-41) years and 29 (20-41) years, respectively. Clarithromycin, 500-mg tablets, was administered orally every 12 h for nine doses. Azithromycin, 500 mg on day 1followed by 250 250-mg capsules, was administered orally, mg daily for four doses. Prior to the first dose administration, a blood sample was collected.

Bronchoalveolar Lavage Standardized bronchoscopy and bronchoalveolar lavage (BAL) were performed at 4, 8, 12, and 24 h after the last dose of medication administration. Subjects were required to fast at least 6 h before the bronchoscopy. A total of four 50-m1 aliquots of normal saline were instilledinto the right middle lobe and each was immediately aspirated into a trap. The first aspirate was processed separately (first aspirate) fromthe second through the fourth aspirates, which were pooled (pooled aspirate).The volumes of the first and pooled aspirates were measured and recorded. Aliquots from the first and pooled aspirates were sent to clinical laboratoryfor cell count and differential. The measured volumes of the first and pooled aspirates werekeptoniceuntilcentrifugation at 400 g for 5 min. The supernatants were immediately removed fromthe cell pellet and then frozen at -70°C until assay. A small aliquot of the supernatant was frozen separately for urea assay.

Specimen Handling Blood samples were spunat lo00 g for min and the plasma separated and frozen until assay. An aliquot of plasma on the day of bronchoscopy was frozen separately for urea assay. The cells were resuspended with a potassiumphosphatebuffer,pH8.0, to a total volumeof 10% of the recoveredlavagefluidvolume. The cellsuspension was freeze-thawed three times and sonicated for 2 min before drug assay.

Drug Assay Clarithromycin (CLA), 14-hydroxyclarithromycin (14-OH CLA), and azithromycin (AZ) were assayed utilizing high-performance liquid chromatography (HPLC) techniques.

Bronchopulmonary Pharmacokinetics

of CLA and AZ

441

HPLC Instrumentation: Estimation of Epithelial Lining Fluid (ELF) Volumes and Determination of Drug Concentration in ELF and Alveolar Macrophage (AM) Cells The urea in plasma samples was measured by the clinical laboratory at Hartford Hospital. ELF volumewas determined by the urea dilution method (2). The volume of AM cells inthe cell pellet suspension was determined from the cell count and differential performed the on BAL fluid. Cell count was performed using a hemocytometer, andthe differential countwas performed after centrifugation of the specimen in a cytocentrifuge

STATISTICAL ANALYSIS Statistical evaluation of the effect of site sampled (AM cells, AM cells/ plasma ratio, and plasma) and time sampled (4, 8, 12, 24 h) on measured drug concentrations was performed using an analysis of variance method with a Sheffe’ F-test for multiple parameters. A P value <.05 was regarded as significant for all tests performed.

RESULTS Forty-two subjects were enrolled and 41 successfully completed the bronchoscopy procedure. Five subjects were placedinto each of the four time groups for both CLA andAZ with the exception of the 12-h CLA group in which six subjects were enrolled.No serious adverse events werereported by the volunteers. The plasma concentrations of CLA, 14-OH CLA, and AZ are presented in Table 1. At 4 and 8 h, both clarithromycin and 14-OH clarithromycin had significantly higher concentrations than azithromycin. Concentrations of three subjects in the azithromycin treatment groups (one in each of the 8-, 12-, and 24-h groups) were below the limit of assay sensitivity. The concentrations of CLA and AZ in the ELF samples are represented in Table 2. Clarithromycin concentrations from several subjects’ BAL fluid were belowthe assay level of detection. The mean ELF/plasma ratios for CLAare also presentedin Table 2.The concentrations for 14-OH CLA were not measurable dueto lidocaine interferencein the assay. The concentrations of azithromycin inthe recovered BAL of subjects from all groups were notably low; onlythe pooled aspirate from two subjects (one in the 8-h and one in the 12-h group) had detectable levels. Therefore, calculation of ELF concentrations were not possible fromthe azithromycin-treated subjects.

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Bronchopulmonary Pharmacokinetics

CLA and AZ

443

Table 2 Concentration of Clarithromycin and Azithromycin in Epithelial Lining Fluid and ELFPlasma Ratio

Time of sample after collection dose last (h)

CLA (Ccdml) (Ccdml)

AZ

CLA ELFlplasma ratio

2 f

2 2 ~~

~

~

~~

aBelow limit of assay. bFour of five subjects had concentrations belowlimit of assay (0.01 pg/ml); one subject had ELF concentration of 1.93 pg/ml. CFour of five subjects had concentrations below limit of assay (0.01 pg/ml); one subject had ELF concentration of 1.76 pg/ml.

Concentrations versus time profiles of CLA, 14-OH CLA, and AZ in AM cells are presented in Table CLA concentrations in the AM cells were significantly higher than AZ at 8 h after the last dose (p<.05). The CLA 8-h time point includes data on the subject who received doses of CLA instead of the 9 doses required by the protocol. The concentrations were not different than those observed inother subjects for the same time point; consequently,the data were includedin the analysis. Also included on Table is the AM cells/plasma ratio of CLA and AZ. The AM cells/ plasma concentration ratio of AZ is significantly higher than CLA at all time periods.

DISCUSSION Only a few investigations have been conducted and published regarding the pharmacokinetic propertiesof the newer macrolides in human pulmonary sites like alveolar macrophages and epithelial lining fluid (4-6). In addition, in vitro studies have shown that the macrolides are not tightly bound to cellular components and readily diffuseinto the extracellular space(7). Our investigation confirmsthe findings of Conte et al. (5) that theconcentrations of CLA and 14-OH CLA are high and persist throughout the sampling period in both ELF and AM cells. CLA concentrations in ELF and AM cells are very high at the earlier time points and decrease over time, followingthe half-life of the drug in plasmaand indicating that equilibrium exists betweenthe extracellular and intracellular compartments. Azithromycin concentrations inthe plasma and ELF were less than half that of CLA. These results mightbe explained by the higher volumeof

Patel et al.

444

Table 3 Clarithromycin, 14-Hydroxyclarithromycin,and Azithromycin Concentrations in Alveolar Macrophage cellsand AM CellsPlasma Ratio ~

Time of sample collection after CLA Pdml) dose(Pdml) (Pdrnl) last (h) 4 8 12 24

~~~

AM cells/

(n)

14-OH CLA (n)

AZ (n)

plasma ratio

AZ 1996 f 2539 (5) 703 f 235 531 f 299 (6) 405 +- 299 (5)

317 & 310 (4) 256 f 45 (2) 124 f 103 (5) 117 f 90 (4)

450 +- 741 (5) 388 f 53 (5) 380 573 (5) 393 +- 364 (5)

*

543 465 1041 1265

1292 15,237 12,807 14,386

.Statistically significantly different thanAZ, p < .05.

distribution of AZ in comparison to CLA. Both CLA and A Z achieved high concentrations inthe AM cells; however, the concentrations of A Z in the A M cells increase slightly over the 24-h samplingperiod. This isprobably due to the slow efflux of AZ from the cellular compartment into the extracellular fluid (8,9). Many investigators have employedthe penetration ratios of antimicrobials into various body sites and fluids to assess the clinical utilityof these agents. Indeed, AZ has a significantly higher A M celllplasma ratio than CLA, which isdue largely inpart to thelow concentrations observed in the plasma of AZ in comparison withCLA. However, whenone examines the actual concentrations in the AM cells, both agents have high concentrations. Thus, the clinical utilityof penetration ratios is somewhat misleading because bacterial eradication is a function of drug concentrationat the site of infection rather than penetration ratios. Both CLA and AZ have been successfully used in the treatment of respiratory tract infections caused by typical bacterial pathogens and atypical intracellular microorganisms. The excellent clinical response for both agents against intracellular pathogens is not surprising, thefor intracellular concentrations of both drugs are exceedingly high in this compartment. In contrast, whereasCLA and its metabolite achieve effective concentrations in serum, AZ concentrations in serum and ELF are below typical minimal inhibitory concentrations forStreptococcus pneurnoniae and Haernophilus influenzae; however, clinical data indicate that AZ is effective clinically against these extracellular bacteria. This remains somewhat enigmatic. Fur ther study is required for a more complete understanding of this phenomenon. The data clearly show that there are pharmacokinetic differences between these drugs. Penetration ratios do not necessarily imply superior microbiological data in vitroor greater efficacy in vivo. Therefore, pharma-

Bronchopulmonary Pharmacokinetics

of CLA and AZ

445

cokinetic and microbiologicaldata as well as clinical data need to be integrated to optimize therapeutic decision making.

ACKNOWLEDGMENTS This work was supportedby a grant fromAbbott Laboratories. We thank David P. Nicolau, Pharm. D., and the physicians at Lung Physicians of Central Connecticut, P.C.,including Robert E. Mueller, R. Frederic Knauft, Michael M. Conway, and Eric T. Shore, as well asthe staff of the Hartford Surgical Center for their invaluable assistance.

REFERENCES 1. Lalak NJ, Morris DL. Azithromycin clinical pharmacokinetics. ClinPharmacokinet 1993; 25:370-374. 2. Rennard SI, Basset G, Lecossier D, O’Donnell KM, Pinkston P, Martin PG, Crystal RG. Estimation of volume of epithelial lining fluid recovered by lavage using urea as.a marker of dilution. J Appl Physiol1986; 60(2):532-538. 3. Wilcox M, Kervitsky A, Watters LC, King TE, Jr. Quantification of cells recovered by bronchoalveolar lavage. Am Rev Respir Dis 1988; 138:74-80. 4. Baldwin DR, Wise R, Andrews JM, Ashby JP, Honeybourne D. Azithromycin concentrations at the sites of pulmonary infection. Eur Respir J 1990; 3~888-890. 5. Conte, JE, Jr, Golden JA, Duncan S, Mckenna E, Zurlinden E. Intrapulmonary pharmacokinetics of clarithromycin and of erythromycin. Antimicrob Agents Chemother 1995; 39:334-338. 6. Honeybourne D, Kees F, Andrews JM, Baldwin D, Wise R. The levels of clarithromycin and its14-hydroxy metabolite in the lung. Eur Respir J 1994; 7~1275-1280. 7. Ishiguro M,Koga H, Kohno S, Hayashi T, Yamaguchi K, et al. Penetration of macrolides into human polymorphonuclear leukocytes. J Antimicrob Chemother 1989; 24:719-729. Johnson JD, Hand WL, Franis JB, King-Thompson N, Corwin RW. Antibiotic uptake by alveolar macrophages. J Lab Clin Med 1980; 95429-439. 9. Panteix G, Guillaumond B, Harf R, Desbos A, etal. 1993. In-vitro concentraJ Antimicrob Chemother tion of azithromycin in human phagocytic cells. 1993; 31 (suppl E):1-4.

RECOMMENDED READING Baldwin DR, Honeybourne D, Wise R. Pulmonary disposition of antimicrobial agents: invivo observationsandclinicalrelevance. Antimicrob Agents Chemother 1992; 36:1176-1180. Chu S-Y, Wilson DS, Deaton R L , Mackenthun AV, Eason CN, Cavanaugh JH.

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Single and multiple dose pharmacokinetics of clarithromycin, a new macrolide antimicrobial. JClin Pharmacol 1993; 33:719-726. Eliopoulous GM, Reiszner E, Ferraro MJ Moellering RC. Comparative in vitro activity of A-56268 (TE-031), a new macrolide. J Antimicrob Chemother 1988; 21~671-675. Fraschini F, Scaglione F, Demartini G. Clarithromycin clinical pharmacokinetics. Clin Pharmacokinet1993; 25:189-204. Foulds G, Shepard RM, Johnson RB. The pharmacokinetics of azithromycin in human serum and tissues.J Antimicrob Chemother25 (suppl A):73-82. Gladue RP, Bright GM, Isaacson RE, Newborg MF. In vitro andin vivo uptake of azithromycin (CP-62,993)by phagocytic cells: possible mechanisms of delivery and release at sites of infection. Antimicrob Agents Chemother 1989; 33:277-282. Hardy DJ, Swanson RN, Rode RA, Marsh K, et al. Enhancement of the in vitro and invivo activities of clarithromycin against Haemophilus influenzae by 14hydroxyclarithromycin, itsmajor metabolite in humans. AntimicrobAgents Chemother 1990; 34:1407-1413. Honeybourne D, Baldwin DR. The site concentrations of antimicrobial agents in the lung. J AntimicrobChemother 1992; 30:249-260. Mandell GL. Delivery of antibioticsby phagocytes. Clin Infect Dis 1994; 19922-925. Metropolitan Life Insurance Company. 1983 Metropolitan height and weight tables. Stat Bull 1983; 64:2-9. Prokesch RC, Hand WL. Antibiotic entry into human polymorphonuclear leukocytes. Antimicrob Agents Chemother1982; 21:373-380. Steinberg "H. Cellular transport of drugs. Clin Infect Dis 1994; 19:916-921.

Kinetics of Dirithromycin: Concentrations in Tonsils, Bronchial Mucosa, and Secretions. Multiple-Dose Studies C. Muller-Serieys, E. Bergogne-Berezin, F. Lemaitre, A. and P. Gehanno Bichat-Claude Bernard University-Hospital Paris, France

M. Derriennic Lilly France St. Cloud, France

C. Le Royer andJ. Clavier University-Hospital Brest, France

INTRODUCTION Serumpharmacokinetics of antibioticsconstitute the firstavailableapproach to their potential in vivo efficacy against infecting pathogens, and pharmacokinetic propertiesof antibiotics are often considered good predictors of extravascular distribution of the drug. A large number of models have been used to study the tissue distribution of macrolides and then provide useful informationon their tissues and fluid penetration In humans, various specimens obtained at surgery or other procedures have been analyzed for local concentrations and kineticsof reference (erythro447

448

Muller-Serieys et al.

mycin) or newer macrolides such as dirithromycin (3). We present the results of single- and multiple-dose studiesof dirithromycin concentration and kinetics in bronchial tissues, bronchial secretions, and tonsils.

PATIENTS AND METHODS

Early Studies 1-1. Bronchial tissues (biopsies) were obtainedat thoracotomy: In study 1, after oral administration of250mg: in 17 patients

after single dose (SD); 28 in patients after multiple doses (MD). Sampling times were 4, 12,or 24 h postdose. In study 2, samples were taken after750 mg in 36patients (SD), or 500 mg in 5 patients (SD) and in 17patients (MD). Sampling times were 12or 24 h after the second dose. 1-2. Bronchial secretions were collected by means of fiberoptic bronchoscopy during exploratory procedures in 9 patients after 750 mg (SD) and in 16 patients after 250 mg (MD). 1-3. Tonsils were obtained from 20 patients undergoing tonsillectomy for chronic tonsillitis after a 500-mg SDat (10 12 h, 10 at24 h postdose). In two groups of six and seven patients receiving two two500-mg doses, specimens were taken at 3-5 and 13-16 h, respectively.

Current Studies 2-1. Bronchial mucosa bronchial secretions. In a multicenter open, randomized study, samples were collected from46 patients with superinfection of chronic bronchitis who were undergoing fiberoptic bronchoscopy for diagnostic purposes. Treatment was continued for 5 days, 500 mg once daily, and specimens wererandomly taken at 24 & 1h, 48 & 1h, or 72 2 1h after the last dose. Group A Group B Group C

Evaluable patients Sampling time after the end of treatment (h)

2-2 Tonsils.

(n = 24

(n = 48

(n = 20) 72

multicenter, open-label, randomized study included 39 patients (25 females, 14 males), mean age 25.95 2 6.48 years, undergoing tonsillectomy for recurrent tonsilitis. Treatment was a single 500-mg dailyoral doseof dirithromycin for 5 days.At enrollment, patients were randomly divided into four groups, A, B, C, and D, according to time intervals(2,3,4, and 5 days, respectively)

Kinetics

Dirithromycin: Multiple-Dose449 Studies between the end of treatment andthe start of surgery. The left and right palatine tonsils were excised, weighed, and dividedinto two fragments, one for antibiotic measurement and the other for pathology analysis. All samples were storedat -80°C until assay. 2-3. Plasma levels werecollectedbeforedosingandattime of surgery.

Antibiotic Assay Concentrations of dirithromycinweremeasured by bioassayin early studies aswell as in current studies; a high-performance liquid chromatography (HPLC) assaywas used in study2. For each lymphoid tissue one frozen fragment was crushed in liquid nitrogen; dirithromycin was then extracted with methanol. All samples (plasma and tissue) were assayed four timesthe andmean concentration was calculated. The limitof detection of dirithromycinwas 0.06 m&,with a coefficient of variation of 7% for plasma samples and 4% for tissue samples.

Pathology Analysis Pathology analysis includedthe following: Crypts: evaluation of squamous cell contents, bacterial inoculum and epithelialstate Lymphoid tissue: size oflymph follicles, density, and interfollicle elements Tonsil lodge: fibrosis and inflammatory condition Endocryptic content and fibrosis

NOTE: All studies were approved by the institutional ethics committee and all patients gave their informed consent.

RESULTS Plasmaconcentrations. Inallstudies,plasmaconcentrations of dirithromycin were very low: They did not exceed 0.02-0.05 m& h after a single dose (Table 1) and 0.17 m& h after multiple doses (Table 2). Inbronchial concentrations of dirithromycin(Study 1) were not significarzly different (0.50-1.7 mg/kg) from those determined by bioassay m&) (Table 1) when measured with HPLC. In bronchial secretions, dirithromycin levels were approxi-

450

Muller-Serieys et al.

+I +l +I +I

+I +I

l I I m

I I I wwmwrIw

m s lI l

Kinetics

Dirithromycin: Multiple-Dose

451

Studies

Table 2 Current Studies: Concentrationsof Dirithromycin (mg/kg or m a )

Sampling times(h) Serum concentrations( m a ) Bronchial mucosaa(mgkg) Bronchial secretions (mg/L)

C

B

A

48

24 0.17 2 6.51 f 1.44b f 0.31

72 f 0.03

6.61 2 0.61 f

0.05 f

5.67 0.84

'Only specimens of > 5 mg. b13.17 13 including discrepant figures (> 30 mgkg) takenbymeans bronchoscopy.

f f

1.02

of fiber-optic

mately similar to those in tissues (0.41-1.04 m&) and increased as a function time after several days of administration (Fig.1). In tonsils, concentrations weremuch higher after two doses 5.01 mgkg) than after a single dose(0.60-1.06 mgkg). Incurrentstudies, concentrations of dirithromycinmeasuredin bronchialmucosaandsecretionswerehigherthan those cited above. Similarly in tonsils, as shown in Fig. 2,2 days after the end of treatment, the mean dinthromycin level was 4.44 mgkg and decreased slowly, persisting at2.85 mgkg at the fifth day after the end the treatment.

1.2

-

1. 0.8 . (n

- 0.6 -

f

Bronchlai secretions

.

0" 0.2

.

Serum A .

+ 0

1

+

1

2

+ 2

3

+ 3 4

+

4

5

5 Time (days)

Application

Figure I Concentrations of dirithromycin in serum and bronchial secretions (250mg multiple doses).

452

Muller-Serieys et al.

8-

7"

T

4 'S -m

t Mean concentrations Lowest C. A Highest C.

g 4.E

8 2" 1-* I

Groups

I

I

I

C

Figure 2 Kinetics of dirithromycin in tonsillar tissue following the last intake.

Pathology analysis camed out in tonsils did not show any significant correlation between the crypt state and dirithromycin concentrations.

DISCUSSION Many convergent figures for dirithromycin tissue concentrations were obtained. Several points should be emphasized:

1. Dirithromycin exhibits excellent tissue distribution in selected tissues where it persists for more than 72 h after the last dose. In these studies sustained concentrations of antibiotic were found in bronchial tissue, bronchial mucosa, bronchial secretions, and tonsils. In all plasma and tissue samples, the measurement of antibiotic activity included that of actual erythromycylamine concentrations. single 750-mg dose wassuperior to a single 500-mg dose 12 h after administration. The concentrations the antibiotic achievedintissuesaftermultipledoses of dirithromycin were higher than after a single dose. 2. slow decrease of tissue concentrations is consistent with the long terminal half-life of 20-50 h calculated for this compound, and persistent tissue levels in tonsils and bronchial mucosa up to 272 h confer upon dirithromycin prolonged in vivo activity after administration.

453

Kinetics of Dirithromycin: Multiple-Dose Studies

"1 PI

'E 15 -

-

I

10C

50-

I

Improvement

Failure

Figure Correlation with clinical outcome: evolution of mean concentration of dirithromycin in bronchialmucosa in relationto clinical efficacy.

Regarding the clinical significance of these high tissue concentrations, two criteria are consistently predictive of the therapeutic efficacy of dirithromycin: (i)the inhibition quotient calculated as the ratio tissue concentrations to the MIC, (minimal inhibitory concentration) of predominant respiratory pathogens; (ii) correlation with clinical outcome, as shown in Fig. 3, is excellent in groups A and B (p = showing a significant correlation with cure and mucosal dirithromycin level.

CONCLUSION In contrast to low plasma concentrations, dirithromycin achieves excellent penetration into tonsils and bronchial tissues. The significant and persistent concentrations that exceed the MIC for most common pathogens in these settings (4), including Streptococcus pyogenes, S. pneumoniae and M o r a ella catarrhalis,could allow a shorter duration of dirithromycin administration in patientswith mild respiratory infections.

REFERENCES 1. Baughman RP, DeSante KA, Lanier TL, Conforti PM, Sides GD. The penetration of dirithromycin into bronchoalveolar lavage fluid and alveolar macrophages. J Antimicrob Chemother

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Bergogne-BCrCzin E, VallCe E. Pharmacokinetics of antibiotics in respiratory tissues and fluids. In: Pennington JE, ed. Respiratory Infections: Diagnosis and Management, 3d ed., New York: Raven Press Ltd, 1994:715-740. 3. Bergogne-BCrCzin E. Tissuedistribution of dirithromycin:comparisonwith erythromycin. J Antimicrob Chemother 1993; 31 (suppl C):77-87. 4. Bauernfeind A. In vitroactivity of dirithromycinincomparisonwithother newandestablishedmacrolides J AntimicrohChemother 1993;31(suppl C):39-49.

A Human Model of Local AbscessUsing Skin Chambers: The Penetrationof Azithromycin and the Chemiluminescence Response of Neutrophils (PMN) K. Takahashi, V. Duchateau, M. Husson, A. M. Bourguignon, and F. Crokaert Universit6 Libre deBruxelles Brussels, Belgium

INTRODUCTION Many investigators have reported significant accumulation of azithromycin (AZ) into humanpolymorphonuclearleukocytes (PMNs) and have discussed its influence on PMN function (1-4). However, most of their experiments were based on circulating PMNs or animal models and did not investigate the relationship between PMN function and AZ penetration at the human local inflammatory site. We assessed this drugPMN relationship with the use of a forearm skin chamber as a model of local inflammation(5,6).

455

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Takahashi etal.

MATERIAL AND METHODS Volunteers Under double-blind control, eight volunteers received500 mg of Az PO for 3 days (days2,3, and 4) andthe other eight volunteers received placebo tablets accordingto thesame schedule.

Skin Chambers Blisters were raisedat two points on each forearm using mild suction (30 cm Hg) for 2 hs. After aspiration of the fluid, the blisters were unroofed and four skin chambers were implanted. The chambers were filled with 1200 p1 of one of the following fluids: phosphate buffered saline (PBS), autologous serum (serum, v/v with PBS), heat-killed S. cerevisiae (yeast, v/v with autologous serum),and heat-killed Haemophilus influenzae (Hae, v/v withautologous serum).The final concentrationof yeast and bacteria in the chamber were 4.5 lo7 yeast/ml and 5.0 lo7 bacteria/ml, respectively. Chamber fluids were collected 18 h after their implantation (T18) for the first sampling.The second was done 4 h (T18+4) after refilling chambers with fresh fluids. The above procedures were performed before (day 1) and after drug administration (day4, eight volunteers asthe short-delay group; day9, eight volunteers asthe long-delay group).

AZ Concentration Azithromycin concentrationwas measured in circulating white blood cells (WBCs), serum, blister fluid, and chamber fluids by bioassay usingSarcina lutea ATCC 9341as the test organism and antibiotic medium No. 11(BBL) as the test agar. WBCs were separated by layering onto the gradient medium NI" and centrifugation.

Chemiluminescence Response Circulating and recruited PMNs (1 X 106/ml) were stimulated by PMA (3 and luminol-enhanced chemiluminescence (CL) was recorded by an automatic computer-driven luminometer (425 nm; Model 1251 LKB-Wallac).

RESULTS The AZ concentrations are summarized in Table 1. The CL response of circulating PMN wasnot influenced by the high accumulation of AZ (Fig. 1). The CL response of recruited PMNs were changed to some degree in

Penetration

AZ and the CL Response of PMNs

80 00

458

Takahashi et al.

II

LONGAZ SHORTAZ

0

i ~

SHORT PLACEBOPLACEBO DRUGGROUP

before administration after admlnlstratlon

*: CL response of D l was set to be 100%.

Figure I Percentage CL response of circulating PMNs after drug administration. CL response of circulating PMNs was not influenced in both azithromycin and placebo group. The CL response of Dl was set at 100%.

the Hae chamber after AZ administration. However, we could not derive any definite trends because similar normal fluctuations were also seen in the placebo group (Fig.2). The same results were obtained from yeast and serum chambers.

DISCUSSION Azithromycin accumulated highly in circulating WBCs and was retained long after serum concentrations had become undetectable. This led to a high accumulation in the chamber fluid, but the actual concentration was always higher than the expected concentration andAZ could be detected even in blister fluid, which did not contain any cells. There might be other factors than PMNs that account for delivery and accumulation of AZ in local inflammatory sites. Glaude and Snider reported that fibroblasts might be an important drug-loading system(7). As Bonnet andVan der Auwera showed for circulating PMNs the presence of AZ in both circulating and recruited PMNs did not influence the CL responseof these cells.

CONCLUSION Azithromycin penetrates well and accumulates in PMNs despite low serum levels. This high accumulation does not influence the CL responseof either circulating or recruited PMNs. It is suggested that good penetration of AZ

Penetration of AZ and the CL Response of PMNs

459

CL T18 before' CL T18+4 before'

m

CL T i 8

after..

CL T18+4 after" *: beforeadministration *:

LONGAZ

SHORT AZ

after administration

PLACEBO

Figure 2 The CL response of recniited PMNs inthe Hae chamber. Although some

changes in CL response wereseen after drug administration, a definitetrend could not be derived.

into PMNs plays a role as a drug delivery system, but other factors might affect accumulation in the local inflammatorysite.

REFERENCES 1. Van der Auwera P, Matsumoto T,Husson M. Intraphagocytic penetration of antibiotics. J AntimicrobChemother 1988; 22:185. 2. Mcdonald PJ, PruulH. Phagocyte uptake and transport of Azithromycin. Eur J Clin Microbiol Infect Dis1991; 10:828. 3. Bonnet M, Van der Auwera P. In vitro and in vivo intraleukocytic accumulation of Azithromycin (CP-62, 993) and its influence on ex vivo leukocyte chemiluminscence. Antimicrob AgentsChemother 1992; 36:1302. 4. Gladue RP, Bright GM, Isaacson RE, Newborg MF. In vitro and in vivo uptake of Azithromycin (CP-62,993) by phagocytic cells: possible mechanism of delivery and release at sites of infection. Antimicrob Agents Chemother 1989; 33:277. 5. Dubertret L, Lebreton C, Touraine R. Neutrophil studiesin psoriasis: in vivo migration, phagocytosis and bacterial killing. J Invest Dermatol 1982; 79:74. 6. Freeman CD, Nightingale CH, Nicolau DP, Belliveau PP, Banevicius MA, Quintiliani R. Intracellular and extracellular penetration of Azithromycin into inflammatory and noninflammatory blister fluid. AntimicrobAgents Chemother 1994; 38:2449. 7. Glaude-RP, Snider ME. Intracellular accumulation of Azithromycin by cultured human fibroblasts. Antimicrob AgentsChemother 1990; 34:1056.

Separation of Presystemic and Post-Absorptive Influences on the Bioavailability of Azithromycin in Cynomolgus Monkeys G. Foulds, A. G. Connolly, J. H. Fortner, and A. M. Fletcher Pfizer, Inc. Groton, Connecticut

INTRODUCTION Both presystemic and postabsorptive factors may limit the bioavailability of orally administered agents. Although presystemic effects might be overcome by specific controlled release oral formulations, postabsorptive effects are usually not amenable to remediation. Therefore, we wanted to differentiatebetweenpresystemic and postabsorptive causes of limited azithromycin bioavailability. In a previous study in monkeys, the mean bioavailability of azithromycin (approximately33% following a lO-mg/kg dose) was similar to mean bioavailabilityof in man(1);therefore, the monkey may be an appropriate model in whichto examine possible causes of limited azithromycin bioavailability in man. Separationof presystemic from postabsorptive effectson bioavailability in cynomolgus monkeys was made by comparing oral bioavailability with the bioavailability following administration directly into the portal vein through an implanted portal vein cannula.

l

460

Bioavailability of Azithromycin in Monkeys

461

METHODS Surgical Implantation of the Portal Vein Cannula Following anesthesia, the monkey was prepared for aseptic surgery. An access port was secured subcutaneouslyon the back between the shoulder blades and the catheter was routed beneath the skin to theabdomen. The vascular accessport catheter was inserted into an isolated mesenteric vein, advanced into the portal vein, and secured. Following routine closure, preand post-operative carewas provided as per accepted veterinary practices. Postanesthetic analgesia was provided as required. The patency of the catheter was determined and maintained at the end of surgery and once weekly thereafter by withdrawal of the contents of the port and catheter (-0.5 mL), flushing with 5 m1 sterile normal saline, and injection of approximately 0.5 m1 sterile heparinized saline solution to fill the port septum and catheter.

Dosing and Sampling of Animals After a recovery period (>2 weeks) following surgery, the animals were administered azithromycin in a three-way crossover pattern. The treatments included anoral dose of 20 mgkg in solution administeredby stomach tube, a portal dose of mgkg given by slow infusion through the portal vascular accessport, and an intravenous doseof mgkg given by 2-min infusion intothe right saphenous vein. Portal infusions were administered over 30-60 min. Following each dose, blood samples were collected from the femoral triangle at times up to 72 h after dosing. Serum was prepared and stored frozen at -70°C until assay for azithromycin.

Assay of Serum Samples Azithromycin serum concentrations were determined by high-performance liquid chromatographyEC utilizing amperometric detection(2). The lower limit of quantification was approximately20 ng/ml.

Calculations The maximum observed serum concentrations(C,,) and the time of C,, (Tmm)were determined by inspection of the data. Area under the serum concentration versus time curve(AUC) was calculated by the trapezoidal method for the interval of predose to the last timeat which allthree modes of administration produced measurable concentrations of azithromycin. Absolute bioavailability was estimated as AUC,,,/AUC&. Bioavailability following intraportal administration was estimated asAUCp,/AUC,.

Foulds et al.

462

RESULTS A N D DISCUSSION The serum pharmacokineticsof azithromycin in cynomolgus monkeys following oral, intraportal, and intravenous administrationare summarized in Table 1. Slow infusion of drug into the portal vein was used in an attempt to mimic slow absorption of drug from the lumen of the intestine into the portal vein following oral administration. Following a 30-min intraportal infusion (monkeyAl),the C, was 1.51 mg/ml, avalue approximately fold C, followingoral administration. Following the l-h intraportal infusion, the mean C, was about 1.4 times (range:0.87-2.6) the mean oral.If the rate of first-pass elimination is linear over the range of concentrations Table I Serum Pharmacokineticsof Azithromycin in Monkeys with Surgically Implanted Portal Vein Cannulas

~~

Oral Administrationof 20 mgkg 3.33' Alb 1.0 0.479 2.69d A2b 1.0 0.804 2.27d

B

2.0

0.282 0.947

C

40.0

36.11.0

Mean 65.3

4.19d 34.7

Portal Administration of 10 mgkg 0.5

1.509

A2 B 56.3 C 3.27d

30

60 60 60 0.75

0.696 0.731 1.462

1.070.5 1.0

2.37d 1.53d

Mean

34.1 54.3

Intravenous Administration of 10 mgkg 0.1

A1 5.076 A2 2.037 B4.401 C5.81d

2 2 2 0.1 2

0.1 0.1

3.36d 4.49d

6.471

Corrected for differences in dose among treatments. "Animal A was used for two series, separated by approximately 6 months. 9-48 h. do-30 h.

56.8 74.2 64.1

Bioavailability

Azithromycin in Monkeys

463

in the portal vein, the failure to mimic peak concentrationsfollowing oral administration is not important. T,, following oral administration in this study was 1-2 h. In a previous study; T,, was between and 1h following oral administrationof 10 mg/kg. Following portal administration, T,, was 0.5-1 h. The results suggest that 1 h was a suitable duration for the portal infusions. However, the apparent extent of delivery to the blood following a 0.5-h intraportal infusion was similarto that observed following a l-h intraportal infusion. The mean absolute bioavailability following oral administration was 34.7%, in good agreement with a previous estimate in cynomolgus monkeys, indicating that the surgery didnot adversely affect bioavailability. Bioavailability followingportal administrationwas 54.3%, indicating that first-pass elimination removed approximately45.7% of the drug that reached the portal blood. The results suggestthat the modest oral bioavailability in the monkey is related in part to postabsorptive effects, such as first-pass excretion or metabolism. The results are consistent with absorption from the intestine of approximately of the dose, followed by firstpass eliminationof approximately 46% of the absorbed dose. This study indicates that low bioavailability in the monkey results in part from postabsorptiveeffects, thereby limiting the potential for remediation with new dosage forms.

ACKNOWLEDGMENTS We acknowledge the assistance of Ms. S. Hawkins, Mr. R. Lariviere, and K. Foss of the Animal Resources Department and Mr.A. Lurding, Ms. R. Destito, and Mr. M. Pol10 of the Drug Metabolism Department.

REFERENCES 1. Foulds G, Shepard R M , Johnson RB. The pharmacokineticsof azithromycin in human serum and tissues. J Antimicrob Chemother 1990; 25 (suppl A):73-82. 2. Shepard RM, Duthu GS, Ferraina RA, Mullins MA. High-performance liquid chromatographic assay with electrochemical detection for azithromycin in serum and tissues. J Chromatog BiomedAppll991; 565321-337.

Clinical Pharmacology of Azithromycin Given at Various Sites Along the Gastrointestinal Tract in Healthy Subjects David R. Luke, G. Foulds, Hylar L. Friedman, and William J. Curatolo Pfizer Central Research Groton, Connecticut

Joseph Scavone Medical and Technical ResearchAssociates Incorporated Boston, Massachusetts

INTRODUCTION

Like other drugs of its class (e.g., erythromycin and clarithromycin), the oral use of the azalide antibiotic azithromycin is associated with mild gastro intestinal side effects-namely, nausea, abdominal cramping, and emesis. The incidence of gastrointestinal side effects is markedly reduced with equivalent dosesof azithromycin when given intravenously (1,2), suggesting that the primary mechanism(s) of the side effects associated withoral dosing is locally mediated. Clearly, systemic exposures after equal doses of intravenous azithromycin far exceed those obtained after oral dosing with few, if any, dose-limiting adverse events.

Site-Spec$c Absorption of Azithromycin

465

Little is known of the disposition of oral azithromycin within the gastrointestinal lumen of man. The bioavailability estimate of approximately 40% suggeststhat azithromycin iseither poorly absorbedor undergoes significant first-pass elimination. An understanding of the absorption characteristics of azithromycin at sites along the gastrointestinal tract may permit development of dosage forms with increased bioavailability and reduced incidence or severity of side effects. The aim of our study was to characterize sitesof azithromycin absorption in man.

METHODS A total of 11 healthy male subjects (mean age, 28 years; age range, 2039 years; mean weight, 75.2 kg; weight range, 64.9-89.4 kg) completed this study. Subjects were determined to be in good health by means of physicalexaminationandstandardclinicallaboratorytests.Theyreceivednoknownprescription or nonprescriptiondrugs for atleast2 weeks prior to participation and throughout the study period. Subjects fasted overnight for 12 h prior to each dose of azithromycin and for 4 h afterward. The following 500-mg azithromycin treatments were assigned in a randomized, balanced, open-label, four-way crossover design: Treatment A: 1mg/ml in 0.9% saline infused intravenously over 1 h Treatment B: 2 X 250-mg capsules orally Treatment C: 10 mg/ml solution directlyto duodenum over5 min Treatment D: 10 mg/ml solution directlyto ileal-cecal junction over 5 min Fourteen daysseparated the treatments. A 2-lumen,4.5-m-longnasoenteric tube was used for thosetreatments requiring nasoenteric infusions. The tube was placed the evening prior to dosing and placement confirmed immediately prior to infusion. Blood samples were collected for azithromycin analysis immediately priorto and 12, 24, 48,72, 96, and 120 h afteroral dosing or the start of infusion on each dosing day. Serum azithromycin concentrations were determined by highperformance liquid chromatography with electrochemical detection at BAS Analytics (West Lafayette, IN). Peak concentration (Cma) and the corresponding time (Tma) were determined by observation of the serum concentration-time profile for each subject. Area under the serum concentration versus time curve (AUC) from predose to 48 h postdose was esti-

Luke et al.

466

mated by trapezoidal summation. Bioavailability(F)was estimated as the ratio of AUC following oral, duodenal, or ileal-cecal route to the AUC following intravenous administration in each subject.

RESULTS The results are summarized in Fig. 1and Table 1.

DISCUSSION Little isknown about the absorption kinetics of azithromycin in man, although the primary site is thought to be limited to thesmall intestine.The

E . 1

u

E

S

5

aE

0.01

0.001

c

I

0

Time

Figure I Meanserumazithromycinconcentrationsfollowinga l-h intravenous infusion oral dosing (2 250-mg capsules) ( +), directly to the duodenum and directly to the ileal-cecal junction (A) of 500-mg azithromycin in 11 healthy male volunteers.

467

Site-Specific Absorption of Azithromycin

Tab& I Azithromycin Pharmacokinetic Parameters After Single 500-mg Doses Given Intravenously over1 h, Orally asTwo 250-mg Capsules, and As Solutions Directly to the Duodenum or Ileal-Cecal Junction in 11 Healthy Male Volunteers Route Intravenous Oral Duodenal Ileal-cecal

Cm,

(Ccg/ml) Wml) AUC Tm, (h)

2.82 f 0.347 f 0.095 0.842 f 0.328' f 0.426

0.8 20.2 1.9 0.9 1.2 21.1 0.7

F 8.14 f 1.77 3.58f 1.22 4.02f 0.96 3.04 f 1.46

0.438 f 0.107 0.499 f0.091 0.367 f 0.123

Note: Data are mean values SD. AUC = area under serum concentration-time curve;C,, = maximum serum concentration; Tmu = time to reach C-; F = absolute bioavailability. ap < .W1when compared with oral routeof administration.

oral bioavailability of azithromycin inthis study following administration of capsules was which is consistent with published data for the oral capsule (43). Azithromycin undergoes acid degradation to the descladinose metabolite, thus accounting for some of loss bioavailability (6). However, the amount of descladinose azithromycin (approximately 13% of the oral dose) is not the major cause for lossof bioavailability (7). Both firstpass metabolism and incomplete absorption likely account for thebioavailability losses(8). The aim of our study was to identify the dominant site of absorption (if one existed), assessthe effect of stomach acidson azithromycin bioavailability, and possibly differentiate the relative contributionsof the small and large bowels in the absorption of azithromycin. From these data, we can assume that azithromycin is equally absorbed throughout the upper gastrointestinal tract with no apparent specificity for any section of the small intestine. WhereasTm, may have beendependent on the routeof administration, the extent of absorption, as measured by the areaunder the serum concentration-time curve, was similar whether the drug was taken orally or directed to the duodenum or ileal-cecal junction. These results suggestthat azithromycin deliveredto theduodenum or ileal-cecal junction is absorbed as well as the drug delivered orally. These results are in contrast to data obtained when azithromycin was administered to the rectum, following which only 3% was bioavailable (9).

REFERENCES 1. Luke DR, Foulds G, Cuddigan FM, Levy B, Cohen SF, ShoupRE. Azithro-

mycin safety, toleration, and pharmacokinetics after intravenous administration. Proceedings of the 35* Interscience Conferenceon Antimicrobial Agents and Chemotherapy, San Francisco,1995.

468

Luke.et al. Luke DR, Foulds G, Friedman HL, Going PC, Scavone J,Curatolo WJ. Sitespecific toleration and absorptionof azithromycin. Proceedingsof the International Conference on the Macrolides, halides, and Streptogramins, Lisbon, Shepard R M , Duthu GS, Ferraina RA, Mullins MA. High-performance liquid chromatographic assay with electrochemical detection for azithromycin in serum and tissues. J Chromatog Biomed Appll991; Coates P, Daniel R, Houston AC, AntrobusJHL, Taylor T. An open study to compare the pharmacokinetics, safety, and tolerability of a multiple-dose regimen of azithromycin in young and elderly volunteers. Eur J Clin Microbiol Infect Dis RB;The pharmacokinetics of azithromycin in Foulds G, Shepard RM, Johnson human serum and tissues. J Antimicrob Chemother (suppl Fiese EF, Steffen SH. Comparison of the acid stability of azithromycin and erythromycin. J Antimicrob Chemother Luke DR, Foulds G, Going P, Connolly A.Oral absorption profile and disposition of azithromycin in ileostomy subjects. Proceedings of the International Conference on the Macrolides, halides, and Streptogramins, Lisbon, Foulds G, Connolly AG, Fortner JH, Fletcher AM. Separation of presystemic andpost-absorptiveinfluences on the bioavailability of azithromycinin Cynomolgus monkeys. Proceedings of the International Conference on the Macrolides, Azalides, and Streptogramins, Lisbon, Luke DR, Going PC, Foulds G, Melnik G. Bioavailability of azithromycin when delivered as a rectal solutionto healthy subjects. Proceedingsof the International Conference on the Macrolides, halides, and Streptogramins, Lisbon,

Effect of Food and Formulationon Bioavailability of Azithromycin G. Foulds, David R. Luke, S. A. Willavize, William J. Curatolo, Hylar L. Friedman, M. J. Gardner, R. A. Hansen, R. Teng, and J. Vincent Pfzer Central Research Groton, Connecticut

INTRODUCTION Based on a studywith a research capsule formulation of azithromycin, the labeling for azithromycin capsules reads “ZITHROMAX (azithromycin) should be given at least 1 hour before or 2 hours after a meal.” We now report studies of the influence of high-fat breakfasts on the bioavailability of azithromycin fromtablets, from a suspension, and from a sachet. These studies indicate that decreased bioavailability of azithromycin in the fed state is avoidedby the use of these formulations.

METHODS All protocols were reviewed and approvedby institutional review boards. All studies were open, randomized, crossover designs in normal volunteers. In all studies, azithromycin was given following an overnight fast and following a high-fat breakfast. The standard high-fat breakfast usually consisted of two eggs fried in butter, two strips of bacon, 6 oz. of hashbrown 469

Foul& et al.

470

potatoes, two piecesof toast with 2 teaspoonsof butter, and 8 of whole milk, ingested within a 20-min period. This meal contains at least50 g of fat. Azithromycin was takenimmediatelyfollowingcompletionof the meal. Fasted and nonfasted volunteers were allowed astandard meal 4 h following the doses. One studyalsoexamined the influence of alight breakfast of cereal (Cheerios@) and milk on the bioavailability of the powder for oral suspension formulation (POS) utilized for administration of azithromycin to children. Following each dose regimen, blood was obtainedfor preparation of serum, which was stored at -70°C until assay for azithromycin by highperformanceliquidchromatographywithelectrochemicaldetection (1). Assays for all studies exceptthe research capsule study were performedat BAS Analytics (West Lafayette, IN). The dynamic range was 0.010 mg/L to either 1.01 mg/L or 1.21 mgL. Validity of standard curveswas assessed by assay of quality control samples, run in duplicate at of each three concentrations during the assays of samples from the studies. In all assay runs from which data were utilized, at least five of six quality control samples fell within 15% of nominal concentrations. The parameters of maximum observed serum concentration (C,,) and the area under the serum concentration-time curve(AUC) were utilizedfor examination of the effect of food on bioavailability. Terminal-phase rate constants were not calculated because the duration of concentrations above the lower limit of quantification of the assay duringthe terminal phaseof the serum concentration curves was too short, relative to the 3-day terminal halflife of azithromycin (2,3), to permit calculation. AUCs following high-fat breakfasts were compared with those following the overnight fastby means of an analysis of variance (ANOVA) of the natural-log transform of the AUCs. Following verificationthat there were no sequence or period effects, treatment effects were examined. Means 90% and confidence boundson the ratio of AUC(fed)/AUC(fasted) were estimated. Similar calculations were performed for C,=.

RESULTS AND DISCUSSION The results are summarized in Fig.1and Table 1. Following administrationof the research capsule andthe commercial capsule formulations with a meal, substantial decreasesin AUC and C, were observed. Following administration of two 250-mg tablets or two 600mg tablets with a meal, mean valuesof AUC and C,, were little changed. The mean AUC ratios were97% and 104%for 250-mg and 600-mg tablets, respectively. Following administration of the POS or sachet formulations, mean values of AUC were little changed. However, mean values of C,

Food Formulation and

;_I I

of Azithromycin Bioavailability

"_"

"""""""""""

-.'0

"

~

""-

_"" " " "

471

"_ "_

1"_

""

Mean (290% confidence limits) relative AUC and C,, for azithromycin when administered with and without a high-fat meal to normal volunteers [BCereal (Cheerios@) and milk.]

Figrue I

were increased with mean ratiosof 146% following administration of the the l-g sachet with a high-fat meal and 161% following administrationof the POS formulation with a high-fat meal. Administration of the POS formulation with a light breakfastof cereal and milk did not affect bioavailability (mean AUC ratio 104%) but decreased C,, (mean ratio 81%). The cause of the difference in the effect of food on bioavailability of azithromycin capsules, relativeto the lack of effect on tablets and suspensions, is unknown. Differences in dissolution rates may play a role. Delivery of different amountsof azithromycin to different sites within the gastrointestinal tract isunlikely to play arolebecause the bioavailability of azithromycin is only slightly affected by delivery to various sites withinthe small bowel .(4,5). A direct effect of the azithromycin within the capsules on the capsule shells, causing incomplete dissolution, is unlikely because the absolute bioavailabilityof azithromycin capsules is similar to that of the other formulations. Because the delivery of azithromycin to abscesses i s related to the total dose administered and the not frequency of dosing (6) and AUC/MIC (minimal inhibitory concentration), not time aboveMIC, is correlated withthe

Foulds et al.

472 Table I

Summary of FedIFasting Studies of Azithromycin in Humans No. of

(90%

Capsule (research)

11

55% (42%-68%)

22% (6%-84%)

Capsule

11

69% (53%-89%)

(67%-96%)

113% (88%-l

46%)

97% (82%-113%)

131% 03%-166%)

1 04% (81%-135%)

(commercial) Tablet

12

(250mg) Tablet (600mg)

POS

POS

12 (1 6

103% (74%-147%)

100% (79%-128%)

6

81YO (59?'0-117%)

104% (82%-133%)

28

(40mg/ml) Sachet

80%

(1 12

l61Yo 34%-194%)

146% (1 16%-1859!0)

(1

1 13% 03%-124%)

1 12% (99%-127%)

efficacy of azithromycin in treatment of infection in neutropenic animals AUC is the relevant parameter for examination of the effect of food on the bioavailability of azithromycin. Changes inC,, are unimportant. Only when azithromycin was administered in capsules did a meal a significant decrease in bioavailability. The bioavailabilitiesof azithromycin 250-mg tablets, 600-mg tablets, 40-mg/ml suspension, and 1000-mg sachet were not significantly decreased by administration immediately following a high-fat breakfast. Thus, these tablet, sachet, and suspension formulations of azithromycin may be administered without regardto meals, further enhancing the convenience of the once-per-day dosage regimens.

REFERENCES 1. Shepard RM, Duthu GS, Ferraina RA, Mullins MA. High-performance liq-

uid chromatographic assay with electrochemical detection for azithromycin in serum and tissues.J Chromatogr Biomed Appl1991;

Food Formulation and 2. 3. 4. 5.

6. 7.

of Azithromycin Bioavailability

473

Gardner MJ, Ronfeld RA. Interpretatiodcharacterizationof the pharmacokinetics of azithromycin in man. Proceedings of the 8th Mediterranean Congress of Chemotherapy, Athens, 1992; abstr Luke DR, Foulds G, Levy B, Going PC. Toleration of increasing concentrations of intravenous azithromycin infusates in healthy subjects. Proceedings of the 3rd ICMAS, Lisbon, 1996;abstr 4.20. Luke DR, Foulds G, Scavone J, Friedman H, Cuddigan M. Clinical pharmacology of azithromycin givenat various sites alongthe gastrointestinal tract in healthy subjects. Proceedings of the 3rd ICMAS, Lisbon, 1996; abstr 4.21. Luke DR, Foulds G, Friedman H, Going PC, Scavone J, Curatolo WJ. Sitespecific toleration and absorption of azithromycin in healthy subjects. Proceedings of the 3rd ICMAS, Lisbon, 1996; abstr 4.22. Girard D, Bergeron JM, Milisen W B , Retsema JA. Comparison of azithromycin, roxithromycin, and cephalexin penetration kinetics in early and mature abscesses. J Antimicrob Chemother 1993; 31 (suppl. E):17-28. Craig W. Postantibiotic effects and dosing of macrolides, azalides andstreptogramins. Proceedings.of the 3rd ICMAS, Lisbon, 1996; plenary session abst 3.

Rectal Azithromycin in Healthy Subjects David

Luke, G . Foulds, and PadenC. Going Pfizer Central Research Croton, Connecticut

Melnik and Valerie Lawrence University of Texas Health Science Center San Antonio, Texas

INTRODUCTION Azithromycin is an azalide antibiotic, called becauseof the addition of a nitrogen atom to the macrolide aglycone ring (1,2). Azithromycin has been approved for most community-acquired infections, including infections involving the upper and lower respiratory tract and skin and skin structure, and some sexually transmitted diseases. The oral bioavailability of azithromycin is 37% after a 500-mg oral dose in healthy subjects, with a peak serum concentration of 0.4 pg/ml at approximately 2 h after dosing (3). In a recent crossover studyin normal volunteers, 500mg of azithromycin was givenintravenously over1h, orally as two 250-mg capsules and as a solution directlyinto the middle portion of the duodenum and to the ileal-cecal junction (4). The mean bioavailability of oral, duodenal, and ileal-cecal and administrations were 4 4% (range: 29-67%), 50% (range: 37-66%), and 37% (range: 18-54%), respectively. The differences were relatively small and of no apparent clinical significance. The aim of our

Rectal Azithromycin

475

study was to.assess the absorption of azithromycin when administered as a solution directlyinto the rectum of normal volunteers.

MATERIALS AND METHODS

Six healthy male volunteers with no evidence of clinically active gastrointestinal disease, documented allergic reactionsto macrolides or cardiovascular, hepatic, or neurological diseases were enrolled in the study. Subjects were determined to be in good health by means of physical examination and clinical laboratory tests. Subjects were restricted from all standard prescription and nonprescription drugs for 2 weeks priorto participation and throughoutthe study period. Subjects also abstained from alcohol and caffeine-containing products for 3 days prior and throughout the study. All subjects provided informed consent before participating the in study. Subjects were randomly assigned to either a 500-mg azithromycin solution administeredby intravenous infusion over60 min or intrarectally (12.5 m1 of a 40-mglml solution) over 5 min. After a2-weekwashout period, each subjectwas crossed-overto the alternate route of administration. Vital signs, assessmentsof side effects, and laboratory safety parameters were determined throughoutthe two study periods. Blood samples for preparation of serum were collected predose and 0.25,0.5,0.75,1,2,3,4, 6,8,12,18, and 24 h and2,3,4, and 5 days following the beginning of the infusion or rectal dose. Serum concentrationsof azithromycin were determined by high-performance liquid chromatography with electrochemical detection at BAS Analytics (West Lafayette, IN) (5). Pharmacokinetics were assessedby noncompartmental methods.Maximum serum concentrations of azithromycin (C,,) and corresponding times(T-) were obtained visually from serum concentration-time plots. The area under the serum concentration-time curve (AUC) was calculated using trapezoidal summation. The absolute bioavailability of azithromycin given rectally was estimated by the ratio of AUC after rectal administration to the AUC after intravenous administration. RESULTS Mean serum concentration-time profiles of azithromycin given intravenously and rectally are depicted in Fig. 1. Azithromycin when administered intravenously produced a geometric meanC, value of 3.993 pglml and a geometric meanA U G t value of 10.009 p g h/ml. Rectal administration of azithromycin resulted in a geometric meanC, value of 0.111 m1 and a geometric meanAUC,,, value of 0.310 pg h/ml. Thus, the mean bioavailabilityforrectaladministrationwasestimated at 3.2%(range:

476

Luke et al. 10 l 0.1

T

T

0.01

T

0.001

I

Figure Z Mean serum concentrations of 500 mgof azithromycin when administered intravenously or rectally (0)to normal volunteers(N= 6).

0.2-19.4%). Both rectal and intravenous administrationsof azithromycin were well tolerated.

I

DISCUSSION Azithromycin absorption appears to be nonselective inthe upper gastrointestinal tract. Equal amounts of azithromycin given to various sites of the upper gastrointestinal site resulted in comparable absorption of 35-45%. The present study assessedthe absorption of azithromycin when delivered to the lowergastrointestinal tract. Similar to other xenobiotics (649, azithromycin was poorly absorbed the in lower gastrointestinaltract with a mean bioavailability of approximately In contrast to nonspecific, yet incomplete absorptionin the upper gastrointestinaltract, spanning an area from the duodenum to the ileal-cecaljunction,azithromycindoesnot appear to be absorbed from rectal mucosa.

CONCLUSION The absolute bioavailabilityof azithromycin solution administeredinto the rectum was highly variable and low. This may due be to limited absorption from the rectum, the delivery medium,andor the inherent chemical properties of azithromycin.

REFERENCES 1. Bright GM, Nafel AA, Bordner J, Desai HA, Dibring JN, Nowakowska J, et al. Synthesis, in vitro and in vivo activity of novel 9-deoxo-9a-aza-9a homo-

Rectal Azithromycin

2. 3. 4. 5.

6. 7. 8.

477

erythromycin A derivatives: a new class of macrolide antibiotics,the azalides. J Antibiot 1988;41:1029-1047. Rodvold KA, Piscitelli SC.New oral macrolide and fluoroquinolone antibiotics: an overview of pharmacokinetics, interactions, and safety. Clin Infect Dis 1993; 17:S192-S199. Foulds G , Shepard RM, Johnson RB. The pharmacokinetics of azithromycin in human serum and tissues.J Antimicrob Chemother 1990;25:73-82. Luke DR, Foulds G , Scavone J, FriedmanHL, Curatolo WJ. Oral absorption of azithromycin.Proceedings of the 3rd International Conference on the Macrolides, halides, and Streptogramins, Lisbon,1996. Shepard RM, Duthu GS, Ferraina RA, Mullins MA. High-performance liquid chromatographic assay with electrochemical detection for azithromycin in serum and tissues.J Chromatog Biomed Appll991; 565:321-337. Appelbaum SJ, Mayersohn M, Dorr RT, Pemer D. Allopurinol kinetics and bioavailability. Cancer ChemotherPharmacoll982; 8:93-98. Magnussen I, Oxlund H E ,Alsbirk KE, Arnold E. Absorption of diazepam inmanfollowingrectaland parenteral administration.ActaPharmacol Toxicol 1979; 45: 87-90. Jonsson, T, Christensen CB, Jordening H, Frolund C. The bioavailability of rectally administered morphine. Pharmacol Toxicol 1988; 62:203-205.

Comparative Pharmacodynamicsof Clarithromycin and Azithromycin Against Respiratory Tract Pathogens A. Bauernfeind,

Jungwirth, and E. Eberlein

Max yon Pettenkofer-Znstitut Munich, Germany

INTRODUCTION The newer macrolides demonstrate only minor differences their in activities in comparison with erythromycin (1). However, they differ from erythromycin withrespect to the incidenceof unwanted side effects (e.g., gastrointestinal) and in their pharmacokinetics. Elimination half-lives of many ofthe new macrolidesare prolonged in comparison with erythromycin, (e.g., for clarithromycin to 3.5-4.9 handforazithromycin to about 40 h) (2). Clarithromycin reaches higher values for maximum concentration (C,,,=) [2.1 mg/L after 500 mg, 0.94 mg/L after mg, for 14hydroxyclarithromycin and 0.46, respectively (3)] in comparison with azithromycin mg/L (4) to 0.4 mgL ( 5 ) after 500 mg). Therefore, azithromycin and clarithromycin represent two rather contrary pharmacokinetic profiles within the same classof antibiotics. We investigated the pharmacodynamicsof azithromycinand clarithromycin during exposure of bacterial cultures to concentrations of azithromycin and clarithromycin which follow their concentration gradients in human sera. Data from these experimentsallow an answer to thequestion 478

Pharmacodynamics

CLA and AZZ

479

of whether pathogens exposed to these gradients of concentrations are killed to a measurable extent when they enter the bloodstream. In addition, the risk for the selection of mutants with reduced susceptibility during exposure to macrolides with different pharmacokinetic profiles was compared.

MATERIAL AND METHODS Strains Clinical isolates of hemolytic streptococci group A and B, Streptococcus pneumoniae, Staphylococcusaureus,Haemophilus influenzae, and Moraxella catarrhalis were collected. Representative strains of these species with variable susceptibilityto clarithromycin or azithromycin as judgedby their minimal inhibitory concentrations(MIC,, or MI&) (Table 1) as well as paired strains with identical MICs for clarithromycin and azithromycin were selected.

Pharmacodynamic Model For investigation of the pharmacodynamics of clarithromycin and azithromycin, the bacterial cultures were exposedto a concentration gradient of the compounds which simulated human serum pharmacokinetics as described previously (6). The bactericidal kinetics were followed by counting the number of survivors before each change of concentration over a period of 26 h (12 h for S. pneumoniae). Serum pharmacokinetics published by others were selected(3,4).

Sensitivity Testing Minimal inhibitory concentrations were determined technique as described (1).

by an agar dilution

Antibiotics Antibiotics used were clarithromycin(Abbott, Wiesbaden, Germany) and azithromycin (Pfizer, Karlsruhe, Germany).

RESULTS Antibacterial Activity Killingofhumanrespiratorytractpathogenswasachievedbothwith clarithromycin and azithromycin (Table 2, Fig. 1). The rate and extent of killing is dependent on the concentration of the macrolide reached in hu-

Bauernfeind et al.

480

x-x

-

mg oid

mg/l

mg mg

mg/l mg/l

A -A A-A

-U:

\ la-\ 1

l o "

-

l@

\A

>

\A I 2

I

I

I

la

26

hours

I Bactericidal kinetics of Streptococcus pneumoniae exposed to variable concentrations following the serum pharmacokineticsof macrolides.

Pharmacodynamics of CLA and AZZ Table l

481

MICs of Macrolides

MIc ( m a ) Species (n) S. aureus S. pneumoniae

PEN-S S. pyogenes S. agafactiae

H.injhenrae AMP-S M.catarrhalis

Compound

MI%

Range

Azithromycin Clarithromycin Azithromycin Clarithromycin Azithromycin Clarithromycin Azithromycin Clarithromycin Azithromycin Clarithromycin Azithromycin Clarithromycin

0.25 0.06

0.06

0.5

man serum (C-) and its relation to the MIC of the respective pathogen (Table 1).Azithromycin kills a lower proportion of the initial population in comparison with clarithromycin (Table 2). Regrowth of the cultures after reduction of the initial inoculum[lo6CFU (colony-forming units) per milliliter] was observed during exposureto azithromycin (Table 2).

Reduction of Susceptibility to Macrolides An increase in MICs(8-16 times) was observed whenbroth cultures of all species included were exposed to a gradientof azithromycin concentrations following its serum pharmacokinetics (Table 2). Under identical conditions, no reduction in sensitivity occurred the in presence of clarithromycin. The selection of resistant mutantsby azithromycin was restricted to strains with MICs below 1m&. Strains with an MIC of 1mgL prior to exposure to azithromycin (e.g., S. aureus, H.infzuenzae) did not further increase their MIC inthe presence of azithromycin (Table 2).

DISCUSSION Serum concentrationsof antimicrobials have been regarded as a major and rational parameter for cutoff points and therapeutic perspectives. Evidence has accumulated for therapeutic efficacy of compounds found intracellulary not at all or only at very low concentrations(e.g.,P-lactams, aminoglycosides). Whether drugs with high intracellular but very low extracellular absolute concentrations (proportions may be misleading) are

et

482

Bauernfeind

al.

Table 2 Kinetics of Killing of Respiratory Tract Pathogens by Macrolides

Dosage

MIC (m@)

Minimum number achievable after time exposure of CmJ/ml

CFU/ml Organism h Compound End Initial (mg) hem. Streptococci hem. Streptococci hem. Streptococci S. pneumoniae S. pneumoniae S. aureus S. aureus S. aureus S. aureus H . influenzae H . infruenzae M.catarrhalis M.catarrhalis

26 h

CLA AZI AZI CLA AZI CLA CLA AZI AZI CLA AZI CLA AZI

2 1 .l 2X 1 1 1 1 1 1 1 1 1

250

500 250

500

500

0.03 0.06 0.13 0.06 0.13 0.25 1 0.25 1 1 1 0.13 0.03

0.03 1 2 0.06 1 0.25 1 2 1 1 1

110 2.0 lo4 16 1.0 I d 5.8 16 6.0 10 4.2 104 8.0 16 1.5 lo6 2.5 io4 1.3 lo6 510 4.7 Id

510 84.4 106 68.0 lo' 12 2.0 102, 64.6 lo6" 26. 6.0 26 4.2 104 62.7 lo8 2. 7.2 lo8 18 3.0 io4 28.0 lo8 24 110 108.4 104

'After 12 h.

safe in infections in which pathogens do not obligatorily reside only intracellularly remains a major issue. Although the macrolides show similar chemical structure (7), they are very dissimilar in their pharmacokinetic profiles. For some of them, the proportion between intracellular and extracellular concentration is much higher [e.g., azithromycin (2,8)] compared with others [e.g, roxithromycin (2)]. However, the therapeutic indication of none of the compounds is restricted to infections caused only by obligatory intracellular pathogens. In a pharmacodynamic model, both azithromycin and clarithromycin (excluding its 14-hydroxy metabolite) demonstrate bactericidal activity at concentrations reached in human blood at recommended dosages (azithromycin 500 mg qd, clarithromycin250 mgbid). For clarithromycin, boththe rate and the extent of killing are higher than for azithromycin. In both aspects, the clarithromycin 500-mgqddosingis superior to the clarithromycin 250-mg bid dosing. Mutants with between 8 and 16times reduced susceptibility in compari son withthe initial strain were selected during exposure to azithromycin(not to clarithromycin) with all strains with an initial MIC10.25 m a . No mu-

Pharmacodynamics of CLA and

483

tants with increased MICs were selected from strains with higher initial MICs (e.g., H . influenzae and S. aureus with MIC 1mg/L). These populations are able to proliferate unimpaired as their MICs are above the concentration of azithromycin achievable in the serum. Therefore, there is no selective advantage for mutants with increased MICs. Consequently, no selective enrichmentof mutants is observed.The selection of mutants with reduced susceptibilityto macrolides is not a phenomenon restricted to the in vitro model but has been observed during therapy of infections caused [e.g., by H . influenzue Helicobacter pylori (lo), Streptococcus pyogenes (11,12), Streptococcuspyogenes (11,12), and S. pneumoniue (13).

CONCLUSION The therapeutically predictive impact of these in vitro data is difficult to interpret; however, the findings might be relevant in cases where pathogens enter the bloodstream of patients with impaired defenses (neutropenia) in which the postulatedantibioticdelivery for internalizeddrugs via the phagocytic system becomes less effective.

REFlERENCES 1. Bauernfeind A. In vitro activity of dirithromycin in comparison with other new and established macrolides.J Antimicrob Chemother 1993; 31 (suppl C): 39-49. 2. Sorgel F, Kinzig M, Naber KG. Physiological disposition of macrolides. In: Bryskier A, Butzler JP, Neu HC, Thlkens PM, eds. Macrolides-Chemistry, pharmacology and Clinical Uses. Pans: 1993:421-435. 3. Kees F, Wellenhofer M, Grobecker H. Serum and cellular pharmacokinetics of Clarithromycin 500 mg q.d. and250 mg b.i.d. involunteers. Infection 1993; 23:168-172. 4. Mazzei T, Surrenti C, Novelli A, Crisp0 A, Fallani S, Carla V, Surrenti E, Petri P. Pharmacokinetics of azithromycin in patients with impaired hepatic function. J Antimicrob Chemother 1993; 31 (suppl. E):57-63. 5. Foulds G, Shepard RM, Johnson RB.The pharmacokineticsof azithromycinin human serum and tissues.JAntimicrob Chemother 1990; 25(suppl. A):73-82. Bauernfeind A, Jungwirth R,Petermuller C. Simultaneous simulationof the serum profilesof two antibiotics and analysis of the combined effect against a culture of Pseudomonas aeruginosa. Chemotherapy(Basel) 1982;28:334-340. 7. Kirst HA, Sides GD. New directions for macrolideantibiotics: structural modifications and in vitro activity. Antimicrob Agents Chemother 1989; 33: 1413-1418. Lode H, Boeckh M, SchabergT. Human pharmacokineticsof macrolide antibiotics.Bryskier A, Butzler JP, Neu HC, ThlkensPM, eds. Macrolides-

484

9. 10. 11. 12. 13.

Bauemfeind et al. Chemktry, Pharmacology and Clinical Uses.Paris: Arnette Blackwell, 1993: 409-420. Davies BI, Maesen FPV, Gubbelmans R. Azithromycin (CP-62,993) in acute exacerbations of chronic bronchitis: an open clinical,microbiological and pharmacokinetic study. J Antimicrob Chemother1989; 23:743-751. Glupczynski Y, Burette A. Failure of azithromycin to eradicate Campylobacter pylon from the stomach becauseof acquired resistance duringtreatment. A m J Gastroenteroll990; 8598-99. Seppala H , Nissinen A, Jarvinen H , et al. Resistance to erythromycin in group A streptococci.N Engl J Med 1992;326:292-297. Seppala H , Klaukka T,Lehtonen R, et al. Outpatient use of erythromycin: link to increased erythromycin resistance in group A streptococci. Clin Znfect D~.s1995; 21~1378-1385. Shah PM, Bryskier A. Epidemiology of tesistance to macrolide antibiotics. Bryskier A, Butzler JP, Neu HC, Tulkens PM, eds. Macrolides-Chemistry, Pharmacology and Clinical Uses.Paris: Arnette Blackwell, 1993:143-166.

VI HELZCOBACTER PYLON, CAMPYLOBACTER

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Comparison of Clarithromycin Efficacy in Ferrets and Humans for Treatment of Helicobacter-Induced Gastritis P. Ewing, J. D. Alder, M. J. Mitten, A. Conway, A. Oleksijew, K. Jarvis, L. Paige, and S. K. Tanaka Abbott Laboratories Abboft Park,Illinois

INTRODUCTION Helicobacter pylon infection of the gastric mucosa causes chronic gastritis and peptic ulcer diseasein humans (1).Eradication of H. pylori produces more rapid healing of ulcers and a decrease in ulcer recurrence. Clinical trials indicate that Helicobacter-associated ulcers can be successfully treated with combination therapy of antibacterial agents and proton pump inhibitors such as omeprazole. Animal modelsof Helicobacter-induced gastritisare useful for evaluating efficacy of both experimentaltherapeutic agents and modificationsof established therapeutic combinations. Ferrets have anatural infection with Helicobacter rnustelae, which results in gastric.pathology similar to that of humans infected with H. pylori (2). The purpose of this investigation was twofold: (1) to determine efficacy of clarithromycin andother antibacterial therapiesin a ferret model of ,

487

488

Ewing et al.

Helicobacter-induced gastritis and (2) to compare the efficacy to results achieved clinically in humans.

MATERIALS AND METHODS In Vitro Tests Minimal inhibitory concentration(MC) values were determinedfor clarithromycin, amoxicillin, omeprazole, and metronidazole against H.pylori and H. mustelae by agar dilution method (3).

In Vivo Evaluations Ferrets were inoculated on study day 0 with 107-CFUs(colony-forming units) of H.mustelae ATCC 43773 by oral gavage. Therapy was initiated 21 days after inoculation. Antibiotics were administered once daily for 14 days (days 21-34) and omeprazolewas administered once daily for 28 days (days 21-48). To determine gastric H.mustelae burden during the trials, gastric mucosal biopsy specimens weretaken from anesthetizedferrets on day 35 (end of antibacterial therapy) or day 49 (end of omeprazole therapy). On day ferrets were sacrificed and sections of pylorus and fundus were excised for determination of H. mustelae burden and histopathologic evaluation.Hematoxylinandeosin(H&E)-stainedsections of pylorus and fundus were graded 0-5 for acute inflammation (polymorphonuclear cell density) and chronic inflammation (mononuclear cell density) according to criteria defined by the modified Whitehead scale. Warthin Starry (WS)stained sectionsof stomach were graded0-5 for Helicobacter density using the modified Whitehead scale. The following trials were conducted: Comparison of monotherapy with 1. Double Versus Triple Therapy: omeprazole to double therapy with clarithromycidomeprazole (C/O; clarithromycin days 21-34, omeprazole days 21-48)or triple therapy with amoxicillidmetronidazole/omeprazole amoxicillidmetronidazoledays 21-34, omeprazole days21-48) 2. Length of Therapy Trial: Comparison of 1week clarithromycinl4 weeks omeprazole therapy to 2weeks clarithromycid4 weeks omeprazole therapy Triple Therapy Trial: Comparison of triple therapy with clarithromycin/metronidazole/omeprazole( W O ; clarithromycidmetronidazole days 21-34, omeprazole days 21-48) to monotherapy with omeprazole (days21-48)

of Gastritis

Clarithromycin Treatment Efficacy in

489

4. PharmacokineticTrial: Single-dose and multiple-dose pharmacokinetic analyses

RESULTS Minimum Inhibitory Concentration The MIC of clarithromycin againstH . mwtelae was 0.015 pg/ml, which was twofold to fourfold lowerthan the MIC values against H . pylori pg/ml). The MIC value of amoxicillin against H . mwtelae was 0.5 pg/ml, which was one to two logs higher than the MIC value range against H . pylori pg/ml). MIC valuesfor metronidazole and omeprazole against H . mustelae were 4 and pg/ml, respectively, compared to MIC values of 1-4 and pg/ml, respectively, againstH . pylori.

Double Versus Triple Therapy Data aresummarized in Table1. Table Z Helicobacter Burden and Histopathology Scoresof Gastric Samples from Ferrets Treated with AntibacteriaYOmeprazole Combinations

Mean log Helicobacter & SD (No. positive1total)’ Mean histopathology scores, Group

Day 35 biopsy

0.00 f 0.W Clari/omec Fundus (014) (014) h o d m e t / 0.49 2 0 . 6 9 omee v41 Omef 3.35 -C 0.47 (414) 5.49 Untreated 3.33 -C 0.73 Fundus (414) (414)

Day 63 pylorus 0.00

0.W

day 63b Region Pylorus

MNC

1.0

1.0 1.0 2.5

0

2.5 2.0

4.8 4.0

0.5

4.43 f 1.74e Pylorus Fundus 3.5 1.5 (414) 5.90 f 0.24 Pylorus Fundus (414) 2 0.73 Pylorus

Helico

PMN

3.8 1.0 3.8 1.0 3.8 4.0 4.0 1.3

0 5.0

5.0

1.8

‘Log counts per 1 l-mm biopsyor per 2 2-mm section of stomach. bHistopathologyscores based on modified Whiteheadscale (0-5). CCladome = clarithromycin 50 mgkg days 21-34, omeprazole 10 mglkg days 21-48. dAmox/met/ome = amoxicillidmetronidazoleSot75 rngikg days 21-34, omeprazole 10 mgikg days 21-48. CSignificantly different (p S .OS) than untreated controls. fOme = omeprazole 10 mglkg days21-48.

Ewing et al.

490

Table 2 Helicobacter Burden and HistopathologyScores of Gastric Samples from Ferrets Treated with ClarithromycidOmeprazole Combinations

Mean log Helicobacter 2 SD (No. positive/total)” Mean histopathology scores, Day 49 GroupMNCPMN Region pylorus biopsy Clari/omec (214weeks) Cladornee (1/4weeks) Omef (4weeks)

Day 63

day 63b ,

‘Helico

1.21 2 1.86d 2.85 2 3.0Sd . (418) 1.93 .+ 2.24d 4.70 2 2.24d . (4/8) 5.13 2 0.30 6.40 0.52 I

1.9 1.3 2.3 Pylorus Fundus 1.6 1.0 1.0 3.3 2.3.2.8 Pylorus Fundus 3.4 4;3 Pylorus‘. Fundus 2.8 2.8 1.3

.

1.0 2.0 4.5’

.Log counts per 1 X l-mm biopsy or per 2 X %mm section of stomach. bHistopathologyscores based on modified Whitehead scale (0-5). CClari/ome (U4 weeks) = clhrithromycin 50 mgkg days 21-34, omeprazole 10 mgkg days 21-48. dSignificantly different(p .05) than omeprazole monotherapy. Cladome (U4 weeks) = clarithromycin 50 mgkg days 21-27, omeprazole 10 mgkg days 21-48. [Ome = omeprazole 10 mgilcgdays 21-48.

Length of Therapy Trial Data aresummarized in Table 2.

Triple Therapy Trial Data aresummarized in Table 3.

Recovery of Resistant Isolates from Length of Therapy Trial The MIC values for clarithromycin against the H.mustelae ATCC strain used for inoculation and isolates recovered from ferrets prior to treatment (day 21) were similar (0.008 pg/ml). After therapy (day 63),three of four ferrets with recoverable H . mustelae yielded isolates with an increase in clarithromycinMICvalues to 32 pg/ml.MICvalues for amoxicillin, metronidazole, and omeprazole remained unchanged.

Pharmacokinetic Analyses Following four daily oral dosesof clarithromycirdomeprazole(50/10 mgkg per day), the 24-h plasma C, and C, levels of clarithromycin were 5.90

of Gasfrifis

Clarithromycin Treatment EfJicacy in

491

Table Helicobacter Burden and Histopathology Scoresof Gastric Samples from Ferrets Treatedwith Clarithromycin/Metronidazole/OmeprazoleCombinations

Mean log Helicobacter f SD (No. positive/total)8 . Day 35

Day 49

Mean histopathology scores, day 63b

Day 63

PMN Region pylorus biopsy biopsy Group

MNC Helico

2.52 f 1.78d 4.70 2 2.22 Pylorus ClarVmetl 0.60 f Fundus 2.5 1.5 omec (3/12) (10112) (9112) 3.2 Omee 4.10 f 0.52 4.19 2 0.42 5.58 f 0.82 Pylorus Fundus (1112) (1112) (12/12) '

3.7 3.4 0.6 3.7 0.9 3.3 2.1

'

3.6 4.1

.Log counts per 1 l-mm biopsy or per 2 2-mm section of stomach. bHistopathology scores based on modified Whiteheadscale (0-5). CClari/met/ome = clarithromycidmetronidazole 50/75 mglkg days 21-34, omeprazole 10 mglkg days 21-48. dsignificantly different(p .OS) than omeprazole monotherapy. eOme = omeprazole mgkg days 21-48.

and 2.35 pglml plasma, respectively. Thearea under the curve (AUC,,+,) was 112.89 pg h/ml. Following a single 50-mglkg oral dose of clarithromycin given with10 mg/kg of omeprazole, the 24-h plasma C, and C, of clarithromycin were 8.01 and 1.62 pglml, respectively. The A U G wh was 83.66 pg Wml.

DISCUSSION The C/O therapy was more effectivethan for treatment of Helicobacter mustelae-induced gastritis. Amoxicillin-based therapy may not be effective inthe ferret model due to the relatively high MIC value (0.5 ml) againstH . mustelae. Two weeks of clarithromycin therapywas more effectivethan 1week of therapy. Triple therapy with clarithromycin was more effective than omeprazole monotherapy at decreasing Helicobacterburden. Successful treatment of Helicobacter-induced gastritis with clarithromycin was more difficultto achieve in ferrets than in humans. Eradication rates were generally lower and more variable (25-100%) ferrets in than in humans, even though plasma concentrations of clarithromycin in ferrets exceeded levels achieved in humans. The ferret model may best serve to compare relative efficacies of experimental therapies withinthe same drug class andto determine optimal dose schedule.

Ewing et al.

492

REFEXENCES 1. Lee A, Fox Hazel1 S. The pathogenicity of Helicobacter pylori: a perspective. Infect Immunoll993; 61:1601-1610. 2. Fox Correa P, Taylor NS,et al. Helicobacter mustelae-associated gastritis in ferrets: an animal model of Helicobacter pylori gastritis in humans. Gastroenterology 1990;99:352-361. Hardy D, Swanson R, Hensey D, et al. Comparative antibacterial activities of temafloxacin hydrochloride (A-62254) and two reference fluoroquinolones. Antimicrob Agents Chemother 1987; 31:1768-1774.

-

AzithromycidRanitidine Combined Treatment of Helicobacterpylori in Patients with Duodenal Ulcer and Chronic Gastritis: A Pilot Study B. Desnica, V. Burek;

N. Makek

University Hospital of Infectious Dbeases "DrFran MihaljeviC" Zagreb, Croatia

INTRODUCTION Peptic ulcer disease is a chronic inflammatory condition of the stomach and duodenum that affects as many as 10% of people at some time in their lives. Althoughthe disease has relatively low mortality, it results in substantial human suffering and its wide prevalence underscores the high economic cost of the .illness. The association with Helicobacter pylori as a major pathogenic.factorin peptic ulcer disease was established in1983. It is now generally accepted that H . pylori is a major causeof gastric and duodenal ulcer and antral gastritis as well.The eradication of the organism is necessary for optimal therapyof the disorder. The gold standards of establishing the diagnosis of H. pyloninfection are invasive tests which include endoscopy followed by gastric biopsy and histological demonstration of organisms, biopsy with direct direction of urease activity, and biopsy with culture of H.pylon. Excellent diagnostic sensitivity and specificity can be obtained with serologic kits for IgG anti493

Desnica et al.

494

bodies to H . pylori. Because of their technical limitations and because antibody levels decrease rather slowly following successful eradication of H . pylori infection, serologic tests may not be useful in diagnosing infections after antimicrobial therapy unless repeated during a longer follow-up period. Azithromycin, administered in combination with ranitidine, bismuth salts, or omeprazole, is well tolerated and safe in doses up to 1 g daily. The aimof our studywas to evaluate the efficacyofcombined azithromycidranitidinetreatment o,n the healing and recurrenceof chronic gastritis typeB and duodenal ulcer disease associated with H . pylori and to establish the correlation with seronegativity achieved after antimicrobial therapy.

PATIENTS AND METHODS Patients of both sexes, aged between 17 and 72 years, with clinical symptomssuchasepigastricpain,dyspepsia, heartburn, or anorexia,were enrolled in this open, noncomparative study. They were included in the study on the basisofendoscopicfindings(endoscopic appearance of duodenal ulcer, measuring between 5 and 20mmin longest dimension, or endoscopic appearance of antral erosions, spotty erythema of antral mucosa, pale areas, goose-pimple-like appearance of antral mucosa, fine spotty erythema of the body of the stomach), histologically defined gastritis (by the Sidney system), and HP seropositivity (ELISA IgG > 1.0 U). Patientswereexcluded if theyhadreceivednonsteroidalantiinflammatorydrugtherapy,corticosteroids,antimicrobialdrugs, or bismuth salts within4 weeks prior to entry or.antiulcer medications within 2 weeksprior to entry.Patientswithevidence of chronicrenal or liver disease,gastricsurgery or vagotomy,pregnancy,andchronicalcohol abuse were also excluded. After discontinuation of all previously employed therapy and serum bilirubin, AST, ALT, g-GT, AP tests performed, 44 patients who met the entry criteria and gave their written informed consent were.treated with azithromycin mg bid for 6 days and ranitidine mg bid for days. Endoscopic evaluations were done in all' patients after 1'month. A new endoscopic evaluation was performed in unhealed patientsafter 2-months of therapy with ranitidine.ELISA was repeated after and 180 days at follow-up. Histological evaluation was performed patients in with macroscopic appearanceof antral gastritis after days and in unhealedpatients after 2-months of therapy.IgG ELISA anti-HP test (DPC, USA)was performed in all patients, andIgM and IgA antibodies were determined as well, but their values were considered insignificant forthe purpose of the

AzithrornycinlRanitidine Treatment of Helicobacter pylori

495

study. Although this test is considered secure from FW interference, each specimen was re-tested by Western blotting. Patients with IgG antibody titer >1.0 U were considered elective for study. Reduction in antibody titer below 0.5 U was defined as seronegativity. Four antral biopsy specimens were assessed by hematoxylin and eosin stain and graded using the Sidney system in casesof antral gastritis. Pathologists were blindto the treatment and serological results.

RESULTS Ulcer healing was.obtained in 23/28 patients with duodenal ulcer (baseline characteristics of patients are presented in Table 1). Clinical and endoscopic cure was followed by IgG seronegativity or a dec1ine;of IgG antibodies (Fig. 1). Three patients returned to seropositivity after 6 months although they were asymptomatic. W Opatients complained of dyspepsia and abdominal discomfort in spiteof complete healing of the ulcer after 2 months of therapy and a decline of IgG antibody titer. Resolution of endoscopic, histological, andELISA evidence of antral gastritis (baseline characteristics of the patients presented in Table1) was obtained in 11/16 patients (Fig. 2). Three patients had. dyspepsia after treatment although they had a histological improvement and turned to seronegativity. W Opatients had a relapse, confirmedby ELISA, although they had undergone a 2-month treatment. relevant adverse events were noticed. Four patients complainedof mild nausea during the first week of treatment. However,bloodchemistrytestsandurinalysisrevealedno pathologic findings. Table I Baseline Characteristics of Patients

Duodenal ulcer (n = 28) Age (mean 2 SD in years) Gender (W) First duodenal ulcer No. of ulcers present (mean 2 SD) Size of largest ulcer (mean -t SD) Gastritis B (n = 16) Age (mean 2 SD in years) Gender (MiF) Intestinal metaplasia Alcohol use

43.2 2 11.5 17/11 36% 1.0 2 0.2 9.1 2 3.6 48.3 2 12.1 11/5 63% 75%

496

Desnica et al.

0 days

30 days

60 days

180 days

Figure Z Healing of duodenal ulcer.

CONCLUSION There was improvement in the healingduodenal ulcer and antral gastritis associated withHelicobacter pylori infection with combined azithromycid ranitidine therapy. The precise role azithromycin in eradicatingHelicobacter pylori should be evaluated in a study including urease activity tests and culture.

100

-

......

90

.

seropositivity ... m Endoscopic cur( IClinical cure

80 70

.c"

60

," 50 n

40

30 20 10

0 0 days

30 days

Figure 2 Healing of antral gastritis.

60 days

180 days

AzithromycinlRanitidine Treatment of Helicobacter pylori

497

BIBLIOGRAPHY Malfertheiner P et al. The year of Helicobacter pylori 1995. Curr Opin Gastroenteroll995; 11(suppl 1). Peters DH et al. Azithromycin: A review of its antimicrobial activity, pharmacokintics properties and clinical efficacy.Drugs 1992; Shonova 0, Petr P. Concomitant and independent treatment with Surnamed" and Jatrox". Its effects on Helicobacter pylon infection in vivo. Workshop on Helicobacter pylon and the New Concepts in Gastroduodenal Diseases, Prague, 1992.

Azithromycin as a Promising Part of Helicocidal Regimens 0. Shonovh, P. Petr, and

Hausner

Regional Hospitalof Southern Bohemia Budweis, CzechRepublic

INTRODUCTION Helicocidal regimens have been reported recently, targeting short-term, low-dose, and high compliance. The aim of this trialwas to assess the effect of omeprazole and azithromycin in Helicobacter pylori positive patients with peptic ulcerof duodenum and aboral stomach.

PATIENTS AND METHODS

An open, uncontrolled, noncomparative clinical trial was performed inan outpatient gastroenterology unit. Patientsof both sexes, aged18-70 years, with endoscopically diagnosed peptic ulcer of duodenum or aboral stomach, measuring at least 5 mm in size, nonmalignant (Diagnoses and of OMED classification), and the presence of H . pylori (confirmed by at least two out of three methods used), were enrolled into the study. Exclusion criteria were pregnancy, women with childbearing potential not taking adequate contraceptive measures, malignant diseases (includinggastricmalignancy),mentalincapacity,administration of antiulcer drugs within the last 6 weeks (bismuth within the last 12 weeks) and/or 498

AzithromycinRegimens in Helicocidal

499

antibiotics therapy within 6 weeks, and/or nonsteroidal anti-inflammatory drug medication within6 weeks priorto entry.

Treatment The combined therapy with azithromycin (1 X mg dailyfor 1week) and omeprazole mg for weeks)wasgiven to all patients with endoscopically diagnosed peptic ulcer (minimal size5 mm) of the duodenum or aboral stomach.

Efficacy Assessment Endoscopy, including gastric biopsy for direct microscopy, culture, and urease test, was performed before the treatment. Control endoscopy and biopsy were performed4 weeks afterthe treatment (i.e., 5 weeks after the end of antibiotic treatment) and the presence of H . pylori was assessed.

Microbiological Assessment Three biopsyspecimens,takenfromantrum of each patient ateach endoscopy, were slightly pressed onto cultivation media as described below and transported to the microbiological laboratory within h, using no special transport medium. The specimens were homogenized and divided in three equal portionsfor each test.

Direct Microscopy The modified Gram-stain method was used, with prolonged fuchsin exposure (H. pylori absorbs the dyes poorly).

Urease Test Christensen fluid medium was used. The results were evaluated and h after inoculation. A semiquantitative three-level simple scalewas used to grade positive findings froml'to

Culture All specimens were cultured on three media, for H . pylori isolation and one (blood agar)for concomitant bacterial flora.The following media were used forH . pylori isolation: (a) Brain-heart infusion (BHI), with addition (per liter): Supplement A (for Neisseria gonorrhoeae cultivation) Yeast

m1 5g

500

(ram)

Shonov& et al. Starch Horse serum 50 m1 Active 2g Sheep 5% (b) Brucella agar, with additionof supplements as above

Vancomycin, trimethoprim, colistin, and amphotericin were added to the selective medium. H . pylori plates were incubated in microaerophilic conditionsat 35°C and assessed after 5 days (time span:3-7 days). Identification of H . pylori was made by typical morphology of the colonies, oxidase, and catalase activity and Gram-stain microscopy. H . pylori presence before treatment was defined by positive findings inat least two of the three methods used. H . pylori presence after treatment was defined by positive findings in at least two the three tests or by positive culture only.

Susceptibility to Azithromycin Sensitivity of all H . pylori isolates to azithromycin was tested using the disk-diffusionmethod.Azithromycin(15 pg) testingdisks(ImunoloSki zavod Zagreb, Croatia) were employed.

RESULTS In this study, 19 adults (11 men and 8 women), aged 21-70 years (mean age: 43 years), with endoscopically confirmed peptic ,ulcer (minimal size5 mm) were included. Before treatment, H . pylori presence was confirmed in all patientsby allthree described methods. Four weeks after the treatment, the ulcer was healed in19 patients (100%) andH.pylori was eradicated in 16 patients (85%) (Table 1). No drop outs and no adverse events were recorded, either in spontaneous referenceor in active interviewing. Sensitivity testing was performed on 20 consecutively isolated H. pylori strains and 19 strains (95%) were sensitive, with an average inhibition zone of mm (176% of mm, regarded as safe marker for susceptibility). Table Z Efficacy of Azithromycin and Omeprazole inthe Treatment of H.pyloriPositive Patients with Peptic Ulcer Disease

Ulcer H . pylon eradication

No. of patients

Rate

19 16

100% 85%

501

AzithromycinRegimens in Helicocidal

CONCLUSION Combined therapy with azithromycin and omeprazole was well tolerated and effective in ulcer healing and inH . pylori eradication. The advantage of this therapy is the short duration of treatment and low dose both of which should provide good compliance.

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ANAEROBES, ENTEROCOCCI, HAEMOPHILUS INFLUENZAE

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Comparative In Vitro Activity of Five Macrolides Against Gram-Positive Cocci, Campylobacter Species, and Anaerobes M. Marina and K. Ivanova National Center of Infectious and Parasitic Diseases Sofia, Bulgaria

N. Hadjieva University Hospital “QueenJoanna” Sofia, Bulgaria

A. Urumov National Drug Institute Sofia, Bulgaria

INTRODUCTION Erythromycin, whichis an alternative to penicillininpenicillin-allergic patients and shows a good antibacterial activity against gram-positive bacteria, has some disadvantages. Several new macrolide compounds with improved pharmacokinetic properties have been introduced, such as josamycin, roxithromycin, clarithromycin, and azithromycin. The purpose of this study was to compare the in vitro activities of these macrolides against gram-positive cocci,Campylobacter sp. and some anaerobes. 505

506

Marina et al.

MATERIALS AND METHODS Bacterial Strains All tested strains were clinical isolates identified by the methods outlined in the Manual of Clinical Microbiology, 1991: gram-positive cocci 237, most ofwhichweremultiresistanthospitalstrains: S. aureus (87 strains), coagulase-negative staphylococci (92strains), and enterococci (58strains); Campylobacter sp. (156 strains): C . jejuni (112 strains), C. coli (40 strains), and C . fetus (4 strains); obligately anaerobic bacteria(134 strains, chosen among those species with greater tendency of erythromycin resistance: Bacteroides fragilis group (86 strains); Prevotella sp. (26 strains), including P . oralis, P . oris, P. intermedia, P . melaninogenica; Fusobacterium sp. (10 strains); Bilophila wadsworthia (2 strains), andClostridium sp. (10 strains).

Antimicrobial Agents The following antibiotics were tested: erythromycin, josamycin, roxithromycin, clarithromycin, and azithromycin.

Susceptibility Testing The minimal inhibitory concentration (MIC) was determined using the NCCLS-recommended agar dilution methods with Mueller-Hinton agar for the cocci and Campylobacter, and the Wadsworth agar dilution method with Brucella blood agar for the anaerobes.

RESULTS Table 1 summarizes the susceptibility of the aerobic gram-positive cocci, Campylobacter species,andanaerobicbacteria to erythromycin,josamycin, roxithromycin, clarithromycin and azithromycin. Data on the last two groups of organisms are also illustrated graphically in Figs. 1 and 2.

DISCUSSION Almostall of the testedmacrolideshadsimilar.activitiesagainst the staphylococci except for a slight predominance of clarithromycin (forboth) and josamycin (for the coagulase-negative staphylococci) (see Table 1). Clarithromycinandjosamycinweremoreactivethan the other drugs against enterococci-29% and 25%of susceptible strainsat 8 pg/ml, respectively.Azithromycinandroxithromycinwere the mostactiveagainst Campylobacter sp.; 90% of the strainsinhibited at 0.5@m1 for azithromycin and at 4 pg/ml for roxithromycin. Josamycin and clarithro-

507

Macrolides Against Gram-positive Bacteria

Table Z Susceptibility of Gram-positive Cocci, Campylobacter Species, and Some Obligately Anaerobic Bacteriato Erythromycin, Josamycin, Roxithromycin, Clarithromycin, and Azithromycin Percentage strains inhibited at (pglml)

MIC (Clglml)

Organism (No.) and Range antimicrobial agents

90%

S. aureus

Erythromycin Josamycin Roxithromycin Clarithromycin Azithromycin Coagulase-negative staphylococci (92) Erythromycin Josamycin Roxithromycin Clarithromycin Azithromycin Enterococci Erythromycin Josamycin Roxithromycin Clarithromycin Azithromycin Campylobacter sp. Erythromycin Josamycin Roxithromycin Clarithromycin Azithromycin Anaerobic bacteria Erythromycin Josamycin Roxithromycin Clarithromycin Azithromycin

OS-> 50.5” OS-> OS->

> > >

os->

>

50.5-> OS-> OS-> OS->

60

40

>

> > > > > >

OS-> 50.5->

2

0.5

os-> OS-> OS-> 50.5->

> > > >

Marina et al. %

90

80 70 60

50 40 30

20 10

0.5

1

2

4

l 64

32

128

MIC /pg/ml Figure l

Campylobacter: Inhibited strains versus MIC.

mycin were intermediate with 96% and 91%, respectively, of susceptible strains at 8 pg/ml. Erythromycin had the lowest activity and the highest percent of resistant Campylobacter strains (11.5%) at the same concentration. Josamycinandclarithromycinwere the mostactiveagainst the anaerobes-83% and 80%, respectively, of susceptible strains at 4 &ml. Erythromycin and azithromycin had similar activity and inhibited 41% of strains at this concentration. Roxithromycin showed the lowest activity, with only 16.4% of susceptible strainsat 4 pg/ml. Our results are comparable to those of other authors (1-7).

CONCLUSIONS There is a trend toward increasing erythromycin resistance in Campylobacter sp., and the new macrolides can be a good alternative for the treatment of severe campylobacteriosis. They can be used in infections

509

Macrolides Against Gram-positive Bacteria YO 100

90 80

70 60 50

40 30 20 10

0.5

1

2

4

8

l 6

32

64

128

MZC &/m1 Figure 2 Anaerobic bacteria: Inhibited strains versusM C .

caused by gram-positive cocci or anaerobes but only after in vitro testing of their activity.

REFERENCES 1. Arguedas AG, et. al, Comparativeactivity of josamycin,roxithromycin, clarithromycin and azithromycin against erythromycin resistant staphylococci. 1st Scientific Meetingof the European Society of Chemotherapy, Budapest, 1993; abstr P2. 2. Citron D, etal. Activity of ampicillidsulbactam, ticarcillidclavulanate, clarithromycin, and eleven other antimicrobial agents against anaerobic bacteria isolated from infections in children. Clin Infect Dis1995; 20 (suppl 2):S356. 3. Dubreuil L, et al. Activitt in vitro de la roxithromycine, nouveau macrolide semisynthetique envers les anaerobies stricts. Pathol Bioll986; 34:440. 4. Garcia-Rodriguez JA, Garcia-Sanchez JE, Prieto-Prieto J. Josamycin alone and in combination against anaerobic bacteria. Drugs Exp Clin Res 1982; VI11 (3):285. 5. Gomes-Garces JL, Cogollos R, Alos JI. Susceptibilities of fluoroquinoloneresistant strains of Curnpylobacterjejuni to 11oral antimicrobial agents.Anti microb Agents Chemother 1995;39542.

510

Marina et al.

6. MaskellJP,Sefton AM, Williams JD. Comparative in-vitro activity of azithromycin and erythromycin against Gram-positive cocci, Haemophilus influenzae and anaerobes. J AntimicrobChemother 1990; 25 (suppl A):19. 7. Spangler SK, Jacobs MR, AppelbaumPC.Comparison of the E test and oxyrase methods in susceptibility testing of201 Gram-negative and Grampositive anaerobes to erythromycin, azithromycin, clarithromycin and roxithromycin. 7th European Congress of Clinical Microbiology and Infectious Diseases, Vienna, 1995; abstr 171.

Prophylactic Effect of Azithromycinon Experimentally Induced Intraabdominal Infection in Rats N. Panovski,

Mil&vski, P.

N. Labaeevski

University “Sv. K i d i Metodij” Skopje, Republic of Macedonia

INTRODUCTION Azithromycin is not recommended for the treatment of intraabdominal infections becauseof the relative resistanceof common bacterial pathogens at this site mean minimal inhibitory concentration (MI(&,) is m& for Escherichia coli and 11 mgL for Bucteroidesfrugilis (the range: mgL) (1). However, azithromycin has been shown to.be effective in several in vivo models of localized infections induced withE. coli or B. frugifis (2). The purpose of the present studywas to evaluate the prophylactic effectof azithromycin, in comparison with clindamycin and ciprofloxacin, on the development of acute peritonitis and on the formation of intraabdominal abscess in an animal model using Wistar rats.

MATERIALS AND METHODS From one strain each of B . fragifis and E . coli, clinical isolates were obtained from a patient with a perforated appendix. The MICs of azithromycin, clindamycin, and ciprofloxacin were 16, and <0.125 m&, 511

512

Panovski et al.

respectively, for E. coli and 8, 2, and 4 m&, respectively, for B . fragilis. Schaedler blood agar enriched with a kanamycin-vancomycin mixture(Bio Merieux) was used for isolation of B. fragilis and blood agar was used for isolation of E. coli. Challenge inocula were grown in brain-heart infusion broth (Bio Merieux). Wistar rats of both sexes (Institute of Pharmacology and Toxicology, Medical Faculty,Skopje), 200-350 g, were used.

Made1 of Acute Infection-Peritonitis Group A perforated gelatin capsule containing artificial fecal material (minced beef and cellulose fiber) with more than lo9CFU (colony-forming units)of E . coli and CFU of B . fragilis were surgically implantedinto the peritoneal cavity of the rats. All animals that survived the first day of bacterial challenge were sacrificed48 h after the challenge. Pathomorphological and bacteriological examinations were performed in all animals (deceased and sacrificed).

Model of Late Complications-Intraabdominal Abscess Group, A nonperforated double gelatin capsule containing artificial fecal material with lo' CFU of E. coli and lo8CFU ofB. fragilis were surgically implanted into the peritoneal cavityof the rats. All animals were sacrificed 15 days after the implantation of the capsule and pathomorphological examinations were performed. Bacteriological examinations were done in animals in each group. Filled capsules, without bacteria, were implanted into the peritoneal cavity of five rats to serve as a negative control of the abscess model.

Antimicrobial Treatment Both the peritonitis and intraabdominal abscess groups were divided into four subgroups. The firstsubgroupreceived no antimicrobialsand the others were treated with azithromycin (70 m a g daily PO), clindamycin (50 mgkg daily IM), or ciprofloxacin (100 mgkg daily PO), respectively. Antimicrobial treatment was initiated 2 days before and continued for 2 days after the challenge in the acute peritonitis group and5 days after the challenge inthe abscess group.

Determination of Antibiotic Activity (Concentrations) The activity of antibiotics was determined in serum, peritoneal fluid, and abscess using a bioassay method with a referent strain of Micrococcus luteus ATCC 9341. Sera were collected duringthe bacterial challenge,3 h

Prophylactic Effect

513

Azithromycin

after the administration of the third dose of drugs. Drug concentrations were determinedby standard curves obtainedby in vitro additionof different known concentrations of the used antibiotics to the serum of an untreated animal. Paper disks(6 mm in diameter) were dipped into peritoneal fluid during the operation and in the deceased or sacrificed animals at defined time intervals after discontinuation of drug administration. The activity of azithromycin inthe abscess was determined in30 mg of pus.

RESULTS AND DISCUSSION Model of AcuteInfection-Peritonitis Group 'All untreated animals developed acute peritonitis, but only eight died (Table 1). Treatment with azithromycin and ciprofloxacin significantly decreased the incidence of acute peritonitisin relation to untreated (p<.OOl) and clindamycin-treated animals Relatively low mortality rates did not allow relevantstatisticalanalysis of early death rates between the groups. Bacteriological findings in the peritoneal cavity were negative in 75% of azithromycin-treated rats and 50% of ciprofloxacin-treated rats. E . coli was present inallclindamycin-treated rats, B . fiagilis in 6% of azithromycin-treatedrats and 33% of ciprofloxacin-treated rats; both microorganisms were present in alluntreated rats, 19% of azithromycin-treated rats, and 17% of ciprofloxacin-treated rats.

Model of Late Complications-Intraabdominal Abscess Group pathomorphological changes were found inthe negative control group (group of five rats implanted with capsules without bacteria). The use of azithromycinandclindamycinmarkedlydecreased the incidence of inTable Z Pathomorphological Findings inRats from the Peritonitis Group

Cause of death (24 h after bacterial challenge)

Presence of peritonitis (48 h after bacterial challenge)

Absent Treatment Present Peritonitis Other Azithromycin Clindamycin Ciprofloxacin None

4 2

8

2 0 1 0

Total No. of rats

0

11

16

8 0 16

6

18

9

12 24

0

Panovski et al.

514

Table 2 Pathomorphological Findingsin Rats from the Intraabdominal Abscess

Group Presence of intraabdominal abscess days after bacterial challenge) Present

Treatment Azithromycin Clindamycin Ciprofloxacin None

25

Total no. of rats

8

1

traabdominal abscess formation, but ciprofloxacin didnot have that effect (Table 2). Bacteriological findings in the abscess pus were negative in 30% azithromycin-treated rats and 10% of ciprofloxacin-treated rats.E . coli was present in 20% of azithromycin-treated rats and 40% of clindamycintreated rats, B . fragilis in 10% of azithromycin-treated rats and 70% of ciprofloxacin-treatedrats;bothmicroorganismswerepresentinalluntreated rats, 40% of azithromycin-treatedrats, 60% of clindamycin-treated rats, and 20% ciprofloxacin-treated rats.

Determination of Antibiotic Activity (Concentrations) The mean serum concentration of azithromycinat the third dayof administhe tration was 1.1m@. However, its activity inthe peritoneal fluid and in abscess pus was marked even 10 days after drug discontinuation (Table Table 3 The Activityof Azithromycin in Peritoneal Fluid (Paper Disk Bioassay) and in AbscessPus

Day after treatment discontinuation Standard diskb Peritoneal disk

Diameter of growth inhibition zone (mm)a 30 mg of pus

aMicrococcus luteus ATCC 9341. b15 of azithromycin.

-

Prophylactic Effect of Azithromycin

515

The other two antibiotics, despite high concentrations in serum (particularly clindamycin), showed no activity of the peritoneal disks collected 2 days after drug discontinuation.

DISCUSSION The observed effects of clindamycin and ciprofloxacin could be explained by in vitro efficacy of the two drugs against E. coli and B . fragilis. The prophylactic effect of clindamycin on the abscess formation was considerably lower than reported by other authors (3,4), probably due to the high minimal inhibitory concentration (MIC) for the B. fragilis strain used inthe experiments (2 m a ) . However, azithromycin showedbetter in vivo activity than comparative agents, despite high MICs for the microorganisms tested. The mean serum concentrations of azithromycin at the time of bacterial challenge were8-16 times lower thanthe MICs for the challenge microorganisms, but its activity in the peritoneal fluid and in abscess pus was marked even days after treatment discontinuation. This might explain the better effect of azithromycin in vivo.

CONCLUSION Azithromycin significantly decreasedthe development of acute peritonitis, exhibiting a similar effect to that of ciprofloxacin. The effect of azithromycinon the reduction ofabscess formation wassimilar to that of clindamycin. The observed prophylactic effect of azithromycin on experimentally induced intraabdominal infections inrats was significant and deserves further investigation.

REFJ3RENCES 1. Peters DH, Friedel HA, McTavish D. Azithromycin: A review of its antimicrobialactivity,pharmacokineticpropertiesandclinicalefficacy.Drugs 1992; 44:750. 2. Girard AE, Girard D, Retsema JA. Correlation of the extravascular pharmacokinetics of azithromycin with in-vivo efficacy in modelsof localized infection. J Antimicrob Chemother1990; 25 (suppl A):61. 3. Onderdonk BA, Cisneros LR, Finberg R. Animal model systemfor studying virulence and host response to Bacteroides fragilk. Rev Infect Dis 1990; 12 (suppl2):S169. 4. Ingham HR, Sisson PR, Selkon Current concepts of the pathogenic mechanisms of non-sporinganaerobes:chemotherapeuticimplications. J Antimicrob Chemother 1980;6:173.

Are Macrolides Active Against Haemophilus influenzae? Are In Vitro Tests Reliable? F. Crokaert, M. Aoun, V. Duchateau, P. %renier, A. Vandermies, and J. Klastersky Institute J . Bordet Brussels, Belgium

H.Goossens Universitair Ziekenhuis Antwerpen Edegem, Belgium

INTRODUCTION Streptococcus pneumoniae, Haemophilus influenzae, and M. catarrhalis are leading respiratory pathogens.H . influenzae is responsible for a quarter ofall acute otitis mediain adultsandchildren (1) and for severe infections in adults (2-5). Resistance to ampicillin has been described for 20 years and ranges from 10% to 45%. It is mainly due to P-lactamase production, whereas ampicillin-resistant p-lactamase negative strains remain uncommon (6). The activity erythromycin toward H . influenzae is generally poor and highly dependent on experimental conditions when tested in vitro (7). There are no specific breakpointsto test erythromycin, roxithromycin, and dirithromycin in the NCCLS guidelines, whereas breakpoints for azithro516

Are Macrolides Active Against H. influenzae?

517

mycin and clarithromycin have been proposed, based on higher intrinsic activity (azithromycin) and tissue concentrations for both drugs (8).

MATERIAL AND METHODS Clinical Samples

Bacteria

One hundred sixty-four strains of H.infruenzae were isolated from purulent sputum produced by ambulatory and recently hospitalized patients with clinically documented respiratory tract infections. A Gram-stained smear was required with more than 20 polymorphonuclear cells, less than 10 epithelial cells at low power field (10 lo), and visible small pleiomorphic gram-negative bacilli (averageof 10). The identity was confirmed by X and V factor dependenceon TSA medium (9).

Agar Dilution Antimicrobials were kindly provided as powders: azithromycin (Pfizer), erythromycin, clarithromycin and hydroxy-clarithromycin (Abbott), dirithromycin (Eli-Lilly), and roxithromycin (Roussel-Hoechst). Stock solutions were prepared accordingto manufacturer’s instructions and NCCLS guidelines and stored at -80°C (8). Stock solutions were diluted in water on the day of useto prepare HTM agar plates(pH 7.3) containing twofold dilutions from to 0.25 for erythromycin, roxithromycin, clarithromycin, and hydroxy-clarithromycin. Azithromycin and dirithromycin were tested by the agar dilution method against 54 strains (32to 0.12). Spots containing IO4 CFU (colony-forming units) were applied with a Steers’ inoculator.A subset of 94 strains were blindly testedby one the authors (HG) against erythromycin, roxithromycin, and clarithromycin.

E-Test Strips were availablefor azithromycin, erythromycin, roxithromycin, dirithromycin, and clarithromycin (Biodisk, Sweden) and were applied onto the surface of PDM agar (Ils, Belgium) inoculated with a suspension of organisms adjusted to 0.5 McFarland. A subset of 30 strains was tested in parallel on PDM agar, HTM (homemade), and commercial HTM (Ils, Belgium).

Disk Diffusion Paper disks were used to measure the inhibition zones obtained on HTM agar plates inoculated with suspensions matched with a 0.5 McFarland standard (10).

518

Crokaert et al.

Incubation The plates were incubated overnightat 37°C in 5% carbon dioxide.

Breakpoints The breakpoints were those of NCCLS when available (11) and those of NCCLS for other microorganisms than H . influenzae when not.

P-Lactamase Activity was determined using nitrocefinpaper disks.

RESULTS Fourteen percent of then H . influentae produced a P-lactamase.The minimum inhibitory concentrations (MICs) of the macrolides were indentical for P-lactamase-producing and -nonproducing strains.The distribution of MIC valuesfor 164 strains are shown in Figs. and 2. The MIC, and MIC, were lower withthe E-test than the agar dilution method: 4 mgL and 8 mg/ L with the E-test versus 8 and with agar dilution for erythromycin, 13 and 32 versus and 32 for roxithromycin, 7 and versus and 32 for clarithromycin, 192and versus 32 and 32for dirithromycin, and 2.3 and 4 versus 2 and 8 for azithromycin. The MIC, and MIC, for hydroxy-

IO0

80

60 40 20

0

03

1

2

4

8

16

32

64

H.influenzae: MIC distribution (agar dilution)(E = erythromycin; C = clarithromycin; R = roxithromycin;OH-C = hydroxy-clarithromycin;Y axis : number of strains; X axis : MIC values).

Figure I

Are Macrolides Active Against H. influenzae?

519

0-1.5 8.5-16 2-3.5 4-5.5 6-8 16.5-19.5 20-30.5 31-128 IlErythro OClarithro lRoxithro

Figure 2 H.in@enzae: MIC distribution (E-test) (Y axis : M C values).

I : number of strains; X

clarithromycin could be tested only with agar dilution and4 were mgL and 8 m&, respectively. The MIC values of ATCC 49766 and 49247 were in the expected range. There was an excellent agreement between both laboratories: Concordance or minordiscordanceswereobservedin 98% of the tests with erythromycin, 97% with roxithromycin, and 98% with clarithromycin; two major discordances occurred with erythromycin, three with roxithromycin, and five with clarithromycin. Poor growth and contamination accounted for 2.1% of E-tests on homemade HTM,
520

Crokaert et al.

Are Macrolides Active Against H. influenzae?

521

that showed poor growth or contamination. The MIC valuesare dependent not only on the medium but also onthe method itself;the results expressed as categoriesfor these 30 strains are shown in Table 1.More than50% of the discordances were observed between agar dilution and disk diffusion.

DISCUSSION Erythromycin cannotbe considered a first-line drug for infections due to H. influenzae because of its poor in vitro activity and pharmacokinetic properties aswell asits gastrointestinal adverse effects (12). New macrolides such as roxithromycin and clarithromycinare better tolerated and exhibit more favorable pharmacokinetics. Clarithromycin, through hydroxy-clarithromycin exhibits higher activity against H. influenzae and better tissue penetration over erythromycin. Azithromycin has high and sustained penetratissue tion andpenetrates well into polymorphonuclear cells. Azithromycin is also more active in vitro than erythromycin, roxithromycin, and clarithromycin. We confirm the in vitro activity of macrolides against H. influenzae is asfollows:azithromycin > hydroxy-clarithromycin > clarithromycin = erythromycin > dirithromycin. Dirithromycin is easily and rapidly hydrolyzed to erythrocylamine, which accounts for much ofthe antimicrobial activity(13). We were unable the E-test in a to establish anti-H. influenzae activity of dirithromycin using carbon dioxide atmosphere. pH values before and after incubation were stable (pH 7.3). We also showed the influence of the medium (E-test MICs on PDM were lower than on I"), methods (E-test MICs were lower than agar dilution MICs), and expression of results (MICsor categories). Categories are dependent on defined breakpoints, whereasMIC, and MIC, are representative of MICdistribution.Diskdiffusionis inappropriate to test macrolides againstH. influenzae and the expression of sensitivity by categories is inadequate.

CONCLUSION H . influenzae is pharmacologically resistant to erythromycin, poorly susceptible to roxithromycin (resistant to the concentrations obtained in vivo), whereas it is pharmacologically more susceptible to clarithromycin (susceptible to the concentrations obtained in vivo) and both pharmacologically and microbiologically more susceptible to azithromycin. There are major pitfalls associated with in vitro testing but they can be overcome. The Etest is an interesting alternativeto the agar dilution method. Disk diffusion is not appropriate to test the activity of macrolides against H. influenzae.

522

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REFERENCES 1. Klein JO. Selection of oral antimicrobial agentsfor otitis media and pharyngitis. Clin Infect Dis 1994;19:823-833. 2. Early D. Purulent pericarditis causedby Haemophilus influenzae.Antimicrob Infect Dis Newslett 1994; 13:29. 3. McDonald CL, Crafton EM, Covin FA. Pericarditis: a probable complication of endocarditis due to Haemophilusinfluenzae. Clin Infect Dis 1994; 12548-649. 4. Moxon ER, Wilson R. The role of Haemophilus infruenzae in the pathogenesis of pneumonia. Rev Infect Dis1991; 13 (suppl6):S518-S527. Najm W, C", Spurgeon L. Bacteremia due to Haemophilus infections: a retrospective studywith emphasis on the elderly. Clin Infect Dis 1995; 21~213-216. 6. Scriver SR, Walmsley SL, Kau CL. Determination of antimicrobial susceptibilities of Canadian isolates of Haemophilus influenzae and characterization of their p-lactamases. Antimicrob AgentsChemother 1994; 38:1678-1680. . 7. Barry AL, Fernandez PB, Jorgensen JH. Variability of clarithromycin and erythromycin susceptibilitytests with Haemophilus influenzae in four different broth media and correlation with the standard disk diffusion test. J Clin Microbioll988; 26:2415-2420. 8. National Committeefor Clinical LaboratoryStandards. Methods for Dilution Antimicrobial Susceptibility Testsfor Bacteria That Grow Aerobically. Third Edition, Villanova, PA: Approved Standard M7-M. National Committee for Clinical Laboratory Standards, 1993. 9. Manual of Clinical Microbiology, 6thed. Washington, D C American Society for Microbiology, 1995. 10. National Committee for Clinical Laboratory Standards. Performance Standards for Antimicrobial Disk Susceptibility Tests-Fifth Edition, Approved standard M2-AS. Villanova, PA: National Committeefor Clinical Laboratory Standards, 1993. 11. National Committee for Clinical Laboratory Standards. Performance Standards for Antimicrobial Susceptibility Testing. Approved Standard M100-S3. Villanova, PA: National Committeefor Clinical Laboratory Standards, 1994. 12. Steigbigel NH. Macrolides and clindamycin. In: Mandell GL, Douglas RG, Bennett JE eds. Principles and Practices in Infectious Diseases, 4th ed. New York: Churchill Livingstone,1995:334-346. 13. Kirst HA, Creemer LC, PaschalJW.Antimicrobial characterizationand interrelationships of dirithromycin and epidirithromycin. Antimicrob Agents Chemother 1995; 39:1436-1441.

Macrolide Susceptibility of Isolates of Haemophilus inpuenzae, Streptococcus pneumoniue, and Moraxella catarrhalis from Patients with Community-Acquired Lower Respiratory Tract InfectionResults of an International Multicenter Study (the Alexander Project), 1992-1994 D. Felmingham, J. Linares, and the Alexander ProjectGroup University CollegeLondon Hospitals London, England Hospital de Bellvitge Barcelona, Spain

INTRODUCTION Antimicrobial choice for the empirical therapy of pulmonary infection is threatened by the increasing prevalence of resistance among communityand hospital-acquired pathogens (1). Therefore, determination of local, national, and worldwide antimicrobial susceptibility patterns, which may vary with time, is of fundamental importance. In early 1992, a prospective, international, multicenter collaborative study of the susceptibility of 523

524

Felmingham et al.

community-acquiredlowerrespiratorytractpathogens-theAlexander Project-was established. We report on the macrolidesusceptibility of isolates of H . influenzae, S. pneumoniae, and M. catarrhalis examined in the study duringthe period 1992-1994.

MATERIALS AND METHODS

Fifteen centers were enrolled into the study, including the following: London/ Belfast, UK; Toulouse/Paris,France; BarcelonaMadrid, Spain; Weingartemunich, Germany; GenodCatania, Italy; New YorkNorcester, MMortland, OWCleveland, OWJohnson City, TN, USA. Each center collected up to 400 isolates from patients with community-acquired lower respiratory tract infection and sent to them the central laboratory in London for susceptibility testing.After reidentification, minimal inhibitory concentrations (MICs) of the macrolides were determined using a microbroth inco poration techniquewith an inoculum of approximately 2 X lo4CFU (colonyforming units) contained in 50-p1 Mueller-Hinton broth, supplemented 2% v/.) and NAD (final concenwith lysed horse blood (final concentration tration 10 m a ) for isolates of H . influenzae and S. pneumoniae. Breakpoint MICs publishedby the NCCLS (2) were used for qualitative interpretation of susceptibility.

RESULTS The MICs of the macrolides for4155 isolates of H . influenzae were distributed unimodally (Table 1). No significant differences in mode MICs were observed among isolates fromthe 3 years or among centers. No high-level resistance was detected. All strains were inhibitedby 5 4 mgL of azithromycin and 97.2% of the isolates were susceptible to clarithromycin at 5 8 m&. No interpretativeerythromycin MIC breakpoints are published bythe NCCLS; therefore, no comment can be made regarding this compound. A total of 1193 isolates of M. catarrhalis were tested. No significant differences in mode MICs were observed among isolates from the 3 years or among centers (Table 2). High-level macrolide resistance was not detected. However, examination of the distribution of MICs for this species shows a trailing downslope of increasing MIC which may indicate incremental changes in susceptibility in a small proportion of isolates. A total of 2829 isolates of S. pneumoniae were tested.The prevalence of high-level macrolide resistance varied widely from country to country, being highest in France and Spain the in European Union with evidenceof increasing prevalence in one center in Italy (Genoa) and one in the United States (Cleveland, OH) (Table 3). Cross-resistance to all three macrolides

Macrolide Susceptibility

Bacteria in RTI

525

526

et

Felmingham

al.

Table 2 Distribution of Macrolide MIC (m@) for 1193 Combined EU and USA Isolates ofM.catarrhah-Alexander Project 1992-1994

No. of isolates inhibited at 0.25 0.12 50.06" Antimicrobial

Erythromycin Clarithromycin Azithromycin

0.5

154 989 1167

865 176 14

143 4 18 3 4

22 3 6

4 1

2

3

2 3 1

1

1

-

'In 19!Z and 1993,0.06 m& was the lowest concentration tested. In 1994, mode MICs were as follows: erythromycin,0.12 m&; clarithromycin, 0.06 m&; azithromycin, m&.

was apparent and was associated with p-lactam resistance. However, this association was notcomplete. Thus, in Toulouse and Genoa, erythromycin resistance among penicillin-susceptible strains (28.8% and 12.3% in 1994, respectively) was considerably higher than in Barcelona (1.5% in 1994), where some of the highest rates of penicillin resistance were found, indicating other selective pressures. Table 3 High-Level Macrolide Resistance Among 2829 Isolates of S. pneumoniae-Alexander Project 1992-1994 '

Center

UK:

2.3

London (1) Belfast (2) France: Toulouse (3) Paris (4) Spain: Barcelona (5) Madrid (6) Germany:Weingarten (7) Munich (8) 58 Genoa Italy: (9) Catania (10) USA: YorkNew (11) 38 14 Worcester 43 (12) Portland 2 (13) 37 49 Cleveland 26 (14) Johnson City 12 8 (15)

No. of isolates tested

% Resistance

1992

1993

1994

1992

1993

1994

128 38 89 80 267 2

105 45 152 73 228 52

1.6 0 27 17.5 9

2.8 0

2 1.4 3 79

110 42 147 42 11.4 263 51 16 17 128 10 51

31

18

0.9 0 45.6 31 18.6 45.1 6.3 0 14.1 0 2 2.6 5.6

60 44 70

46

-

1.7 0

25 45.2 19.2 0 -a

5.2

-

-

0 0 0 0 10.8 4.1 8.3 8.7

-

Note: High-level resistance breakpoint MIC greater than or equal to 4 m& erythromycin. 'No data, % calculated only when total isolates examined 210.

Macrolide Susceptibility of Bacteria inRTI

527

CONCLUSIONS High-level macrolide resistance was not detected among the isolates of H. influenzae and M. catarrhalis tested. Establishmentof MIC breakpoints to guide clinicians inthe choice of any one of the marolides, rather than another, for the treatment of infections caused by H . influenzae can only be achieved by considering controlled clinical response data for defined infections. The breakpoints referredto in this study, or lack of them inthe case of erythromycin, may not be appropriate. High-level cross-resistance to the macrolides among isolatesof S. pneumoniae is wellestablished worldwide, but the prevalence varies greatly depending on geographical originof strains and, to some extent, with the passage of time. Although strongly associated with penicillin resistance, macrolide-resistant penicillin-susceptible strains occur with high prevalence in some countries.

REFERENCES 1. Felmingham, D. Antibioticresistance. Do we need new therapeutic approaches? Chest 1995; 108:70S-78S. NationalCommittee for ClinicalLaboratory Standards. PerformanceStandards for Antimicrobial Susceptibility Testing. Fifth Informational Supplement Document M100-SS. Villanova, PA, NCCLS; Vol. 14, No. 16.

Resistance of Common Respiratory Pathogens to Erythromycin and Azithromycin Milan CiZman University Medical Centre Ljubljana Ljubljana, Slovenia

Ana Zlata Drag& and Katja Seme Institute of Microbiology Ljubljana, Slovenia

Andreja OraZem and Metka Paragi Institute of Public Health Ljubljana, Slovenia

INTRODUCTION

Macrolides account for 10-15% of the worldwide oral antibiotic market(1). In the period 1991-1994,macrolide/azalideprescriptions for nonhospitalized patients in Slovenia increased from 4.8% to 12.7% of all antibiotic prescriptions, or from 0.89 to 1.87 defined daily doses (DDD) per 1000 inhabitants per day. A DDD for erythromycin is 1 g and that for azithromycin (AZM) is g. The greatest increase in prescriptions was documented for AZM (from 0.0703 to 0.7997 DDD/1000 inhabitantsper day) (2). A correlation between erythromycin consumption and erythromycinresistant pathogens has been observed in some countries (3,4). to This le 528

Respiratory Pathogen Resistance to Erythromycin and Azithromycin

529

examine the frequency of erythromycin and AZM resistance in common respiratory pathogensin Slovenia.

MATERIALS AND METHODS Throat swabs were obtained from inpatients and outpatients with acute respiratory tract infection treated at the Department of Infectious Diseases, University Medical Centre Ljubljana. Bacterial isolates were performed at the Institute of Microbiologyof the Faculty of Medicine in Ljubljana. Additional strains of S. pneumoniae isolated from the respiratory tract (predominantly upper) or sterile body fluids and H.injluenzue isolated from sterile body fluidsof patients in different Slovenia hospitals were included in the study as well. Susceptibility tests were performed at the Institute of Public Healthof Slovenia. Antimicrobial susceptibility testing of S. pyogenes, group B, C, and G streptococci, S. pneumoniae, H. injluenzae, H. parainjluenzae, and B . catarrhalis was performed routinely using NCCLS disk-diffusion methods (5). Erythromycin (15 pg) and AZM (15 pg) susceptibility testing disks (Becton Dickinson Microbiology Systems, Cockeysville, USA) were employed. S. pyogenes, S. pneumoniae, and B . catarrhalis isolates were cultured on Mueller-Hinton agar supplemented with 5% defibrinated sheep blood (100-mm plates). For H.injluenzae and H . parainfluenzae isolates 100-mm agar plates of Haemophilus Test .Medium (Oxoid, Basingstoke, B. England) were used. The agar plates (except those inoculated with catarrhah) were incubated at 35°C in 5-7% CO, for 18-24 h before measuring the zones of inhibition. The organisms were reported as either susceptible, moderately susceptible, or resistant to erythromycin andAZM according to thefollowing zone diameter interpretative standards: Moderately Susceptible susceptible Resistant organisms organisms organisms 14-22 Erythromycin 513 218 Azithromycin The Fisher exact test and the chi-square test were used for statistical analysis. p-Values below .05 were considered to indicate statistical significance.

RESULTS The nearly triple increase in macrolidehzalide prescriptions recorded in 1991-1994 did not have a major impact on the resistance rates of common

&fman et al.

530 Table Z In Vitro Antimicrobial Resistance

Clinical Isolates to Erythromycin

1994

No. of isolates tested Pathogen S. pyogenes S. agalactiae

Group C streptococci Group G streptococci S. pneumoniae Invasive strains Noninvasive strains H . injluenzae Invasive strains Noninvasive 1.9 strains H . parainfluenzae B . catarrhalis

112 10 12

1995

% of resistant isolates

No. of isolates tested

10

13

11

11

4.8

16.6b 108 5.2 4.2

0 0

0.74

0.6

10 62 19 28

p-Valuea

4.1

21 242

92

25 61

96 0.09 4 ' 17

%

resistant isolates

0.50 0.65

8

0.12

0.60 0.68

.Incidence of resistance, 1995 versus 1994.

b7% moderately susceptible isolates. CModerately susceptible isolates.

respiratory pathogensto erythromycin andAZM in the year 1994 (Tables 1 and 2). Aninsignificant (p = .12-.74) increasein the resistance of S. pneumoniae, S. pyogenes, and B . catarrhalis to erythromycin was observed in the year 1995.

DISCUSSION The increase in macrolide/azalide prescriptions in Slovenia did not have a nificantimpact on the resistance ofcommon respiratorypathogens to erythromycin andAZM during the 4-year period our survey. The results are in agreement with the data of Barry and Jones(6), who found that the introduction a numberof antibiotics in U.S. hospitals was not followed by substantial changes in the incidence of resistant strains 3-5 years later. In Slovenia, macrolide/azalide consumption in 1994 was below the rates re-

Respiratory Pathogen Resistance to Erythromycin and Azithromycin

531

T&& 2 In Vitro Antimicrobial Resistance of Clinical Isolates to Azithromycin 1994

Pathogen S. pyogenes S. agalactiae Group C

streptococci Group G streptococci S. pneumoniaeb H . influenza8 H . parainfluenzae B. catarrhalis

No. of isolates resistance tested

1995 % of

No. of

isolates

isolates tested

% of resistant isolates

3.2

12

93 4 17

13

11

0

92

21 39 14 12

0

112

108 25 61

4.2

p-Valuea

0

nIncidence of resistance, 1995 versus 1994. bNoninvasive strains.

ported by anumber of countriesfor the previousdecade. In 1983, erythromycin was consumed atrate a of 4.1 DDD/1000inhabitants per day in the United States and 2.3 DDD/1000 inhabitants per day in France, whereas in 1989, a consumption rate 3.0 DDD/1000 inhabitants per day was recorded in Finland Despite the high erythromycin consumption inthe 5% of United States, no more than of S. pneumoniae isolates, less than S. pyogenes isolates, and a very small proportion of Moraxella catarrhalisisolates were resistantto erythromycin duringthe 1980s (8). In contrast, about 4% of S. pyogenes iso20% of S. pneumoniae isolates in France and upto 4 lates in Finland showed resistanceto erythromycin, although the estimated usage of macrolides in thosetwo countries was comparatively low (3,9). The correlation between antibiotic use and bacterial resistance is not straightforward, however, and many other parameters besides antibiotic consumption, such as infection control practices, presence or absence of resistance genes, selectivity and transmissibilityof these resistance genes, and antibiotic dosages, warrant consideration (10).

CONCLUSION Close monitoringof antibiotic prescriptions and frequent determinationof the susceptibility common respiratory pathogens to macrolides is needed in the era of macrolide renaissance.

532

REFERENCES 1. Kirst HA. New macrolides. Expanded horizons for and old classof antibiotics. J Antimicrob Chemother 1991; 28:787-790. 2. Institut za varovanje zdravja republike Slovenije. Ambulantno predpisovanje v letu 1994, I1 del. Zdrav Var 1995; zdravil v Sloveniji in zdravstvenih regijah 39 (suppl5):6-13. 3. Seppala H, Nissinen A, Jiirvinen H, et al. Resistance to erythromycin in group A streptococci. N Engl J Med 1992; 326:292-297. 4. Westh H. Influence of erythromycin consumptionon erythromycin resistance in Staphylococcus aureus in Denmark. APUA Newslett 1995;13:1-4. 5. National Committee for Clinical Laboratory Standards. Performance Standards for Antimicrobial Disk Susceptibility Tests. Fifth Edition Approved standard M2-A5. Villanova, PA: National Committeefor Clinical Laboratory Standards, 1993. 6. Barry AL, Jones R. Bacterial antibiotic resistance before and after clinical application in the United States. BullNY Acad Med 1994; 63:217-230. 7. Col NF, O’Connor RW. Estimating worldwide current antibiotic usage:report of Task Force. Rev Infect Dis 1987;9 (suppl3):S232-S243. 8. Jacobi GA. Prevalence and resistance mechanisms of common bacterial respiratory pathogens. Clin Infect Dis 1994;18:951-957. 9. Acar JF. Bun-Hoi AY. Resistance patterns of important gram-positive pathogens. J Antimicrob Chemother 1988; 21 (suppl C):41-47. 10. PCchere JC. Antibiotic resistance is selected primarily in our patients. Infect Control Hosp Epidemiol 1994;15472-477.

Efficacy of Clarithromycin and Azithromycin at Human Pharmacological Dosage Against Experimental Haemophilus injluenzae Pulmonary Infection J. D. Alder, M. J. Mitten, A. Conway, A. Oleksiew, K. Jarvis, L. Paige, and S. K. Tanaka Abbott Laboratories Abbott Park,Illinois

INTRODUCTION The macrolide antibiotics clarithromycin and azithromycinare commonly used for the treatment of respiratory tract infections. These two drugs have modest minimal inhibitory concentration (MIC) values against Hemophilus influenzae, and the serum C, concentrations oftendo not attain the MIC value. Clinically,H . influenzae strains with different MIC valuesappear to respond as a uniform population to clarithromycin or azithromycin therapy due in part to intracellular concentrationof the macrolides. However, the NCCLS susceptibility breakpointfor clarithromycin 8 &ml) arbitrarily bisects the population at approximately whereas the breakpoint for azithromycin 4 pg/ml) covers99.5% of the isolates. rat model of pulmonary H . influenzae infection was used to test clarithromycin and azithromycin efficacyat pharmacological dosages. The

533

al.

et

534

Alder

Table Z Plasma and Lung Concentrations of Clarithromycin and Azithromycin in Rats Following Oral Dosing Plasma concentrations concentrations Lung C-

Cm,

AUG-24

Druddosage (Pdml) (Pdml) Clarithromycin mgkg mgkg Azithromycin 25 mdkg mgkg

Cm, cm, (PdN (Pdml)

AUG24

0.06

Note: Clarithromycin 100 mgikg represent near human pharmacologicaldosage in rat; 150mg/ kg represents a slight (1.3-2x) overdosage. Azithromycin 25 mgikg represents dosage moderately higher (2.5X) than attained in human; 100mg&g represents a large overdosage (4-5x).

Table 2 Efficacy of Clarithromycin and Azithromycin Versus H.infruenzae Rat Lung Infection

H.injluenzae Druddosage Clarithromycind mgkg 100mg/kg Azithromycine mg/kg mg/kg Untreated

H.in.uenzae

H.injluenzae

(log count/lung)a

(log count/lung)b

(log count/lung)e

f

f f

f f

f f

f

f

f

f

f

f

f

~

f

~ ~ ~ _ _ _ _____ _ _

Note: Clarithromycin yielded5.31-5.69 log reduction at 150 mgikg,and 3.51-4.11 log reduc-

tion at 100 mgikg. Azithromycin yielded 1.06-4.12 reduction log at 25 mgikg (50 mgikg day 0) and 0.4-2.33 log reduction at 12.5 mgikg(25 mgikg day 0). 'Mean log count f SD; H.influenme 43095 clarithromycin MIC = 4, azithromycin MIC = 1. bMean log count f SD;H.influenme 1435 clarithromycin MIC = 8, azithromycin MIC = 2. CMean log count f SD;H.influenzue 43095 clarithromycin MIC = 8, azithromycin MIC = 2. dclarithromycin dosage bid days0-2. CAzithromycin dosage qd days 1-2; 2X strength dosage day 0.

Pharmacology Dosage of Clarithromycin and

Azithromycin

535

Efficacy of Clarithromycin and Azithromycin Versus H.influenzae Rat Lung Infection Table

H.influenzae Drugdosage (log

count/lung)8

H.influenzae H.influenzae count/lung)b (log counflung)” (log

Clarithromycind mgkg mg/kg

2 f

f 2

f 0.00

f f

f 2 f

2 2 f

f

Azithromycine mg/kg mg/kg

Untreated

f

Note: Clarithromycin yielded3.18-6.33 log reduction at 150 mgkg, and 1.39-5.30 log reduction at 100 mgkg. Azithromycin yielded0.65-2.51 log reduction at25 mgkg (50 mgkg day 0) and 1.07-1.29 log reduction at12.5 mgkg (25 mgkg day 0). .Mean log count SD;H . influenzae 3598 clarithromycin MIC = 8, azithromycin M C = 2. bMean log count f SD;H.influenzae 3643 clarithromycin MIC = 16, azithromycin MIC = 4. CMeanlog count f SD;H . influenzae 3558 clarithromycin M C = 16, azithromycin MIC = 4. dClarithromycin dosage bid days0-2. CAzithromycindosage qd days 1-2; 2 x strength dosage day0.

goal was to determine the relation between clarithromycinefficacy and H . influenzae MIC.

MATERIALS AND METHODS The MIC values against H . influenzae isolates were determined by broth dilution using standard NCCLS guidelines. A single oral dose pharmacokinetic trial was conducted in g Sprague-Dawley rats. The rats were bled and 24 h postdosing. Drug concentration was determined by bioassay using Micrococcus leuteus as the detecting organism. For the efficacy trials, rats were inoculated intratracheally with logs of H . influenzae in molten agarose. All treatments were initiated 5 h postinfection with dosages to model clinical pharmacokinetics. Clarithromycin was administeredPO, bid on days Azithromycin was administered PO, qd, strength dosage day0, and 1 X strength dosage days 2. Lungs were harvested h after the last dosage. Bacterial burden was determined by dilution platingon chocolate agar.

RESULTS The results of the pharmacokinetic trialare shown in Table The results of the efficacy trials are summarized in Tables and

536

Alder et al.

DISCUSSION H . infruenzae pulmonary infection was established rats in using strains with clarithromycin MIC valuesof and azithromycin MIC values of 1-4. Clarithromycin or azithromycinwas administered to rats on schedules and dosages to produce human pharmacological concentrations in plasmaand tissue. At dosagesthat produced pharmacological concentrations in plasma, clarithromycin (C- = pg/ml; area under the plasma concentration versus time curve (AUC) = pg Wml) produced greater reductions of H . infruenzae in lung tissuethan azithromycin (C,,,= =.0.74 pg/ml; AUC = pg Wml). Clarithromycin was effective against H . infruenzae strains with MIC values of Clarithromycin therapy produced up to a log reductions in bacterial burden against H . infruenzae strains with MIC= which is greater than the established MIC breakpoint. The lung concentrationsof clarithromycin (C- = 43.4 pg/ml) and azithromycin (Crn== 22.7 pg/ml) werein great excess of the MICvalues of all H . infruenzae strains. Clarithromycin therapy demonstrated efficacy against H . infiuenzae strains with MIC values of indicatingthat successful therapy was independent of NCCLS breakpoint considerations.

MYCOBACTERIA AND OTHER INFECTIONS IN HIV-INFECTED PERSONS

This Page Intentionally Left Blank

Clarithromycin 500 mgBID as Prophylaxis for MAC Disease-A Follow-up Review John T. Sinnott, A. Holt, Sally H. Houston, Gary Bergen, Pamela Sakalosky, JulieA. Larkin, and Richard Oehler University of South Florida College of Medicine Tampa, Florida

BACKGROUND In the last several years, the as prevalence of Mycobacterium avium(MAC) infection amongAIDS patients has increased, antiretroviral and prophylactic therapies have been introduced to delay the onset of AIDS-defining events andto prolong survival (1). Clarithromycin is a macrolide antimicrobial agent which is highly potent against a varietyof aerobic and anaerobic gram-positive and gramnegative organisms with activity equal to or greater than that of erythromycin for most strains tested.It has also demonstrated antimicrobial activity against MAC in vitro [minimal inhibitory concentration (MIC) = 0.254 pg/ml] and in vivo. Clarithromycin has been shownto effectively inhibit the intracellular replication of MAC within human macrophages (2), as well as to decrease and eradicate MAC from the blood of AIDS patients (3Y4).

Clarithromycin is approved for treatment and for prevention of disseminated MAC(DMAC).SuccessfulprophylaxisofDMACinfection 539

tY

a

-3

m

&

m

E

d

8

a

cl

m m

a 4

Sinnott et al.

542

with clarithromycin therapy could have a significant impacton the morbidity and mortalityof AIDS patients.

STUDY OBJECTIVE The objective was to evaluate retrospectivelythe efficacy of clarithromycin in preventing the onset of disseminated MAC infection in patients with

METHODS Study Design Retrospective studyof patients with AIDS Clarithromycin mg bid

Evaluation Parameters Patient demographicdata Baseline and during prophylaxis CD, counts Number of MAC infection-free days Occurrence of adverse events

RESULTS The mean CD, countat or prior to initiation of clarithromycin was cells/mm3(N= (range During clarithromycin prophylaxis, one patient received concurrent clofazamine, three received concurrent ethambutol, and two received concurrent rifabutin. The mean duraton on clarithromycin (as of September was days (range days), with no symptomatic evidence or blood culture evidenceof DMAC infection. No untoward side effects werenoted. Although diarrhea was present in several patients before therapy had begun and developed on therapy in others, it did not warrant drug discontinuation. Eleven patients died of AIDS-related complications and all were MAC-free at timeof death, with a mean of days of prophylaxis (range: days). Six patients continue to receive clarithromycin prophylaxis witha current mean CD, count of cells/mm3 (down froma mean of cells/mm3) and remain MAC-free for almostyears.

Clarithromycin as Prophylaxis for MAC Disease

543

CONCLUSION Clarithromycin is of benefit in preventing DMAC infections and is well tolerated in persons with AIDS. Clarithromycin at a doseof 500 mg twice daily appears to be efficacious in preventing disseminated MAC infections and shouldbe considered a good potential agent for MAC prophylaxis.

REFERENCES 1. Horsburgh Jr CR. Mycobacterium avium complex infection in the acquired immunodeficiency syndrome.N Engl J Med 1991; 324:1332-1338. 2. Gikas A, Perrone C, Truffot C, et al. Inhibition of Mycobacterium aviumInintracellulare (MAIC) by antimicrobialsinhumanmacrophages.29th terscience Conferenceon Antimicrobial Agents and Chemotherapy, Houston, 1989; abstr 885A. 3. Dautzenberg B, Legris S, Truffot C, Grosset J. Double-blind studyof efficacy of clarithromycin versus placebo in Mycobacterium avium-intracellulareinfection in AIDS patients. J AmThorac SOC 1990; 141:S615. 4. Data on file. Abbott Laboratories, Abbott Park, IL, 1992.

Clarithromycin Prophylaxis for Disseminated Mycobacterium avium Infection

,

T. M. File, Jr., D. C. Claypoole, S. J. Longstreth, D. J. Signs, W.H.Ruby, and A. S. Indorf Summa Health System Akron, Ohio

INTRODUCTION Disseminated Mycobacterium aviumcomplex (DMAC) is a common AIDSrelated opportunistic infection and increases morbidity and mortality in AIDS patients. One prospective studyof over lo00 patients with advanced HIV infection revealed a40% risk of becoming bacteremic withMycobacterium avium complex after 2 years (1).Previous trials have demonstrated that rifabutin (300 mg PO daily) can reducethe incidence of DMAC infection by approximately 50% (2). The effect on survivalwas less clear, however. Clarithromycin has been shown to be effective in vitro against the Mycobacterium avium complex A recent study by Pierce et al. of 682 patients treated in the United States or Europe to evaluate the efficacy of clarithromycin demonstrated a statistically significant reduction in DMAC as well as a survival benefit in patients treated with clarithromycin versus placebo (4). Further clinical experience in the community will be helpful further in evaluating the utility of clarithromycin. The aim of this study wasto evalu544

Clarithromycin Prophylaxis

DMAC

545

ate the efficacy and safety of clarithromycin as prevention for DMAC in HIV-infected patients in a community setting.

PATIENTS AND METHODS Charts of patients followed at an HIV unit associated with a teaching community hospital were retrospectively reviewed. All patients who received clarithromycin 500 mg bid for MAC prevention were included (including two patients who also received rifabutin at some time concomitantly with clarithromycin). Blood cultures were not routinely done prior to clarithromycin.

RESULTS Fourteen patients were identified who received clarithromycinfor prophylaxis. Patient characteristics and summary of clarithromycin useare shown in Tables1and 2, respectively. The mean CD4 countat or prior to initiation of clarithromycin was 60 cells/mm3( n = 14; range: 2-262). Elevenpatients had CD4 counts of 4 0 0 cells/mm3. The mean duration of clarithromycin was 340 days (range: 132-720). lhelve patients (86%) remained free of DMAC, including 10 patients who diedof other causes. lho patients developed DMAC (at 135 and 167 days, respectively); both hadlow CD4 at the time of clarithromycin initiation (5 and 23 cells/mm3, respectively). adverse effects secondaryto clarithromycin werereported.

DISCUSSION The United States PublicHealthService/InfectiousDiseaseSociety of America Guidelines currently recommended that prophylaxis for DMAC should be considered for HIV-infected adults and adolescents who have a CD4 count less than 75/p1(5).Current data indicate that clarithromycin is effective for this purpose. We found that 12/14 patients (86%) remained free of DMAC during our evaluation-including 10 who died of other causes. patients developed DMAC(at 135 and 167 days, respectively) after initiation of clarithromycin. Both had low CD4 counts (5 and 23, respectively)andpossiblyhadasymptomaticinfection atthe time of clarithromycin initiation. For this reason, the guidelines recommend that DMAC should be ruled out (by a negative blood culture) before prophylaxisis started. Susceptibility data werenotavailable for either of the isolates fromour two patients who developed DMAC.

File et al.

546 Tabk I

PatientCharacteristics

baseline Agelgender Patient

018

47M

10

KS

Cryptococcus 6oM 4

60

CMV

retinitis, PCP 5

5 m PCP

Concurrent therapyb

TMP-SXT, Anti-Retro, IJW, Flu Anti-Retro, Flu, W - S X T Anti-Retro, TMP-SXT Anti-Retro, ganciclovir, Flu, Clinddprimaquin Flu W-SXT,

Flu TB, m, nephropathy KS, PCP PCP, KS PCP, Thrush

59/M

Candidiasis 4uM

5

10 58

CMV retinitis Thrush

"P-SXT, Flu, Anti-Retro Anti-Retro, pentamidine "P-SXT,

Flu Anti-Retro, Flu, W - S X T , rifabutin TMP-sm, Anti-Retro, Flu TMP-SXT, ganciclovir, Flu Anti-Retro, dapsone, Flu, rifabutin Anti-Retro, TMP-SXT, Flu

'opportunistic infections prior to baseline. Toncurrent therapy while receiving clarithrornycin (Anti-Retro = antiretroviral agents; Flu = fluconazole;TMP-SXT = trimethoprimhlfarnethoxazole).

Clarithromycin Prophylaxis

547

for DMAC

n

0

%

0

0

N

File et al.

548

CONCLUSIONS Clarithromycin was well tolerated in these patients. Clarithromycin appears to be effective in preventing DMAC disease. DMAC was observed two in patients who had advancedHIV at the time of initiation of clarithromycin.

REFERENCES 1. Nightingale SD, Byrd LT, Southern PM, et al. Incidence of Mycobacterium avium-intracellulare complex bacteremia in human immunodeficiency viruspositive patients. J Infect Dis 1992; 1651082-1085. 2. Nightingale S, Cameron DW, Gordin FM, et al. ' b o controlledtrials of rifabutinprophylaxisagainst Mycobacteriumavium complexinfection in AIDS. N Engl J Med 1993; 329:828-833. 3. Fernandes PB, Hardy DAYMcDaniel D, Hanson CW, Swanson RN. In vitro and in vivo activities of Clarithromycin againstMycobacterium avium. Antimicrob Agents Chemother 1989; 33:1531-1534. 4. Pierce M,Crampton S, Henry D, Craft C, Notario G . The effect of MAC and its prevention or survival in patients with advanced HIV infection. 35th Interscience Conferenceon Antimicrobial Agents and Chemotherapy, FranSan cisco, 1995; abstr LB-18. 5. USPHS/IDSA Guidelines for the Prevention of Opportunistic Infections in Persons Infectedwith Human Immunodeficiency Virus. Morbidity and Mortality Weekly Report 1995; 44:No. RR-8.

+

Azithromycin + Pentamidine Pyrimethamine in the Prophylaxisof Opportunistic Infections in IHV-Positive Patients withCD4 Count Less Than 200 G. Barbarini, G. Garavelli,

P. Alcini

IRCCS S. Matteo-University of Pavia Pavia, Italy

G. Barbaro University La Sapienza Roma, Italy

B. Division of Infectious Diseases0 O . R R . Foggia ,Italy

A. Lucchini Ser. T. of Gorgonzola Gorgonzola,Italy

Lopez Ser. T. of Abbiategrasso Abbiategrasso,.Italy

Del Buono Ser. T. of Voghera Voghera, Italy Ed0

Ser. T. of Vigevano Vigevano,Italy

549

550

Barbarini et al.

INTRODUCTION Pneumocystis carinii pneumonia (PCP) and toxoplasmic encephalitis are two of the most common opportunistic infections that define the diagnosis of AIDS. In Italy, PCP occurred in 1983-1989,the asAIDS-defining diagnosis before primary PCP prophylaxis was standardized. in about 65% of all cases, After 1989 toxoplasmic encephalitis became the AIDS-defining diagnosis in about 25% of all cases, and todaythe appropriate prophylaxis against this opportunistic infection is also not standardized. W Odrugs have proved effective in primary prophylaxis against PCP [TMP-SMZ (trimethoprid sulfamethoxazole) and aerosolized pentamidine] and they are extensively used today (1).The combination of pyrimethamine and sulfadiazinerepresents the most effective therapyfor cerebral toxoplasmosis; pyrimethamine plus clindamycin has been also reported as successful (2). The risk of a primary episodeof PCP in HIV-positive patients presenting withthan less200 CD4 cells is about 70% and the risk ofcerebral toxoplasmosis about is 30% if they have antibodies against T . gondii. Obviously, the primary preventionof these opportunistic infections is desirable for high-risk patients. Azithromycin, a new azalide(3) with a long half-life and high and sustained tissue concentration without toxicity, has been shown to concentrate in phagocytic cells (4), which could act as a vector to deliver azithromycin to the site of infection. On the basis of studies in animals, azithromycin was considered a candidate for the prevention of cerebral toxoplasmosis in HIV-positive patients and it was included in many primary prophylaxis protocols (5). It was also demonstrated that azithromycin can reduce rates of Mycobacterium avium complex (MAC) infections (6) by about 50% and it may be efficacious against cryptosporidiosis: (7): These two opportunistic infectionsare common in HIV-positive patients presenting with less than CD4 100 cells.

OBJECTIVE The primary endpointof our work wasto verify if primary prophylaxis with azithromycin plus pyrimethamine plus aerosolized pentamidine may reduce the incidence of PCP and cerebral toxoplasmosis in asymptomatic HIV-infected patients presenting with 100-200 CD4 cells. The secondary endpoint was to prove if long-term therapy with azithromycin is safe and tolerable for patients because there is very limited experience with longterm administration.

SUBJECTS AND METHODS Ninety asymptomatic HIV-positive patients (68 males and 22females) presenting with 100-200 CD4 cells and positive toxoplasma serology wereen-

Azithromycin + Pentamidine Table I

+ Pyrimethamine

H N Patients

551

Prophylaxis Dosage Groups

Group 30 Subjects (22 M and 8 F) Mean age: 31 years (range: 20-39) Mean CD4 cells count: 152 (range: 121-194)

Aerosolized pentamidine:300 mg monthly Pyrimethamine: 50 mg daily orally Azithromycin: 500 mg daily orally Group 2 30 Subjects (23 M and 7 F) Mean age: 28.5 years (range:20-35) Mean CD4 cells count: 139 (range: 111-186)

Aerosolized pentamidine:300 mg monthly Pyrimethamine: 50 mg daily orally Azithromycin: 500 mg every 2 days orally Group 3 30 Subjects (23 M and 7 F) Mean age: 29.3 years (range: 23-37) Mean CD4 cells count: 146 (range: 121-187)

Aerosolized pentamidine:300 mg monthly Pyrimethamine: 50 mg daily orally Azithromycin: 500 mg daily for 3 days, followedby 2 drug-free days rolled from June 1994 to March 1995, in a multicentertrial (Pavia, Roma, Foggia, Gorgonzola, Abbiategrasso, Vigevano, and Voghera). All subjects received 50 mg pyrimethamine orally dailyand 300 mg aerosolized pentamidine monthly. Azithromycin was administered orally at three different dosages: (1) mg daily (30 subjects), (2) 500 mg every 2 days (30 subjects), and 500 mg daily for days followed by two drug-free days (Table 1). Thepatients were followedeither to termination of the study (6 months) on 30 September 1995,or longer.

RESULTS All enrolled patients completed at least 6 months of therapy; 25 were treated for 1year and8 for morethan 1year. Therapy was well tolerated in Groups 2 and In Group 1, no patient was able to tolerate the daily administration of azithromycin longer than months, at which time they reverted to thedosage used in Group 2. With this change,the drug was well

Barbarini et al.

552

tolerated and no further stoppage for gastric intolerance was reported. No clinically significant changes in liver function testsor hematology parameters were observed as a consequenceof azithromycin plus pyrimethamine treatment. We did not observe any PCP, MAC, or toxoplasma infection; among treated patients we diagnosed one CMV infection (Group 2), one Kaposi’s sarcoma (Group 2), one esophageal candidiasis (Group 2), and one lymphoma (Group

DISCUSSION Our preliminaryexperiencewith pyrimethamine-azithromycin-pentamidine prophylaxis suggeststhat it is well tolerated if azithromycin is not administered daily and may effectively prevent for 6 months the development of PCP; cerebral toxoplasmosis, and MAC infection in asymptomatic HIV-positive patients with 100-200 CD4 cells. Because longer controlled trials are warranted to establish the definitive efficacy of this prophylaxis, starting fromJune 1995we are conducting a controlled trial in asymptomatic HIV-positive subjectswith 100-200 CD4 cells with pyrimethamine, azithromycin, and pentamidine, and a controlled trial on HIV-positive subjects with >l00 CD4 cells with pyrimethamine, azithromycin, pentamidine, plus rifabutin.

REFERENCES 1. Centers forDiseasesControl.Recommendationsforprophylaxisagainst Pneumocystis carinii pneumonia for adults and adolescents infected with human immunodeficiency virus.MMWR 1992; 41:l-ll. 2. Danneman B, McCutchan JA, Israelski D, et al. Treatment of Toxoplasmic encephalitis in patients with AIDS. A randomized trial comparing pyrimethamine plus clindamycin to pyrimethamine plus sulphadiazine. Ann Inter Med 1992; 116:33-43. 3. Girard AE, Girard D, Englisha R, et al. Pharmacokinetic and in vivo studies with azithromycin, a new macrolide with an extended half life and excellent tissue distribution. 4. Gladue RP, Snider ME. Intracellular accumulation of azithromycin by cultured human fibroblasts. Antimicrob Agents Chemother 1990; 34:1056-1060. 5. Chang HR. Thepotential roleof azithromycin in the treatment on prophylaxis of Toxoplasmosis. Intern J STD AIDS 1996; 7 (suppl1):18-22. of 6. Inderlied CB, KolonoskiPT, Wu M, Young LS. In vitro and in vivo activity azithromycin against the Mycobacterium avium Complex. J Infect Dis 1989; 159:994-997. Intern J STD AIDS 1996; 7 (suppl 7. Hoepelman IM. Human cryptosporidiosis. 1):28-33.

Ix SPECIAL PATHOGENS

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Human and Animal Bite Wounds: Bacteriology, Macrolide Susceptibility, and Therapeutic Potential Ellie J. C. Goldstein and Diane M. Citron Santa Monica-UCLA Medical Center and theUCLA School of Medicine Los Angeles, California

INTRODUCTION The bacteriology of infected bite wounds is diverse and includes aerobic and anaerobic organisms from veterinary, environmental, and skin sources (1-6). Human and animal bites can result in infectious complications including cellulitis, septic arthritis, and osteomyelitis (1,5). Annually they account for approximately 1% of emergency department visits and numerous physician office visits and hospital admissions, often as a result of infection and related problems. Streptococci, Staphylococcus aureus, Staphylococcus intermedius, Pasteurella multocida, Capnocytophaga canimorsus, and oral anaerobes of the Prevotella and Porphyromonas species are frequently isolated from animal bite wounds (Fig. Streptococci, S. aureus, Haemophilus influenzae, Eikenella corrodens, and oral anaerobes are often isolated from human bite wounds. Empirical treatment of infected human and animal bite wounds should include broad coverage against these common pathogens. Erythromycin has been used in the penicillin-allergic patient as an alternative therapy, but it has relatively poor activity against certain bite 555

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Pasteurella Streptococci

SIOphylococcus Fusobacterium Bacteroides Porphyromanas Prevotella Peptostreptococci Enterococci Klebs~e//a/En~r Others

Figure I Approximate frequency of various bacteria growing in infected cat and dog bite wounds. (Adapted fromRef. 1.)

would isolates such as P . multocida, E. corrodens, and many oral anaerobes (6). Previous studies of the activity of the macrolides clarithromycin androxithromycinand the azalideazithromycinagainstcommonbite wound isolates have been reported (2-4). This study reviews and assesses their comparative therapeutic potential and effectiveness as alternatives to erythromycin.

MATERIALS AND METHODS Bacterial strains were isolated from wounds causedby the bites of dogs, cats,andhumans,as wellas other animalsandsaved at -70°C’ for various periods of time in the R. M. Alden Research Laboratory culture collection. Isolates were taken from frozen stock and transferred twice to assurepurityandadequacy of growth.Agardilutiontestingwas performedaccording to NCCLSguidelines;Brucellabloodagarwasused for mostisolates.Antimicrobialagents,azithromycin,clarithromycin, erythromycin, and roxithromycin, were suppliedby the respective manufacturers and reconstituted according to the manufacturers’ instructions into serial twofold dilutions. The plates were inoculated using a Steers replicator and incubated for 24-48 h in the appropriate atmosphere. The minimum inhibitory concentration (MIC) was defined as the lowest concentration of an antibiotic that yielded no growth, a barely visible haze, or one discrete colony.

Bite Wounds: Bacteriology, MacrolideSusceptibility,Therapeutics

557

Table l Activities of Erythromycin, Clarithromycin, Roxithromycin, and Azithromycin Against Clinical Bite-Wound Isolates(MI%, pglml) Species (No. of isolates) ERYTH CLARa

ROXb

2.w

Streptococcur and Enterococcus spp. Staphylococcus aureus Coagulase-negativeStaphylococcus spp. Pasteurella spp. EF-4 Eikenella corrodens Miscellaneous gram-negative bacilli Peptostreptococcus spp. Fusobacterium spp. Prevotella, Porphyromonas, and Bacteroides

AZITH

NIAd 0.5

NIA

NIA NIA

NIA NIA 0.25

“IC,values against Streptococcus spp. (23),S. aureus (13), P. multocidu (16), EF-4 (13), E. corrodens (16), Peptostreptococcus spp. (12), Fusobacterium spp. (20), and Prevotella and Porphyrornonas spp. (29). (From Ref. 3.) bMIC, valuesagainst S. aureus (17), P . multocidu (22), E. corrodens (12), miscellaneous gram-negative bacilli (U), Fusobacterium spp. (H), and nonpigmented (18) and pigmented (16) Bacteroides. (From Ref. 4.) =Does notinclude Enterococcus spp. dNot available. =IncludesEF-4 (7), Actinobacillus actinomycetemcomituns (U), and Haemophilus spp. (3). Source: Adapted from Refs. 2-4.

RESULTS The results of the susceptibility studies are shown in Table 1 as MIC, values. Erythromycin demonstrated limited activity againstP . multocida, E . corrodens, Peptostreptococcus, and Fusobacterium species and limited activity against staphylococci. Clarithromycin more was active than erythromycin against P . multocida (MC, 52.0 and E . corrodens, as well as Prevotella, Porphyromonas,and Bacteroides species, but showed poor activity against Peptostreptococcus and Fusobacterium species. Roxithromycin was less active than erythromycin against all species on a weight basis. Azithromycin was more active against P. multocida ( M I C 4 2 . 0 ml), E . corrodens, and species of Prevotella, Porphyromonas, and Bacteroides and was 4-16-fold more active than erythromycin against Peptostreptococcus and Fusobacterium species.

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DISCUSSION Whereas p-lactam agents remain attractive for the primary therapy of human and animal bite wounds, our in vitro data suggest that the new azalide, azithromycin, has improved in vitro activity comparedto erythromycin against a broad spectrum of bite wound pathogens. Azithromycin and clarithromycin have similar activities against P. multocida, E . corPrevotella and rodens, and EF-4. Althoughbothwereactiveagainst Porphyromonas species, clarithromycin showed a lower MIC, (0.25 pg/ml versus 1.0 pg/ml). Azithromycin, but not clarithromycin, had improved activity againstPeptostreptococcus species andFusobacterium species. Azithromycin appearsto merit further clinical evaluation in the treatment of bite wounds.

REFERENCES 1. Goldstein EJC, Citron DM, NesbitCA, Talan DA, the Emergency Medicine Animal Bite Infection Study Group. Prevalence and characterization of anaerobic pathogens from 50 patients with infectedcat and dog bites. Proceedings of the Society for Anaerobic Microbiology (British), 1995. (in press). 2. Goldstein EJC, Citron DM, Vagvolgyi AE, Finegold SM. Susceptibility of bite wound bacteria to seven oral agents including RU-985, a new erythromycin: Considerations in choosing empiric therapy. AntimicrobAgents Chemother 1986; 29556. 3. Goldstein EJC, Nesbit CA, Citron DM. Comparative in vitro activities of azithromycin, Bay y 3118,levofloxacin,sparfloxacinand 11 other oral antimicrobial agents against 194 aerobic and anaerobic bite wound isolates. Antimicrob Agents Chemother1995; 39:1097. 4. Goldstein EJC, Citron DM. Comparative susceptibilitiesof 173 aerobic and anaerobic bite wound isolates to sparfloxacin, temafloxacin, clarithromycin, and older agents. Antimicrob AgentsChemother 1993; 37:1150. 5 . Goldstein EJC. Bite wounds and infection. Clin InfectDis 1992; 14:633. 6. Goldstein EJC, Citron DM, Richwald GA. Lack of in vitro efficacy of oral forms of certain cephalosporins,erythromycinandoxacillinagainst Pasteurella multocida. Antimicrob AgentsChemother 1988; 32:213.

Azithromycin in the Treatmentof Pertussis in Children: A Pilot Study A. Bate and N. Kuzmanovit University Hospitalof Infectious Diseases "Dr. Fran MihaljeviC" Zagreb, Croatia

T.ZmiC PLNA d.d. Pharmaceuticals Division Zagreb, Croatia

INTRODUCTION National epidemics of pertussis have been controlled in Croatia for over three decades (since 1958) by the universal immunization of infants and children. The vaccination program for pertussis used in 1991 and 1992 resulted in 87.4% and 86.7% coverage, respectively (1). Because of the presence of unvaccinated and incompletely immunized children as well as infants up to months, outbreaks and regional epidemics continue to occur. Thus, pertussis is still a noticeable disease in Croatia with 310 cases reported to the Epidemiologic Service, Croatian National Institute of PublicHealthin1994(2).Erythromyciniscurrentlyrecommendedin the treatment of pertussis.Although a 14-dayerythromycincourseis very effective, the relatively low tolerability and inconvenient dosing of erythromycin prompt further efforts to improve antibiotictreatment of pertussis. 559

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The pilot study was conducted to assess efficacy and safety of azithromycin inthe treatment of pertussis in children.

PATIENTS AND METHODS

Fifteen children of both sexes aged months with symptoms and signs consistentwithpertussis-whoopingcough,presenceofleukocytosis (> with lymphocytosis-were included in the study. Clinical diagnosis of pertussis was confirmed by presence Bordetellapertussis of in culture from nasopharyngeal or pharyngeal swabsor nasal suction aspirates. Only children whose parents provided an informed consent were enrolled. Patients with hypersensitivity to macrolides, severe renal or hepatic impairment, or gastrointestinal tract disturbances which could affect drug absorption were excluded, as were patients who received any antibiotic within h prior to entering the study. Azithromycin (Surnamed, Pliva)was administered as anoral suspension once daily for5 days: mgkg on day followed by 5 mgkg on days Azithromycin was taken h before or 2 h after a meal. All patients were hospitalized. Clinical responseto therapy was assessed at days and after the start of therapy. It was classified as a success (completeor partial disappearance of all baseline signs and symptoms of infection) or failure (no change or worsening of initial condition). Bacteriological examinations were performedat baseline andat days and after the start of therapy. Samples for bacteriological culture were obtained with nasopharyngeal swabs, pharyngeal swabs, and nasal suction aspirates. All specimenswereculturedonRegan-Lowemedium.Bacteriologicalfindings were classified as elimination, elimination with relapse, or persistence of pathogen. All adverse eventsthat occurred duringtreatment were recordedand followed up. Adverse events were scored for severity, duration, and relationship to treatment. Laboratory safety tests (hematology, blood biochemistry, and urinalysis) were undertaken at baseline and at days and after the initiation of treatment.

RESULTS Fifteen hospitalized children, boys and girls, aged months (mean age: months) were included in the study. All children had acute an form of pertussis. B . pertussis was isolated in of patients included inthe study. In all of them, at least one specimen was positive (Table Clinical response to azithromycin therapy is presented in Table

Azithromycin for Children Pertussis in

561

Table Z Baseline Bacteriological Findings in Children with Pertussis

Specimen No. of patients ~~

Nasopharyngeal swab

Nasal suction aspirate

Pharyngeal swab

~

-a

positive specimen positive specimens positive specimens

+ + + +

Total ~~

~

'Absence of B. pertussis in culture. bPresence of B. pertussis in culture. positive In examined specimens.

Eight children missed the final checkup visit (at day 21), probably because they were free of symptoms. The results of the bacteriological findings are presented in Table Seven days after initiation of azithromycin therapy, all bacteriological cultures(taken from nasopharynx,pharynx, and nasal suction aspirate) were negative. At the end of the follow-up period, no relapses or reinfections were observed. Tolerance of azithromycin was very good. side effects were recorded. Laboratory parameTable 2 Clinical Response to Azithromycin in the Treatment of Children with Pertussis

No. of patients Pretreatment Symptoms General Condition Good Moderately altered Cough Weak Moderate Intensive Whooping Cyanosis Vomiting

8

Day 7 (n=15)

Day

Day (n=7)

5

6 8

9

9

6 9 7

2

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Bade et al.

Table 3 Eradication of B. pertussis in ChildrenTreated with Azithromycin

No. of patients Positive culture for B. pertussis Nasopharyngeal swab Pharyngeal swab Nasal suctionaspirate

Pretreatment (n=15)

Day 7 (n=15)

Day 14 (n=14)

13 3 8

0

0

Day 21 (n=7)

ters were within reference limits with the exception of a slight and transient increase in liver enzymes in6 children.

DISCUSSION Azithromycin showed excellent bacteriological efficacy with eradication rate of B . pertussis within 7 days after the start of therapy. Assuming the natural course of pertussis, the reported clinical results could be also considered as successful. The drug was well tolerated. Although the incidence of liver function abnormalitieswas higher than usuallyreported for azithromycin, a causal relation to azithromycin is uncertain, as the majority of patients (10 of 15) already had slightly elevated baseline ALT values. According to our experience, a slight increase of liver enzymes is not uncommon in children with pertussistreated with erythromycin.

CONCLUSION Related to highefficacy,goodtolerability,and short dosageregimen, azithromycin could be a promising agent in the treatment of pertussis in children.However, the observedresultsshouldbeverifiedinalarger comparative study.

REFERENCES 1. Bejuk D, Begovac BaCe A, KuzmanoviC N, Aleraj B. Culture of Bordefeffa pertussis from three upper respiratory tract specimens. Pediatr Infect Dis 1995; 14 (l):@-65. 2. BorEiC B, DobrovSak-Sourek V. The impact of compulsory vaccination against some diseases on their incidence in Croatia. In: Hajsig, D, ed. Vaccinology Today and Tomorrow. Zagreb: Croatian Society for Microbiology, 19954246.

The Useof New Macrolides in Experimental Brucella melitensisInfection R. Lang,D. Torten, B. Shasha, and Ethan Rubinstein Sheba Medical Center and Meir Hospital, Sackler Schoolof Medicine Tel-Aviv,Israel

INTRODUCTION The most effective and least toxic therapy for brucellosis remains an unsettled issue.The frequently used combinations of tetracycline and streptomycin or tetracycline with rifampin usually achieve excellent results. Nevertheless, a variable percentage of failures and recurrencesare reported (relapse rates 8-12%). The new azalides present an opportunity for single-drug therapy for brucellosis, with hopes of shortening the duration of therapy and diminishingthe number of antibiotics administered.

MATERIALS AND METHODS The Brucella melitensis standard smooth strain 16M was used. The strain was inoculated into nonstudy mice to confirm viability and pathogenicity and purify the strain. The organism was cultured brucella agar (Difco) at with 5% CO, to the logarithmic growth phase and stored at until use.

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Susceptibility Testing MICs and MBCs Minimal inhibitory concentrations (MICs) were tested both with the broth microdilution method in microtiter trays and with the agar dilution method usingbrucellaagarcontaininggradedconcentrations of antibiotics at inocula of 105 CFU (colony-forming units) per milliliter. Agarplate results were used to finalize MICs. Minimal bactericidal concentrations (MBCs) were determinedby plating on brucella agar MIC values plus samples from two higher concentrationwells.

Animals Adult white male ICR miceaveraging 20-40 g were used. Mice were housed according to therapeutic groups and were provided with food and water adlibitum.

Inoculation The B . melitensis smooth strain 16M was grown in brucella broth for 4872 h to the logarithmic growth phase. The culture was adjusted (on the basis of viable counts) to yield 4 x 10 to 8 x lo8 CFU/ml. Inoculation was performed by injecting 0.5m1 of culture containing2x105: to 4X lo5organisms in saline, intraperitoneally(IP). Mice were then randomly assignedto treatment and control groups.

Therapeutic Groups Followinganincubationperiod of 7days,animalswereadministered azithromycin clarithromycin (50 or 75 mgkg per day) for 7 or 14 days. Both were administered IP in one (azithromycin) or two divided (clarithromycin) daily doses. Treatment was started 1 week after inoculation. Animals were randomizedto be studied for primary cure or relapse, as well as for control groups.

Primary Cure Mice were sacrificed 7 days after the last antibiotic dose to assure clearance of the drugs which might cause false-negative culture results. Therapy was continued for 7or 14 days.

Relapse Group Azithromycin, 50 mgkg per day, was givenfor 1 or 2 weeks, and75 mgkg per day for 2 weeksto 12,15, and 14 animals, respectively. Clarithromycin,

Macrolides melitensis in B.

Znfection

565

120%

mcm

S

80%

c

60%

LOG

.y

20%

0% Figure I

+ LOG cm 4.46

Cllarithrom 50 mgKgl

Cure rate 7 days following completion of a 7-day course of therapy.

50 mgkg per day, was givenfor 1 and 2 weeks and 75 mgkg per day for 2 weeks to 14 animals in each group.

Assays At the conclusion of the scheduled therapy period, treated and control mice were weighed and sacrificed under ether anesthesia, and their spleens were aseptically removed, weighed, and homogenized in 1.0 m1of sterile saline. The homogenates (0.1 ml) were diluted in saline at decimal dilutions and plated brucella agar. Plates were incubated at with 5% CO, for 48-72 h and B. melitensis colonies were counted. Each procedure was performed in triplicate andthe results averaged and expressed as a decimal logarithm.

Data Evaluation Cure was defined assterilization of spleens and reductionof the log CFU of Brucellae cultured fromthe nonsterile homogenized spleens.

Lang et al.

566

STATISTICAL ANALYSIS The cure rate was evaluated statistically by use of the chi-square test. A comparativeanalysis of meanlog CFU was performedbetweenmice treated with the different regimens andnontreated mice.

RESULTS Therapeutic Outcome by Groups Primary Cure: One of 26 untreated animals sacrificed parallel to thethera-

peuticgroupswascured(cure rate: 3.8%). All animals treated with azithromycin 50 mgkg per day, for 1week or for 2 weeks (15 in each group) were cured. This represents a100%cure rate (Figs. 1 and 2). Following 1 week of treatment with clarithromycin 50 mgkg per day, 10of 15 animals were cured (66.6% curerate), and following 2 weeksof the same regimen, 11 of 13 mice were cured (cure rate: 84.6%). The log CFU of Brucellae

120% LQGCFU

100%

k E

80%

60% 40%

20%

0% 50 mg/Kg/day

50 mg/Kg/day

Figure 2 Cure rate 7 days following completion of a 14-day course of therapy.

Macrolides in B. melitensis Infection

567

120%

100%

S

80%

W

.c,

60%

a 40%

20%

0% SO mg/Kg/;lay

50 mg/Kg/day

Figure 3 Cure rate 4 weeks following completion of a 7-day course of therapy.

isolated fromthe spleen, averaged 3.24 for animalstreated for 1 week and for animals treated for 2 weeks. Relapse groups: Tivo of nine (22.2%) control animals were cured. In the azithromycin 50-mgkg per day group, 11 of 12 animals sacrificed 4 weeks after completion 7 daysof therapy were cured(91.6% cure rate) and 14 of 15 animals treated by the same dose for 14 days were cured (cure rate: 93.3%). The mean log CFUs of infected spleens were1.6 and 2.3, respectively. In the azithromycin 75-mgkg per day group 13 of 14 animals were cured at the relapse end point (92.9%), and the log CFU of the single infected animalwas 1.6. (See Figs. and 4.) In the clarithromycin 50-mgkg per day l-week therapeutic group, 4 of 14 had sterile spleens (28.6% cure rate). The mean log CFU of the infected animalswas 3.48. In the clarithromycin 50-mg/kgper day 2-week therapeutic group, all14 animals were infected4 weeks after completion of therapy (0% cure rate). The mean log CFU was 3.56. Clarithromycin 75 mgkg per dayfor 14 daysresulted in no cure (100% infection)when animals were sacrificed 4 weeks after terminationof therapy. The mean log CFU was 3.46.

h n g et al.

568

120% LOG

100%

8.

80%

W

E

60%

40%

20%

0% AzithromycinClarithromycin

Figure 4 Cure rate 4 weeks following completion of a 14-day course of therapy.

DISCUSSION AND SUMMARY The nearly 100%efficacy of azithromycin 50 mgkg per day in the primary cure of brucellosis following1and 2 weeks of therapy, demonstratedin the present experiment, has not yet been reported with any single antibiotic agent. This therapeutic effect was maintained for 1month and prevented relapses, an effect hitherto undescribed. In view of these results and the therapeutic failures with other macrolides and fluoroquinolones, plusthe drug’s known safety and tolerance profiles,we suggest a possible role for azithromycin in the treatment of human brucellosis with special emphasis on the potentialmanagement of brucellosisininfantsandpregnant women.

X NONANTIBACTERIAL EFFECTS

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In Vivo Effectof Azithromycin Subinhibitory Concentrations on the Mortality of Experimental Pseudomonas aeruginosa Sepsis M. N. Marangos, M. E. Klepser, D. P. Nicolau, Charles H. Nightingale, and Richard Quintiliani Hartford Hospital Hartford, Connecticut

INTRODUCTION Pseudomonas aeruginosa remains one of the most prevalent and clinically significant pathogens involved in nosocomial infections. Owing to its synthesis of extracellular products (e.g., exotoxin A, proteases, cytotoxins, hemolysins), P . aeruginosa has the ability to colonize, break down physical barriers, impair host defenses, and damagethe host. Previous in vitro and in vivo studies have shown that different antibiotics inhibitthe expression of the exoenzymes of P . aeruginosa Molinari et al. have demonstratedthat macrolideshzalides as a class share the potential to downregulate the expression of these virulence factors in vitro at sub-MICs(minimalinhibitoryconcentrations). Of thisclass,azithromycin,anazalide,exhibited the highestinhibitoryeffectonenzyme expression (3,4). 571

Murungos et al.

572

The aim of this study was to investigate the effect of azithromycin sub-MICs on mortality in an experimental P . ueruginosu sepsis model.

MATERIALS AND METHODS Antibiotics The following antibiotics were used: azithromycin (&er Inc., New York, and ceftazidime (Eli Lilly& Company, Indianapolis, IN).

Bacteria Ten clinicalisolates of P.ueruginosu acquired atour institution were evaluated for the production of exoenzymes.

Susceptibility Testing The MIC and MBC (minimal bactericidal concentration) were determined for eachantimicrobial by the microdilutiontechniqueusingcationsupplemented Mueller-Hinton (MH) broth (Difco Laboratories, Detroit, MI).

Determination of Exoenzyme Production Enzyme activities and pyocyanin production were qualitatively evaluated employing separate agar plates by spotting a 10-p1 inoculum from broth cultures. Lecithinase production was tested by adding 10% (v/v) egg yolk enrichment to tripticase soy agar plates (Difco). A white precipitate around or beneath the inoculum spot indicated lecithinase activity. Elastase activity was measured on nutrient agar plates containing 1%elastin. Plates were incubated at 37°C for 48 h and then transferredto room temperature for 3 additional days. Clearingof the opaque medium aroundthe inoculum spot was taken to indicate elastase activity. DNAse production was detected on DNAse plates. After 72 h of incubation at 37"C, the plates were flooded with 1N HCl and a clear zone around the growth area manifested DNAse activity. Hemolysins were detected after 72 hof growth at room temperature on MH agar plates containing5% sheep red cells. Gelatinase production was determined on MH agar plates containing 0.4% gelatin. After incubation, the plates were flooded withsaturated ammonium sulfate and examined for zones of clearingaround the inoculum.Pyocyaninand mucoid production were evaluatedby examining the color and consistency of the colonies on MH agar plates after 48 h of incubation.

Effect of Azithromycin on Mortality of P. aeruginosa

573

Animals Swiss Webster mice (20-25 g) were obtained and caredfor according to the guidelines providedby the U.S. Department of Health andHuman Services.

Determination of the Minimum Lethal Dose Groups of15-30mice were infected intraperitoneally with a range of inocula in a 3% mucin solution. A total of 0.5 m1of organism suspension was injected and the minimum lethal dose (MLD) was the dose that resulted in 100% mortality within36 h of inoculation.

Determination of the Protective Dose One hour followingthe intraperitoneal inoculationof P . aeruginosa at the MLD, groups of 10 mice were injected with 0.2 m1 of ceftazidime subcutaneously (SC) in various dosing regimens. The dosing regimen that produced a survival rate of 25-40% on day 6 following inoculation wasto be selected asthe regimen in the comparative trial. Ceftazidimeat 375 mgkg SC 2 dosesat 4-h intervals protected25% of the animals fromthe MLD.

Comparative Survival Study inoculum of lo7 CFU (colony-forming units) in a 3% mucin solution was determined to be the MLD and was injected intraperitoneally. Onehour postinoculation,mice were randomizedto receive ceftazidime(CFZ, 375 mgkg SC 2 doses) aloneor in combination withAZ (20 mgkg SC 1dose). llvo other groups received either AZ alone or no treatment (control). All mice were observed, and mortality was recorded every 12 h over a 72-h period. In addition, another group of 24 mice received a single 2O-mgkg SC dose of AZ todetermine the pharmacokinetic profileof the agent. Animals were sacrificed at times between 0.25 and 6 h postdose. Serum concentrations were determined using a validated high-performance liquid chromatographic assay.

RESULTS One of the 10 isolatesthat displayed activity of all enzymes was selected for the in vivo study.The MIC/MBC values for this nonmucoid isolateto AZ and CFZ were 32/64 and 4/4 pg/ml, respectively. As shown in Table1, all animals in the untreated control and AZ alone groups died within 24 hof the intraperitoneal inoculation with P . aeruginosa. However, as displayed in Fig. 1, a significant(p < .01 usingthe logrank test) decrease in mortality

Marangos et al.

574

Tabk l Percent Survival After Treatment with CFZAlone, AZ Alone, AZ, or No Treatment (Control)

+

Timea (h)

60 Control AZ Alone CFZ Alone + AZ

0

0 0

'Postinoculation.

rate occurred during the first h postinoculation in animalstreated with CFZ plus AZ compared to CFZ alone. This beneficial effectof AZ treatment wasno longer evident atthe 48-h observation period. Serum concentrations of AZ obtained from animals receiving the single 20-mgkg SC dose revealed a mean peak concentration of 1.47 pg/ml at 0.25 h postdose and a mean concentration of 0.14 &m1 at 6 h postdose. Thus, serum concentrationsof AZ were well belowthe MIC (32 pg/ml)of the test organism.

I

0

12

24

36

48

60

72

T h e (hours)

Figure l Percentage survival after treatment with CFZ alone and in combination with AZ.

Effect

Mortality of P. aeruginosa

Azithromycin on

575

DISCUSSION When given alone in the P . aeruginosa peritonitis-sepsis model, azithromycin does not improve survival overuntreated controls and providesno measurable antibacterial effect. However, when azithromycin is given at sub-MICs with ceftazidime, there appears to be a significant reduction in mortality rate compared with animalstreated with ceftazidime only. These observations suggest that the suppression of pseudomonal exoenzymes by azithromycin, as reported by Molinari et al., might be responsible for the improvement in survival when the animals are treated with the combination The protective effect shownby azithromycin also might berelated to factors other than exoenzyme. suppression (e.g., the host's defense tem). Further studies are required to clarify the protective mechanisms. .

CONCLUSION

Our data indicate a potential adjunctive role for azithromycinthe in treatment of P. aeruginosa infections despite a lack of specific activity for this pathogen. Our observations underscore the complexity of the interactions among the organism, sub-MICs .of azithromycin, and the need for additional studyto elucidate the mechanism responsiblefor these observations.

REFERENCES 1. Grimwood K, To M, Rabin HR, Woods, DE. Inhibition of Pseudomonas aeruginosa exoenzyme expression by subinhibitory antibiotic concentrations. Antimicrob Agents Chemother 1989; 33:41-47. 2. Kita E, Sawaki M, Oku D, Hamuro-'A, Mikasa K, Konishi M, Emoto M, Takeuchi S, Narita N, Kashiba S. Suppression of virulence factors of Pseudomonas&eruginosa by erythromycin. J. AntimicrobChemother1991; 27: 273-284. 3. Molinari G, Guznlan' A, Pesce A, Slchito GC. Inhibition of Pseudomonas aeruginosa virulence factors by subinhibitory concentrationsof azithromycin and other macrolide antibiotics. J Antimicrob Chemother 1993; 31:681-688. 4. Molinari G, Paglia P, SchitoGC.Inhibition of motility of Pseudomonas aeruginosa and Proteus mirabilis by subinhibitory concentrations of azithromycin. Eur Clin Microbiol Infect Dis 1992; 11:469-71.

Clarithromycin Reduces Cl-Dependent Transepithelial Potential Difference in Tracheal Mucosa of Anesthetized Rabbits J. Tamaoki, H. Takemura, E. Tagaya, Y. Takeda, K. Konno Tokyo Women’sMedical College Tokyo, Japan

INTRODUCTION Long-term administrationof eiythromycin is effective inthe treatment of diffusepanbronchiolitisprobablythroughactions other than its antimicrobial properties (1). Although the mechanism of the effect is uncertain, immunomodulatoryactiononinflammatorycells,inhibition of mucus secretion and inhibition of airway epithelial Cl transport and subsequentwatersecretion havebeenproposed.However, the effect ofnewlydevelopedmacrolides on Clsecretionisunknown and, previous in vitro findings do not necessarily reflect ion transport function in vivo because of the lack of innervation and blood supply. Therefore, to determine whether the newmacrolideinhibitsClsecretionin vivo,we studied the effect of clarithromycin(CAM) on Cldiffusion potential difference (PD) (4) across the tracheal mucosa in anesthetized rabbits; 576

Reduction

Cl-PD in Tracheal Muscosa

with Clarithromycin

577

MATERIALS AND METHODS Measurement of PD of Tracheal Mucosa Male Japanese white rabbits were anesthetized, and a polyethylene tube was canulated 5 mm above the carina, through which artifical ventilation was performed. Cartilage rings of the upper trachea were incised transaxially and the surface of the membranous portion was fully exposed. The exploring bridge was placed the on surface of the tracheal mucosa(5), and contactwith the tracheal surface was ensured by continuous perfusion (0.3 mumin) through the bridge with Krebs-Henseleit (KH) solution. The perfusion reservoir was connected to a calomel half-cell via a polyethylene tube filled with KH solution in agar. The reference bridge, a 21-gauge needle that contained KH solution in agar, was inserted intothe subcutaneous space of the right anterior chest wall. Each bridge was connected by a calomel halfcell to a high-impedance voltmeter, and the electrical signal (transmembrane PD) was monitored.

Effect of CAM on Cl-PD

To measure Cl-PD of the tracheal mucosa, superfusing KH solution was switched to one that contained amiloride (10-4M), a Na channel blocker. This maneuver decreasesPD, and the remaining PD (Cl-PD) is an index of epithelial cellular and paracellular paths available for Cl diffusion. When C1PD became stable, the amiloride-containing solution was changed to a similar solution that contained CAM and 10-4M), or CAM (10 m a g ) was administered throughthe jugular veinby a bolus injection. To determine the dose-response relation, several doses of CAM (1,3, 10, and30 m a g , IV) or its vehicle (saline) were administered in a cumulative manner; in every case, the next dose of CAM was given 5 min after the response of Cl-PD to a given dose plateaued. To test the effects of other antibiotics, erythromycin (EM), aminobenzyl penicillin (ABPC), cefazolin(CEZ), or amikacin (AMK) at 10 mg/ kg was intravenously administered, andthe change in Cl-PD was continuously recorded. RESULTS demonstrated inFig. 1, the baseline value of in vivo PD of rabbit tracheal mucosa was 17.8 -t 1.9 mV (n =14), lumen negative. Application of amiloride to KH solution reduced PDto 11.0 -t 1.2 mV (n = 14), which isreferred to asCLPD.Subsequentapplication ofCAM ator M did not significantly alter the Cl-PD. Onthe other hand, intravenous admin-

578

Tamaoki et al.

20 -

-

-\,

CAM (perfusion)

n a. 10

-

CAM (i.v.)

1 min

Figure I Representative tracing of rabbit tracheal potential difference (PD) in vivo. Addition of amiloride(AML, M) to Krebs-Henseleit solution perfusing the tracheal mucosa decreased PD, which referred to as Cl diffusion PD (Cl-PD). When Cl-PD became stable, clarithromycin (CAM) was added to the perfusion media at lod5M or administered by bolus injection from jugular vein at m a g .

*** ***

"

Control

1

'0

CAM ( m g W

Figure 2 Dose-dependent effect of clarithromycin (CAM) on Cl diffusion potential difference (Cl-PD)of rabbit tracheal mucosain vivo. CAM was intravenously administered in a cumulative manner, the andplateau valueof Cl-PD in response to each dosewas determined. Values are expressed asthe decrease in Cl-PD fromthe baseline values. Data are means SE; n = 9 for each point. **p < ***p < significantly different from control values.

*

Reduction

579

Cl-PD in Tracheal Muscosa with Clarithromycin

Table l Effects of Antibiotics on Cl Diffusion Potential Difference Across Rabbit Tracheal Mucosa

Cl diffusionpotential difference

(mV) Before Control CAMa EMa ABPCa CEZa AMP

After

f

2

f

f f

f f

*

f 0.5

f f

Difference f

* f f

*

Note: Each drug was intravenously administered at10 m a g . In the control experiment, the same volume of saline alone was given. Values are means2 SE; n = 8 for each group. ***p < .001,significantly different from valuesfor placebo. C A M , clarithromycin; EM, erythromycin; ABPC, aminobenzyl penicillin; CEZ,cefazolin; A M K , amikacin.

istration of CAM (10 m a g ) rapidly decreased Cl-PD from 10.8 2 0.7 to 6.9 0.4 mV (p < .001, n = 7) within 2 min. As shown in Fig. 2, intravenous CAM reduced Cl-PD in a dose-dependent fashion, the maximal decrease in Cl-PD fromthe baseline value andthe apparent dose requiredto produce a half-maximal effect (EC,,) being 5.6 2 0.9 mV (p < .001, n = 9) and 2.7 mg/kg, respectively, whereas administration of the vehicle alone had no effect. Effects of several antibiotics (10 m a g , IV)on Cl-PD are shown in Table 1. Each CAM and EM dose decreased Cl-PDby 5.1 2 mV (p < .001, n =S) and 5.6 2 0.7 mV (p < .001, n = 8), respectively, but other antibiotic agents did not significantly alter Cl-PD.

DISCUSSION Our present studieson the transepithelial bioelectricproperty provide indirect evidence that CAM may inhibit Cl secretion across rabbit tracheal mucosa in vivo. This notion is based on the finding that Cl-PD, apotential difference between the submucosal space and the mucosal surface of the trachea recorded under open-circuit conditions the presence in of amiloride, was decreased by CAM. The inhibitory effect on Cl-PD was likewise observed with another macrolide antibiotic agent EM, but other types of antibiotic suchas ABPC,

Tamaoki et al.

580

and AMKhadnoeffect.Thesefindings are inaccordancewith previous observationsin vitro suggesting that the effect may be specific for macrolides. Because the inhibition of C1 secretion resultsin the reduction of subsequent movement of water toward the lumen, the effect of CAM may be associated with the inhibition of water secretion, thereby causing a decrease inthe amount of airway secretions.

CONCLUSION Macrolide antibiotics specifically decrease Cl-PD of tracheal mucosa in vivo, an effect that mayresultin the corresponding decrease in water secretion from the submucosa toward the airway lumen, thereby possibly implicating one of the mechanisms ofthe efficacy of macrolides on patients with chronic airway diseases who have copious amounts of sputum.

REFERENCES 1. Kadota J, Sakito 0,Kohno S , et al. A mechanism of erythromycin treatment in patients with diffuse panbronchiolitis. Am Rev Respir Dis 1993; 147:153-159. 2. Goswami SK, Kivity S, Marom Z. Erythromycin inhibits respiratory glycoconjugate secretion from human airways in vitro. Rev Respir Dis 1991; 141~72-78. 3. Tamaoki J, Isono K,Sakai N, et al. Erythromycin inhibits Cl secretion across canine tracheal epithelial cells.Eur Respir J 1992; 5234-238. 4. Roszkowski K, Beuth J, KO HL, et al. Comparative study onthe macrolides erythromycin and clarithromycin: antibacterial activity and influence on immune responses. Zbl Bakt 1990; 273518-530. 5. Boucher RC, Bromberg PA, GatzyJT. Airway transepithelial electric potential in vivo: species and regional differences. J Appl Physioll980; 48:169-176.

Inhibition of Adherence of KZebsieZla pneumoniue Strains to Intestine-407 Cell Lines by Roxithromycin S. Favre-Bonte, C. Forestier, A. Darfeuille-Michaud, C. Rich, J. Sirot, and B.Joly Universitt!#Auvergne-Clermont I Clermont-Ferrand,France

INTRODUCTION Klebsiella pneumoniue accounts for 10% (among the Enterobacteriacae family) of nosocomial infections in intensive care units. It is responsiblefor urinary and respiratory tract infections, septicemia, and meningitis (1). Epidemiological studies have shown that infection is precededby intestinal colonization (2). Mucosal colonizationby bacteria is always linked to adhesion processes K.pneumoniue adherence can be reproduced in vitro using intestinal cells in culture. This study was designedto determine the effect of subinhibitory concentrationsof roxithromycin on the adhesion of K.pneumoniue strains to Intestine-407 cell lines.

MATERIALS AND METHODS Bacterial Strains K . pneumoniue CF504 and K . pneumoniue LM21 were obtained from clinicalisolatesinvolvedinnosocomialinfections(collection

of J. Sirot). 581

582

et

Favre-Bonte

al.

K . pneumoniae CF504 adheres with a diffusepattern in which the adhesion CF29K is involved (4).K . pneumoniae LM21 presents an aggregativepattern characterized by the formation of a bacterial clusteron the intestinal cell surface (5). Cell Cultures The Intestine-407 (Int-407) cell line derived from human embryonicjejunum and ileum was used. The cells were cultured in Eagle medium supplemented with 10% heat-inactivated fetal calf serum.

Adherence Assays Bacteria were suspended in the cell culture medium containing 2% D mannose. lo8 bacteria were addedto confluent monolayers of Int-407 cultured in 24-well plates and incubated for 3 h at 37°C. After incubation, each well was rinsedthree times in phosphate-buffered saline and released by the addition of 0.5% Triton X-100. Adherent bacteria were quantified by plating appropriate dilutions on Luria Bertani agar medium.

Adhesion Inhibition Tests The inhibitory effectof roxithromycin was studied following two protocols.

Protocol A: Roxithromycin was added to the culture medium of the bacteria. Bacteria weregrown for 18 h at 37°C in brain-heart infusion medium containing subinhibitory concentrations of roxithromycin. The bacteria were then centrifuged, washed, and allowed to adhere as described earlier. ProtocolB: Roxithromycin was added to the culture mediumof the Int407 cells. Bacteria were suspendedin the cell culture medium containing subinhibitory concentrationsof roxithromycin and addedto the Int-407 cells. Adherence assays were performed as above.

RESULTS The results are summarizedinFig. 1. Whenadded to the cell culture medium (protocol B), roxithromycin inhibits adhesion of K.pneumoniue CF504 from 80% for minimal inhibitory concentration (MIC)/2 to 46.5% for MIC/8 (Fig. 1A). This result is similar forK . pneumoniue LM21 (Fig. lB), for which adherence inhibition ranges from 96.75% (MIC/2)to 42% (MICB). According to protocol A (when roxithromycin was addedto the culture mediumof bacteria), adherenceof the K.pneumoniae strains is diminished by more than 50% only for MIC/2 (Figs. 1A and 1B).

Adherence of K.pneumoniae to Intestine Cell

Lines

583

A. Adherence of K. pneumoniae CF504

l

Control

MIC12

MIC14

MlC10

MIC116 MIC132

B. Adherence of K. pneumoniae LM21

l 100

5

80 60

(I)

40

20

0 Control

MIC12

MIC14

MlC18

MIC116 MIC132

Adherence of Klebsiella pneumoniae strainsinthepresence of subinhibitory concentrationsof roxithromycin.CFU = mean numberof bacteria adhering per c m 2 of tissue culturewell. live assays were performedin duplicate.

Figure l

DISCUSSION significant adherence inhibition ofK.pneumoniae by subinhibitory concentrations of roxithromycin was obtained only whenthe drug is added to cell culture medium containing the bacteria (protocol B). Furthermore, this effect occurs independently of the type of adherence factor because it is the same with K.pneumoniae CF504 (which presents a diffuse adherence

584

Favre-Bonte etal.

pattern) and with K. pneumoniue LM21 (which presents an aggregative adherence pattern). Therefore, we speculate that the effect could be related to a modification of the surface or the metabolism of eucaryotic cells by roxithromycin. We obtained similar results when the adherence assays were performed with Caco-2 cellsthat produced microvilli after 15 days of culture (6). Thus, adherence inhibition of K.pneumoniae by roxithromycin is not limited to bacteria adhering to Int-407. Moreover, because microvilli produced by Caco-2 cells have the same properties as human enterocytes, we can postulate that adherence inhibition also could be produced in vivo. Thus, it may be possible that roxithromycin could prevent colonizationof the gut by K . pneumoniae.

CONCLUSIONS Whenadded to Int-407cells(Protocol B), roxithromycinhasa strong inhibitory effect (more than 40%) on the adherence of two K.pneumoniue strains at concentrations ranging fromMW2 to MIC/8. This effectof subinhibitory concentrationsof roxithromycin could be related to a modification of the INT-407 cells because it occurs when roxithromycinis added in the culture medium of the Int-407 cells and it is independent of the pattern of adhesion. Because the same effect was obtained with cultured Caco-2 cells (which have the same properties as human enterocytes), it can be postulated that roxithromycin could prevent the in vivo adherence of K. pneumoniae to enterocytes, whichis the initial step in the gut colonization.

REFERENCES Markowitz SM, Veazey JM, Macrino FT+ Mayhall CG, Lamb VA. Sequencia1 outbreaks of infection due to Klebsiella pneumoniaein a neonatal intensive care unit: implication of a conjugative R plasmid. J Infect Dis De Champs C,Sauvant MP, Chanal C, Sirot D, Gazui N, Malhuret R, Baguet JC, Sirot J. Prospectivesurvey of colonization and infectioncaused by extended-spectrum-P-lactamase-producingmembers of the family Enterobacteriaceae in an intensive care unit. J Clin Microbiol Finlay BB, Falkow S. Common themes in microbial pathogenicity. Microbiol Rev Darfeuille-Michaud A, Jallat C, Aubel D, Sirot D, Rich C, Sirot J, Joly B. Rplasmid-encoded adhesive factor Klebsiella in pneumoniae strains responsible for human nosocomial infections. InfectImmunoll992; 60:44-55.

Adherence

K.pneumoniae to Intestine Cell Lines

585

5. Favre-Bontk S, Darfeuille-Michaud A, Forestier C. Aggregative adherence of Klebsiella pneumoniae to human Intestine-407 cells. InfectImmunol1995; 63~1318-1328. 6. Joly B, Darfeuille-Michaud A, Rich C, Sirot D, Sirot J, Cluzel R. Inhibition of Klebsiella pneumoniae adherence to human intestinalcells by roxithromycin. 19th ICC, Montrkal, 1995.

Clinical and Immunological Studyof Roxithromycin on Infectious Diseasesin Obstetrics and Gynecology K. Izumi, H. Mikamo, K. Kawazoe,

T. Tamaya

Gifu University Schoolof Medicine Gifu, Japan

INTRODUCTION Evidence accumulated in recent years has shown that macrolide antibiotics have a wide varietyof pharmacological effects.In particular, the activities of erythromycin (EM) have been extensively studied, demonstrating that EM reduces the motility and the production of oxygen species in polymorphonuclear leukocyteswhile it enhances phagocytosis by macrophages and cytokine productionin leukocytes It also has beenreported that along-termadministration ofEM reduces the severity of bronchial hyperresponsiveness in bronchial asthma Roxithromycin (RXM), one ofnew macrolides,hasbeenused therapeuticallybecauseitseffects are similar to those of EM. In this study, we investigated the effect of long-term administration of RXM on clinicalresponsesandimmuneresponses(changes of neutrophil count and Interleukin-8 level) in patients with chronic intrauterine infection or pyometra. 586

Roxithromycin on Infectious Diseases in OBIGYN

587

MATERIAL and METHODS Subjects Eighteen patients with intrauterine infections diagnosed by transvaginal ultrasound tomography, uterine tenderness, and bacterial presence and 5 healthy womenweresubjected to the study.Nowomanreceivedany antimicrobial drug before this trial.

Drug Administration One hundred fifty milligrams of RXM was administered orally once a day for months to the infected patients.

Sampling Method disposable Nelaton catheter connected to a microsyringe was inserted into the uterine cavity, bluntly or after cervical dilatation. Sequentially, 5-m1 aliquots of phosphate-buffered saline (PBS) were instilled, followed by immediate aspiration,which wasdone twice. Bacteria were cultured and identified inthe aspirate fluid.

I a 300

200

5

-

100 0

0

Normal

Pyometra

Figwe I Neutrophil percentage (right) and Interleukin 8 (left) in the lavage fluid of the uterine endometrial cavity.

588

Izumi et al.

Assessment Each patient was completely evaluated clinicallyfor temperature, subjective (pelvic pain) and objective (uterine tenderness) findings, white blood cell count ( W C ) , erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP). The clinical responses were classified as excellent, good, and poor. Excellent or good were defined as resolution or improvement, respectively, of all clinical symptoms and laboratory findings at the end of treatment and during the postantibiotic follow-up period. Either inapparent or incomplete clinicalresponse to therapy or relapsewasconsideredpoor. For bacteriological evaluation, bacteriological response was graded as follows: (a) eradicated, (b) decreased, (c) unchanged, and(d) replaced.

Investigation Materials Neutrophil counts and Interleukin-8 (IL-8) levels were determined in lavagefluid of the uterineendometrialcavityobtained from 5 healthy women and the 18 infected patients.

'

I

betore

Figure 2 Neutrophil percentage (right)and Interleukin 8 (left) in the lavage fluid of the uterine endometrial cavity obtained frompatients with pyometra beforeand

after roxithromycin treatment.

Roxithromycin on Infectious Diseases in OBIGYN

589

0

Neutrophil In the lavagefluid (%)

Figure Correlation between the count of neutrophils accumulated and the level of Interleukin-8 inthe lavage fluid.

RESULTS Of the 18 patients, 16 (88.9%) experienced excellent or good improvement, showing significant improvements in WBC count, ESR, and CRP levels. %o of 18 (ll:l%) or 10 of 18 (55.6%) patients showed bacterial eradication or decrease, respectively. The results of neutrophil counts and Interleukin-8 determination in lavage fluid fromthe uterine endometrial cavity are summarized in Figs. 1 and 2. There was a close correlation between the neutrophil count and IL-8 levels in the lavage fluid from infected patients (Fig.

DISCUSSION Our study demonstratedthat patients with intrauterine infection had significantly higher percentages of neutrophils in pretreatment endometrial lavage fluids than did normal women. The neutrophil percentagewas significantly decreasedwith RXM treatment.

Izumi et al.

590

The accumulation of neutrophils in the inflammatory uterine cavity predicts neutrophil oxidative and proteolytic products,which are capable of producing endometrial damage in the uterine cavity(4,5). number of novel chemotactic cytokines are becoming increasingly recognized as important participants that contribute to the migration of specific inflammatory cells fromthe peripheral blood to inflammatory sites.Recent observations have demonstrated that each chemotactic cytokine carries a specificity for the individual movement of immune/inflammatory cells. IL-8 has been identified as a neutrophil chemotactic factor (6). These results indicatethat RXM decreases uterine endometrial cavity inflammation through a reduction in neutrophil migration to the inflammatory sitesand is effectiveon chronic intrauterine infection.

REFERENCES Kadota J, Sakito 0, Kohno S , Sawa H, Mukae H, Oda H, Kawakami K, Fukushima K, Hiratani K, Hara K. A mechanism of erythromycintreatment in patients with diffuse panbronchiolitis.Am Rev Respir Dis Yagyu Y. Analysis of the long-term treatment with erythromycin in chronic lower respiratory tract infections: I. Effects on human polymorphonuclear leukocyte functions. J Nara Med Assoc Mikami M.Clinical and pathophysiological significance of neutrophil elastase in sputum andthe effect of erythromycin in chronic respiratory diseases. Jpn J Thorac Dis 4. Nelson S, Summer WR, Terry PB, Warr GA, Jakab GJ. Erythromycininduced suppressionof pulmonary antibacterial defences: a potential mechanism of superinfection in the lung.Am Rev Respir Dis 5. Hirata T, Matsunobe S, Matsui Y, Kado M, Mikiya K, Oshima S. Effect of erythromycin on the generation of neutrophil chemiluminescencein vitro. Jpn J Thorac Dis Kunkel SL, Standiford, T, Kasahara K, Strieter RM. Interleukin-8 (IL-8): The major neutrophil chemotactic factor in the lung. Exp Lung Res

XI CLINICAL STUDIES: OTITIS MEDIA

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Five-Day Treatment of Acute Otitis Media in Children with Clarithromycin Dimitris A. Kafetzis, Theodore Bairamis, Dimitra Dinopoulou, Stamatina Vlachou,and Nicholas Apostolopoulos University of Athens “A. Kyriakou” Children’s Hospital Athens, Greece

INTRODUCTION Acute otitis media is common a infectious disease and is a major reason for seeking medicalattention during early childhood. Duringthe preantibiotic era, resolution required myringotomyor followed spontaneous perforation of the tympanic membrane, whereas severe intracranial complications developed in approximately of cases. In recent decades, antibiotics are usually prescribed for a 10-day treatment period, and complications have been almost eradicated with an incidence of lessthan 0.15% (1). Seven-day antibiotic therapy also has beenwell accepted and proven effective In some studies, however, it has been shown that even ashorter duration of therapy could be as effective(3,4). We report the results of an open, randomized clinical study comparing the efficacy and safety of clarithromycin with cefuroxime-axetil, each given orallyfor 5 days forthe treatment of pediatric patients suffering from acute otitis media.

593

Kafetzis et al.

594

PATIENTS AND METHODS Patients were seen in the outpatient clinic of the “P. & A. Kyriakou” Children’s Hospital in Athens and were eligible for the study if they had a diagnosis of acute otitis media with fever > 38”C, otalgia, or irritability, with otoscopic findings ofan erythematous bulging and opacified tympanic membrane. To be considered eligible for the study, a parentof each patient had to give hisher informed consent. Patients were excluded from the study if they had received any antibiotic treatment during the last 2 weeks before enrollment, had an underlying disease that might affect the pharmacokinetics of the medication or the outcome of the study, had a perforated tympanic membrane or if complication of otitis mediawas present atthe time of enrollment. Each patientwas randomly assignedto either antibiotic for 5 days and was reevaluatedat the end of treatmentandfollowed for 30days. Wpanocentesis was performed when patients failed to respond to treatment or if there was arecurrence of the infection. In all patients, tympanometry was performed priorto treatment and posttreatment. Forty patients were enrolledin the study andtheir demographic characteristics are shown in Table1.

RESULTS

No significant differences were found between the treatment groups. Patients in both treatment groups were similar with regard to their medical histories, physical examination results, and baseline otoscopy and tympan metry results. A clinical response to therapy could be determined for all Table I

Patient Demographics Cefuroxime-axetil Clarithromycin

No.of Patients

20

Males Females Age median (months) Range Mean weight (kg) Dosage (mgkg) (every 12h) Prior history of otitis media episodes 1episode >l episodes

12

20 11

21 6-48 12.1 7.5

22 6-52 12.4 15

4

5 12

Treatment of Acute

OtitClarithromycin is with

595

Tub& 2 ClinicalResults Cefuroxime-axetil Clarithromycin 17 14

Cured clinically Posttreatment pathologic tympanometry results Failed clinically Recurrence of infection Side effects Nausea

16

14 4 2

1

patients. Two and three patients in the clarithromycin and cefuroximeaxetil study group, respectively, required an extensionof therapy in order to achieve a successful result. Clinical results are shown in Tables2 and

DISCUSSION Treatment of uncomplicated acute otitis media with amoxicillinklavulanate for 5 days has been found to be as effective as 10 days (4). Similarly, we have found that a 7-day treatment with either clarithromycin or cefuroximeaxetil is effective in almost 95% of cases (5). This study demonstratesthat both clarithromycinor cefuroxime-axetil are effective for 5-day treatment as well. Results showthat clarithromycin and cefuroxime-axetil achieved clinical cure ratesof 85% and SO%, respectively, when administered to children suffering from clinicallyor tympanoscopy-documented casesof acute otitis media. Persistence of middle ear fluid at posttreatment tympanometry did Tab& 3 Treatment of Clinical Failures Cefuroxime-axetil Clarithromycin Clinical failures Isolated organism

Recurrences Isolated organism Amoxicillidclavulanate Amoxicillidclavulanate Treatment

Extension of treatment:2 Treatment changed: H.influenzae:

Extension of treatment: 4 Failed after 10 days: 1 H.influenzae: 1 S. pneumoniae: H . influenzae + Strept. pneumoniae: 1

H.influenzae:

H . influenzae:1 Strept. pneumoniae: 1

596

Kafetzis et al.

not differ betweenthe two groups(14 and 14 patients, respectively). Additionally, although five patients (two and three cases in each study group, respectively) requiredfurther treatment in order to achieve a95% satisfactory result, the decision to continue treatment beyond 5 days on the fifth treatment day was reasonable and effective.

CONCLUSION 5-day regimen of clarithromycin for treatment of uncomplicated acute otitis media is safe and effective. Patients should be evaluated on the fifth day of therapy to determine whether additional therapy is warranted.

REFERENCES 1. Bass J W , Cashman TM, Frostad A L , et al. Antimicrobials inthe treatmentof acute otitis media.A second clinical trial. Am J Dis Child. 1973; 125:397-403. 2. Chaput de Saintonge DM, LevineDF, Templae Savage et al. Trialof threeday and ten-day courses of amoxicillin in otitis media. Br Med J 1982; 284: 1078-1081. 3. Meistrup-Larsen KI, Sorensen H, Johnson NJ, et al. ?kro versus seven days penicillin treatment for acute otitis media. Acta Otolaryngol (Stockh) 1983; 96:99-104. 4. Hendrickse WA, KusmieszH, Shelton S, et al. Five versusten days of therapy for acute otitis media. Pediatr Infect Dis J 1988; 7:14-23. 5. Kafetzis DA, Malaka-Zafiriou C , Bairamis T, et al. A comparison of the safety and efficacy of clarithromycin suspension and cefuroxime axetil suspension in the treatment of acute otitis media. Unpublished.

Azithromycin Versus Amoxicillin for Acute Otitis Media Prophylaxis Nicola Principi, Paola Marchisio, Emanuela Sala, Luisa Lanzoni, and Stefania Sorella University of Milan Milan, Italy

INTRODUCTION Recurrent acute otitis media (AOM) is common in infants and young children. Because of the morbidity and possible long-term sequelae, prevention is the main goal. Among the various approaches (chemoprophylaxis, immunoprophylaxis, surgery, and control of environmental risk factors), chemoprophylaxis is still considered the best medical option. However, there is some concern about the possible role of chemoprophylaxis in the emergence of resistanceamongrespiratorypathogens. In our previous study (l),we demonstrated that once-daily administration of low-dosage amoxicillin for 6 months, is as effective as trimethoprim-sulfamethoxazole (TMPBMX) and superiorto placebo in reducingthe occurrence of AOM: of otitis-prone childrentreated either with amoxicillin or TMP/SMX developed AOM compared to of patients given placebo. However, compliance with such a long-term administration canbe a problem, especially in younger children. Intermittent prophylaxis theoretically could be more suitable, but several studies(2-4) have demonstratedthat intermittent administration of 597

598

Pnncipi et al.

recommended drugs such as amoxicillin is less effective than a continuous prophylactic regimen.A possible alternative could the be use of long-acting antibiotics. Azithromycin, an azalide compound structurallyrelated to the macrolides, is characterizedby a broad spectrumof activity which includes all the bacteriausuallyencounteredinAOMandpeculiarpharmacokinetics which allows high concentrations in polymorphonuclear leukocytes and respiratory fluids (including middle ear effusion) for a long period after a single dose. Our study compares the efficacy and safety of prolonged administration of continuous low-dosage amoxicillin with that of intermittent azithromycin in a groupof otitis-prone children.

PATIENTS AND METHODS Children aged 9 months to 6 years with a history of recent recurrent AOM, defined as three or more AOM in the preceding 6 months, were included. Children were randomly assignedto (1) amoxicillin, 20 mgkg per day, (2) azithromycin, 10 mgkg followed by 5 mgkg once a week, or azithromycin, mgkg once a week. All patients weretreated for 6 months and were examined at entry and subsequently at intervals of4-6 weeks and whenever they developed symptoms upper respiratory tract illness or suggesting AOM.At each visit, an interval history was obtained, and pneumatic otoscopy and tympanometric testing were performed.At entry into the study, at completion of the third month, andat the end of the treatment period, a nasopharyngeal swab was obtained to detect the presence of S. pneumoniae, H.influenzae,Moraxella catarrhalis,and beta hemolytic streptococcus and their resistance to p-lactams andto azithromycin. Occurrence of AOM duringthe 6-month periodwas calculated for all groups.

RESULTS Patient characteristicsare summarized in Table1. The number of children enrolled in the 5-mgkg per week azithromycin arm is lower than the other two groups because the inclusion was prematurely interrupted due to the high incidence of AOM in this group: Overall, 55.5% of the children had new episodes of AOM and 419 (44.4%) had two episodes of AOM in the first months of prophylaxis and were thus discharged fromthe study and no longer analyzed. The occurrence of AOM duringthe 6-month study period is reported in Table2: Azithromycin 10 mgkg once a week was superior to amoxicillin in preventing AOM. Both drugs were safe and well tolerated. In none of

599

Azithromycin Versus Amoxicillin for AOM Table I

Patient Characteristics Azithromycin (5 mg/kg once

Azithromycin (10 mg/kg once

Amoxicillin week) a week) a

No. of patients Sex Male Female Age 9 months-3 years >3-6 years Initial ear status No OME OME bilateral OME monolateral Season at entry October-March April-September Day care attendance Yes

No Most recent AOM <30 days >31 days

70

9

74

39 (55.7%) 31 (44.3%) 49 (70.0%) 21 (30.0%)

3 (33.3%) 6 (66.7%) 2 (22.2%) 7 (77.8%)

43 (58.1%) 31 (41.9%) 43 (58.1%) 31 (41.9%)

17 (24.2%) 40 (57.2%) 13 (18.6%)

1(11.1%) 6 (66.7%) 2 (22.2%)

40 (54.1%)

52 (74.3%) 18 (25.7%)

8 (88.9%) 1(11.1%)

61 (82.4%) 13 (17.6%)

40 (60.6%) 26 (39.4%)

7 (77.8%) 2 (22.2%)

50 (73.5%) 18 (26.5%)

53 (75.7%) 17 (24.3%)

4 (44.4%) 5 (55.6%)

49 (66.2%) 25 (33.8%)

15 (20.2%) 19 (25.7%)

Note: No statistical difference between azithromycin 10 mgkg once a week and amoxicillin.

Table 2 Occurrence of Acute Otitis Media During 6-Month Study Period

No. of children with AOM No. of episodes of AOM Mean No. of episodes per patient

Amoxicillin (20 mgkg per day) (n=70)

Azithromycin (5 mgkg per week) (n=9)8

Azithromycin (10 mgkg perweek) (n=74)

22 (31.4%)

5 (55.5%)

11(14.8%)b

28

7

16

0.4

0.7

0.21

T h e 5-mgkg once-a-week azithromycin group was prematurely interrupted (and not further analyzed)because of thehighoccurrencerate of AOM: Childrenwerethenrandomizedonly to amoxicillin or azithromycin 10 mgkg. bp < .05 versus amoxicillin and azithromycin5 mgkg once a week.

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600

Table 3 Nasopharyngeal Colonization According to Prophylactic Regimen

Baseline nasopharyngeal culture Amoxicillin Negatives (n=60)(92.3%)

End of prophylaxis nasophar. culture

End of prophylaxis nasophar. culture

(negative)

(positive)

54

6 S . pyogenes H.influenzae 2 (1 R to azithromycin)

Strep beta hemol group 1C Positive (n=5)(8.7%)

5

0

S. pneumoniae S. pyogenes 1

H.influenzae 3 Azithromycin Negative (n=62)(93.9%) 4Positive (n=4)(6.1%)

61

1 (S. pyogenes) 0

S. pneumoniae 1

H.influenzae .Negative or positivefor S. pneumoniae, H . influenzae, S. pyogenes, and Moraxella catarrhalh. Only resistanceto D-lactams and azithromycinis shown.

the two prophylactic regimens was any remarkable modification of the nasopharyngeal flora demonstrated, both regardingthe prevalence of the bacteria and their susceptibility to p-lactams and macrolides (Table 3).

CONCLUSION Intermittent administration of azithromycin 10 m a g per week (but not at 5 m a g per week) is more effective than continuous low-dosage(20 m a g per day) amoxicillin in preventing AOM in children with recent recurrent AOM. As far as the possible role of chemoprophylaxis in determiningthe emergence of resistant bacteria,we did not note any substantial modification in the small proportionof children harboring nasopharyngeal pathogens. Thus, azithromycin appearsto be suitable for the prophylaxisof acute otitis media in children with a recent historyof recurrent acute episodesof otitis media. In particular, azithromycin could be suggested in (1) those children, suchas infants, inwhom compliance with a continuous long-term treatment could be problematic, (2) in children with p-lactam allergy, (3) when amoxicillin-resistant, new macrolide-susceptible bacteria are highly prevalent inthe population.

Azithromycin Versus Amoxicillin for AOM

601

REFERENCES Principi N, Marchisio P, Massironi E, et al. Prophylaxis of recurrent acute otitis media and middle ear effusion. J Dis Child Peterson L, Peterson KE, Peterson LR. Intermittent antimicrobial prophylaxis for recurrent acute otitis media.J Infect Dis Berman S, Nuss R, Roark R, et al. Effectivenessof continuous vs intermittent amoxicillin to prevent episodes of otitis media. Pediatr Infect Dis J 4.

Heikkinen T, Ruuskanen 0, Ziegler T, et al. Short-term use of amoxicillinclavulanate during upper respiratory tract infection for prevention of acute otitis media. J Pediatr

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XII CLINICAL STUDIES: BRONCHITIS

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Azithromycin for the Treatmentof Community-Acquired Bronchitis: A Large Multicenter Efficacy-Tolerance Trial F. Raffi Centre Hospitalier Rkgional Universitaire de Nantes Nantes, France

INTRODUCTION Antibiotic therapymay be needed for persistent acute bronchitis (AB) and acute exacerbations of chronic bronchitis(AECB), particularly in high-risk patients. Azithromycin is a new azalide (nitrogen-containing macrolide) antibiotic andmight proveto be useful as first-line treatment in these indications. The open, multicenter study described was performed the withobjective of evaluating the efficacy and safety of azithromycin when administered once-daily for 5 days in everyday practice in a large adult population suffering fromAB or AECB. Particular attention was paidto rare adverse events.

METHODS Patients included were adult outpatients presenting with either persistent AB or AECB based on the following criteria:

A B : Persistent progression with mucopurulent sputum accompanied by a risk factor for bacterial secondary infection such as heavy 605

smoking (more than packdyear) or chronic disease (alcoholism, heart disease, arterial disease, diabetes, autoimmune disease, obesity, etc.). AECB: Mucopurulent or purulent sputum and increase in dyspnea and/or volumeof sputum (Anthonisen typesI and I1 criteria). The exclusion criteria were the lack of informed consent, concomitant pneumonia, acute sinusitis, stage V dyspnea (not excluding minor deviations), cystic fibrosis, bronchial dilatation, and concomitant administration of another antibiotic. If concomitant pneumonia was suspected, a chest x-ray was prescribed andthe result documented withinthe first 48 h following inclusion. Patients received500 mg of azithromycin (2 250-mg capsules) on the first day and then 250 mgof azithromycin daily for the next 4 days. Patients hadto be seen again at day 2 1 and day 14 for the final evaluation.

ASSESSMENT CRITERIA Assessment of Efficacy in Patients with AB Recovery with complete disappearance of symptoms was defined by a combination of the following four criteria: cessationof all expectoration, disappearance or return to previous state of dyspnea, disappearanceof fever (if initially present), and disappearanceof cough. The result was regarded as an improvement when physical symptoms improved frequent but coughing or mucous or mucopurulent sputum persisted. Treatment was regarded as a success in patients whoeither recovered or showed considerable improvement. Treatment was considered a failure when the final evaluation revealed continued coughing, severe fits of coughing, purulent sputum, temperature equal to or greater than 38"C, or if the lack of anyimprovement or the aggravation of symptoms during the trial had warranted a change in therapy.

Assessment of Efficacy in Patients with AECB Recoverywasdefinedby a combination of the following four criteria: disappearance of purulence of sputum, disappearanceor decrease (return to previous state) in dyspnea, disappearance of fever (if initially present), and decrease of sputum or cessation of expectoration. An improvement was defined as a decrease in physical symptoms but without the disappearance of all clinical signs or a return to the previous state. Failure was defined asthe persistence or an increase inthe volume of purulent sputum or signs of infection requiring an alteration in antibiotic therapy.

Azithromycin for Bronchitis

607

Assessment of Safety All patients who received at least one capsule of azithromycin were included inthe safety assessment which was based on clinical data or questioning, together with any additional tests requested for documentation. The analysis included 7947 patients entered between 1 September 1994 and 15 May 1995 and distributedon the basis of diagnosis as follows: A B , 4817 cases (60%); and AECB, 3130 cases (40%).

RESULTS Efficacy was evaluated (Table 1) In terms of the total sample sizeof patients included (“intention to treat”); that is, n = 7904 (4791 AB and 3113 AECB) In terms of the samplesize of patients whocompliedwith the protocol(“perprotocol”)[i.e.,n=7286(4444ABand2842AECB)], after excluding 661 patients forthe following reasons (severalreasons might be involved for any given patient): mucopurulent or purulent sputum absent at day (n= 87), dyspnea absent at day (n=42),type I11 AECB accordingto the Anthonisen classification (n= 46), radiological diagnosisof infectious pneumoniaor,pleural abnormality (n= 50), final evaluation too early (less than 12 days after day ) (n= 430), and data missing for the global evaluation of efficacy (n= 43).

EVALUATION OF SAFETY The “intention-to-treat” analysis (entire patient population) showed that 97.2% (7728) did not exhibit any adverse event (A.E.). The A.E.s in2.8% of the patients included diarrhea(0.7%), nausea ( O S % ) , gastralgia (0.5%), and abdominal pain(0.2%). Of the rare A.E.s, rash was noted in 0.06% of cases (n=6) and dizziness in 0.06% of cases (n=5). Fewer than 0.5% of patients (38/7947)had to discontinue the trial because of an A.E. An analysisof safety inthe various subgroups showedthat more than 4% of patients with one of the following risk factors presentedat least one A.E.: renal or hepatic impairment (8.3%), history of cardiorespiratory decompensation diabetes (5.6%), heart disease, valvular disease or arterial disease (4.3%), alcoholism (4.2%), o r obesity (4.1%). Patients over 70 years of age did not constitute a subgroup at risk in terms of adverse reactions by comparisonwith the general population: 2.7% of patients over 70 presented at least one A.E. (the same frequencyas in the general population).

608 Tabh

Evaluation of Efficacy “Intention-to-treat” analysis

“Per protocol” analysis

AB Success rate Recovery Improvement Failure rate AECB Success rate Recovery Improvement Failure rate

%

CONCLUSION The trial was carried out in routine practical conditions and confirmedthe satisfactory results obtained using 5 days of once-daily azithromycinin the treatment of AB and AECB in adults A success rate of 96.6% and 95.6% was achieved in compliant patients with AB and AECB, (Anthonisen typesI and II), respectively. A large numberof patients were studied, thereby allowing an accurate estimate of the incidence of adverse events and confirmingthe absence of rare adverse events. Gastrointestinal symptoms werethe most frequent A.E.s, but still accounted for less than 2%. A combination of a low incidence of A.E.s and once-daily treatment for 5 days contribute to goodcompliancewith treatment and the successof azithromycin in these indications.

REFERENCES Balmes P, Cerc G , Dupont B, et .al. Comparative study of azithromycin and amoxicillidclavulanic acid in the treatment of lower respiratory tract infections. Eur J Clin Microbiol Infect Dis Feldstead SJ. Azithromycin LTRI Study Group. Double blind comparison of azithromycin and amoxicillin in the treatment of lower respiratorytract infections. Proceedings of the 6th International Congress of Infectious Diseases,

Safety and Efficacy of Clarithromycin Compared with Cefpodoxime Proxetil in the Treatment of Acute Exacerbation of Chronic Obstructive Pulmonary Disease P. IRophonte and J. P.Chauvin Hospital Rangueil Toulouse, France Abbott France Laboratories Rungiv, France

INTRODUCTION Chronic obstructive pulmonary disease (COPD) is characterized by a reduc-' tion of the expiratory output together with clinical symptoms associated with chronic bronchitis. Infection is a frequently encountered problem in the everyday treatment of this disorder. Infection in patients with COPD entails an immediate riskof pulmonary decompensation and potential aggravation of chronicbronchitis.Rapidactionin the form of antibiotic therapy is recommended, particularly in the face of worsening clinical symptoms (dyspnea and increase a n d or purulent sputum).At the present time, p-lactams are the treatment of choice. Various studies have revealedthat in approximately 50% of cases, a bacteriological profile exists that includes Huemophilus influenzae or Strep609

610

Ltophonte and Chauvin

tococcus pneumoniae and, in a few cases, Moraxella catarrhalis. For the remaining 50% of cases, no microbiological confirmation is apparent, although Mycoplasma pneumoniae or Chlamydia pneumoniae infection cannot be ruled out, given that little is known about the incidence of such microorganisms in this disorder. As clarithromycin is active against microorganisms that are responsible for lower respiratory tract infections, it should be considered inthe treatment of these infections.

STUDY OBJECTIVE The objective was to compare the safety and efficacy of clarithromycin with cefpodoxime proxetil in the treatment of acute bacterial exacerbationsof COPD as administered under everyday general practice conditions.

PATIENTS AND METHODS Study Design Multicenter, open-label, comparative randomized study. lbelve regionalcoordinatingpneumologistsandinfectiousdiseasespecialistsactedaslocalscientificadvisors to 80 general practitioners. Efficacy wasevaluated onthe basis of the nature of patient sputum ondays12-15(success = absendmucus;failure = purulent/ mucopurulent) and resolution of signs ofexacerbation (e.g., aggravation of dyspnea, increase in sputum).

Inclusions Male or female outpatients aged 18-80 years. Presenting COPD: History of chronic bronchitis (cough and sputum every day for a continuous periodof months for 2 consecutive years) Confirmedbronchialobstruction,practicallyirreversiblewith &stimulants Demonstrating an acute exacerbation of COPD at time of inclusion definedby Modification of sputum color or consistency indicative of acute bacterial infection (e.g., change to yellow/green color, increased tenacity of sputum) andthe presence of at least one of increased volume of expectoration worsening of dyspnea No use of antibiotics within 15 days priorto the study.

ry

Clarithromycin Versus Cefpodoxime Proxetil for COPD Table I

611

Evaluations Visit 1 Evaluation (day

1)

Medical

Visit 2 Day

X X X

Physical examination Clinical examination

Clinical pulmonary evaluation Prescription for standard Cytobacterial examination Clinical response Adverse drug events

(days 12-15)

Day

X X X

X X X X

X X

X X

.Telephone consultation; if necessary, the investigator could request patient consultation. Welephone consultation to determine clinicalevolution (especially infectious relapse).

suspicion of active tuberculosis, severe respiratory failure, associated parenchymal infection, or signs of respiratory decompensation that could necessitate hospitalization. hypersensitivity to p-lactams or macrolide antibiotics. severe renal or hepatic impairment. Written informed consent.

Drug Administration 332 patients were randomizedto receive 1or 2 treatments, each of which was to be taken by mouth for a 10-day treatment period. Treatments were as follows Clarithromycin (nclar): 250 mg 2 bid (n =51 179) Cefpodoxime proxetil (Cefodox): 100mg 2 bid (n = 153)

Evaluation The visits are summarized in Table1.

RESULTS Demographic Data The study included 332 patients with a mean age of 60 years (sex ratio M/F = 1.8). The mean history of the pulmonary disease was up to 10 yearsfor 55% of patients.

Ltophonte and Chauvin

612

Table 2 Clinical success at Days 12-15 by Treatment Group

proxetil Clarithromycin Cefpodoxime Success Failure Missing data

1361168 (81.0%) 321168 (19.0%) 11

115/149 (77.2%) 341149 (22.8%) 4

Clinical success defined as absenceof mucus expectorate.

The Anthonisen score atday 1was equal to 1in of patients in both groups. Spirometric data: the average VEMS was at 60% of the theoric value. There was nodifferencein the two groupsconcerningdemographic data and infectionstatus of enrolled patients.

Clinical Evaluation Table 2 shows that the clinical success defined as the absence of mucous expectorate on days 12-15 was not significantly different in the two groups. Regardingclinicalfailure, there werefewerclarithromycinpatients who required anew course ofantibiotic (7.9% for clarithromycin group, 15% for cefpodoxime proxetil,p = .0048).

Adverse Events Most adverse events were mild to moderate in severity in each treatment group. Excluding taste perversion reported exclusively in the clarithromycin group, there was no statistically significant difference in the two groups.

DISCUSSION

In this clinical trial, analysis of lung respiratory tract sampleswas not very helpful for therapeuticdecisions. The high frequency of poor quality expectorated sputum or the presence of normal respiratory flora makes cytobacterial examination of expectorated sputum less reliable. Treatment of acute exacerbationof chronic bronchitis in patients with chronic obstructive pulmonary disease still remains basedtheonmost likely etiologic organisms in COPD. This study concludedthat the clinical efficacy of clarithromycin compared to that of a third-generation cephalosporin is equivalent inthe treatment of COPD although clarithromycin incurs lower cost.

Clarithromycin Versus Cefpodoxime Proxetil for COPD

613

CONCLUSIONS Tbice-daily treatment with 500 mg of clarithromycin resulted in a high rate of clinicalsuccesscomparable to that obtained with twice-daily 200 mg of cefpodoxime proxetil in the treatment of acute exacerbationof chronic obstructive pulmonary disease. Overall, both drugs werewell tolerated, although the incidence of drug-related effectswas higher in the clarithromycin group due to the increased frequencyof taste perversion. Fewer clarithromycin patients required a new course of antibiotic therapy (p = .048).

REFERENCES 1. Anthonisen NR. Antibiotic therapy in exacerbations of chronic obstructive pulmonary disease. Ann Intern Med 1987; 106:196-204. 2. Guay DRP, Craft JC. Comparative safety and efficacy of clarithromycin and of ampicillin inthe treatment of out-patients with acute bacterial exacerbation chronic bronchitis. J Intern Med 1992; 231:295-301.

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XIII CLINICAL STUDIES: PNEUMONIA

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Randomized Comparative Trial of the Safety, Efficacy, and Cost of Intravenous Cefuroxime Plus Intravenous Erythromycin Versus Intravenous Cefuroxime Plus Oral Clarithromycin in the Therapy of CommunityAcquired Pneumonia D. Skupien, A. Margulis, Kaczander, S. Jaworski, A. Eklebeny, J. Pypkowski, and M. J. Zervos Wayne State University School of Medicine and William Beaumont Hospital Royal Oak, Michigan

INTRODUCTION Pneumonia occurs in overthree million persons per year and accountsfor 500,000 hospitaladmissions. It is the fourth leading cause of death in persons overthe age of 65 in the United States and the number 1cause of death from infectious diseases. The cost of hospitalization alone exceeds 1.5 billion dollars per year. Despite diagnostic efforts, the detection of all causative pathogens is limited. It is often not possible to obtain a sputum specimen, or cultures do not yield a specific organism; therefore, etiology is only known 50% of the time in community-acquired pneumonia (CAP). 617

618

,

et

Skupien

al.

Frequently encountered pathogens suchMycoplasma as pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, and respiratory viruses are not detected at allbyGram stain and routine culture. According to a concensus statement by the AmericanThoracicSociety, it isbecauseof these diagnostic limitations that initial empiric therapy of a second- or third-generation cephalosporin and a macrolide with activity against common pathogensisnecessary(1).Erythromycinisoftenincludedwith cefuroxime as empiric therapy because of its excellent coverage of M . pneumoniae and L. pneumophila, but it is considered ineffective in vitro against H . influenzae. Clarithromycin, a newer macrolide, also includes coverage for C . pneumoniae and H . influenzae, common pathogens in CAP patients (2). The present studywas undertaken to determine a cost-effectivetreatment regimen for CAP. We also sought to evaluate in a randomized, comparative study the safety and efficacy of cefuroxime plus erythromycin versus cefuroxime.plus clarithromycin in the treatment of CAP.

PATIENTS AND METHODS .

,

Patients

Between August 1994 and June 1995, patients admitted to William Beaumont Hospitalwith a primary diagnosis of community-acquired pneumonia were eligible for inclusion in this study. [William Beaumont Hospital is a 975-bed community teaching hospital in Royal Oak, Michigan with over 40,000 admissions annually. In 1994,863 patients with an admission diagnosis of community-acquired pneumonia weretreated at William Beaumont Hospital with an average (hospitalization) length of 14.2 days.] Adult hospitalized patients with diagnosis of community-acquired bacterial pneumonia met inclusion criteriaby presenting with a newor radiographically demonstrated changing infiltrate associatedwith fever, cough, and physical findings compatible with pneumonia, which could include purulent sputum production and leukocytosis (leukocytes > 11O , OOcm3). Patients must have been suitable candidates for oral antibiotic therapy based on the presence of a functioning gastrointestinal tract. Exclusion criteria includedthe following:evidence of sepsis or septicshock,empyema,lungabscess, or aspiration pneumonia, neutropenia (< 1500/mm3 WBC) or an immunocompromising condition, anyother infection which necessitates use of concomitant systemic antibiotics, a historyof hypersensitivity to macrolide or p-lactam antimicrobials, or prior presumably effective intravenous antibiotic therapy for more than24 h. No females who were planning pregnancy or wereactivelypregnant or breastfeedingwereincludedin the study population. Informed written consent was obtained from all patients.

Comparative Study

619

CAP

Therapy Patients were chronologically randomized to a nonblinded dosing schedule to receive cefuroxime 750 mg intravenously every 8 h plus erythromycin 500 mg intravenously every 6 h (cefuroxime/erythromycin)or cefuroxime 750 mg intravenously every8 h plus clarithromycin 500 mg every 12 h orally (cefuroximeklarithromycin). Both groups were subsequently convertedto clarithromycin 500 mg PO every12h.No other antibiotics were given during or after administration of the study drug. The duration of antibiotics, time of conversion to oral therapy, all other forms of therapy, and hospital discharge were at the discretion of the attending physician caring for the patient.

Bacteriological Investigations In patients able to produce sputum, Gram stains of sputum smears and sputum cultures were performed. Bacteriologic blood cultures were obtained on all patients; Gram stains were reviewed and correlation with culture results was needed for the organism to be considered a pathogen. Identification of pathogen@) and antibiotic susceptibilities (erythromycin, cefuroxime,andclarithromycin)weredetermined by the microdilution technique, Microscan (WalkAway Baxter, West Sacramento, CA), using NCCLS standards.

Evaluation of Efficacy and Safety Clinical response to treatment was determined by the following criteria: cure, as definedby the disappearance of clinical symptomatology andtheir continued absenceat the end of a 1-2 week patient follow-up period, and faiZure as defined byno significant improvementor clinical worsening while on the study treatment. All adverse events were recorded along with severity, and outcome and determined causality to the study medication. Microbiological efficacy was determinedby using the following criteria: eradication, defined as the elimination of the principal pathogen(s) at the endof therapy and continued absence through 1-2 weeks of patient follow-up, and persistence, defined asthe continued presenceof the principle pathogen at the end of therapy. Statistical comparisons were made using Student’s t-test for continuous variables, and Fisher’s exact test or Chi-square analysis withthe Yates correction was used for dichotomous variables.

Evaluation of Cost Cost comparisons were made between the two groups basedon duration of treatment, drug acquisition costs, and intravenous administration costs.

620

Skupien et al.

Drug acquisition costs of intravenous cefuroxime were $17/day, intravenous erythromycin $7/day, and oral clairthromycin $5/day. Other antibioticsnecessary to treat the studied episode of pneumonia were also considered in the total costs incurred by both groups. Antibiotic costs were providedby the hospital pharmacy and determined based on unit-dose drug acquisition costs. Intravenous administration charges were estimated at $Ydose. Hospitalization charges were estimated at $120/day.

RESULTS A total of 60 eligible patients were included in this study. Thirty patients were randomly assigned to receive cefuroxime and erythromycin andto30 receivecefuroximeandclarithromycin,withconversion to oral clarithromycin in both groups. Patients inthe two treatment groups were similar with respect to demographics and underlying disease history (Table 1). Table 2 summarizes the bacteriological findings in the two groups. Overall, the most frequently isolated organisms were H.infruenzae (10%) and S. pneumoniae no pathogen was isolated in 25% and 31.7% were unableto produce sputum. Adverse events occurred in 16 patients (Table Ten cefuroxime/ erythromycin patients (33.4%) experienced adverse events, six cefuroximel clarithromycin patients (20%) experienced adverse events. The most frequent adverse events were gastrointestinal disturbances (five cefuroxime/ erythromycin patients, three cefuroxime/clarithromycinpatients). Phlebitis was seen intwocefuroxime/erythromycinpatients. One cefuroxime/ clarithromycin patient experienced an allergic reaction to cefuroxime. Three Table Z Demographic and Clinical Characteristics of Patients in theTwo Study Groups at Presentation ~~

group Characteristics

evaluable No. of patients 30 No.of males/females 16/14 (range) yearsin age Mean No. (%) receiving prior abx. No. (%) of underlying disease: COPD Cardiovascular disease Genitourinary Diabetes Total

Cefuroxime/erythromycin (n=30) 30 12/18 (21-93) 70 (13.3%) 5 (16.7%) 4 8 (26.7%) 3 (10%) 9 (30%) 20 (66.7%)

~

~

Cefuroxime/clarithromycin group (n=30)

71 (31-93)

11(36.7%) 4 (13.3%) 1(3.3%) 15 31 (100%)

roup

621

Comparative Study of CAP Table 2 Bacteriologic Findings (Numberof Strains Isolated) inthe Two Study Groups

Cefuroxime Cefuroxime plusplus clarithromycin erythromycin Organism Streptococcus pneumoniae flaemophilus injluenzae Staphylococcus aureus Klebsiella pneumoniae Moraxella catarrhalis Klebsiella ozaenae Enterobacter spp. Proteus spp. Serratia marcescens Pseudomonas aeruginosa Normal oral flora Unable to produce sputum

group

0

1

Table 3 Outcome of Therapy and Clinical Course

Cefuroxime plus erythromycin group Clinical outcome No. (%) cure No. (%) failure Bacteriologic outcome No. (%) Eradication . No. (%) Persistence No. (%) Indeterminate Mean duration (range) of fever in days Mean duration (range) of leukocytosis in days Adverse events No. (%) gastrointestinal No. (%) allergy No. (%) phlebitis No. (%) ICU admissions No. (%) death .Second to cefuroxime.

Cefuroxime plus clarithromycin group

622

al.

Skupien et

cefuroxime/erythromycin patients and two cefuroxime/clarithromycinpatients expired but deaths were not related to the study medication. One cefuroxime/clarithromycinpatient was withdrawn from study for the following reasons: failure, side effects, and antibiotic resistance. One cefuroximel clarithromycin patient was withdrawn from study because of the isolation of a resistant Enterobacter cloacae. Table illustrates the difference in the cost of treatment of the two groups. The cefuroxime/clarithromycingroup shows a shorter duration of treatment and lower drug acquisition costsfor both study medicationsand additional antibiotics. In the cefuroxime/erythromycin group, intravenous erythromycin was associatedwithincreasedintravenousadministration costs, as well asthe use and costof nonstudy antibiotics.

DISCUSSION The present study was designedto compare the efficacy, safety, and cost of two antibiotic regimens for the treatment of community-acquired bacterial pneumonia in persons requiring hospitalization for acute care intravenous therapy yet suitablefor oral antibiotics. Demographic features andclinicalandlaboratoryfindingsin our study population were similarin the two groups and to those described in earlier reports on community-acquired bacterial pneumonia Adprior antibiotic treatment with no response vanced age (mean age 2 and underlying cardiorespiratoryor metabolic disease were common. Of the studied patients, (three in cefuroxime/erythromycin,two in cefuroxime/clarithromycin)died. This figure is comparable to the mortality reported in patients with CAP in other studies Overall successful treatment rates of for cefuroxime/clarithromycin group, and for cefuroxime/erythromycin group were observed. The rate of eradication of the pathogen was similar the in two groups and respectively). Serious side effects and allergy were infrequent in both groups. The cefuroxime/erythromycingroupincurredmorecost than the cefuroxime/clarithromycin group. The financialburden of the use of erythromycin inthe cefuroxime/erythromycin group is apparent not only in the actual costof drug but also in its intravenous administration costs. The cefuroxime/erythromycin group also spent more on the use of other antibiotics and their administrationdue to more treatment failures, side effects, and antibiotic resistance. The final cost analysis shows that less was

Comparative Study of CAP Table 4 Cost Analysis of Two Study Groups

Cefuroxime plus Cefuroxime plus clarithromycin erythromycin group (n=30) group (n=30) Duration of hospitalization in days mean (range) Duration of cefuroxime in days mean (range) Duration of erythromycin in days mean (range) Duration of clarithromycin in days mean (range) Drug acquisition costs Cefuroxime Erythromycin Clarithromycin Other antibiotics failures, side effects, antibiotic resistance Intravenous administration costs Erythromycidcefuroxime Nonstudy antibiotics Total antibiotic costs

7.6

7.5 4.1 (2-12)

NA 10.2 (3-12) $2397 $840 $1410 $1974

$2091

$3900 $840

$1950 $360

$11,361

$6,777

NA $1530 $846

spent in the cefuroxime/clarithromycingroup, representing a40% savings over the cefuroxime/erythromycin group. The new oral macrolide antibiotics provide a cost-effective alternative to.standard treatment in elderly patients with underlying disease suitable for oral antibiotic treatment. We also concludethat for some patients hospitalized with community-acquired pneumonia, only a short course of intravenous antibiotic therapy isnecessary. The. resultsof this study show that both intravenous cefuroxime plus intravenous erythromycin intraand venous cefuroxime plus oral clarithromycin are effective and safe for the treatment for community-acquired bacterial pneumonia, but cost savings were demonstrated with cefuroxime plus clarithromycin.

ACKNOWLEDGMENT This study was supported in part by the William Beaumont Hospital Research Institute and by Abbott Laboratories, Abbott Park, Illinois.

Skupien et al.

REFERENCES 1. Berntsson E, Blomberg J, Lagergard T, Trollfors B. Etiology of communityacquired pneumonia in patients requiring hospitalization. Eur J Clin Microbiol 1985; 4:268-272. 2. Bently DW. Bacterial pneumonia in the elderly: clinical features, diagnosis, etiology, and treatment: Gerontology 1984; 30:297-307. 3. Berk SL, Wiener SL, Eisner LB, DuncanJW, SmithJK. Mixed Streptococcus pneumoniae and gram-negative bacillary pneumonia the in elderly. South Med J 1981; 74:144-146. 4. Dorff GJ, Rytel MW, Farmer SG, Scanlon G. Etiologies and characteristic features of pneumonias in a municipal hospital. Am J Med Sci 1973; 266: 349-358. 5. Ebright JR, Rytel MW. Bacterial pneumonia inthe elderly. J Geriat Soc 1980; 27:220-223. 6. Griffith DE. Pneumonia in chronic lung disease. Infect Dis Clin North 1991; 5467-484. 7. Garb JL, Brown RB, Garb JR, lhthillRW. Differences in etiology of pneumonias in nursing homes and community patients. Am J Med Assoc 1978; 240: 2169-2172. 8. Garibaldi RA. Epidemiology of community-acquired respiratory tract infections in adults. Am J Med 1985; 78(suppl6B):32-37. 9. Karnad A, Salvador A, Berk SL.Pneumonia causedby gram-negative bacilli. Am J Med 1985; 79(suppl lA):61-67. 10. Klimek JJ, Ajemian E, Fontecchio S, Giacewski J, Nemas B, Jimenez L. Community-acquired pneumonia requiring admission to hospital. J Infect Contr 1983; 11:79-82. 11. MacFarlane JT, Finch RG, Ward MJ, MacRae AD. Hospital study of adult community-acquired pneumonia. Lancet1982; ii:255-258. 12. Sullivan RJ, Dowdle W B , Marine MW, Hierholzer JC. Adult pneumonia in a general hospital. Arch Intern Med 1972; 129:935-942. 13. Verghese A, Berk S. Bacterialpneumoniain the elderly.Medicine1983; 62~271-285. 14. White RJ, Blainey AD, Harrison W,Clark SKR. Causes of pneumonia presenting to a district general hospital. Thorax 1982; 36:566-570. 15. Karalus NC, Cursons RT, LengRA, Mahood CB, Rothwell RPG, Hancock B, Cepulis S, WawataiM,Coleman L. Communityacquiredpneumonia: aetiology and prognostic index evaluation. Thorax 1991; 46:413-418. 16. Fang GD, Fine MJ, Orloff J, Arisumi D, Yu VL, Kapoor W, Grayston JT, Wang SP, Kohler R, Muder RR, Yee YC, Rihs JD, Vickers RM. New and emerging etiologiesfor community acquired pneumonia with implications for therapy. Medicine 1990; 69:307-316. 17. Donowitz GR, Mandell GL. Empiric therapy for pneumonia. Rev Infect Dis 1993; (SUPPI 5):40-51. 18. Larsen RA, Jacobson JA. Diagnosis of community-acquired pneumonia: experience of a community hospital. ComprehensiveTher 1984; 10:20-25.

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19. Farr BM, Sloman AJ, Fisch UT. Predicting death in patients hospitalized for community-acquired pneumonia.Ann Intern Med 1991; 115:428-436.

BIBLIOGRAPHY Engle JC, Lifland PW, Schleupner CJ. Comparison of ceftazidimewithcefamandole for therapy of community-acquired pneumonia. Antimicrob Agents Chemother 1985; 28:146-148. Fekety FR, Caldwell J, Gump D, Johnson JE, Maxson W, Mulholland J, Thoburn R. Bacteria, viruses, and mycoplasmas in acute pneumonia in adults. Am Rev Respir Dis1971; 104:499-507. Fiala M. A study ofthe combined roleof viruses, mycoplasmas in acute pneumonia in adults. Am J Med Sci 1969; 257:44-51. Finch R, MacFarlane JT, Selkon JD, Watson J, White RJ, Winter JH, Woodhead MA. Guidelines for the management of community-acquired pneumonia in adults admitted to hospital. Br J Hosp Med 1993; 49:346-350. Foweraker JE, Cooke NJ, Hawkey PM. Ecology of Haemophilus influenzae and Haemophilus parainfluenzaein sputum and saliva and effects of antibiotics or their distribution in patients withlowerrespiratory tract infection. Antimicrob Agents Chemother 1993; 37:804-809. Lode H. Initial therapy of pneumonia. J Med 1986; 80 (suppl5C):70-74. McCabe WR, Jackson GG. Gram-negative bacteremia. I: etiology and ecology, Arch Intern Med 1962; 110:847-855. Murray PR, Washington JA. Microscopic and bacteriologic analysis of expectorated sputum. Mayo Clin Proc 1Y5; 50:339-344. National Committeefor Clinical Laboratory Standards. Performance Standards for Clinical laboratory Standards. Approved Standard M7-A2. Villanova, PA: NCCLS, 1990. National Committeefor Clinical Laboratory Standards. PerformanceStandards for Microdilution Susceptibility Tests. Approved Standard M2-A4. Villanova, PA: NCCLS, 1990. Rhind GB, Gould GA, Ahmad F, Croughan MJ, Calder MA. Haemophilus parainfluenzae and Haemophilus influenzae respiratory infection: comparison of clinical features. Br Med J 1985; 291:707-708. Wallace RJ, Niefield SL, WatersS, Waters B,Awe RJ, Wiss K, Martin RR, Greenberg SB. Comparative trial of cefonicid and cefamandole in the therapy of community-acquired pneumonia. Antimicrob Agents Chemother 1992; 21: 231-235. WeberDJ,CalderwoodSB,KarchmerAW,Pennington JE. Ampicillinversus cefamandole as initial therapy for community acquired pneumonia. Antimicrob Agents Chemother 1987; 31:876-882.

Azithromycin in the Treatment of Community-Acquired Pneumonia K. Golec, M. Rzeszutko-Grabowska, D. Bukowska-Nierojewska District Hospital No. 2 Rzesrbw, Poland

INTRODUCTION Most cases of community-acquired pneumonia (CAP) in adults can be treated effectively without hospitalization. Considering the variety of causative pathogens and the necessity of immediate administration of antimicrobial therapy,the selection of an appropriate drug forthe treatment of CAP is often based on empirical premises. The drug of choice should be well tolerated, easily administered, and highly effective against all common pathogens involved in CAP. Azithromycin, an azalide antibiotic with distinct pharmacokinetic and pharmacodynamic properties, seems to comply with these requirements and offers a considerable -advance inthe outpatient treatment of CAP. The aim of the study wasto assess the efficacy of 5day azithromycin therapy inthe management of CAP in adults.

PATIENTS AND METHODS Patients of both sexes withCAP were included in an open, noncomparative study. CAP was defined as an acute illness with typical clinical manifes626

Azithromycin in Treatment

CAP

627

~3

Failure (5%)

(95%) Figure Z Clinical efficacyof azithromycin in the treatmentof community-acquired

pneumonia. tations, radiologicalfindingsindicatingpneumonia,andhematological changes in peripheral blood suggesting bacterial infection (leukocyte count >lO,OOO/mm3 with >60% neutrophilic granulocytes). Bacteriologicaltests (sputum culture with identification of isolates and drug-sensitivity determination according to NCCLS standards) were performed on all patients. Azithromycin was given once daily for 5 days, 500 mg on the first day, followed by 250 mg on the subsequent 4 days. Clinical examination and bacteriological tests were repeated and 9-10 daysafter the beginning of treatment.

RESULTS A total of 40 patients, aged 15-85 years, were included inthe study. In 38 of them (95%), no clinical symptoms of disease were found on days 9-10 (Fig. 1).In cases, significant clinical improvement with disappearance of fever was obtained by the third day of treatment. Baseline sputum culture was positive for respiratory tract pathogens in 32 patients. Isolated pathogens and their eradication ratesare presented in Table1. In three patients Escherichia coli coexistedwith Streptococcus pneumoniae. In 26/32patients, the pathogen was eradicated from sputum within days, in 3 cases within 7 days, and in1case within 9 days after the initiation of azithromycin therapy. In eight patients, sputum specimens were not suitable for evaluation due to contamination with oral organisms. These patients might have had infection with organisms that do not grow on routinely used media (e.g., Chlamydia sp. or Mycoplasma sp.). However, in all of them, the treatment was successful. There were two treatmentfailures:apatientwith Huemophilus paruinfluenzae who was hospitalizeddue to generalized deterioration and a second patient who needed additional treatment dueto persistence of the

628

Golec et al.

Table Z Bacteriological Efficacy Azithromycin in the Treatment of Community-Acquired Pneumonia

n eradicated / n isolated

Pathogen

17/17 515 414 U2 112 111 111 011 111 111

Streptococcus pneumonia@ Escherichia colia Haemophilus inffuenzae Moraxella catarrhalis Klebsiella pneumoniae Streptococcus agalactiae Staphylococcus aureus Haemophilus parainjluenzae Pseudomonas aeruginosa Serratia spp.

(94%)

Total ~~~

~

.In three patientsE. coli coexisted with S. pneumoniae.

initial pathogen (Klebsiella pneumoniae).Azithromycin was well tolerated and only one patient complained of slight dizziness.

CONCLUSION The results indicate that azithromycin, given once daily for 5 days, is clinically and bacteriologicallyeffective and well tolerated in the treatment of community-acquired pneumonia.

Azithromycin: 3-Day Versus 5-Day Dosage Regimen for Community-Acquired Pneumonia in Children B. Ficnar andN. Huzjak Pediatric University Hospital Zagreb, Croatia

I. Klinar and M. Matrapazovski Pliva d.d. Pharmaceuticals Division Zagreb, Croatia

INTRODUCTION Azithromycin,anazalideantibiotic,isan appropriate choice for the initial treatment of community-acquired pneumonia because it is active against all common causative pathogens (i.e. , Streptococcus pneumoniae, Haemophilus influenzae, and Mycoplasma pneumoniae). In contrast to other oral wide-spectrum antibiotics,the unique pharmacokineticproperties of azithromycin enable simple and short dosage regimens, once daily for or 5 days, a particular advantage in pediatric practice. The aim of thisstudy was to compare the efficacyandsafety of 3-day and 5-day azithromycin courses inthe treatment of community-acquired pneumonia in children.

629

630

Ficnar etal.

PATIENTS AND METHODS open, randomized, multicentric study was conducted in 24 study centers, placed in 16 Croatian towns, between February 1994 andJune 1995. The study protocol was approved by local ethics committees. Children of both sexes, aged 6 months to 12 years, with signs and symptoms consistent with pneumonia [cough and/or auscultatory findings (rales or evidence of pulmonary consolidation)with or without fever or leukocytosis (blood leukocyte count >lOX106/L with neutrophils or >7% band forms)] were included. Clinical diagnosis of pneumonia was confirmed by the presence of new infiltrate on chest x-ray. Before inclusion, informed consent was obtained from the patient's parent. Patients with hypersensitivity to macrolides, severe renal or hepatic impairment, gastrointestinal tract disturbances which affect drug absorption, acute viral infection and fibrocystic disease were excluded as were immunocompromised patients and patients who received any antibiotic within 24 h prior to entering the study or depot-penicillin in the past 2 weeks. Azithromycin (Surnamed, Pliva) was administered orally, once daily, 1 hbefore or 2 hafterameal.Patientswererandomized to receive azithromycin for 3 days, 10mgkg daily (3-day group)or for 5 days, 10mg/ kg on day 1,followed by 5 mgkg from days2-5 (5-day group). Clinical examination was performed at baseline and 72 h, 6 days, 10 days, and 3 weeks afterthe start of treatment. Chest x-ray was performed at baseline and optionally 10 days after the start of treatment. When possible, an appropriate specimen for microbiological studies (sputum, endotracheal aspirate, pleural fluid, or blood) was obtained at baseline, 72 h, and 10 days after the start of treatment. Pneumonia due to Mycoplasma pneumoniae wasidentifiedby at least a fourfold rise in serum titer of complement-fixing antibodies. Clinicalresponsewasdefined cure (complete disappearance of clinical symptoms of pneumonia within 5 days after the start of treatment with partial or complete regression of infiltrate on control chest x-ray)or failure (allother outcomes). Bacteriological responsewas defined as eradication of initial pathogen, presumed eradication (posttreatment cultures were not performed due to the complete .resolutionof clinical signs and symptoms), persistence,or relapse. All. adverse events were recorded at each visit and classified according to severity 'and causative relationship to the study drug. Laboratory safety tests (hematology, biochemistry) were undertaken at baseline and 10 days after the start of treatment.

Azithromycin: 3-Day Table I

Versus 5-Day Regimen for CAP

631

BaselinePatientCharacteristics 3-Day azithromycin

Total no. of patients Femalelmale Mean age (years) Age range Mean weight (kg) Weight range (kg)

70 38/32 6.6 8 months-l2 years 22.1 9-74

5-Day azithromycin 85 41/44 9 months-l2 years 23.8 9-61

Clinical and bacteriological response between the two groups was compared by Cochran-Mantel-Haenszel statistics based on Ridit scores and the incidence of side effects and laboratory abnormalities by Fisher’s exact test (two-tailed). Differences were considered to be significant if p<.os.

RESULTS Among 155 children who were included into the study, 70 were randomized to the 3-day group, and 85 to the S-day group. Both groups were comparable regarding baseline demographic data (Table 1). patients who missedthe checkup visits and two who violated the entry criteria were excluded from evaluation of efficacy. Clinical efficacy was evaluated in 151 children. In general, patients became afebrile approximately 48 h after the initiation of treatment. Cure was achieved in 65 (97%) and 81 (96%) patients in the 3-day and 5-day groups respectively (Table2). There were no significant differences in clinical response between treatments(p = 342). Bacteriological efficacy was evaluated in 16 patients in each group. Eradication or presumed eradicationof initial pathogenwas accomplished in all. M.pneumoniae infection was confirmed in 16/38 and 10/36patients in 3-day and S-day groups, respectively. Allone but(from the 5-day group) Table 2 Clinical Response to 3-Day and 5-Day Azithromycin in Children with Pneumonia

Clinical No. of evaluable patients Cure Failure

67 65 (97%)

2 (3%)

81 (96%) 3 (4%)

Ficnar et al.

632

Table Nature and Incidenceof the Side Effects in Children Treatedwith 3- and 5-Day Azithromycin

Side

5-Day

Nausea Vomiting Abdominal pain Diarrhea Skin rash

1 1

-

-

Total

5 (7%)

4 (5%)

1 2

2 1

were cured. Although in the remaining child treatment was evaluated as failure (shewas subfebrile for 10 days), all signs and symptoms of pneumonia disappearedby day 14 without additional antimicrobial treatment. Tolerance ofazithromycinwas evaluated in 153 children. Patients tolerated azithromycin well. Side effects, mostly mild gastrointestinal disturbances, were observed infive (7%) patients in the 3-day and four (5%) patients in the 5-day group (Table 3). There were no treatment discontinuations due to sideeffects. Clinicallysignificantlaboratoryabnormalities were recorded in nine patients: eosinophilia twoinand six patients in the 3day and 5-day groups, respectively, and thrombocytosis inone child from the 5-day group. There were no statistically significant differences in incidence side effects (p = .732) or laboratory abnormalities (p = .186).

DISCUSSION

The results of several clinical studies confirm that 3- or 5-day azithromycin courses are as equally effective and well tolerated as 10-day courses with other macrolides in the treatment of children with pneumonia (1-3). In comparison with erythromycin and josamycin, faster recovery has been observed in azithromycin-treated patients (2,3). The efficacy and safetyof 3-day and 5-day azithromycin have already been compared in children with tonsillitis/pharyngitis (4) and in adults with atypical pneumonia (5). In both studies, 3- and 5-day azithromycin courses were equivalent. Therefore, our results correspond with others in the literature. Considering that azithromycin has been widely used in Croatiafor several years,the results probably reflect the effectivenessof azithromycin in common pediatric practice.

CONCLUSION Either 3-day and 5-day azithromycin courses are very effective and well tolerated in the treatment of community-acquired pneumonia in children.

Azithromycin: 3-Day

Versus 5-Day Regimen for CAP

633

However, the 3-day regimen is preferable becauseis itmore convenient for children and parents.

REFERENCES 1. Verona E, &ak

B, Klinar I. Macrolides and azalide in the treatment of pneumonia in children. 9th Mediterranean Congress of Chemotherapy, Milan, 1994; abstr 99. 2. Principi N,Marchisio P, Biasini G, Brunelli A, Caramia G , Osimani P, Cascio A, Cascio G, Chiodo F, Manfredi G, Manfredi R, Longo L, Schiavone R, Seven F, Marseglia G , Schettini F, Amendola F, Spada A, Rocca M. Azithromycin versus erythromycin in the treatment of pediatric communityacquired pneumonia.Eur J Clin Res 1993; 4:127. Ronnchetti R, Blasi F, Grossi E, Pecori A, The Azithromycin Pediatric Research Group. The role of azithromycin intreating children with communityacquired pneumonia.Curr "her Res 1994; 55:965. 4. Principi N, Ambrosioni G, Bianco R, Blatto M, Caramia G, Cocuzza S , D'Angelo G , Raggi M, Reali E. Randomized multicenter study of azithromycin vs. erythromycin in pediatric patients with acute pharyngotonsillitisdue to group A beta-haemolytic streptococci,The 2nd International Conference on the Macrolides, halides and Streptogramins, Venice,1994; abstr 239. 5. Schonwald S, Skerk V, PetriEevie I, Car V, Majerus-MiSiC Lj, GunjaEa M. Comparison of three-day and five-day courses of azithromycin in the treatment of atypical pneumonia. Eur J Clin Microbiol Infect Dis 1991; 10:877.

Single-Dose Azithromycin in the Treatment of Atypical Pneumonia: A Pilot Study S. Schonwald, I. Kuman, and V. Car University Hospital Infectious Diseases “Dr. Fran MihaljeviP Zagreb , Croatia

J. culig and K. OreSkoviC Pliva d.d. Pharmaceuticals Division Zagreb , Croatia

INTRODUCTION Azithromycin is a rational choicefor the treatment of atypical pneumonia because it shows great in vitro activity against common etiologic agents: Mycoplasma pneumoniae, Chlamydia pneumoniae, Ch. psittaci, Ch. trachomatis, Coxiella burnetii, and Legionella pneumophila. Distinct pharmacokinetic propertiesof azithromycin enable short-termtreatment and it has been establishedthat azithromycin in atotal dose of 1.5 g over or 5 days is effective in the treatment of atypical pneumonia Considering the pharmacodynamic and pharmacokinetic characteristicsof azithromycin as well excellent experience with single-dose azithromycin treatment of chlamydial urethritis, it could be proposedthat 1.5 g might be given as a single dose inthe treatment of atypical pneumonia. 634

Single-Dose Azithromycin for Atypical Pneumonia

635

PATIENTS AND METHODS Patients of both sexes, older than 16 years, with atypical pneumonia who did not require hospitalization and who gave informed consent were included. Atypical pneumonia was defined asthe presence of new infiltrate on chest x-ray andthe presence of fever (238°C) with or without nonproductive or poorly productive cough. Exclusion criteria were pregnancy and lactation, hypersensitivity to macrolides, severe renal or hepatic impairment, gastrointestinal tract disturbances which affect drug absorption, the presence of signsand symptomsof typical acute bacterial pneumonia, pneumonia inpatients with underlying pulmonary disease, immunocompromised patients, andpretreatment with any macrolideor tetracycline or with more thanone daily doseof any other antimicrobial agent within1 week prior to entering the study. All patients weretreated with a single 1.5-g oral dose of azithromycin (Surnamed, Pliva) that was given at least 1 h before or 2 h after a meal. For the purpose of the study patients were hospitalized and their clinical condition was monitored daily. Chest x-ray was repeated 10-14 days after the initiation of treatment. Cure was defined as resolution of fever within72 h and disappearanceof other signs and symptomsof pneumonia within 10 days afterthe initiation of treatment with partial or complete regression of the infiltrate on control chest x-ray. All other outcomes were considered as failures. All adverse events were recorded. Laboratory safetytests(hematology,bloodbiochemistry,andurinalysis)were performed at baseline and 10-14 days after the initiation of treatment.

RESULTS Ten patients, aged 16-66 years, with symptoms and signs of atypical pneumonia were included inthe study. None of them required hospitalization. Baseline patient characteristics are presented in Table 1. Eight patients became afebrile within24 h, one within 36h, and one within 72 h after the initiation of treatment (Fig. 1). In all patients, clinical signs and symptoms of pneumonia disappeared within1week with totalor partial regression of the pneumonic infiltrate. Toleranceof azithromycin was very good. There were no side effects, and mild, clinically insignificant increase of ALT was noticed in one patient.

DISCUSSION A single 1.5-g dose of azithromycin was effective in patients all in this pilot study. We did not establish the etiological diagnosis of pneumonia and it

Schonwald et al.

636

Table I Baseline Characteristicsof the Patients with Atypical Pneumonia Treated with Single 1.5-g Dose of Azithromycin

Age Auscultatory Infiltrate Fever Sexx-raya Cougha findingsa chest (“C) (years)

Initials

N.Z. B.M. 21V.M. 16 S.V. 31 S.N. D.D. KM. S.M. B.A.

M M F F F M M M F M

S.J. a+

25 39

31 17 24 22 66

40.0 39.0 39.2 38.3 38.0 39.0 39.6 39.5 38.4 39.2

on

-

+ + + + + + + +

+ + + + + + + + + +

-

+ + -

+ -

+

present; - absent.

36

0

12

24

36 40 after initiationof heatment

60

72

Figure I The regression of fever in 10 patients with atypical pneumonia treated with single 1.5-g doseof azithromycin.

Single-Dose Azithromycin

for Atypical Pneumonia

637

can be argued that some of our patients had viral pneumonia. However, besides the low incidence ofthe viral pneumonia inadults, the characteristicclinical presentations andprompteffects of antimicrobial therapy strongly support our presumption that pneumonia was caused by atypical bacteria in all patients.

CONCLUSION The results of the pilot study justifyfurther evaluation of single 1.5-g dose of azithromycin for the outpatient treatment of atypical pneumonia.

REFERENCES 1. Schonwald S, GunjaEa M, KolaEny-BabiC L, Car V,GoSev M. Comparison of azithromycin and erythromycin in the treatment of atypical pneumonias. J Antimicrob Chemother 1990; 25 (suppl A):123. 2. Schonwald S, Skerk V, PetriEeviE I, Car V, Majerus-MiSiE Lj, GunjaEa M. Comparison of three-day and five-day courses of azithromycin in the treatment of atypical pneumonia. Eur J Clin Microbiol Infect Dis 1991; 10:877. Schonwald S, BarSiC B, Klinar I, GunjaEa M. Three-day azithromycin compared with ten-day roxithromycin treatment of atypical pneumonia. S a n d J Infect Dis1994; 26:706.

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CLINICAL STUDIES: OTHER RESPIRATORY INFECTIONS

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Azithromycin in the Treatment of Patients with Upper Respiratory Tract Infections, Comparison of 3-Day and 5-Day Regimens P. Dolehl

M . KroSlSk

Postgraduate Schoolof Medicine Bratislava, Slovakia

J. Klaeanskf ENT Department of University Hospital Bratislava, Slovakia

The purpose of this studywas to compare the clinical resultsof 5-dayand day regimen dosages in the therapy of ENT infections.

INTRODUCTION Azithromycin isan azalide antibiotic with a wide spectrum of antimicrobial activity. Its pharmacokinetics indicatethat the minimal inhibitory concentration (MIC)for important respiratorypathogensisachievedintracellularly and its long half-life permits once-daily dosing (1).In vitro studies had shown that azithromycin accumulates in human phagocytic cells, resulting in antimicrobial agent concentrated at the site of infection Using the same total dose (1.5 g), pharmacokinetic comparisonof a 3-day (500 mg once-daily) regimen with 5 days (500 mg as a single dose on day 1, 641

DoleZal et al.

642

then 250 mg days 2-5 perorally) indicates a trend in the direction of higher accumulationof azithromycin overthree days (4). Reports from the literature (5-10) about 3-day courses of azithromycin administered once daily led us to revise our experience with this drug (11) and to compare both dosages.

PATIENTS AND METHODS Clinical examinationof the 5-day regimen of azithromycin wasdone from September 1989 to February 1990 at the ENT clinicof the University Hospital in Bratislava.A total of 51 patients aged18-67 (mean 41.5) years were entered into the randomized study. Otitis media was present in 19 and 32 presented with upper respiratory tract infections. The drug was administered to 26 patients with acute disease and 25 with acute exacerbation of chronic inflammation (Table 1). Separate protocols were established for each diagnosis. The main data were case history, local findings, bacteriology specimens, blood picture,ESR, blood biochemistry, and x-rayin some cases. After the completion of therapy the 11th day, these examinations were repeated. Clinical examination of the 3-day regimen of azithromycin was performed from February to November 1995 at the same outpatient department. A total of 40 patients aged 10-75 (mean 29) years wereentered into this randomized study, 9 with otitis media and 31 with upper respiratory tract infections. Azithromycin, 500 mg, as a single daily dose was administered to 34 patients with acute otitis media, sinusitis, or tonsillopharyngitis and to 6 patients with relapses of chronic tonsillopharyngitis. The same evaluation criteria were usedasin the firstgroup of patients. Repeat

Tabk I

Results After 5-Day Treatment with Azithromycin Not

proved Cured Diagnosis Otitis media, acute Otitis media, chronic 1 Maxillary, sinusitis acute Tonsillopharyngitis, acute Tonsillopharyngitis, chronic Total

6 4

10

11

0

12

3

41 (80.4%)

9

1 3

1 1

6

0

11 15

(15.7%)

2 (3.9%)

51 (100%)

Azithromycin: 3-Day

and 5-Day Regimen for Upper RTI

643

Table 2 Results After 3-Day Treatmentwith Azithromycin

Diagnosis Otitis media, acute Sinusitis, acute

Tonsillopharyngitis,

Cured

Improved 1

Not improved

19

1

0 0 0

3

2

1

3

Total 9 5

acute

Tonsillopharyngitis, chronic Total

33

(15%)

40 (100%)

examination was performed on the seventh day after the start of therapy (Table 2).

RESULTS The results of treatment according to diagnosis are shown in Tables1and Satisfactory responses were deemed “cured.” After completion of treatment, clinical symptoms abated, examinations normalized, and subjective complaints disappeared. Cures occurred in 41 patients (80.4%) in the 5-day group and in (82.5%) in the 3-day group. Patients deemed “improved” were those in whom the elimination of the microorganism’ led only to partial improvement of subjective complaints and objective findings, or those inwhom the bacterial specimens remained positive. This occurred in eight patients (15.7%) in the first group and in 6 patients (15%) of the second group. In only three patients was no improvement observed after treatment.

Side Effects The drug was welltolerated in both dosage regimens. In the 5-day regimen,

no side effectswere observed.In the 3-day regimen,two patients had a mild stomach ache, which did not result in discontinuation of therapy. Liver enzymes were elevated inone patient, but no follow-up was required.

CONCLUSIONS In our group of patients, the results of 3-day azithromycin therapy were similar to results with 5-day therapy. It can be explained by the greater number of chronic diseases in the first group. Failure rates were low in

644

et

DoleZal

al.

both regimens. This study confirmed that 3-day azithromycin was as effective and well tolerated as S-day azithromycin in the treatment of ENT infections including acute otitis media, tonsillopharyngitis, and sinusitis.

REFERENCES 1. Schonwald S, Kovaliic D, Ajhler T. Pharmacodynamic and pharmacokinetic comparison of macrolides with a special attention to azithromycin. Medicus 1994; 3(2/3):119-128. 2. Foulds G, Shepard RM, Johnson RB.The pharmacokineticsof azithromycin in human serum and tissues. J Antimicrob Chemother 1990: 25(suppl A):73-82. 3. Retsema JA, Girard Schelkly W, Manousos M, Anderson M, Bright G. Spectrumandmode of action of azithromycin(CP-62,993),anew15membered-ring macrolide with improved potency against Gram-negative organisms. Antimicrob Agents Chemother 1987;31:1939-1947. 4. Foulds G, Johnson RB. Selection of dose regimens of azithromycin. J Antimicrob Chemother 1993;3l(suppl E):38-50. 5. Bradbury F. Comparison of azithromycin versus clarithromycin in the treatment of patients with lower respiratory tract infections. J Antimicrob Chemother 1993; 3l(suppl E):132-136. 6. Daniel RR. Comparison of azithromycin and co-amoxiclav the in treatment of otitis media in children. J Antimicrob Chemother 1993; 3l(suppl E):65-72. 7. Manfredi R, Januzzi C, Mantero E, Longo L, Schiavone R, Tempesta A, Pavesio D, Pecco P, Chiodo F. Clinical comparative study of azithromycin versus erythromycin in the treatment of acute respiratory tract infections. J Antimicrob Chemother 1992;30(6):364-370. 8. Myburg J, Nagel GJ, Petschel E. The efficacy and tolerance of a three-day course azithromycin in the treatment of community-acquired pneumonia. J Antimicrob Chemother 1993; 31:153-162. in treatment 9. Muller 0. Comparison of azithromycin versus clarithromycin the of acute upper respiratory tract infections. J Antimicrob Chemother 1993; ~ ~ ( S UE):137-146. PP~ 10. Pukander J. Penetration of azithromycin into middle ear effusions in acute and secretory otitis media, Proceedings from 2nd ICMAS, Venezia, 1994. 11. DoleZal P, KroSlAk M. Report about the clinical examinationof the antibiotic Surnamed (azithromycin). Lek Obzor 1993; 15(5):270-273.

Clinical and Bacteriological Evaluation of Clarithromycin (50-mg Tablets) in Children with Lower Respiratory Tract Infection Yoshikiyo

Toyonaga

Yamanashi Red Cross Hospital Yamanashi, Japan

Acute lower respiratory tract infection in children aged 1year or older is generally caused by Mycoplasma pneumoniae, Chlamydia pneumoniae, Haemophilus influenzae, pneumococci, or Moraxella catarrhalis. Clarithromycin (CAM) is approved for use against infectious diseases caused by these organisms. In this study,we evaluated the clinical and bacteriological efficacyof clarithromycin in pediatric patients with pneumonia and bronchitis.

SUBJECTS Subjects were 123 children aged between 4 years, 2 months and 11years, 6 months, weighing 15-36 kg, who were able to take clarithromycin tablets orally. The diagnoses were bronchitis in 15 patients, pneumonia in 63, and mycoplasma pneumonia in45, as shown in Table1. 645

646

Toyonaga

fficacy

Clarithromycin: ClinicalBacteriological and Evaluation

647

The dosage regimen employed wastablet (50 mg) to patients or tablets mg) to patients, each given three times daily. The average daily doses were mgkg per day and mgkg per day for eachgroup, respectively. days, although most The administered duration in80 patients was of the patients with mycoplasmal pneumonia were treated for more than days.

CLINICAL EFFICACY The results are summarized in Table The clinical efficacy rate was 80% for bronchitis, for pneumonia, for mycoplasmal pneumonia, and 90% when all patientsare considered. Causative organisms were identified in patients and the efficacy rate of clarithromycin was

BACTERIOLOGICAL EFFICACY Causative organisms were detected in out of patients with bronchitis or bacterial pneumonia. A total of patients had infection caused by a single organism and by multiple organisms, but in causative organisms were unknown. In four patients with bronchitis and five withpneumonia, causative organisms were not eradicated during treatment. One hundred seven strains were isolated from the patients and their eradication rates are listed in Table

Z'uble 2 Clinical Effect Classified by Diagnosis Causative organisms unknown

Causative organisms identified Total Efficacy No. of cases Diagnosis Pneumonia 88.9 Bronchitis 85.7 Mycoplasma pneumonia Total 87.8

54 13 7 74

'Not detected by cultivation.

Efficacy rate

(%l

No. of cases

100 50.0 84.6 94.7

9 2 3ga 49

rate

(%l

90.2 93.9

No. of cases

rate

63 15 45

90.5 80.0 93.3

123

(%l

Toyonaga

648

Table 3 Bacteriological Effect Classifiedby Causative Organisms (Pneumonia and Bronchitis)

No.of strains

Bacteriological effect Eradicated

Unchanged Unknown

Eradication rate (%)

H.injluenzae H. parainfluenzae B. catarrhalis S. pneumoniae S. aureus S. pyogenes Total

2

SUSCEPTIBILITY DISTRIBUTION The minimal inhibitory concentrations (MICs) of clarithromycin against clinically isolated H . influenzae, B . catarrhalis, and S. pneumoniae were compared with those of erythromycin and roxithromycin. The susceptibility distribution of clarithromycinwas similarto erythromycin, showing MICs of pg/ml against H . influenzae and pg/ml against B . catarrhalis. Some pneumococci were resistantto these macrolides.

CONCLUSION Clarithromycin possesses antibacterial efficacy against nearly all organisms which can cause lower respiratory tract infection. Currently available lactams fail to treat mycoplasmal or chlamydial disease. Clarithromycin appears to be a reasonable drug for empiric therapy of respiratory tract infections beforethe identificationof causative organisms. Clarithromycin, 50-mg tablets, are used clinically in pediatrics in Japan. In this study, infantsweighing less than 20 kg tolerated the tablets in preference to granules withoutany difficulties.

Experience with Improved Compliance of Clarithromycin Granules in Children Keisuke Sunakawa The 2nd Tokyo National Hospital Tokyo, Japan

Hironobu Akita St. Marianna University Schoolof Medicine Tokyo, Japan

Satoshi Iwata Kasumigaura National Hospital Tokyo, Japan

Yoshitake Satoh Ohta General Hospital Tokyo, Japan

Tatsuo Aoyama Kawasaki Municipal Hospital Tokyo, Japan

Ryochi Fujii Research Instituteof Chemotherapy Mothers and Children Tokyo, Japan

649

Sunakawa et al.

650

INTRODUCTION The ease of administration of antibiotics is a key factor in selecting drugs for children. Approximately one-fourth of children given antibiotics as outpatients do not take all of the prescribed drugs. The reasons include reluctance to take medication, discontinuation by parents who concluded the children were cured, or parents’ forgetfulness (Fig.1). Clarithromycin(CAM),whichhasbeenmarketedsince 1991 in Japan,ismarkedlyresistant to acid,well absorbed, andhasagood tissuedistributionprofile. CAMalsohasstrongbactericidalactivity against most etiologic bacteria of respiratory infections, and its efficacy against pertussis and chlamydial pneumonia has been proven in Japan, with the result that it is widely used in hospitals. However, because the currently marketed granules have an unpleasant taste, a large number of childrenrefuseto take them.In the pediatricfield, 50-mg tablets for children account for the largest proportion of prescriptions, and granules account for only 4.5%. Recently,taste-improvedCAMgranuleshave been developed. We report our clinical experience with this preparation of CAM. This preparation is different from other granule preparations in that it is suspended in water before use and it is defined in Japan as a “dry syrup.”

Reasons that children do not take all the drugs. Tokyo National Hospital.)

Figure l

(Data from The 2nd

Compliance of Clarithromycin Granules in Children

651

SUBJECTS General Clinical Trial Of the patients who visited or more of the facilities participating in this trial from June to March patients whose etiologic agents were estimated or were identified by antigen detection or increases in antibody titers and from whom parental informed consent was obtained were enrolled inthe trial. Some of the subjects also underwent pharmacokinetic study.

Clinical Trial in Patients with Pertussis Bacterial eliminationrates were compared between pertussis patients given CAM (5 mg/kg, a dayfor 7 days) and those given erythromycin (EM) mgkg per day for days). W Oage-, sex-, and vaccine historymatched patients were separately selected for each patientgiven CAM and were treated with EM. A case control study was conducted with the two groups.

-0-

10mg/kg

= 1)

U

5mg/kg

= 12)

Cmax2.26 f 0.42 L.cg/ml Tmax1.60 f 0.10h T1/23.89 0.52h Auc 13.48 1.93 L.cg*h/ml

2

0

16

5 3 Time (hours)

Figure 2 Changes in blood concentrations.

4

652

Sunakawa et al.

400

.-E

40

l00

0

0 2-4 Time (hours)

0-2

4-6

Figure 3 Urinary concentration and cumulative recovery rate.

Table l Clinical Response Rates According to Diagnosis Diseases

23 respiratory Upper infection, bronchitis 100 Bacterial pneumonia 7 Mycoplasma pneumonia6 1 Chlamydia pneumoniaa 2 Pertussisa 3 Dermal soft tissue 6 infection Campylobacter enteritis

Efficacy

No. of patients Excellent Moderate Poor rate

(%) 5

14

33 3 5 9 3 90

7 22

1

11

95.7 100

5 2 61

'Including patients diagnosed by serumantibody titer or antigen.

1 1

98.9

653

Compliance of Clarithromycin Granules in Children

(U

m

Sunakawa et al.

654 Tabk 3 AdverseEvents

No. of

ence patients Symptoms

in

(%)

Diarrhea Vomiting Dizziness

2

Sum

4

No. subjects

2.4

Laboratory Abnormalities Increase Increase in GPT

RESULTS Changes in CAM blood concentrations in 12 patients (6.1 2 1.4 years) given 5 mg/kg are shown in Fig. 2. The new preparation showed similar absorption as the conventional granules. Concentrations in urine and cumulative urinary excretion rates in seven patients (9.2 +1 1.1years) given5 mg/ kg are shown in Fig. 3. The new preparation was effective for all 90 patients with respiratory infections, skin infections, and enteric infections except one for pharyngolaryngitis patient who did not respondto the treatment. The efficacy rate was 99% (Table 1). Table 2 shows bacteriological efficacy according to the etiologic bacteria.Haemophilus influenzae and pneumococci showed a low elimination rate; however, allother strains were eliminated. Safety was assessed in all 169 patients given the granules (Table Diarrhea, vomiting, and dizziness occurred in 2, 1, and 1patients, respectively.Abnormallaboratory data includedincreasesinacidocytes and GPT. Ease of administration was evaluated in five categories: taken willingly (very easy to take-11%); all the granules taken without reluctance (easy to take-64%); all the granules taken with reluctance (taken without comment-23%);smallamount taken withreluctance (hard to take-1%); and could not take at all (very hard to take-1%). Seventyfour percent of the childrenscored the medication“easy to take” or higher. In patients with pertussis, the bacterial elimination rate was compared in patients given CAM at a dosageof 10 mg/kg for 7 days andthose given EM 40-50 mgkg for 14 days. Elimination rates after 1week were919 and 16/18 for CAM and EM, respectively. W Oweeks after treatment, absence of bacteria was noted in bothtreatment groups.

Compliance of Clarithromycin Granules in Children

655

SUMMARY The newly improved formulation had excellent ease of administration, efficacy, and safety. We conclude that this preparation has potential efficacy for the treatmentofpediatricinfectious diseases causedbyclarithromycinsusceptible organisms.

Efficacy and Tolerabilityof Azithromycin Versus Josamycin in the Treatment of Children with Lower Respiratory Tract Infections St4pBn Kutflek, JozefHoza, Daniela Markov& and Milan Bayer Charles University Prague, Czech Republic

INTRODUCTION Azithromycinisanazalideantimicrobialagentstructurallyrelated to erythromycin. Its mechanism of action is interference with bacterial protein synthesisby binding to the 50s ribosomal subunit(1).The spectrum of activity includes gram-positive and gram-negative microorganisms as well as intracellular pathogens(1). The results of numerous clinical trials have shown that azithromycin, administered once daily for or 5 days, is equally efficaciousin the treatment of respiratory tract infections as other commonly used antibiotics (amoxicillin, erythromycin, penicillin) that are administered two to four times a day for 7-14 days (1-4). The aim of our study was to compare the efficacyandtolerance ofazithromycinandjosamycinin childrenwith lower respiratory tract infections. 656

Azithromycin Versus Josamycin in Childrenwith LRTI

657

PATIENTS, MATERIALS, METHODS The study was open and comparative. Thirty-five children aged months (mean age: months) with lower respiratory tract infections were included in the study. Inclusion criteria were body weight above kg, temperature above measured axillary, cough, and/or dyspnea, and bronchitis or bronchopneumonia confirmedby auscultation and/orby x-ray. Exclusion criteria were renalor hepatic impairment, treatment with more than one dose of any antibiotic during h preceding the study, immunosuppressive treatment, malabsorption, sepsis, severe hypoxia with need for artificialventilation,terminaldisease withlifeexpectancyless than months. Patients were randomized to receive azithromycin mgkg once daily) for days or josamycin (50 mgkg divided in three daily doses)for 8 days. lkenty patients girls and boys) with a mean age of months (range: months)withbronchitis children) and bronchopneumonia children) were treated with a single daily dose of azithromycin mgkg) for consecutive days. Fifteen patients girls and boys) aged months(meanage: months)withbronchitis or bronchopneumonia received josamycin(50 mgkg per day inthree daily doses) for days. All patients were examined by a pediatrician at baseline andon days and The body temperature was monitored by the parents three times daily until it declined below Chest x-rays were performed on baseline in children andon day in children. Erythrocyte sedimentation rate (ESR), blood count, basic biochemistry (S-Na, K, urea, creatinine, bilirubin, AST, ALT, ALP, GMT, albumin, glucose) and microbiological evaluationof nasal pharyngeal swabs were performed on days and in all children.

RESULTS In the azithromycin-treated group, the body temperature declined below within (average: days, pathologic auscultatory findings disappeared within (average: days, and ESR normalized by day in children In the josamycin group, the body temperature declined below within (average: days, pathologic auscultatory findings disappeared within (average: days, and ESR normalized by day in children All patients were cured. Bacteriological findings are presented in Table Both drugs were very well tolerated, and only intwo azithromycin-

658

Kutflek et al.

Table Z Elimination of Pathogens Isolated fromNasaYPharyngeal Swabs in Children Treated with Azithromycin or Josamycin

Isolated/eliminated elimination rate (%) Pathogen Klebsiella pneumoniae Staphylococcus aureus Haemophilus influenzae Enterobacter sp. Escherichia coli

414 (loo) U1 (50)

1/1 (loo)

(loo) 1/1 (100) 111 (loo)

treated children was mild dyspepsia reported. There were no treatmentrelated laboratory abnormalities.

DISCUSSION In spring 1994, when we commenced our study, there were no reports comparing the efficacy and tolerance of azithromycin and josamycin in pediatric patients with lower respiratory tract infections. When evaluating the results, we focused on clinical and laboratory data. We observed similar clinical success in both groups.The results are in accordance with previous studies that in patients with upper and lower respiratory tract infections, otitis media, or skininfections, or 5-daytherapywithonce-daily azithromycin was equally effective as 7-14-day courses with other antibiotics (penicillin, amoxicillin, ampicillin, cefaclor, erythromycin, dicloxacillin) administered two to four times daily (1-6). However, azithromycin should be considered more convenient because a comparable clinical effect is achieved after a shorter treatment course and with only one daily dose.

CONCLUSION

.

Azithromycin given once daily for days is as effective as josamycin administered three times daily for 8 days in the treatment of lower respiratory tract infections in children. Azithromycin was well tolerated.

REFERENCES 1. Ballow CH, Amsden GW. Azithromycin: the firstazalide antibiotic, Ann Pharmacother 1992; 26:1253-1261.

Azithromycin Versus Josamycin Children in

with LRTI

659

Mohs E, Rodriguez-Solares A, Rivas E, El Hoshy Z. A comparative studyof azithromycin and amoxicillin in paediatric patients withacute otitis media. J Antimicrob Chemother1993; 31 (suppl E):73-79. 3. Hamill, J. Multicentre evaluation of azithromycin and penicillin V in the J treatment of acutestreptococcalpharyngitisandtonsillitisinchildren. Antimicrob Chemother 1993; 31 (suppl E):89-94. in 4. Weippl G . Multicentre comparisonof azithromycin versus erythromycin the treatment of paediatric pharyngitis or tonsillitis caused by group A streptococci. J Antimicrob Chemother 1993; 31 (suppl E):95-102. S. Rodriguez-Solares A, Perez-GutiCrrez F, Prosperi J, Milgram E, Martin A. A comparative study of the efficacy, safety, and tolerance of azithromycin, dicloxacillin, and flucloxacillin in the treatment of children with acute skin and skin-structure infections. J Antimicrob Chemother 1993; 31 (suppl E): 103-110. of azithromycininchildren. J Hopkins S. Clinicalsafetyandtoleration Antimicrob Chemother 1993; 31 (supplE):lll-118.

Azithromycin in the Treatmentof Respiratory Tract Infections in Children K. Galova, I. Marinova, and H.Cintalanova Childrens Hospital KoSice KoSice, Slovak Republic

J. Hractova Postgraduate Medical School, Old Town Hospital Bratislava, Slovak Republic

Kukova and S. Sufliarska Commenium University Bratislava, Slovak Republic

S. W i n and A. Nogeova University of Tmava Tmava, Slovak Republic

INTRODUCTION Azithromycin is a derivativeof erythromycin which belongs to a new subclass of macrolides, the azalides. Azithromycin is characterized by high and sustained tissue concentrations and extended spectrum of activity, which includes gram-positive, gram-negative,and intracellular pathogens ( The bim of this multicentric studywas to verify the efficacy and safety of azithromycin inthe treatment of respiratory tract infections in children. 660

Azithromycin

RTI in Children

Table l Baseline Patient Characteristics

patients No. of Maledfemales Age range Age distribution < year: years: years: > years:

months-l4 years children children children children

PATIENTS AND METHODS Children of both sexes aged 2 months to 14 years with signsand symptoms of acute upper or lower respiratory tract infection were included in the study. Patients with known hypersensitivity to macrolides or with severe infection that required parenteral treatment were excluded. Azithromycin was administered orally, once daily, foreither 5 days mgkg on day 1 and 5 mgkg ondays 2-5) or days mg/kg daily). Clinical examination was performed at baseline, 48 h, and 8 days after the initiation of treatment. Laboratory( C m , W CESR) , and microbiological tests were performed before and after therapy. Clinical efficacy was evaluated at day 8 and classifiedas cure (disappearanceof all baseline signs and symptoms of infection), improvement (partial disappearance of baseline signs and symptoms),or failure (no change or worsening of initial condition). The occurrence of adverse events was recorded during treatment and follow-up. Adverse events were scored for seventy, duration, and relation to treatment. Table 2 Clinical Efficacy of Azithromycin in the Treatment of Acute Respiratory Tract Infections in Children

Diagnosis Pharyngitis Tonsillitis Otitis media Sinusitis Bronchitis Pneumonia Total

evaluable success Clinical No. of (curepatients

+ improvement) (100%) 12 (92%) 10 (91%) (95.5%)

Galova et al. Table 3 Bacteriological Efficacy of Azithromycin in the Treatmentof Acute Respiratory Tract Infections in Children

Pathogen Haemophilus injluenzae Staphylococcus aureus Streptococcus pneumoniae Streptococcus pyogenes Haemophilus spp. Bordetella catarrhalis Streptococcus agalactiae Escherichia coli Pseudomonas aeruginosa Klebsiella spp. Proteus morganii Enterobacter Corynebacterium hoffmanii

Gram-negative bacilli Totai

n eradicated / n isolated 15/16 11/12 7/7 515 314 314 313

112 111 111 111 111 111 111 54/59 (91.5%)

RESULTS A total of 101 children were enrolled in the study. Baseline demographic data are presented in Table 1. The majority of patients (92 children) were treated for 5 days; only 9 received a 3-day course. Eighty-nine patients wereevaluableforclinicalefficacy.Cure was achievedin children (86.5%), improvement in and therapy failed 4in(4.5%). Clinical success rates for each diagnosis are presented in Table 2. Pretreatment culture was positive in46/65 children. Isolated pathogens and their eradication rates are presented in Table 3. In total, 91.5% of baseline pathogens were eradicated. Adverse events were observed in nine children (8.9%): gastrointestinal side effects (diarrhea, vomiting or nausea) in four, skin rash in one, eosinophilia inthree, and elevated liver enzymes in one. Our results confirm high clinical efficacy and safety of azithromycin in the treatment of respiratory tract infections in children and correspond well with the findings fromother studies (3,4).

CONCLUSION Short-course azithromycin therapy is effective and well tolerated in the treatment of acute respiratory tract infections in children.

Azithromycin for RTI in Children

663

REFERENCES 1. Foulds G, Johnson REI. Selection of doseregimens of azithromycin.J Antimicrob Chemother 1993; 31 (suppl E):39. J Med 1991; 91 (suppl 2. Neu HC. Clinical microbiology of azithromycin. A):3. Bull AP. Azithromycin in the treatment of lower respiratory tract infections. Rev Pharmacother 1994; S:329. 4. Manfredi R, Jannuzzi C, Mantero E, Longo L, Schiavone R, Tempesta A, Pavesio D, PeccoP, Chiodo F. Clinical comparative study of azithromycin versus erythromycin in the treatment of acute respiratory tract infections in children. J Chemother 1992; 4:364.

Efficacy of Roxithromycin in Elderly and Middle-Aged Patients with Respiratory Tract Infections S . Yakovlev Moscow Medical Academy, Municipal HospitalN 7 Moscow, Russia

INTRODUCTION Respiratory tract infection isone of the most frequent bacterial infections in all age groups. One of the difficulties in treatment is that the causative pathogens are changing, with new pathogens as Mycoplasma, Legionella, and Chlamydia being isolated more frequently These intracellular organisms are not accessibleto many antibacterial agents such as p-lactam antibiotics and others which have poor penetration through the cell wall. Treatment is further complicated especially in elderly patients with chronic respiratory tract diseases, by the increasing incidenceof resistant strains of Staphylococcus aureus, Haemophilus influenzae, and Moraxella catarrhalis whichproducep-lactamases.Thefrequency of penicillinresistant strainsof Streptococcwpneumoniae also has increased by Macrolide antibiotics have been used the fortreatment of respiratory tract infectionsformorethan years, the mostwellknownbeing erythromycin. However, erythromycin has the disadvantagesof insufficient stability at low pH, a relatively narrow spectrum of activity, and a high 664

Roxithromycin the in Elderly and

Middle Aged with RTI

665

incidence of gastrointestinal side effects.The new generation of macrolide antibiotics have improved chemical, biological, and pharmacokinetic properties, together with improved clinical efficacy and safety. Among them is roxithromycin, anew semisynthetic macrolidewhich differs fromerythromycin by the presence of a 2-methoxy-ethoxy-methoxyamino group in the ninth position.We studied the effects of roxithromycintreatment in elderly patients with acute and chronic bronchitis.

MATERIALS AND METHODS Patients A total of 26 patients with community-acquired bronchitis were included in this open study. Sixteen patients were over60 years of age (mean: f6 years; range:64-81) and patients were less than 50 (mean: f 8 years; range: 28-46 years). Twelve patients had acute bronchitisand 14 had exacerbations of chronic obstructive pulmonary disease. Patients with a history of hypersensitivityto macrolides, with clinical signs and symptoms of viral infections, renal or hepatic failure, or patients treated with antibiotics in the previous 3 days were excluded. Diagnosis of bronchitis was based on typical clinical signs (productive cough for more than 5 days with purulent sputumor fever), confirmed by physical examination, chestx-ray, laboratory and hematological tests,and bacteriological examination. Hematological examinations and bacteriology of sputum (if obtained) were performed beforetreatment and within 48 h after the end of treatment.

Treatment Roxithromycin was given orally at a dose of mg bid. Patients were instructed to take medicine,before meals with a sufficient quantity of water. The duration of treatment was days, depending on the severity of infection and the clinical response.

Assessments A satisfactory clinical response was excellent (complete disappearance of signs and symptoms of the disease in days) or good (disappearance within days, or incomplete disappearance of symptoms during treatment). Response was poorif clinical signs of infection remained or worsened. Bacteriological responsewas considered satisfactory if the microorganism isolated fromthe sputum was eradicatedon posttreatment culture, or if the patient improvedto such an extentthat no culturable materialwas

Yakovlev

466

available at the end of treatment. It was unsatisfactory if the causative pathogenremained on posttreatment culture, or a newmicroorganism appeared with evidenceof a new infection.

RESULTS All patients completed the study and were suitable for analysisof efficacy and tolerance. Beforetreatment, pathogens were isolated from elderly (44%)and 5/10 middle-aged (50%) patients. ?he most commonly isolated pathogens were S. pneumoniue (six), H . infruenzae (four), and S. aurem (three); in cases, two causative pathogens were isolated. After treatment, sputum was obtained from elderly and middle-aged patients. Posttreatment bacteriological examination was positive in onlytwo cases. The duration of roxithromycin treatment was days in five elderly patients, days in eight patients, and days in three patients. Five middle-aged patients received roxithromycin for days, the other five for days. The total clinical efficacyrate was in patients over and in patients less than 50 years. Bacteriological eradication was achieved in elderly patients and in 4/5 middle-aged patients. The results of treatment in the two age groups of patients were similar. Only one case of mild diarrhea, probably related to treatment, was noted during the study. significant changes were observed in laboratory tests at the end of treatment.

DISCUSSION The results of our study are in agreement with data from numerous trials of roxithromycin in lower respiratorytract infections. The clinical efficacy of roxithromycininpatientswithacutebronchitisandexacerbation of chronic obstructive pulmonary disease (COPD) was Similar results were shown in the study by Ludwig et al. The clinical efficacy rate was in elderly patients, together with a low incidence of side effects. Thus, roxithromycin is an effective and safetreatment for elderly patients at the recommended doses based on available pharmacokinetic data (5). dosage adjustment of roxithromycin in elderly patients appears to be necessary. Bacteriological examinationof sputum in lower respiratory tract infections is oftenof little use in identifying the causative pathogens due to the difficulties of obtaining adequate specimens of sputum from elderly patients. Identification of some pathogens (Mycoplasma, Legionella, Chlamydia) requires special techniques not available in ordinary medical prac-

Roxithromycin the in

Elderly and Middle Aged with RTI

667

tice. The rate of positive bacteriological examination of sputum in our study is which agrees withother data (6). It appears worthwhileto use an empiric approachto thetreatment of lower respiratory tract infections in elderly patients, taking into consideration the spectrum of antibiotic activity, pharmacokinetic properties (high concentration of drug in sputum and intracellularly), side-effect profile, and frequency of drug administration.On this basis,the macrolides, in general, and roxithromycin, in particular, be canconsidered inthe empiric treatment of community-acquired respiratorytract infection in elderly patients.

CONCLUSION Roxithromycin at a daily doseof 150 mg bid is a safe, well tolerated, and effective treatmentfor elderly and middle-aged patients with acute bronchitis and exacerbations of COPD.

REFERENCES 1. MoelleringR.Introduction:revolutionarychangesinthemacrolideand azalide antibiotics.Am J Med 1991; 91:l. 2. Neu H.Roxithromycin-an overview. Br J Clin Pract 1988;42:l. 3. Markham A, Faulds D. Roxithromycin. update of its antimicrobial activ-

ity, pharmacokinetic properties and therapeutic use. Drugs 1994; 48:297. 4. Ludwig E, Kovacz G, Arr M,Szekely E. Once-a-dayadministrationof roxithromycin in young and elderly patients. Proceedings of the 18th ICC, Stockholm, 1993,p. 418. 5. Young R A , Gonzales JP, Sorkin EM. Roxithromycin. A review of its antibacterialactivity,pharmacokineticpropertiesandclinicalefficacy.Drugs 1989; 37%. 6. Tilyard MW, DoveySM.Arandomizeddouble-blindcontrolledtrialof roxithromycin in the treatment of acute respiratory tract infections in general practice. Diagn Microbiol Infect Dis 1992; 1597.

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xv OTHER CLINICAL STUDIES

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Experience with Azithromycin as Prophylaxis in Transrectal Biopsy of the Prostate M. Kvarantan University Hospital Center Re bro Zagreb, Croatia

C . Dohoczky Pliva d . d . Research Institute Zagreb, Croatia

INTRODUCTION Diagnosis of prostate cancer is based on digitorectal examination, determination of PSA serum values, and transrectal ultrasound of the prostate. However, diagnosis should be verified by histological examination of tissue samples obtained by needle biopsy of the prostate. Complications of transrectal needle biopsy of prostate include hematuria, enterorrhagia, hematospermia, and infections such as prostatitis, urinary infections, sepsis, and so forth. The development of infectious complications due to the instrumental procedures (puncture, biopsy, etc.) is generally prevented by rigorous asepsis. As it is almost impossible to achieve aseptic conditions at the site of needle entry, the use of periprocedural antibiotic prophylaxis remains an open question. Most authors recommend the use of antibiotics prior to such procedures. The aim of this study was to assess the efficacy of 671

Kvarantan and Dohoczky

672

azithromycinin the prophylaxis of infectiouscomplicationsfollowing transrectal biopsy of the prostate.

PATIENTS AND METHODS The study design was approved bythe IRB of the Clinical Hospital Rebro. Adult malepatients with suspectedprostate cancer, undergoingtransrectal biopsy of the prostate under ultrasound control were included. Patients received 1 g of azithromycin(Surnamed,Pliva)divided in twodoses: 500 mgorally,12 h and 2 h prior to biopsy. Transrectal biopsyof the prostate was performed under ultrasound control. A Tru cut needle, 1.2 mm in diameter, was inserted into the prostate through the rectum. Four to eight tissue samples were taken from variousareas of the prostate. Specimens were taken for histopathologic diagnosis and determination of azithromycin concentrationin prostatic tissue. After the biopsy all patients were monitored for the occurrence of complications (hematuria, enterorrhagia, hematospermia, and infection).

RESULTS A total of 48 patients were included in the study. Six to eight punctures were performed in each patient (a total of 312, or punctures per patient). The type and incidence of observed complications is presented in Fig. 1. Hematuria and enterorrhagia, lasting most often from 3 to 4 days, was observed in all patients. Short-lasting hematospermia wasobserved in two patients, whereas only onepatient developed a urinary tract infection which was successfullytreated.

"

Hematuria Enterorhagia Hernatospermia Infection

Complications of transrectal prostate biopsy in patients pretreated with azithromycin.

Figure I

Azithromycin as Prophylaxis for Prostate Biopsy

673

DISCUSSION According to theliterature which indicated the importance of antibiotic prophylaxis during surgical procedures, we pretreated patients undergoing transrectal biopsy of the prostate with azithromycin. Azithromycin was administered in two doses, and h prior to biopsy. The first dose was given to obtain high prostatic tissue concentrations of azithromycin. fourteen h after the 500-mg oral dose of azithromycin, the mean prostatic tissue concentration is2.5 &g and remain elevatedfor several days (prostatic tissue half-life is h) This concentrationis above the minimal inhibitory concentrations (MICs) for microorganisms causing soft tissue infections and sexually transmitted diseases. Moreover, it is overthe M I 0 for some microorganisms found in the rectal flora [e.g., Bacteroides spp. (MICs range to m@) or Peptostreptococcus (MI% = mg/ml)]. Similar high concentrationsof azithromycin are also found inejaculate (5). Prostatic tissue concentrations exceeded plasma concentrations by a factor of 20. Mean peak plasma concentrations of m@ are obtained approximately h after a500-mg oral dose of azithromycin

CONCLUSION According to our experience prophylactic use of azithromycin for transrectal needle biopsy of the prostate is associated with a low rate of infection. Other complications (hematuria, enterorrhagia) are mild and do not require additional treatment.

REFERENCES 1. Burke JF. Surgery1961;50:161. 2. Cruse PJE, Foord R. Surg Clin North Am 1980; 60:27. 3. Foulds G, Shepard RM, Johnson RE%. JAntimicrob Chemother 1990; (suppl A):73. 4. Foulds G , Madsen P, Cox C, Shepard RM, Johnson RB. Eur J Clin Microbiol Infect Dis 1991; 102368. 5. Le BelM,Bisson C, Allard S, Vallee F. 33rd Interscience Conference on Antimicrobial Agents and Chemotherapy,New Orleans, 1993; abstr 730.

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Author Index

Acar, J. F., Agouridas, C., Akita, H., Akova, M., Alcini, P., Alder, J. D., Andriole, V.T., Aoun, M., Aoyama, T., Apostolopoulos, N., Appelbaum, P. C., BaCe, A., Bairamis, T., Bajaksouzian, S., Barbarini, G., Barbaro, G., Bauernfeind, A., Bayer, M., Benedetti, Y., Bergen,, G., Bergogne-Berezin, E., Bonaly, R., Bonnefoy, A., Bouanchaud, D., Bourguignon, A. M., Bryskier, A., Bukowska-Nierojewska,D.,

Burek, V., Butzler, J.-P., Campbell, M., Car, V., Cerwinka, S. L., Chaloupka, M., Chantot, J.-F., Chaslus-Dancla, E., Chauvin, J. P., Chmurny, J., Cimpennan, J., eintalanova, H.,660 Cipriani, P., Citron, D. 555 M., &man, M., 528 Clavier, J., Claypoole, D.C., Cocito, C., Collette, P., Connolly, A. G., 460 Conway, A., Craft, J, Craig, W. A., Crokaert, F., b l i g , J., Curatolo, W. J.,

675

676 Dajek, Z., Darfeuille-Michaud, A., Debbia, E. A., Del Buono, G., DeLollis, R.D., Denis, A., Derevianko, I. I., Demennic, M., Desaulty, A., Desnica, B,, Di Giambattista, M., Dinopoulou, D., Dohonky, C., Dolei?al, P., DragaS, A. Z., Duchateau, V., Dunne, M. W., Eberlein, E., Edo, S., Eklebemy, A., Enogaki, K., Erman, M., Ewing, P., Fasola, E.L., Favre-Bonte, S., Felmingham, D., 523 Ferianec, V., Ficnar, B., File, T.M., 544 Finance, C., Finch, R. G., Flek, J., Fletcher, A. M., Forestier, C., Fortner, J. H., Foulds, G., Friedman, H. L., Fujii, R., Furneri, P. M., Galova, K., Garavelli, G., Gardner, M. J.,

Author Index Gasser, R., Gehanno, P., Gendrel, D., Gesu, G. P., Going, P. C., Goldstein, E. J. C., 555 Golec, K., Gomberg, M. A., Goossens, H., GoriSek, J., Grayston, J. T., Grba, V., Grenier, P., Grisorio, B., Gur, D., Hadjieva, N., 505 Hammerschlag, M. R., Hansen, R.A., Hausner, O., Hayran, M., Herman, V., Hidaka, T., Hilali, L., Hoepelman, I. M., Holoman, K., Holt, D. A., Houston, S. H., Hoza, J., Hractova, J., 660 Huovinen, P., Husson, M., Huzjak, N., Indorf, A. S., 544 Iniguez, J.-L., Ivanova, K.,505 Iwata, S., h m i , K., Jacobs, M. R., Jarvis, K., Jaworski, S., Joly, B., Jungwirth, R.,

Author Index Kaaander, N., Kafetzis, D., Kalifa, G., Kawazoe, K., KlaEnski, J., Klamo, J., Klastersky, J., Klepser, M. E., Klinar, I., Kobal, B., Kolokathis, A., Konno, K., Krtisny, B., Krifan, S., KroSlBk, M., Kukova, 660 Kundsin, R. B., KUO,C.-C., Kutilek, Kuzman, I., KuzmanoviC, N., Kvarantan, M., LabaEevski, N., Labbe, G., Labro, M.-T., Lafont, J.-P., Lang, R., Lanzoni, L., Larkin, J., Lawrence, V., Le Royer, C., Lebon, P., Lemaitre, F., Uophonte, R., Linares, J., Longstreth, S. J., Lopez, S., Loppinet, V., LotriE-Furlan, S., Lucchini, A., Luke, D. R., Makek, N., Marangos, M. N.,

677 Maraspin, V., Marchisio, P., 597 Margulis, A., Marina, M., 505 Marinova, I., 660 Markov& D., Martel, J.-L., Mashkilleyson, A. L., Matrapazovski, M'. Mauvais, P., Mayer, K. H., McCracken, G. H., McCutchan, J. A., McGowan, J. E., MCgraud, F., Melnik, G., Melo-Cristino, J., Menozzi, M. G., Meulbroek, J. A., Mikamo, H., MiloSevski, P., Mitten, M. J., Miura, K., Moellering, Jr., R. C., Moulin, F., Muller-Serieys, C., Musher, D. M., Muto, H., Nakayama, I., Nani, E., Nicolau, D. P., Nicoletti, G., Nicolosi, V., Nightingale, C. H., Nogeova, D. A., Nyssen, E., Oehler, R., Ogawa, M., Oksman, A., Oleksijew, A., Ordem, A., OreSkoviC, K., Ortisi, G.,

678 Padk, D., Paige, L., Pankuch, G. A., Panovski, N., Paragi, M., Patel, K.B., Peters, G. , Petr, P. , Phillips, I., Poulin, S. A., Powell, M. , Principi, N., Pypkowski, J., Quintiliani, R., Raffi, F., Raymond, J., Ravilly, S., Reisinger, E. C., Remington, J. S., Rich, C., Rigoli, R., Roblin, P. M., Rogl, J., Rose, L.M., Rubinstein, E., Ruby, W. H., 544 Ruskin, J., Russomanno, J. H., Rzeszutko-Grabowska, M. , Schonwald, S., Sakalosky, P., Sala, E., Satoh, Y. , Scavone, J., Schaad, U. B., Schachter, J. , Schito, G. C., Segev, S., Seme, K., Serra, R. , Shash, B.,

Author Index Shimada, K., Shimooka, K., Shonovfi, O . , Shortridge, V. D., Signs, D. J., Sinnott, J. T. , Sirot, J., Skupien, D., Sloos, J. H., Sokolovskaya, N., Sorella, S., 597 Sorgel, F., Spangler, S. K., Stano, F. , Stratton, C., Strle, F. , Sufliarska, S., Sunakawa, K., Tagaya, E., Takahashi, K., Takeda, Y., Takemura, H., Tamaoki, J., Tamaya, T. , Tanaka, S. K., Tarlow, M. J., Tempera, G., Teng, R., Tessier, P. R., TomaStik, P., Toni, M., Torten, D., Toyonaga, Y.,

Unal, S., Urban, M., Urumov, A. , van Asselt, G. J. , Vandermies, A., Vannuffel, P. , Vigan6, F. E., Vincent, J.,

679

Author Index Vlachou, S., 593 von Eiff, C. ,402 Wendelin, I. ,299 Willavize, S. A., 469 Xuan, D., 439

Yakovlev, S., Yamaji, E., 415 Yokoyama, S., 415 Youssef, N.,286 Zervos, M.J., 617 ZmiC, T.,559

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Subject Index

Alexander Project, lower respiratory tract infection, macrolides and, 523-27 Alfentanil, effect of macrolides on, 186 Amoxicillin, vs. azithromycin, forotitis media, 597-601 Animal bite wounds, bacteriology, macrolide susceptibility,55558 Animal uses, macrolides for,117-19 growth promotion, 117-18 resistance, 119-23 Actinomyces pyogenes, 123-24 bacterial pathogens, susceptibility in, 119-21 genetic support, 121-22 mechanisms of, 122-23 Pasteurella, 123-24 therapy, 118-19 veterinary problems, 123-24 Asthma prevention, clarithromycin, 343-48 halides bartonellosis, 271-72 bites, wound infections,267-68 brucellosis, 269 Chlamydia pneumoniae,70 Corynebacterium jeikeium,268-69

[halides] dosing interval, effect onin vivo efficacy, 31-32,32t gonorrhea, 272 Haemophilus influenzae, 70 human immunodeficiency virus, 25765

legionella, 70,209-12,21Ot Listeria monocytogenes, 268-69 malaria, 270-71 microbiologic activity, 67-69,68t Moraxella catarrhah, 70 Mycobacterium avium prophylaxis of, 259-61,.260t treatment of, 257-59 neutrophils, effecton in vivo efficacy, 32-33,33t opportunistic infections,189-204 overview, 67-73 pertussis, 270 pharmacokinetics, 69,69t postantibiotic effects, 27-38,29-30t, 30-31 sub-MIC effects,28,29t salmonellosis, 270 Azithromycin, 13-16 vs. amoxicillin, for otitis media, 597601

681

682 [Azithromycin] bioavailability effect sf food, presystemic, post-absorptiveinfluences on, bronchitis, Chlamydia pneumoniae, pneumonia, Chlamydia trachomatis, chlamydial infection, genital, clarithromycin bronchopulmonary pharmacokinetics, compared, respiratory tract pathogens, comparative pharmacodynamics, erythema migrans, gastrointestinal tract administration, pharmacology, Helicobacter pylon, Hemophilus influenzae, human immunodeficiency virus, intraabdominal infection, vs. josamycin, respiratory tract infection, in children,

Subject Index [Azithromycin] with ranitidine, combined treatment duodenal ulcer, chronic gastritis, Helicobacter pylori, duodenal ulcer, chronic gastritis, rectal, bioavailability and, respiratory tract infection, in children, resistance, sexuallytransmittedinfections, skin infections, soft tissue infections, Streptococcus pyogenes, susceptibility testing, syphilis, tonsilitis, toxoplasmic encephalitis, trachoma, transrectal biopsy of prostate,

Bacteremia, quinupristiddalfopristin, resistant gram-positive bacteria, Bacterial protein synthesis, streptogramins,

Legionnaires’ disease, Bartonella, immunocompromised paLyme disease, tients, Mycobacterium aviumcomplex infec- Bartonellosis, tions, Biopsy of prostate, azithromycin, nongonococcal urethritis, in males, Bite wounds pediatric infection, infections, penetration macrolides, Bordetella pertussk, microautoradiographic studies, Borrelia burgdorferi Lyme disease, neutrophil, pertussis, in children, roxithromycin, temperature effects, pneumonia, Bromocriptine, effectof macrolides on, Chlamydia pneumoniae,in children, Bronchitis, (See also RespiraPseudomonk aeruginosa, tory tract infection)

Subject Index

683

[Clarithromycin] Helicobacter pylori, 18 Hemophilus influenzae, 533-36 indications, 79-80 mycobacterial infections,17-18 Mycobacterium avium, 539-43,54448 pediatric, 132-43 Campylobacterjejuni, 237-49 compliance, inchildren, 649-55 Campylobacter species, 505-10 otitis media, in children, 593-96 Carbamazepine, effect of macrolides pertussis, in children, 651 on, 182-84,183t respiratory tract infection, in chilCefpodoxime proxetil, clarithromycin, dren, 645-48 compared, chronic obstructive peptic ulcer disease, 81-83,82pulmonary disease,609-13 Chemiluminescence, penetration rerespiratory tract infection, 1 6 1 7 sponse, neutrophil, 455-59 skin infections, 17 Chlamydia soft tissue infections,17 genital, azithromycin,352-53,354-57 Streptococcus pneumoniae, 411-14, RU 004,400 429-31 Chlamydia pneumoniae,133,142,219Streptococcus pyogenes, susceptibility 23 testing, 407-10 azalides, 70 toxoplasmosis, azithromycin, 322-25 transepithelial potential difference, benzoxazinorifamycin, 317-21 tracheal mucosa,576-80 KRM-1648,317-21 urogenital infections, 17 macrolides, 317-21 Clindamycin resistance, susceptibility Chlamydia trachomatis testing methods,variations, azithromycin, 349-51 369-75 KRM-1648,318 Chronic obstructive pulmonary disease, COPD (See Chronic obstructive pulmonary disease) cefpodoxime proxetil, Corynebacterium jeikeium,268-69 clarithromycin, compared, Cryptosporidium, immunocom609-13 promised patients, 194-95 Clarithromycin, 1618,76-83,78-79t Cyclosporine, effectof macrolides on, asthma prevention, 343-48 184,185t azithromycin Cystic fibrosis,Pseudomonas bronchopulmonary aeruginosa, bacterial pharmacokinetics, compared, glycocalyx of, macrolides sup439-46 pression, 91-100 respiratory tract pathogens, comparative pharmacodynamics, Digoxin, effectof macrolides on, 184 478-84 Dirithromycin, kineticsof, 447-54 cefpodoxime proxetil,compared, chronic obstructive pulmonary Disopyramide, effectof macrolides on, 186 disease, 609-13

[Bronchitis] azithromycin, 605-8 clarithromycin, cefpodoxime proxetil, compared, 609-13 Brucella melitensis, macrolides, 563-68 Brucellosis, 269

684

Subject Index

Drug interactions, (See also under specific drug) Duodenal ulcer, azithromycin, ranitidine, combinedtreatment, Helicobacter pyloriy Elderly, respiratorytract infection in, roxithromycin, Enterococci overview, RPR Enterococcus faecium, quinupristid dalfopristin, Enterococcus species RPR RPR ratio, RPR

RPR

ratio,

Ergot alkaloids, effectof macrolides on, Erythema chronicum migrans azalides, macrolides, spirochetes, Erythema migrans, azithromycin, Erythromycin, pediatric infection, respiratory pathogen resistance, sparfloxacin and, antipneumococcal activity, Streptococcus pyogenes, susceptibility testing, Gastritis, chronic, azithromycin, ranitidine, combined treatment, Helicobacter pylori, Genitalia (See also Sexuallytransmitted infections) chlamydial infection, azithromycin, roxithromycin,

Giardiasis, immunocompromised patients, Gonorrhea, Growth promotion, macrolidesand, Haemophilus influenrae, azalides, macrolides, respiratory tract infection, macrolides, RPR RPR ratio, RPR RPR ratio, Helicobacter pylori, antibiotic regimens, azithromycin, azithromycidranitidine combined treatment, duodenal ulcer, chronic gastritis, clarithromycin, gastritis, macrolides, MICs, resistance to macrolides, roxithromycin, HIV (See Human immunodeficiency virus) Human immunodeficiency virus azalides, azithromycin, clarithromycin, Mycobacterium avium, macrolides, mycobacteria, Mycobacterium avium-intracellulare infection, opportunistic infections, streptogramins, pentamidine, Pneumocystis cariniipneumonia, roxithromycin, pyrimethamine, streptogramins, toxoplasmosis, roxithromycin,

Subject Index [Ketolides] Immunocompromised patients (See akro Human immunodefi[RU Mycoplasma species, 400 ciency virus) respiratory pathogens, 395-401 opportunistic infections stability, in acidic medium, 282, bartonella, 199t, 199-200 283 cryptosporidium, 194-95 Staphylococcus aureus, 280, 281 Enterococcus faecium, structure, 280, 281 vancomycin-resistant,201 RU 64004,43-47,44-46 giardiasis, 192 Klebsiella pneumoniae, roxithromycin, legionella, 19Ot, 190-91 581-85 Mycobacterium avium complex, KR"1648, Chlamydia pneumoniae, 195-99 318 prophylaxis, 196-97 azithromycin, 196-97 clarithromycin, 196 Legionella treatment, 197-99 azalides, 70,209-12,210t clarithromycin, 197-99 azithromycin, 295-98 Pneumocystis cariniipneumonia, clinical presentation, 208 191 immunocompromised patients, 19Ot, pneumonia, 200,200t 190-91 toxoplasmosis, 192-94,193t laboratory diagnosis, 208 Inflammation macrolides, 209-12,210t macrolides, 102-3 RPR 106972,61 antibacterial agents and, 103-4 RU 004,400-401 phagocyte function, macrolide modifitherapy, 208-9 cation of, 108-9 Leukocytes, effectof macrolides, 101Intraabdominal infection, 16 azithromycin, 511-15 Lincosamide resistance,in Streptococcus pneumoniae. 369-75 Josamycin, azithromycin,contrasted, Listeria monocytogenes, 268-69 lower respiratorytract infecLower respiratorytract infection tions, in children,656-59 Alexander Project, macrolides, 52327 Ketolides, 39-49,41-42 azithromycin, 13-14 RU 004,279-85,421-24 vs. josamycin, in children, 656-59 atypical bacteria, 400-401 clarithromycin, 16-17 chlamydia, 400 in children, 645-48 gram-negative bacteria, 400 Haemophilus influenzae, macrolides, gram-positive bacteria, 398 523-27 hepatic cytochrome P-450, effects josamycin, azithromycin,contrasted, on, 282-85,284 in children, 656-59 human monocytic cells,uptake by, Moraxella catarrhalis, macrolides, 282 523-27 legionella, 400-401 roxithromycin, 19 MIC, effect of pH on, 282,283

Subject Index [Lower respiratorytract infection] Streptococcus pneumoniae, macrolides, 523-27 Lyme disease, 212-14 Macrolides alfentanil, effect on, 186 animal bite wounds, animal uses, 117-29 growth promotion, 117-18 resistance, 119-23 therapy, 118-19 antibacterial agents and, 103-4 bartonellosis, 271-72 bite wounds, 267-68,555-58 bromocriptine, effect on, 184 Brucella melitensis, 563-68 brucellosis, 269 Campylobacter jejuni,23740,238t Campylobacter species, 505-10 carbamazepine, effect on, 182-84, 183t Chlamydia pneumoniae,317-21 clarithromycin, 76-83,78-79t indications, 79-80 peptic ulcer disease, 81-83,82-83t usage, 79-80 clinical use overview, 3-26 Corynebacterium jeikeium,268-69 cyclosporine, effect on, 184, 185t development of, 4-21 digoxin, effect on, 184 disopyramide, effecton, 186 dosing, 27-38 effect on in vivo efficacy, 31-32, 32t drug interactions, 177-88 mechanism of, 178 ergot alkaloids, effecton, 186 erythromycin, 75-76 future of, 86 gonorrhea, 272 Haemophilus influenzae, 516-22 respiratory tract infection,523-27 Helicobacter pylori, 240-46

[Macrolides] [Helicobacterpylori] MICs, 241t, 241-42 resistance, 24244,243 human immunodeficiency virus, 25765 inflammation, 101-16 isolation from soil sample, Nocardioi'des strain, 286-91 ketolides, RU 004,279-85 legionella, 209-12,210t leukocytes, effecton, 101-16 Listeria monocytogenes, 268-69 malaria, 270-71 methylprednisolone, effect on, 186 Moraxella catarrhah, respiratory tract infection, 523-27 Mycobacterium avium prophylaxis of, 259-61,260t treatment of, 257-59 neutrophils, effect on invivo efficacy, 32-33,33t nonantibiotic effectsof, 91-100 opportunistic infections,in immunocompromised patients, 189204 pediatric infection, 131-43 azithromycin, 132-43 clarithromycin, 132-43 erythromycin, 132-43 pneumonia, Mycoplasma pneumonia, 335-38 roxithromycin, 132-43 peptic ulcer disease,81 pertussis, 270 phagocyte function, modificationof, 108-9 pharmacodynamics, 233-35 pharmacokinetics, 10-12,33-35,34, 36, 233-35 other drugs, effect on, 180-87 stomach, 242 postantibiotic effects,27-38, 29-3Ot, 30-31 and dosingof, 27-38

Subject Index [Macrolides] [postantibiotic effects] sub-MIC effects,28,29t prokinetic drugs, 83-86,84, 85t Pseudomonas aeruginosa, bacterial glycocalyx of, suppression of, with cystic fibrosis, 91-100 resistance Streptococcus pneumoniae, 369-75 streptogramin, 390-94 RU 004,279-85 salmonellosis, 270 Streptococcus pneumoniae, respiratory tract infection, 523-27 Streptococcus pyogenes, 380-84 structure activity, 4-5 terfenidine, effect on, 187 theophylline, effecton, 180-82,181t warfarin, effect on, 184 Malaria, 270-71 Methylprednisolone, effectof macrolides on, 186 Moraxella catarrhalis, 139 azalides, 70 respiratory tract infection, macrolides, 523-27 RPR 106972,60 Mycobacteria species clarithromycin, 17-18 human immunodeficiency vi^^, 53752 RPR 106972,61 Mycobacterium avium azithromycin, 15-16 clarithromycin, 53943,544-48 immunocompromised patients, 19599 prophylaxis, 196-97 azithromycin, 196-97 clarithromycin, 196 treatment, 197-99 clarithromycin, 197-99 Mycobacterium avium-intracellulareinfection, human immunodeficiency virus, 257

687 Mycoplasma pneumonia, 133,142,143, 219-23 pediatrics, pneumonia, macrolides, 335-38 RPR 106972,61 Mycoplasma species, RU 004,400 Neisseria species, RPR 106972,60 Neutrophils azithromycin, penetration response, 455-59 chemiluminescence, penetration response, 455-59 effect on in vivo efficacy,32-33,33t streptogramins, effecton in vivo efficacy, 32-33,33t Nocardioi'des strain, macrolide isolation, from soil sample,286-91 biological properties, 287 fermentation, 287 medium conditions, 287 purification, chromatographic methods, 287 structural analysis,288-89

Opportunistic infections,261-63 bartonella, 199t,199-200 cryptosporidium, 194-95 Enterococcus faecium, vanwmycinresistant, 201 giardiasis, 192 legionella, 190-91 Mycobacterium avium complex, 19599 prophylaxis, 196-97 azithromycin, 196-97 clarithromycin, 196 treatment, 197-99 clarithromycin, 197-99 Pneumocystis carinii pneumonia, 191 pneumonia, 200,200t toxoplasmosis, 192-94, 193t Otitis media,591-601 azithromycin, vs. amoxicillin for, 597-601 clarithromycin, in children,593-96

688

Subject Index

Pasteurella species, streptogramins,60 [Pneumonia] Pediatrics Chlamydia pneumoniae,azithroazithromycin, mycin, vs. josamycin, lower respiratory in children, tract infections, immunocompromised patients, Chlamydia pneumoniae,azithromycin, Mycoplasma pneumoniae,macroclarithromycin, lides, in children, compliance, Streptococcus pneumoniae, erythromycin, clarithromycin, macrolides, Pristinamycin, otitis media, clarithromycin, Prostate, azithromycin, in transrectal bipertussis OPSY Of, azithromycin, Protein synthesis, streptogramins, anticlarithromycin, biotic inhibitors, pneumonia, Mycoplasma pneumoPseudomonas aeruginosa niae, macrolides, azithromycin, respiratory tract infection bacterial glycocalyx of, macrolides azithromycin, suppression of, with cysticficlarithromycin, brosis, roxithromycin, macrolides, streptogramins, Streptococcus pneumoniae, Pyrimethamine, human immunodefirokitamycin, ciency virus, Ureaplasma urealyticum, with respiratory problems, Quinupristiddalfopristin, Pentamidine, human immunodeficiency virus, animal models, Peptic ulcer.disease, clarithromycin, antipneumococcal activity, Peptidyltransferase center, antistaphylococcal activity of, streptogramins, bacteremia, resistant gram-positive Pertussis, bacteria, azithromycin, in children, clinical use, clarithromycin, children, vs. Enterococcus faecium, Phagocyte function, macrolide modifica- resistance to, tion of, vs. Staphylococcus aureus, Pharmacodynamics, (See also methicillin-resistant, 54t, under specific drug) 55 Pharmacokinetics, (See vs. Streptococcus pneumoniae, 55also under specific drug) Pneumocystis cariniipneumonia immunocompromised patients, Ranitidine, azithromycin, combined roxithromycin, treatment, Helicobacter Pneumonia, pylori, azithromycin, Resistance cefuroxime, with erythromycin, Actinomyces pyogenes,macrolides,

Subject Index [Resistance] bacterial pathogens, susceptibilityin, macrolides, 119-21 genetic support, macrolides, 121-22 macrolides, 119-23 Pasteurella, macrolides, 123-24 Streptococcus pneumoniae macrolides, 390-94 RPR 106972,390-94 streptogramin, 390-94 Streptococcus pyogenes, 376-79 veterinary problems, macrolides, 123-24 Respiratory tract infection azithromycin, 641-44 lower Alexander Project, macrolides, 523-27 azithromycin, 13-14 vs. josamycin, in children,65659 clarithromycin, 16-17 in children, 645-48 Haemophilus influenzae, macrolides, 523-27 josamycin, azithromycin, contrasted, in children, 656-59 Moraxella catarrhalis, macrolides, 523-27 roxithromycin, 19 Streptococcus pneumoniae, macrolides, 523-27 roxithromycin, in elderly,664-67 upper azithromycin, 13,641-44 clarithromycin, 16 roxithromycin, 18-19 Rokitamycin, Streptococcus pneumonine, in children, 432-35 Roxithromycin, 18-20 Borrelia burgdorferi, temperature effects, 299-302 genitalia, 586-90 Helicobacter pylori infections, 19 Klebsiella pneumoniae, 581-85 pediatric infection, 132-43 '

689 [Roxithromycin] Pneumocystis carinii pneumonia, 261 respiratory tract infection,18-19 in elderly, 664-67 skin infections, 19 soft tissue infections, 19 Streptococcus pyogenes, susceptibility testing, 407-10 toxoplasmosis, 261 urogenital infection, 20,361-65 RP 59500 (See Quinupristinl dalfopristin) RPR 106950, RPR 112808 combination, synergism,61-62 ratio, 62 Enterococcus species, 62-63 Haemophilus influenzae, 63 Staphylococcus aureus, 62 Streptococcus pneumoniae, 62 in vitro bactericidal activity,6263 RPR 106972,59-63 anaerobes, 61 enterobacteria, 61 enterococci, 60 Haemophilus influenzae, 61 Legionella species, 61 Moxarella catarrhalis, 60 Mycobacteria species, 61 Mycoplasma, 61 Neisseria species, 60 RPR 112808 ratio, 62 in vitro bactericidal activity,6263 RPR 106950, ratio, 62 Enterococcus species, 62-63 Haemophilus influenzae, 63 Staphylococcus aureus, 62 Streptococcus pneumoniae, 62 synergism, 61-62 in vitro bactericidal activity, 6263 synergism, 61-62 staphylococci, 59 streptococci, 59-60

690 [RPR Streptococcus pneumoniae, macrolide resistance, in vitro bacteriostatic activity, RPR RPR ratio, Enterococcus species, Haemophilus infiuenzae, Staphylococcus aureus, Streptococcus pneumoniae, in vitro bactericidal activity, RPR combination, synergism, RU atypical bacteria, chlamydia, conformational analysis, gram-negative bacteria, gram-positive bacteria, hepatic cytochrome P-450,effects on, human monocytic cells,uptake by, ketolide, respiratory pathogens, legionella, MIC, effect of pH on, Mycoplasma species, physicochemical properties, respiratory pathogens, stability, in acidic medium, Staphylococcus aureus, structure, in vivo antibacterial activity, RU Salmonellosis, Sexually-transmitted infections azithromycin, chlamydial infection, clarithromycin, roxithromycin, Sexually transmitted infections, azithromycin, Skin infections aiithromycin,

Subject Index [Skin infections] clarithromycin, roxithromycin, Soft tissue infections azithromycin, clarithromycin, roxithromycin, Soil sample, macrolide isolation from, Nocardioi'des strain, biological properties, fermentation, purification, chromatographic methods, structural analysis, Sparfloxacin, antipneumococcal activity, Spirochetes Borrelia burgdorferi, Lyme disease, diseases causedby, erythema chronicum migrans, Lyme disease, syphilis, Staphylococcus aureus, methicillin-resistant, quinupristid dalfopristin, 54t, RPR RPR ratio, RPR RPR ratio, RU Staphylococcus pneumoniae, Streptococcus pneumoniae,

clarithromycin, lincosamide resistance, macrolide resistance, quinupristiddalfopriistin, respiratory tract infection, macrolides, rokitamycin, in children, RPR RPR ratio, RPR macrolide resistance, RPR ratio,

691 [Streptogramins] Streptococcus pyogenes, 225-31,226t postantibiotic effects,27-38 macrolides, 380-84 sub-MIC effects,28,29t resistance, 376-79 in vitro, 28,29t susceptibility testing in vivo, 28-31,29-30t, 30-31 azithromycin, 407-10 pristinamycin, 59-63 clarithromycin, 407-10 protein synthesis, antibiotic inhibierythromycin, 407-10 tors, 150-51 roxithromycin, 407-10 Pseudomonas aeruginosa, 61 Streptogramins bacterial protein synthesis, metabolic quinupristiddalfopristin,54-59 antistaphylococcal activityof, pathway, 14649,14749, 402-6 148t vs. Enterococcus faecium, 56-58 bartonellosis, 271-72 resistance to, 58-59 biological effectsof, 151-53,152, vs. Staphylococcus aureus, 152t, I54 methicillin-resistant, 54t,54bites, wound infections, 267-68 55 brucellosis, 269 vs. Streptococcus pneumoniae, 55clinical use overview,3-26 56,56t Corynebacterium jeikeium,268-69 RPR 106972,59-63 development of, 4-21,8-l0 anaerobes, 61 dosing interval, effecton in vivo effienterobacteria, 61 cacy, 31-32,32t enterococci, 60 gonorrhea, 272 Haemophilus influenzae, 61 human immunodeficiency virus,257Legionella species, 61 65 Moraxella catarrhalis, 60 Listeria monocytogenes, 268-69 Mycobacteria species, 61 malaria, 270-71 Mycoplasma, 61 molecular mechanism of action, 145Neisseria species, 60 72 RPR 112808, RF'R 106950, ratio, Mycobacterium avium 62 prophylaxis of, 259-61,260t Enterococcus species, 62-63 treatment of, 257-59 Haemophilus influenzae, 63 neutrophils, effecton in vivo effiStaphylococcus aureus, 62 cacy, 32-33,33t Streptococcus pneumoniae, 62 opportunistic infections,189-204 synergism, 61-62 azalides, 189-204 in vitro bactericidal activity,62in immunocompromised patients, 63 189-204 staphylococci, 59 macrolides, 189-204 streptococci, 59-60 Pasteurella species, 60 in vitro bacteriostatic activity, 59peptidyltransferase center, 166-68, 61 167 salmonellosis, 270 pertussis, 270 Streptococcus pneumoniae, RPR pharmacokinetics, 10-12 106972,390-94 in vivo efficacy,correlation, 33macrolide resistance,390-94 35,34,36

692

Subject Index

[Streptogamins] structure activity,

me A

mechanism of action, I58 Qpe B, synergism between,

'Qpe B, mechanism of action, I59-6I Ureaplasma species, in vitro activity, Syphilis, azalides, azithromycin, macrolides, Terfenidine, effect of macrolides on,

[Toxoplasmosis] immunocompromised patients, roxithromycin, Trachoma, azithromycin, Upper respiratory tract infection azithromycin, clarithromycin, roxithromycin, Ureaplasma species, streptogramins, Ureaplasma urealyticum, children, with respiratory problems, Urethritis, nongonococcal, in males, azithromycin, Urogenital infection clarithromycin, roxithromycin,

Theophylline, effect of macrolides on, Tick-borne disease (See Lyme disease) Tonsils dirithromycin kinetics, tonsilitis, azithromycin, Toxoplasmic encephalitis, azithromycin, Toxoplasmosis clarithromycin,

Vancomycin, Enterococcus faecium resistance, immunocompromised patients, Veterinary problems, macrolidesand, Warfarin, effect of macrolides on, Whooping cough (See Pertussis) Wound infection, animal bite,

About the Editors

Stephen H. Zinner is Professor of Medicine, interim Chairman of the Department of Medicine, and Director, Division of Infectious Diseases, Department of Medicine,BrownUniversity,Providence, Rhode Island; Head, Division of Infectious Diseases, Roger Williams Medical Center, Providence; Head of the Division of Infectious Diseases at Rhode Island Hospital, Providence; Consultant in Infectious Diseases at Womenand Infants Hospital of Rhode Island, Miriam Hospital, and the Veterans Administration MedicalCenter; and a Lecturerin Medicineat Harvard Medical School, Boston, Massachusetts. The author or coauthor of over 200 articles, book chapters, and abstracts, and the coeditor of The New Macrolides, Azalides, and Streptogramins: Pharmacology and Clinical Applications and New Macrolides,Azalides, and Streptogramins inClinical Practice (both titles, Marcel Dekker, Inc.), Dr. Zinner is a Fellowof the American College of Epidemiology, the Infectious Diseases Societyof America, the American Collegeof Physicians, andthe American Heart Association, and a member of the American Society for Clinical Investigation and many other organizations. He received the B.A. degree (1961) from Northwestem University, Evanston, Illinois, and the M.D. degree (1965) from the University of Pennsylvania School of Medicine, Philadelphia. Lowell S. Young is Director of the Kuzell Institute for Arthritis and Infectious Diseases, Medical Research Institute of San Francisco; Chief of the Division of Infectious Diseases at the California Pacific Medical Center, San Francisco; and Clinical Professor of Medicine at the University of California, San Francisco. He is also Adjunct Professor of Pharmacy atthe University of the Pacific, San Francisco. The author or coauthor of more

than 300 articles and the coeditor of The New Macrolides, Azalides, and Streptogramins: Pharmacology and Clinical Applications and New Macrolides, Azalides, and Streptogramins in Clinical Practice(both titles, Marcel Dekker, Inc.), Dr. Young isa Fellowof the American Collegeof Physicians and the Infectious Diseases Society of America and a member of the American Societyfor Clinical Investigation, among others. He received the A.B. degree (1960) from Princeton University, Princeton, New Jersey, and the M.D. degree(1964) from Harvard Medical School, Boston, Massachusetts.

Jacques F. Acar is Professorof Microbiology, UniversitCde Paris VI, Pierre et Marie Curie, and Head of the Department of Clinical Microbiology and Infectious Diseases, Fondation H6pital Saint-Joseph, both in Paris, France The author or coauthor of more than 400 articles andthe coeditor of New Macrolides, Azalides, and Streptogramins inClinical Practice (Marcel Dekker, Inc.), Dr. Acar also serves as an editorial board member for several journals and isthe Editor-in-Chief of Clinical Microbiology and Infection, the official journal of the European Society of Clinical Microbiology and Infectious Diseases.A consultant atthe World Health Organization, heis a member of the Infectious Diseases Society of America, the American Society for Microbiology, the SociCttFranqaisedeMicrobiologie,and the SociCtC de Pathologie Infectieuse de Langue Franqais. He is also aformer President of the International Societyof Infectious Diseases andthe European Society of Clinical Microbiology and Infectious Diseases. Dr. Acar received the M.D. degree (1954) from the University of Paris, France. He was a Fellowat Harvard Medical School, Boston, Massachusetts, a visiting professor at Brown University, Providence, Rhode Island, and an invited lecturer at several American universities. Harold C. Neu is Professorof Medicine and Pharmacologyat the Columbia University College of Physicians & Surgeons, New York, New York. The author or coauthor of over 800 journal articles, reviews, and book chapters and the editor or coeditor of over books, includingThe New Macrolides, Azalides, and Streptogramins: Pharmacology andClinicalApplications and New Macrolides, Azalides, and Streptogramins in Clinical Practice (both titles, Marcel Dekker, Inc.), Dr.Neu isa Fellow ofthe American Academy of Microbiology, the New York Academy of Medicine, and the New York Academy of Sciences, a masterof the American Collegeof Physicians, and a member of several other societies. He received the A.B. degree (1956) from Creighton University, Omaha, Nebraska, and the M.D. degree(1960) from the Johns Hopkins University, Baltimore, Maryland.