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Frontiers in Anti-Infective Drug Discovery Bentham Science Publishers Ltd. http://www.bentham.org/fiaidd
Volume 1 Contents Preface
i
Contributors
ii
Strategies for Effective Naked-DNA Vaccination Against Infectious Diseases Pieranna Chiarella, Emanuela Massi, Mariangela De Robertis, Vito M. Fazio and Emanuela Signori
1
Light Activated Compounds as Patented Antimicrobial Agents David A. Phoenix and Frederick Harris
17
Proteases and Kinases: Attractive Targets for Combating Infectious Diseases Mona Arabshahi, Usha Bughani, Surya N. Vangapandu, Ritu Aneja, Ramesh Chandra, Daniel Kalman and Harish Joshi
49
Antibacterial Therapy in the Elderly Ayman M. Noreddin and Walid F. El-Khatib
70
SARS Coronavirus Anti-Infectives Tommy R. Tong
83
Probiotics as Drugs Against Human Gastrointestinal Pathogens Yolanda Sanz, Inmaculada Nadal and Ester Sánchez
107
Insights into the Treatment of Helicobacter pylori Infection Campo Salvatore Maria Antonio, Hassan Cesare, Burza Maria Antonietta, Ridola Lorenzo, Cristofari Francesca, Morini Sergio and Zullo Angelo
124
Inhibitors of Bacterial Efflux Pumps as Adjuvants in Antibacterial Therapy and Diagnostic Tools for Detection of Resistance by Efflux Françoise Van Bambeke, Jean-Marie Pagès and Ving J. Lee
138
Drugs Candidates in Advanced Clinical Trials Against Tuberculosis Marcus Vinícius Nora de Souza, Marcelle de Lima Ferreira and Raoni Schroeder B. Gonçalves
176
Drug Effects on Drug Targets: Inhibition of Enzymes by Neuroleptics, Antimycotics, Antibiotics and Other Drugs on Human Pathogenic Amoebas and their Anti-Proliferative Effects Raúl N. Ondarza
202
Macrophage Inflammatory Protein 1 and CCR5 as Potential Therapeutic Targets for HIV Infection and Acquired Immunodeficiency Syndrome Tsuyoshi Kasama, Ryo Takahashi, Michihito Sato and Kuninobu Wakabayashi
227
Small-Molecule Inhibitors of Raf for Treatment of Malignant Diseases Li Li, Shuhong Wu, Wei Guo and Bingliang Fang
238
Tigecycline: A New Treatment Choice Against Acinetobacter baumannii Virginia Bosó-Ribelles, Eva Romá-Sánchez, Jorge Carmena, Cristina Cáceres and Daniel Bautista
251
Cefepime and its Role in Pediatric Infections Sukhbir K. Shahid
261
Antibacterial Properties of Organosulfur Anti-Infectives: A Review of Patent Literature 1999-2009 Monika I. Konaklieva and Balbina J. Plotkin
269
A Review of the Carbapenems in Clinical Use and Clinical Trials Tze Shien Lo, Justin M. Welch, Augusto M. Alonto and Eileen Anne R. Vicaldo-Alonto
279
Anti-Infective Quinone Derivatives of Recent Patents Junko Koyama
294
Targets and Patented Drugs for Chemotherapy of Chagas Disease Vilma G. Duschak and Alicia S. Couto
323
Recent Patents on Development of Nucleic Acid-Based Antiviral Drugs against Seasonal and Pandemic Influenza Virus Infections Edward G. Saravolac and Jonathan P. Wong
409
Author Index
426
Preface
Frontiers in Anti-Infective Drug Discovery, 2010, Vol. 1
i
PREFACE The scientists invited to contribute short reviews to this first volume of book series, “Frontiers in Anti-Infective Drug Discovery” were selected on the basis of their own original contributions in this important field of health research. The chapters contain in this volume are the updated version of articles published in Recent Patents on Anti-Infective Drug Discovery. The reviews cover key aspects of infections caused by a variety of organisms.
With the enhanced understanding of diseases at cellular and molecular levels, the search for anti-infective agents is now more rational and strategically based on new tools and novel techniques. Most of the reviews address the molecular mechanisms of infections and development of inhibitors of these mechanisms. Several groups of existing anti-infective agents have also been reviewed. The topics range from vaccines to clinical trials of exploratory drugs, treatment by age-old anti-infective agents, and reviews on recent patents. Each review is well-written and extensively referenced. We hope that volume 1 of this series will be welcomed by of students and researchers and that it will lead to a broader understanding of the current status of this subject.
We wish to express our profound gratitude to all authors for their excellent contributions. We are also grateful to the management and the staff of the Bentham Science Publishers (the Netherlands), especially Mr. Mahmood Alam (Managing Director), Ms. Samina Khan (Senior Manager), Ms. Taqdees Malik (Assistant Manager) and Ms. Sadaf Idrees Khan (Composer), for their help in compilation of the first volume of this important series of books.
Prof. Dr. Atta-ur-Rahman, FRS Prof. Dr. M. Iqbal Choudhary Editors
ii Frontiers in Anti-Infective Drug Discovery, 2010, Vol. 1
Contributors
Contributors Pieranna Chiarella
Laboratory of Molecular Medicine and Biotechnology, CIR, Campus Bio-Medico University of Rome, Via Alvaro del Portillo 21 - 00128 Rome, Italy; Institute of Neurobiology and Molecular Medicine, CNR, Via Fosso del Cavaliere 100 - 00133 Rome, Italy
Emanuela Massi
Laboratory of Molecular Medicine and Biotechnology, CIR, Campus Bio-Medico University of Rome, Via Alvaro del Portillo 21 - 00128 Rome, Italy; Institute of Neurobiology and Molecular Medicine, CNR, Via Fosso del Cavaliere 100 - 00133 Rome, Italy
Mariangela De Robertis
Laboratory of Molecular Medicine and Biotechnology, CIR, Campus Bio-Medico University of Rome, Via Alvaro del Portillo 21 - 00128 Rome, Italy; Institute of Neurobiology and Molecular Medicine, CNR, Via Fosso del Cavaliere 100 - 00133 Rome, Italy
Vito M. Fazio
Laboratory of Molecular Medicine and Biotechnology, CIR, Campus Bio-Medico University of Rome, Via Alvaro del Portillo 21 - 00128 Rome, Italy; Laboratory of Oncology, Research Department, IRCCS H "Casa Sollievo della Sofferenza" - 71013 S. Giovanni Rotondo (FG), Italy
Emanuela Signori
Institute of Neurobiology and Molecular Medicine, CNR, Via Fosso del Cavaliere 100 - 00133 Rome, Italy
David Andrew Phoenix
Deputy Vice Chancellor, University Lancashire, Preston, PR1 2HE, UK
Frederick Harris
School of Forensic and Investigative Sciences, University of Central Lancashire, Preston, PR1 2HE, UK
Mona Arabshahi
Department of Cell Biology, Emory University School of Medicine, Atlanta, GA, USA
Usha Bughani
Department of Cell Biology, Emory University School of Medicine, Atlanta, GA, USA
Surya N. Vangapandu
Department of Cell Biology, Emory University School of Medicine, Atlanta, GA, USA
Ritu Aneja
Department of Cell Biology, Emory University School of Medicine, Atlanta, GA, USA
of
Central
Contributors
Frontiers in Anti-Infective Drug Discovery, 2010, Vol. 1
iii
Ramesh Chandra
Department of Chemistry and Dr. B. R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi, India
Daniel Kalman
Department of Pathology, Emory University School of Medicine, Atlanta, GA, USA
Harish Joshi
Department of Cell Biology, Emory University School of Medicine, Atlanta, GA, USA
Ayman M. Noreddin
School of Pharmacy, Hampton University, Hampton Roads Bridge Tunnel, VA 23668, Hampton, USA
Walid F. El-Khatib
College of Pharmacy, University of Minnesota, 4-101 Hanson Hall 1925 Fourth Street South Minneapolis, MN 55455, USA
Tommy R. Tong
Department of Pathology, Montefiore Medical Center, 600 E 233 Street, Bronx, New York 10466, USA
Yolanda Sanz
Instituto de Agroquímica y Tecnología de los Alimentos (CSIC), P.O. Box 73 46100, 46100 Burjassot, Valencia, Spain
Inmaculada Nadal
Instituto de Agroquímica y Tecnología de los Alimentos (CSIC), P.O. Box 73 46100, 46100 Burjassot, Valencia, Spain
Ester Sánchez
Instituto de Agroquímica y Tecnología de los Alimentos (CSIC), P.O. Box 73 46100, 46100 Burjassot, Valencia, Spain
Campo S. M. Antonio
Gastroenterology and Digestive Endoscopy, “Nuovo Regina Margherita” Hospital, Rome, Italy
Hassan Cesare
Gastroenterology and Digestive Endoscopy, “Nuovo Regina Margherita” Hospital, Rome, Italy
Burza Maria Antonietta
Gastroenterology and Digestive Endoscopy, “Nuovo Regina Margherita” Hospital, Rome, Italy
Ridola Lorenzo
Gastroenterology and Digestive Endoscopy, “Nuovo Regina Margherita” Hospital, Rome, Italy
Cristofari Francesca
Gastroenterology and Digestive Endoscopy, “Nuovo Regina Margherita” Hospital, Rome, Italy
Morini Sergio
Gastroenterology and Digestive Endoscopy, “Nuovo Regina Margherita” Hospital, Rome, Italy
iv Frontiers in Anti-Infective Drug Discovery, 2010, Vol. 1
Contributors
Zullo Angelo
Gastroenterology and Digestive Endoscopy, “Nuovo Regina Margherita” Hospital, Rome, Italy
Françoise Van Bambeke
Unité de Pharmacologie Cellulaire et Moleculaire, Université Catholique de Louvain, Brussels, Belgium
Jean-Marie Pagès
EA2197 Enveloppe Bactérienne, Perméabilité et Antibiotiques, Faculté de Médecine, Université de la Méditerranée, Marseille, France
Ving J. Lee
Adesis, Inc., New Castle, DE 19720, USA; Limerick BioPharma, Inc., South San Francisco, CA 94080 USA
Marcus Vinícius Nora de Souza
Instituto de Tecnologia em Fármacos-Far-Manguinhos, Rua Sizenando Nabuco, 100, Manguinhos, 21041-250 Rio de Janeiro-RJ, Brazil
Marcelle de Lima Ferreira
Instituto de Tecnologia em Fármacos-Far-Manguinhos, Rua Sizenando Nabuco, 100, Manguinhos, 21041-250 Rio de Janeiro-RJ, Brazil
Raoni Schroeder B. Gonçalves
Instituto de Tecnologia em Fármacos-Far-Manguinhos, Rua Sizenando Nabuco, 100, Manguinhos, 21041-250 Rio de Janeiro-RJ, Brazil
Raúl N. Ondarza
Department of Biochemistry, Faculty of Medicine, National Autonomous University of Mexico (UNAM), University City, Mexico 04510, USA; Center of Research on Infectious Diseases, National Institute of Public Health, Cuernavaca, Morelos, Mexico 62508, USA; Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California, San Diego (UCSD), La Jolla, CA 92093-0204, USA
Tsuyoshi Kasama
Division of Rheumatology, Department of Medicine, Showa University School of Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8666, Japan
Ryo Takahashi
Division of Rheumatology, Department of Medicine, Showa University School of Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8666, Japan
Michihito Sato
Division of Rheumatology, Department of Medicine, Showa University School of Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8666, Japan
Kuninobu Wakabayashi
Division of Rheumatology, Department of Medicine, Showa University School of Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8666, Japan
Contributors
Frontiers in Anti-Infective Drug Discovery, 2010, Vol. 1
v
Li Li
Department of Laboratory Medicine, First Hospital of Shanghai, Shanghai, China
Shuhong Wu
Department of Thoracic and Cardiovascular Surgery, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA
Wei Guo
Department of Thoracic and Cardiovascular Surgery, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA
Bingliang Fang
Department of Thoracic and Cardiovascular Surgery, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA
Virginia Bosó-Ribelles
Pharmacy Department, Hospital La Fe, Valencia, Spain
Eva Romá-Sánchez
Pharmacy Department, Hospital La Fe, Valencia, Spain
Jorge Carmena
Infectious Diseases Department, Hospital Dr. Peset, Valencia, Spain
Cristina Cáceres
Infectious Diseases Department, Hospital Dr. Peset, Valencia, Spain
Daniel Bautista
Preventive Medicine Department, Hospital Dr. Peset, Valencia, Spain
Sukhbir K. Shahid
Consultant Pediatrician and Neonatologist, Mumbai-400 077, India
Monika I. Konaklieva
Department of Chemistry, American University, 4400 Massachusetts Avenue, NW, Washington, DC 200168014, USA
Balbina J. Plotkin
Department of Microbiology and Immunology, CCOM, Midwestern University, 555 31 St., Downers Grove, IL 60515, USA
Tze Shien Lo
Section of Infectious Diseases, Veterans Affairs Medical Center, Fargo, ND, USA
Justin M. Welch
Pharmacy Department, Veterans Affairs Medical Center, Fargo, ND, USA
Augusto M. Alonto
Section of Infectious Diseases, MeritCare Medical Center, Fargo, ND, USA
vi Frontiers in Anti-Infective Drug Discovery, 2010, Vol. 1
Contributors
Eileen Anne R. Vicaldo-Alonto
Department of Internal Medicine, University of North Dakota School of Medicine & Health Sciences, Fargo, ND, USA
Junko Koyama
Faculty of Pharmaceutical Sciences, Kobe Pharmaceutical University, Higashinada-ku, Kobe 6588558, Japan
Vilma G. Duschak
Instituto Nacional de Parasitología “Dr. Mario Fatala Chabén”, ANLIS-Malbrán, Ministerio de Salud. Av. Paseo Colon 568 (1063), Buenos Aires, Argentina
Alicia S. Couto
CIHIDECAR (CONICET) Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, CP 1428, Argentina
Edward G. Saravolac
Formulation Technology Consulting, 3 Essex St., Footscray, Victoria, 3011 Australia
Jonathan P. Wong
Defence R&D Canada - Suffield, Biotechnology Section, Box 4000 Main Station, Medicine Hat, Alberta, T1A 8K6 Canada
Frontiers in Anti-Infective Drug Discovery, 2010, 1, 1-16
1
Strategies for Effective Naked-DNA Vaccination Against Infectious Diseases Pieranna Chiarella1,2, Emanuela Massi1,2, Mariangela De Robertis1,2, Vito M. Fazio1,3 and Emanuela Signori*,2 1
Laboratory of Molecular Medicine and Biotechnology, CIR, Campus Bio-Medico University of Rome, Via Alvaro del Portillo 21 - 00128 Rome, Italy, 2Institute of Neurobiology and Molecular Medicine, CNR, Via Fosso del Cavaliere 100 - 00133 Rome, Italy, 3Laboratory of Oncology, Research Department, IRCCS H "Casa Sollievo della Sofferenza" - 71013 S. Giovanni Rotondo (FG), Italy Abstract: To date, vaccination is an active area of investigation for its application to a great variety of human diseases including infections and cancer. In particular, naked-DNA vaccination has arisen as effective strategy in the preventive medicine field with promising future prospects. The ability of plasmid DNA to activate the humoural and the cellular arms of the immune system against the encoded antigen have resulted in intensive study of new strategies aimed at increasing the DNA vaccine immunogenicity. Nevertheless, plasmid-based vaccines emerged as a safer and advantageous alternative with respect to viral vector vaccines. Recent advances in both the immunological and biotechnological research field made it possible to enhance significantly the DNA vaccine potency. Most of these approaches are based on both the discovery of novel delivery systems and the implementation of plasmid constructs, achieved through genetic engineering. In this review, we will describe some of the most relevant patents issued in the last ten years, supporting the progress made in naked-DNA vaccination against infectious diseases.
Keywords: DNA vaccines, human infectious diseases, gene therapy, naked DNA, delivery systems, adjuvant, plasmid vector. INTRODUCTION Vaccination is historically one of the most important methods for preventing infectious diseases in humans and animals. Vaccines containing whole killed or live attenuated microorganisms and purified components of the pathogen, namely sub-unit vaccines, have been used effectively as the best defensive tool against numerous bacterial and viral agents. However, despite the encouraging results obtained with these conventional approaches, substantial progress has also been made in the vaccination field. Recent scientific advances have increased our understanding of the biology of the immune system and now allow the more rational design of vaccines. These advances include new delivery technologies, aimed at improving the safety and immunogenicity of traditional vaccines and at introducing *Corresponding author: Tel: +390649934232; Fax: +390649934257; E-mail:
[email protected] Atta-ur-Rahman / M. Iqbal Choudhary (Eds.) All rights reserved – © 2010 Bentham Science Publishers.
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entirely new methods of vaccination such as those based on gene transfer. Genetic vaccination originated from gene therapy. The aim of genetic vaccination is to transfer a gene in the host encoding for the disease target antigen to induce a specific immune response, whereas the goal of gene therapy is to ensure production of a protein which is lacking or defective in the host. To date, the vast majority of gene therapy clinical trials have addressed cancer (66.5%), cardiovascular diseases (9.1%) and infectious diseases (6.5%). For infectious diseases, a total of 85 gene therapy trials have been carried out, the majority of these trials being performed on human immunodeficiency virus infection, tetanus, cytomegalovirus and adenovirus infections [1]. Current techniques of gene transfer in mammals include packaging the DNA into recombinant viral vectors such as retrovirus, vaccinia virus or adenovirus [2-4]. In viral vectors, the genes coding for viral proteins are removed so that the vaccine is nonpathogenic for humans. The vaccine can be designed with the gene encoding the antigen of interest which is put into the virus particle. Viral vectors are usually immunogenic and can cause an inflammatory reaction which can be beneficial for the host immune response elicited against the desired antigen. However, the potency of vaccines based on viral vectors is limited by two major drawbacks. The first is related to a pre-existing immunity of the host against the virus; the second is related to a safety concern of live vector systems [5, 6]. In 1990, Wolff and collaborators found that bacterial plasmid DNA encoding a reporter gene could result in in vivo expression of the encoded protein after simple intramuscular injection without the need for more complex vectors [7]. Following Wolff’s findings a new era of vaccination started. Naked-DNA vaccines are for definition vectors based on bacterial plasmids engineered to express the disease-specific antigen using promoter elements active in mammalian cells, without the addition of surrounding chemicals or a viral coat. The mode of action of plasmid DNA vaccines is dual. Firstly, the antigen encoded by the plasmid is produced in host cells, either in professional antigen presenting cells (APCs) that lead to direct priming of immune responses, or in non-professional cells from where the antigen can be transferred to APCs so resulting in cross-priming. Secondly, because DNA plasmids are derived from bacteria, they stimulate the innate immune system by interacting with Toll-like receptor 9 [8]. This non-specific immune response augments the antigen-specific immune response. The main advantages of naked DNA vaccines are safety, flexibility in design, production in large amount, as well as stability at different temperatures. They are also likely to be attractive from a health economics perspective: they are relatively easy to manufacture in large quantities and do not require any special transportation or storage conditions that could hinder their widespread distribution. On the other hand, a major disadvantage of plasmid DNA vaccines is their poor immunogenicity when administered as unformulated intramuscular injection. Large quantities of DNA are required to induce only modest immunogenicity and many efforts have focussed on the development of new technologies aimed at increasing the DNA vaccine potency [9]. In this review, we highlight the mechanism of action of DNA vaccines and the most relevant patented strategies recently developed to enhance the plasmid DNA vaccine immunogenicity against infectious diseases.
NAKED-DNA VACCINES: MECHANISM OF ACTION It was not until early 1990 that the first publications on DNA immunisation focussed new attention on the importance of this approach for vaccine and immune therapeutic
Naked-DNA Vaccination
Frontiers in Anti-Infective Drug Discovery, 2010, Vol. 1
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development. The concept behind genetic immunisation is very simple. The gene encoding an antigen of a particular pathogen is cloned into a plasmid vector which is administered to the recipient individual. Once the DNA is taken up by host cells, the gene-encoded protein is produced, then processed and presented appropriately to the immune system, inducing a specific immune response. Immunisation with DNA thus mimics aspects of live infection, with pathogen proteins synthesised endogenously by host cells. DNA vaccines are commonly delivered by simple intramuscular injection and their mechanism of action is quite intriguing. Transfected muscle cells clearly express antigen and behave as target of immune effector cells. Apparently, they could also up-regulate expression of MHC class II and co-stimulatory molecules, accompanied by production of cytokines and chemokines, contributing to the DNA vaccine immunogenicity [10].
Fig. (1). Mechanism of action of naked DNA vaccine. Viral antigen sequence is inserted in a bacterial plasmid. After massive production of plasmid in bacteria the naked DNA vaccine can be delivered by intramuscular injection. Plasmid enters in the nucleus of muscle cells, where the gene is transcribed, followed by protein production in the cytoplasm. Transfected muscle cells have the potential to activate T cells through direct presentation (A) as well as through APCs activation. Proteins secreted by muscle cells can be presented in association with MHC-II molecules or MHC-I molecules (cross-presentation) (B). Furthermore, secreted proteins (C) can induce the production of antibodies that will react with and eliminate virus. Professional APCs can directly uptake DNA vaccine (D), present peptides in context of the MHC-I and activate killer cells which lyse virus-infected cells.
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However, for induction of high-level immunity, professional APCs can uptake antigen released by skeletal muscle apoptotic bodies and activate efficient cross-presentation of the same [11-13]. This route allows stimulation of both MHC-I and MHC-II restriction pathways. The capture of exogenous antigens by dendritic cells results in both types of presentation, leading to simultaneous stimulation of CD4+ T-helper and CD8+ cytotoxic T lymphocytes. Therefore, extracellular antigens can also have access to the MHC-I compartment through endoplasmic reticulum, leading to stimulation of the humoural as well as the cytotoxic immune response, (Fig. 1). In this way, genetic immunisation confers the same broad immunological advantages as immunisation with live, attenuated vaccines does, without the accompanying unsafety concerns associated with live infection, such as reversion to the virulent form and/or incomplete inactivation of live vaccines.
STRATEGIES TO ENHANCE DNA VACCINE IMMUNOGENICITY Live vaccines generally possess a natural adjuvant effect based on the ability of the immune system to recognise many features of potentially dangerous pathogen agents; as such, they have contributed immeasurably to the control of disease. Although some live vaccines might have undesirable characteristics, they remain able to reduce significantly the impact of associated diseases. In contrast to live or attenuated vaccines, plasmid DNA vaccines, although extremely safe and easy to manufacture, suffer from a weak intrinsic immunogenicity [9, 11]. For this reason there is a growing demand in the scientific and industrial field for novel strategies directed at increasing the DNA capacity of stimulating the immune system [14, 15]. This fundamental goal is now being reached through development of innovative DNA delivery systems as well as through sophisticated DNA design and engineering that arise from manipulation of the plasmid vector. A list of the patents related to the strategies employed for optimising naked-DNA vaccines and described below, is showed in Table 1. Delivery Systems Traditionally, DNA vaccine delivery systems have been classified as viral and non-viral vector-mediated systems. Currently, because of their highly evolved and specialised components, viral systems are at present the most effective means of DNA delivery, achieving high levels of efficiency, estimated around 90%, for both gene delivery and expression. In 2000, around 75% of recent clinical protocols involving gene therapy used recombinant virus-based vectors for DNA delivery [16]. However, the use of viral vectors as antigen delivery systems has numerous drawbacks including toxicity, recombination, precedent host immunity, higher immunogenicity in comparison to the target antigen, limited DNA carrying capacity, and high production costs [17, 18]. For all these reasons, nonviral systems, especially those based on plasmid DNA in association with nanocarriers and other delivery systems, have become increasingly desirable in both basic research laboratories and clinical settings. Compared to viral systems, nonviral systems are considered to be safe, cheap, and with option for multiple delivery [19]. In the following section, we illustrate patents describing the use of different methods for enhancing DNA vaccine delivery into host cells.
Naked-DNA Vaccination
Table 1.
Frontiers in Anti-Infective Drug Discovery, 2010, Vol. 1
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Strategies for Optimisation of Naked-DNA Vaccines Against Infectious Diseases Strategy
Microparticulates
Biolistic delivery Delivery system
Electroporation
Plasmid backbone
Codon usage
Plasmid Vector Engineering Antigen optimisation
Genetic adiuvants
Patent Number
Reference
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[21]
WO0203961
[25]
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[27]
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[28]
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[31]
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[42]
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Microparticulates Most of naked DNA delivery systems operate at one of two general levels: DNA condensation and complexation in particles and facilitation of DNA entry into recipient
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cells. As far as DNA complexation in small particle is concerned, this system relies on DNA adsorption to or entrapment in biodegradable microparticles such as poly-lactide-coglycolide (PLG) or chitosan, or complexed with non-ionic block copolymers or polycations such as polyethyleneimine (US2007248679) [20, 21]. The major advantage of particulate delivery is that antigen and adjuvant are delivered to the same cell and that synthetic microparticles have excellent potential for targeting cells of the immune system and stimulating antigen uptake. It has been demonstrated that particles of 1-3 m in diameter, are readily internalised by phagocytic cells of the immune system, this resulting in enhanced antigen presentation to the immune effector cells. Furthermore, microparticulates appear to improve delivery of DNA to APCs by facilitating trafficking to the local lymphoid tissue via the afferent lymph and antigen uptake by dendritic cells [22-24]. Patent WO0203961 [25] relates to the DNA encapsulation into biodegradable microspheres for DNA vaccine delivery. A tubercolosis-associated antigen (TbH9) was encoded by the DNA. According to this invention, at least 50% of the DNA in the microspheres comprised supercoiled DNA, and at least 50% of the DNA was released from the microspheres after 7 days at a temperature of 37°C. The microspheres of the invention preferably comprised a biodegradable polymer, such as polylacto-co-glycolide, polylactide, polycaprolactone, or polyhydroxybutyrate copolymers. The described delivery system can include also an aminoalkyl glucosaminide 4-phosphate (AGP) adjuvant in order to increase the immunogenicity of the formulation. Microparticulate adjuvants are currently being tested in some clinical trials against human immunodeficiency virus (HIV), hepatitis B virus (HBV) and influenza [26]. Biolistic Particle Delivery As described in patent US6004287, a biolistic apparatus is generically used for accelerating micro-projectiles into intact cells or tissues [27]. Application of this strategy to DNA vaccines resulted in the invention of a new DNA delivery technology that made it possible to move naked DNA plasmid into target cells on an accelerated particle carrier. This specific delivery system is based on the use of the gene gun device that, under pressurized helium, is capable of delivering plasmid DNA-coated gold beads to the epidermal layer of skin (US6436709) [28]. Because the DNA carrier is introduced directly into the skin cells, delivery of plasmid DNA vaccines using this strategy reduces the amount of DNA needed to induce immune responses. Robust immunogenicity has been shown in many different pre-clinical models and in clinical trials predominantly for infectious diseases [26]. In contrast to intramuscular or intradermal injection by needle, the gene gun delivery system releases plasmid DNA directly into the cells of the epidermis [29]. Intradermal injection is becoming increasingly popular, as the dense network of antigenpresenting cells in the skin, absent in muscle, provides a favourable environment for induction of antigen uptake. This network of Langerhans cells (LCs) can help in the priming of both cellular and humoural immune responses. Epidermal immunisation by gene gun has been shown to lead to the transfection of both skin cells and cells with APC-like morphology [30]. Importantly, direct transfection of Langerhans cells is carried out with very small doses of plasmid DNA (i.e. 1-10g), suggesting that minimum amounts of vector are required to induce the immune response. The advantage of using low doses of plasmid DNA is particularly attractive for prophylactic vaccines against infectious diseases, where a simple and rapid delivery is the main pre-requisite. Gene gun delivery has recently been used with success in a trial against the influenza virus, inducing sero-protective levels of antibody and is currently being used in trials against HBV and HIV infections [26]. In
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particular, patent CN1628858 discloses a method for inducing Th-1 type immune response after inoculation of HBs DNA vaccine through HBV core protein reinforced gene gun. In this patent, the co-expression of HBV core gene is used as adjuvant simultaneously while inoculating plasmid HBs DNA vaccine with a gene gun. The experiment carried out in animals, show that the invention can be applied to enhance the Th-1 type immune response induced by gene gun HBs DNA vaccine eliciting antibody response, CTL activity and IFN- production [31]. A further implementation of the biolistic delivery was obtained also by creating improved injection device suitable for humans. Patent US6730663 describes a method of targeting transient gene expression and stable gene expression from the exogenous administration of a DNA sequence into human tissues. Object of this invention are an improved jet injection needle for the deep injection of DNA within tissues, a flexible multi-needle injector device with a wide surface area as well as a modified injector device to be used for injection through an endoscopic device [32]. Electroporation Electroporation emerged as a suitable physical tool for introducing DNA into muscle cells. This technique exposes the cell membrane to high-intensity electrical pulses that can cause transient and localised destabilisation of the cell surface. During this alteration of the cell permeability, exogenous molecules such as DNA can easily enter the skeletal muscle cells. Electroporation has been shown to increase both the number of transfected cells and also the number of plasmids that permeate into each cell [33]. For vaccination purposes, electrical stimulation of skeletal muscle with a pulse generator is applied immediately after intramuscular injection of DNA [34-36]. Application of electric fields leading to pore formation on the cell membrane, allows the increased passage of plasmid into muscle cell, and causes local tissue damage which has been shown to play a role in enhancing the antigen-specific immune response [37]. The outcome is a dramatic enhancement of humoural and cellular immune responses to the vaccine [38-41]. The advantage of DNA electrotransfer is therefore dual. On the one hand, a high number of muscle cells are transfected with the DNA vaccine; on the other hand the damaged muscle cells release danger signals that favour antigen presenting cell recruitment, thus enhancing the immune response [39]. Electrically-mediated delivery technology has been applied to DNA vaccines against HIV virus and substantially higher immune responses have been achieved in mice and rabbits following vaccination with DNA encoding HIV genes. Vaccines were administered with constant electric current or constant electric voltage causing up to twenty-fold higher immune responses in comparison to the application of DNA vaccines alone (WO0045823) [42]. Electroporation has been used also to deliver Ad6 vectors and a nucleic acid containing an inactive NS5B RNA-dependent RNA polymerase region. The nucleic acid is particularly useful as a component of an adenovector or DNA plasmid vaccine providing a broad range of antigens for generating a specific cell-mediated immune response against hepatitis C virus (HCV) (WO03031588) [43]. Therefore, electroporation is now regarded as a promising delivery system for plasmid DNA vaccination and is used not only on small animals but also on large animals and humans [44, 45]. This novel delivery technology is currently being tested with successful results against highly pathogenic avian influenza virus in non human primates [46] as well as in a number of clinical trials against HIV, tuberculosis and cancer [47, 48]. However, electroporation is used not only for delivering DNA into muscle but also into skin. A patent published recently, describes electroporation devices and methods for introducing biomolecules into skin such as intradermic or
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subcutaneous tissue (WO/2008/048632). This invention presents a skin EP device, which produces a pulse of energy and delivers the same to the skin tissue using a skin electrode array and maintains a constant current in the same tissue allowing the storage and acquisition of current waveform data [49]. Another patent, issued in 2007 (US7171264) [50], relates to the combination of needle-free injection and electroporation demonstrating that this non-invasive strategy is sufficient to introduce the agent into cells in skin, muscle or mucosa. The DNA vaccine can be introduced in a form suitable for electrotransfer into a region of tissue of the subject. A portion of the tissue can be contacted with two oppositely charged injectors, one acting as the donor electrode and one acting as the counter electrode, or a single injector and one or more electrodes can be used. This needle-free injection may be used in combination with suitable non-invasive electrode configurations.
PLASMID VECTOR ENGINEERING Construction of plasmid vectors for vaccination is very simple. Since DNA is a highly flexible molecule, the basic construct can be manipulated in several ways by genetic engineering in order to increase antigen expression, immunogenicity and uptake by recipient cells. As described in patents US2001006950A1, WO9941369 [51, 52] all these modifications can affect both the vector backbone and the gene sequence incorporated into the plasmid, which can include adjuvant-like sequences with stimulating activity on the immune system. Therefore, DNA vaccines not only carry the genetic information for the target antigen, but also deliver an adjuvant effect due to the presence of immuno-stimulatory CpG motifs within the plasmid backbone [53]. The use of targeting moieties has also been explored as a means to enhance DNA vaccination. By using this strategy, enhanced antigen specific immune responses were observed, suggesting that this could be a general method for targeting antigen to selected cell types. In the following section, we classify the various strategies for enhancing the plasmid DNA vaccine potency in two main categories: a first group aimed at improving the plasmid backbone e.g. through codon and antigen optimisation; then a second group aimed at increasing the antigen immunogenicity by addition of genetic adjuvants and fusion to T-cell epitopes. Plasmid Backbone Optimisation Key elements of an expression vector for genetic vaccination generally include a promoter/enhancer sequence, the gene of interest, a polyadenylation/transcriptional terminator sequence and the elements necessary for propagation of DNA plasmid in bacterial cells such as a kanamycin resistance gene for plasmid selection and an origin of replication (Ori) which makes it possible to achieve high plasmid copy numbers. The power of a plasmid DNA vector to drive gene expression is due to an optimal combination of all these elements, and the possibility of introducing various modifications into the plasmid backbone. The most widely used promoters are cytomegalovirus immediately-early promoter (CMV) [54], simian virus SV40 early promoter (SV40) and Rous sarcoma virus promoter (RSV). The CMV promoter is the most commonly used, as it drives expression in a wide range of cells and tissues. Plasmid DNA vectors can also contain tissue specific, synthetic and regulatable promoters, whose sequences are identified by either highthroughput screening or by rational design. If selective expression of a specific gene is desirable in certain types of tissues, it is possible to use promoters which control expression in a cell- or tissue-specific manner. Two examples are offered by the liver-specific promoter [55] and the alpha skeletal muscle actin promoter which is specifically activated in skeletal
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muscle cells (US5374544, EP1310561) [56, 57]. Moreover, various cell-specific promoters exist and are often used, allowing gene transcription when the vector is within the nucleus of the target cell. The use of promoters generally derived from genes encoding abundant cellular proteins, e.g. beta-actin [58] or ubiquitin [59], is particularly advantageous, as they are able to confer long-term in vivo expression. The flexibility of DNA vector allows investigators to exploit the concept of gene expression optimisation by creating synthetic promoters, such as enhancer/promoters composed of numerous combinations of various regulatory sequences [60, 61]. One of these regulatory synthetic sequence, the hybrid CMVUb promoter, was found to have higher expression than the natural muscle promoters [62]. In order to display gene expression kinetic, naturally regulated systems were developed by incorporating sequence elements that respond to the local environment of the given cell or tissue or that are regulated by small molecule drug [63]. Transgene expression can thus be regulated by modulating expression of these transcriptional factors or by altering their activity through drug administration. However, regulation of in vivo transgene expression by such approaches is unreliable, mainly due to the low levels of control associated with the complexity of these systems. Appropriate choice of regulatory elements and vector backbone can lead the gene expression kinetics from a few days to several months. From a safety perspective, one of the drawbacks of DNA vaccines is the presence of antibiotic resistance genes into the vector which are included into the backbone in order to induce plasmid retention by the bacteria during propagation. Unfortunately this may cause concern over the spread in the host of antibiotic resistance to microrganisms previously amenable to antibiotic therapy. Therefore, as described in patent WO 2006017857, a new advance in DNA vector engineering for vaccination purposes is represented by the generation of antibiotic resistance-free DNA vaccines. Here methods of generating an antibiotic resistance gene-free plasmid are described [64]. Another attempt to overcome the biosafety problem related to the use of plasmid-based DNA vaccines is represented by the use of DNA minicircle. Minicircles DNA for nonviral gene transfer contain only the therapeutic expression cassette and besides their advantages for biosafety, show improved gene transfer and bioavailability properties due to the small size [65]. In patent US20070031378 the method for producing a minicircle DNA comprising the prokaryotic origin of replication, the marker sequence, a multiple cloning site and the gene for the sequence specific recombinase is depicted [66]. Codon Usage Optimisation Plasmid DNA vaccines exclusively utilise host cell molecules for protein transcription and translation. Since bacterial codon usage differs from mammalian usage, codon optimisation in DNA vaccine is necessary in order to favour antigen expression and, consequently, the immunogenicity of the gene inserts in DNA vaccines [67]. Various codon usage approaches are now commonly exploited in both non-human primate studies and clinical trials. In patent US7316925 a synthetic expression plasmid is constructed and used to replace codons that contained detrimental sequences, but do not affect the final gene product. The plasmid of this invention has reduced components, and has been optimised to increase efficacy, and reduce adverse reactions in vivo [68]. The codon optimisation strategy has been successfully used to implement the sequence of the human papilloma virus (HPV) (EP1301614A1) and to produce DNA vaccines against the gag protein of HIV (WO03025003) [69, 70].
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Antigen Optimisation Plasmid DNA vaccines are designed to encode for one single antigen as well as multiple antigens belonging to the same pathogen. Patents US20020177569 and US7022320 refer to vectors encoding for different Mycobacterium tuberculosis antigens [71, 72]. As specific examples for suitable antigens cited in this patent we report Ag85B (p30) [73], Ag85A [74] and ESAT-6 [75-77]. Patent US5736524 also describes a method for making a polynucleotide vaccine against tuberculosis [78]. Genes encoding Mycobacterium tuberculosis proteins were cloned into eukaryotic vectors to express the specific proteins in mammalian muscle cells in vivo. This vaccine elicited a good Th-1 immune response to Ag85A antigen in vaccinated mice. For the treatment of the Japanese encephalitis (JEV), patent US20070190617 describes a DNA vaccine encoding a membrane protein and an envelope protein of JEV, prM and E respectively, which enhance antigenic stability and induce a high level of immune response. The vaccine, similarly to most of the DNA constructs used for vaccination, contains a CMV promoter sequence, an enhancer sequence, a chimeric intron, a bovine growth hormone polyadenylation sequence and a kanamycin resistance gene [79]. Since the main drawback of plasmid DNA vaccines is the low immunogenicity of the target antigen, different strategies have been developed to enhance a stronger immune response. Among the approaches explored to improve antigen immunogenicity some have emerged as particularly useful. These involve the increase of intracellular degradation of the antigen, targeting the antigen towards the MHC molecules or to the proteasomal compartment by antigen fusion to ubiquitin. Fusion of antigen with the cytosolic region of LIMP-II (lysosomal integral membrane protein II) (US5633234) [80] or ubiquitin [81] facilitates antigen targeting to the lysosome or proteasome for degradation, resulting in an increase of epitope presentation by MHC class II and I molecules. For antigens released as intact proteins from living transfected cells (i.e. via secretion process), a further improvement is shown by including a secretion signal sequence, namely a leader peptide [82], or a sequence that is directed to the intracellular compartments. As inefficient entry of DNA into the nucleus is one of the main obstacles of transgene delivery, especially in nondividing cells, the vector can be engineered to encode for a nucleus localizing signal sequence (NLE) so as to enhance the plasmid transport into the nucleus (US5827705) [83]. Another factor influencing the short life of transgene is the topological form of the DNA. For this reason, some approa-ches investigated the use of linear DNA in transfection. It was observed that linearised DNA presented higher transgene expression levels in comparison with circular plasmid DNA [84]. A major advantage of DNA vaccines is the possibility of including in the same plasmid several minigenes, encoding selected antigenic epitopes belonging to different pathogens. This type of vaccination, referred to as the multi-epitope approach, is particularly useful for inducing protection against many infectious diseases in the population. Furthermore, the same antigenic epitope sequence can be optimised in order to increase either the binding to MHC or the interaction with the T lymphocyte TCR, as described in patent US20020177569, where a vaccine encoding peptide of the Mycobacterium domain is able to elicit MHC class I-restricted CD8+ T cells responses [71]. To date, plasmid DNA vaccine carrying antigens of influenza virus, HIV, HBV and HCV have been utilised in clinical trials against these infectious diseases [1].
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Linking Antigen to T Cell Epitopes The design of plasmid vectors to maximize the generation of T-cell epitopes from encoded gene products is another valuable strategy of vaccine optimization for improving the presentation of antigen derived-peptides through MHC class I and class II molecules [85, 86]. A consequence of this method is the opportunity to induce both cellular and humoral responses, respectively. A simple and efficient means to improve humoural responses is based on the fusion of a small-size T-helper epitope with the sequence coding for the antigen of interest. Well-defined universal T-epitopes derived from tetanus toxin were extensively used. These epitopes were previously shown to stimulate T cells not only in many mouse strains [87] but also in humans with different genetic backgrounds [88]. Significant T cell responses were induced after immunisation with plasmid DNA vaccines encoding multiple T cell epitopes derived from pathogens such as influenza, HIV and Plasmodium falciparum [89, 90]. Another approach used for induction of a multi-specific immune response against numerous antigen conserved epitopes is described in Patent US6534482. Here a DNA vaccine containing target epitopes of several pathogens has been developed in a string-of-beads fashion. The result is a multi-epitope vaccine for the treatment of HBV, HCV, HIV and CMV infections [91, 92]. Genetic Adjuvants The low immunogenicity of naked DNA vaccines is due to the lack of danger and proinflammatory signals present in live and attenuated vaccines. Therefore, when DNA vaccines are administered as unformulated intramuscular injection, the vaccination’s effect is quite poor. An approach aimed at enhancing low antigen and vector immunogenicity is based on the identification of immunostimulatory sequences which, following insertion into the plasmid through vector engineering, can directly or indirectly modulate the immune response (US2001006950A1) [51]. The additional sequences can code for a variety of molecules; they are not necessarily immunogenic themselves, but can have various immunological properties or functions. Among immunostimulating proteins, we can mention cytokines [93], chemokines [94], costimulatory molecules (B7-1, B7-2) [95-97], granulocyte-macrophage colony-stimulating factor (GM-CSF) [98], and ubiquitin [99]. The strategies used to optimize DNA vaccines by addition of immunomodulator genes are described in patents US7141651, EP1518927, WO9941369 [52, 100, 101]. The best way of ensuring the coordinate expression of a specific antigen and immunomodulatory molecules would be to insert both genes into the same plasmid. A family of plasmids designed for in vivo naked DNA transfer, which is used for two cDNAs co-expression, are commonly utilised in DNA vaccination [102]. The coadministration strategy of the immunostimulatory sequence and antigen has been widely exploited to enhance immunogenicity of plasmid DNA vaccination. Co-delivery of cytokines in the DNA vaccine formulation has been extensively used to confer protection against a wide range of infectious diseases. These genetic adjuvants can be included in the same vector encoding the specific antigen or in a different plasmid. For treatment of infectious diseases US7141651 patent shows a method of making multiple cytokine fusion proteins. In this method cytokine-encoded genes are fused to a target moiety such as a region of the Immunoglobulin that binds to the target antigen [100]. Similarly, US2007036752A1 patent describes a method for producing DNA constructs encoding cytokine fusion proteins, such as the immunoglobulin fused to IL-2 (Ig-IL-2), which is able to bind to more than one cell type and exhibits a longer circulating half-life in the patient's
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body than the corresponding natural cytokine [103]. Plasmid DNA encoding Interleukin 2 (IL-2) has also been utilised in murine models to augment immune response to the DNA vaccine expressing hepatitis C virus core protein [104], hepatitis B virus surface antigen [105], and HIV-1 gp120 [106]. In patent WO/2007/103048-PCT/US2007/005004 DNA conjugates which contain a TLR/CD40/ agonist as immune adjuvants and optional antigen combination are used as vaccines for treatment of various chronic diseases such as HIV infection [107]. Co-immunisation by plasmid encoding IL-15 and a DNA encoding the 144 amino acids of the N-terminus of HBV core gene, induces effective cell immunity and enhances the longevity of a specific memory CD8+ T cells [108]. Plasmid encoding GMCSF has been shown to increase antibody and T-cell response in DNA vaccination against HIV-1 [109, 110], herpes simplex virus type 2 (HSV-2) [111] and hepatitis C [112].
CURRENT AND FUTURE DEVELOPMENTS After a long period of experimentation, DNA vaccines have become an important tool to prevent diseases in animal models although they still show low immunogenicity when tested in human clinical trials. To make their use possible as general method of immunisation in humans, significant efforts must be done. The potential combination of the several approaches reviewed here, could lead to development of more effective vaccination protocols in comparison to the classic vaccination methods based on attenuated or killedpathogen vaccines. A promising strategy of vaccination against infectious diseases seems to be the genetic immunisation with plasmid DNA vectors coupled to innovative adjuvants and delivery systems. Although it is impossible to identify universal genetic adjuvants for all infectious diseases, particular attention should be paid to find efficacious and non-toxic vaccine formulations able to induce long-term, potent and safe immune response. However, recent developments in the genetic engineering and in the immunology research field have resulted in notable improvements of the DNA vaccine potency. In the next future, we will assist to the advent of successful DNA vaccines for infectious diseases that will significantly improve the quality of human life.
ACKNOWLEDGEMENTS The authors thank “Energy for Research”, sponsor group of the Laboratory of Molecular Pathology and Experimental Oncology, CNR-INMM. This work was partly supported by MIUR FIRB 2006 RBIP0695BB.
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Harth G, Lee BY, Wang J, Clemens DL, Horwitz MA. Novel insights into the genetics, biochemistry, and immunocytochemistry of the 20-kilodalton major extracellular protein of Mycobacterium tuberculosis. Infect Immun 1996; 64: 3038-3047. Huygen K, Content J, Denis O, et al. Immunogenicity and protective efficacy of a tuberculosis DNA vaccine. Nat Med 1996; 2: 893-898. Sorensen AL, Nagai S, Houen G, Andersen P, Andersen AB. Purification and characterization of a lowmolecular-mass-T cell antigen secreted by Mycobacterium tuberculosis, Infect Immun 1995; 63: 17101717.[76] Andersen P, Andersen AB, Sorensen AL, Nagai S. Recall of long-lived immunity to Mycobacterium tuberculosis infection in mice. J Immunol 1995; 154: 3359-3372. Harboe M, Oettinger T, Wiker HG, Rosenkrands I, Andersen P. Evidence for occurrence of the ESAT-6 protein in Mycobacterium tuberculosis and virulent Mycobacterium bovis and for its absence in Mycobacterium bovis BCG. Infect Immun 1996; 64: 16-22. Content, J., Huygen, K., Liu, M.A., Montgomery, D., Ulmer J.: US5736524 (1998). Wu, C., Tao, M.: US20070190617 (2007). August, T.J., Pardoll, D.M., Guarnieri, F.G.: US5633234 (1997). Wang QM, Sun SH, Hu ZL, et al. Epitope DNA vaccines against tuberculosis: spacers and ubiquitin modulates cellular immune responses elicited by epitope DNA vaccine. Scand J Immunol 2004; 60: 219225. Xu W, Chu Y, Zhang R, Xu H, Wang Y, Xiong S. Endoplasmic reticulum targeting sequence enhances HBV-specific cytotoxic T lymphocytes induced by a CTL epitope-based DNA vaccine. Virology 2005; 334: 255-263. Dean, D.A.: US5827705 (1998). Chen ZY, Yant SR, He CY, Meuse L, Shen S, Kay MA. Linear DNAs concatemerize in vivo and result in sustained transgene expression in mouse liver. Mol Ther 2001; 3: 403-410. Sette AD, Oseroff C, Sidney J, et al. Overcoming T cell tolerance to the hepatitis B virus surface antigen in hepatitis B virus-transgenic mice. J Immunol 2001; 166: 1389-1397. Chiarella P, Massi E, De Robertis M, Signori E, Fazio VM. Adjuvants in vaccines and for immunisation: current trends. Expert Opin Biol Ther 2007; 7: 1551-1562. Kumar TR, Fairchild-Huntress V, Low MJ. Gonadotrope-specific expression of the human folliclestimulating hormone beta-subunit gene in pituitaries of transgenic mice. Mol Endocrinol 1992; 6: 81-90. Panina-Bordignon P, Tan A, Termijtelen A, Demotz S, Corradin G, Lanzavecchia A. Universally immunogenic T cell epitopes: promiscuous binding to human MHC class II and promiscuous recognition by T cells. Eur J Immunol 1989; 19: 2237-2242. Hanke T, Schneider J, Gilbert SC, Hill AV, McMichael A. DNA multi-CTL epitope vaccines for HIV and Plasmodium falciparum: immunogenicity in mice. Vaccine 1998; 16: 426-435. Thomson SA, Sherritt MA, Medveczky J, et al. Delivery of multiple CD8 cytotoxic T cell epitopes by DNA vaccination. J Immunol 1998; 160: 1717-1723. Sette, A., Fikes, J.D., Chesnut, R.W., Hermanson G.G., Ishioka, G.Y., Livingston, B.: US20036534482 (2003). Kuhrober A, Wild J, Pudollek HP, Chisari FV, Reimann J. DNA vaccination with plasmids encoding the intracellular (HBcAg) or secreted (HBeAg) form of the core protein of hepatitis B virus primes T cell responses to two overlapping Kb -and Kd -restricted epitopes. Int Immunol 1997; 9: 1203-1212. Du X, Zheng G, Jin H, et al. The adjuvant effects of co-stimulatory molecules on cellular and memory responses to HBsAg DNA vaccination. J Gene Med 2007; 9: 136-146. Kim SJ, Suh D, Park SE, et al. Enhanced immunogenicity of DNA fusion vaccine encoding secreted hepatitis B surface antigen and chemokine RANTES. Virology 2003; 314: 84-91. Conry RM, Widera G, LoBuglio AF, et al. Selected strategies to augment polynucleotide immunization. Gene Ther 1996; 3: 67-74. Corr M, Tighe H, Lee D, et al. Costimulation provided by DNA immunization enhances antitumor immunity. J Immunol 1997; 159: 4999-5004. Iwasaki A, Stiernholm BJ, Chan AK, Berinstein NL, Barber BH. Enhanced CTL responses mediated by plasmid DNA immunogens encoding costimulatory molecules and cytokines. J Immunol 1997; 158: 45914601. Sedegah M, Weiss W, Sacci JB Jr, et al. Improving protective immunity induced by DNA-based immunization: priming with antigen and GM-CSF-encoding plasmid DNA and boosting with antigenexpressing recombinant poxvirus. J Immunol 2000; 164: 5905-5912. Wu Y, Kipps TJ. Deoxyribonucleic acid vaccines encoding antigens with rapid proteasome-dependent degradation are highly efficient inducers of cytolytic T lymphocytes. J Immunol 1997; 159: 6037-6043. Gillies, S.D., Lo, K.M.: US20017141651 (2001). Punnonen, J., Stemmer, W.P.C., Whalen, R.G., Howard, R.J.: EP1518927 (2005).
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Light Activated Compounds as Patented Antimicrobial Agents David Andrew Phoenix*,1 and Frederick Harris2 1
Deputy Vice Chancellor, University of Central Lancashire, Preston, PR1 2HE, UK 2 School of Forensic and Investigative Sciences, University of Central Lancashire, Preston, PR1 2HE, UK Abstract: Microbial pathogens with resistance to conventional drugs are a problem of global proportions and in response, photodynamic antimicrobial chemotherapy (PACT) has been developed. PACT involves the delivery of a non-toxic photo-sensitiser (PS) to the site of a microbial infection, which is then taken up by the pathogen. Illumination of the PS by light at an appropriate wavelength can lead to inactivation of the pathogen through the production of highly reactive free radical species, which induce oxidative damage to microbial targets such as lipid, proteins and DNA. Here we briefly review light sources for PACT, the desirable electronic and physiochemical properties of PS, and the photochemical and photophysical steps underlying PS antimicrobial action. With reference to recent patents, we then illustrate uses of PACT agents, including: 5-aminolevulinic acid, phenothiazinium based compounds, psoralens and organorhodium complexes.
Keywords: Photodynamic antimicrobial chemotherapy, photodynamic therapy, photosensitiser, light, type I mechanism, type II mechanism, singlet oxygen, viruses, bacteria, protozoa, blood, aminolevulinic acid, phenothiazinium, psoralen, organorhodium. INTRODUCTION It was believed that the introduction of antibiotics into health-care would lead to the mastery of infectious diseases and it is beyond dispute that these agents have saved countless lives. However, bacterial infections remain the leading cause of mortality worldwide and globally the treatment of infectious diseases has become an intolerable strain on healthcare services. A number of factors appear to have negatively affected the epidemiology of infectious diseases with social, political, economic and environmental factors all impacting on the spread of infection. However, the biggest single contributor to the current pandemic of infectious diseases has been the emergence of pathogenic bacteria with multiple antimicrobial drug resistance. In response, there has been extensive research into new antimicrobial agents with novel mechanisms of action [1, 2]. Penicillins and cephalosporins are among the most widely prescribed antibiotics but antimicrobial resistance to these compounds is becoming widespread. The core structure of these antibiotics is the β-lactam ring system and increasingly, many bacteria have acquired the ability to express β-lactamases, which are able to hydrolyze this ring system thereby *Corresponding author: Tel: +1772 892504; E-mail:
[email protected] Atta-ur-Rahman / M. Iqbal Choudhary (Eds.) All rights reserved – © 2010 Bentham Science Publishers.
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rendering many established β-lactam antibiotics ineffective [3]. A number of strategies, which have been developed to overcome this problem, have led to patents: In a recent study, it was found that a novel β-lactam antibiotic could be isolated from a fermentative culture of a protoplast fusion strain of Penicillium chrysogenum and Cephalosporium acremonium. This antibiotic exhibited potent activity against both Gram-positive and Gram-negative bacteria and was patented as a potential antibacterial agent [4]. Another study led to a patented methodology in which the administration of gemifloxacin, a broad spectrum quinolone antibiotic, with a variety of β-lactams provided an antibacterial regimen that had a broader spectrum of activity than either agent alone. In particular, it was found that such combina-tions exhibited synergistic activity that was effective against infections of the respiratory and urinary tracts in immunocompromised patients [5]. Defence peptides are naturally occurring antibiotics that are produced by a diverse range of creatures and organisms, and their therapeutic potential has been the focus of much recent research. The antimicrobial action of these peptides does not generally involve receptors but rather, involves multiple targets including: intracellular sites of action and membranes. In most cases, defence peptides appear to induce the death of microbial cells by partitioning into membranes, which leads to cell lysis and the leakage of cellular contents. The relatively non-specific nature of such antimicrobial action makes the emergence of acquired microbial resistance to defence peptides an intrinsically complex process, unlike traditional antibiotics where such resistance can arise from only a few mutations [6]. Defence peptides are thus attractive propositions for development as novel antibiotics and a number of these compounds have featured in recent patents. Halocidin is a defence peptide that was isolated from the body fluid of the tunicate, Halocynthia aurantium, and along with derivative peptides, it was found to have potent, broad-range antibacterial activity. Based on these findings, halocidin and its daughter peptides were patented for their potential to act as novel antibiotics in the treatment of infections and as antibacterial additives to food, cosmetics and ointments [7]. A patent was also taken out on rattusin, which is a synthetic peptide derived from the C-terminal region of a murine defence peptide. Rattusin was found to possess broad-spectrum antibacterial activity that was insensitive to salt and the presence of divalent cations. These properties make the peptide an attractive drug candidate for the treatment of systemic infections, which are resistant to other antibiotics. In addition, the salt-insensitive activity of rattusin give it potential for the treatment of cystic fibrosis and Crohn's disease [8]. Bacterophages are viruses that infect bacteria and a recent line of patented research identified the first of these bacterial viruses, which is lytic for the genus Methylobacterium and its close relative, the Human Blood Bacterium (HBB). A number of potential agricultural and therapeutic uses for the bacteriophage were put forward, including: the elimination of Methylobacterium from the seeds and other parts of a plant and decontaminating blood from HBB, which is associated with auto-immune disorders in humans such as rheumatoid arthritis [9]. Another patented antibacterial strategy based on the use of bacteriophages involved the administration of one or more purified preparations of these later organisms, which are specific for the bacterial infection under treatment. It was envisaged that this technique may be used to treat infection by a number of pathogens including vancomycinresistant Enterococcus and multi-drug resistant Pseudomonas species [10]. Photosensitisers (PS) are light activated compounds whose antimicrobial properties were first noted in dyes that were used for the histological staining of cellular components. In the 1930s, it was reported that phages and viruses, which had been stained with such dyes, were photosensitive, and in the early 1960s, quantitative studies on the ability of these dyes to
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photoinactivate viruses and bacteria were reported [11]. Since then, in addition to these latter organisms [12-14], eukaryotic microbes such as yeasts, fungi and protozoa have been shown to be susceptible to dyes and other PS [15, 16]. When applied in a therapeutic context, this use of PS has become known as photodynamic antimicrobial chemotherapy (PACT) with the underlying ethos that if, in a human subject, a live microbe can be selectively demonstrated by a stain, which is also a PS, then it should be possible to destroy the stained microbe upon illumination [17, 18]. Over the last few decades, PS have attracted increasing attention as antimicrobial agents with therapeutic potential. Here, we present an overview of the photophysics and photochemistry involved in the antimicrobial action of PS, and illustrate the therapeutic uses of this action with reference to a number of PACT agents that have featured in recent patents. These agents include: 5-aminolevulinic acid, which is a prodrug that induces the production of endogenous PS, phenothiazinium based compounds, which are the most studied of the PACT agents, psoralens and organorhodium complexes. PACT PHOTOCHEMISTRY It is well established that the photodynamic action of PS can be directed against a number of cellular components, primarily: DNA, proteins and membrane lipid [19-24]. The primary photochemistry involved in this action appears to be similar for all PS and a schematic representation of the possible photochemical / photophysical steps involved in PDT and PACT is shown in Fig. (1). A PS has two electrons with opposite spins in its ground state (1PS0). After the absorption of a photon of light (hv), 1PS0 undergoes an electronic transition, which promotes an electron to the first excited singlet state, 1PS*, with the electron retaining its spin. This is a short-lived species with a lifetime of nanoseconds and may lose its absorbed energy by electronic decay (fluorescence) or by internal conversion into heat, and thus return to the ground state. This fluorescence has been utilised to quantify the level of PS in cells or tissues, and for in vivo measurement of their pharmacokinetics and distribution in living animals and patients. However, 1PS* can also undergo an electronic rearrangement known as intersystem crossing whereby the spin of the excited electron inverts to give the first excited triplet state, 3PS*, in which electron spins are in parallel. The 3PS* state is relatively long-lived with a lifetime of microseconds, which is due to the fact that the loss of energy through electronic decay (phosphorescence) back to the 1PS0 state is a “spin-forbidden” process and thus has a low probability of occurring. This 3 PS* state may pass its excitational energy onto other molecules by either of two mechanisms, which are defined as type 1 and type II mechanisms, both of which facilitate photodynamic action [25-28]. Type I mechanisms involve direct interaction between 3PS* and a molecule in the immediate vicinity with hydrogen abstraction or electron transfer between these molecules yielding radical anions and cations respectively, which are generally termed reactive oxygen species (ROS). Superoxide anions (O2-) are often an initial product of type I pathways, yielded by electron transfer from 3PS* to molecular oxygen, 3O2. These anions are not particularly toxic to biological systems but tend to undergo dismutation – a process, which can be catalysed by superoxide dismutase and involves reaction between superoxide anions themselves to produce 3O2 and hydrogen peroxide, H2O2. This latter molecule is important to biological systems because it can readily pass through cell membranes and is necessary for the function of many enzymes. O2- is also important to biological systems by playing a key role in the Fenton reaction where the anion reduces cellular metal ions, typically Fe3+ to Fe2+. The reduced form of the metal catalyses cleavage of the O-O bond in H2O2 to produce
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the highly reactive hydroxyl radical, OH*, which can then interact with superoxide to generate singlet oxygen, 1O2. OH* also readily diffuses through membranes and can add on to biomolecules (R), yielding hydroxylated adducts, or oxidise substrates by electron abstraction. In each case, the reaction product is itself a radical and can react with other molecules in a chain reaction. As an example, interaction with ground state oxygen produces a peroxyl radical, RCOO*, which is also highly reactive and can interact with another substrate molecule in a chain reaction. A variety of other highly reactive radicals such as nitric oxide, NO-, and peroxynitrite, OONO-, can also be produced in type I pathways, which initiate chain reactions, and collectively these ROS can inflict high levels of photo-damage to cells and tissues. In contrast, type II mechanisms proceed via energy transfer processes during a collision of 3PS* with 3O2 and involves electron spin exchange between the two molecules, yielding 1PS0, 1O2. The latter molecule has a lifetime of the order of microseconds, which is relatively long in relation to other ROS, and it may diffuse up to 20 nm from its site of generation before reacting with a biomolecule, again leading to further reactions and cellular damage [25, 29, 30].
Fig. (1). The photochemistry / photophysics of PS antimicrobial action. Fig. (1) was adapted from [40] and shows a schematic representation of the possible photochemical / photo-physical steps involved in the photodynamic action of PACT agents. In Fig. (1), a PS absorbs a photon of light (hv) and undergoes an electronic transition, which promotes an electron from its ground state 1PS0 to the excited singlet state, 1PS*. If 1PS* is relatively stable, the PS may undergo an electronic rearrangement to give the excited triplet state 3PS*. An electron in this state can pass its excitational energy onto other molecules by either type I and type II mechanisms, which can then lead to photo-oxidative cellular damage. Under some circumstances, a PS in either the 1PS* or 3PS* state can also engage in covalent adduct formation. Higher excited states or electron energy levels can be reached using high intensity pulsed radiation of excitation at two wavelengths [25, 29, 30].
PACT PS AND THEIR PROPERTIES There are a number of desirable characteristics for PS to serve as efficient PACT agents. In terms of economic viability, synthesis of PS should be relatively simple and the starting materials readily available so that large-scale production is feasible, cost-effective and widely applicable. In terms of physical properties, the PS should be a pure compound with a constant composition, a stable shelf life, and ideally, it should be water soluble or soluble in a non-hazardous aqueous solvent mixture [31-33]. There are a number of parameters that determine the photosensitizing capability of a PS and amongst the most important are: the triplet-state yield (Φt), which is the probability that after absorbing a quantum of light a PS converts to the 3PS* state, the singlet oxygen yield
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(ΦΔ), which is the probability that after absorbing a quantum of light a PS converts to the 3 PS* state then transfers its excess energy to 3O2, thus causing the formation of 1O2. SΔ is the efficiency of energy transfer and relates ΦΔ and Φt according to the equation: ΦΔ = SΔ Φt. Other parameters used to characterise the efficacy of PS include the lifetime ( ! t) and the energy (ΔEt) of the 3PS* state, which is defined as the difference between the energies of this state and the 1PS0 state [34]. To be therapeutically useful, the light adsorption properties of PS are of primary importance. Ideally, the maximum wavelength of absorption, λmax of the PS will lie within the therapeutic window of 600-800 nm. Also, the PS will possess a high coefficient of absorption for that wavelength, εmax > 20,000 -30,000 M−1 cm−1, to minimize the dose of PS needed to achieve the desired effect. The PS should absorb light at wavelengths sufficiently long so that the therapeutic effect of PACT is maximised in terms of tissue penetration and that the light used causes minimal photosensitization of healthy tissues. However, the absorption wavelengths of the PS should not be too long as this could render ΔEt too low for efficient 1O2 formation and decrease the photo-stability of the PS. Moreover, to minimise the risk of generalized photosensitivity due to sunlight, it is desirable that the PS should not strongly absorb light with wavelengths in region 400-600 nm [29]. To exhibit a high photosensitising efficiency, the PS should have a high value of Φt, show a ΔEt that is larger than ΔEΔ (the energy difference between 1O2 and 3O2, which is 94 kJ mol−1) and demonstrate energy-transfer efficiency for the formation of 1O2. Moreover, the PS should not show significant self-aggregation in the body as this decreases both Φt and ΦΔ. For PS that generate singlet oxygen, high values of ΦΔ are desirable and are often used as a gross performance indicator of PS photosensitising ability although this will also be determined by a multitude of other factors such as pharmacokinetics and physiochemical properties of the PS [25]. When administered to patients, PS should have low levels of dark toxicity and a low incidence of administrative toxicity such as hypotension or allergic reaction. In addition, PS should be selectively enriched in the target cells and be eliminated from the body sufficiently quickly to avoid generalized skin photosensitization. Ideally, the pharmacokinetic elimination of PS from the patient should less than one day to avoid the necessity for post-treatment protection from light exposure and prolonged skin photosensitivity. Moreover, a short interval between administration and illumination is desirable to facilitate outpatient treatment that is both patient-friendly and cost-effective [30]. The lipophilicity of a PS, log P, is a measure of its relative solubility in water compared to that of some reference organic solvent such as n-octanol This provides an indication of the relative hydrophobicty / hydrophilicity possessed by the PS and can suggest a preference for a lipid / aqueous environment and thereby cellular locations. As an example, a strongly hydrophobic PS (log P >> 0) may have a preference for a membrane location. The concentration of 3O2 in membrane lipid is higher than in the surrounding aqueous phase and these conditions could favour a type II mechanism of photo-damage and membrane attack [3537]. A concept related to lipophilicity, and also an important physiochemical property in relation to PS efficacy as a PACT agent, is amphiphilicity, which considers the segregation of hydrophilic and hydrophobic regions within a molecule. Amphiphilicity can act as a determinant of the ability of a PS to orientate in membranes, thus affecting levels of membrane penetration [17, 38]. Membranes themselves are amphiphilic structures and the hydrophobic regions of a PS will seek the central hydrophobic core region of the membrane
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whereas the hydrophilic regions of the photosenstiser will prefer to preside in the membrane interfacial region and engage in electrostsatic interactions with charged moieties in the lipid headgroup region [39]. The hydrophilic characteristics of a molecule primarily depend upon the nature of its constituent atoms and chemical groups, which in turn are reflected by chemical properties such as pKa, and for most PS, the oxidation state of its chromophore. These properties govern the overall charge possessed by a PS, which can be of fundamental importance in terms of its ability to target the anionic membranes of microbial pathogens [17, 38, 40]. PS AND PACT Clearly, there are three major choices to be made for the successful application of PACT, which are: a suitable PS, the mode of delivery of these PS to the target site and the delivery of light from an appropriate source at the optimal wavelength for the chosen PS [17, 41, 42]. In addition, appropriate dosages for light and PS dosages have to be selected for PACT application and PDT has shown that these dosages are affected by a number of variable factors, which makes their selection difficult. For example, local PS concentration will vary between sites in the body, from individual to individual, and as a function of time, whilst the penetration of light into target tissue will depend on the specific optical properties of that tissue [41]. However, in general, dosimetry determination is beyond the scope of this review and will not be discussed further. In relation to PS delivery, the study of PDT has shown that these agents can be administered orally, topically or parenterally by intravenous injection, and due to the disordered metabolism and blood flow peculiar to dysplastic or neoplastic tissue, photosensitizing levels of PS accumulate in the target lesions relatively quickly [43, 44]. A number of strategies to improve the targeting efficiency of these PS have been explored, including: antibody conjugation [45], attachment of polycationic peptides [46], employing bacteriophages [47] and the use of nanoparticle technology [48]. Nonetheless, as yet, only a few PS have been approved for systemic administration in PDT [45-48] and there are still technical problems associated with systemic light delivery. Taking all these observations into consideration, it seems highly probable that for the present, the use of PS in PACT would be limited to the topical treatment of accessible or localized microbial infections. This could include the treatment of: wounds and burns, rapidly spreading and intractable soft-tissue infections and abscesses, infections in body cavities such as the mouth, ear, nasal sinus, bladder and stomach, and surface infections of the cornea and skin [15, 17, 40, 49, 50]. In terms of light administration, laser systems are becoming the standard light source for both PACT and PDT [15, 51, 52], and those in current use include: argon / dye lasers, helium-neon lasers and KTP:YAG / dye lasers [53-55]. Most recently, diode lasers systems have been developed, which are semiconductor light sources and offer distinct advantages over other laser systems used in PACT in terms of light delivery, optical dosimetry and target cell photo-inactivation [56, 57] with the result that they have gained approval in the US and Europe for use in PDT [58]. However, light emitting diodes (LEDs) seem likely to supersede diode lasers as they have much higher power outputs than these latter lasers but a similar efficiency. In addition, arrays of LEDs can be readily configured into different irradiation geometries, which permits the direct irradiation of easily accessible tissue surfaces [59]. It has been predicted that future light source developments for PDT and PACT include: multi-wavelength laser diode systems, novel light source technologies, such as organic LEDs, and chemiluminescence-based sources [22, 41, 59, 60].
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In respect to PS development, the first generation of PS is generally considered as compromising haematoporphyrin and its derivatives and up to the early 1980s, these PS were the most commonly used PS in PDT research. However, these PS exhibited a number of serious disadvantages, including chemical heterogeneity, poor selectivity for tumours and weak light absorbance at therapeutically desirable wavelengths [34, 52]. In response, a myriad of compounds, which are generally regarded as second generation PS, have been synthesised over the last twenty-five years [25, 34, 61-65]. In general, these PS have been developed to strongly absorb light in the therapeutic window of 600-800 nm and to exhibit high values of ΦΔ, Φt, ΔEt and τt, thereby possessing the ability to generate 1O2 efficiently. Other criteria for the development of these PS include: photo-stability, low dark toxicity, high tumour selectivity and strong affinity for target sites [1, 23, 25, 26, 30]. Most major classes of second generation PS have members that are either clinically approved or in clinical trials for PDT [65] (http://www.clinicaltrials.gov.) or patented (http://www.uspto. gov/patft/index.html). Major examples of such PS are porphyrins (Photofrin®) and chlorins (Visudyn®, Foscan®, Temoporfin®), along with the prodrug, 5-aminolevulinic acid (Levulan®) [25, 34, 61-65]. Compared to PDT, PS clinically tested for PACT are relatively few, which underlies the infancy of this approach to antimicrobial therapy. An eclectic group of PS active against bacteria, viruses, fungi and parasites have been described and include: tricyclic dyes such as merocyanine 540, riboflavin (vitamin B2), acridine orange, proflavine, fluorescein, eosine, erythrosine and rose bengal, and tetrapyrroles, including: porphyrins, chlorophyll, phylloerythrin, phthalocyanines and chlorins. In addition, there is a variety of other structurally diverse PS with microbial photo-sensitizing ability, which include: furocoumarins and fullerenes [12-14, 30-35, 38, 40, 41, 49, 50, 53, 54, 59-61, 6676]. However, the diversity of PS used in PACT is aptly illustrated in terms of structure, mechanisms of antimicrobial action and clinical application by reference to four classes of these compounds that have recently featured in patents. THE INDUCTION OF ENDOGENOUS PS 5-Aminolevulinic acid (ALA) has featured in a number of patents (http://www.uspto. gov/patft/index.html), which derive from a novel approach to PDT. In this approach, ALA is not administered as a PS per se, but serves to induce the in situ build up of endogenous PS in target tumour cells [30, 34, 77, 78]. ALA is the first intermediate in the heme biosynthetic pathway (Fig. 2) and its synthesis is regulated by negative feedback from heme levels. Thus, the uptake of exogenous ALA by tumour cells causes this feedback mechanism to be bypassed, which leads to the accumulation of photodynamically active, protoporphyrin IX, and other porphyrin intermediates (Fig. 2). Appropriate photo-activation of these endogenous PS then leads to tumour cell death [79-81]. The notion that ALA could be used in PACT was first suggested by Kennedy et al., [82] and since then, a number of studies have shown that the ALA-mediated induction of porphyrins could be used to kill fungi [14]. In particular, ALA based PACT has recently been shown to kill Trichophyton rubrum and Trichophyton interdigitale, which can cause infections of the fingernails and toenails, known as onychomycosis [83, 84]. Based on these results, it was suggested that ALA based PACT may be developed for the treatment of the disease and in this capacity, a patent for the technique has been applied for. The patent application describes the design of a bio-adhesive strip, which releases ALA into the matrix of toenails and fingernails so that subsequent irradiation of the infected areas leads to the photo-inactivation of the infecting fungi [85]. However, the major antimicrobial focus of
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Fig. (2). The haem biosynthetic pathway Fig. (2) was adapted from [77]. Fig. (2A) shows the haem biosynthetic pathway, which is highly conserved across organisms except for the initial step of ALA synthesis. In non-plant eukaryotes, ALA-synthetase is used to catalyse the production of ALA from glycine and succinyl CoA (Fig. (2Ba) whereas in prokaryotes, glutamate-1-semialdehyde transferase catalyses the formation of ALA from glutamate-1-semialdehyde (Fig. (2Bb). The introduction of exogenous ALA bypasses the negative feedback step of the pathway and promotes the production of protoporphyrin IX, a potent PS.
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ALA based PACT has been directed to treating infections caused by medically relevant bacteria. A number of studies have demonstrated that ALA based PACT is able to kill a variety of Gram-positive and Gram-negative bacteria [15, 86-91]. Recently, it was shown that ALA and its methyl ester were able to induce the accumulation of a range of porphyrins in members of these bacterial classes. Subsequent photo-killing of the parent organisms appeared to involve singlet oxygen and type II mechanisms of action with rates of photokilling dependent upon the distribution and level of induced porphyrins. In general, Grampositive bacteria were highly susceptible to this form of PACT but Gram-negative bacteria showed higher levels of resistance, probably due to the barrier posed by the outer membrane of these latter organisms to ALA uptake [92, 93]. Propionibacterium acnes and Heliobacter pylori inherently accumulate high levels of porphyrins [94, 95], which renders them particularly susceptible to ALA-mediated photokilling and PACT. H. pylori is a Gram-negative endemic pathogen, causing chronic gastritis and gastric ulcer in humans, and is a risk factor for gastric adenocarcinoma. The organism is susceptible to a range of PS and is usually found on the superficial mucosa of the upper gastrointestinal tract where it is readily accessible endoscopically [96]. More recent studies showed that when ALA was orally administered to humans that were positive for the organism, subsequent application of laser and endoscopic light to zones of gastric antrum led to the death of high levels of H. pylori, thereby showing the potential for clinical development [97]. P. acnes is a common Gram-positive skin organism that normally inhabits human sebaceous glands and contributes to acne, a chronic inflammatory disease of the pilosebaceous unit. Acne is a multi-factorial condition, where excessive sebum production, brought about by hormonal changes, is followed by abnormal desquamation of follicular corneocytes. The resulting mixture of cells and sebum leads to blockage of the sebaceous duct and proliferation of P. acnes with subsequent release of chemotactic factors by the organism generating an inflammatory response [98]. P. acnes has been shown to produce enhanced levels of endogenous porphyrins after ALA uptake [99] with subsequent illumination leading to death of the organism [100] and a number of studies have shown acne vulgaris to be successfully treated by ALA-mediated photodynamic action [101, 102]. Moreover, ALA is known to be preferentially taken up by the pilosebaceous units, leading to a build up of PPIX with light activation generating singlet oxygen and photo-damage to mitochondria, nuclei and cell membranes. ALA-mediated photodynamic action thus has the potential to treat acne by both selectively damaging the pilosebaceous unit, thereby removing blockages from the sebaceous duct, and killing P. acnes [103, 104]. Based on these observations, a patent for the treatment of acne vulgaris using topical application of ALA followed by light irradiation of the affected tissue was recently granted [105] and a subsequent patent application has been submitted for a modified form of the patented treatment [106]. Nonetheless, it was recognised that a major limitation to the use of ALA is its high hydrophilicity at physiological pH and within the terms of this patent, ALA was taken to include pharmacologically equivalent forms of the drug, including previously patented ALA esters. A number of novel esters of ALA have been recently synthesised with improved penetration of the skin's natural permeability barrier [87, 107] and the general chemical structure of these compounds is featured in a pending patent, along with their use as PS in photo-chemotherapy [108]. It has been predicted that some of these novel ALA derivatives may find use in the treatment of acne [109, 110].
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PHENOTHIAZINIUM BASED PS Methylene blue (MB) was amongst the first PS to feature as a PACT agent in a successful patent when Swartz [111] patented a method for inactivating viruses, bacteria and other microbes, which involved the concurrent application of an electric field and light for PS activation. MB is now the prototype of phenothiazinium based PS (PhBPs) [112], which are the most extensively researched PACT agents and amongst those most frequently featured in patents (http://www.uspto.gov/patft/index.html). PhBPs are cationic dyes with a core structure consisting of a planar tricyclic heteroaromatic ring system and in its oxidized form, this ring system constitutes the phenothiazinium chromophore. The general structure of MB (3,7-bis(dimethylamino)phenothiazine-5-ium) and other major phenothaziniums is shown in Fig. (3) whilst those of typical analogues such as PYY (N-6-(dimethylamino)-3Hxanthene-3-ylidene-N-methylmethanaminium) and NR (3-amino-7-dimethylamino-2methylphenazine) are shown in Fig. (4). The chromophore of PhBPs can efficiently Table 1. Photodynamic Properties of PhBPs Maximal Absorption in the Range 500 nm – 750 nm (λ max)
Relative Singlet Oxygen Yield
Lipophilicity
(Φ Δ )
(Log P)
MB
656
1.0
0.1
NMB
648
1.35
1.2
DMMB
630
1.22
1.01
TBO
625
0.86
-0.21
AA
625
0.77
0.7
AB
546
0.77
0.7
AC
647
0.77
0.7
NR
623
0.18
0.16
PYY
536
0.05
-0.5
PhBPs
Table 1 was adapted from [40]. The lipophilicity of PhBPs was measured as the partition coefficient, log P, of a dye between water and n-octanol. The singlet oxygen yield, ΦΔ, of PhBPs was determined relative to ΦΔ, = 1 for MB, and was measured by monitoring the decolourisation of 1,3 diphenylisobenzofuran at 410 nm. Form a long-lived 3PS* state and can strongly absorb light, with values of λmax that generally lie within the therapeutic window of 600-900 nm and high values of εmax (Table 1). In general, PhBPs exhibit high values of ! Δ (Table 1) and thus are efficient inducers of singlet oxygen and utilise type II mechanisms of photo-oxidation [18, 35, 40, 80]. Nonetheless, some PhBPs such as pyronine Y (PYY) appear to use type I mechanisms [113] and in what appears to be the first reported observation, photo-activated MB was recently found to form a covalent adduct with DNA when acting in concert with other PS [114]. The first reported synthesis of MB was in 1876 [112] and today, the dye and its analogues are usually prepared via oxidative cyclisations of p-phenylenediaminethiosulphonic acids and aniline
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Phenothiazinium
R1
R2
R3
R4
R7
R8
R9
MB
H
H
NMe2
H
NMe2
H
H
AA
H
H
NH2
H
NMe2
H
H
AB
H
H
NHMe
H
NMe2
H
H
AC
H
H
NH2
H
NHMe
H
H
Th
H
H
NH2
H
NH2
H
H
TBO
H
Me
NH2
H
NMe2
H
H
NMB
H
Me
NHEt
H
NHEt
Me
H
DMMB
Me
H
NMe2
H
NMe2
H
Me
27
Fig. (3). The chemical structure of phenothiaziniums Fig. (3) was adapted from [118] and shows the general structure of major phenothiaziniums. Given in the table are the specific peripheral moieties for: methylene blue (MB), azure A (AA), azure B (AB), azure C (AC), thionin (Th), toluidine blue O (TBO), new methylene blue (NMB) and dimethyl methylene blue (DMMB) where Me = methyl and Et = ethyl.
derivatives or via the oxidative amination of 10H-phenothiazine. These derivatives are structurally based on the nature of substituted amino group in the aniline precursor and thus it is possible to design or ‘customize’ their characteristics by a suitable choice in starting material [115]. Such functionalisation usually involves varying the peripheral atoms and groups of the phenothiazinium ring system and can be used to optimise physical properties like lipophilicity (log P), which is able to affect both the distribution of PhBPs within target cells, and their mechanism of uptake by these cells [115, 116]. Indeed, a number of PhBPs that are taken up by cells are routinely used as systemic / vital stains in surgical procedures, also showing that these dyes have low cytotoxicity [11, 15, 17]. Taken together, these characteristics show that PhBPs have many of the properties associated with therapeutically acceptable PS and led to investigation of these dyes as PACT agents. PhBPS AND THEIR MICROBIAL TARGETS PhBPs have been shown to photo-inactivate a wide range of microbes [49, 50] including bacteria [40, 57, 117], viruses [40, 54, 118-120], fungi [40] and protozoa [121]. Earlier studies suggested that PhPBs targeted solely the genetic material of organisms but it is now known that these dyes use a number of sites of action although nucleic acids are recognised as major targets [24, 122-124], especially in the case of viruses [11, 40, 54, 118, 120].
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Photo-activated MB has been shown to induce 8-hydroxy-guanosine formation in RNA, either in the isolated polymer [125] or within the genome of phage Qβ [126, 127]. This base modification was shown to be important to the MB-mediated photo-inactivation of Qβ [128] although more recent work has indicated a major role for cross-linking between viral proteins and regions of double stranded Qβ RNA in this process [118, 126, 127]. MB has been shown to cause photo-damage to the envelope of human immunodeficiency virus type 1 (HIV-1) [129] whilst more recent work has shown the dye to photo-attack reverse transcriptase (RT), viral core proteins and viral RNA [130]. Loss of reverse transcriptase activity was suggested to play a major role in the MB-mediated photo-inactivation of HIV-1 [54] but recent studies have shown that this event significantly precedes the inactivation of RT activity [118]. Moreover, this latter work has also suggested that MB-mediated crosslinking of RT to HIV-1 RNA is unlikely to make a major contribution to photo-inactivation of the virus by the dye. To better understand the ability of MB to photo-inactivate HIV, direct comparison was made to the corresponding ability of TBO. The latter dye was significantly more effective than MB and it was suggested that this may be due in part to better penetration of the viral envelope by TBO [40]. PhBPs are able to induce photo-damage to DNA that can involve strand breakage both in viruses [118, 120, 131] and bacteria [132]. MB is known to cause strand breaks in E. coli DNA [133] and a series of PhBPs were recently shown to inflict high levels of photodamage on the DNA of Staphylococcus aureus that was consistent with multiple breaks in the polymer (Fig. 4). Nonetheless, base modification appears to be the major mechanism of photo-damage involved in the action of these dyes against DNA [120]. MB mediated type II attack on DNA is known to produce high levels of 8-oxodG [134, 135] and similar results have been observed for other PhBPs with the order of efficacy MB > AB > AA > TBO [136] although recent studies suggested that this guanosine derivative was not a major product when PhBPs were directed against phage DNA [132]. Within the PhBPs, the ability of MB to photo-damage viral DNA is the best characterised [118, 120]. In the case of simplex virus type 1 (HSV-1), MB treatment led to no significant photo-damage to the viral envelope and the virus was still able to penetrate host cells but intracellular replication was completely inhibited [137]. In contrast, when phage PM2 was photo-inactivated by MB, DNA taken from the phage was found to remain infective, implying an absence of damage to nucleic acids. However, binding of treated PM2 to host cells was unimpaired, suggesting that the site(s) of photo-damage used by MB may be an internal viral protein or lipid of the viral envelope [138]. PhBPs are well known to interact with the bacterial envelope whose negatively charged outer surface, either promotes the selective uptake of cationic PhBPs or functions as a site of photodynamic action per se [117, 139]. When directed against Gram-negative organisms, PhBPs show a correlation between their efficacy and both their lipophilicity [140] and levels of interaction with lipopolysaccharide (LPS), the major component of the outer membrane [141]. Several studies have suggested that these interactions might involve multimeric species of PhBPs [142] and structural changes in LPS [143], thereby reducing the barrier function of the molecule. Most recently, PhBPs were found to be highly photo-toxic to E. coli [113] but showed to significant photolysis of E. coli cells and no ability to induce photo-oxidative damage to the organism’s membrane lipid [116]. Based on these results, it was suggested that E. coli membranes were not photodynamic targets of PhBPs and that these dyes may be taken up by the organism to attack intracellular targets such as DNA [117]. It has been previously suggested that at the low pH of the bacterial membrane surface, PhBPs may adopt their neutral lipophilic quinonemine forms, thereby facilitating cellular uptake in a photodynamically inactive form with the possibility of subsequent
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intracellular regeneration of the cationic form of the parent dye [15, 17]. Nonetheless, other studies on E. coli strains have shown the photodynamic action of PhBPs to be directed
Fig. (4). The ability of PhBPs to photo-damage DNA Fig. (4) was adapted from [117] and shows S. aureus DNA, after incubation with photo-activated PhBPs under the conditions of a Comet assay. The results are shown are for (a) DMMB, (b) PYY, and (c) NR. The 'tails' observed indicate the presence of fragmented DNA, implying damage resulting from interaction with PhBPs. The control (d), with no PS present, shows no such 'tail'. Also shown are the corresponding PS structures.
against either the cell envelope [144] or both the cell envelope and the DNA of the organism [145]. In comparison to bacteria and viruses, studies on the action of PhBPs against eukaryotic microbes are relatively few. Earlier investigations have shown that Candida albicans is susceptible to the photodynamic action of TBO and MB, which appears to involve perforation of the cell wall and membrane with subsequent translocation to mitochondria and the induction of cell death via apoptosis [146]. MB and a number of other PhBPs have been shown to possess potent intrinsic toxicity to strains of Plasmodium falciparum [121, 147, 148], a protozoan parasite that multiplies in human erythrocytes and is an etiological agent of malaria [149]. The mechanisms underlying this toxicity are largely
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unclear [121, 147, 148] and it has been variously suggested that MB-mediated inactivation of the parasite may involve: DNA intercalation, food vacuole alkalinisation, inhibition of haem polymerization and perturbation of the parasite’s redox cycle through interference with processes catalysed by the glutathione system [121]. PhBPS AND THEIR CLINICAL APPLICATION AS ANTIMICROBIAL AGENTS In terms of clinical application, the safe use of MB in human therapy goes back almost a century and includes: oral administration as an antiseptic, disinfectant, and antidote for nitrate poisoning, and the treatment of ifosfamide-induced encephalopathy [150]. MB has also been successfully administered to humans in clinical trials as an anticancer agent [151, 152] but along with other PhBPs, the most investigated clinical applications of these dyes have been as PACT agents. PhBPs and Eukaryotic Microbes MB was the first synthetic antimicrobial compound produced and based on its inherent toxicity to P. falciparum, was used as an antimalarial drug in the early 1890s [112, 121]. In the late 1930s, MB was found to exhibit inherent toxicity to Trypanosoma cruzi, which is another protozoan parasite and the etiological agent of Chagas disease found primarily in Latin America [153]. Most recently, the application of MB as an antimalarial agent has been revived for the presence of the dye has been found to sensitise P. falciparum to the action of chloroquine. Previously, chloroquine has been used as a highly effective antimalarial drug but the appearance of resistant strains of the organism has necessitated the search for new antimalarial agents [154]. More recent studies have shown that P. falciparum exhibits no cross-resistance between MB and chlorquine and that in combination these compounds form a potential treatment for malaria, which could be used in endemic regions [155]. Most recently, an in vivo investigation found that MB exhibited activity against the rodent malaria parasites, Plasmodium berghei and R yoelii nigeriensis, which was much higher than that shown by chloroquine [156]. A further benefit from the use of MB may arise from its ability to prevent methemoglobinimia, a serious complication associated with malarial anaemia [155]. Accordingly, several groups have used MB as a lead compound and synthesised a range of novel PhBPs, which show the potential to act as potent antimalarial compounds [157]. MB has also shown clinical potential as an antifungal agent for treatment of oral candidiasis. This condition has become one of the most common manifestations of HIV infection and within the HIV-infected population, there is a high resistance to the azole antifungal agents normally administered [158]. In response, a recent study investigated the ability of MB to photoinactivate an azole-resistant strain of C. albicans in a SCID (severe combined immunodefieciency disease) murine model, which lacks T and B cells, along with natural killer cells [59]. These infected mice provided a mimick of AIDS related oral candidiasis in humans and it was found by this latter study that phototreatment using topical administration of the dye at therapeutically acceptable levels completely eradicated C. albicans from the murine oral cavity and thus, may be of use in treating oral candidiasis in immunodeficient human patients.
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PhBPs and Bacteria In view of the global problems caused by drug resistant bacteria, PhBPs have been extensively investigated as antibacterial agents [1, 40, 117]. The dyes show great potential in this capacity although that of MB is somewhat limited as bacterial enzymes are known to reduce the dye to an inactive leucobase [17]. PhBPs have been shown to be highly effective against a range of Gram-positive bacteria [113, 159, 160] at therapeutically acceptable levels [117]. Antibiotic resistant pathogens of this bacterial class have become an increasing problem over the last decade, particularly those of nosocomial origin [161, 162]. In response, PhBPs have been tested as clinical agents against these pathogens and were shown to be highly photo-toxic to epidemic strains of methicillin-resistant Staphylococcus aureus [163] and to vancomycin-resistant Enterococcus faecalis and Enterococcus faecium [164]. In these cases, PhBPs often exhibited an antibacterial an efficacy that was significantly greater than that of vancomycin, currently regarded as one of the last lines of defence in treatment regimes against these pathogens [165]. Given the urgent nature of the threat posed by these pathogens, it was proposed that PhBPs could find an immediate use as general microbial disinfectants within hospital environments [1, 40]. PhBPs have also been shown to be effective against a range of Gram-negative bacteria [113, 159, 160], including Helicobacter pylori, Haemophilius influenza, Pseudomonas aeruginosa, and Klebsiella pneumoniae [117, 140, 166]. Of particular clinical interest, photo-activated PhBPs have been shown to be effective against a number of pathogenic Gram-negative strains such as Yersina enterocolitica, which is a blood-borne organism and problematic to transfusion services [167] and E. coli O157:H7, which is a food-born pathogen with acquired resistance to many conventional antibiotics [159]. Moreover, the efficacy of PhBPs against Gramnegative organisms is comparable to that observed for Gram-positive bacteria [113, 160], behaviour that contrasts strongly with that of many other classes of PS, which generally show low efficacy against Gram-negative bacteria. This decreased efficacy is primarily due to the barrier function provided by the negatively charged outer membrane of these organisms, which contributes to the multi-drug resistance of many Gram-negative pathogens [1, 18]. A number of strategies to overcome this barrier have been investigated [117], including the use of ALA, described above, but the inherent ability of PhBPs to partition into membranes, coupled with their ability to target the negatively charged outer membrane [116] makes them attractive propositions to combat Gram-negative pathogens [117]. As an example, TBO was recently shown to be highly active against multi-drug resistant P. aeruginosa, which has become increasingly recognized as an emerging opportunistic pathogen of clinical relevance. The pathogen causes a variety of systemic infections, particularly in patients with severe burns and in cancer and AIDS patients who are immunosuppressed, with a fatality rate approaching 50 percent [168]. Photo-activated PhBPs clearly have the potential to act as broad spectrum antibacterial agents, which lends itself to a number of therapeutic applications. Recent dermatological studies have shown that MB is able to photo-inactivate a range of bacterial species, which were representative of those encountered on the skin in both health and disease states, including S. aureus, S. epidermis, Streptococcus pyogenes, Corneybacterium minutissimum and P. acnes [169]. The levels of MB used in these latter studies were at therapeutically acceptable levels and more recent in vitro studies have shown corresponding levels of the dye to exhibit insignificant cytotoxicity [170] and genotoxicity to keratinocytes [171]. It was suggested by these latter authors that MB-mediated PACT could be applied in vivo as an alternative and / or adjuvant to antibiotics and antiseptics for the treatment of microbe associated skin diseases, or to produce asepsis prior to surgery or other clinical procedures. In this latter capacity, TBO was found to be highly photo-active against biofilms of S.
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aureus and Staphylococcus epidermidis, which are the most common causes of osteomyelitis, endocarditis, and catheter- and orthopedic-implant-associated infections in hospitalized patients [172]. The broad-spectrum photo-bactericidal activity of PhBPs has also made them well
suited to the treatment of dental plaque-related diseases such as caries, gingivitis and periodonitis [53]. These diseases are amongst the most common bacterial infections found in humans and are strongly associated with a variety of Gram-positive pathogens such as Streptococcus sanguis and Gram-negative pathogens such as Porphyromonas gingivalis [173]. When colonising the oral cavity, these pathogens adhere to oral surfaces to form multi-species biofilms, which are generally highly refractive to conventional antibacterial agents [174]. However, in vitro studies have shown that these biofilms, and their individual component organisms, are readily susceptible to the photodynamic action of PhBPs [174, 175]. Moreover, ampicillin-resistant bacterial biofilms were broken down by the photodynamic action of NMB with this action including both photo-bactericidal activity and photo-damage to polysaccharides of the extracellular polymeric substance (EPS), which stabilise the biofilm matrix [176]. This dual ability to attack biofilms is not observed with conventional antibacterial agents and clearly gives an advantage to the use of PhBPs in killing bacteria that use this form of colonisation [53]. Photo-activated PhBPs and other such agents could thus be used to disinfect root canals, periodontal pockets, cavity preparations and sites of peri-implantitis [58]. A major step in this direction was taken when photo-activated TBO was recently used in vivo to kill P. gingivalis, a major causative agent of periodonitis, in the oral cavities of rats with no apparent effects on adjacent healthy tissue [177]. It has been suggested that PhBPs may find use in the treatment of other accessible localised bacterial infections, such as those associated with burn therapy [117, 143, 178]. Strongly, supporting this suggestion, recent in vivo studies showed a photoactivated cationic photosensitiser able to kill E. coli [179] and P. aeruginosa [180], which were infecting excisional wounds in mice. In each case, the PS was administered topically and the treated wounds healed normally with no apparent photo-damage to the host tissue. PhBPs and Viruses In the early 1960’s, PhBPs including MB, TBO and NR were tested with limited success as antiviral agents in the production of vaccines [181] and a decade later, NR was clinically tested for the treatment of herpes simplex virus (HSV) [182, 183]. However, these latter trials were terminated due to side-effects involving the transformation of healthy cells [184] although this has since been questioned [120]. Currently, the major antiviral use of PhBPs is the photo-decontamination of blood and blood fractions and thus their microbial targets may be in suspension (plasma), cell-associated or intracellular (platelets or RBCs). This makes targeting pathogens in blood fractions a complex task although it is simplified, by the fact that the primary site of action used by PhBPs is genetic material and red blood cells (RBCs), platelets and plasma do not contain viable nucleic acids [185]. MB is already used by a number of European transfusion services to photo-disinfect plasma [33, 41, 186, 187] and the dye shows particular efficacy for the photo-inactivation of enveloped viruses found within this blood fraction [188, 189], including HIV and the West Nile virus (WNV) [121, 190]. WNV is an RNA virus, which has become a significant global threat since the turn of the century [191, 192], and little is known about its mode of infection or propagation [193] although dissemination between humans via the transfusion of infected blood has been demonstrated [194]. In contrast, non-enveloped viruses have a more diverse spectrum of susceptibility to photo-activated MB with those such as the human B19 parvovirus showing low levels of resistance and those such as the poliovirus, porcine virus and hepatitis A virus showing high levels of resistance [187-189]. In response, the use of other PhBPs has been
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proposed, including: thionin (Th), AA, AB and AC, each of which have shown the potential for the photo-inactivation of plasma borne viruses [38, 195]. It is established that the MB mediated photo-disinfection of plasma can produce collateral damage, generally manifested as a reduced coagulation capacity through losses in clotting factors [196], but technical advances have minimised these effects to therapeutically acceptable levels [187]. It is also established that MB and other PhBPs show a significant ability to photo-inactivate viruses that infect blood fractions containing cellular components such as platelets and erythrocytes. However, the ability of PhBPs to photo-decontaminate these blood fractions is generally compromised by collateral damage arising from uptake of these dyes by platelets and erythrocytes [33, 38, 185, 186]. Studies on blood platelets showed that significant levels of collateral damage were induced in these bodies by the photodynamic action of Th, MB, TBO AA, AC and AB when measured in terms of hypotonic shock and platelet activation [197]. Nonetheless, the lowest levels of collateral damage and highest levels of virus photo-inactivation seen in these latter studies were those exhibited by Th, which led to further investigations into the potential of the dye to photodecontaminate these blood fractions. These investigations showed photo-activated Th, potentially able to inactivate viral contaminants of platelet concentrates, including enveloped and non-enveloped viruses, leucocytes and bacteria with studies ongoing [198]. Research into the photo-decontamination of blood fractions containing RBCs by PhBPs has been limited [33, 38, 185, 186]. Recently, in vitro studies showed MB to photoinactivate Dengue virus [199], which is an emerging pathogen, and transfusion-transmission of Dengue through RBCs has been reported [185]. MB mediated photo-treatment of RBCs was found to inactivate extracellular viruses but also to induce significant damage to the erythrocyte membrane, which was exacerbated by refrigerated blood storage. Moreover, under conditions found to kill extracellular viruses, the MB mediated photo-treatment of RBCs was generally ineffective against intracellular viruses and bacteria [200]. It was suggested that this ineffectiveness may arise from the inability of hydrophilic MB to transverse the membranes of these pathogens [112], which led to the testing of more hydrophobic PhBPs for the ability to photo-inactivate intracellular viruses in RBCs. Methyl violet (MV), which is structurally similar to MB but differs by virtue of its neutral charge status and higher lipophilicity [118] was investigated in this capacity [201]. These latter studies found that the neutral nature of MV allowed the dye to access to the interior of RBCs where it showed a strong ability to photo-inactivate the intracellular vesicular stromatitis virus, contrasting to MB, which was ineffective against the virus. However, the virucidal action of MV was inhibited in the presence of plasma, which appeared to be due to high levels of binding between plasma lipoprotein and the strongly lipophilic dye molecule [201]. DMMB is amongst the most strongly lipophilic of the PhBPs [115] with a greater affinity for nucleic acids than MB and was tested as a photo-virucidal agent in contaminated RBCs [189, 200, 202, 203]. Taken with other investigations [204, 205], these latter studies showed DMMB to photo-inactivate a range of RNA and DNA viruses, both intracellular and extracellular, enveloped and non-enveloped phages, and leukocytes more efficiently than MB, apparently without deleterious effects on RBCs [202, 203]. It was suggested that the dye may be of use as a photo-decontaminating agent for RBCs but later studies indicated that excessive levels of red cell membrane damage and haemolysis could be associated with DMMB and MV, questioning their potential as such agents [38, 200]. Recent studies have shown that when quinacrine, a planar tricyclic compound with structural similarities to DMMB, was used as a competitive inhibitor to limit PS binding to the membranes of RBCs, photo-induced haemolysis stemmed from DMMB both in free solution and membranebound [206]. Based on these studies, the use of additives to prevent colloidal-osmotic
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haemolysis of RBCs and the use of novel flexible dyes that function as PS only when bound to the erythrocyte membrane are two techniques that currently are under investigation for reducing damage to blood fraction of RBCs [200]. PhBPs as PACT Agents Featured in Patents Awareness to the full potential of PhBPs as PACT agents came towards the end of the last century, which led to the inclusion of these dyes in a number of patents related to blood decontamination over the period 1980 to 2000 (http://www.uspto.gov/patft/index.html). For example, one patent is directed to methods for reducing the level of active pathogenic contaminants, including viruses, bacteria and parasites, frequently found in whole blood and blood components, such as red blood cells, platelets and plasma. [207]. However, it is clear from the above discussion that there has been extensive research into the antiviral capabilities of PhBPs over the last five years primarily because viruses pose the major current threat as blood supply contaminants [185]. Over this same period, PhBPs have featured in a number of successfully obtained patents that relate to antiviral therapy [208] and blood disinfection [207]. These patents clearly recognise the potential of PhBPs as broad range PACT agents in that their target microbes are defined to include not only established and currently emerging viral pathogens but also those of bacterial and protozoan origins, which in some cases have not been tested for susceptibility to PhBPs. The importance of PhBPs as PACT agents is further reinforced by the fact that a patent for these dyes as biologically active molecules per se has been recently granted. This patent includes a number of roles for PhBPs but in relation to PACT, refers to the use of these dyes in the treatment and prevention of microbial infections and in photo-disinfection or photosterilisation [209]. Several other patent applications that feature PhBPs as PACT agents for decontaminating blood and body fluids are pending [210, 211]. Given the likely emergence and recurrence of blood borne pathogens [185] and the amenability of PhBPs to functionalisation [115], further applications for patents featuring PhBPs as PACT agents seems a distinct possibility. PSORALENS Psoralen (furanocoumarins) are now known as PACT agents but a scan of the US patents website (http://www.uspto.gov/patft/index.html) shows that a number of these compounds feature in patents that have been granted in relation to photophoresis, or extracorporeal chemotherapy. This form of light-based therapy constitutes a major use of psoralens and their derivatives and is primarily used in the treatment of T-cell lymphoma [212]. Essentially, the patient is dosed with the PS, which enters the white blood cell nuclei and intercalates with DNA. Extracorporeally, these white blood cells are then exposed to ultraviolet A light (UVA), which leads to the cross-linking of DNA and unwinding of the biopolymer during transcription. UVA light damages abnormal T-cells, rendering them more immunogenic and subsequent re-infusion of these altered T-cells into the patient causes an immunological reaction that targets T-cells carrying the same surface antigens [213, 214]. It was this strong affinity shown for nucleic acids that led to psoralens and their derivatives being developed as antimicrobial agents [17, 41, 185]. Psoralens are plant-derived PS and are aromatic tricyclic compounds, consisting of a furan ring fused to a coumarin moiety (Fig. 5). The molecules of these PS are thus planar in nature and taken with their general hydrophobicity, this facilitates intercalation with genetic
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material [215]. With subsequent illumination by UV light in the range 200–350 nm,
Fig. (5). Photochemical action of psoralens on nucleic acids Fig. (5) was adapted from [187] and shows the structure of 4’-aminomethyl-4,5’,8-trimethylpsoralen (AMT) but the structure of amotosalen hydrochloride (S-59) is unavailable. Also shown above are the possible adducts produced by the generalized interaction of an intercalated psoralen with nucleic acids.
psoralens are able to utilise type I and type II mechanisms and induce photo-oxidative damage at their site of action [41]. However, when excited in situ, these PS can also undergo [2+2] cycloaddition reactions with olefinic moieties in nucleotide bases such as cytosine. The formation of mono or bis adducts can occur, either of which damages the nucleic acids, causing cross-linking in the latter case (Fig. 5). It is well established that the formation of mono-adducts from the furan side of these PS derives exclusively from the 1 PS* state of the molecule whereas pyrone-adducts result predominantly from the 3PS* state [54, 215, 216]. This covalent modification of genetic material is an attractive mechanism of photo-toxicity in that the damage inflicted efficiently forms a replication block to the
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cellular repair systems of target cells [215]. Although cycloaddition with nucleic acids is the primary photo-reaction of psoralens, the furocoumarin nucleus of these PS will undergo this reaction with other olefinic moieties such as unsaturated fatty acids [217-219]. A number of studies have shown that illuminated psoralens and derivatives such as 8methoxypsoralen (8-MOP) can inactivate viruses and bacteria through nucleic acid crosslinking and base modification [220, 221]. Recently, novel derivatives of 8-MOP, such as 4’aminomethyl-4,5’,8-trimethylpsoralen (AMT) (Fig. 5) have been synthesized, which show a strong ability to photo-inactivate a range of pathogenic microbes [41]. AMT demonstrated a very high affinity for DNA, which promoted concerns for increased mutagenicity, but the PS proved to be a viable lead compound in the development of psoralens as antimicrobial agents [219-221]. This led to the production of amotosalen hydrochloride (S-59), for which the full structure has not been released but is described as an aminoalkylated psoralen derivative [222]. The PS was shown to intercalate with nucleic acids and when activated by UVA light, to form covalent adducts with pyrimidine bases in both DNA and RNA [223]. These adducts then form both interstrand or intrastrand cross-links within the genetic material, thereby blocking replication and transcription with cell death resulting. S-59 has undergone clinical trials as a blood decontaminant [222] and was found to photo-inactivate a broad range of microbes in blood fractions, including: enveloped single-stranded and double-stranded RNA and DNA viruses, and their non-enveloped counterparts [224-227], Gram-positive and Gram-negative bacteria [224, 228, 229] and the protozoan parasite T cruzi [230]. Clinical studies have demonstrated that S-59 is non-toxic and that treated plasma and platelets have acceptable functional characteristics. However, the PS was found to be ineffective for pathogen inactivation of RBCs due to light absorbance by haemoglobin and the viscosity of packed RBCs [231-236]. Based on its ability to intercalate with nucleic acids and low toxicity, S-59 has featured in a number of patents applied for over the last three years, which relate to use of the PS in either photophoresis to treat lymphomia (http://www.uspto.gov/patft/index.html) or as an antimicrobial agent in the decontamination of biological fluids such as blood and blood components [237]. Underlining the importance of psoralens as PACT agents, novel members of this PS class with have primary amino substitutions on the 3-, 4-, 5-, and 8-positions of the psoralen ring system have been recently patented per se along with their potential use as photo-decontaminants of blood and blood products [238]. ORGANORHODIUM COMPLEXES PS with a transition metal ion coordinated at the centre of their ring systems such as metalloporphyrins and metallophthalocyaines [25, 34] are well established as potential PACT agents [12, 41, 62, 74] and in this capacity, have featured in a number of patents (http://www.uspto.gov/patft/index.html). In general, these PS have high values of ! t , ! t and ! Δ when their central transition metal is diamagnetic such as Zn2+, but lower values when the metal is paramagnetic such as Cu2+ [25, 34]. Since the 1960s, there has been an increasing interest in the potential of inorganic transition metal complexes to function as PACT PS [239]. It has been shown that when irradiated with UV light, many of these metal complexes, particularly those based on ruthenium(II), are efficient producers of 1O2 with values of ! Δ comparable to MB [25, 240]. Moreover, when illuminated, many inorganic transition metal complexes show a strong nuclease activity and / or a high capability to form covalent bonds with nucleic acids, which led to the suggestion that these complexes could be used as nucleic acid probes and as agents for nucleic acid inactivation [241, 242]. Based on these observations, photo-activated transition metal complexes have been investigated as
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agents for viral inactivation and antitumour therapy, and in this respect, bisbipyridyl complexes of rhodium(III) showed therapeutic potential [239]. The photo-activated octahedral rhodium complex, cis-dichlorobis(1,10-phenanthroline) rhodium(III) chloride [cis-Rh(phen)2Cl2+; BISPHEN] (Fig. 6A) was found to have a low ! Δ of 0.087 [243] and a strong tendency to form covalent adducts with RNA and DNA, and cross-link the bases of both biopolymers [244]. The use of non-oxygen dependent pathways to target nucleic acids is a highly desirable mechanism of photo-toxicity in therapeutic scenarios where hypoxic conditions exist as most PS require the presence of oxygen for photodynamic action [25, 34]. Studies on photo-activated BISPHEN showed that incubation with calf thymus DNA, either aerobically or anaerobically, or with 2′-deoxyguanosine (dG) aerobically, led to the metal complex binding with high selectivity to the N1 position of dG [245, 246]. It was proposed that this binding involved an electron transfer mechanism in which the reaction is initiated by reduction of the metal complex excited state by dG [245, 247, 248]. However, the incubation of photo-activated BISPHEN with dG under anaerobic conditions showed the metal complex to preferentially bind to the N7 position of the base with electron transfer from dG to the excited complex apparently initiating a chain reaction [249]. The photoactivated metal complex has also been shown to exhibit toxicity to single stranded DNA, double stranded DNA and isolated infectious DNA from phages [250]. These latter studies indicated that BISPHEN may utilise some mechanism of photo-toxicity other than covalent bond formation whilst other investigations have suggested that the rhodium complex may use multiple mechanisms of nucleic acid photo-inactivation, including breaks in the strand of DNA [239, 251, 252]. There have been a number of attempts to improve the characteristics of BISPHEN as a therapeutically viable PS [239]. One potential limitation to the therapeutic use of BISPHEN is the occurrence of its λmax in the UV region. The photo-activated metal complex was shown to efficiently kill viruses in platelet suspensions but was restricted in its potential as a blood decontaminant due to negligible absorption at wavelengths within the therapeutic window of 600-900 nm [253]. In response, it was shown that the irradiation of BISPHEN with visible light in the presence of MB initiated a synergistic interaction between the two PS that led to that to the covalent binding of both dye and metal complex to DNA. Moreover, this synergistic action was found to operate only under anaerobic conditions, which suggested that the photo-activated BISPHEN / MB system may be of use in hypoxic environments [114]. More recently, it was found that BISPHEN could be photo-activated by light at the red end of the spectrum (λ > 520 nm) and covalently bind DNA in the absence of MB. This binding was inhibited by the presence of molecular oxygen, indicating that the excited state responsible for the photochemistry of the metal complex at longer wavelengths was different to that populated when excitation of the complex with UV radiation occurred [254]. Other limitations to the use of BISPHEN as a therapeutic agent have been the low hydrophobicity shown by the metal complex, thereby reducing its ability to cross cell membranes and attack DNA, and its low ground state association with the biopolymer. In response, a number of groups have attempted to prepare analogues of BISPHEN for which these characteristics have been improved. An octamethylated analogue of BISPHEN, cisdichlorobis (3,4,7,8-tetramethyl-1,10-phenanthroline) rhodium(III) chloride (OCTBP) was synthesised [255]. Similarly to BISPHEN, OCTBP formed covalent bonds with dG when the two were irradiated with UV light and showed some photo-toxicity to target cells when irradiated in their presence with light at the red end of the spectrum (λ > 500 nm). In contrast to BISPHEN, OCTBP was found to exhibit significant levels of hydrophobicity and was able to cross membranes for uptake by target cells. OCTBP was also found to show higher levels of photo-reactivity and ground state association with double stranded DNA
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than BISPHEN. In relation to the parent compound, OCTBP showed higher levels of to higher levels of photo-toxicity to target cells although there was some evidence that the analogue acted as a prodrug and after cell uptake, the metal complex was photolysed to cischloroaquoOCTBP, which was the active phototoxic agent [255].
(A)
(B)
Fig. (6). The chemical structures of organorhodium compounds Fig. (6) was adapted from [243] and shows the chemical structures of the octahedral organorhodium complexes (A) BISPHEN and (B) DPPZPHEN.
In another attempt to prepare analogues of BISPHEN with enhanced hydrophobicity, one of the phen ligands was replaced by the dipyridol[3,2a-2’,3’c]phenazine (dppz) moiety to give cis-Rh(dppz)(phen)Cl2+ (DPPZPHEN) [243] (Fig. 6B). Contrasting to BISPHEN, it was found that DPPZPHEN was taken up by a range of target cells and showed high levels of ground state association with DNA. The photoactivated analogue appeared to inactivate DNA using a dual mode of action, both covalently binding to the biopolymer and causing nicks or strand breakage. Of particular significance to PACT, DPPZPHEN was found able to directly target the genome of the Sinbis virus (SINV), which is an enveloped RNA virus and closely related to the Flaviviridae, know to be significant human pathogens. The analogue was able to penetrate the protein layers and the lipid bilayer of SINV and inflict extensive photo-damage on the genome of the pathogen. Moreover, although DPPZPHEN used multiple mechanisms of action to photo-attack nucleic acids, the complex showed a low ! Δ of 0.068 and these mechanisms were found to be independent of the presence of oxygen, thus eliminating the involvement of ROS. It was observed that DPPZPHEN and its analogues may be of use as a blood decontaminant for a major disadvantage of many PS currently used in this capacity is that the 1O2 generated by their photodynamic action can cause indiscriminate damage to blood components [243]. Based on these observations, a patent for the use of bisbipyridyl rhodium(III) compounds as microbial photo-decontaminants in blood and other bodily fluids was recently applied for [256]. CURRENT AND FUTURE DEVELOPMENTS In response to the urgent need for novel antimicrobial strategies [1, 18, 29], it is clear from this review that current developments in PACT offer great potential. The induction of endogenous PS by ALA and its derivatives has broad range antibacterial potential and forms the basis of successful treatments for acne. Both PhBPs and psoralens are highly photo-
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active against a broad range of pathogens including bacteria viruses and parasites, which has led to clinical trials for these compounds as blood decontaminants with MB now successfully used in this capacity. PhBPs also show high potential as broad range antibacterial agents, with particular potential in combating Gram-negative pathogens, and as antimalarial compounds. Organorhodium complexes show efficacy as antiviral agents with DPPZPHEN exhibiting particular potential for use as an antimicrobial agent in hypoxic environments. For the future, each class of PS discussed have a demonstrated amenability to functionalisation, offering the opportunity to synthesis PACT agents with therapeutically desirable photochemical characteristics and physiochemical properties. This facility has already led to the production of PhBPs that can be considered as third generation PS, only functioning as photosensitising molecules when bound to the cell membrane [200]. Moreover, it seems likely that the design of third generation PACT PS will benefit from PDT research, which is currently generating a wide range of photosensitising molecules with novel structure / function relationships [34]. As a final comment, the four classes of PACT PS discussed here have all featured in patents relating to their antimicrobial function. It is perhaps self evident that successful pharmaceuticals, particularly those of a commercial nature, will lead to patents. What appears to be less obvious is the fact that patents are a rich source of information for they are rarely cited in the literature. Thus, in addition to highlighting the great potential of PACT, this review can also serve to draw attention to the existence of a greatly underused data resource. REFERENCES [1] [2] [3] [4] [5] [6] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17]
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Aznar JA, Molina R. Montoro JM. Factor VIII/von Willebrand factor complex in methylene blue–treated fresh plasma. Transfusion 1999; 39: 748-750. Mohr H, Bachmann B, Klein-Struckmeier A, Redecker-Klein A, Muller TH. Virus inactivation of blood products by phenothiazine dyes and light. Photochem Photobiol 1997; 65: 441-445. KleinStruckmeier A, Mohr H. Photodynamic virus inactivation of thrombocyte concentrates by phenothiazine dyes. Transfusionsmedizin 1997; 34: 43-47. Huang Q, Fu W-L, Chen B, Huang J-F, Zhang X, Xue Q. Inactivation of dengue virus by methylene blue / narrow bandwidth light system. J Photochem Photobiol B: Biol 2004; 77: 39-43. Wagner SJ, Skripchenko A. Investigation of photosensitizing dyes for pathogen reduction in red cell suspensions. Biotech Histochem 2003; 78: 171-177. Skripchenko A, Robinette D, Wagner SJ. Comparison of methylene blue and methylene violet for photoinactivation of intracellular and extracellular virus in red cell suspensions. Photochem Photobiol 1997; 65: 451-455. Wagner SJ, Skripchenko A, Robinette D, Mallory DA, Hirayama DA, Cincotta L. The use of dimethylmethylene blue for virus photoinactivation of red cell suspensions. In: Developments in Biologicals; Advances in transfusion safety. Brown F, Vyas GN, Eds. Basel, Karger, 2000; 102: 125-129. Skripchenko AA, Wagner SJ. Inactivation of WBCs in RBC suspensions by photoactive phenothiazine dyes: Comparison of dimethylmethylene blue and MB. Transfusion 2000; 40: 968-975. Wagner SJ, Skripchenko A, Pugh JC, Suchmann DB, Ijaz MK. Duck hepatitis B photoinactivation by dimethylmethylene blue in RBC suspensions. Transfusion 2001; 41: 1154-1158. Hirayama J, Wagner SJ, Gomez C, et al. Virus photoinactivation in stroma-free hemoglobin with methylene blue or 1,9-dimethylmethylene blue. Photochem Photobiol 2000; 71: 90-93. Wagner SJ, Skripchenko A, Thompson-Montgomery D. Use of a flexible thiopyrylium photosensitizer and competitive inhibitor for pathogen reduction of viruses and bacteria with retention of red cell storage properties. Photochem Photobiol 2002; 76: 514-517. Wagner, S.J., Cincotta, L.: US6030767 (2000). Floyd, R.A., Schinazi, R.F.: US6346529 (2002). Brown, S.B., O'Grady, C.C., Griffiths, J., Mellish, K.J., Vernon, D.I.: US7371744 (2008). Meserol, P., Acker, J., Prodell, R., Lenart, L., Schenck, R., Meserol, S.: US20040256329A1 (2004). Wagner, S.J., Skripchenko, A.: US20010046662A1 (2001). Oliven A, Shechter Y. Extracorporeal photopheresis: a review. Blood Rev 2001; 15: 103-108. Aubin F, Salard D, Pouthier F, Herve P, Humbert P. Extracorporeal photochemotherapy. Med Sci 1999; 15: 983-989. Edelson RL. Photopheresis: a clinically relevant immunobiologic response modifier. Ann N Y Acad Sci 1991; 30: 154-64. Bethea D, Fullmer B, Syed S, et al. Psoralen photobiology and photochemotherapy: 50 years of science and medicine. J Dermatol Sci 1999; 19: 278-288. Cimino GD, Gamper HB, Isaacs ST, Hearst JE. Psoralens as photoactive probes of nucleic acid structure and function: organic chemistry, photochemistry, and biochemistry. Ann Rev Biochem 1985; 54: 11511193. Zarebska Z, Waszkowska E, Caffieri S, Dall’Acqua F. PUVA (psoralen plus UVA) photochemotherapy: processes triggered in the cells. Farmaco 2000; 55: 515-520. Specht KG, Kittler L, Midden WR. A new biological target of furocoumarins: photochemical formation of covalent adducts with unsaturated fatty acids. Photochem Photobiol 1998; 47: 537-541. Caffieri S. Furocoumarin photolysis: chemical and biological aspects. Photochem Photobiol Sci 2002; 1: 149-157. Marley KA, Larson RA, Davenport R. Alternative mechanisms of psoralen phototoxicity. Light-activated pest control. ACS Symp Ser 1995; 616: 179-188. Corash L. New technologies for the inactivation of infectious pathogens in cellular blood components and the development of platelet substitutes. Best Prac Res Clin Haematol 2000; 13: 549-563. Pamphilon D. Viral inactivation of fresh frozen plasma. Br J Haematol 2000; 109: 680-693. Wollowitz S. Targeting DNA and RNA in pathogens: mode of action of amotosalen HCl. Transfus Med Hemother 2004; 31: 11-16. Lin L, Cook DN, Wiesehahn GP, et al. Photochemical inactivation of viruses and bacteria in platelet concentrates by use of a novel psoralen and long-wavelength ultraviolet light. Transfusion 1997; 37: 423435. Lin L. Inactivation of cytomegalovirus in platelet concentrates using Helinx technology. Semin Hematol 2001; 38: 27-33. Lin L, Alfonso R, Behrman B, et al. Photochemical treatment of platelet concentrates with a novel psoralen and UVA to enhance the safety of platelet transfusions. Infus Ther Transfus Med 1998; 25: 3948.
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Lin LL, Hanson CV, Alter HJ, et al. Inactivation of viruses in platelet concentrates by photochemical treatment with amotosalen and long-wavelength ultraviolet light. Transfusion 2005; 45: 580-590. Knutson F, Alfonso R, Dupuis K, et al. Photochemical inactivation of bacteria and HIV in buffy-coat– derived platelet concentrates under conditions that preserve in vitro platelet function. Vox Sang 2000; 78: 209-216. Lin L, Dikeman R, Molini B, et al. Photochemical treatment of platelet concentrates with amotosalen and UVA inactivates a broad spectrum of pathogenic bacteria. Transfusion 2004; 44: 1496-1504. Van Voorhis WC, Barrett LK, Eastman RT, et al. Trypanosoma cruzi inactivation in human platelet concentrates and plasma by a psoralen (amotosalen HCl) and long-wavelength UV. Antimicrob Agents Chemother 2003; 47: 475-479. Ciaravino V. Preclinical safety of a nucleic acid-targeted Helinx compound: a clinical perspective. Semin Hematol 2001; 38: 12-19. van Rhenen DJ, Vermeij J, Mayaudon V, et al. Functional characteristics of S-59 photochemically treated platelet concentrates derived from buffy coats. Vox Sang 2000; 79: 206-214. van Rhenen D, Gulliksson H, Cazenave JP, et al. Transfusion of pooled buffy coat platelet components prepared with photochemical pathogen inactivation treatment: The euroSPRITE trial. Blood 2003; 101: 2426-2433. Hambleton J, Wages D, Radu-Radulescu L, et al. Pharmacokinetic study of FFP photochemically treated with amotosalen (S-59) and UV light compared to FFP in healthy volunteers anticoagulated with warfarin. Transfusion 2002; 42: 1302-1307. McCullough J, Vesole DH, Benjamin RJ, et al. Therapeutic efficacy and safety of platelets treated with a photochemical process for pathogen inactivation: The SPRINT trial. Blood 2004; 104: 1534-1541. Murphy S, Snyder E, Cable R, et al. Transfusion of INTERCEPT platelets vs. reference platelets at doses ≥3 × 1011 results in comparable hemostasis and platelet and RBC transfusion requirements. Results of the SPRINT trial. Blood 2003; 102: 815a. Veome, E.A., Vermeiren, C., Fredericks, C.L., Jhonson, S.E., Rabe, I., Merkourioun, S., Berthiaume, K.: US20040088189A1 (2004). Wollowitz, S., Nerio, A.: US6455286 (2002). Loganathan D, Morrison H. ‘Photocisplatin’ reagents. Curr Opin Drug Dis Dev 2005; 8: 478-486. Tfouni E. Photochemical reactions of ammineruthenium(II) complexes. Coord Chem Rev 2000; 196: 281305. Hudson BP, Barton JK. Solution structure of a metallointercalator bound site-specifically to DNA. J Am Chem Soc 1998; 120: 6687-6888. Moucheron C, Kirsch-De Mesmaeker A, Kelly JM. Photoreactions of ruthenium (II) and osmium (II) complexes with deoxyribonucleic acid (DNA). J Photochem Photobiol B: Biol 1997; 40: 91-106. Menon EL, Perera R, Navarro M, et al. Phototoxicity against tumor cells and sindbis virus by an octahedral rhodium bisbipyridyl complex and evidence for the genome as a target in viral photoinactivation. Inorg Chem 2004; 43: 5373-5381. Morrison H, Harmon H. Hot Spots” Associated with the photoinduced binding of cis-dichloro bis(1,10 phenanthroline)rhodium(iii) chloride to hiv-1 and c-raf DNA. Photochem Photobiol 2000; 72: 731-738. Mahnken RE, Billadeau MA, Nikonowicz EP, Morrison H. Toward development of photo cis-platinum reagents. Reaction of cis-dichlorobis(1,10-phenanthroline)rhodium(III) with calf thymus DNA, nucleotides, and nucleosides. J Am Chem Soc 1992; 114: 9253-9265. Mahnken RE, Bina M, Deibel RM, Luebke K, Morrison H. Photochemically induced binding of Rh(phen)2Cl2+ to DNA. Photochem Photobiol 1989; 49: 519-520. Billadeau MA, Wood KV, Morrison H. Reductive photochemistry of cis-dichlorobis(1,10-phenanthroline) rhodium(III) chloride. Inorg Chem 1994; 33: 5780-5784. Harmon H. An investigation of the anaerobic photoinduced binding of cis-dichlorobis (1,10) phenanthroline rhodium (III) chloride to DNA and nucleosides. PhD dissertation Purdue University, West Lafayette, IN 1996. Harmon HL, Morrison H. Anaerobic photoinduced N7 binding of cis-dichlorobis(1,10phenanthroline)rhodium(III) chloride to 2′-deoxyguanosine: a one-electron-transfer chain process. Inorg Chem 1995; 34: 4937-4938. Mohammad T, Tessman I, Morrison H, Kennedy MA, Simmons SW. Photosensitized inactivation of infectious DNA by urocanic acid, indoleacrylic acid, and rhodium complexes. Photochem Photobiol 1994; 59: 189-196. Mohammad T, Chen C, Guo P, Morrison H. Photoinactivation of nucleic acids by cis-Rh(phen)2Cl2+ and cis-Rh(phen)(phi)Cl2+ involving multimodal mechanisms. Photochem Photobiol 1998; 67: 95S. Mahnken RE. An investigation of the photochemical and non-photochemical interactions of rhodium(III) polypridyl complexes with DNA. Ph.D dissertation. Purdue University, West Lafayette, IN 1991.
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Proteases and Kinases: Attractive Targets for Combating Infectious Diseases Mona Arabshahi1, Usha Bughani1, Surya N. Vangapandu1, Ritu Aneja1, Ramesh Chandra2, Daniel Kalman3 and Harish Joshi*,1 1
Department of Cell Biology, Emory University School of Medicine, Atlanta, GA, USA 2
Department of Chemistry and Dr. B. R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi, India
3
Department of Pathology, Emory University School of Medicine, Atlanta, GA, USA Abstract: Infectious diseases have haunted the human population for thousands of years. Although many breakthroughs have been made in the discovery of various treatments and cures for these diseases, multiple complexities enable them to continue to cause illness, disease and death. Evolution, for example, brings about mutations that cause the emergence of new pathogenic species. In addition, resistant species may emerge due to selective pressure of existing powerful antibiotics. The imminent possibility of new pathogenic strains forming and eventually threatening the human population verifies the urgent need for new, innovative strategies in fighting these pathogens. Here we review various important patents that have been licensed for drug development pertaining to protease and kinase inhibitors. Rather than being comprehensive, we have been selective in which patents to include. The goal is to inform the public at large of these new inventions in the pipeline and the status of development of these technologies into drugs to ultimately be used in a clinical setting.
Keywords: Patents, anti-infective agents, protease inhibitors, tyrosine kinase inhibitors, RAF kinase inhibitors, pharmaceutical industry. INTRODUCTION Infectious disease continues to pose as one of the most substantial dilemmas to threaten the global community throughout human history. It is important to realize that the current battle against the imminent threat of disease upon human life is far from reaching an end. Successes, such as the elimination of small pox and the impending elimination of polio, are few and far between. The catastrophic potential exhibited by infectious diseases has been attested by several instances; most notably by the 14th century Black Death pandemic and the 1918 influenza pandemic, both of which resulted in the deaths of at least 50 million people [1, 2]. The existence of further records depicting human suffering and death urges for a “comprehensive, global effort for the eradication, elimination, or control of infectious *
Corresponding author: Tel: +1 404 727 0435; Fax: +1 404 727 6256; E-mail:
[email protected] Atta-ur-Rahman / M. Iqbal Choudhary (Eds.) All rights reserved – © 2010 Bentham Science Publishers.
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disease” [3]. The growing level of interest expressed by the public in reducing the worldwide burden of disease has helped to facilitate many remarkable advances in medical research and treatment strategies during recent years. Despite the achievement of considerable progress in disease control afforded by improved sanitation, immunization, and antimicrobial therapy, infectious diseases continue to be a common and significant problem facing modern medicine. Infectious disease remains as one of the leading causes of death worldwide due to three reasons: (1) emergence of new infectious diseases, (2) re-emergence of old infectious diseases, and (3) persistence of intractable infectious diseases [4]. Progresses in diagnostic and detection techniques have led to numerous discoveries of previously unidentified pathogens that are now recognized as the causes of various pandemics observed in the past few decades (Table 1). Emerging diseases observed in recent and past years have Table 1. Significant Pathogens Recognized in the Past 30 Years Year
Microorganism
Type
Disease
1973
Rotavirus
Virus
Major cause of infantile diarrhea globally
1976
Cryptosporidium parvum
Bacterium
Acute and chronic diarrhea
1977
Ebola virus
Virus
Ebola hemorrhagic fever
1977
Leginoella pneumophilia
Bacterium
Legionnaires’ disease
1980
Human T-lymphotrophic virus
Virus
T-cell lymphoma/leukemia
1981
Toxin-producing Staphylococcus aureus Bacterium
Toxic shock syndrome
1982
Escherichia coli O157:H7
Bacterium
Lyme disease
1983
Human Immunodeficiency Virus (HIV)
Virus
Acquired Immuno-deficiency Syndrome (AIDS)
1983
Helicobacter pylori
Bacterium
Peptic ulcer
1989
Hepatitis C
Virus
Liver infection
1992
Vibrio cholerae O139
Bacterium
New strain associated with epidemic cholera
1993
Henta virus
Virus
Adult respiratory distress syndrome
1994
Cryptosporidium
Protozoa
Enteric disease
1995
Ehrlichiosi
Bacterium
Severe arthritis
1995
Human herpes virus-8
Virus
Associated with Kaposi sarcoma in AIDS patients
1996
nvCJD
Prion
New variant Creutzfeldt-Jakob disease
1997
Avian Influenza [Type A (H5N1)]
Virus
Influenza
1999
Nipah
Virus
Severe encephalitis
2003
SARS coronavirus
Virus
SARS – Severe Acute Respiratory Syndrome
2009
Influenza A (H1N1) Virus
Virus
Influenza
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demonstrated serious potential for devastating a vast portion of the human population. The current H1N1 influenza pandemic reveals the reality of this possibility. The emergence of new infectious diseases is influenced by several factors including rapidly changing human demographics, global travel, changes in land use patterns, and ecological, environmental, and technological changes. Social and environmental factors can also provoke the reemergence of previously existing diseases. Re-emergence of infectious disease is largely influenced by dynamic interactions between rapidly evolving pathogens and changes in the environment and host behavior [5]. The unstable nature of infectious agents enables them to rapidly evolve into new genetic variations. Human behavior can greatly influence the process of pathogenic evolution into new genetic strains. Increased use of antimicrobial drugs and pesticides, for example, has resulted in the development of pathogenic strains exhibiting drug resistance for former treatments that were successful in fighting against the disease. This allows diseases that were previously controlled through treatment innovations (e.g. tuberculosis, malaria, nosocomial and food-borne infections) to re-emerge as a threat to humanity once again. Resistance to antibiotics due to inappropriate prescribing habits of physicians or poor adherence by patients to treatment is a significant emerging public health issue. Additional threats of infectious disease on the civilian population result from the lack of adequate disease control in developing countries, as well as in the utilization of disease as a weapon in bioterrorism. Fig. (1) reveals the significant impact of infectious diseases on the number of annual deaths worldwide.
Fig. (1). Leading causes of death worldwide. Figures published by the World Health Organization (see http://www.who.int/whr/en).
The identification of specific microbes as the causative agents of a wide variety of infectious diseases has led to enormous progresses in the development of vaccines and antimicrobials for treatment therapies. Successful drug therapies for treating infectious disease will provide an invaluable resource in combating and controlling disease now and in the future, making drug development an extremely attractive area of research. Proteases and kinases represent two promising agents in combating infectious disease due to the extensive amount of knowledge available on the enzymatic mechanisms and structural repertoires of these enzymes. Thus, targeting proteases and kinases seems to be an easily amenable area of attack against infectious disease.
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This review highlights patents on promising new anti-infective agents. It describes the developments in protease inhibitors, tyrosine kinase inhibitors, raf kinase inhibitors and miscellaneous agents and their stage of clinical development as potential new medicines. Much of the information has been obtained through patent databases, literature search, and Clinical Trials Databases such as Pharmaprojects, Annual Reports in Medicinal Chemistry, recent conference abstracts, company websites and press releases. This review also describes our view on the future promise of these patents to mature into FDA approved drugs based upon the following patents on protease and kinase inhibitors. PROTEASE INHIBITORS Proteases, also known as peptidases are very important proteolytic enzymes, accounting for ~2% of the genes in humans and infectious organisms. They regulate most physiological processes significantly by controlling the activation, synthesis and turnover of all proteins. Consequently, they are pivotal regulators of conception, birth, growth, maturation, ageing, disease and death of afflicted humans. Genetic and environmental factors can disturb the balance of protease-catalyzed human physiology leading to abnormal development, poor health, and fatal disease. Proteases are also essential for replication/transmission of viruses, parasites and bacteria that cause infectious diseases in mammals. Thus, given the importance in both health and disease, protease inhibitors have already been developed into blockbuster drugs and diagnostics with many others in clinical trials (see Table 2 and 3).
Fig. (2). Schematic diagram showing substrate/inhibitor residues (P) and protease binding sites (S). Prime and non-prime designations distinguish C-versus N-sides respectively of cleavage site.
Proteases are categorized into five groups based on the catalytic residue present in the active site of the enzyme: aspartic, serine, cysteine, metallo and threonine. All proteases bind their substrates in a groove or cleft, where peptide bond hydrolysis occurs. Amino acid side chains of substrates occupy enzyme sub-sites in the groove, designated as S3, S2, S1, S1′, S2′, S3′, that bind to corresponding substrate/inhibitor residues P3, P2, P1, P1′, P2′, P3′ with respect to the cleavable peptide bond (Fig. 2) [6,7]. More than 1500 crystal structures of proteases are available the protein database (pdb) alone. The shape-compatibility to the peptide β-strand backbone conformation is of extreme importance in the recognition of proteases with their inhibitor ligands [8, 9]. This property should inspire the development of new approaches to β-strand mimics as protease inhibitors [10, 11]. Inhibitors of such proteases could potentially be useful in the treatment of diseases as diverse as cancer [1214], parasitic, fungal and viral infections (e.g. schistosomiasis [15,16], malaria [17,18], C. Albicans [19,20], HIV [21-23], Hepatitis [24,25], Herpes [26,27]), inflammatory, immunological, respiratory [28-31], cardiovascular [32] and neurodegenerative disorders like Alzheimer's disease [33,34]. Many protease inhibitors have displayed promising therapeutic activities in preclinical trials in animals and in early clinical trials in humans for
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viral and parasitic infections, cancer, inflammatory, immunological, and respiratory conditions, cardiovascular and degenerative disorders [35]. However, despite a great deal of research over the last two decades, there are relatively few protease inhibitors that have successfully progressed through clinical trials and are currently available as relatively safe and effective medicines for humans. They include angiotensin converting enzyme (ACE) inhibitors for treating high blood pressure, HIV-1 protease inhibitors for treating HIV/AIDS, thrombin inhibitors for treating stroke and an elastase inhibitor for treating systemic inflammatory response syndrome (SIRS). We further discuss below the recent and most significant patents out of seventeen new patents that have been granted as protease inhibitors. A) Aspartic Protease Inhibitors Various aspartic protease inhibitors that are in clinical development are shown in Table 2. Aspartic proteases tend to use two catalytic aspartic acid residues to catalyze the hydrolysis of polypeptide substrate, hexa-deca peptide segments of which normally bind within the active site of the protease [36, 37]. Most inhibitors have been derived from such substrate segments by first replacing the cleavable peptidic bond with a transition state isostere. Most aspartic proteases have one or more flaps that close down on top of the inhibitor, forming the active site with pockets or indentations on both sides of the catalytic residues. There are hundreds of crystal structures now deposited in the pdb database for inhibitor-bound and uncomplexed aspartic proteases including HIV-1, HIV-2 and related viral proteases SIV, FIV [38]. The most recent and significant patents in this area are discussed below. Weinstein and Weinstein [42] disclosed the invention that provides methods and compounds for treating and/or preventing HIV infection. It features the use of poxviruses such as vaccinia virus, which utilizes CCR5 chemokine receptor (CCR5, HIV-1 co-receptor) for entry into a cell, thus interferes with HIV-1 infection for therapy, prophylaxis and diagnosis, as well as for any other medical or veterinary use associated with HIV or homologous viruses. The invention also provides the use of poxviruses in the discovery of new agents to prevent and/or treat HIV infection. In particular, reverse transcriptase activity of PBMC cells from vaccinated vs. non-vaccinated subjects infected with the macrophage (CCR5) tropic HIV is provided [42]. In US 6841381, Robinson et al., [43] claimed a method of immunizing a vertebrate with DNA encoding a desired antigen or antigens resulting in their expression. This then elicits humoral or cell-mediated immune responses or both for protection against infectious agents and this provides an anti-tumor or contraception response. Arimilli et al., have disclosed preparation of phosphonate analogs of HIV protease inhibitors and methods for identifying anti-HIV therapeutic compounds [44]. The invention relates to phosphonate-substituted carbamates and cyclic ureas that inhibit reverse transcriptase activity and have improved intracellular half-life compared to analogs not having the phosphonate or phosphonate prodrug. Libraries of such compounds were screened using the novel enzyme GS-7340 ester hydrolase. In addition, extensive biological data regarding PBMC uptake and metabolism, serum stability and alkaline phosphatase protease inhibitor (ALPPI) activity of selected phosphonate-substituted prodrugs is also presented. For instance, a 9-step synthesis of compound 1 (Ki ≤10 pM for ALPPI activity) starting from N-tert-butoxycarbonyl-O-benzyl-L-tyrosine is provided, which involves multiple protection and deprotection along with coupling reactions using isobutyl amine,
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(3R,3aR,6aS)-hexahydrofuro[2,3-b]furan-2-yl-4-nitrophenyl hydroxymethylphosphonate.
Arabshahi et al.
carbonate,
and
dibenzyl
Table 2. Aspartic Protease Inhibitors in Clinical Development [2, 39-41] Drug Name
Company Name
Indication
Target
Clinical Status
Ritonavir, Ritonavir soft gel
Abbott
HIV/AIDS Infection
HIV-1 Protease
Launched
Lopinavir
Abbott
HIV/AIDS Infection
HIV-1 Protease
Launched
Nelfinavir Mesylate
Pfizer
HIV/AIDS Infection
HIV-1 Protease
Launched
Atazanavir Sulfate
Bristol-Myers Squibb
HIV/AIDS Infection
HIV-1 Protease
Launched
Saquinavir, Saquinavir soft gel
Hoffmann-La Roche
HIV/AIDS Infection
HIV-1 Protease
Launched
Crixivan
Merck & Co.
HIV/AIDS Infection
HIV-1 Protease
Launched
Fosamprenavir Calcium
GlaxoSmithKline
HIV/AIDS Infection
HIV-1 Protease
Launched
Tipranavir
Pfizer
HIV/AIDS Infection
HIV-1 Protease
Phase III
KNI-272
Japan Energy
HIV/AIDS Infection
HIV-1 Protease
Phase II (No Dev)
TMC-114
Johnson & Johnson
HIV/AIDS Infection
HIV-1 Protease
Phase II
SPI-256
Sequoia
HIV/AIDS Infection
HIV-1 Protease
Phase I
PL-100
Procyon BioPharma
HIV/AIDS Infection
HIV-1 Protease Preclinical (No Dev)
C Sixty
C Sixty
HIV/AIDS Infection
HIV-1 Protease
Preclinical
SM-309515
Sumitomo
HIV/AIDS Infection
HIV-1 Protease
Preclinical
GS-9005
Gilead Sciences
HIV/AIDS Infection
HIV-1 Protease
Preclinical
protease inhibitor
Zapaq
HIV/AIDS Infection
HIV-1 Protease
Preclinical
LY-450139
Eli Lilly
Alzheimer’s Disease
BACE
Phase II
TGCN-001
The Genetics Company
Alzheimer’s Disease
BACE
Preclinical
β-secretes inhibitor
Acetilon
Alzheimer’s Disease
BACE
Preclinical
β-secretase inhibitor
Astex Technology
Alzheimer’s Disease
BACE
Preclinical
β-secretase inhibitor
De Novo
Alzheimer’s Disease
BACE
Preclinical
β-secretase inhibitor
GlaxoSmithKline
Alzheimer’s Disease
BACE
Preclinical
β-secretase inhibitor
Locus
Alzheimer’s Disease
BACE
Preclinical
β-secretase inhibitor
NeoGenesis Pharma
Alzheimer’s Disease
BACE
Preclinical
β-secretase inhibitor
Sunesis
Alzheimer’s Disease
BACE
Preclinical
β-secretase inhibitor
Zapaq
Alzheimer’s Disease
BACE
Preclinical
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Fig. (3). Aspartic protease inhibitors.
Colonno et al. disclosed a method of treating HIV infection in atazanavir resistant patients using a combination of atazanavir and another protease inhibitor [45]. The resistance to atazanavir in the human is manifested by the existence of the signature mutation consisting of I50L mutation in the HIV protease. Flentge et al., have disclosed preparation of imidazolidine derivatives as HIV protease inhibitors [46]. They described preparation of compound 2 (Fig. 3) in 62% yield that has showed antiviral activity against Wild-Type HIV with EC50 in the range of 1 nM to 100 nM. Gillim-Ross et al., disclosed multiplex reverse transcription-PCR and susceptible cell lines for detecting severe acute respiratory syndrome (SARS) coronavirus [47]. The invention also provides screening of anti-SARS coronavirus agents and vaccines for reducing infection with plus-strand RNA viruses. A series of human cells derived from lung, kidney, liver, and intestine tested to be suitable for productive infection and replication of SARS-CoV, such as monkey kidney cells pRhMK and pCMK, Mv1Lu, and human HEK-293T and Huh-7 were disclosed. In contrast, cells permissive to other corona viruses, such as serogroup 1 and 2 corona viruses, are not susceptible to SARS-CoV, suggesting SARS-CoV binds an alternative receptor. Furthermore, protein inhibitors are required for these cell lines since transgenic cells expressing aminopeptidase N are not permissive to SARS-CoV [47]. Jeong et al., disclosed protease inhibitors for use in treatment of bone loss, excessive cartilage degradation and parasite infections [48]. This invention relates to substituted 8oxo-5,8,9,10,11,13-hexahydro-7H-[1,2]diazepino[1,2-b]phthalazine amides as protease inhibitors. These compounds may be used to treat osteoporosis, rheumatoid arthritis and infections with various parasites such as Plasmodium falciparum, Trypanosoma, Giardia lamblia, etc. Compound 3 (Fig. 3) is an inhibitor of cathepsin K (no data). Emini et al., disclosed therapeutic immunization of HIV-infected individuals with controlled viremia using adenoviral vectors [49]. It provides an improved method for eliciting a therapeutic immune response in an individual infected with HIV and the method comprises administering an adenoviral vaccine composition expressing an HIV antigen to an individual with controlled viremia. Immunization of infected individuals in this manner elicits a cell-mediated immune response against the virus that is significant in both the level and the breadth of the response. The therapeutic immune response that ensues is capable of effectively maintaining low titers of virus and thus offers the prospect of reducing individual dependency on antiviral therapy. Experiments in rhesus macaques indicate that adenovirusmediated immunization in infected individuals exhibiting controlled viremia, can provide high levels of both CD4-positive and CD8- positive T cell responses of a broad nature. Pfizer Inc. [50] disclosed a series of piperazine derivatives that have a therapeutic use in the treatment of HIV infection. The invention reported piperazine derivative 6 (Fig. 4) that was prepared via an amidation reaction of (2S)-2-(quinolin-5-yloxy)propionic acid sodium salt with (3R)-(3-methylpiperazin-1-yl)phenylmethanone using N-[(dimethylamino)(3H1,2,3-triazolo(4,5-b)pyridine-3-yloxy)methylene]-N-methylmethanaminium
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hexafluorophosphate (HATU) and Et3N in DMF under an N2 atmosphere for 14 h. These piperazine derivatives were tested for their inhibition of binding interaction of the CD4 binding site of gp120 of HIV-1 with CD4 cell surface receptor sites.
Fig. (4). Synthesis of piperazine derivative (6) as anti-HIV agent.
Vanderipe Donald [51] in his disclosure describes a method of use of the radioisotope Xenon-133 for the treatment of AIDS, other viral and non-viral infections. The Xe-133 is formulated in a carrier gas comprising of oxygen, xenon-131 and air or nitrogen. The treatment entails inhalation of a gas mixture containing the radioactive beta-particle emitting Xe-133 gas for providing superior targeting and destruction of the AIDS vector in the blood, lymph and body water when compared against external beam X-ray therapy. Maziasz Timothy [52] disclosed a patent demonstrating a method for the treatment of HIV infection and its associated diseases and disorders. More particularly, the invention provides a combination therapy for the treatment of HIV infection comprising the administration of an anti-human immunodeficiency virus agent in combination with a cyclooxygenase-2 selective inhibitor. Chiron Corporation [53] disclosed some novel polynucleotides that encode HIV Env polypeptides, e.g. gp120, gp160 and gp140. In particular, the disclosure relates to genetic or viral vectors containing sequences derived from HIV strain Botswana MJ4 encoding Env polypeptides. The disclosure also reveals compounds comprising these polynucleotides and methods of their usage. B) Serine Protease Inhibitors A well-accepted classification system for serine proteases is based on the nature of the P1 residue in their peptide substrates [54-57]. Three major classes are designated as ‘trypsin-like’ (positively charged residues Lys/Arg preferred at P1), ‘elastase-like’ (small hydrophobic residues Ala, Val at P1) or ‘chymotrypsin-like’ (large hydrophobic residues Phe/Tyr/Leu at P1) [58]. A catalytic triad of residues, Ser195, His57 and Asp102, (the numbering system has been adapted from chymotrypsin) is responsible for peptide bond hydrolysis. Various serine protease inhibitors that are in clinical development are shown in Table 3. Recently, Shapiro et al. [59] disclosed the invention of a serine protease inhibitor, α1-antitrypsin and thus a method for treating and preventing bacterial diseases. The invention provides compounds and methods for inhibition of Gram negative, Gram positive and acid fast bacilli in general and tuberculosis (TB), Mycobacterium avium complex (MAC) and anthrax, in particular. The invention relates to modulation of cellular activities including macrophage activity and the inhibitory compounds comprising naturally occurring and synthetic inhibitors of serine protease. TYROSINE KINASE INHIBITORS We discuss below the most significant patents out of thirty seven new patents that have been granted recently as kinase inhibitors. Jin et al., [62] disclosed in their WO 2005016966, the isoforms of receptor tyrosine kinases including intron fusion proteins and
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their pharmaceutical compounds. It also provided methods of identifying and preparing isoforms of cell surface receptors including receptor tyrosine kinases. WO 2005009973 Table 3. Serine Protease Inhibitors in Clinical Development [2, 60, 61] Drug Name
Company Name
Indication
Target
Clinical Status
Argatroban
Mitsubishi Pharma
Arterial Thrombosis
Thrombin
Launched
Venous Thrombosis
Thrombin
Phase III
Dabigatran/Etexilate Boehringer Ingelheim MCC-977
Mitsubishi Pharma
Thrombosis, general
Thrombin
Phase II
Flovagatran
PAION AG
Thrombosis, general
Thrombin
Phase II
SSR-182289
Sanofi-Aventis
Thrombosis, general
Thrombin
Phase I
AZD-0837
Astra Zeneca
Thrombosis, general
Thrombin
Phase II
E-5555
Eisai
Thrombosis, general
Thrombin
Phase II
LB-30870
LG Life Sciences
Venous Thrombosis
Thrombin
Preclinical
DX-9065a
Daiichi
Thrombosis, Angina
Factor Xa
Phase II
DPC-906
BMS
Venous thrombosis
Factor Xa
Phase II
CI-1031
Berlex Biosciences
Thrombosis
Factor Xa
Phase II
JTV-803
Japan Tobacco
Venous thrombosis
Factor Xa
Phase II
BILN-2061, Ciluprevir
Boehringer-Ingleheim
Hepatitis C Virus Infection
NS3-protease
Phase II
VX-950
Vertex
Hepatitis C Virus Infection
NS3-protease
Phase I
Sivelestat, Elaspol
Ono
SIRS, Inflammation,
Elastase
Launched (Japan)
Midesteine
Media Research
COPD
Elastase
Pre-registration (Italy)
AE-3763
Dainippon
COPD
Elastase
Pre-clinical
R-448
Roche
COPD
Elastase
Phase I
Nafamostat, FUT175
Japan Tobacco
Pancreatitis, Inflammation
Broad-Spectrum
Launched
Camostat mesilate
Ono
Pancreatitis
Broad-Spectrum
Launched
WX-UK1
Wilex
Cancer, Gastrointestinal
Urokinase
Phase II
NK-3201
Nippon Kayaku
Restenosis
Chymase
Preclinical
LAF-237
Novartis
Diabetes Type II
DPP IV
Phase III
MK-0431
Merck
Diabetes
DPP IV
Phase II
P32/98 (P3/01)
ProBiodrug
Diabetes
DPP IV
Phase I
T-6666
Tanabe Seiyaku
Diabetes
DPP IV
Phase I
NN-7201
Novo-Nordisk
Diabetes
DPP IV
Phase I
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disclosed the preparation of 5-membered heterocyclic compounds as p38 kinase including p38α and p38β kinase inhibitors [63]. For example, reaction of 3-amino-N-methoxy-4methylbenzamide with NaNO2 in the presence of SnCl2, and cyclization of the hydrazine with 2-(3-iodobenzoyl)-3-phenylaminoacrylonitrile to afford compound 9 (Fig. 5). Selected compounds of this series displayed IC50 values <1 µM in a p38α kinase inhibition assay. These compounds are useful for the treatment, prevention and amelioration of one or more symptoms of p38 kinase mediated diseases and disorders, e.g. inflammatory disease, autoimmune disease, etc.
Fig. (5). Synthesis of imidazole derivative (9) as p38 kinase inhibitor.
Pharmacia Corporation [64] disclosed β-carboline series of novel compounds and methods for inhibiting mitogen activated protein kinase-2. The method involves using a βcarboline MK-2 inhibiting compound 10 (Fig. 6) or its pharmaceutically acceptable salt. Over fifty synthetic examples describe preparation of representative compounds of βcarboline series. For example, a multi-step synthesis of 7-methoxy-3,4,5,10-tetrahydro-1H2,5-methanoaze-pino[3,4-b]indole-1-carboxylic acid hydrochloride (10), starting from 5methoxyindole, was given. These novel compounds are capable of inhibiting mitogen activated protein kinase-2, IC50 values were given for over 200 compounds and the most active compound of this series is 10 (Fig. 6). These compounds are useful in treating or preventing a TNFα mediated diseases (e.g. pain, inflammation). The invention also described kits that contain these pharmaceutical compounds. Lang et al., [65] disclosed preparation of pyrimidines and related compounds as P-38 kinase inhibitors. For example, cross coupling of 5-cyclopropylaminocarbonyl-2-methylboronic acid with N-(4methoxybenzyl)-4-amino-2-methyl-mercaptopyrimidine-5-carboxy-amide, prepared from ethyl 4-amino-2-mercaptopyrimidine-5-carboxylate in 3 steps, afforded compound 11 in 14 % yield. The IC50 value of compound 11 was <1 µM in p38α kinase inhibition assay. These compounds are claimed to be useful in the treatment of inflammatory disease, autoimmune disease, etc.
Fig. (6). Tyrosine kinase inhibitors [64,65].
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In another invention, Weiner and Muthumani [66] have disclosed methods of treating HIV infected individuals. Of the methods disclosed, some comprise administering an amount of (a) p38 inhibitor effective to inhibit FasL expression; (b) p38 inhibitor effective to inhibit HIV replication; (c) p38 inhibitor effective to inhibit HIV replication without inhibiting T cell activation; (d) one or more other anti-HIV compounds in combination with a p38 inhibitor. They also disclosed methods of identifying compounds that inhibit Nef mediated upregulation of FasL expression and p38 pathway. Axxima Pharmaceuticals have disclosed preparation of pyrazines as protein kinase, especially pUL-97 kinase inhibitors for treatment of infectious diseases, particularly human cytomegaloviral infections [67]. These compounds are useful in the prophylaxis and/or treatment of infectious diseases including opportunistic diseases, prion diseases, immunological diseases, autoimmune diseases, bipolar and clinical disorders, cardiovascular diseases, cell proliferative diseases, diabetes, inflammation, transplant rejections, erectile dysfunction, neurodegenerative diseases and diseases caused by human cytomegalovirus (HCMV). It also described preparation of compound 12 (Fig. 7) by monoacylation of 2,6dichloropyrazine with 1-(4-pyridinyl)piperazine and coupling of the chloride with (4aminocarbonylphenyl)boronic acid. It has an inhibitory effect on the protein kinase activity of various protein kinases such as pUL-97, EGFR, etc. It is a potent inhibitor of HCMV replication in cell cultures and in HFF cells (IC50 <3 µM). It did not show any or low toxicity up to concentrations of 10 µM in HFF cells. In another invention from Axxima Pharmaceuticals, Herget Thomas disclosed phenyl(quinazolinyl)amines as UL 97-kinase inhibitors for the prophylaxis and/or treatment of human cytomegaloviral and other herpesviral infections, including opportunistic infections, and diseases induced by human cytomegalovirus (HCMV, a highly specific β-herpesvirus) [68]. Choidas et al., [69] disclosed preparation of pharmaceutically active 4,6-disubstituted aminopyrimidine derivatives as modulators of protein kinases for use in the prophylaxis and/or treatment of infectious diseases, including opportunistic diseases, prion diseases, immunological diseases, autoimmune diseases, bipolar and clinical disorders, cardiovascular diseases, cell proliferative diseases, diabetes, inflammation, transplant rejections, erectile dysfunction, neurodegenerative diseases and stroke. The invention is also related to a medium comprising at least one of these compounds (e.g. compound 13, Fig. 7) in an immobilized form and its use for enriching, purifying and/or depleting nucleotide binding proteins. They have an inhibitory effect on the protein kinase activity of various protein kinases such as Abl, CDK1, CDK5, etc. and some of them have an inhibitory effect on CDK9 and CDK2 with IC50 values in the range of 1 to 1000 nM. They are potent inhibitors of HIV and HCMV replication in cell cultures, for example, 13 showed inhibition of HCMV replication in HFF cells.
Fig. (7). Protein kinase inhibitors.
Tinsley et al., have identified macrophage inflammatory protein-4 (MIP-4) as an endogenous ligand for chemokine (C-C motif) receptor-like 2 (CCRL2) and sequences of
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human MIP-4 and CCRL2 [70]. MIP-4 is also known as DC-CKI, CCL18 and PARC. AntiCCRL2 antibody was blocking MIP-4 and synovial fluid induced monocyte chemotaxis, whereas anti-MIP-4 antibody was also blocking RA synovial fluid induced monocyte chemotaxis. This demonstrates that MIP-4 is a major mediator of monocyte induced chemotaxis found in RA synovial fluid. CCRL2 modulators, such as antibodies against CCRL2 or MIP-4, are useful in treating an inflammatory disease or an infection. Burns et al., have disclosed preparation of pyrazine derivatives as kinase inhibitors. They prepared compound 14 by coupling of (6-chloro-pyrazin-2yl)-(1-benzyl)-amine with benzimidazole [71]. The activity of 14 and related compounds was evaluated to found an inhibition capacity of 50% or greater at a concentration of 20 µM and should prove useful in the treatment of diseases such as, but not limited to, rheumatic, viral and cardiovascular diseases. In another invention, they reported preparation of pyrazines and related compounds as tubulin inhibitors [72]. For example, Pd catalyzed coupling reaction of chloride derivative with 4-{[(ethylamino)carbonyl]amino}-3-methoxylphenylboronic acid pinacol diester afforded compound 15 (Fig. 8). In tubulin assays, compound 15 inhibited tubulin polymerization by greater than 50% at 50 µM and is claimed useful for the treatment of cancer, infection, etc. Kroemer et al., have disclosed identification of Puma, phosphorylated p53 and p38 MAP kinase as markers for HIV-1-induced apoptosis in circulating T cells of infected patient and drug screening, diagnostic and prognostic uses [73]. It relates to an in vitro method for the screening of anti-HIV compounds for predicting the progression of HIV related diseases or for determining the efficiency of an anti-HIV-1 treatment based on the expression level of Puma and on phosphorylated p53 and/or the p38 MAP kinase protein level in circulating white blood cells of a sample from a HIV-1 infected patient. It was shown that NF-κB and p53 are the dominant apoptosis-inducing transcription factors elicited by the HIV-1 envelope. They demonstrated essential role of p53 phosphorylation by p38 MAPK in apoptosis induction by the HIV-1 envelope. The invention further comprises a method and kits for the diagnosis of HIV infection in a patient. Hunter et al., have disclosed implantable sensors and pumps in combination with an anti-scarring agent (e.g., a cell cycle inhibitor) in order to inhibit scarring that may otherwise occur when the pumps and sensors are implanted within an animal [74]. For example, a drug device was coated with a heparin by dipped into a solution of heparin-benzalkonium chloride complex in isopropanol and airdried for use. Furthermore, Hunter et al., disclosed polymer compositions comprising an anti-fibrotic or an anti-infective agent [75] and soft tissue implants for use in cosmetic or reconstructive surgery and to compositions to make the implants resistant to growth by inflammatory scar tissue [76]. For instance, a silicone gel containing paclitaxel was used as a filling in breast implant. Schepartz et al., disclosed a method for grafting amino acid residues into avian pancreatic polypeptide (aPP) for construction of miniature proteins that exhibit specific protein binding [77]. The invention provides a protein scaffold such as aPP that can be modified by substitution of two or more amino acid residues on the α-helix domain of the polypeptide. Because of its small size and stability, aPP is an excellent protein scaffold for protein grafting of α-helical recognition epitopes. By grafting various combinations of residues on the solvent-exposed α-helical face or domain of aPP, which are essential to binding of GCN4 to the CRE half site, a series of polyproline helix-basic region molecules containing most or all of the DNA-contact residues of GCN4 and folding residues of aPP are generated. This procedure generated three positions (Tyr-27, Leu-28, and Val-30), where essential DNA-contact and aPP-folding residues occupied a single position on the
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helix. Miniature proteins are also designed whose DNA binding properties mimic those of the CCAAT/enhancer protein C/EBP-δ, or whose protein binding properties mimic those of protein kinase A or CREB transcription factor [77]. Veas et al., disclosed agonists of the formyl peptide receptors (FPR) family and formyl peptide receptors-like 1 (FPRL1) as biomarkers of resistance to HIV-infections in humans and their biological applications [78]. Despite being repeatedly exposed to HIV-1 certain individuals remain uninfected. To study the molecular mechanisms underlying the resistance to HIV infection, the inventors performed a comparative study of CD3/CD28activated T cells (to enhance signaling and gene expression) and plasma (to study their soluble proteins) from cohorts of HIV-1-exposed uninfected individuals (EU), their infected sexual partners and healthy controls (HC). The invention thus relates to the use as biomarkers of resistance to infections in humans of one or several agonists of the FPR family and FPRL1. First results of transcriptome analysis by serial analysis gene expression (SAGE) method showed that EU were found to overexpress Th1 cytokine interleukin-22 and SOCS-1 and HIV+ individuals underexpressed granzyme B compared to EU and HC. Power blot analysis of proteins from T cells of three cohorts showed the expression of STAT3 as well as the increase in a soluble protein molecular weight approximately 8.6 kDa. The results allowed the identification of a cascade of events starting with interleukin-22 that favor the innate host resistance to HIV infections characterizing EU. These biomarkers are useful in diagnostics, prophylaxis, and therapeutics. Schulze et al., disclosed preparation of thiazolidinones as polo like kinase (PLK) inhibitors [79]. For example, condensation of pyrrolidine-1-carboxylic acid (4aminophenyl)amide and thiazolidinone ethyl ether, afforded compound 16. In PLK-1 inhibition assays, eighteen compounds of this class exhibited IC50 values ranging from 233100 nM and are claimed to be useful as polo like kinase inhibitors. Luecking et al., disclosed preparation of pyrimidinylaminoarylsulfoximines as cyclin dependent kinase (CDK) and/or vascular endothelial growth factor (VEGF) inhibitors [80]. The compound 17 (Fig. 8) inhibited MCF7 cell proliferation with IC50 = 0.06 µM. Beck et al., disclosed preparation of imidazopyrazines as tyrosine kinase, particularly IGF-1R inhibitors [81]. For example, compound 18 (Fig. 8) was prepared by cyclocondensation of amide in methylene chloride in the presence of POCl3 and ammonolysis of the chloride in methanol. In an in vitro tyrosine kinase assay, these compounds showed efficacy and activity by inhibiting IGF-1R with IC50 <15 µM. and are useful for the treatment of cancer. Showell et al., [82] disclosed silylated oxazolylethenylthiazolamine derivatives as potential cyclin-dependent kinase inhibitors in therapy of cancer, alopecia, neurodegenerative disorders, viral and fungal infections (no data). The derivatives were prepared by Wittig-Horner olefination of 2-amino-5-thiazolecarboxaldehyde by 5-silylated 2-diethoxyphosphinyloxazole, followed by acylation or carbamoylation of the thiazole-2-amine group. Saturated 1,2-ethanediyl analogs were prepared by Pd/C hydrogenation of the 1,2-ethenediyl moiety. Bristol-Myers Squibb Company disclosed preparation of pyrimidine and pyridine derivatives useful as HMG-CoA reductase inhibitors [83]. For instance, compound 19 is prepared in 5 steps from a substituted pyrimidine, 2-methyl-2H-[1,2,4]triazol-3-ylamine and a homochiral dihydroxy acetonide derivative. These compounds are HMG-CoA reductase inhibitors and are active in inhibiting cholesterol biosynthesis, modulating blood serum lipids, for example, lowering LDL cholesterol and/or increasing HDL cholesterol and treating hyperlipidemia, dyslipidemia, hormone replacement therapy, hypercholesterolemia, hypertriglyceridemia and atherosclerosis as well as Alzheimer's disease and osteoporosis [no data]. Wilson et al., [84] disclosed methods and compositions for producing increased
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antigenic response using adenosine A1 receptor-activating agents in an amount sufficient to increase the antigenic response of the antigen-presenting cell to the antigen. The invention further provides methods, compositions, combination therapies, imaging techniques and diagnostic kits that may improve the diagnosis, prognosis and/or survival of cancer patients, pathogen-infected patients, and infectious or non-infectious immune-deficient patients.
Fig. (8). Protein kinase inhibitors.
Astrazeneca [85] disclosed preparation of thiophene compounds as CHK1 inhibitors. For example, amidation of compound 20 with dimethylaluminumbutyloxy carbonyl-(S)-3aminopiperidine, e.g., in-situ prepared by reaction of (S)-3-aminopiperidine-1-carboxylic acid tert-butyl ester with trimethylaluminum, followed by acidic deprotection afforded the title compounds. In CHK 1 (checkpoint kinase 1) inhibition assays, these compounds showed an IC50 value of 10 nM and are claimed useful for the treatment of cancer and infection. RAF KINASE INHIBITORS Merck [86] disclosed a preparation of malonamide derivatives as raf-kinase inhibitors (no biological data). For instance, malonamide derivative 21 (Fig. 9) was obtained via amidation of 3-[(4-chloro-3-trifluoro-methylphenyl)amino]-2-oxo-propionic acid by 4-(4pyridinyl-oxy)phenylamine with 57% yield. Merck also disclosed [87] a preparation of benzimidazole amides and are useful for the treatment of cancer. For instance, compound 22 (Fig. 9) is prepared from the corresponding 2-aminoimidazole and carboxylic acid (DMF,
Fig. (9). RAF kinase inhibitors.
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TBTU, HOBt, iPr2NEt). In another disclosure Merck claims a preparation of benzimidazolecarboxamide derivatives [88], for example, compound 23 (Fig. 10) was prepared via amidation of 5-chlorobenzimidazolecarboxylic acid by 4-(4-pyridinyloxy)phenylamine with 75% yield. The preferred compounds of the invention are raf-kinase inhibitors and showed IC50 values in the range of 100 µM or below. MISCELLANEOUS DRUGS OF OTHER CLASSES UNDER DEVELOPMENT Abbott Laboratories [89] disclosed in their patent WO 2005019191 a series of compounds having the formula 24 as hepatitis C (HCV) polymerase inhibitors. The disclosure also claims a composition and method for inhibiting HCV polymerase, processes for making the compounds and synthetic intermediates employed in the processes. Masayuki et al., [90] claimed a process for synthesis of phenylalanine derivatives and are prepared as HCV replication inhibitors for the treatment of viral infectious diseases, especially liver diseases attributable to HCV infection. For example, the compound 25 was prepared in a multi-step synthesis and found to inhibit replicon with IC50 value of 0.002 µM in cow. Mavromara and Niki [91] disclosed a novel form of core+1 protein of Hepatitis C virus (HCV), designated shorter form core+1 protein. The shorter form core+1 protein of Hepatitis C virus is the product of translation of a coding sequence consisting of all or part of a nucleotide sequence extending from nucleotide 598 to nucleotide 920 within the core+1 ORF of HCV. The invention also provides methods for detecting infection by Hepatitis C virus, methods of screening compounds that interact with viral propagation in HCV infected cells and their use for the preparation of drug candidates useful as anti-viral agents. Ph O
O R5
N
S
R6 R1
O
HO2C HO2C
Ph NH
R7 N
R8
HO2C O
N
OH
Me O
R2
N
O 24
25
Br
26
Fig. (10). HCV polymerase inhibitors.
Virochem Pharma Inc., [92] disclosed preparation of spiro compounds for the modulation of chemokine receptor activity. The compounds, for example 26 (Fig. 10), were prepared for their modulation of CCR5 chemokine receptor activity. A multi-step synthesis of compound 26 starting from tert-butyl 1-oxo-2,8-diaza-spiro[4,5]decane-8-carboxylate and 4bromobenzyl bromide, was given. These compounds have been found to have activity in binding to the CCR5 receptor, generally with IC50 values of <25 µM. Certain compounds of this series have also been tested in an assay for HIV activity and generally having an IC50 values of <1 µM. 3M Innovative Properties Company [93] disclosed methods of providing prophylaxis against an infectious agent using immune response modifier (IRM) compounds. In general, the methods include topically administering an immune response modifier to the respiratory tract of rats to reduce infection. Rats treated with IRM compound, N-{2-[4amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl]-1,1-dimethylethyl}methane sulfonamide, 4 hours or 24 hours before intranasal infection with humanized, non-lethal influenza virus showed reduced viral titers in nasal lavage fluid and whole lung homogenates.
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Me
Me OH
O
N
O H2N N
O
H N
N
NaOH OO
Me
O N S
27
S O
28
29
O
O
O
Fig. (11). Synthesis of tetrahydro-2H-thiopyran-4-carboxamide (29) as an anti-herpes virus agent.
Yamanouchi Pharmaceutical Co., disclosed preparation of tetrahydro-2H-thiopyran-4carboxamides [94] as anti-herpes virus agents. These novel compounds are found to be useful for the prophylaxis or therapeutic treatment of various diseases involving infections with viruses of the Herpesviridae family, specifically various herpes virus infections such as varicella (chicken pox) via varicella zoster virus, herpes labialis and herpes encephalitis via HSV-1 and genital herpes via HSV-2 infection. These compounds have potent anti-virus activity and are orally active. The compound 29 (Fig. 11) was prepared by treating ethyl {(2,6-dimethylphenyl)[(1,1,-dioxo-tetrahydro-2H-thiopyran-4-yl)carbonyl]amino} acetate (27) with NaOH solution followed by reacting the crude acid with [4-(1,3-oxazol-4yl)phenyl]amine (28) afforded compound 29 (Fig. 11) which showed inhibitory activity in HSV-1 infected nude mice model (93%) and EC50 of 0.075 µM in anti-VZV activity assay. Another class of genetic drugs based on the immense information following the completion of the Human Genome Project (HGP) provides a rich resource for the design of antisense drugs. Several antisense drugs are now in late stage clinical development (Fig. 12) and a key milestone for antisense therapeutics was the 1998 US FDA approval of Isis' first antisense drug Vitravene®. Vitravene® caters to the niche market of eye infections caused by HIV-induced cytomegalovirus and is marketed in the US and Europe. Antisense Pharma
Fig. (12). Antisense Therapeutics’ R&D Pipeline. Figures published by Antisense Therapeutics (see www.antisense.com.au).
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disclosed an antisense oligonucleotide for the inhibition of the expression and/or functional activity of melanoma inhibitory activity (MIA) protein and its use for the prevention or the treatment of neoplasms, infections and immunosuppressive disorders [95]. The disclosure also provided an antisense oligonucleotide selected from the group of the sequence 5'- TTG CAT AAA CCC AAG GAG - 3' and a fragment having at least eight nucleotides of the sequence 5'-TTG CAT AAA CCC AAG GAG - 3'. They show effective inhibition of the expression and/or function of MIA, thereby eliciting effective inhibition of tumor metastasis and for effective stimulation of immune system. Hybridon Inc., [96] disclosed optimized methods and compounds for enhancing the immune response caused by immunostimulatory compounds used for the treatment of cancer, autoimmune disorders, asthma, respiratory allergies, food allergies and infectious diseases. The optimized methods, according to the invention, provide synergy between the therapeutic effects of immunostimulatory oglionucleotides and immunogenic compounds and the therapeutic effect of cytokine immunotherapy and/or chemotherapeutic agents and/or radiation. CURRENT AND FUTURE DEVELOPMENTS There has been extensive research activity and resources targeted to the development of potent and selective protease inhibitors. Although many compounds have entered clinical trials over the past 5 years, most have not progressed as expected. Even those that have survived the rigors of clinical evaluation and entered the market, like inhibitors of HIV protease, thrombin, ACE and elastase are not without side effects that warrant a new generation of improved candidates. It is clear that potent inhibitors can now readily be obtained for most proteases given sufficient time and resources, but target selectivity is still a major problem. Some of the larger pharmaceutical companies have been building libraries of all known human proteases specifically to screen in vitro for non-selective actions of putative protease inhibitors. This may assist in improved selection of clinical candidates, although there is no real way of accurately predicting in vivo selectivity for proteases. Similarly selectivity is difficult to predict over other targets, the in vivo human side effects of ACE, HIV protease and thrombin inhibitors were not realized during their early stage development. Drug delivery is another serious concern. For example, although promising new predictive methods for ADMET properties are being developed, it remains practically very difficult to predict oral bioavailability and other pharmacokinetic and pharmacodynamic parameters in humans. There is a further issue of development of drug resistance in the face of a build up of substrate pressure, and selection of catalytically active mutant or other salvage proteases that do not have complementarity for carefully designed inhibitors of wild type proteases. Despite these drawbacks, a great deal has been learned about drug design and development. The future appears to still hold considerable promise for enzyme inhibitors. We can anticipate new, overexpressed proteases from genomic/biochemical comparisons made between normal/diseased cells, host/pathogen, healthy/unhealthy subjects leading to more effective and efficient validation of proteases as drug targets. New advances in structural biology (crystallography, NMR spectroscopy) will produce faster and more accurate inhibitor-protease structures providing automatic faster screening methods for selective inhibitors using robots and chip-arrays. Advances in solid phase organic chemistry are producing structurally more diverse chemical libraries for inhibitor screening, and faster and more reliable predictive in silico methods are enabling better inhibitor design with
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desirable ADMET properties. These advances, together with more careful attention to inhibitor conformation, mechanism of action, and drug-like composition are expected to result in more potent, more selective, more bioavailable inhibitors with a higher probability of success in the clinic. In our view, the most significant and promising drug candidates patented during last six months include HIV protease and RAF kinase inhibitors. SELECTED LIST OF WEBSITES RELATED TO INFECTIOUS DISEASES Centers for Disease Control and Prevention-Parasite Image Library http://www.dpd.cdc.gov/dpdx/HTML/Image_Library.htm Howard Hughes Medical Institute-Virtual Immunology Lab http://www.hhmi.org/grants/lectures/1996/vlab/ The National Institutes of Allergy and Infectious Diseases http://www.niaid.nih.gov/final/immun/immun.htm National Cancer Institute http://www.mfmdesign.com/NCI_WEBSITE/PATIENTS/ INFO_TEACHER/bookshelf/NIH_immune/ index.html Brown University--Development of Vaccines to Infectious Disease http://www.brown.edu/Courses/Bio_160/ World Health Organization-Disease Outbreak News http://www.who.int/emc/outbreak_news/ Centers for Disease Control and Prevention-Morbidity & Mortality Weekly Report http://www.cdc.gov/epo/mmwr/mmwr.html US State Department-Early Warning and Surveillance of Infectious Disease http://www.state.gov/www/global/oes/health/task_force/early.html Food and Drug Administration http://www.fda.gov/ National Institutes of Health http://www.nih.gov/ National Science and Technology Council-Task Force on Emerging Infectious Diseases http://www.state.gov/www/global/oes/health/task_force/index.html PubMed-National Library of Medicine/Medline http://www.ncbi.nlm.nih.gov/PubMed/ American Association for the Advancement of Science http://www.aaas.org/ American Public Health Association http://www.apha.org/ American Society of Tropical Medicine and Hygiene http://www.astmh.org/ Infectious Diseases Society of America
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UNAIDS-AIDS and HIV Resources http://www.us.unaids.org/highband/link.html World Health Organization Office of HIV/AIDS and Sexually Transmitted Diseases http://www.who.int/asd/home.htm
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Antibacterial Therapy in the Elderly Ayman M. Noreddin*,1 and Walid F. El-Khatib2 1
School of Pharmacy, Hampton University, Hampton Roads Bridge Tunnel, VA 23668, Hampton, USA and 2College of Pharmacy, University of Minnesota, 4-101 Hanson Hall 1925 Fourth Street South Minneapolis, MN 55455, USA Abstract: As our aged population increases, the unique spectrum of infections presented by this elderly population, particularly those residing in long term care facilities will challenge our ability to maintain an effective battery of antibiotics. The inability to clear drug from the body due to declining lung, kidney/bladder, gastrointestinal and circulatory efficiency can cause accumulation of standard antibiotic doses in the body. Accordingly, there is a heightened risk of reaching toxic drug levels as well as an increased chance of unfavorable interactions with other medications. On the other hand, we can predict problems that arise in treating elderly patients who may have a history of previous antibiotic treatment and exposure to resistant organisms from multiple hospitalizations. Furthermore, the elderly often acquire infections in tandem with other common disease states such as diabetes and heart disease. Thus, it is essential that optimized dosing strategies be designed specifically for this population using pharmacodynamic (PD) principles that take the unique circumstances of the elderly into account. Rational and effective dosing strategies based on pharmacodynamic breakpoints and detailed understanding of the pharmacokinetics of antibiotics in the elderly further the goal of achieving complete eradication of an infection in a timely manner. Specific PD information on isolates and drug susceptibility profiles as well as patient pharmacokinetic (PK) information along with a history of prior antibiotic treatment is imperative for the rational design of specific treatment for an infection in the elderly. Attention must be paid to the PK/PD of the chosen drug in order to ensure maximum bacterial eradication. In addition, this strategy also seeks to prevent the selection of drug resistant bacteria as well as the minimization of toxic effects in the elderly patient. For elderly patients, antibacterial agents with high tissue penetration, lack of interaction with many drugs commonly prescribed to the elderly and whose clearance is not affected by decline of kidney function, may be a preferred choice for the elderly population.
Keywords: Pharmacokinetics, Pharmacodynamics, Adverse Drug Reactions, Elderly, Aminoglycosides, β-Lactams, Macrolides, Glycopeptides, Fluoroquinolones, Oxazolidinones, Ketolides. SPECIAL CONSIDERATIONS IN THE TREATMENT OF INFECTED ELDERLY PATIENTS Elderly patients not only have a higher rate of hospitalizations but also longer hospital stays, often followed by entrance or return to a long term care facility [1]. This population is
Atta-ur-Rahman / M. Iqbal Choudhary (Eds.) All rights reserved – © 2010 Bentham Science Publishers.
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*Corresponding author: E-mail:
[email protected]
plagued with multiple chronic diseases combined with significant physical impairments. A German survey study found a median of 3 concomitant diseases among the 125 nursing home patients they studied, with cardiovascular disease and neurological disease/psychiatric disease the two most commonly occurring at 98 and 86% respectively, followed by diabetes at 42% [2]. Nosocomial infections are frequent among these elderly patients. They generally have a history of infection with drug resistant bacteria and often act as transmitters, carrying bacterial infections acquired in the hospital setting with them back to the long term care facility. Thus, outbreaks of infection with multi-drug resistant strains are common in long term care facilities. For any particular patient, every day spent in such an environment increases the risk of nosocomial infection and with it an increased risk of infection related death [1-4]. Symptoms of infection in the elderly may not include fever and chills but instead, may mimic normal signs of aging or symptoms of concomitant disease further complicating the decision to administer antibiotics to elderly in long term care facilities. Even communityacquired pneumonia in otherwise healthy elderly may present first as confusion, without obvious classical signs of respiratory infection. Conversely, patients may have fever without apparent infections [1, 3, 5-8]. Choice of treatment is often empirical, despite availability of information for more rational and precise choices. Whenever possible, particularly in the elderly population, pharmacodynamics of a drug should be assessed against the offending isolate and the information combined with pharmacokinetic information based on results of individual patient data; most importantly, creatinine clearance and the monitoring of free drug levels after initiation of treatment [9]. Publications discussing specific criteria for rational decision making when initiating antibiotic treatment, either in long term care facility patients or in community-acquired illness, are available and the reader is referred to the literature as this discussion is beyond the scope of this review [10-13]. PHARMACOKINETIC CONSIDERATIONS Normal physiological changes that occur with aging require attention when determining a course of antibiotic treatment. While there does not appear to be evidence for significant changes regarding antibiotic absorption in the elderly, there are significant changes in distribution and clearance of antibiotics. Pharmacokinetics of renally cleared antibiotics are altered due to changes in fat/muscle composition and declined renal function, as measured by decreased creatinine clearance rate. Elderly patients are therefore at risk for accumulation of antibiotics resulting in plasma free drug concentrations in excess of recommended levels, even at standard doses. While this may have the potential benefit of achieving therapeutic concentrations at a lower dose, there is a heightened risk of increased adverse drug reactions (ADR) [3, 5, 7, 9, 14-17]. Pharmacokinetic changes of antibiotics in the elderly are presented in Table 1.
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Table 1. Pharmacokinetic Changes of Antibiotics in the Elderly Physiologic Change
Result
Increased proportion of adipose tissue
Increased solubility of lipophilic drugs
Decrease in total body water and lean mass
Decreased solubility of water soluble drugs
PK Parameter Volume of Distribution
Edema Dilution of standard doses at infection site and in plasma
PK Effect
Refs.
Prolonged drug half-life Increased plasma concentration
[14] [9]
Standard dose is inadequate
[8]
Increased proteinuria or decreased albumin production due to chronic disease
Decreased plasma albumin
Volume of Distribution
Decrease in protein-bound drug fraction (inactive) and increase in free drug in plasma (active)
[17] [9] [18]
Physiological aging Liver disease
Decreased hepatic blood flow or decreased function
Drug Metabolism
Increased half-life of hepatically cleared drugs
[17]
Polypharmacy (concomitant disease)
Competition for P450 metabolism
Drug Metabolism
Inhibition of metabolism of competing drug-accumulation of unmetabolized form Enhanced metabolism of competing drug-increased drug activity Competition for albumin binding sites-accumulation of drug not preferentially bound to albumin
[5]
Reduced renal function: Physiological aging Renal disease
Decreased blood flow Decreased glomerular filtration rate
Renal drug elimination
Increased drug half-life, inability to remove drug from the plasma, accumulation of drug in the plasma
[19]
Dose adjustment for some drugs based on type of therapy
[20]
Renal Replacement Therapy
Increased drug removal
ADVERSE DRUG REACTIONS CONSIDERATIONS Antibiotics are a significant cause of ADRs in the elderly with as many as 10% of patients from geriatric units dying and up to 20% of readmissions to hospitals the result of ADR [17]. The two reactions of greatest concern are gastrointestinal effects and CNS effects. CNS related reactions are particularly troublesome because they are often indistinguishable from normal decline such as confusion and muscle weakness [3, 5, 9, 14]. Regarding elderly patients suffering from cardiovascular disease, chronic obstructive
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pulmonary disease (COPD), diabetes, cancer, orthopedic injury, autoimmune disease or other chronic diseases common in the elderly population, design of a successful antibiotic treatment regimen is more complicated due to the likelihood of Polypharmacy [1, 5, 14, 19]. . Of particular note, is the fact that a large proportion of drugs are metabolized via the cytochrome P450 enzyme pathway. There is limited capacity to metabolize multiple drugs by this pathway and many common antibiotics act as inhibitors of P450 enzymes contributing to the potential for a toxic combination [17, 14]. An example of antibiotic ADR with dire consequences is dysglycemia caused by gatifloxacin. Park-Wylie, et al., 2006 studied the medical records of 1.4 million elderly Canadian patients, over 3,000 of which were treated as outpatients with antibiotics and subsequently hospitalized for dysglycemia. Gatifloxacin was compared to moxifloxacin, levofloxacin and ciprofloxacin as well as macrolides and second generation cephalosporins regarding hospitalization of elderly patients, on antibiotics, for dysglycemia. Gatifloxacin is somewhat unique in causing opposing effects of both hypoglycemia and hyperglycemia. Elderly patients treated with gatifloxacin were four times more likely than controls to be hospitalized with hypoglycemia. Furthermore, patients hospitalized for hyperglycemia were 17 times more likely to have been treated with gatifloxacin than macrolides, with no evidence of hyperglycemia in any patient treated with the other fluoroquinolones or cephalosporins. This group recommended the use of alternate, and safer antibiotic choices for this population. However, original recommendations for gatifloxacin included reduction of dosage based on age and/or renal function particularly in elderly patients prone to disturbances in serum glucose levels. Ambrose et al., 2003 used clinical data and in vitro methods to predict the ability to attain bacterial killing with the reduced dosage in the elderly. However, it was unclear at that time whether the reduced dose would encourage production of bacterial resistance [21-23]. PHARMACODYNAMIC CONSIDERATIONS Pharmacodynamic parameters describe the relationship between serum concentration and the extent to which the drug is able to bind or interact with its specific bacterial target and cause cell growth inhibition or death as measured by MIC (minimum inhibitory concentration) [24, 25]. MIC measurements provide useful information on the inhibition or killing of a pathogen at a measured endpoint. However, they are static measurements that do not provide data on the time course of antimicrobial action such as the duration of drug exposure necessary for bacterial eradication, the rate of bactericidal activity or persistent effects of the antimicrobial agents [24, 26]. Antibiotics display two types of antibacterial activity, either concentration-dependent killing or time-dependent killing. The time-dependent group of antibiotics includes the βlactams as well as vancomycin and clindamycin. Bacterial killing assessed for antibiotics in the time-dependent group correlates poorly with the peak serum concentration and instead, the best predictor of clinical success is the amount of time during which the plasma antibiotic concentration exceeds the MIC for the organism. Since the primary parameter influencing clinical success is time at or over MIC, these agents are optimized by providing smaller, frequent doses or constant infusion [25, 27]. The most important parameter for antimicrobial agents that kill bacteria in a concentration-dependent manner is the concentration achieved in the patient plasma. The rate and extent of bactericidal activity of these drugs increases proportionately as the drug concentrations increase, even when the levels achieved are substantially above the MIC of the target organism. The aminoglycoside
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and quinolone drug classes are examples of antibiotics that display concentration-dependent killing [25, 27]. PHARMACOKINETICS/PHARMACODYNAMICS (PK/PD) PK/PD ratio predicts the therapeutic response of microorganisms to antimicrobials by correlating free drug (ƒ) exposure (area under the plasma concentration-time curve over 24 hours of dosing [ƒAUC24]- defined as achievable serum and tissue concentrations in patients) to measures of drug potency (MIC). Thus, the pharmacokinetic/pharmacodynamic parameters of interest in predicting clinical outcomes are the ƒCmax/MIC, the ƒAUC024/MIC (also sometimes referred to as the area under the inhibitory plasma concentrationtime curve [AUIC]), and the time above the MIC (T/MIC) [25, 27]. As an example, clinical data for fluoroquinolones therapy indicate that a peak serum concentration to MIC ratio of 10:1 or greater and AUC24/MIC ratios in the range of 100 to 125 maximize bacterial eradication and prevent resistance in critically ill patients with nosocomial lower respiratory infections caused by gram-negative bacilli (eg. P. aeruginosa) [28, 29]. However, in outpatients with community acquired respiratory infections such as acute exacerbations of chronic bronchitis and community acquired pneumonia caused by S. pneumoniae, animal, in-vitro and clinical data support AUC24/MIC of ≥25 as being predictive of bacterial eradication [30-33]. Optimizing dosage regiments using the PK/PD principles will be demonstrated in the following discussion of important drug classes considerations. DRUG CLASS CONSIDERATIONS Aminoglycosides Aminoglycosides such as gentamicin are one of a few drug classes that may still be administered intramuscularly in the elderly as absorptions of other drugs may be impaired due to the decreased muscle mass [9]. Aminoglycosides are concentration-dependent drugs and as such the Cmax/MIC and ƒAUC24/MIC ratios are the critical parameters [7]. Aminoglycosides have a prolonged post-antibiotic effect such that once daily dosing may be appropriate. While aminoglycosides may be appropriate for elderly patients with a variety of severe infections in the hospital setting where constant monitoring is available, they are generally not optimal for treatment of respiratory infections in the elderly. This is due to the combination of poor penetration into the lung tissue, high potential of nephrotoxicity and ototoxicity and a lack of evidence for benefit to this population over relatively safer and more effective drugs, such as the fluoroquinolones or macrolides, which also have prolonged post-antibiotic effect (PAE) but fewer ADR and good penetration into the lung [5, 7, 15, 34, 38]. When used, dosages should be reduced in patients with reduced creatinine clearance. However, in patients with edema, the loading dose may have to be increased [18]. Aminoglycosides may play a role in treatment of Pseudomonas aeruginosa infections when used in conjunction with β-lactams. However, this is dependent on local resistance profiles. A large survey study performed at the All India Institute of Medical Sciences in 2004 found that among hospitalized patients, aminoglycosides even in combination with β-lactams would most likely not be adequate treatment for soft tissue infections caused by the most common trio of bacteria: MRSA, E. coli and Pseudomonas spp [39]. β-Lactams
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β-lactams (penicillins, cephalosporins and carbapenems) are strictly time-dependent so that increasing dose more than 5 fold of MIC is ineffective. The critical PK/PD parameter for β-lactams is T/MIC such that trough levels must remain above MIC to prevent bacterial recovery and regrowth and to prevent the emergence of resistant strains [7, 18, 25]. Thus, dosing strategy for this class of drug requires many smaller doses per day-up to six. However, patients given this therapy may be better served by continuous infusion so that compliance is assured and the likely hood of dropping below MIC values is low. Continuous infusion in the elderly also avoids absorption problems due to muscle loss for IM dosages. The preference for continuous infusion of β-lactams is supported by some clinical studies that appear to be predictive of the need to increase the PD target for β-lactams over previous predictions by in vitro studies. In fact, time over MIC (T/MIC) must be 100% for the dosing interval in some cases to achieve successful eradication of infection, particularly against gram negative bacteria [36]. Macrolides The Macrolides have traditionally been considered concentration-independent agents, however recent data suggest that azithromycin may be concentration dependant. The critical PK/PD parameters for macrolides then are T>MIC and ƒAUC0-24/MIC [36, 40]. For example, to explain treatment success with azithromycin despite serum concentrations that do not achieve levels above the recommended MIC with standard dosing strategies, one must take into account azithromycin’s PK property of accumulating in phagocytic cells that move into the interstitial spaces of the tissue where the infection lies, so that the concentration of the drug in other tissues and body fluids, such as epithelial lining fluid, exceeds the plasma concentration [41]. Thus, for azithromycin, the concentration of drug in the epithelial lining fluid for patients with lung disease may be a better predictor of clinical outcome than serum concentrations [7, 41]. Azithromycin has the potential advantage of a single dose providing 5 days of antibiotic therapy directly at the infection site with typical side effects experienced with antibiotics and no adjustment necessary for renal impairment due to elimination primarily via feces [18]. Indeed for many antibiotics, the trend is moving toward studies involving increased dosage of antibiotic over shorter time periods designed to give maximal antibacterial effect and minimize exposure of the bacterial population to the drug, decreasing the chance for relapse due to appearance of endogenous resistant clones [42-44]. Glycopeptides Glycopeptides such as vancomycin are time-dependent antibiotics. Thus, as for βLactams, ideal treatment means continuous infusion to maintain T/MIC at the optimal levels. However, glycopeptides are nephrotoxic when infused and have poor penetration into the lung [35]. Although vancomycin is the standard choice for MRSA which can cause infections in the elderly both in lung and soft tissue, a high mortality rate in patients with ventilator associated pneumonias make this class overall a poor choice for treatment of respiratory infections in the elderly [7]. With intermediate and resistant MRSA emerging problems worldwide, newer broad spectrum antibiotics such as the glycylcycline tigecycline are recently approved alternatives. While tigecycline has the disadvantage of requiring intravenous infusion every 12 hours, it has superior tissue penetration with high levels achievable in Plymorph Nuclear Leucocytes (PMNs), significantly longer half life and does not require dose adjustment based on age or renal function [34, 45].
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Fluoroquinolones The fluoroquinolones levofloxacin, ciprofloxacin, gatifloxacin, moxifloxacin and gemifloxacin are among the first-line therapies for UTI and respiratory infections in the elderly regardless of etiology [6, 24, 46]. Some clinical studies indicate that these drugs are regarded as tolerated by the elderly population at least as well as non-fluoroquinolone therapy, with excellent therapeutic results when compared to other drug regimens [47]. Quinolones, overall, are considered to be a concentration-dependent drug class and the ƒAUC0-24/MIC and ƒCmax/MIC ratios are considered to be the major parameters predictive of bacteriological eradication and clinical efficacy. However, clinical trials appear to indicate that a high ƒCmax/MIC helps prevent selection of resistant bacterial strains, and thus may be the more important parameter if there is a significant risk of emergence of resistant subpopulations [7, 48-50]. Evaluation of optimal ƒAUC0-24/MIC in a patient population with nosocomial lower respiratory tract infections demonstrated a breakpoint for bacterial killing at approximately 100, significantly higher than the accepted 25-40 for typical gram -positive infections. While ƒAUC0-24/MIC ratios greater than 175 are associated with more rapid bacterial killing, more recently, in vitro studies as well as a study by Jumbe, et al. in the mouse thigh model conclude that for gram -negative bacteria, suppression of resistance as well as treatment success requires drug AUC0-24/MIC of 157 and 190 respectively [27,36, 51-53]. This parallel in vitro analyses indicating that ƒAUC0-24/MIC ratio can be significantly lower for gram-positive pathogens. While previous breakpoints of 30-40 for fluoroquinolones versus Streptococcus pneumoniae were considered adequate, new evidence indicates that may not be the case. An investigation into the activity of moxifloxacin against S. aureus and β-hemolytic streptococci reported optimal antibacterial effects with ƒAUC0-24/MIC ratios of 150-200 [27]. With rising S. pneumoniae MIC values worldwide, several groups have concluded that AUC0-24/MIC ratios in the range of 100 to 400 maximize bacterial eradication. More importantly, the new targets may prevent second step resistance development out of populations already containing the parC first step mutation [27, 36, 37]. Regarding fluoroquinolones, the clinician has two issues complicating the choice of optimal target ƒAUC0-24/MIC ratios for fluoroquinolones; (1) the longer-acting quinolones display comparable antimicrobial effects at much lower ratios due to their longer half-lives and (2) the variability within the class to penetrate tissues at the site of infection. The target ratio should thus be chosen based on MIC determined against several specific fluoroquinolones versus the offending isolate(s) and the potential for tissue penetration at the site of infections, as well as the probability of achieving that target in an elderly patient without reaching toxic levels [15, 38, 54, 55]. The fluoroquinolone class is a good example of a pharmacodynamic property referred to as post-antibiotic effect (PAE); defined as the continued suppression of an organism’s growth that persists after antimicrobial exposure. Most relevant to the elderly in long term care situations is that fluoroquinolones display prolonged PAEs for gram-negative bacilli. The PAEs for quinolones for both gram-positive and gram-negative isolates are generally in the range of 1.5-2.5 hours. Prolonged PAEs protect against bacterial regrowth during troughs when serum levels fall below the MIC value. Post-antibiotic leukocyte enhancement (PALE) is also important in the persistence of antimicrobial action as it describes the
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observed characteristic increase in the susceptibility of bacteria to leukocyte phagocytosis that occurs in the post-antibiotic phase. This added bacterial susceptibility to intracellular killing doubles the duration of the PAE of quinolones for gram-negative bacilli. These prolonged pharmacodynamic effects allow large, infrequent doses of fluoroquinolones. Thus, one high daily dose confers a rapid, high level attack on the bacterial population. The high AUC and Cmax levels provide optimal eradication times and more importantly, a decreased selection of resistant mutants [27]. In clinical practice for elderly patients, optimization by the achievement of high concentrations must be balanced with the risk of toxicity and potentially serious adverse drug effects. Most fluoroquinolones (e.g. ciprofloxacin and levofloxacin) are renally cleared and as such, the already long half-life of fluoroquinolones is considerably extended in elderly patients who may be assumed to have decreased renal function, such as those over 80 years of age or exhibiting decreased muscle mass. Although, dosages can be adjusted downward for the elderly, a creatinine clearance should be obtained if possible so that doses are optimized based on patient creatinine clearance, rather than directly as a matter of age [47]. Of the fluoroquinolones, moxifloxacin may be the preferred choice for elderly patients, particularly those with comorbidity, for the treatment of respiratory ailments. It is completely absorbed through the gastrointestinal tract and has multiple elimination pathways with only 20% or less of the drug eliminated in the urine unchanged [56]. The long elimination half-life of 11-15 hours after a single dose as well as good bioavailability allows once daily dosing for easier compliance. Furthermore, even critically ill patients on continuous renal replacement therapy do not require adjustment of moxifloxacin dosages in order to attain the accepted target ratio of 30-40 in plasma [57, 58]. Standard moxifloxacin dose of 400mg once daily provides higher penetration into infection sites such as epithelial lining fluid, vitreous and aqueous humor and alveolar macrophages without accumulation in the plasma of impaired patients as is observed for other fluoroquinolones such as levofloxacin [ 20, 27, 38, 47, 59]. Moxifloxacin is not metabolized by the cytochrome P450 pathway, as such there are no or very rare adverse reactions when moxifloxacin is combined with digoxin, warfarin, NSAIDS, glyburide, theophylline or probenecid. Similar plasma peak volumes, volume of distribution and attainment of target PK/PD with moxifloxacin between healthy young and elderly patients and elderly patients with comorbidity can be demonstrated using Monte Carlo simulations and in vitro studies [6062]. Furthermore, for cardiac patients, moxifloxacin has no greater risk of adverse effects on QTc interval than levofloxacin but with approximately three times the concentration of levofloxacin penetrating into the lung [63]. In a Spanish study, moxifloxacin was shown to provide high cure rates of 100%, 95.7% and 96.4% against Streptococcus pneumoniae isolates resistant to penicillin, macrolides and with multi-drug resistance, respectively [64]. and >90% cure in a U.S. study against all isolates, all with a comparable safety level to levofloxacin [65]. The primary caution for use of moxifloxacin in an elderly population, either recently exposed to antibiotics or in a long term care/hospital situation, is that while active against gram-negative and gram-positive isolates, including many drug resistant Streptococcus pneumoniae isolates and atypical organisms like mycoplasma, Pseudomonas aeruginosa, a frequent pathogen of these populations, may be unaffected by this drug [66]. Oxazolidinones
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Oxazolidinones are a new class of synthetic narrow spectrum drugs, represented by linezolid, the first marketed member. Antibacterial spectrum of linezolid includes Grampositive organisms, some Gram-negative anaerobic species, but not Gram-negative aerobes [67]. Linezolid is bacteriostatic against some species (enterococci) and bactericidal against others (pneumococci). Linezolid has almost 100% bioavailability and the AUC is identical after oral and intravenous administration [68]. Linezolid shows excellent penetration in lung tissue and epithelial lining fluid; moreover its pharmacokinetic properties are not affected by age. Since the drug has both renal and non renal elimination no dose adjustment is necessary in patients with mild-to-moderate renal function or liver disease [69, 70]. The most common adverse reactions to linezolid are gastrointestinal disturbances (nausea, diarrhoea), followed by headache and rash. The most serious adverse effect reported in phase III clinical trials is thrombocytopenia, being recorded in 2–4% of linezolid recipients [71, 72]. There is a need to well designed clinical studies aimed to study safety of linezolid in the elderly. Ketolides Ketolides has been developed with the goal of overcoming the problem of macrolideresistance with telithromycin as the first one to be approved by the FDA [73]. The pharmacokinetic profile reveals that telithromycin penetrates rapidly into lung tissues and fluids, reaching concentrations higher than that found in serum. The drug does not require dose reduction in elderly patients, including those with hepatic impairment and can be administered effectively once daily [74, 75]. Telithromycin related adverse events are mild or moderate in intensity. Ketolides could represent substitutes for macrolides in the empiric treatment of Lower Respiratory Tract Infections (LRTIs) in the elderly population, especially in areas with high rates of Grampositive isolates resistant to the macrolides. CONCLUSION Pharmacokinetic parameters of most antibacterial agents are altered when assessed in the elderly. Consequently, treatment outcome in the elderly patients can be influenced by decreased renal function and alteration in volume of distribution leading to treatment failure and/or increased risk of adverse drug reactions. Additional problems arise in treating elderly patients who may have a history of previous antibiotic treatment. These patients may already harbor resistant bacterial strains created from previous inadequate treatments. As with other infections of the elderly, antibiotic resistance is a major concern. Long term care facilities often harbor endogenous multi-drug resistant bacteria which can spread rapidly from patient to patient within such a facility. Furthermore, these patients often acquire infections in tandem with other common disease states such as heart disease and diabetes so that interference with multiple other treatments can cause changes in the availability of active antibacterials.
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For elderly patients, antibacterial agents with high tissue penetration, lack of interaction with many drugs commonly prescribed to the elderly and whose clearance is not affected by the decline of renal function, may be a preferred choice for the elderly population. Attention must be paid to the PK/PD of the chosen drug in order to ensure maximum bacterial eradication and to prevent development of resistant bacterial strains. Specific PD information on isolates and drug susceptibility profiles as well as patient PK information along with a history of prior antibiotic treatment is imperative for the rational design of specific treatment for an infection in the elderly. ACKNOWLEDGEMENTS The authors have no conflicts of interest relevant to the content of this review. REFERENCE [1] [2] [3] [4] [5] [6] [7] [8] [9] [10]
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SARS Coronavirus Anti-Infectives Tommy R. Tong* Department of Pathology, Montefiore Medical Center, 600 E 233 Street, Bronx, New York 10466, USA Abstract: Severe acute respiratory syndrome (SARS) emerged in late 2002 and was controlled in July 2003 by public health measures. Its causative agent, SARS coronavirus (SARS-CoV) jumped from an animal reservoir to humans and has the potential to re-emerge. Since then, the world has seen another virus that emerged in 2009, the pandemic influenza A (H1N1)v virus. Following the sequencing of the genetic code of SARS-CoV and the deciphering of some of the functions of its proteins, including the cellular receptors and host proteins that participate in the life cycle of the virus, promising lead drugs and new uses of old drugs have been discovered. Engineered monoclonal antibodies have surmounted the hurdle of provoking antibody enhancement as well as providing broad coverage against various SARS-CoV strains and mutants to prevent viral escape. Protease inhibitors are favored small molecule inhibitors because of possible broad spectrum coverage as well as the ability to be formulated for oral use. RNAi-based therapeutics produced impressive in vitro data and is rapid to develop. Interferon and chloroquine are likely to be effective as nonspecific antivirals with a good safety profile. The development of SARS-CoV anti-infectives is ongoing and will undoubtedly strengthen the infrastructure and know-how in the field of antiviral drug discovery.
Keywords: Severe acute respiratory syndrome, SARS, coronavirus, SARS-CoV, antiinfective, antiviral, main protease, 3CLpro, polymerase, helicase, interferon, interferoninducer, antibody. INTRODUCTION Severe acute respiratory syndrome (SARS) [1-5] is a novel viral pneumonia with 10% fatality rate caused by SARS coronavirus (SARS-CoV). The virus crossed the species barrier from animal reservoirs and became efficient in human-to-human transmission [6, 7]. The disease emerged in late 2002 and had spread to 29 countries within a few weeks, infecting ~8,000 people and causing some 800 fatalities. The disease was eliminated through exemplary international cooperation, preventing a potentially catastrophic pandemic [8]. In the few years since the epidemic, the molecular evolution [9] of the virus has been worked out and additional novel coronaviruses identified in humans and animals, greatly increasing our knowledge of the Coronaviridae and of zoonosis in general.
*Corresponding author: Tel: 1-718-920-9150; E-mail:
[email protected]
Atta-ur-Rahman / M. Iqbal Choudhary (Eds.) All rights reserved – © 2010 Bentham Science Publishers.
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The SARS-CoV genome (Fig. 1A) is among the largest in the world of RNA viruses (27-31 kb). It is single-stranded, sense (+), capped and methylated at the 5´ end, and polyadenylated at the 3´ end. It has 14 predicted open reading frames (ORF) encoding 28
Fig. (1). SARS-CoV genome. A 29-nucleotide stretch is deleted in the humanized strain. Compare with that of bat and civet strains at the bottom.
proteins [10-12]. Through a putative recombination event with an unidentified virus, SARSCoV acquired a receptor-binding domain (RBD) that binds specifically with the noncatalytic region of human angiotensin-converting enzyme 2 (ACE2). It also binds civet ACE2 with avidity, as well as DC-SIGN and DC-SIGNR [13-15]. After attaching to ACE2, a necessary and sufficient cellular receptor, SARS-CoV spike undergoes conformational change at the S2 domain that leads to fusion of its lipid envelope with the host cell membrane. The nucleocapsid enters the cytosolic compartment, where cellular translational machinery begins without delay to produce viral replicase enzymes that self-assemble after auto-proteolytic cleavage of the ORF1 gene product (Fig. 1B). Polyprotein (pp) 1a is translated from ORF1a. Pp1ab is encoded by an overlapping ORF1a and ORF1b, and involves a -1 ribosomal frameshift mechanism. Other ORFs and structural proteins are translated from a nested set of 3´-co-terminal subgenomic mRNAs. Spike (S), envelope (E), and membrane (M) proteins are targeted to intracellular membranes between the
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endoplasmic reticulum and Golgi apparatus. Replicated viral genomic RNA associate with nucleocapsid (N) proteins, which interact with M, triggering viral assembly. This is followed by budding into vesicles, which traffic to the cell surface, where mature virions are released. CLASSES OF SARS-COV ANTI-INFECTIVES Some 40 drugs against different viruses are now available, abolishing the notion that viral illnesses cannot be treated specifically. However, the challenge now is to be able to respond fast enough to emerging viral diseases as well as to reduce the cost of producing drugs that generally are virus specific and subject to viral escape. Viral binding, entry, transcription, replication, maturation, release, and cellular processes usurped by viruses represent possible therapeutic targets. The subject was recently reviewed by De Clercq [16]. Table 1 summarizes the various SAR-CoV anti-infectives discussed below. Table 1. Properties of Some SARS-CoV Anti-Infectives Mechanism
IC501 /EC502
CC503
SI4
Refs.
HR121
Heptad repeat (Entry inhibitor)
4.13 M
-
-
[46]
HR212
Heptad repeat (Entry inhibitor)
0.95 M
-
-
[46]
MDL28170
Cathepsin L inhibitor (Entry inhibitor)
2.5 nM
-
-
[60]
Chloroquine
Entry inhibitor
8.8 +/- 1.2 M
261.3 +/14.5 M
30
[63]
Chloroquine
Entry inhibitor
4.4 +/- 1.0 M
-
-
[64]
AG7088
3CLpro inhibitor
Not effective at 10 M
-
-
[76]
KZ7088
3CLpro inhibitor
-
-
-
[77, 78]
Ritonavir
3CLpro inhibitor
Not effective at 50 M
-
-
[76]
Saquinavir
3CLpro inhibitor
Not effective at 50 M
-
-
[76]
Lopinavir
3CLpro inhibitor
50 M
-
-
[76]
Lopinavir-like compounds 26-36
3CLpro inhibitor
23-40 M
-
-
[76]
Lopinavir
3CLpro inhibitor
1 g/ml (with ribavirn 6.25 g/ml) at viral inoculum of 50 TCID50 or below
-
-
[101]
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(Table 1) Contd….. Mechanism
IC501 /EC502
CC503
SI4
Refs.
MP576
3CLpro inhibitor
2.5 M
> 50 M
-
[81]
Ketoglutamine analogue 8c
3CLpro inhibitor
0.6 M
-
-
[82]
Cinanserin
3CLpro inhibitor
19 M
[83]
Anilides
3CLpro inhibitor
0.06 M
[85]
TL-3 derivatives
3CLpro inhibitor
Ki = 0.073 M
[86]
Nelfinavir
3CLpro inhibitor
0.0484 M
14.6 M
301.6
[106]
GRL0617
PLpro inhibitor
5 M
-
-
[96]
6-thioguanine
PLpro inhibitor
5.0 +/- 1.7 M
-
-
[99]
6-mercaptopurine
PLpro inhibitor
21.6 +/- 1.8 M
-
-
[99]
MAC-5576
3CLpro inhibitor
0.5 +/- 0.3 M
-
-
[80]
MAC-8120
3CLpro inhibitor
4.3 +/- 0.5 M
-
-
[80]
MAC-13985
3CLpro inhibitor
7 +/- 2 M
-
-
[80]
MAC-22272
3CLpro inhibitor
2.6 +/- 0.4 M
-
-
[80]
MAC-30731
3CLpro inhibitor
7 +/- 3 M
-
-
[80]
Tannic acid
3CLpro inhibitor
3 M
-
-
[94]
TF2B
3CLpro inhibitor
7 M
-
-
[94]
TF3
3CLpro inhibitor
<10 M
-
-
[94]
Hesperetin
3CLpro inhibitor
8.3 M
-
-
[95]
Ribavirin
Polymerase inhibitor
0.5-5 mg/ml
0.2-1 mg/ml
<1
[110]
-D-N4-hydroxycytidine
Polymerase inhibitor
5 M
50 M
10
[114]
Aurintricarboxylic acid
Polymerase inhibitor
0.2 mg/ml
37.5 mg/ml
187
[119]
Valinomycin
?
0.85 M
68 M
80
[76]
Reserpine
?
3.4 M
25 M
7.3
[76]
Reserpine derivatives (compounds 19-24)
?
<100 M
-
-
[76]
Aescin
?
6.0 M
15 M
2.5
[76]
Bananins
Helicase inhibitor
<10 M
>300 M
>30
[125]
HE602
Helicase inhibitor
6 M
>50 M
-
[81]
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(Table 1) Contd….. Mechanism
IC501 /EC502
CC503
SI4
Refs.
RNA aptmer ES15
Helicase inhibitor
1.2 nM
-
-
[126]
Glycyrrhizin derivatives (compounds 6, 6, 17 & 18)
?
<100 M
-
-
[76]
Niclosamide
Unknown
1-3 M
250 M
-
[129]
Glycyrrhizin
?
300 mg/l
>20,000 mg/l
>67
[130]
Calpain inhibitor VI
?
3 M (EC90) virus yield reduction assay
-
-
[115]
Calpain inhibitor III
?
15 M (EC90) virus yield reduction assay
-
-
[115]
1
- IC50 – Concentration of a drug that is required for 50% inhibition of viral replication in vitro. - EC50 – Plasma concentration required for obtaining 50% of the maximal effect in vivo. - CC50 - Cytotoxic concentration that reduced cell viability to 50%. 4 - SI (Selectivity index) = CC50/EC50. 2 3
I. VIRAL ENTRY INHIBITORS Prevention of viral entry into cells is a conceptually sound antiviral strategy because viruses are intracellular parasites. Even after a susceptible person is exposed to a virus, a medication that blocks viral entry continues to exert its effect in the multicellular organism, preventing infection of more of the host’s cells. The entry of SARS-CoV into its host cell involves binding to its cellular receptor ACE2, conformational change of S2 and membrane fusion, all of which are targets for therapy. Neutralizing Antibodies Against Spike (S) Prevent Binding to ACE2 Neutralizing convalescent or engineered antibodies have prophylactic and therapeutic potential in SARS-CoV infection [17-23]. MEDI-493 (palivizumab), a humanized monoclonal antibody, is administered prophylactically to prevent neonatal respiratory syncytial virus infections in high-risk children [24]. Within a year of the epidemic, several reports of human monoclonal antibodies against SARS-CoV became available [21, 25, 26]. CR3014 protected the SARS animal model ferret from macroscopic lung pathology [21]. This was also demonstrated in mice using 80R, an engineered IgG1 human monoclonal antibody [20]. These antibodies were derived from non-immune libraries of human single-chain variable region fragments (scFv) displayed on phages [21, 25]. Monoclonal human antibodies, including S3.1, m396 and S110 from SARS-immune patients, were engineered by another group, using CpG 2006 (a CpG oligonucleotide) as polyclonal B-cell activator to improve B-cell immortalization. Employing irradiated allogeneic mononuclear cells, Epstein-Barr virus, and CpG 2006, the authors interrogated the B-cell memory repertoire of an immune SARS patient. S3.1, a neutralizing antibody from one stable B-cell clone was shown to protect mice lungs from SARS-CoV challenge [26]. Although it potentiated infection by civet-SARS-CoV, a similar antibody, S110, did
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not. A total of thirty-five monoclonal neutralizing antibodies were isolated in this study. The drawback of the method is that convalescent patients are required. Human monoclonal antibodies against SARS-CoV have also been generated from transgenic mice (HuMAb-mouse®, XenoMouse®) [27, 28]. Those from XenoMouse belong to the IgG2 subclass, and are less susceptible to antibody-dependent enhancement of viral infection, owing to generally lower binding affinity with Fc receptors on macrophages and phagocytes. They are also more readily available in extracellular fluids and potentiate the alternative but not the classical complement pathway [27]. More than 20 monoclonal antibodies are approved by the FDA and being used in oncology and other diseases. These does not include dozens of antiviral immunoglobulin preparations already in the US market, in clinical trial and being developed throughout the world [29]. Their application in infectious diseases is certain to explode in the near future. The possible role of monoclonal antibody in SARS has been reviewed recently [22, 30]. Chimeric Protein that Neutralizes SARS-CoV Spike Protein The first step of viral entry is attachment of viral surface molecule with its cellular receptor. Soluble decoy receptors that saturate these viral molecules could be used to prevent viral binding to cells. Unmodified decoy receptors have been disappointing in HIV infection [31-33]. However, a chimeric protein CD4-IgG with multiple binding regions for HIV-1 gp41 is currently being tested in human subjects, e.g. ClinicalTrials.gov, identifier NCT 00000876 [34-36]. This chimeric molecule overcomes the problems of soluble CD4, such as low neutralizing activity, enhancement of viral infection, and short half-life in vivo. Jacobson et al. evaluated PRO 542 [37], a CD4-IgG2, in HIV-infected adults in a phase 1 study and reported reductions in plasma HIV RNA and plasma viremia with no dose-limiting toxicities [38]. In another phase 1/2 clinical trial in children with HIV-I infection, PRO 542 was again shown to be well-tolerated besides reducing the viral burden [39]. In a similar fashion, engineered multivalent soluble ACE2 (sACE2)-immunoglobulin might also be efficacious in neutralizing SARS-CoV [40]. sACE2 can conceivably be improved by using residues 90-93 of civet ACE2 [41]. Post-Binding Entry Inhibitors Certain carbohydrate-binding plant lectins prevent coronaviral entry at a poorly understood post-binding stage. They include Galanthus nivalis agglutinin, Hippeastrum hybrid agglutinin and Urtica dioica agglutinin and the non-peptidic mannose-binding antibiotic pradimicin A [42]. Mannose-binding lectin, a component of the innate immune system, proves to play a role in preventing SARS-CoV infection. Deficiency, usually constitutional, is associated with SARS [43]. Formulating these topical agents for systemic administration is being investigated. Membrane Fusion Inhibitors SARS-CoV shares a similar mechanism with HIV-1 in achieving membrane fusion between virus and host cell. Thus, heptad repeats (HR1 and HR2) located in the S2 domain of SARS-CoV spike protein, oligomerize to form a six-helix bundle after viral binding with ACE2. The conformational change approximates the viral lipid envelope and the cell membrane, leading to fusion and entry of the nucleocapsid. Spike protein heptad repeat-
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derived peptides have therefore been predicted [44] and recently shown to inhibit SARSCoV infection of Vero cells [45]. Further efforts resulted in stable recombinant proteins containing HR1 and HR2, having potent inhibitory activities (HR121 and HR212; IC50 values of 4.13 and 0.95 μM, respectively) on entry of the HIV/SARS pseudoviruses [46]. These proteins are also more economical to produce than synthetic peptides. However, they will need to be administered parenterally. In addition to the heptad repeat region, the fusion region of S2 [47] and other regions of S, such as an area involved in trimerization of S monomers [48], have also been successfully targeted. Cathepsin L Inhibitors Active Against Endosomal Entry Phase Cathepsins are host intracellular enzymes belonging to the papain family of cysteine proteases, of which there are over a dozen types. Most of them are activated by the low pH environments of lysosomes and endosomes, in which they function. Better known cathepsins include type A, required to stabilize sialidase and -galactosidase; type B, involved in activation of tissue plasminogen and cancer metastasis [49, 50]; type D, involved in mediating apoptosis [51]; type H, expressed in renal oncocytomas but not carcinomas [52]; type K, involved in degradation of type I collagen (mutated in congenital bone disorder) and cancer-induced osteolysis [53]; type L, required for degradation of li in cortical thymic epithelial cells but not marrow-derived antigen-presenting cells [54] and probably participating in malignant transformation [55]; type N, also with collagenolytic activity [56]; and type S, which is inducible by interferon (IFN)- in MHC-class II expressing cells and is pivotal in the maturation and peptide-binding competency of class II molecules [57]. After SARS-CoV binds to its receptor, endosomal cathepsin L cleaves the receptor binding subunit of S from the membrane-anchored fusion subunit [58]. This cellular protease is not however, required by human coronavirus NL63, which also utilizes ACE2 as receptor [59]. Specific cathepsin L inhibitors (CLI) have now been shown to prevent SARS-CoV infection in vitro [59, 60]. For example, MDL28170 (calpain inhibitor III) had an IC50 of 2.5 nM on substrate cleavage and efficient inhibition of SARS-CoV replication in vitro. Caution should be employed in using CLI because they have been shown to enhance HIV-1 and Leishmania infection in vitro and in laboratory mice, respectively [59, 61]. Chloroquine and Other Entry Inhibitors A potentially useful old drug is Chloroquine (Fig. 2), being tested for its anti-HIV effect in clinical trials [62]. It is a 9-aminoquinoline discovered by German chemist Hans Andersag in 1934 and used for the treatment of malaria, amebiasis, and autoimmune diseases such as rheumatoid arthritis. It has a high selectivity index of 30 against SARSCoV in in vitro studies [63]. At 10 μM concentration, achievable in vivo at dosages used for malaria prophylaxis and treatment, viral inhibition was total by immunofluorescence assay [64]. Chloroquine increases endosomal pH, which explains its similar efficacy as ammonium chloride, another lysosomotropic agent, when given up to 5 hours after infection of cell culture by SARS-CoV [63, 64]. However, it is also effective when given before viral inoculation onto cell culture, probably due to its interference with terminal glycosylation of ACE2 [63]. In addition, novel synthetic organometallic compounds closely mimicking
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hydroxychloroquine were found to have selective effect on SARS-CoV. The cytotoxic effects as expected, are less than the parent ferroquine compound [65]. Recently, chloroquine was shown to also inhibit human coronaviruses OC43 and 229E in vivo and in vitro, respectively [66, 67]. N N Cl HN
Fig. (2). Chloroquine (7-Chloro-4-(4-Diethylamino-1-Methyl-butylamino) Quinoline). This antimalarial probably has multiple actions that prevent SARS-CoV from entering cells.
Apart from repurposing old drugs, natural compounds offer another rich resource. In the case of SARS-CoV, Tetra-O-Galloyl-ß-D-Glucose (TGG), luteolin and emodin are a few examples [68, 69]. II. SARS-COV PROTEASES AS DRUG TARGET SARS-CoV 3CLpro is an attractive drug target because it is conserved and essential to the formation of a functional replication complex by cleaving 11 of the 14 sites in the ORF1 products, polyprotein 1a and 1ab [70-72]. With the availability of the SARS-CoV genome [10, 11, 73], a homology model of SARS-CoV chymotrypsin-like protease (main protease, also called 3CLpro) was constructed, providing a basis for the design of anti-SARS drugs. This model is based on the crystal structures of HCoV-229E main protease (MPRO) and TGEV MPRO in complex with AG7088 [74]. Functional conservation among the coronaviruses suggests that a drug with Gln (Ser,Ala,Gly) specificity ( denotes cleavage site) against SARS 3CLpro may also have activity against the other members [75]. This becomes highly relevant with the recent discovery of several SARS-CoV-like viruses in the wild [6, 7]. This functional conservation was demonstrated by structural homology studies [74]. These results led to the speculation that an anti-rhinoviral drug already in clinical trial, AG7088 (Fig. 3), might be useful against SARS-CoV. It was subsequently found not to O
O
O N
O
N
N
N O
O O
F
Fig. (3). AG7088. This molecule is being tested in clinical trials against rhinovirus. However, it has no in vitro activity against SARS-CoV at a concentration of 10 μM.
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have in vitro activity against the virus at a concentration of 10 μM [76]. However, its derivative KZ7088 (Fig. 4) interacts specifically with the active site of SARS-CoV 3CLpro through six hydrogen bonds [77]. Based on the atomic coordinates obtained by docking KZ7088 with the enzyme’s active site, a pharmacophore virtual screening narrowed down the list of compounds worthy of further experiments to 0.03% of the 3.6 million screened [78]. O
O
O N
O
N
N
N O
O O F
Fig. (4). KZ7088. A derivative of AG7088, KZ7088 interacts specifically with the active site of SARS-CoV 3CLpro.
Recently, proteomic technologies were employed to assist in peptide-based screening of 3CLpro substrate specificity [79]. This information will further assist in drug design. Moreover, this approach using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is readily adaptable to study the substrate specificity of other proteases in a high throughput manner. Compounds with Activity Against SARS Main Protease (3CLpro) Using a quenched fluorescence resonance energy transfer assay (Abz-peptide-Nitrotyrosine) and screening 50,000 drug-like small molecules, Canadian scientists discovered five new molecules with 3CLpro IC50 in the range of 0.5-7 μM (MAC-5576, -8120, -13985, -22272, -30731) [80]. Other competitive 3CLpro inhibitors identified by high-throughput screening include MP576 with IC50 of 2.5 M [81], ketoglutamine analogue with IC50 of 0.6 M [82], cinanserin with IC50 of 19 M for SARS-CoV and 34 M for HCoV-229E [83], hexachlorophene [84], anilides with IC50 of 0.06 M [85] and TL-3 derivatives with Ki of up to 0.073 M [86]. Recently, the non-competitive inhibitors, benzotriazole esters, intermediates in the synthesis of lopinavir, were found to have kinact (maximal rate of enzyme inactivation) of 0.0011 sec-1 and a KI (inhibitor concentration that supports half the maximal rate of inactivation) of 7.5 nM against 3CLpro [87]. Other non-competitive inhibitor leads include E64-d (epoxysuccinyl cysteine protease inhibitor derivative) [88, 89], N3 (a Michael acceptor) [75, 90], WRR 183 (alpha-betaEpoxyketone) [91], and halomethyl ketone inhibitors [92].
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Natural Substances with Activity Against SARS Main Protease (3CLpro) Theaflavins from black tea has activity against bovine rotavirus and coronavirus as shown by in vitro studies. The EC50 against coronavirus was 34.7 micrograms/ml [93]. OH OH OH
OH
TF2B
OH TF1
OH OH
O
HO
O
OH OH
O
O OH
O
HO
O OH OH
OH
O
OH
O
OH OH
OH
OH OH OH
OH
TF3 OH O
O
OH
O
HO
OH OH
O
O O
O OH
OH
OH
OH OH
Fig. (5). Chemical structures of TF1, TF2B and TF3.
Substances in black tea with activity against SARS-CoV were discovered as part of a large-scale screening process involving 720 natural products. These include simple and complex oxygen heterocycles, alkaloids, sequiterpenes, diterpenes, pentacyclic triterpenes, and sterols [94]. Assaying for inhibitory activity against 3CLpro proteolytic activity using HPLC, tannic acid and TF2B were identified as having IC50 at concentrations <10 μM. Additional experiments using well-known ingredients in tea resulted in the findings that the
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gallate group-containing TF2B and TF3 have more potent 3CLpro-inhibitory activity than TF1 (Fig. 5). Thus, tannic acid, TF2B and TF3 join the list of compounds that require evaluation for activity in cell culture. In another study, Isatis indigotica root extract, major compounds from I. indigotica root, and several plant-derived phenolic compounds were tested for anti-SARS-CoV 3CLpro inhibitory activity in in vitro assays and cell cultures [95]. Cleavage assays with 3CLpro demonstrated that IC50 values were in μM ranges for I. indigotica root extract, indigo, sinigrin, aloe emodin and hesperetin. Hesperetin (Fig. 6) dose-dependently inhibited cleavage activity of the 3CLpro, in which the IC50 was 8.3 μM in cell-based assay. Thus hesperetin needs to be further investigated. OH OCH3 OH
O
OH
O
Fig. (6). Hesperetin is a bioflavonoid. It is the aglycone of hesperidin, found in citrus fruits. Hesperetin dose-dependently inhibited cleavage activity of 3CLpro in cell-based assay.
Papain-Like Cysteine Protease Inhibitors SARS-CoV Papain-like cysteine protease (PLpro) is a Zn-ribbon-containing proteinase and conserved with the corresponding PL2pro of other coronaviruses. SARS-CoV does not have a homolog of PL1pro that is present in other family members. PL2pro probably took over the function of cleaving the N-terminal portions of pp1a and pp1ab [12, 96]. Comparative studies of coronaviral PLpro conservation suggested a link with substrate specificities, which SARS-CoV PLpro demonstrates. Thus PLpro recognizes the motif LXRR, which accounts for its deubiquitinating and de-ISGylating activities [97]. This narrow specificity for substrates is an Achilles’ heel that may be exploited for drug O N H
NH2
Fig. (7). GRL0617. PLpro inhibitor drug lead with EC50 of 15 μM.
development [96]. Indeed, a first generation of non-covalent inhibitors that bind to S4-S3 subsites is being developed, with the pharmacophore GRL0617 having EC50 of 15 M [98]. These inhibitors do not nonspecifically inhibit a variety of human deubiquitinating enzymes. Recently, the thiopurine analogues 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) were reported as potential lead compounds for PLpro inhibitors. Both are competitive and selective and have Kis values of 10-20 μM [99].
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Clinical Experience with Protease Inhibitors During the epidemic clinicians in Guangzhou observed that HIV-positive patients on HAART appear to be protected against SARS [100, 101]. In Hong Kong, the utility of lopinavir-ritonavir was investigated in a multi-center retrospective matched cohort study as initial and rescue therapy for SARS [102]. Patients who received this therapy as initial treatment for SARS had better outcome (reduced death and intubation rate) compared with an uncontrolled group, with lower rate of use of methylprednisolone at a lower mean dose. The results were similar to another report of a subset of those patients treated by the same senior researcher [103]. These clinical trials are in agreement with structural studies that predicted the utility of lopinavir, ritonavir, niclosamide and promazine against 3CL pro [104], with lopinavir and nelfinavir also showing in vitro activity [103, 105, 106]. III. SARS-COV POLYMERASE AS DRUG TARGET Widespread interest in coronaviruses is relatively recent. As a result, there is little experimental data on the characteristics of coronaviral RNA-dependent RNA polymerase (RdRp) and a consequent lack of inhibitors for this enzyme. The situation is very different for hepatitis B and C, HIV and herpes viruses, where polymerase inhibitors are very successful clinically. SARS-CoV RdRp, which is very important in the viral life cycle, is therefore high on the list of drug targets [107]. Ribavirin Ribavirin (1-(-D-Ribofuranosyl)-1H-1,2,4-triazole-3-carboxamide) was used extensively during the epidemic (Fig. 8). Derived from D-ribose, it is a long half-life purine nucleoside analog that interacts with viral RNA polymerases, as well as having other activities, such as inhibition of cellular inosinate (IMP) dehydrogenase [108]. In treatment of HCV, ribavirin is thought to act by inhibition of IMP dehydrogenase and by enhancement of Th1 activities [109]. O NH2 N N
OH O
N
HO OH
Fig. (8). Ribavirin. Ribavirin has low selectivity index (<1) against SARS-CoV.
For SARS-CoV however, at a low selectivity index (SI; SI=CC50/EC50) of <1 [110], coupled with an absence of demonstrable clinical benefit in uncontrolled series [111, 112], the role of ribavirin in treatment is in doubt. The side effects include teratogenicity and a dose-dependent but reversible hemolytic anemia [113]. The N-terminal domain of SARSCoV nsp14 is homologous to 3'-to-5' exonulcease (ExoN) and may perform RNA proofreading, repair and/or recombination [12]. This unusual capability among RNA viruses may be responsible for the failure of ribavirin in SARS-CoV therapy. Recently, data emerged that ribavirin and other IMP dehydrogenase inhibitors enhance lung infection in a BALB/c mice model [114]. The continued use of ribavirin alone is not recommended [112,
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113], unless perhaps in combination with an ExoN-inhibitor and supported by experimental results. Other Nucleotides, Nucleosides, and Nucleoside Analogs Despite the attractiveness of this group of drugs, -D-N4-hydroxycytidine (Fig. 9) is the only nucleoside analog among 26 tested that is selective and has an ED90 of 6 μM by virus yield-reduction assay [115]. Its CC50 and EC50 were 50 and 5 μM, respectively (SI=10). It was earlier found to have selective activity against hepatitis C virus [116-118]. NHOH N OH O
N
O
HO OH
Fig. (9). -D-N4-hydroxycytidine. This is the only member among 26 nucleoside analogs tested that has selective activity against SARS-CoV.
Other Drugs that Inhibit SARS-CoV Polymerase Aurintricarboxylic acid (ATA) is a general inhibitor of nucleases found recently to be more potent than IFN- against SARS-CoV [119]. Molecular docking studies suggest that it inhibits SARS-CoV RdRp by binding to a region in the palm domain (754-766), where two of the three catalytic residues (Asp 760, Asp 761) are located [120]. Besides being nonspecific, ATA also interferes with the interferon pathway of antiviral response [121]. IV. SARS-COV HELICASE AS DRUG TARGET This enzyme is another viral enzyme that is worth investigating for drug development. Earlier work has revealed that the viral enzyme unwinds DNA as well as RNA. This property facilitates the development of high-throughput DNA-based helicase assays, which will facilitate the search for inhibitors [96]. That the effort may be worthwhile can be seen in the success of helicase inhibitors currently being developed for herpes viruses [122, 123] and HCV [124]. Recently, several bananins (pyridoxal-conjugated trioxa-adamantanes), including iodobananin, bananin (Fig. 10), eubananin (Fig. 11) and vanillinbananin (Fig. 12) were found to non-competitively inhibit the ATPase activity of SARS-CoV helicase with IC50 values in the range of 0.5-3 μM [125]. In cell culture, bananin has an EC50 of <10 μM and a CC50 of >300 μM (SI of >30). Steric hindrance around the pyridoxal ring of some bananins (ansabananin [Fig. 13] and adenino-bananin) appears to explain why they have no antiSARS-CoV activity. Surprisingly, bananins may aggravate infection when given prophylactically. Other helicase inhibitors identified include HE602 with EC50 of 6 μM on plaque inhibition in Vero cells [81], and an RNA aptamer which inhibits DNA helicase activity by up to 85% with an IC50 of 1.2 nM [126].
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N
CH3
OH
OH O
O O
OH
OH OH
Fig. (10). Bananin. H N+
I
CH3
O-
OH O O
O O
OH
OH OH
Fig. (11). Eubananin. OH OCH3
O
O O
OH
OH OH
Fig. (12). Vanillinbananin. OCH3 O HO ONa5H2O ONa5H2O N
ONa5H2O H3C
N CH3 OH
O O
O O
OH
OH OH
Fig. (13). Ansabananin.
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V. OTHER SARS-COV TARGETS Other SARS-CoV enzymes and proteins have been targeted. Thus, nsp8 RdRp with primase activity was inhibited by 3-dGTP [127], and another human coronavirus, HCoV229E was inhibited by envelope protein inhibitor hexamethylene amiloride in in vitro studies [128]. VI. OTHER SARS-COV ANTI-INFECTIVES Calpain inhibitors have shown some promise, although one of them (calpain inhibitor III) appears to work primarily on cathepsin L rather than the purported main protease 3CLpro [60, 115]. Niclosamide Rational drug design and drug discovery by irrational chemical screening are common strategies but others adopted a strategy of screening old drugs for novel antiviral activities against SARS-CoV [104]. Such efforts resulted in the identification of niclosamide (2',5-dichloro-4'nitrosalicylanilide) (Fig. 14), an antihelminthic agent, as having potent activity [104, 129]. Vero E6 cells preincubated with drug at 10 μM concentration for 1 hour (also effective 3 hours after infection) and infected by SARS-CoV at an MOI (multiplicity of infection) of 0.1 were observed for protection against CPE (cytopathic effect). Immunofluorescent assay determined the EC50 to be between 1 and 3 μM, whereas the CC50 (cytotoxic concentration that reduced cell viability to 50%) was 250 μM (at 48 hours incubation). The mechanism of viral inhibition by Niclosamide is not dependent upon inhibition of entry or anti-3 CLPRO activity and remains to be determined. ON+
O
O
Cl N H OH
Cl
Fig. (14). Niclosamide. This antihelminthic agent has potent in vitro activity against SARS-CoV.
Glycyrrhizin Another such compound is glycyrrhizin [130] and its derivatives [76, 131]. Glycyrrhizin is (Fig. 15) a triterpenoid saponin found in Glycyrrhiza glabra (licorice). It stands out among the inosine monophosphate decarboxylase inhibitors (ribavirin and mycophenolic acid) and orotidine monophosphate decarboxylase inhibitors (6-azauridine and pyrazofurin) as having a high selectivity index (SI=CC50/EC50) of 67 against SARS-CoV. Its mechanisms of action are uncertain and may be due to its effects on protein kinase C (cellular signaling pathway), AP-1, NF-B (transcription factors), and upregulation of inducible nitrous oxide synthase and increased production of nitrous oxide by macrophages. Experimental support of the latter mode of action was provided by the induction of nitrous oxide synthase activity by glycyrrhizin and the fact that addition of nitrous oxide donor (DETA NONOate) inhibits viral replication in Vero E6 cells [130]. Moreover, its effect on lowering plasma membrane
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fluidity and hence impeding viral entry, is consistent with its observed broad antiviral activity [132]. Side effects include hypertension and hypokalemia in some patients after prolonged treatment. COOH
O
O O COOH OO
Fig. (15). Glycyrrhizin. Several studies demonstrated a high selectivity index of this agent against SARS-CoV. Its anti-SARS-CoV actions are likely to be multiple.
Valinomycin Valinomycin (Fig. 16), a dodecadepsipeptide (a macro-cyclic molecule made of twelve alternating amino acids) potassium transporter obtained from the cells of several Streptomyces strains, one of them S. Tsusimaensis, has EC50, based on ELISA, of 0.85 M (SI = 80). The mode of antiviral action is unclear [76]. In the same study, FP-21399, and some saponins have also being identified as being highly effective and worthy of further study. O
O N H
O
O O HN
O
O O
NH O
O NH O O
O
NH
H N
O O
O O
Fig. (16). Valinomycin.
O
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VII. INTERFERONS AND INTERFERON INDUCERS Interferons in SARS Like most metazoan viruses, SARS-CoV targets the interferon (IFN) signaling pathway [133-135], highlighting the evolutionary importance of IFN against viral infections. Not unexpectedly, evidences of IFN efficacy in vitro were readily established during the epidemic. The type-I IFNs (/) but not type-II INF () were found to inhibit SARS-CoV infection and replication [136]. Natural IFN- and IFN- have more potent in vitro activity than recombinant IFN- [105]. Alferon N Injection is the only approved natural, multi-species, alpha-interferon available in the US. In vitro studies demonstrated specific anti-SARS-CoV activity in Vero 76 cell culture. Alferon inhibited SARS-CoV at an EC50 of 5,696 +/- 1,703 (SEM) IU/ml (visual) and 10,740 +/- 5,161 (SEM) IU/ml (neutral red). Viral load reduction by one log10 was 78,000 +/- 22,000 (SEM) IU/ml [137]. The in vivo activity of interferons were confirmed in macaques, which were protected from SARS-CoV by prophylactic pegylated IFN- [138]. Postexposure treatment also produced measurable antiviral efficacy, supporting the rationale of employing IFN for prophylaxis or treatment of SARS. In humans, uncontrolled clinical trials have been reported [110, 139-143]. No double-blind randomized controlled trial has yet been conducted. Interferon Inducers Substances that induce IFN production by dendritic cells and peripheral blood mononuclear cells, such as CpG oligodeoxynucleotide (BW001) yielded supernatant that protected Vero cells from SARS-CoV infection [144]. Hemispherx biopharma recently filed a patent application [137], for treatment of acute and severe viral infections that includes natural human alpha interferons and Ampligen. In vitro cytopathic effect-prevention data on influenzaviruses using various combinations of interferon, ribavirin, oseltamivir and Ampligen were presented. Ampligen (rIn.r(C12U)n., Poly APoly U or rInr(C29,G)n, in which r is ribo) is a mismatched derivative of double-stranded RNAs. It was recently reviewed by De Clercq with the interferons [145]. Mice given Ampligen alone were protected from coxsackie B3 virus-induced myocarditis [146] and flavirus-induced encephalitis [147] but offered only limited protection against lethal pichinde virus challenge [148]. When given with interferon, Ampligen amplifies its effects. It also has synergistic activities with most anti-retrovirals [149] and has generated data in clinical trials [150]. No data is available for SARS-CoV. CURRENT AND FUTURE DEVELOPMENTS It is apparent from this review that anti-SARS-CoV drug development is advancing on many fronts and that many target and molecules have been screened. It is also apparent that much needs to be elucidated, in particular the in vivo activity of these agents in animal studies. However, some of these agents, particularly traditional remedies have well-known safety profiles and should receive priority for further developments. As antivirals come into use, the issue of drug resistance will emerge, as in the case of neuraminidase inhibitor resistance in influenzavirus. Viral escape from drug inhibition could lead to reduced viral
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fitness but is a virtual certainty and needs to be anticipated and monitored. This also applies to monoclonal antibodies. Also needing further development are RNA interference-based therapy, reviewed recently [151-158] and anti-sense therapy [159], as exemplified by US20060063150 [160], which employs uncharged highly stable morpholino oligonucleotides. The specific delivery of such cargoes into target cells has been a challenge. Recently, Song et al. gave us hope that an antibody-mediated delivery via cell-surface receptors may revolutionize this field [161, 162]. The rapid accumulation of anti-SARS-CoV medications is a welcome sign of strong basic research. We are now in bad need for paradigm shifts that would lead to more vigorous, but unglamorous, translational research as well as a business ethic of investing in money-losing products that nevertheless might still benefit the pharmaceutical industry in unimaginable ways. REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14]
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Hoever G, Baltina L, Michaelis M, et al. Antiviral activity of glycyrrhizic acid derivatives against SARScoronavirus. J Med Chem 2005; 48: 1256-1259. Harada S. Broad anti-viral agent glycyrrhizin directly modulates the fluidity of plasma membrane and HIV-1 envelope. Biochem J 2005; 392: 191-199. Cheung CY, Poon LL, Ng IH, et al. Cytokine responses in severe acute respiratory syndrome coronavirusinfected macrophages in vitro: possible relevance to pathogenesis. J Virol 2005; 79: 7819-7826. Law HK, Cheung CY, Ng HY, et al. Chemokine upregulation in SARS coronavirus infected human monocyte derived dendritic cells. Blood 2005; 106: 2366-2374. Spiegel M, Pichlmair A, Martinez-Sobrido L, et al. Inhibition of Beta interferon induction by severe acute respiratory syndrome coronavirus suggests a two-step model for activation of interferon regulatory factor 3. J Virol 2005; 79: 2079-2086. Zheng B, He ML, Wong KL, et al. Potent inhibition of SARS-associated coronavirus (SCOV) infection and replication by type I interferons (IFN-alpha/beta) but not by type II interferon (IFN-gamma). J Interferon Cytokine Res 2004; 24: 388-390. Carter, W.A., Strayer, D.: US0035859A1 (2006). Haagmans BL, Kuiken T, Martina BE, et al. Pegylated interferon-alpha protects type 1 pneumocytes against SARS coronavirus infection in macaques. Nat Med 2004; 10: 290-293. Cinatl J, Morgenstern B, Bauer G, Chandra P, Rabenau H, Doerr HW. Treatment of SARS with human interferons. Lancet 2003; 362: 293-294. Gao ZC, Zhu JH, Sun Y, et al. Clinical investigation of outbreak of nosocomial severe acute respiratory syndrome. Zhongguo Wei Zhong Bing Ji Jiu Yi Xue 2003; 15: 332-335. Loutfy MR, Blatt LM, Siminovitch KA, et al. Interferon alfacon-1 plus corticosteroids in severe acute respiratory syndrome: a preliminary study. JAMA 2003; 290: 3222-3228. Wu W, Wang J, Liu P, et al. A hospital outbreak of severe acute respiratory syndrome in Guangzhou, China. Chin Med J (Engl) 2003; 116: 811-818. Zhao Z, Zhang F, Xu M, et al. Description and clinical treatment of an early outbreak of severe acute respiratory syndrome (SARS) in Guangzhou, PR China. J Med Microbiol 2003; 52: 715-720. Bao M, Zhang Y, Wan M, et al. Anti-SARS-CoV immunity induced by a novel CpG oligodeoxynucleotide. Clin Immunol 2005; 118: 180-187. De Clercq E. Interferon: ten stories in one. A short review of some of the highlights in the history of an almost quinquagenarian. Acta Microbiol Immunol Hung 2005; 52: 273-289. Padalko E, Nuyens D, De Palma A, et al. The interferon inducer ampligen [Poly(I)-Poly(C12U)] markedly protects mice against coxsackie B3 virus-induced myocarditis. Antimicrob Agents Chemother 2004; 48: 267-274. Leyssen P, Drosten C, Paning M, et al. Interferons, interferon inducers, and interferon-ribavirin in treatment of flavivirus-induced encephalitis in mice. Antimicrob Agents Chemother 2003; 47: 777-782. Gowen BB, Barnard DL, Smee DF, et al. Interferon alfacon-1 protects hamsters from lethal pichinde virus infection. Antimicrob Agents Chemother 2005; 49: 2378-2386. Essey RJ, McDougall BR, Robinson WE, Jr. Mismatched double-stranded RNA (polyI-polyC(12)U) is synergistic with multiple anti-HIV drugs and is active against drug-sensitive and drug-resistant HIV-1 in vitro. Antiviral Res 2001; 51: 189-202. Thompson KA, Strayer DR, Salvato PD, et al. Results of a double-blind placebo-controlled study of the double-stranded RNA drug polyI:polyC12U in the treatment of HIV infection. Eur J Clin Microbiol Infect Dis 1996; 15: 580-587. He ML, Zheng BJ, Chen Y, et al. Kinetics and synergistic effects of siRNAs targeting structural and replicase genes of SARS-associated coronavirus. FEBS Lett 2006; 580: 2414-2420. Li B-j, Tang Q, Cheng D, et al. Using siRNA in prophylactic and therapeutic regimens against SARS coronavirus in Rhesus macaque. Nat Med 2005; 11: 944-951. Li T, Zhang Y, Fu L, et al. siRNA targeting the leader sequence of SARS-CoV inhibits virus replication. Gene Ther 2005; 12: 751-761. Shi Y, Yang de H, Xiong J, Jia J, Huang B, Jin YX. Inhibition of genes expression of SARS coronavirus by synthetic small interfering RNAs. Cell Res 2005; 15: 193-200. Tao P, Zhang J, Tang N, Zhang BQ, He TC, Huang AL. Potent and specific inhibition of SARS-CoV antigen expression by RNA interference. Chin Med J (Engl) 2005; 118: 714-719. Wu CJ, Huang HW, Liu CY, Hong CF, Chan YL. Inhibition of SARS-CoV replication by siRNA. Antiviral Res 2005; 65: 45-48. Zhang R, Guo Z, Lu J, et al. Inhibiting severe acute respiratory syndrome-associated coronavirus by small interfering RNA. Chin Med J (Engl) 2003; 116: 1262-1264. Tong TR. Therapies for coronaviruses. Part 2: inhibitors of intracellular life cycle. Expert Opin Ther Pat 2009; 19: 415-431. Schubert S, Kurreck J. Oligonucleotide-based antiviral strategies. Handb Exp Pharmacol 2006: 261-287.
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Probiotics as Drugs Against Human Gastrointestinal Pathogens Yolanda Sanz*, Inmaculada Nadal and Ester Sánchez Instituto de Agroquímica y Tecnología de los Alimentos (CSIC), P.O. Box 73 46100, 46100 Burjassot, Valencia, Spain Abstract: The microbiota that colonizes the gastrointestinal tract confers health benefits to the host by contributing to dietary digestion, regulating immunity and preventing pathogen colonization and invasion. Lactic acid bacteria (e.g. Lactobacillus) and Bifidobacterium constitute the main groups of probiotics commercialized for human consumption. The prevention and treatment of gastrointestinal infections continues to be complex due to the expansion of antibiotic resistances. The possible uses of probiotics and derived products as therapeutic or preventive drugs against gastrointestinal infections have been intensively investigated, as reflected in a large number of published patents. The possible mechanisms of action of probiotics against gastrointestinal pathogens addressed in diverse patent applications include: (i) modification of the environmental conditions, (ii) competition for nutrients and adhesion sites, (iii) production of antimicrobial compounds, (iv) modulation of the immune and nonimmune defense mechanisms of the host, and (v) regulation of the intestinal neuromuscular function. The molecules responsible for the antimicrobial effects of probiotics include cell-wall fractions, surface proteins, nucleic acids, organic and short-chain fatty acids, antimicrobial proteins and other less-well identified soluble factors. The effectiveness of probiotics is supported by human clinical trials on treatment of acute diarrhea, prevention of antibiotic associated-diarrhea and as adjuvant therapy with antibiotics in eradication of Helicobacter pylori infection. Probiotics and their bioactive compounds constitute attractive alternative or adjuvant drugs as they can reduce the use of antibiotics and improve conventional pharmacological therapies. The advances in the knowledge of novel bioactive molecules and the intricate host-microbe interactions within the intestine and extra-intestinal sites will result in the future development of a new generation probiotic-based products targeting a broader range of pathologies.
Keywords: Probiotics, lactic acid bacteria, Lactobacillus, Bifidobacterium, pathogens, gastrointestinal infection, antibiotic, antimicrobial compound, immune system, immunomodulation. INTRODUCTION The gastrointestinal tract is one of the largest surfaces of the human body exposed to the environment. It plays a major role in nutrient digestion and absorption, but also integrates *Corresponding author: Tel: 34 963900022; Fax: 34 963636301; E-mail:
[email protected]
Atta-ur-Rahman / M. Iqbal Choudhary (Eds.) All rights reserved – © 2010 Bentham Science Publishers.
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complex mechanisms to handle with foreign antigens. The epithelium, the local immune system and the commensal microbiota contribute to the intestinal barrier function [1]. These elements orchestrate a network of immunological and non-immunological defenses that protect the host from invasion by pathogenic microorganisms [2]. More than 500 different bacterial species populated the human gut and confer health benefits to their host by helping dietary digestion, regulating immunity, maintaining the microbial balance and preventing pathogen colonization [3]. A large number of probiotics and prebiotics, which favor the prevalence of gut health-promoting bacteria, have been introduced in the market in dietary and pharmaceutical forms. Probiotics are defined as live microorganisms that when ingested in sufficient quantities exert positive effects on the host’s health [4]. Prebiotics are nondigestible dietary ingredients that allow changes, both in the composition and/or activity of the gastrointestinal microbiota that confers benefits on the host’s health [5]. Lactic acid bacteria (e.g. Lactobacillus) and Bifidobacterium constitute the main groups of probiotics commercialized for human consumption. Strains of these genera are natural inhabitants of the human gut and have an excellent record of safety use in food fermentations. Moreover, non-pathogenic Gram-negative bacterial strains (Escherichia coli Nissle) and yeast (Saccharomyces boulardii) are included within the currently commercialized probiotics. The treatment of gastrointestinal infections continues to be complicated due to the expansion of antibiotic resistances. This has led to an intensive search for both new antibiotic treatments and alternative therapies [6]. The potential preventive and therapeutic uses of probiotics against gastrointestinal infections have been the objective of most investigations, as reflected in a large number of patents applications [e.g. 7-9]. The clinical situations studied so far include prevention or treatment of antibiotic-associated disorders, traveler's diarrhea, acute gastroenteritis and infections by pathogenic bacteria such as Helicobacter pylori and Clostridium difficile [10-12]. So far, the effectiveness of probiotics is supported by solid clinical studies mainly on treatment of acute diarrhea (mainly viral gastroenteritis) in children, prevention of antibiotic associated-diarrhea in adults and children and as adjuvant therapy with antibiotics in eradication of Helicobacter pylori infection [10,11]. The mechanisms of action of probiotics against gastrointestinal pathogens include modification of the environmental conditions, competition for nutrients and adhesion sites, production of antimicrobial metabolites and modulation of immune and nonimmune defense mechanisms of the host [13]. Structural components of probiotic microorganisms as well as secreted factors and molecules derived from their metabolism are active players in the fight against infections. Thus, it is considered that the ingestion of these beneficial live microorganisms can contribute to the production and delivery of bioactive molecules to proximal or distal intestinal sites, thereby helping to combat infection [11]. Probiotics and their bioactive compounds constitute, nowadays, attractive alternative drugs that can contribute to reduce the use of antibiotics as well as to improve conventional pharmacological therapies. MECHANISMS OF ACTION OF PROBIOTICS Probiotics and commensal gut microbes can exert therapeutic and preventive effects against gastrointestinal infections by modifying the environment, interfering with the pathogenic mechanisms of harmful microbes and stimulating the host defense mechanisms (Fig. 1). Probiotics might contain structural components and produce compounds active, directly or indirectly, against pathogenic microbes. These bioactive compounds include cellwall fractions (lipopoly-saccharides and lipoteichoic acids), surface proteins, nucleic acids, organic and short-chain fatty acids, antimicrobial proteins and other less-well identified
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soluble factors [13,14]. The mechanisms of action by which these microbes and their metabolites contribute to combat the colonization and invasion of gastrointestinal pathogens are reviewed below. Environmental Changes and Pathogen Interference Changes in Ecological Conditions and Competition for Nutrients Lactic acid bacteria have been utilized as fermenting agents for the preservation of food since ancient times, taking benefit of the inhibitory effects of the low pH and antimicrobial products (organic acids, hydrogen peroxide, carbon dioxide, etc.) resulting from their metabolic activity on carbohydrates (Fig. 1). The same concept applies to probiotic bacteria and to foods that serve as vehicle of these bacteria and their metabolites. The fermentative activity of probiotic strains can induce significant changes in the ecological conditions of the gut, turning them adverse for the survival of pathogenic bacteria. A large number of patents on probiotics active against gastrointestinal pathogens are based on their acidifycation ability, together with other beneficial traits [e.g. 15]. For example, the positive effects attributed to some Lactobacilli against H. pylori infection are mainly due to their acidification ability, and their use has been proposed for the eradication of the infectious agent together with antibiotic therapies applied to these patients [16-18].
Fig (1). Desirable traits of probiotic strains intended to be used as drugs for prevention and treatment of gastrointestinal infections.
Competition for nutrients is also considered to be one of the mechanisms by which some probiotic strains interfere with gut colonization by pathogenic microbes. The repertoire of glycohydrolases synthesized by commensal and probiotic bacteria could be a competitive advantage to survive in the colon, where complex oligosaccharides constitute the main energy source, at expenses of other bacteria such as bacteroides, clostridia and coliforms
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[19,20]. Mutations in carbohydrate metabolic pathways of commensals such as Bacteroides thetaiotaomicron and E. coli have been demonstrated to modify their colonization, revealing the importance of this trait [21]. This is partly the basis of the beneficial effects of prebiotics on maintaining the intestinal balance and counteracting dysbiosis in pathologic conditions. As prebiotics are substrates for probiotic bacteria, their administration is considered a mean to increase their metabolic activity and chances of residence in the gut. A number of recently patented products that claim to have protective effects against gastrointestinal infections are based on the combination of pro- and prebiotics, named synbiotics. For instance, patent No. SI1398369T is based on pure cultures of two strains of Bifidobacterium longum called W11 and W11a, which have been formulated in the form of pharmaceutical or alimentary type preparations, together with prebiotic agents like fructo-oligosaccharides or inuline, and/or vitamins (E, B1, B2, B6 and B12 groups) [8]. These products are claimed to help the good health of the gastrointestinal tract and to restore its altered functionality from gastroenteritis of various origin. Patent No. WO06112714 relates to compositions containing probiotic bacteria and uronic acid oligosaccharides as prebiotic, which is claimed to be suitable for infant nutrition and able to reduce the incidence of infections [22]. Additionally, it is claimed that both probiotic bacteria and the uronic acid oligosaccharides stimulate the systemic immunity. Another nutritional competitive advantage recently identified in Bifidobacterium strains consists of their ability to secrete a siderophore in the intestine that complexes iron and prevents its acquisition by harmful microbes and thereby their colonization and invasion, disclosed in patent No. US2004191233 [23]. Adhesion to Intestinal Mucus The intestinal mucosa consists of a mucous layer formed by mucins linked to cellular and stromal components that participate in the defense of the host [24]. Mucins are high molecular weight glycoproteins that can be subdivided into two groups: secreted gelforming mucins and transmembrane mucins. The oligosaccharide side chains, which comprise up to 80% of the molecular weight of the mucin glycoproteins, consist of hexose, fucose, hexosamine, sialic acid and sulphate ester and show a high variability in structure. Mucins are involved in protection of luminal epithelial surfaces, signal transduction, cellcell recognition and in the first steps of pathogen invasion [25,26]. Inhibition of pathogen adhesion by probiotics is supposed to contribute to the primer line of defense by limiting their proximity to the epithelial layer and possible tanslocation by M cells covering the Peyer´s patches (PPs) or dendritic cells (DCs) (Fig. 1) [27,28]. The adhesion properties of probiotic strains might also determine their own ability to persist in the intestine and interact with epithelial and immune cells regulating the host defenses [13]. The ability of probiotic microorganisms to competitively inhibit the adhesion of pathogenic microorganisms and displace previously adhered pathogens has been mainly assessed in vitro, using immobilized intestinal mucus and mucus-producing epithelial cell lines. The pathogens include Salmonella enterica serovar Typhimurium, S. arizonae, Escherichia coli, Listeria monocytogenes, Enterobacter sakazakii, Clostridium difficile and C. perfringens [29-32]. These models have also been used to demonstrate that some strains such as B. lactis DR10 and Bifidobacterium sp. CA1 and F are able to impair invasion of intestinal cell monolayers by E. coli O157:H7 and S. enterica serovar Typhimurium [33,34]. The adhesion mechanisms of bacteria involved passive forces, electrostatic and hydrophobic interactions, lipoteichoic acids and specific structures, such as lectins and polysaccharides. Adhesion of Lactobacillus strains has been reported to be mediated by surface proteins for instance in L. fermentum 104R, and lipoteichoic acids for instance in L.
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johnsonii La1 [35]. Fujiwara et al. [36] reported that both the production of organic acids and proteinaceous factors by Bifidobacterium sp. BL2928 interfered with the binding of E. coli strains to the intestinal epithelium. They also demonstrated that this strain secreted a bacterium-binding structure similar to that of enterotoxigenic E. coli strains. However, in vitro adhesion studies have demonstrated that adhesion ability of commensal and probiotic bacteria is usually significantly lower than that of well-recognized pathogens and varies widely depending on the strain [32]. In addition, the in vivo protective effects of high adhesive probiotic strains against pathogen translocation across the epithelial layer are less well documented. This has lead to the continuing search for strains with higher adhesion ability and for the molecules responsible for this property. The following patents are interesting examples of recent discoveries in this field. Patent No. WO04022727 describes a composition comprising a Lactobacillus fermentum variant strain VRI 003 derived from L fermentum LMG or a cell component thereof that adheres to mouse PPs due to modifications on expression of cell-wall proteins [37]. It is claimed that the bacterium or a component thereof adheres to PPs inhibiting pathogens both by a direct antagonistic effect and by inducing a Th 1-type response in a subject. Patent No. WO04020467 relates to novel genes and polypeptides, including elongation factor efTu, heat shock protease clpE and pyruvate kinase like proteins of Lactobacillus johnsonii involved in binding of the bacteria to mucins and to the corresponding genes [38]. In particular, the invention pertains to the use of the present genes in the preparation of polypeptides having the capacity to bind to mucins. Patent No. WO03002131 (A1) is based on the strain Lactobacillus fermentum ME-3 DSM 14241 as a novel anti-microbial probiotic [15]. It is stated that the glycocalyx of the cell wall of this strain has a special composition, containing residues of galactose and N-acetylgalactosamine, which can act as adhesins for engaging the receptors of mucosa on the epithelial cells. This is considered a possible mechanism for blocking the mannose-resistant pili of Escherichia coli, which makes this lactobacilli strain applicable in the prophylaxis of infections. Toxin Inactivation and Toxin Binding The proteolytic inactivation of toxins produced by Clostridium difficile has been recognized as one of the mechanisms of action of the probiotic S. boulardii against this pathogen (Fig. 1) [39]. The ability of lactic acid bacteria and probiotics to bind microbial toxins to their surfaces has also been suggested as a possible protective mechanism. Patent No. WO02062360 is a recent example of applications in this field. It includes the use of at least one strain of lactic acid bacteria (e.g. Lactobacillus johnsonii, L. reuteri and L. paracasei) and/or bifidobacteria having hydrophobic surface properties, for the preparation of a composition intended for the prevention or the treatment of endotoxin mediated and/or associated disorders in human and animals [9]. It is proposed that the hydrophobic cell wall of the selected bacteria can scavenge main pro-inflammatory toxins of the Gram-negative bacteria that otherwise may translocate from the lumen of the gut into the blood. The composition is particularly intended for the prophylaxis or the treatment of infections related to Gram-negative bacteria, endotoxin producing bacteria such as Helicobacter spp., and Samonella spp., and also to small intestinal bacterial overgrowth. Production of Antimicrobial Compounds The production of antimicrobial compounds is one of the mechanisms by which probiotics might inhibit pathogenic microorganisms. These include low molecular mass compounds (bellow 1,000 Da), such as organic acids that have a broad spectrum of action,
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and antimicrobial proteins (over 1.000 Da) termed bacteriocins or bacteriocin-like compounds (Figs. 1 and 2) [40,41]. Bacteriocins are proteins or protein complexes that show bactericidal activity against species that are closely related to the producer species. In addition, the term bacteriocin-like compound has been coined to refer antagonistic substances, which do not fit the typical criteria defining bacteriocins and tend to have a broader spectrum of activity [42,43]. These compounds and the producer lactic acid bacterial strains are applied as food preservatives and are of interest to the development of novel therapies and antibiotics to combat infections [43]. As previously mentioned, lactic acid bacteria have a fermentative metabolism that leads to the generation of relative large amounts of organic acids and short-chain fatty acids (e.g. lactic, acetic, propionic and butyric acids). These have been considered to be the main antimicrobial compounds responsible for the inhibitory activity of protective bacteria against pathogenic microorganisms such as S. enterica and H. pylori [44,45]. For instance, in patent No. WO03002131 (A1) a Lactobacillus fermentum ME-3 DSM 14241 strain, as indicated above, is presented as a novel anti-microbial probiotic for use in pharmaceutical and food industry for the prophylaxis and treatment of gastrointestinal and urogenital infections [15]. The high antimicrobial effect of this strain is partly ensured by production of organic acids (acetic acid, lactic acid, and succinic acid) and ethanol. Additionally, this strain has anti-oxidative activities that are claimed to counteract the excessive formation of reactive oxygen species (ROS) that can cause the damage of cells and tissues (Fig. 2). Lactic acid bacteria can also synthesize other low-molecular-weight substances such as phenyllactic acid, p-hydroxyphenyllactic acid, cyclic dipeptides, benzoic acid, methylhydantoin, mevalonolactone, and short-chain fatty acids that contribute to their antimicrobial activity against fungi in food fermentations [46]. These compounds might also play a role in the vaginal and intestinal infections but their production has not been detected yet in probiotic lactic acid bacteria except for short-chain fatty acids. The synthesis of bacteriocins has been described mainly in probiotic Lactobacillus strains [47]. Moreover, a bacteriocin (Bifidocin B) active against Gram-positive bacteria was purified from B. bifidum NCFB 14 [48]. Recently, other bacteriocin-producing bifidobacterial isolates from newborns have been identified as Bifidobacterium thermacidophilum, and B. thermophilum. One strain of B. thermacidophilum RBL70 was shown to be effective in blocking invasion of Listeria on Caco-2 and HT-29 cells [49]. The following patents are examples of recent applications in this field. Patent No. WO0179278 discloses a Lactobacillus johnsonii LMG P-19261 bacteriocin, active against Helicobacter pylori [50]. Patent No. WO06070041 relates to metabolic cellular compounds with antifungal activity from strains of the genus Bifidobacterium [51]. The compounds have a wide range of activity including yeasts and fungi and, among the same, important mycotoxin producers. Patent No. WO06070040 relates to Bifidobacterium strains and to the extracts resulting from the metabolism thereof, which contain antibacterial proteins, which have a wide range of activity including Gram-positive and Gram-negative bacteria [52]. Patent No. KR20040084167 discloses probiotic bacteria that produced antimicrobial compounds that are also the basis of new products for preserving food by inhibiting the growth of harmful enteropathogenic microorganisms. The composition of the product for preserving food contains the culture media obtained by culturing one or more stains selected from a probiotic microorganism comprising Lactobacillus reuteri Probio-016, Lactobacillus
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Fig. (2). Schematic representation of interactions of pathogenic, commensal and probiotic microorganisms with the host immune system in the intestinal ecosystem [2,11,71]. PDG, peptidoglycan; LPS, lipopolysaccharide; LTA, lipoteichoic acids; pathogen cells and toxins, represented as red solid and open circles, respectively; probiotic or commensal cells and soluble factors represented as green solid or open circles, respectively. Pathogen and commensal microbes can be translocated to the lamina propria by dendritic cells (1) or M cells (2) and modulate innate as well as adaptive immunity by interacting with naïve and memory T cells. Microbial antigens can be presented to T cells, leading to either IgA antibody-mediated mucosal immune response, and to proinflammatory and cytotoxic responses to protect against microbial invasion, or to non-inflammatory responses through the induction of regulatory T cells (Treg). Soluble factors of probiotic and commensal bacterial (3) and interactions of GpC DNA motifs with TLR9 (4) can modulate inflammatory responses triggered by the NFκB signaling pathway. Interactions of LTA of commensal and probiotic bacteria with TLR2 (5) can lead to pro-inflammatory responses via the NFκB signaling pathway or to its attenuation. Interaction of pathogen bacterial LPS and PDG with TLR4 (6) and NLRs (7), respectively, or pathogen toxins with the membrane (8) lead to pro-inflammatory responses via the NFκB signaling pathway.
salivarius Probio-037 and Lactobacillus plantarum Probio-038 [53]. The composition additionally contains the culture media of one or more probiotic microorganisms selected from the group consisting of Saccharomyces, Bacillus and Bifidobacterium genera. Patent No. MXPA99007378 discloses probiotic Lactobacillus salivarius strains, isolated from resected and washed human gastrointestinal tract, that inhibits a broad range of Grampositive and Gram-negative microorganisms and secretes a product having antimicrobial activity into a cell-free supernatant that has bacteriocin-like properties [54]. Patent No. US20060165661 provided pediocin-producing Pediococcusacidilactici strains isolated from
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human feces [55]. These strains are claimed to be suitable for use in the gastrointestinal tract of humans to provide benefits against infection by multi-resistant pathogens. Nevertheless, the relevance of the production of bacteriocins in vivo is not known yet. In an attempt to study the role of nisin in the gastrointestinal tract, the effects of pure nisin, a nisin-producing Lactococcus lactis strain and a non-nisin-producing L. lactis on the composition of the intestinal microbiota of human flora-associated rats have been evaluated. However, the profiles of the microbiota from animals dosed with nisin did not differ from the controls, and nisin seemed to be degraded or inactivated in the gastrointestinal tract [56]. Stimulation of Host Defense Mechanisms Effects on Epithelium and Secretions The intestinal epithelium consists of a single layer of epithelial cells that constitutes a physical barrier, which limits the entry of exogenous agents. It also secretes various protective substances such as mucins, enzymes, and antimicrobial peptides, which contribute to the non-specific intestinal defenses. The mucus layer constitutes a first line of the host’s defenses and is formed by mucins (glycoproteins). The polysaccharide structures of mucins come into direct contact with the commensal or pathogenic bacteria and play an important role in cell-cell recognition processes. Probiotic strains as well as members of the commensal microbiota could contribute to the regulation of mucin gene expression, modifying the glycosylation pattern and total production of mucins [57]. Probiotic strains, such as L. plantarum 299v and L. rhamnosus GG, have been shown to increase the expression of mucin MUC-2 and/or MUC3 mRNA in diverse cellular lines [58]. VSL#3 probiotic formula also stimulated colonic mucin secretion and MUC2 gene expression in Wistar rats. In addition, bacterial secreted products contained in the conditioned media stimulated mucin secretion in vitro using colonic epithelial cells [59]. Intestinal epithelial cells and in particularly Paneth cells are the major producers of antimicrobial peptides and proteins with activity in the intestine, such as defensins [60]. These peptides act synergistically and have specialized activities against different microorganisms. Secretion may be stimulated by Gram-negative, Gram-positive and probiotic bacteria, and by bacterial products (e.g., lipopolysaccharide, lipoteichoic acid, and muramyl dipeptide (Fig. 1) [61]. For example, patent No. WO07020884 includes Bifidobacterium and lactic acid bacterium strains having effect on preventing infection via stimulation of expression level of defensin upon infection with pathogenic bacteria, such as E. coli [62]. Cytoskeleton and tight-junction proteins regulate the permeability of the epithelium. These elements constitute a physical barrier preventing the entry of exogenous agents but they are also exploited by many pathogens to proliferate and persist within the host [2]. Probiotic strains have been shown to protect against leakage of tight-junctions detected in infections and inflammatory conditions (Fig. 1). S. boulardii maintained tight-junction structure in T84 cells during infection by an enteropathogenic E. coli stain [63]. Soluble factors secreted to the growth medium by the probiotic mixture VSL#3 and Lactobacillus GG have also been shown to increase the production of heat shock proteins involved in maintaining cytoskeleton integrity by the MAPKs signaling pathway [64,65]. Some probiotics also have trophic functions, involved in cell proliferation, differenttiation and apoptosis (Fig. 1). The regulation of these processes can have an overall impact on the intestinal barrier function [11]. Lactobacillus GG exerted an inhibitory effect on apoptosis by activation of enterocyte signal transduction pathways [66]. S. boulardii was
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also thought to contribute to the release of polyamines that exert trophic effects on the intestine of rats [67]. In addition, some components of commensal microbiota and probiotics have been shown to generate butyrate from complex carbohydrates, which contribute to the intestinal barrier function by supplying energy (60-70%) to the epithelial cells, stimulating the intestinal blood flow, and affecting epithelial proliferation and differentiation [68]. Probiotic strains such as S. boulardii have also been shown to inhibit chloride secretion induce by E. coli and V. cholera toxins (Fig. 1) [63]. Streptococcus thermophilus ATCC19258 and Lactobacillus acidophilus ATCC4356 also reduce chloride secretion induced by enteroinvasive E. coli strains [69]. Effects on Host Immune Functions The gut-associated lymphoid tissue consists of both organized lymphoid tissues, like mesenteric lymph nodes and PPs, and more diffusely scattered lymphocytes in the lamina propria and epithelium, including large numbers of IgA-secreting plasmocytes [70]. Sentinels cells including epithelial cells, macrophages and DCs, sense the environment and coordinate defense mechanisms depending on the kind of stimulant [71]. The mucosal intestinal barrier maintains a local homeostatic response to the resident intestinal bacteria, while protecting the host against enteric pathogens. The interactions between gut microbes and the host epithelial and immune cells are involved in the regulation of both innate and adaptive immunity (Fig. 1) [72]. Pattern-recognition receptors located in epithelial cells and DCs are considered the start point of immunity, sensing the environment and informing the cell to respond to infection. These receptors have the ability to discriminate between harmful pathogens and the harmless members of the commensal flora. They include the Toll-like receptor family (TLRs), which are located at the cell surface and intracellularly, and Nod-like receptors (NLRs), which are located intracellularly [73,74]. The recognition of different microbial signals coming from pathogens, commensals or probiotics by these receptors lead to different responses that involve new gene transcription and production of pro-inflammatory or regulatory cytokines (Fig. 2). For example, TLR2 recognizes lipoteichoic acids form Gram-positive bacteria, TLR4 recognizes lipopolysaccharide from Gram-negative bacteria, TLR9 recognizes special sequences of DNA (unmethylated CpG motives) and NLRs appear to recognize bacterial peptidoglycans [2,11]. TLRs are associated with diverse adaptor molecules, like the adaptor protein MyD88 (myeloid differentiation protein), to trigger the activation of the transcriptional factor NF-κB and the MAP kinase transduction-signaling pathway, as well as other stress responsive transcriptional elements such as AP-1 [2,75]. The activation of the factor NF-κB is considered to be the pivotal event in TLR-mediated stimulation of the innate immune response against pathogen infections, constituting an important therapeutic target [76]. Upon activation, NF-κB is translocated into the nucleus with subsequent activation of inflammatory genes, including those encoding cytokines, cytokines receptors, immunoregulatory proteins, adhesion molecules, stress-associated proteins, defensins and other mediators. These molecules are overall involved in the recruitment of other immune cells (T cells, basophils, neutrophils, dendritic cells and natural killer cells) and in the development of inflammatory responses that could lead to pathogen clearance [2]. Interleukin 8 (IL-8) and tumor necrosis factor-alpha (TNF-α) play a central role in attracting inflammatory cells to the sites of bacterial infection. In this context, commensal bacteria have a mutualistic relationship with the host and produce a transient cellular response and even suppress the proinflammatory activation through NF-κB by ligand interactions with TLR2 or TLR9 [77,78]. In addition to the activation of the innate
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immune responses, TLRs play a pivotal role in linking innate and adaptive immunity through actions on T-cells and particularly on DC that lead to the selection of the appropriate adaptive immune responses in the face of immunological danger [74]. Signaling through TLR stimulates the maturation of DCs with enhanced ability to present antigens and activate T cells, T-cell co-stimulatory molecules (CD80 and CD86) and other activation markers. T-cell differentiation into Th1, Th2 or even Treg cells is thought to depend on the type of TLRs involved (Fig. 2) [71]. Th1 responses are usually associated with inflammatory reactions and clearance of intracellular pathogenic bacteria and virus, Th2 responses with allergic responses and parasite clearance, and Treg cells (Th3 and Thr1) are essential in preventing over-reactions [79,80]. Commensal bacteria and pathogens can also be sampled by DCs or by M cells located at the PPs [2]. Then, the translocated antigens encounter immature DCs, which can drive Tcell responses (Fig. 2). DC may present antigens locally to T-cells, migrate to T-cell zones or to mesenteric lymph nodes or interact with memory B cells [72]. For example, the capsular polysaccharide of the ubiquitous Gram-negative bacteria Bacteroides fragilis can be taken up by CD11c+ DC leading to the production of IL-12 and to the expansion of Th1 cell population [81]. There is increasing evidence that some probiotics can sufficiently stimulate protective innate and acquired immune responses to enhance resistance to microbial pathogens [82]. Probiotic are claimed to modulate the host immune system by both activating the responses devoted to eliminate the pathogens or their toxic compounds and by attenuating excessive proinflammatory cytokine production and cell activation that can lead to tissue damage and chronic inflammatory disorders [14, 83]. Overall, these effects are believed to be mediated through activating non-specific cell phagocytosis, increasing levels of cytokines, increasing natural killer (NK) cell activity and/or increasing levels of immunoglobulins, particularly the synthesis and secretion of polymeric IgA, which coats and protects mucosal surfaces against harmful bacterial invasion [84,85]. The immunomodulatory properties of probiotics for the control of human enteropathogens have been studied in animal models (germen-free and infection/challenge models) and humans. For example, the administration of B. lactis NH019 to S. typhimurium infected mice resulted in a higher production of S. typhimurium pathogen specific antibodies in serum and intestinal mucosa as well as in an increased phagocytic activity of peritoneal macrophages and blood-borne neutrophils [86]. Strains of B. bifidum and B. infantis administrated to rotavirus-infected mouse pups increased virusspecific IgA levels in serum and the gastrointestinal tract [87]. A probiotic Lactobacillus casei ATCC27139 has recently been demonstrated to up-regulate innate immune responses in vivo in mouse models of systemic Listeria monocytogenes infection via NF-κB and p38 MAP kinase-signaling pathways. The detected effects involved the induction of innate cytokine production and the expression of patter recognition molecules such as TLR2 (Fig. 2) [88]. In humans, for example the administration of Bifidobacterium Bb12 together with Lactobacillus acidophilus and Streptococcus thermophilus in the form of a fermented-milk product enhanced the humoral response, inducing the production of higher levels of IgA against attenuated S. typhi vaccine [89]. The oral administration of B. breve YIT4064 to infants increased the production of anti-rotavirus IgA and reduced the frequency of rotavirus shedding in stool samples [90]. In elderly subjects, the ingestion of B. lactis HN019 increased the proportions of total, helper (CD4+), and activated (CD25+) T lymphocytes and NK cells. The consumption of B. lactis HN019 also increased the phagocytic capacity of mononuclear and polymorphonuclear phagocytes [91]. L. fermentum CECT5716 enhanced the immunologic response of an anti-influenza vaccine and provided enhanced
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systemic protection from infection by increasing the Th1 response and virus-neutralizing antibodies [92]. The proposed mechanisms by which probiotic bacteria might interact with host epithelial and immuno-competent cells are similar to those describe for commensal bacteria and summarized in Fig. (2). Association of probiotics with epithelial cells might be sufficient to trigger signaling cascades that ultimately activate underlying immune cells and lamina propria. Probiotics may also release soluble factors that themselves trigger signaling cascades at the level of the epithelium or associated immune system [14,93]. Probiotic bacteria can also activate the immune system after translocation by DCs or by adhesion to M cells of the PPs. These PPs exert a pivotal role for the development of secretory IgA and, as a consequence, probiotics targeting M cells could be selected for bolstering the intestinal immunity [94]. So far, the probiotic molecules that are know to interact with the epithelial and immune cells can be subdivided into three classes: (i) structural components (cell surface proteins, DNA, cell-wall components, etc.); (ii) secreted molecules or soluble factors and (iii) products derived from their growth and metabolic activity in specific media [95,96]. The identification of bioactive probiotic molecules and the mechanism by which they interact with components of the host immune system are the basis of novel patent applications in this field. For example, patent No. EP1615657 discloses a new immunomodulator product obtained from cultures of Bifidobacterium breve I-2219 in a medium containing lactoserum permeate, lactoserum protein hydrolyzate and lactose, and further separated by exclusion chromatography. This product is proposed for regulating the intestinal microbiota and for suppression of potential harmful bacteria like Clostridium perfringens or Bacteroides fragilis [97]. Patent No. WO2004076615 discloses immunomodulating probiotic compounds based on enzymes located on the bacterial surface [98]. The invention relates to methods employing species of Lactobacillus and Bifidobacterium having an altered expression of at least one cell surface polypeptide capable of exerting an immunomodulating effect when binding an epithelial cell or a cell forming part of mucosal associated lymphoid tissue. The cell surface polypeptides are substantially identical to intracellular enzymes acting in glycolytic metabolic pathways, such as enolase or GAPDH, in Lactobacillus and Bifidobacterium. Patent No. US2005032731 and US20060147465 relate to oligoribonucleotides smaller than 10,000 Da (ORN) that alert the immune system of animals to the imminence of microbial infection [99, 100]. They also address the methods for the accumulation and retention of the immune-enhancing, bacterial-derived ribonucleotides. The bacteria release or accumulate ORNs by application of a chemical, physical, or biological stress, or when the pH of the growth medium becomes acidic. It is proposed that administration of these ORNs modulates the immune response by: (i) stimulating macrophages that release pro-inflammatory cytokines (e.g. IL-1, IL-6 and TNF-α) to fight against infections and (ii) down-regulating the CD-14 and CD-16 receptors of macrophages to prevent over-stimulation by endotoxin, leading to the over-production of IL-1, IL-6 and TNF-α, associated with inflammation. Patent No. WO04103083 consists of a probiotic composition comprising at least two specific lactic acid bacterial strains, which have an infection protecting property based on immunopotentiating effects [101]. The protective strains were found to have different abilities of activating the transcription of the NF-κB leading to cytokine production, which was either pro-inflammatory or anti-inflammatory (Fig. 2). Lactobacillus strains, particularly L. paracasei F19, activate the transcription of NF-κB in a macrophage cell line, resulting in the synthesis of the interleukins IL-1 and IL-8 of pro-inflammatory type that could activate defences against pathogens. Patent No US 20040208863 includes lactic acid bacteria that produce soluble factors (such as peptides or
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proteins) that block inflammatory responses in a mechanism that depends on G proteins [102]. The soluble compound produced by the lactic acid bacteria binds to receptors on immune cells and inhibits the cytokine production (TNF-α) and chemokine production (IL8) through inhibitory heterotrimeric G protein activity, without affecting the production of the anti-inflammatory cytokine IL-10. Lactobacilli-conditioned media containing the active compound also decreases TNF-α production of E. coli- and Helicobacter-conditioned media-activated peritoneal macrophages, reducing their excessive inflammatory effects upon infection. Patent No. US 20060182727 provides the strains Lactobacillus ONRIC b0239 and Lactobacillus ONRIC b0240 and compositions containing the bacteria in the form of foods or pharmaceutical products, being capable of stimulating mucosal immunity by promoting IgA production (Fig. 2) [94]. The strains induce IgA production by interacting with PPs in a mouse cell culture system as well as serum IgG, suggesting the activation of both mucosal and systemic immunity. Therefore, it is claimed that these strains can inhibit the invasion of pathogenic microorganisms through mucosa immunity stimulation and that are also expected to be useful in new probiotic applications such as oral vaccines. Patent No. WO 2007040445 discloses the use of probiotic of the Lactobacillus genus and, in particular Lactobacillus plantarum 299v (DSM 9843) and Lactobacillus paracasei 8700:2 (DSM 13434), for the treatment and/or prevention of diverse virus infections, via modulation of immune responses [103]. Intake of lactobacilli activates the innate immune system by expanding the NK T cell population, particularly following the intake of L. paracasei, and by increasing the phagocytosis of the Gram- negative bacteria by granulocytes in human subjects. Intake of lactobacilli also activates T cells and, remarkably, L. plantarum 299v induces a two-fold increase of the expression of the activation marker CD25 on CD8+ T cells (cytotoxic T cells) and a similar trend is reported on CD4+ T cells. Intake of L. plantarum also has a pronounced positive effect on activation and induction of memory T cells. A significant up-regulation of the IL-2 receptor [alpha] chain (CD25) and a strong tendency toward up-regulation of HLA-DR on cytotoxic T cells is reported. A similar trend is observed on helper T cells. Expression of these activation markers indicates that the T cells have started to proliferate in response to a stimulus and that these cells more readily exert their effector functions compared to resting T cells. Expression of the cell marker CD45RO, characteristic of memory T cell, is also increased on helper T cells and a tendency toward up-regulation is also detected on cytotoxic T cells by intake of L. plantarum. The coincident activation of cytotoxic T cells and NK T cell expansion suggests a role of these strains in enhancing immune defence against viral infections. CURRENT AND FUTURE PERSPECTIVES Probiotics and molecules thereof have been demonstrated to be active against gastrointestinal pathogens, and beneficial in different clinical situations. The protective role of probiotics and their bioactive molecules against diverse infectious agents has been patented, addressing general mechanisms of action such as (i) competition for nutrients and adhesion sites, (ii) secretion of antimicrobial compounds and (iii) stimulation of immune defenses. Moreover, the most recent patents on probiotics reflect a progress in the understanding of the molecular mechanism behind their beneficial effects as well as the discovery of novel modes of action. Genome data of commensal bacteria inhabiting the gastrointestinal tract and considered potential probiotics, such as strains of the genus Bifidobacterium, is allowing the identification of molecules involved in their persistence in the gut and in the regulation of functions relevant to the host defenses. For example, the two predicted cell-wall anchored
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proteins (FimA and FimP) identified in B. longum NCC2705 genome share identity with two major fimbriae subunits of Actinomyces found in the oral cavity, and could be involved in the ability of the bifidobacteria to colonize and interact with the host [20]. Another predicted secreted protein found in B. longum NCC2705, which shares similarity to the serine protease inhibitor (serpin) family, is being currently investigated for its possible role in inflammatory responses of the host, for instance during infection, and has been the basis of a recent patent [20, 104]. This information has also lead to patent No. US20040126870, which discloses other potential applications derived from the genome and plasmid sequence of B. longum NCC2705, including a method of producing polypeptides of said strain as well as food and pharmaceutical compositions containing it or its active components for the prevention and/or treatment of diarrhea brought about by rotaviruses and pathogenic bacteria [105]. Advances in the identification of structural constituents, secreted products or metabolites produced by commensal bacteria, which interfere with virulence factors of pathogenic microbes, are also the basis of novel applications of probiotics against gastrointestinal pathogens, as exemplified in patent No. US20070036776 [106]. This patent relates to bacterial signaling molecules (proteins, peptides and amino acids) produced particularly by Lactobacillus strains GR-1 and RC-14, which down-regulate pathogenic bacterial virulence properties. These bioactive compounds act by down-regulating virulence properties of pathogenic organisms and/or enhancing host defenses and, therefore, could be used to treat and prevent microbial-associated infections. The patent shows that presence of lactobacilli cells or their by-products can down regulate fimbria production by pathogenic E. coli strains and modify the expression of exotoxin genes located in the pathogenicity island of the Staphylococcus aureus genome. Novel roles of probiotic strains in gastrointestinal functions (e.g. neuromuscular function) are also being discovered in recent years, thereby broadening their range of applications against infections. In this context, patent No. US2007128178 describes the role of probiotics and, particularly, of the strain Lactobacillus paracasei (NCM I-2116) in the normalization of the post-infectious hypercontractile state of the bowel muscles after nematode infection of the gastro-intestinal tract. Thus, this and other strains may be used for preventing or treating neuro-muscular disorders of intestines, caused by infection of the intestinal tract by pathogens [107]. Combination of probiotic strains with other biologic and pharmaceutical products also constitutes the basis of new patents that intend to improve the efficacy of the probiotic alone. Patent No. WO 2007136719 includes a mutant E. coli probiotic strain that is resistant to an antibiotic that acts as a pharmaceutical carrier [108]. The combination of both therapeutic agents is acknowledged for its improved efficacy against diverse intestinal disorders as compared with the original probiotic. Patent No. WO 2009092810 is based on a similar principle and includes the use of antibiotic and probiotic combinations in a timecontrolled manner in single pharmaceutical composition [109]. The pharmaceutical composition delivers at first step the antibiotic that exerts the effect against the infectious agent and, then, the probiotic. The time-controlled release of the probiotic after the antibiotic enables it rapid multiplication to suppress further pathogenic activity between two pharmacocinetic cycles. All in all, the effects of probiotics against gastrointestinal infections, alone or in combination with other drugs, are promising as shown in the large number of patents that claim these benefits. However, a larger number of well-designed placebo-controlled trials in
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humans are still necessary to provide solid evidence of the in vivo effects of the strains included in patent applications, following the criteria of FAO/WHO [4]. In addition, advance in the knowledge of novel bioactive molecules and the intricate host-microbe dialogues within the intestine and extraintestinal sites will be critical for the future development of a new generation probiotic-based products targeting more specific pathologies and their etiologic agents. ACKNOWLEDGEMENTS This work was supported by grants AGL2008-01440/ALI and Consolider Fun-C-Food CSD2007-00063 from Ministry of Science and Innovation (MICINN, Spain). I. Nadal is recipient of a scholarship from Generalidad Valenciana (Spain) and E. Sánchez was recipient of a contract from the PTR95-0987.OP.01 grant (MICINN, Spain) and a scholarship from DANONE Institute. COMPETING INTEREST STATEMENT The authors declare that they have no competing financial interests. REFERENCES [1] [2] [3] [4]
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Insights into the Treatment of Helicobacter pylori Infection Campo Salvatore Maria Antonio*, Hassan Cesare, Burza Maria Antonietta, Ridola Lorenzo, Cristofari Francesca, Morini Sergio and Zullo Angelo Gastroenterology and Digestive Endoscopy, “Nuovo Regina Margherita” Hospital, Rome, Italy Abstract: Helicobacter pylori infection is a widespread disease causing significant morbidity and mortality, including non-ulcer dyspepsia, peptic ulcer, lymphoma and cancer of the stomach. Moreover, different extra-digestive diseases have been related to such an infection, but only for idiopathic thrombocytopenic purpura and a specific form of iron deficiency anaemia there are reliable data. Several information are available on its pathogenetic mechanisms, as well as on therapeutic approaches. Different regimens have been suggested as first-line treatment, but therapy is still a challenge, no treatment achieving bacterial cure in all cases. New drugs and different treatment schedules have been proposed to cure such an infection. Several new antibiotics have been claimed in the last 5 years, and some of them showed a very powerful antibacterial activity in vitro, even against clarithromycin and metronidazole resistant strains. Among the new compounds, thienylthiazole derivatives, benzamide derivatives and pyloricidins should be regarded as very promising molecules. Novel interesting therapeutic approaches with the highest eradication rate are also described.
Keywords: Antibiotics, Helicobacter pylori, therapy.
1. INTRODUCTION Helicobacter pylori infection is a long-lasting, transmissible, worldwide spread infection which is involved in the pathogenesis of different gastroduodenal diseases, such as chronic active gastritis, duodenal and gastric ulcers, and gastric neoplasia [1]. H. pylori is responsible for virtually all duodenal and gastric ulcers, when non-steroidal anti-inflammatory drugs (NAID)-associated lesions are excluded. Several studies have recognised that H. pylori may cause gastric low-grade MALT-lymphoma in genetic susceptible patients, and a complete regression has been reported following bacterial eradication when such a neoplasia is in the early stage [2]. Furthermore, H. pylori is a proved environmental risk factor for gastric carcinoma, and it has been recognised as a definite type I carcinogen [3]. Although the role of this infection in non-ulcer dyspepsia is still debatable, two comprehensive *Corresponding author: Tel: 0039 06 58446608; Fax: 0039 06 58446533; E-mail:
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reviews showed a small, but significant, benefit of eradicating H. pylori in such patients [4,5]. Moreover, a prospective, very large study found a long-term advantage in curing such an infection in these patients [6]. The clinical manifestations of H. pylori infection mainly depend on the interaction between the host and the bacterium, being a genetic predisposition, an early age of infection, and the presence of concomitant risk factors (i.e. bile reflux in stomach) on one hand, and virulent factors (i.e. CagA cytotoxin), bacterial density or distribution in the gastric mucosa (i.e. confined in the antrum or involving the whole stomach), on the other, important factors involved in the disease development [7-9]. A putative pathogenetic role of H. pylori infection has also been claimed in several extradigestive diseases, such as vascular (ischaemic heart disease, Raynaud phenomenon, headache) autoimmune (Sjogren’s syndrome, Henoch-Schonlein purpura, autoimmune thyroiditis, idiopathic arrhythmias, Parkinson’s disease, non-arterial anterior optic ischaemic neuropathy) and skin disease (chronic idiopathic urticaria, rosacea, alopecia areata), as well as in growth retardation, late menarche, sudden infant death syndrome, and hepatic encephalopathy [10,11]. Nonetheless, excluding both iron deficiency anaemia and idiopathic thrombocytopenic purpura for which the link seems to be reliable [12,13], the available data between the association of H. pylori and these diseases are still largely inconclusive. H. pylori treatment still remains a challenge for the physicians, no therapy being able to cure the infection in all cases. Therefore, perspectives on different therapeutic alternatives to cure H. pylori infection and on the appropriate use of various agents as well as on novel molecules − some of which will be probably available in next future − were reviewed.
2. HELICOBACTER PYLORI TREATMENT PECULIARITIES H. pylori has evolved several efficacious mechanisms to enable the colonization of human gastric epithelium. Due to its production of urease and the presence of 3−7 flagella, it is able to survive over a wide pH spectrum and to efficiently penetrate the gastric mucous layer reaching the underlying gastric epithelium, where it can be found strongly attached to cells and even within cells [14]. Gastric juice has also been shown to contain viable H. pylori organisms, even when pH is below 3.0. These gastric luminal bacteria are transiently attacked with oral antibiotics, but are probably best treated by drugs that are secreted in gastric juice. The pH near the lumen of the stomach is maintained at 2.0, whereas the cellmucus interface is less acid, with a pH of approximately 5.5. Only very few antibiotics are active in both these pH extremes. Indeed, several antibiotics, which own a strong bactericidal efficacy in vitro, are readily inactivated by the low pH values encountered in the stomach. Moreover, oral agents reach very high concentrations in gastric mucus, but levels quickly fall as the stomach empties after a meal. These observations clearly demonstrate how it is mandatory to administer drugs able to significantly increase gastric pH − as a proton pump inhibitor (PPI) − together with antibiotics in order to enhance their efficacy [15]. Although the immune system produces antibodies against the bacterium virtually in all infected patients, immonuglobulin activity is largely inhibited by the gastric acid. Moreover, H. pylori is able to minimise the exposure of antigens by masking flagella with lipopolysaccharide, which mimics host carbohydrates. These abilities can explain, at least in part, the complexity in generating an efficient vaccine [16]. Furthermore, due to its motility, the bacterium may be inaccessible to direct contact with immune cells, such as neutrophils,
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which can migrate only slowly across the gastric mucosa. In addition, its catalase activity is able to destroy lysosomal hydrogen peroxide before it can generate the oxygen radicals necessary for a bactericidal effect [17]. H. pylori resistance towards antibiotics is becoming a cause for concern. Indeed, both primary and acquired bacterial resistance against different antibiotics has been clearly demonstrated, and it is increasing in the last decade in several countries [18]. Some studies have suggested that CagA-negative H. pylori strains seem to be less susceptible towards therapy as compared to CagA-positive bacteria [19]. A co-infection with different H. pylori strains is not infrequent, and a mixture of colonies differing in their susceptibility to antibiotics have been also observed [20]. Of note, it has been observed that there is a frequent discrepancy between antibiotic susceptibility in vitro and bacterial eradication in vivo, so that the role of bacterial culture with antibiotic susceptibility assessment in clinical practice has been questioned [21].
3. ACTIVITY OF ANTIBIOTICS Since its discovery, the bactericidal activity of several compounds has been tested against H. pylori either in vitro or in vivo [22]. The initial attempts to cure H. pylori by using a single drug were largely disappointing, and it became evident that a combination of drugs was required to treat such an infection [23]. There are some agents (penicillins, tetracyclines) that lead to secondary antibiotic resistance less frequently than others (macrolides, nitroimidazoles, quinolones) and, therefore, they may be re-used in the same patients with other drug combination. 3.1. Penicillins Undoubtedly, amoxicillin is the most used penicillin for H. pylori therapy. The high susceptibility of H. pylori to amoxicillin in vitro contrasts with the low efficacy of this antibiotic in vivo, with an eradication rate of less than 20% when administered as monotherapy. However, when amoxicillin is administered together with a PPI for 7−14 days, an eradication rate higher than 50−60% may be achieved [24]. Indeed, high concentrations of amoxicillin are obtained in gastric juice and mucosa during oral or intravenous therapy, the molecule being actively secreted into the stomach, especially through the gastric body mucosa [25]. Moreover, besides its bactericidal effect, amoxicillin offers other advantages for H. pylori therapy. It acts destroying the bacterial membrane, and this effect is of paramount importance, even if the bactericidal effect is not directly achieved. In fact, there is evidence that pre-treatment with amoxicillin reduces the onset of secondary clarithromycin resistance [26]. Other penicillins, such as ampicillin, are largely inactivated in the stomach and, therefore, are largely useless for H. pylori therapy. Similarly, the amoxicillin/clavulanic acid combination does not offer any advantage over amoxicillin alone, since H. pylori is not a beta-lactamase producer. By excluding a specific Italian area (Sardinia) and Korea where an amazing 18.5–26% rate was observed [27,28], both primary and secondary amoxicillin resistance is rare worldwide, being estimated to be as low as 0–1.3% in several countries [19].
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3.2. Tetracyclines Tetracycline is acid stable and active at acid pH, achieving high concentrations in the gastric mucosa and exceeding MIC of H. pylori for several hours. It is ineffective in eradicating this infection, but it is helpful for prolonged bacterial suppression. Some evidences suggest that its efficacy is improved by co-administration of bismuth salts. Such a phenomenon seems to be due to a higher concentration of tetracycline achieved on gastric mucosa during bismuth salts therapy, probably due to a “trapping” effect [29]. Indeed, tetracycline is generally administered together with bismuth salts and metronidazole. Although both primary and secondary tetracycline resistances are infrequent [29], high doses (1.5–2 g) should be employed for H. pylori treatment, so that a large number of tablets is needed. This is a major limitation for this drug, only 250 mg/tablets being available in different European countries. Unfortunately, bismuth salts are no more available in some countries, further restricting the use of tetracycline for H. pylori therapy. Kanamycin and minocycline were found to own powerful bactericidal effect against H. pylori strains in vitro [30]. The efficacy of a minocycline-based triple therapy has been also evaluated in both first- and second-line regimens. Unfortunately, the minocyclineamoxicillin combination achieved an eradication rate as low as 38.5% when administered in patients never previously treated for H. pylori, even if primary minocycline resistance was absent [31]. On the contrary, the minocycline-metronidazole triple therapy achieved an acceptably high (82.5%) cure rate as second-line treatment [31]. 3.3. Nitrofurans Nitrofurans may be used as gastric luminal antibacterial agents. As far as H. pylori infection therapy is concerned, furazolidone and nifuratel have been used in combination with other drugs, generally in quadruple therapy as either first-line or a “rescue” treatment. An acceptable high (>85%) eradication rate has been reported in first-line therapy by using a quadruple combination with PPI, bismuth salts, amoxicillin and either furazolidone or nifuratel [32]. This could be due, at least in part, to low H. pylori primary resistance towards these compounds. Indeed, some studies found a furazolidone resistance rate as low as 0– 8.7% [33,34]. Some studies found a high eradication rate following a 14-day furazolidoneamoxicillin-based quadruple therapy as second-line treatment [35], but cure rates as low as 67.6% and 68.8% have been observed in two recent studies [36,37]. Some preliminary data found a very high eradication rate (88%) by using a 7-day triple therapy with furazolidone and levofloxacin as a rescue therapy, but further data are needed [38]. Nevertheless, some crucial ethical concerns arise now-a-days with the use of furazolidone [39]. This antibiotic was used in the 1980s for parasitic infections. However, different studies raised several concerns about this agent and its potential for causing tumors [40]. Nevertheless, the drug continues to be available in some developing countries such as Iran, Pakistan, India, Mexico and Brazil, while both FDA and EMEA (the equivalent of the FDA in the European Union) withdrew approval for furazolidone in 2005 in the United States and Europe, respectively. Finally nifuratel seems to be better tolerated than furazolidone, a prevalence of side-effects of 3% and 21%, respectively, being reported in a comparative study in children [32]. Nitrofurans offer the advantage of a low cost but, unfortunately, their use is not allowed in European countries.
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3.4. Bismuth Salts Bismuth salts (subcitrate and subsalicilate) were the first molecules successfully used as a monotherapy for H. pylori eradication in peptic ulcer patients [41]. They act detaching the organism from the mucosa and causing their lysis. These compounds have been generally used as a first-line triple therapy with tetracycline and metronidazole for 2 weeks (American therapy) or as a second-line quadruple therapy with the same antibiotics and PPI for 7 days (European therapy) [42]. Because of its short-half life in gastric mucus, bismuth should be administered frequently, at least 3–4 times daily. Unfortunately, bismuth compounds – including ranitidine bismuth subcitrate – are no more available in several European countries due to their potential neurotoxic effect. Indeed, a study found that 9% of patients receiving the quadruple regimen had very high blood bismuth concentrations within the Hillemand alarm level [43]. 3.5. Macrolides Currently, clarithromycin is the most powerful drug against H. pylori, with MIC values as low as 0.01 mg/L [18]. Like to amoxicillin, clarithromycin is actively secreted in the gastric juice [22]. A dual therapy with PPI or ranitidine bismuth subcitrate and clarithromycin 1 g daily was proposed for 2 weeks in the nineties, with a cure rate of 60– 80%. However, it was observed that secondary clarithromycin rapidly developed in all eradication failure patients. Moreover, primary clarithromycin resistance is remarkably increased in the last five years worldwide, being quoted as high as 10% (range: 2−25%) [19], and constantly higher than 15% in more recent evaluations [44], even in children [45]. Therefore, clarithromycin (500 mg twice daily) is currently used only in triple or quadruple combination for 7–14 days. Azithromycin (500 mg u.d for 3 days) has been used instead of clarithromycin as triple therapy in combination with PPI and either amoxicillin [46], tinidazole [47], or levofloxacin [48] administered for 7 days. Moreover, it has been administered as quadruple therapy with PPI, bismuth and amoxicillin. However, all these studies found eradication rate <75%. Therefore, the use of this macrolide does not offer any advantage as compared to clarithromycin, further considering that a 32.3% primary resistance rate has been recently reported [48]. Erythromycin is available as base, stearate, ethylsuccinate, and erythromycin estolate. Although very low MIC values have been observed in vitro, H. pylori eradication rates achieved in vivo were low, mainly due to the high instability of erythromycin at low pH values. It has been used as enteric-coated erythromycin base delivered as small granules within a capsule and erythromycin-base-film-coated tablets, but 8 tablets daily are required to achieve a 80% eradication rate in quadruple therapy [49], heavily limiting the compliance of patients. 3.6. Nitroimidazoles Nitroimidazoles are active drugs for anaerobic infections and differ from each other in dosage and half-life. They act by altering membrane redox potential. Contrarily to other antibiotics, nitromidazoles are quite stable in the gastric juice, and this characteristic is of paramount importance for H. pylori therapy. Among imidazole-derivatives, both metronidazole and tinidazole are largely used for H. pylori treatment as triple or quadruple therapy [15]. As compared to metronidazole, tinidazole has a longer half-life [50], and such
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a pharmacological characteristic seems to be useful for H. pylori treatment [51]. Some data suggest that the clarithromycin-imidazole is more effective than amoxicillin-imidazole combination. However, primary metronidazole resistance is very high, with values ranging from 20% to 40% both in Europe and USA, and with values as high as 70–80% in developing countries, especially in female [18]. This depends on the use of nitromidazoles for parasites or vaginal infection. Finally, imidazoles cause an alcohol intolerance which seems to be independent from a disulfiram-like action [52]. Consequently, alcohol use should be discouraged during eradication therapy including an imidazole. 3.7. Quinolones Several quinolones exert in vitro a bactericidal action against H. pylori. However, some of these, such as ciprofloxacin or norfloxacin, are largely inactivated in gastric juice. Undeniably, the most experienced fluoroquinolone is levofloxacin. It has been used both for first- and second-line therapy, in a triple combination with either amoxicillin, tinidazole or clarithromycin, generally achieving acceptably high eradication rates [53, 54]. Unfortunately, levofloxacin is quite expensive, and H. pylori primary resistance seems to be increasing worldwide, with a prevalence ranging from to 9.7% to 21% [55,56]. Of note, a recent study found that prevalence of levofloxacin resistance was significantly higher in those bacterial strains simultaneously owing either clarithromycin (44% vs. 15%) or metronidazole (40% vs. 13%) resistance as compared to those susceptible [47]. Therefore, levofloxacin-based triple therapy should be reserved as a “rescue” treatment [57], being significantly more effective than standard quadruple therapy [58,59]. Indeed, current Italian guidelines on H. pylori management advise a 10-day levofloxacin-amoxicillin-based triple regimen as a second-line therapy in clinical practice [60]. Moxifloxacin-based triple therapies either with clarithromycin [61], tinidazole or amoxicillin [62] have been also proposed as first-line H. pylori treatment. All these regimens achieved an eradication rate >90%, but data of only two hundred patients, all enrolled in a single centre, are available. Moreover, it should be considered that moxifloxacin is similarly expensive to levofloxacin. Akin to levofloxacin, a moxifloxacinbased triple therapy has been recently tested as second-line regimen with an eradication rate significantly higher as compared to a quadruple therapy [63]. Interestingly, garenoxacin administered alone for 14 days at oral doses of 400 mg demonstrated activity against H. pylori, and it was safe and well tolerated [64], whilst not impressive results have been observed by using a gatifloxacin-amoxycillin combination [65,66]. In addition, gatifloxacin was eventually withdrawn in 2006 for safety reasons and it is no longer available. 3.8. Rifamycines Some rifamycin derivatives, such as rifampin, rifaximin and rifabutin have been shown to exert an in vitro activity against H. pylori isolates [67]. In detail, rifampin showed a high bactericidal activity with MIC90 values ranging 0.032 – 2 mg/l, and no primary bacterial resistance was found among 81 clinical isolates, including those strains harbouring both clarithromycin and metronidazole resistance [67]. Unfortunately, resistant mutants emerged after repeated exposures. Rifaximin was proven to be effective in vitro, although their bactericidal activity was quite low (MIC50 = 4 mg/l), and without a significant synergism with other antibiotics [68]. Indeed, high rifaximin doses (1800 mg/day) gave disappointing
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results when administered for 2 weeks combined with either omeprazole [69] or with omeprazole plus amoxicillin [70]. A therapeutic approach based on a rifabutin–amoxycillin combination has been proposed for patients who failed two or more courses of therapy, with an eradication of 71–86.6% after a 7-day regimen [71-72] and 90% following a 12-day therapy [73]. However, this is an expensive antimycobacterial drug particularly useful for tuberculosis treatment (e.g. in AIDS patients) and, therefore, resistance development for this drug should be avoided as much as possible. Moreover, the onset of myelotoxicity has been recently observed following rifabutin-based regimen for H. pylori eradication, suggesting more caution in this approach [74]. Therefore, such a drug should be employed after failure of other therapeutic approaches, exclusively in patients with multi-resistant strains in whom H. pylori cure is strongly indicated (MALT-lymphoma, complicated peptic ulcer, etc.) [60]. 4. CURRENT HELICOBACTER PYLORI TREATMENTS 4.1. Standard Triple Therapies A 7-day triple therapy, comprising a proton pump inhibitor, clarithromycin and amoxycillin (French therapy) or tinidazole (Italian therapy), is advised as a first-line therapy in the current European guidelines for those geographic areas where primary clarithromycin resistance is lower than 15-20%, whilst a 14-day regimen is suggested if the resistance is higher [42]. Nevertheless, the eradication rate following a standard 7-day triple therapy is falling worldwide, with a cure rate as low as 45% in some studies, and frequently lower than 70% in different countries [75-77]. Moreover, the suggestion of prolonging triple therapy duration does not appear to offer an impressive advantage. Indeed, a recent meta-analysis found that only a minor therapeutic gain can been achieved by prologing the standard triple from 7 to 14 days, the eradication rate being 72.8% and 78.2%, respectively, whilst the cost of therapy is doubled [78]. Since the efficacy of first-line therapy strongly affects the cost of H. pylori infection management − by reducing the need of both second- or even a third-line re-treatment and re-testing [79] − much effort has been performed during recent years in order to identify better therapies. Both concomitant and sequential regimens have been proposed to overcome the decreasing efficacy of standard triple therapies. 4.2. The Concomitant Therapy The concomitant therapy is based on the simultaneous use of clarithromycin, amoxicillin and metronidazole together with a proton pump inhibitor administered for 3−7 days [80]. A recent meta-analysis pooling data of 771 patients found an eradication rate of 89.7% and 92.9% at intention to treat and per protocol analyses, respectively [80]. Possible limitations of such a quadruple therapy are the high number of tablets needed, the high incidence of side-effects, which occur in 27−51% of cases, and 3 cases of anaphylaxis to medication have been observed [80]. Moreover, no data on a second-line treatment in those patients who failed the concomitant therapy are currently available. All these aspects should be opportunely considered when dealing with a widely spread infection. 4.3. The Sequential Therapy In order to improve the eradication rate of standard triple therapies, Zullo et al. conceived a different combination of the available antibiotics consisting in a novel 10-day
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sequential regimen [81]. This schedule is a simple dual therapy (PPI plus amoxycillin) given for the first 5 days followed by a triple therapy (PPI, clarithromycin, and tinidazole) for the remaining 5 days. Such a therapy regimen was proved to be highly effective in different multicenter studies, with an eradication rate constantly higher than 90% in more than 1.800 treated patients [82]. Interestingly, sequential therapy was equally effective and well tolerated in adult, elderly and paediatric patients [82]. Moreover, the cure rate was independent from gastroduodenal disease, smoking habit, and CagA status which are known to lower the efficacy of standard triple therapies [18]. In addition, although primary clarithromycin resistance reduced the eradication rates, the sequential therapy was found more effective than triple therapy even in those patients infected with resistant strains [83]. Such a phenomenon most likely depend on the use of amoxicillin − to which resistance is rare − in the initial therapeutic phase. It is known that bacteria can develop efflux channels for clarithromycin, which rapidly transfer the drug out of the bacterial cell, preventing binding of the antibiotic to the ribosome [84,85]. Because amoxicillin acts on the bacterial cell wall and damages it, the initial phase of treatment may prevent the development of efflux channels by weakening the cell wall of the bacterium (Fig. 1). Moreover, it has been found that regimens containing amoxicillin prevent the selection of secondary clarithromycin resistance [13]. In detail, the sequential therapy is successful even against those clarithromycin resistant strains harbouring the A2143G point mutation which markedly reduces the efficacy of standard triple therapy [86]. Moreover, the cure rate following sequential therapy is not affected by the presence of isolated metronidazole resistance, whilst it seems unsuccessful in the presence of double (clarithromycin and metronidazole) resistance [83]. Recent data suggest that the sequential regimen including tinidazole achieves higher eradication rate as compared to that with metronidazole [51]. In addition, when drug combination used in the sequential therapy has been changed including tetracycline or gatifloxacin instead of clarithromycin, the eradication rates were disappointing [66,87]. Finally, H. pylori eradication has been obtained in more than 85% of patients who failed the sequential regimen following a triple therapy with levofloxacin and amoxicillin [88]. Therefore, the 10-day sequential therapy as first-line regimen and the 10day levofloxacin-amoxicillin as a second-line therapy would appear as an effective therapeutic “package” for H. pylori management in clinical practice.
5. NEW ANTIBIOTICS In the last 5 years, several compounds owing antibacterial activity in vitro against H. pylori have been claimed, and few of these novel antibiotics have been also experienced in vivo either in animal models or humans. In this field, quinolones certainly remain the most investigated drugs. It has been observed that both sparfloxacin and tosulfloxacin exhibit a similar bactericidal activity in vitro against H. pylori, including clarithromycin and metronidazole resistant strains, with the same MIC90 of levofloxacin [54]. Moreover, both sitafloxacin and clinafloxacin were tested for activity, showing MIC90 values as low as 0.008 and 0.12 mg/L, respectively [54]. Finally, a new fluoroquinolone (Y-904) was found to own an in vitro antibacterial activity similar to that of clarithromycin, showing an equal strong activity at pH 5.5 and 7.0 [89]. Interestingly, this drug cleared H. pylori infection in Mongolian gerbils with a potency 30-fold higher than that of clarithromycin and levofloxacin. The in vitro activities of mupirocin, quinupristin/dalfopristin, linezolid, eperezolid, have been tested [90]. Among these drugs, only mupirocin was very active at both pH 7.4 and 5.4
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with MIC90 of 0.25 and 0.12 mg/L, respectively. Among new molecules, the efficacy of novel polycyclic compounds against some H. pylori strains has been investigated in vitro
Fig. (1). Mechanism of action of sequential therapy. (A) When clarithromycin is administered firstly, both point mutations on ribosomal RNA and efflux channel system on bacterial wall contrast its action. (B) If amoxicillin is administered firstly, it destroys bacterial wall and damages pump efflux favouring clarithromycin concentration in the bacterium.
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[91]. These agents showed MIC values of 0.20-0.39 mg/L, exhibiting a similar efficacy of tetracycline. The antibacterial activity of a new benzamide derivative (BAS-118) against H. pylori has been investigated [92]. Of note, this molecule showed in vitro MIC90 values as low as 0.013 mg/L, without a significant difference between strains with either clarithromycin or metronidazole resistance and those susceptibles. It has been demonstrated that a new family of natural antibiotics (pyloricidin A, B, and C) exhibits a potent and highly selective bactericidal activity against H. pylori with a MIC90 value of 0.013 mg/L [93]. Recently, several arylthiazole analogues have been tested showing a potent bactericidal effect against H. pylori. Among these compounds, thienylthiazole derivative-44 exhibited the strongest activity, with MIC90 values as low as 0.0065 mg/L [94]. In addition, such compounds cleared the infection in 60% of Mongolian gerbils upon 7-days, twice daily, oral administration as monotherapy. A novel cephem derivative (FR193879 8a) has been found to exert a potent therapeutic efficacy against H. pylori, showing an excellent safety in dog [95]. Bismuth salt of antibiotics of the moenomycin group (the so called phophoglycolipid antibiotics) have been tested in vitro, but the MIC90 value (0.5 mg/L) against H. pylori has been not impressive.
6. CONCLUSIONS H. pylori infection is a worldwide spread disease which causes several, and potentially lifethreatening, gastroduodenal disease. Although it remains asymptomatic in a large percentage of subjects, several patients undoubtedly need to be currently treated for such an infection. The treatment is mainly based on a combination of PPI together with 2 or 3 different antibiotics. However, the therapy regimens suggested have given disappointing eradication rates in several countries. To find out new drugs in order to improve H. pylori eradication rate is of a paramount importance in primary medical care. Indeed, the management cost of this infection deeply depends on the efficacy of first-line therapy, the “rescue” treatments being generally more expensive and less effective. Prolonging the length of treatment beyond 7 days was aimed at improving the disappointing eradication rates with standard triple therapy. A novel 10-day sequential regimen has been recently received more attention. Interestingly, this simple dual 5 days-therapy regimen (PPI plus amoxicillin) followed by a triple 5 days-therapy (PPI, clarithromycin and tinidazole) was proven to be highly effective for H. pylori eradication. Several new antibiotics have been claimed in the last years, some of which showed a very potent antibacterial activity in vitro. Different molecules among quinolones have been tested, with preliminary interesting results. Among new patents, both benzamide derivatives and pyloricidins should be regarded as very promising molecules, due to their high efficacy even towards clarithromicyn and/or metronidazole resistant strains. Moreover, a thienylthiazole derivative showed a very impressive anti-bacterial activity against H. pylori, showing the strongest activity in vitro. Other molecules have been found to exibhit a similar efficacy at both neutral and pH 5, and this characteristic is a clear advantage for H. pylori therapy, since antibiotics must act in the gastric juice. Finally, some compounds seem to be safe and effective also in vivo, although data are still preliminary. In conclusion, several interesting patents have been claimed in the last 5 years and, therefore, it is foreseable that the arsenal against H. pylori will be increased in the next future.
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Zullo A, Rinaldi V, Winn S, et al. A new highly effective short-term therapy schedule for Helicobacter pylori eradication. Aliment Pharmacol Ther 2000; 14: 715-718. Zullo A, De Francesco V, Hassan C, Morini S, Vaira D. The sequential therapy regimen for Helicobacter pylori eradication. Gut 2007; 56: 1353-1357. Vaira D, Zullo A, Vakil N, et al. Sequential therapy versus standard triple-drug therapy for Helicobacter pylori eradication. A randomized trial. Ann Int Med 2007;146: 566-573. Paulsen IT, Brown MH, Skurray RA. Proton-dependent multidrug efflux systems. Microbiol Rev 1996; 60: 575-608. Webber MA, Piddock LJ. The importance of efflux pumps in bacterial antibiotic resistance. J Antimicrob Chemother 2003; 51: 9-11. De Francesco V, Margiotta M, Zullo A, et al. Clarithromycin-resistant genotypes and eradication of Helicobacter pylori. Ann Intern Med 2006; 144: 94-100. Uygun A, Kadayifci A, Yesilova Z, Safali M , Ilgan S, Karaeren N. Comparison of sequential and standard triple-drug regimen for Helicobacter pylori eradication: a 14-day, open-label, randomized, prospective, parallel-arm study in adult patients with non ulcer dyspepsia. Clin Ther 2008; 30: 528-534. Zullo A, De Francesco V, Hassan C, et al. Second-line treatment for Helicobacter pylori eradication after sequential therapy failure: a pilot study. Therapy 2006; 3: 251-254. Iwao E, Yokoyama Y, Yamamoto K, Hirayama F, Haga K. In vitro and in vivo anti-Helicobacter pylori activity of Y-904, a new fluoroquinolone. J Infect Chemother 2003; 9: 165-171. Sanchez JEG, Saenz NG, Rincon MR, et al. Susceptibility of Helicobacter pylori to mupirocin, oxazolidinones, quinupristin/dalfopristin and new quinolones. J Antimicrob Chemother 2000; 46: 283-285. Careanague D, Shue YK, Wounola MA, et al. Novel structures derived from 2-[[(2-pyridyl)mathyl]thio]1H-benzimidazole as anti-Helicobacter pylori agents, Part 2. Med Chem 2002; 45: 4300-4309. Koboyashi I, Muraoka H, Hasegawa M, et al. In vitro anti-Helicobacter pylori activity of BAS-118, a new benzamide derivative. J Antimicrob Chemother 2002; 50: 129-132. Hasuoka A, Nishikimi Y, Nakayama Y, et al. Synthesis and anti-Helicobacter pylori activity of pyloricidin derivatives II. The combination of amino acid residues in the dipeptidic moiety and its effect on the antiHelicobacter pylori activity. J Antibiot 2002; 55: 499-507. Katsura Y, Nshino S, Inoue Y, et al. Anti-Helicobacter pylori agents. 5.2-(Substituted guanidino)-4arylthiazoles and aryloxazole analogues. J Med Chem 2002; 45: 143-150. Yoshida Y, Matsuda K, Sasaki H, et al. Studies on anti-Helicobacter pylori agents. Part 3: A novel, efficacious cephem derivative, FR193879. Bioorg Med Chem Lett 2004; 14: 2627-2631.
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Inhibitors of Bacterial Efflux Pumps as Adjuvants in Antibacterial Therapy and Diagnostic Tools for Detection of Resistance by Efflux Françoise Van Bambeke*,1, Jean-Marie Pagès2 and Ving J. Lee3, 4 1
Unité de Pharmacologie Cellulaire et Moleculaire, Université Catholique de Louvain, Brussels, Belgium; 2EA2197 Enveloppe Bactérienne, Perméabilité et Antibiotiques, Faculté de Médecine, Université de la Méditerranée, Marseille, France; 3Adesis, Inc., New Castle, DE 19720, USA; 4Limerick BioPharma, Inc., South San Francisco, CA 94080 USA Abstract: Active efflux is a wide-spread mechanism for bacterial resistance to antibiotics, which contributes to poor intrinsic susceptibility, cross-resistance to structurally diverse classes of drugs, or selection of other mechanisms of resistance. Thus, inhibition of efflux pumps appears to be (i) a promising strategy for restoring the activity of existing antibiotics, and (ii) a useful method to detect the presence of efflux determinants in clinical isolates. Structurally dissimilar classes of inhibitors have been patented in the last decade, some are analogues of antibiotic substrates [tetracyclines, quinolones or aminoglycosides] and others are new chemical entities [including substituted indoles, ureas, aromatic amides, piperidinecarboxylic acids, alkylamino- or alkoxyquinolines, peptidomimetics, and pyridopyrimidines]. Their spectrum of activity, in terms of companion antibiotics and bacteria, differ significantly. Narrow spectrum inhibitors are of prime interest as diagnostic tools, while broad spectrum inhibitors are expected for adjuvant therapies. Apart from (i) a peptidomimetic inhibitor of Mex pumps in Pseudomonas aeruginosa (MC-04,124), for which efficacy was evaluated in animal models, and (ii) a piperidinecarboxylic acid inhibitor of fluoroquinolone efflux in Gram-positive (VX-710), which was safely administered to humans, most of these products have only demonstrated their activity in vitro, so further investigations are needed to evaluate their clinical potential.
Keywords: Efflux pumps, resistance, S. aureus, S. pneumoniae, H. influenzae, E. coli, P. aeruginosa, E. aerogenes, reserpine, indoles, ureas, aromatic amides, piperidine-carboxylic acid derivatives, quinolines, peptidomimetics. GENERAL DESCRIPTION OF ANTIBIOTIC EFFLUX PUMPS IN BACTERIA AND IMPACT IN ANTIBACTERIAL THERAPY Active efflux was first described in 1980, as a causative mechanism of resistance to tetracyclines [1]. It has subsequently been found to be a widespread mechanism that confers to both Gram-positive and Gram-negative organisms the capacity to expel antibiotics from all the major structural classes ([2-4] for reviews). More recent studies, however, suggest that antibiotics are only opportunistic substrates of these physiological transporters, with *Corresponding author: Tel: +32-2-7647378; Fax: +32-2-7647373; E-mail:
[email protected] Atta-ur-Rahman / M. Iqbal Choudhary (Eds.) All rights reserved – © 2010 Bentham Science Publishers.
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efflux pumps also playing a major role in the extrusion of poorly diffusible endogenous molecules [5-7] and protection of bacteria against exogenous, potentially harmful, diffusible substances [8-10]. In this context, antibiotics have probably provided the necessary pressure that selects for efflux pump overexpression as a non-specific mechanism of resistance ([11] for a review on the regulation of the expression of efflux pumps by antibiotics and other pump substrates). Phylogenetically, bacterial antibiotic efflux pumps belong to five superfamilies (see
for classification and [12,13] for reviews and application to antibiotic transporters), namely (i) ABC (ATP Binding Cassette), which are primary active transporters energized by ATP hydrolysis, and (ii) SMR (Small Multidrug Resistance subfamily of the DMT [Drug/Metabolite Transporters] superfamily), (iii) MATE (Multi Antimicrobial Extrusion subfamily of the MOP [Multidrug/Oligosaccaridyllipid/Polysaccharide flippases] superfamily), (iv) MFS (Major Facilitator Superfamily) and (v) RND (Resistance/Nodulation/Divison superfamily), which are all secondary active transporters driven by ion gradients. Since these pumps are discussed in details in recent reviews (topology, presence in bacterial species, main substrates [2,3,13,14]), we will focus here on the elements pertinent for the present review, namely antibiotic transport in clinically-relevant pathogens. Table 1 lists the main transporters identified so far in frequently encountered human pathogens, together with the main antibiotic classes they transport. It is clear that MFS and RND are the most abundant pumps, with MFS found in both Gram-positive and Gram-negative bacteria, and characterized by a narrow spectrum (recognizing usually one, and sometimes a few, antibiotic classes), and RND found exclusively in Gram-negative bacteria and displaying an extremely wide spectrum (recognizing usually several classes of antibiotics [from 2 to 7] together with other pharmacological agents like antiseptic compounds, dyes, or detergents [15-17]). Of note, ABC transporters, which play a major role in drug resistance in eukaryotic cells [18], are lesser known in bacteria (MsrD and PatA/PatB in S. pneumoniae [19, 20]; MsrA and Vga in S. aureus [21-23]). Active efflux usually confers a moderate level of resistance (1- to 64-fold increase in MIC upon expression of efflux pumps, both in laboratory mutants and clinical isolates; see [24-29] for a few examples). Nevertheless, it markedly affects the response of bacteria to antibiotics. Potential consequences of antibiotic active efflux have been discussed extensively elsewhere ([13, 16] for reviews) and can be summarized as follows: •
Apparent poor permeability of antibiotics in some bacteria has been attributed to the constitutive expression of efflux pumps, which confers a natural resistance to unrelated antibiotics [30]. This is best exemplified in Pseudomonas aeruginosa, in which disruption of the gene encoding the MexB efflux pump makes these mutants hypersusceptible to chloramphenicol, fluoroquinolones, tetracyclines or β-lactams [31].
•
Cross-resistance to unrelated antibiotic classes can be observed in bacteria expressing pumps with broad substrate specificity, like RND [32]. Thus, exposure to a given antibiotic may select resistance to other classes by triggering the overexpression of these pumps. Further, efflux pumps can transport antiseptic compounds, with similar consequences in terms of cross-resistance or selective pressure [15,33]. In addition, common regulators for independent mechanisms of resistance have been described, so that exposure to an antibiotic that is not subject to
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Table 1.
Van Bambeke et al.
Principal Efflux Pumps Expressed in Selected Human Pathogens and their Main Antibiotic Substrates (Adapted from [3] and [13])
ABC
MdeA
+
NorA
+
NorB
+
+ + +
MsrD
+
PatA/PatB MFS
+
MefA
+
MefE
+
PmrA
+
Tet K-L H. influenzae
E. coli
+
MATE
hmrM
MFS
TetB,K
RND
AcrAB-TolC
ABC
MacAB-TolC
MATE
YdhE
MFS
Bcr
+
Dep
+
+ + +
+
+
+ +
ErmAB-TolC
+
+
+ +
+
Fsr MdfA
Chloramphenicol
+
Tet K-L, Tet38 S. pneumoniae
Sulfamides
+
Vga MFS
Trimetoprim
MsrA
Tetracyclines
ABC
Lincosamides / streptogramin A
S. aureus
Macrolides
Efflux Pump
Fluoroquinolones
(Super) Family
Aminoglycosides
Organism
β-lactams
Antibiotics
+ +
+
+
+
+
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(Table 1) Contd…..
SetA
+
Tet A-E
+
Ycel
+
YidY
+
YebQ RND
AcrAB-TolC
+ +
AcrAD-TolC AcrEF-TolC
P. aeruginosa
ErmE
MFS
Tet A, C, E
+
+
+
+
+
+
+
+
+
+
+
+ +
YegN SMR
+
+
CmlA RND
+
MexAB-OprM
+
+
+
+
+
MexCD-OprJ
+
+
+
+
+
+
+
+
MexEF-OprN
+
MexJK-OprM MexXY-OprM E. aerogenes
Chloramphenicol
Sulfamides
Trimetoprim
Tetracyclines
Lincosamides / streptogramin A
Macrolides
Efflux Pump
Fluoroquinolones
(Super) Family
Aminoglycosides
Organism
β-lactams
Antibiotics
+
+
+
+
+
+
+
+
MFS
CmlB
+
RND
AcrAB-TolC
+
+
+
+
EefABC
+
+
+
+
efflux can trigger overexpression of efflux pumps. As an example, the expression level of the marA regulator, which is involved in the genetic control of membrane permeability via porin and AcrAB-TolC efflux pump expression, can be affected by imipenem in Enterobacter aerogenes, so that exposure to this carbapenem, which is not a substrate for the pump, is accompanied by a loss in susceptibility to quinolones, tetracycline, and chloramphenicol [34].
142 Frontiers in Anti-Infective Drug Discovery, 2010, Vol. 1
Van Bambeke et al.
•
Wide spectrum or high level resistance can be observed in bacteria in which active efflux and other mechanisms of resistance function synergistically. This is exemplified in an Escherichia coli strain that concomitantly expresses β-lactamase and efflux pumps, and is therefore insensitive also to β-lactams resisting enzymatic hydrolysis [29]. Likewise, the combination of target mutations and of active efflux increases the level of resistance to fluoroquinolones [35]. Combination of poor influx (due to modification of porins) and increased efflux is also responsible for a significant loss of antibiotic susceptibility [36].
•
Selection of mutations can be favored in bacteria overexpressing efflux pumps, because antibiotic targets become exposed to subinhibitory concentrations. This has been demonstrated in Pseudomonas aeruginosa, in which disruption of the three main RND efflux pumps is required in order to reduce the appearance of first-step mutants in fluoroquinolone targets (from 10-7 to < 10-11 [28]). Few epidemiological surveys, however, document the respective contribution of efflux and mutations in resistance of clinical isolates. What can be concluded at the present stage is that it is highly variable, depending on the bacteria, the antibiotic class, and the geographic area examined, as exemplified in a study of macrolide resistance in 8 European countries [37].
Natural genetic recombination facilitates the dissemination of efflux-mediated resistance. The expression of resistance usually appears upon mutation(s) in the corresponding regulatory system (see [2] for review) but may also occur following mutations altering substrate specificity of transporters or acquisition of mobile genetic elements expressing non-native pumps (see [38] for review). Genetic elements encoding pumps and their regulators can be located on plasmids or on conjugative or transformable transposons [39]. Moreover, these determinants can be transferred between disparate bacterial species [40]. On these basis, it is not surprising that epidemiological surveys, although often limited to specific populations or geographic areas, report on the high prevalence of efflux pumps in clinical isolates [27,37,41-44]. Accordingly, the importance of efflux as a resistance mechanism in the clinics is acknowledged in various review papers [30,38,45-48]. Thus, strategies aimed at overcoming resistance by efflux are compelling, like the combination of β-lactamase inhibitors with β-lactams to combat resistance in β-lactamase producing pathogens [49]. STRATEGIES TO OVERCOME RESISTANCE BY EFFLUX Bypassing Efflux Pump Mechanisms Even though the molecular determinants responsible for the recognition of antibiotics by efflux pumps have not yet been fully elucidated, differences in transport can be observed between structural analogues within an antibiotic family. In this respect, it is interesting to note that the newer molecules developed from the main antibiotic classes are less susceptible to efflux than older ones, as demonstrated for the third and fourth generation quinolones versus first and second generation quinolones, for ketolides versus macrolides, or for glycylcyclines versus tetracyclines ([13,16] for reviews). Optimizing the structure of a molecule within an antibiotic class by taking into account susceptibility to resistance mechanisms is thus an important design element.
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Biological Inhibition of Active Efflux A strategy to inhibit efflux pump activity could consist of blocking either the proteins themselves, using neutralizing antibodies, or the corresponding genes, by means of antisense approaches. The latter employs antisense oligonucleotides or small interfering RNA (which selectively prevent the transcription of the gene coding for the pump), or other nontraditional antisense molecules, which can interfere with the transcription or the translation of that gene of that RNA. This patented strategy was exemplified for the inhibition of the AcrAB efflux pump in E. coli [50], but its application could be broadened to every pump of known sequence or regulatory mechanism, or for which antibodies can be produced. The usefulness of this strategy is based on the demonstration that deletion of the acrAB gene in E. coli restores its sensitivity to a series of antibiotics [51], while a mutation in its Mar regulator has the opposite effects [52]. This approach is primarily a tool to study the role of efflux pumps in pathogens on antibiotic exposure in vitro, not applicable for therapeutics. Pharmacological Inhibition of Active Efflux A more widely exploited strategy is the development of inhibitors of efflux pumps ([53,54] for recent reviews), which are intended for adjunctive therapy with specific antibiotics. Conceptually, pharmacological inhibition of efflux pumps can be attained by different mechanisms [55]. The dissipation of the energy gradient that drives an efflux pump is a non-specific strategy that will not be discussed here in details. Notable example is the energy decoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP), used for in vitro studies with bacteria efflux pumps, being also extremely toxic to eukaryotic cells. The creation of a perturbation in the outer membrane channel or the assembly of the three proteins constituting the efflux system are strategies restricted to Gram-negative bacteria, where efflux pumps consist of a tripartite protein complex working in concert (the pump itself is located in the inner membrane, and is connected to a channel crossing the outer membrane by an adaptor protein; [56] for review). The induction of a flux-competition in the pump it-self is therefore probably the more general mechanism of action for pump inhibitors. At the present stage, however, few reports are available that study the mode of action of inhibitors with efflux pumps, but the situation should change in the near future, because the first crystal structures of efflux pumps were recently obtained [57-59]. Figs. (1 and 2) show the general structure of the main classes of inhibitors that have been patented so far, and Table 2 lists the most active compounds from various chemotypes and their spectrum of pump inhibitory activity. The first efflux pumps inhibitors were fortuitously discovered from existing drugs. The most popular one is reserpine (1) [60-62], but similar effects were described with the phenothiazines (2) [63], calcium channel antagonists (3) [63,64], selective inhibitors of serotonin re-uptake (4) [65], or proton pump inhibitors (5) [66,67]. A major limitation for combining these drugs with antibiotics is that they need to be used at concentrations significantly higher than that used to exert their pharmacological effects, which makes them unviable for safety reasons. Derivatives devoid of the pharmacological activity of the parent compound are now produced, as described for inhibitors of serotonin reuptake [65,68] or of omeprazole (6) [69,70]. Likewise, natural products-derived inhibitors, such as 5’methoxyhydrocarpin (7) [71,72] or totarol (8) [73] have been reported ([74] for review and [71,75-78] for other examples), but their therapeutic index is sometimes questionable, and their purification, laborious and time-consuming. Semi-synthetic derivatives of these natural
144 Frontiers in Anti-Infective Drug Discovery, 2010, Vol. 1
Van Bambeke et al.
Antibiotic
Tetracyclines
HO
CH3
Patent Authors; Applicants [ref]
Inhibitors
N(CH3)2
R1
OH
R2
N(CH3)2
NH2 OH
OH OH O
O
Levy;
OH
The Trustees of Tufts College [93]
NH2
O
OH
tetracycline
OH OH O
O
O
OH R
O
HO HO
HN
H N
NH O HO
O
OH
R
R R
O
Aminoglycosides
NH
H2N
O
Nelson & Alekhsun;
R
NH2
NH2 O HO
O
HO HO
O
HO HO
OH
HN
OH
R2
OH
OH
OH
H2N O
R3
O
O
NH2
HO HO
O
NH2
Paratek pharmaceuticals, Inc. [97]
NH2
paromomycin
O
HO
O OH
O
OH NHR1 NH2 OR4
O OH NH2
O
Quinolones
F
R5
COOH
R4
R6
N
N
HN
R7
X
N R1
R4a
R2
De Souza et al.; Wockhardt Limited [98]
Y
ciprofloxacin
Fig. (1). General structure of analogues of antibiotics used as inhibitors of bacterial efflux pumps. The figure shows the chemical structure of antibiotics on the left, and the general structure of inhibitors on the right. The parts common between antibiotics and inhibitors are highlighted in bold characters.
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145
products with improved activity have also been described, as exemplified for piperine analogues (9,10) that are 2-4-fold more potent than the parent compounds at 8-fold lower concentrations [79,80], stimulating further research in that direction [81]. Patent Authors; Applicants [ref]
Families Patented R3
R7
R2
R4
R4
R8
R6
R1 N
R5
R9
O
N H
N H
R10
R6
R2
R5
R3
R3
R2
R4
R1
O
N H
R6
R5
R1
Br H3C
O
O
H3C
HN
Marham et al.;
S NH
HN
OH
Influx, Inc. [100] O
INF 392 (40)
INF 277 (39)
R2
R1
Lemaire et al.; Université C. Bernard Lyon; Ecole national supérieure de chimie de Paris; CNRS [104]
R3
R4
S
B J
N
J
E
A
N
O
O
B
K A
K
D
(CH2)n
O Y R 1 R2
Z
D
Grossman; A
B O
K
J
N
X
N O
J
(CH2)n
B
K
R1 D O
O
X
N O
O
Vertex Pharma [106]
C
D
M
Pages et al.; R3
R2 R1
N
CNRS, INSERM, Univ. droit, économie, sciences; Univ. de la méditerranée [118]
146 Frontiers in Anti-Infective Drug Discovery, 2010, Vol. 1
Van Bambeke et al. (Fig. 2) Contd…..
W
R2 N
R O
W
O NHX
R2
R3
N
R
R1
O
W
R4
R OH
R1
Chamberland et al.;
R1 N
Z
R2
R4
R3
Microcide Pharmaceuticals, Inc. [123-125,184]
R1
Nakayama et al.;
N R2 S
N
W N
R4 W2
R3
Q
Daiichi Seiyaku Co, Essential Therapeutics Inc. [176]
O
Fig. (2). General structure of classes of inhibitors of bacterial efflux pumps corresponding to new chemical entities that have been patented so far. Parts of the molecules appearing in bold correspond the skeleton of the inhibitors shown in Table 2.
The convincing demonstration of the in vitro capacity of these pharmacological agents or of natural molecules to restore antibiotic activity in strains encoded with efflux-mediated resistance has however stimulated research for new inhibitors that are free of pharmacological activity on eukaryotic cells. A first category of original inhibitors are chemotypes of clinically-used antibiotics, with low intrinsic antibacterial effects. Three main families have been patented so far, namely analogues of tetracyclines, aminoglycosides, and quinolones, which minimize efflux of the corresponding antibiotics. The second category consists of inhibitors that are structurally unrelated to known antibiotics, and totally new entities. Some of them inhibit pumps that efflux multiple classes of antibiotics. Based on empiric observations on the properties of these inhibitors, one can conclude the following: •
The chemical structure of the various inhibitors (Table 2) has several recurrent structural features, namely (i) aromatic rings, which are present in all molecules (except aminoglycoside analogues) and ionizable moieties, which are found in many (but not all) of the putative inhibitors. This is consistent with the fact that efflux pumps preferentially transport amphiphilic substrates [82] and possess affinity binding pockets presenting at their surface amino-acid side chains prone to establish hydrophobic, aromatic stacking and van der Waals interactions [83].
•
Some of the inhibitors also modulate eukaryotic multidrug transporters like P-glycoprotein, MRP, or BCRP, as demonstrated for verapamil (3) [84], VX-710 (22) [85], VX-853 (23) [86], and GF120918 (34) [87,88] (note that these inhibitors are not specific for ABC transporters in bacteria as they are in eukaryotic cells; see the data shown in Table 2). Since antibiotics are also substrates for eukaryotic efflux pumps ([18] for review), this property is possibly advantageous. Indeed, efflux pumps expressed by eukaryotes can modulate (i) the pharmacokinetic profile of the
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147
antibiotics (absorption, distribution, elimination), and concomitantly their serum level ([18] for review), and (ii) their cellular accumulation, which impacts their activity in intracellular infections ([89-91] for examples). In contrast, other inhibitors like MC207,110 (28) do not interact with eukaryotic transporters [92]. This favors a specificity of action and minimizes untoward effects due to inhibition of physiological functions of eukaryotic efflux pumps. ANALYSIS OF THE MAIN CLASSES OF EFFLUX PUMPS INHIBITORS Tetracycline Analogues (See Patent [93]) Inhibitors of tetracycline efflux were identified by their ability to reduce tetracycline efflux in inverted membrane vesicles enriched in one of the efflux resistance determinants. Structure-activity relationships have shown that most effective inhibition is obtained for 6(alkylthio)methyldoxycycline analogues (11,12) [94,95]. These derivatives are usually more potent inhibitors of class A or B efflux determinants (found in E. coli) than of class K or L (found in Gram-positive organisms), producing synergistic effects with tetracyclines in Gram-negative, but additive effects in Gram-positive [96]. However, they show an intrinsic antibacterial activity on Gram-positive, with MIC close to those of doxycycline in non-resistant strains as well as in resistant strains due to ribosomal protection (TetM) [96]. This unexpected observation suggests that, in Gram-positive, these analogues are able to inhibit the pump and also bind, probably differently than conventional tetracyclines, to the tetracycline binding site on the ribosome. This may pave the way to the design of new compounds endowed with a higher intrinsic activity, encompassing strains that are resistant due to efflux or ribosomal protection. Aminoglycoside Analogues (See Patent [97]) Aminoglycosides have been historically considered as poor substrates for efflux pumps, because of their highly hydrophilic nature. Recently they were shown to be transported by (i) a few narrow spectrum efflux pumps of the MFS superfamily, which also transport sugars, and (ii) wide spectrum efflux pumps of the RND superfamily, like the AcrAD-TolC pump of E. coli or the MexXY-OprM pump of P. aeruginosa (Table 1). Accordingly, the patent claims the use of analogues (13) of the aminoglycoside paromomycin as inhibitors of efflux pumps, based on studies with Haemophilus influenzae. The analogues tested show a higher intrinsic activity (1 to 4-fold decrease in MIC) against Acr-disrupted H. influenzae than against the wild-type strain, suggesting a competitive mode of inhibition. These analogues also increase the susceptibility of wild-type strains and clinical isolates to gentamicin and tetracyclines. Notably, the efflux of aminoglycosides has not yet been documented (neither positively, nor negatively) in H. influenzae. Fluoroquinolone Analogues (See Patent [98]) These modified fluoroquinolones (or ester derivatives) are able to increase the activity of these antibiotics in Gram-positive and Gram-negative organisms overexpressing wellcharacterized efflux pumps. Optimal targeting to a given bacterial species (or a given transporter) can be obtained by modifying the substituents in position 1, 7, or 8 (14-17). Quite intriguingly, some of these inhibitors also restore macrolide activity in streptococci overexpressing Mef pumps. In the absence of any detailed publications on these inhibitors,
[63]
Phenothiazines
Ca antagonists
[63,64]
[60,62]
Alkaloids
2+
Refs.
Type of inhibitor
No patent
No patent
No patent
Substrates
H3CO
H3CO
O
OCH3
O
O
OCH3
CH3
N
verapamil (3)
CN
CH(CH3)2
chlorpromazine (2)
S
N
reserpine (1)
H3CO
(H3C)2N
N H
N
OCH3
OCH3
OCH3
OCH3
OCH3
Structure of the most active compounds in the series a
Pharmacological agents
Bacteria
Claimed spectrum of activity in corresponding patents
isoniazid
Tetracyclines
Tetracyclines
Fluoroquinolones
Substrates
M. smegmatis
E. coli
E. coli
S. pneumoniae S. aureus
Bacteria
Demonstration of activity
120 µg/ml 25 µg/ml
45 µg/ml
20 µg/ml
Typical active concentrations
Table 2. Most Active Inhibitors of Efflux Pumps, with Substrates and Bacterial Species in which their Activity has been Demonstrated and the Spectrum of Activity Claimed in the Corresponding Patents
148 Frontiers in Anti-Infective Drug Discovery, 2010, Vol. 1 Van Bambeke et al.
Refs.
[65]
[66,67]
[69,70]
Type of inhibitor
Phenylpiperidine selective serotonin reuptake inhibitors
Proton pump inhibitors
Pyrrolo[1,2-a] quinoxaline analogues of omeprazole
(Table 2) Contd…..
No patent
No patent
No patent
Substrates
Bacteria
Claimed spectrum of activity in corresponding patents
Cl
H
H3C N
O
O
HN
N
N
N
N
H N
O
OCH3
OCH3
Fluoroquinolones
Fluoroquinolones
Norfloxacin ethidium bromide tetracycline
Substrates
Space for Table 2
(6)
SO
omeprazole (5)
SO
CH3
paroxetine (4) OCH3
N H
F
Structure of the most active compounds in the series a
S. aureus
S. aureus
E. coli
S. aureus
Bacteria
Demonstration of activity
128 µg/ml
100 µg/ml
20 µg/ml
Typical active concentrations
Inhibitors of Bacterial Efflux Pumps Frontiers in Anti-Infective Drug Discovery, 2010, Vol. 1 149
Refs.
[72]
[73]
[79]
Type of inhibitor
Flavonolignans
Phenolic diterpenes
Piperine analogues
Bacteria
No patent
No patent
HO
O
O
O
O
O
R
H3CO
H3CO
N
SK20 (9) SK56 (10)
C2H5
O
totarol (8)
OH
5’-methoxyhydnocarpin (7)
OH
O
OCH3
OH
Structure of the most active compounds in the series a
Natural products and semi-synthetic derivatives
No patent
Substrates
Claimed spectrum of activity in corresponding patents
OCH3
OH
Ciprofloxacin
Erythromycin norfloxacin tetracycline ethidium bromide
Norfloxacin ethidium bromide
Substrates
S. aureus
S. aureus
S. aureus
Bacteria
Demonstration of activity
6.25 µg/ml
1 µg/ml
10 µg/ml
Typical active concentrations
(Table 2) Contd…..
150 Frontiers in Anti-Infective Drug Discovery, 2010, Vol. 1 Van Bambeke et al.
Space for Table 2
Refs.
[93,94]
Type of inhibitor
Tetracyclines
(Table 2) Contd…..
Tetracyclines
Substrates
Tetracyclineresistant bacteria
O
O
EtSCH2
OH
R=
OH
RSCH2
OH
OH
(12)
OH
OH
(11)
OH
OH
O
O
O
OH
N(CH3)2
O
OH
N(CH3)2
Structure of the most active compounds in the series a
Analogues of substrates
Bacteria
Claimed spectrum of activity in corresponding patents
NH2
CH2
NH2
Tetracyclines
Tetracyclines
Substrates
E. coli
E. faecalis E. coli
S. aureus
Bacteria
Demonstration of activity
16 µg/ml
1-2 µg/ml
Typical active concentrations
Inhibitors of Bacterial Efflux Pumps Frontiers in Anti-Infective Drug Discovery, 2010, Vol. 1
Space for Table 2
151
Refs.
[97]
[98]
Type of inhibitor
Aminoglycosides
Fluoroquinolones
Bacteria
Very wide spectrum
Very wide spectrum
Substrates
All antibiotic classes
Fluoroquinolones macrolides tetracyclines linezolid novobiocin
Claimed spectrum of activity in corresponding patents
HO
R
R=
HO HO
HN
HO HO
N
F
N
O
HN H N
S
COOC2H5
OH
OH
R
R
Fluoroquinolones
Tetracyclines, gentamicin
Substrates
Space for Table 2
(14)
F
(13)
R
NH
O
O
NH O
O
Cl
O
R
HO
OH
Structure of the most active compounds in the series a
S. aureus
H. influenzae
Bacteria
Demonstration of activity
< 4-20 µg/ml
?
Typical active concentrations
(Table 2) Contd…..
152 Frontiers in Anti-Infective Drug Discovery, 2010, Vol. 1 Van Bambeke et al.
Type of inhibitor
(Table 2) Contd…..
Refs. Substrates
Bacteria
Claimed spectrum of activity in corresponding patents
But
O
HN
O
N
F
N
(17)
CH3
(16)
N
F
(5)
OCH3
N
O
N
O
N
O
COOH
COOH
COOH
E. coli
S. pneumoniae
Bacteria
Fluoroquinolones P. aeruginosa
Fluoroquinolones
Macrolides
Substrates
Space for Table 2
H3CO
H2N
H3C
F
Structure of the most active compounds in the series a
Demonstration of activity
< 4-20 µg/ml
< 4-20 µg/ml
< 4-20 µg/ml
Typical active concentrations
Inhibitors of Bacterial Efflux Pumps Frontiers in Anti-Infective Drug Discovery, 2010, Vol. 1 153
Refs.
[100,101]
[100,101]
[100,101]
Type of inhibitor
Indoles
Ureas
Aromatic amides
E. faecalis E. coli P. aeruginosa M. smegmatis S. marcescens
Staphylococci Streptococci
E. faecalis E. coli P. aeruginosa M. smegmatis S. marcescens
Staphylococci Streptococci
E. faecalis E. coli P. aeruginosa M. smegmatis S. marcescens
Staphylococci Streptococci O
O
N
O
N
O N H
O
IFN240 (20)
N H
IFN271 (19)
N H
IFN55 (18)
New chemical entities
Bacteria
OCH3
Structure of the most active compounds in the series a
Ciprofloxacin, ethidium bromide
Ciprofloxacin, ethidium bromide
Ciprofloxacin, ethidium bromide
Substrates
S. aureus S. pneumoniae
S. aureus S. pneumoniae
S. aureus S. pneumoniae
Bacteria
Demonstration of activity
Space for Table 2
Fluoroquinolones
Fluoroquinolones
Fluoroquinolones
Substrates
Claimed spectrum of activity in corresponding patents
2.5 µg/ml
2.5 µg/ml
2.5 µg/ml
Typical active concentrations
(Table 2) Contd…..
154 Frontiers in Anti-Infective Drug Discovery, 2010, Vol. 1 Van Bambeke et al.
Refs.
[104,105]
[106,117]
Type of inhibitor
Thiophene or benzothiophene
Piperidine-carboxylic acid derivatives
(Table 2) Contd…..
H3CO
O
Very wide spectrum
N O
OCH3
O
O
novobiocin
ethidium bromide
S. aureus S. pneumoniae E. faecalis
S. aureus
Bacteria
100 µg/ml
25 µg/ml
Typical active concentrations
Frontiers in Anti-Infective Drug Discovery, 2010, Vol. 1
VX-710 (22)
OCH3
N
(21)
R=H; specific to NorA; R=Cl; non specific
Fluoroquinolones gentamicin
Fluoroquinolones macrolides rifamycins tetracyclines chloramphenicol gentamicin, linezolid penicillin, amoxicilin ceftriaxone imipenem mupirocin
N
Fluoroquinolones macrolides
R
Antibiotics antifungal agents S
Staphylococci Streptococci Enterococci B. subtilis E. coli, P. aeruginosa H. influenzae S. cerevisiae C. albicans CHO
Substrates
Bacteria
Structure of the most active compounds in the series a
Demonstration of activity
Space for Table 2
Substrates
Claimed spectrum of activity in corresponding patents
Inhibitors of Bacterial Efflux Pumps 155
Refs.
[118,120]
Type of inhibitor
Alkylaminoquinolines
Bacteria
Enterobacteriaceae
Substrates
Quinolones tetracyclines chloramphenicol macrolides
Claimed spectrum of activity in corresponding patents
O2N
H3CO
O
H3C
OCH3
O
OCH3
O
N
CH3
733 (25)
N
HN
814 (24)
CH3
N
HN
VX-853 (23)
N
N
N
O
N
Chloramphenicol
Chloramphenicol norfloxacin tetracycline
novobiocin
ethidium bromide
Fluoroquinolones gentamicin
Substrates
Bacteria
E. aerogenes
E. aerogenes
S. aureus S. pneumoniae E. faecalis
Space for Table 2
O2 N
Cl
N
Structure of the most active compounds in the series a
Demonstration of activity
330 µg/ml
60 µg/ml
6 µg/ml
Typical active concentrations
(Table 2) Contd…..
156 Frontiers in Anti-Infective Drug Discovery, 2010, Vol. 1 Van Bambeke et al.
Refs.
[118,119]
[118,121]
Type of inhibitor
Alkoxyquinolines
Thioalkoxyquinolines
(Table 2) Contd…..
Bacteria
Enterobacteriaceae
Enterobacteriaceae
Substrates
Quinolones tetracyclines chloramphenicol macrolides
Quinolones tetracyclines chloramphenicol macrolides
Claimed spectrum of activity in corresponding patents
CH3
7d (27)
N
Bacteria
Chloramphenicol
E. aerogenes
Chloramphenicol E. aerogenes tetracycline K. pneumoniae norfloxacin
Substrates
Space for Table 2
N
S
N
CH3
905 (26)
N
O
Structure of the most active compounds in the series a
Demonstration of activity
280 µg/ml
270 µg/ml
Typical active concentrations
Inhibitors of Bacterial Efflux Pumps Frontiers in Anti-Infective Drug Discovery, 2010, Vol. 1 157
Very wide spectrum
Very wide spectrum
All antibiotic classes
All antibiotic classes
[92,127,171, 184-188]
[128]
Bacteria
Peptidomimetics
Substrates
Refs.
Type of inhibitor
Claimed spectrum of activity in corresponding patents
N H
N H NH
N H O
H N
MC 02,595 (29)
O
NH2
N
MC 207, 110 (28)
O
O
Bacteria
Levofloxacin
fluoroquinolones quinolones chloramphenicol
P. aeruginosa
E. aerogenes E. coli
Y.enterocolitica S. enterica
Fluoroquinolones P. aeruginosa chloramphenicol erythromycin carbenicillin tetracycline ethidium bromide B.pseudomallei spectinomycin clarithromycin A. baumannii nalidixic acid S. maltophilia
Substrates
Space for Table 2
H2N
H2N
H2N
H N
Structure of the most active compounds in the series a
Demonstration of activity
10 µg/ml
10 µg/ml
Typical active concentrations
(Table 2) Contd…..
158 Frontiers in Anti-Infective Drug Discovery, 2010, Vol. 1 Van Bambeke et al.
[173,176]
[27,129]
Refs.
Fluoroquinolones -lactams
Very wide spectrum
All antibiotic classes
(Expressing MexAB OprM)
P. aeruginosa
Bacteria
Substrates
Claimed spectrum of activity in corresponding patents
H2N
S
N
H N N H O
H N
O
(31)
N O
N
MC 04,124 (30)
O
N HN N
N
N
Structure of the most active compounds in the series a
Levofloxacin aztreonam
Fluoroquinolones
Substrates
(Specific to MexABOprM)
P. aeruginosa
P. aeruginosa
Bacteria
Demonstration of activity
Space for Table 2
Pyridopyrimidines
Type of inhibitor
(Table 2) Contd…..
2.5 µg/ml
10 µg/ml
Typical active concentrations
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Refs.
[132,134]
[135,136]
Type of inhibitor
Disiloxanes
Lindans
Bacteria
No patent
Patented as an adjuvant to anticancer chemotherapy
Substrates
Claimed spectrum of activity in corresponding patents
F
C2H5
F
O Si
N
N
N
CH3
CH3
Ro 07-3149 (33)
H3C
HC
HO
SILA 421 (32)
Si
CH3 CH3
N
Structure of the most active compounds in the series a
Tetracyclines
Bacteria
S. aureus
M. tuberculosis
Space for Table 2 (Active alone)
Substrates
Demonstration of activity
3 µg/ml
Typical active concentrations
(Table 2) Contd…..
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No patent
No patent
[65]
[68,144]
Arylpiperidines
No patent
[143]
Acridine carboxamides
Substrates
Refs. Bacteria
Claimed spectrum of activity in corresponding patents
Type of inhibitor
(Table 2) Contd…..
OCH3
N H
O
N H
F
O
O
GF 120918 (34)
N H
O
N H
F
(36)
Cl
Br
NNC 20-7052 (35)
O
N
CH3O
Structure of the most active compounds in the series a
OCH3
Space for Table 2 Linezolid Ethidium bromide 1-
Norfloxacin ethidium bromide tetracycline
tetracycline
Fluoroquinolones
Substrates
E. coli S. aureus
E. coli
S. aureus
S. aureus
Bacteria
Demonstration of activity
32 µg/ml 6 µg/ml
20 µg/ml
10 µg/ml
Typical active concentrations
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a
[147-150]
[151]
Arylpiperazines
9-formyl-5-methyldeca-2,8-dienoic acid amides No patent
No patent
Substrates
Bacteria
(38)
COCH3
O
NMP (37)
N
NR1R2
NH
Structure of the most active compounds in the series a
parts of the molecules shown in bold correspond to the common core of the whole family of inhibitors, as illustrated in Fig. (1 and 2).
Refs.
Type of inhibitor
Claimed spectrum of activity in corresponding patents
Ciprofloxacin
Bacteria
S. aureus
E. coli A. baumanii E. aerogenes K. pneumoniae C. freundii
Space for Table 2 Linezolid levofloxacin clarithromycin oxacillin rifampicin chloramphenicol tetracycline
Substrates
Demonstration of activity
25 µM
50 µg/ml
Typical active concentrations
(Table 2) Contd…..
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it is difficult to rationalize this observation in the cited patent. Noteworthy, dimeric piperazinyl-linked fluoroquinolones display potent antibacterial activity against S.aureus, including resistant strains due to NorA pump activity as well as mutations in topoisomerase IV [99], inferring that they combine a high intrinsic activity and a low affinity for NorA. Indoles, Ureas and Aromatic Amides (See Patent [100]) Markham et al. screened a library of compounds by an uptake assay for ethidium bromide in NorA-overexpressing S. aureus, with 399 (4 %) molecules demonstrating activity and belonging to four chemotypes, namely indoles (18) (note the indole moiety also present in reserpine), biphenylureas (19), aromatic amides (20), and molecules bearing a trichloromethylaminal group [101]. Two other active compounds (INF 277 (39) and INF 392 (40)), not structurally similar with the above chemotypes, were also mentioned in the patent (Fig. 2). Further molecules in the indole series have been produced recently, with similar activities [102,103]. The broad structural diversity of inhibitors therefore suggests that the inhibited transporters have low structural specificity for substrate/inhibitor recognition. All active products synergize the uptake of ethidium bromide and ciprofloxacin, and also reduce the selection of resistant mutants (at least 50-fold). Their inhibitory profile typically showed activity with homologous transporters, like Bmr from Bacillus subtilis, and, for some of them, PmrA of Streptococcus pneumoniae [101]. The structural diversity of molecules showing activity increases confidence that some pharmacophores will have appropriate safety profile and can be used to construct molecules usable in adjunctive therapy. For example, leads with the trichloromethylaminal group have been abandoned [101], and INF 392 (40) and INF 240 (20) have significantly different cytotoxicity profile (INF 392 (40) showing the highest, and INF 240 (20) the lowest selectivity for bacterial cells [100]). Arylbenzo[b]thiophenes and Diarylthiophenes (See Patent [104]) Based on the observation that the activity of INF55 was more dependent on the 2arylindole moiety than on the nitro substituant [101], sulfur analog of this molecule were produced, giving raise to arylbenzo[b]thiophenes and diarylthiophenes. These were tested for their capacity to restore ciprofloxacin activity in NorA producing and of erythromycin in MsrA producing strains of S. aureus and for their safety towards eucaryotic cells [105]. Most active molecules belong to the aryl benzothiophenes; the nature of the benzyl substituents affects the spectrum of activity (specificity to NorA or broader spectrum). Piperidine-Carboxylic Acid Derivatives (See Patent [106]) This class of molecules was patented [85,107-110] as inhibitors of P-glycoprotein and of MRP-1, with VX-710 (22, biricodar) progressing through Phase II clinical trials [111] as adjuvant for the treatment of cancer by paclitaxel, mitoxantrone or anthracyclines [112114]. Since its pharmacokinetic and toxicity profile in humans was already established in the above studies [115,116], it may expedite its profiling for combination use with antibiotics. Since a broad range of structural variations are disclosed in the patent (Fig. 2), it is probable that molecules selective for inhibition of prokaryotic or eukaryotic transporters can be identified in the future. Simultaneous inhibition of both eukaryotic and prokaryotic transporters is indeed disadvantageous. Dual inhibitors could alter the pharmacokinetics of antibiotics or cause toxicity when used as adjuvants to antibiotics, or, on the contrary,
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indirectly select bacteria acquiring resistance to them when used in combination with anticancer agents. While the efficacy of VX-710 (22) and VX-843 (23), in combination with fluoroquinolones [117], has been demonstrated so far in Gram-positive organisms, the patent claims encompass a range of bacteria and classes of antibiotics belonging to different classes which need further validation. Alkylaminoquinolines, Thioalkoxyquinolines, Alkoxyquinolines (See Patent [118]) These compounds were found to increase the accumulation and the activity of chloramphenicol in AcrAB-TolC-positive clinical isolates of Enterobacter aerogenes, and were selected for their selectivity, a negligible intrinsic activity and no permeabilizing effect on the membrane [119,120]. Optimal structure-activity relationships of the alkylaminoquinolines were found with piperidino- (24) or morpholino- (25) substituents [120], and that the alkoxyquinolines (26) and thioethers (27) were comparable [121]. Methylation of the pendant unit of the alkoxyquinolines further increases activity [120]. The data suggests that the alkylamino moieties on the quinoline backbone play a strategic role in recognition by the pump and competition for transport. Mallea et al., [120] have calculated that the maximal exclusion space of alkylaminoquinolines is 20 Å, which could fit into the central pore of the inner membrane protein AcrB, which is thought to play a major role in the transport function of the protein [122], and with the restricted region of this pore in particular [57]. This suggests that inhibition could occur either on the inner membrane protein itself, or at the inner pump-outer channel junction, where this restriction is located. Again, additional studies are needed to determine the spectrum of activity of these inhibitors, with other clinically-relevant Gram-negative bacteria expressing broad-spectrum RND transporters. Peptidomimetics (See Patents [123-125]) MC-207,110 (28) was selected as lead compound, after screening a library of 150K natural products and synthetic molecules, for synergism with levofloxacin towards P. aeruginosa [92,126]. Mechanistic studies have shown that it specifically increases the activity of antibiotics that are substrates for Mex pumps without perturbing proton gradients [127]. These studies suggest that it is also a substrate for efflux pumps, since it displays low intrinsic activity only in bacteria in which the genes coding for the main efflux pumps have been disrupted. This activity seems to be due to disruption of membrane integrity [127]. Additional structural modifications have provided derivatives for in vivo evaluations. The initial goal consisted of improving the proteolytic stability of the inhibitors in biological media, which was achieved by structural permutations, including using D-amino-acids, exemplary is MC-02,595 (29) [128]. The second goal focused on enhancing the therapeutic indices and pharmacokinetic-pharmacodynamic profile of the molecular class for in vivo applications. A balance of these features is present in the conformationally-restricted analogues like MC 04,124 (30) [129,130]. In parallel studies, structure-activity relationships have shown that the peptidic backbone present in these three inhibitors is not essential for inhibitory activity [131]. Substituted Disiloxanes (See Patent [132]) SILA 421 (38) is a potent inhibitor of efflux pumps in cancer cells and in multidrug resistant E. coli [133]. Interestingly enough, it shows antibacterial activity against multidrug
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resistant Mycobacterium tuberculosis at concentrations that are not toxic for eucaryotic cells [134]. Since this effect is obtained with SILA 421 alone, it is unlikely to result from efflux pump inhibition. Other Original Derivatives (Not Patented) Five other structural classes of inhibitors have been reported, but no associated patents or patent applications have been cited. Ro 07-3149 (33) increases the accumulation of tetracyclines in S. aureus by noncompetitive inhibition of the TetK transporter [135]. Interestingly, it loses it activity when TetK is expressed in E. coli, probably due to insufficient permeability of the outer membrane of this Gram-negative to the compound [135]. In contrast with the derivatives lacking the hydroxypropyl side chain, Ro 07-3149 does not affect the energy state of the pump [136]. Similar to VX-710 (22) or VX-843 (23), GF120918 (34) [137] was evaluated as an inhibitor of P-glycoprotein and BCRP [66,67]. It underwent Phase I studies, in combination with anthracyclines [138,139] in several animal studies, to demonstrate modulation of the pharmacokinetic profile of anticancer agents [140] and some antivirals [141,142]. It was more recently shown to also markedly increase the effectiveness of fluoroquinolones, and marginally that of macrolides and tetracyclines against S. aureus [143]. However, the effective concentration required to modulate active transport in bacteria is significantly higher than the human toxicity levels [144]. The arylpiperidines are topologically similar to some serotonin reuptake inhibitors (4). The paroxetine isomer NNC 20-7052 (35) is equipotent as paroxetine inhibiting MFS(NorA and TetK) and RND-class (AcrB) pumps but much less potent as an inhibitor of serotonin reuptake [65], suggesting that absolute stereochemistry maybe unimportant as far as pump inhibition is concerned and that structural congeners may combine reasonable safety profile and potency. Among them, a dihalo analog (36) was effective in restoring linezolid accumulation in E. coli [145], even though linezolid has not yet been documented as potential substrate for efflux pumps in general ([146] for a preliminary report). Similarly 1-(1-naphthylmethyl)-piperazine (37) facilitated the accumulation of levofloxacin in E. coli and the activity of several antibiotics [147]. It also reverses antibiotic resistance in A. baumanii [148] It is however moderately active to restore fluoroquinolone activity in clinical isolates of E. coli and other enterobacteriaceae [149,150]. Citral-derived amides [151] were designed based on the observation that piperine [152], a major constituent of Piper nigrum, and semi-synthetic derivatives thereof are potent inhibitors of NorA [79-81]. The most potent molecules belong to the 9-formyl-5-methyldeca-2,8-dienoic acid group of amides (38) and cause a 4-fold reduction in MIC at 25 µM [151]. This remains slightly inferior to the SK-20 (9) or SK-56 (10) analogs of piperine, which caused a 8-fold reduction in MIC at 6.25 µM [79]. Hybrid Molecules An elegant way to facilitate the potential use of efflux pump inhibitor is to develop hybrid molecules that combine both the antibiotic moiety and an inhibitor of its efflux. A priori, this should permit simultaneous delivery of both molecules to the target site of infection. Two examples illustrating this strategy have been published so far.
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The first one consists in a hybrid of berberine, a natural antibacterial agent, with INF55 (18) [153] or simplified derivatives thereof [154]. These compounds are > 300 fold more active than berberine alone against NorA overproducers; however berberine is not approved for human use. Another example are the fluoroquinolones conjugated via position 7 with an efflux pump inhibitor [155]. These compounds maintain the inhibitory activity of the inhibitor alone but show higher MICs than the parent fluoroquinolones. Potential Uses of Efflux Pumps Inhibitors (see also [156] for Review) The first application of these inhibitors would obviously be restoration of antibiotic activity against bacteria that encode a mechanism of resistance by efflux. Since the compelling inhibitors described herein lack intrinsic antibacterial activity, they need to be used in combination with antibiotics, similar to the β-lactamase inhibitor-β-lactam combinations. At the present time, data exists for the efficacy and safety of such combinations from animal studies. A preliminary report discusses the potentiation effect of MC-04,124 (30) (Table 2) with levofloxacin in mouse models of P. aeruginosa infections (thigh infection and sepsis), and that of azithromycin in a mouse model of E. coli pyelonephritis [157]. Except for the above studies, other examples of this strategy are based on in vitro data that demonstrate synergy between inhibitors and antibiotics. The latter is accompanied by a shift of MIC to lower values, which makes the whole population more susceptible to antibiotics (as an example, the MIC90 of a P. aeruginosa population to levofloxacin shifted from 8 to 0.5 mg/L in the presence of MC-207,110 (28) [126]). Importantly also, this synergy may reduce the selection of resistant mutants, based on the observation that resistance to quinolones by target mutation is difficult to select in strains lacking functional efflux systems [158]. A same effect was demonstrated for (i) reserpine and quinolones in S. aureus [159] and (ii) MC-207,110 (28) and quinolones in P. aeruginosa ([127]; in this case, the probability to select resistant mutants falls to a same level as upon disruption of the genes encoding efflux pumps [28]). Increasing antibiotic concentration in a bacteria above the MPC (Mutation Prevention Concentration), the concentration that corresponds to the minimal concentration to prevent enhancement of resistant mutants, is important. MPC values will vary depending on the antibiotic class and the bacteria, but is typically 5-10 times higher than the MIC (see [160] for a review of the concept). Of note, a recent study suggests that exposure to an efflux pump inhibitor like resepine can trigger overexpression of efflux pumps in S. pneumoniae [161], suggesting that resistance to inhibitors can also develop. Considerable debate exists on whether efflux pumps are expressed in vivo. Indirect evidence exists from studies in Gram-negative bacteria. For example, P. aeruginosa multidrug transporters are involved in the secretion of virulence factors and quorum-sensing molecules and are therefore needed for host invasion [6]. Moreover, mechanisms of regulation are common between efflux pumps and virulence genes [162]. Interestingly enough, a cystic fibrosis epidemic strain displays an enhanced virulence (by up-regulation of its quorum-sensing system) and an increased antimicrobial resistance associated to mutations in efflux pump genes [163]. In enteropathogens, efflux pumps are essential for survival in the gut, since they expel bile salts present in this hostile environment [10,164]. In Gram-positive organisms, in contrast, the physiological roles of efflux pumps have not yet been established. The only evidence of their potential clinical importance in the clinics is that their overexpression is evidenced in clinical isolates of Gram-positive organisms [165167], as it is in clinical isolates of Gram-negative organisms [29,168-170].
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A second application of pump inhibitors is their use as diagnostic tools. Reserpine is commonly used for Gram-positive pathogen profiling [166,167] and MC- 207,110 for Gram-negative bacteria [27,168,170,171], but the absence of specificity of these inhibitors does not allow for classification of the active efflux pumps. The results reported from the search of specific inhibitors (31), as done for the MexAB-OprM pump in Pseudomonas [172-175] (patent [176] and Fig. (2) and Table 2 for structure) are instructional. When other mechanisms of resistance are present, which mask the effect of the inhibitor, false-negative results can occur in such studies. This is particularly critical for broad-spectrum pumps in multi-resistant organisms, for which a single substrate is usually used as reporter of efflux pump activity [177]. CURRENT AND FUTURE DEVELOPMENTS In a world of increasing bacterial resistance to antibiotics, the search of therapeutic alternatives to currently existing drugs appears is a priority. This challenge can be met in two ways [178]. The first one consists in the discovery of antibiotics directed against new pathogen targets (reviewed in [179]), which are therefore not affected by existing mechanisms of resistance. This strategy is daunting because (i) the discovery of such new entities is laborious and (ii) development of resistance to these new antibiotics is inevitable. Lessons can be learned from the post-approval events of linezolid, the only novel class of antibiotics introduced in the last decade [180,181], in which resistance was rapidly observed [see [182] for a recent survey]). An alternative, and maybe more rewarding pathway towards new antibacterial therapies, embraces the development of inhibitors of resistance mechanisms, which allows extending the utility of existing antibiotics with well known pharmacological and toxicological properties. Efflux pump inhibitors belong to this second strategy. The present review highlights inhibitors of bacterial efflux pumps, which have shown promise in vitro. They can be used as diagnostic tools for detection of active efflux in pathogens as a mechanism of resistance. For this application, narrow-spectrum inhibitors will be preferred which allow gross identification of the transporters that are expressed. At the present time, phenotypic analysis approach is limited to epidemiological surveys, or characterization of resistant mutants in research laboratories; detection of resistance by efflux is not yet implemented in routine clinical laboratories. The concomitant development of genotypic methods, in combination with phenotypic methods, allows for a more precise identification of the pump [177,183] will probably be adopted in the near future. In sharp contrast, developing combinations of efflux inhibitors with antibiotics is a continuing challenge. A priori, broad-spectrum inhibitors have substantial potential for clinical applications. The selection could be possibly oriented towards inhibitors targeting several pumps in a given organism (to be added to antibiotics for empiric therapy) or targeting transporters of a given class of antibiotics in different bacterial species (to be used in combined formulations). In this context, inhibitors of pump functioning may have broader spectrum than competitive inhibitors, but their use in vivo is unlikely because they would also affect eukaryotic transporters. Most of the inhibitors described in this manuscript were recently tested in vitro by small companies or isolated laboratories, which have limited preclinical and clinical capabilities. While the major pharmaceutical firms have reduced their interest in antibacterial
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therapeutics [179], they acknowledge interest in this approach to rejuvenate the activity of current antibiotics [38,45]. Corroborating this idea, Mpex Pharmaceuticals recently licensed the Microcide Pharmaceuticals efflux portfolio, and one of the leads is in Phase Ib clinical trial as an aerosol drug candidate in cystic fibrosis (CF) patients (see “news” page on the web site of the company at
). This encouraging news suggests the interest of extensive in vivo studies aimed at evaluating the pharmacological properties, safety profile, and efficacy in models of infection by resistant organisms of other efflux pumps inhibitors. ACKNOWLEDGEMENTS F.V.B. is Maître de Recherches of the Belgian Fonds National de la Recherche Scientifique. REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13]
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Drugs Candidates in Advanced Clinical Trials Against Tuberculosis Marcus Vinícius Nora de Souza*, Marcelle de Lima Ferreira and Raoni Schroeder B. Gonçalves Instituto de Tecnologia em Fármacos-Far-Manguinhos. Rua Sizenando Nabuco, 100, Manguinhos, 21041-250 Rio de Janeiro-RJ, Brazil Abstract: Tuberculosis (TB) is an important public health problem worldwilde due to AIDS epidemic, the advent of multidrug resistant strains (MDR) and the lack of new drugs in the market. This disease was responsible for almost 1.8 million deaths in 2007, according to WHO (World Health Organization), which declared tuberculosis a global health emergency in 1993. In spite of this problem, there is a lack of development of new TB drug. For example, it has been nearly 40 years since the introduction of a new class of compounds for the treatment of TB. Thus, there is an urgent need for new drugs to fight this disease. Considering that, this review aims to present promising drug candidates in final clinical studies against TB.
Keywords: Tuberculosis, drugs, clinical studies. 1. INTRODUCTION Nowadays, tuberculosis (TB) is becoming a worldwide problem. This contagious disease is transmitted through the air and it is caused by the Mycobacterium tuberculosis, which can attack different organs of human body. However, it most commonly affects the lungs, being responsible for more than 75 percent of cases. The common symptoms of this disease are prolonged cough, chest pain, and hemoptysis, fever, chills, night sweats, appetite loss, weight loss, and easy fatigability [1]. Different factors are responsible for the resurgence of TB, such as people infected with HIV virus, immigration, war, famine, homelessness, the lack of new drugs and multi-drug-resistant tuberculosis (MDR TB) due to inconsistent or partial treatment. Considering TB problems, the World Health Organization (WHO) declared this disease a global health emergency in 1993 [2]. According to statistics, one-third of the world population is currently infected with the TB bacillus. In 2007, WHO estimated 9.27 million first episodes of TB and 13.7 million prevalent cases world-wide. The most affected country was India, with 21.6% of the total numbers of incident cases, followed by China (14%), Indonesia (5.7%), Nigeria (4.9%) and South Africa (4.9%). In spite of the mortality decline since 2000, about 1.8 million people died from TB in 2007, among them 456 000 were HIV-positive people [3]. Statistical dates demonstrate that without a coordinated control effort, tuberculosis will infect an estimated 1 billion people by 2020, killing 70 million [2].
*Corresponding author: E-mail: [email protected] Atta-ur-Rahman / M. Iqbal Choudhary (Eds.) All rights reserved – © 2010 Bentham Science Publishers.
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2. MULTI-DRUG RESISTENT (MDR) AND EXTENSIVE DRUG RESISTANT (XDR) TB Multi-Drug-Resistent (MDR) TB is normally defined as a patient who has active tuberculosis with resistant bacilli at least to both rifampicin and isoniazid. This patient needs to be treated intensively and for up to 24 months with regimen based on others antituberculosis drugs. The factors that contribute to MDR-TB are interrupted, erratic or inadequate therapy, as well as an inadequate public health system. The cost to treat MDRTB is 1400 times the cost of regular treatment. According WHO, 511 000 cases of MDR-TB occurred in 2007 (4.9 % of all cases). Among these cases, 289 000 were new cases and 221 000 were cases that had previously treated for TB [3]. The drugs normally used to treat MDR-TB are amikacin (AMK), capreomycin (CAP), ciprofloxacin (CIP), cycloserine (CYC), ethionamide (ETA), kanamycin (KAN), ofloxacin (OFX), p-aminosalycilic acid (PAS) and protionamide (PTO) [2,4]. Recently, another important factor in the worldwide TB treatment is the advent of Extensive Drug Resistant or Extreme Drug Resistance TB (XDR-TB), which is commonly defined as strains resistance to at least rifampicin (RIF) and isoniazid (INH) from the first line anti-TB drugs in addition to resistance to any fluorquinolone, and to at least one of three injectable second-line anti-TB drugs used in TB treatment (CAP, KAN and AMK) [5]. By the end of 2008, 55 countries and territories reported at least one case of XDR-TB [3]. WHO estimates that 19% of MDR cases are in fact XDR-TB and the cure is possible for up to 50-60% of the affected people [5]. Taking into account the present panorama of TB, it is very important the development of new drugs, which can be used to reduce the total duration of treatment, to treat MDR TB and XDR TB, with less side effects, and to provide more effective treatment of latent tuberculosis infection [6,7]. However, in spite of the worldwide problem caused by TB, unfortunately the development of new drugs for the treatment of tuberculosis has been slow [8-10]. Due to the importance of new drugs in this field, the aim of this review is to highlight the promising drugs candidates against TB, which are in final clinical trials [2] (Table 1, 2) (Fig. 1).
Table 1. New promising Drugs Candidates in Clinical Trials Against TB Compound
Class of Compound
Developer
Clinical Phase
Moxifloxacin
Fluoroquinolone
Bayer
Phase III
Gatifloxacin
Fluoroquinolone
BMS
Phase III
OPC 67683
Nitroimidazo-o-xazole
Otsuka Pharmaceutical
Phase II
TMC207
Diarylquinoline
Johnson and Johnson
Phase II
PA-824
Nitroimidazol
PathoGenesis Inc
Phase I
SQ-109
Diamine
Sequella Inc.
Phase I
LL-3858
Pyrrol
Lupin
Phase I
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Table 2. New Promising Drugs Candidates in Clinical Trials Against TB Compound
MIC (μg/mL)
Mechanism of action
Sponsor/Coordinator
Moxifloxacin
0.06-0.5a
Inhibition DNA replication
Bayer; TB Alliance; Transcription CDC; University College London; John Hopk.U.
Gatifloxacin
0.03-0.12a
Inhibition DNA replication transcription
European Comission; IRD; WHO/TDR; Lupin
OPC 67683
0.006-0.012
Inhibition of cell wall biosynthesis
Otsuka Pharmaceutical
TMC207
0.03-0.12
ATP depletion and imbalance biosynthesis In pH omeostasis
Johnson and Johnson (Tibotec)
PA-824
0.015-0.25
Inhibition of protein synthesis Inhibition of cell wall lipids synthesis
TB Alliance
SQ-109
0.16-0.32
Inhibition of cell wall biosynthesis
Sequella Inc
Data not available
Lupin
LL-3858 a
0.125-0.25
b
b
MIC50; MIC90; Effect on bacterial cell: Bactericidal
Fig. (1). Mechanism of action of anti-TB substances, including the first and second-line agents (black), and the new drug candidates (red).
3. NEW PROMISING DRUGS AGAINST TB 3.1 Moxifloxacin The fluoroquinolone moxifloxacin (MXF) (Fig. 2) (BAY12-8039) is a drug developed by Bayer [11,12], which possesses excellent activity against a variety of different types of
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bacteria. MXF has also excellent oral bioavailability and the half-life time elimination of the drug in man is about 12 hours in comparison with 1 to 2 hours for INH. O F
CO2H
H N
N OMe NH H Moxifloxacin
Fig. (2). MXF structure.
3.1.1 Mechanism of Action The inhibition of bacterial replication caused by fluoroquinolones is in general due to the inhibition of two bacterial enzymes: DNA gyrase (topoisomerase II) and topoisomerase IV enzymes. DNA gyrase is an essential enzyme involved in the replication, transcription and reparation of the bacterial DNA. In the case of topoisomerase IV enzyme, it is responsible for decatenation, that it is removing the interlinking of daughter chromosomes, thereby allowing segregation into two daughter cells at the end of the replication round. For the Gram-negative organisms DNA gyrase is the primary target, while in the Gram-positive bacteria topoisomerase IV is the most affected. The four-generation fluoroquinolone such as, MXF and gatifloxacin (GAT) show a greater efficacy compared to the earlier fluoroquinolones, due to their dual activity inhibiting both DNA gyrase bacterial type II and topoisomerase IV, which limits also the emergence of fluoroquinolones resistance [13,14]. 3.1.2 Anti-TB activity of MXF Since the earliest studies, MXF have displayed excellent activities in vitro and in animal models against M. tuberculosis. Its MIC range is 0.12-0.50 against several strains of this bacterium, including the reference strain H37Rv, MDR strains [15] and the highly virulent strain CSU93 [16]. When tested in infected mice, MXF activity was comparable to INH against susceptible strains and it was more active than this drug against persisters strains [15, 17-20]. In addition, MXF showed a considerable sterilizing activity [21], which is defined as the ability to prevent relapse, killing all bacilli remained after the initial phase of therapy [22]. When compared with others fluoroquinolones, such as levofloxacin (LFX) and OFX, MXF is much more effective against M. tuberculosis, with better bactericidal and sterilizing activity [23]. Due to these important results, the efficacy of MXF was evaluated in different combinations with drugs actually used in TB treatment. 3.1.3 MXF Combined with Anti-TB Drugs It was verified in vitro that combinations of INH with MXF were more active than each compound alone. Other important result was founded when MXF was combined with RIF. At low concentrations of RIF, the combination was more lethal than RIF alone, however, at high concentrations of the first-line drug, a signifcant decrease of the activity was observed.
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Probably, this result is related to the fact that RIF inhibits the expression of a protein involved in MXF mechanism of action. Tests combining MXF with the first line drug ethambutol (EMB), showed a decrease in 80% of the fluorquinolone activity. It was also verified that EMB interfered with the bacteriostatic activity of MXF [23]. More important results about the biological activity of MXF could be founded in in vivo tests, carried out in mice. For example, in a study performed by Nuermberger and coworkers, it was verified that the replacement of INH by MXF in a 6-month standard treatment composed of INH, RIF and PYZ (INH, RIF and PYZ in the first two months and INH and RIF in the last months), led to a high increase in the bactericidal activity, allowing a substantial reduction in treatment time [24]. In subsequently investigations, no relapse was observed in mice treated with MXF, RIF and PYZ during 4 months (two months with MXF, RIF and PYZ and two months with MXF and RIF). It is important to mention that, this time is two months earlier than the time required to reach the same result in the treatment with INH, RIF and PYZ [25]. Because of the excellent results observed by MXF in animal models, the evaluation of its efficacy in humans has great importance. In the first study, the early bactericidal activity (EBA) of MXF was compared to INH in 17 patients (9 treated with INH and 8 treated with MXF). Both substances showed similar activities [26]. This result was later confirmed by Gosling and co-workers [27], which compared the EBA of MXF with INH and RIF in 43 patients with smear-positive pulmonary tuberculosis, among them, 5 HIV-positive. In despite of the similar efficacy with INH, MXF was more active than RIF. When MXF was combined with INH, none significant increase in EBA was observed [28]. Aiming to find shorter treatment regimens, the introduction of MXF in the first-line standard treatment was verified in phase II clinical trials. In 2006, Burman and co-workers [29] published results of a study where EMB was substituted by MXF and the efficacy of this drug combined with INH, RIF and PYZ was evaluated in the first 2 months of pulmonary tuberculosis treatment. This study showed that MXF or EMB, combined with INH, RIF and PYZ, had a similar effect on the first 2 months sputum culture status, suggesting that the substitution did not allow a significant shortening in the treatment. However, in the first month, the MXF group had a higher rate of culture negativity. Patients treated with MXF related more nausea, nevertheless, this drug was well tolerated. In another phase II study, MXF was utilized replacing INH during the first two months of treatment [30]. The rate of patients who were culture negative after two months was very similar in both treatment regimen suggesting that the substitution of INH by MXF in the first line standard treatment will not lead to a reduction in treatment duration. However, MXF can be considered a good INH substitute in patients who are INH intolerant or infected with INHresistant strains. Because of the high activity of MXF against M. tuberculosis resistant strains, this drug could be very useful in MDR and XDR-TB treatment, specially combined with second-line antituberculosis drugs. Lu and Drlica [23] evaluated the activity of MXF in combination with CAP and CYC. In the first case, the combination was more active than each drug alone, however, in the second one, a little improvement on MXF activity was observed. Table 3 summarizes the most important results obtained from combining MXF with others anti-TB drugs.
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Table 3. MXF Combined with Other Anti-TB Drugs Tests
Drugs
Results
in vitro
MXF + INH
Improvement in the activity of both drugs [23].
MXF + RIF
Low concentrations of RIF increase MXF activity. High concentrations of RIF decrease MXF activity [23].
MXF + EMB
Decrease MXF activity [23].
MXF + CAP
More active than either drug alone [23].
MXF + CYC
Little effect in MXF activity [23].
in mice
MXF + RIF + PYZ
Treatment time reduction [24].
in humans
MXF + INH
Kept the activity unchanged [28].
MXF + INH + RIF + PYZ
Higher rate of culture negativity in the first month than the standard regimen but similar effect after 2 months [29].
MXF + EMB + RIF + PYZ
The activity was similar than the standard regimen [30].
3.1.4 Clinical Studies By the end of 2009, five clinical trials using MXF were in course: a phase II clinical trial combining MXF, RIF, PYZ and INH, a phase III study combining MXF, EMB, INH, PYZ and RIF, a phase II study verifying the efficacy of MXF instead of EMB in the standard first-line treatment and a phase I study, evaluating the pharmacokinetics of MXF alone versus combined with RIF [31]. 3.1.5 MXF Synthesis The synthesis of MXF (Scheme 1) [32] is based on the acid chloride 1 as start material, which was transformed into the -Ketoester 2 and subsequently converted to its O F
COCl KO
OEt F O
F
F
O F OEt triethyl orthoformate
O F
Et3N
OCH3
O
F
Ac2O
OCH3 2
1
F
F
OEt
OCH3 3 NH2 EtOH
H NH
6
Moxifloxacin HCl
CO2Et
O
N H H 1) (S,S)
F
2) HCl
F
2) H+ N OCH3 5
Scheme 1. Synthesis of MXF.
O CO2Et
F CO2H 1) NaF, DMF F
F OCH3 4
NH
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ethoxyacrylate derivative 3. Treatment of this ethoxy-acrylate derivative with the cyclopropylamine gave the enamine 4. Cyclisation of the enamine and hydrolysis led to the 4oxoquinoline-3-carboxylic acid 5. The substitution of fluorine atom at the C-7 position by byciclo 6, which was prepared (Scheme 2) using pyridine-2,3-carboxylic acid 7 as start material furnished MXF. O
CO2H Ac2O N
CO2H
O N
7
8
O BnNH2
NBn N
O
9
O 1) H2 - Pd-C 2) LiAlH4
H NH N H H (S,S)
1) L-(+)-tartaric acid 2) crystalization 3) (mother liquors), NaOH 4) L-(-)-tartaric acid crystalization 5) NaOH 6) H2 - Pd-C
H NBn N H H (+ - ) cis 10
6
Scheme 2. Synthesis of bicyclo 6 present in MXF structure.
3.2 Gatifloxacin Another 8-methoxy fluoroquinolone with promising perspectives against TB is gatifloxacin (GAT) (Fig. 3). This substance presents similar activities to MXF against Gram-positive bacteria and similar maximum plasma concentration [33]. The half-life of GAT is 8.4 h [33], and its elimination route is renal, with 77% of the oral dose excreted in urine [34]. GAT is well tolerated when administrated in the advised dose (400 mg), nevertheless, dysglycemia was reported in patients using this substance [35-36]. O F H3C
CO2H
N HN
N OMe
Gatifloxacin
Fig. (3). GAT structure.
3.2.1 In vitro Studies In the first in vitro study, GAT had its activity available against 45 M. tuberculosis strains. The MIC50 of GAT (0.1 g/mL) was higher than the MIC50 of INH ( 0.05) and RIF
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( 0.05) against non-MDR strains. However, when tested against MDR strains, GAT displayed a MIC50 of 0.39 g/mL whereas the MIC50 of RIF and INH was 50 and 12.5 g/mL, respectively. GAT was also more active than RIF and INH against LFX resistant strains with a MIC50 of 0.78 g/mL, whereas RIF showed a MIC50 of 12.5 g/mL and INH 25 g/mL [37]. The high activity of GAT was later confirmed by Alvirez-Freites and coworkers [38], which utilized 23 M. tuberculosis strains, four resistant to INH or PYZ and one resistant to both drugs. GAT showed a MIC range of 0.007-0.12 g/mL, being more active than MXF (MIC range of 0.031-0.12 g/mL) and LFX (MIC range of 0.12-1 g/mL). Other important result was founded by Dong and colleagues [39], which determined the mutant prevention concentration (MPC) of GAT. MPC is the drug concentration that allows no mutant to be recovered from a susceptible population of more than 1010 cells. To be therapeutically useful, MPC must to be below than the concentration achievable in serum (Cmax) or tissue with safe doses of antibiotic. The MPC of GAT against the M. tuberculosis strain TN6515 was 1.5 g/mL and its rate MPC/ Cmax was 0.41, lower than to MXF (0.55) and ciprofloxacin (1.8). The importance of GAT in TB treatment was also verified combining this drug with the first line agent EMB, which raises the MIC of GAT from 0.03 to 0.052 [23]. 3.2.2 In vivo Studies In murine tuberculosis model, GAT showed a similar activity to that of MXF and INH [38]. When GAT was combined with EMB in vivo, no improvement was verified in GAT activity but, the combinations INH-RIF-GAT and RIF-GAT were more active than GAT alone and showed a similar activity compare to INH-RIF [38,43]. A very interesting result was founded when GAT was combined with ethionamide (ETA). GAT-ETA was more active than GAT or INH alone. It was also verified that the addition of PZA or PZA plus EMB did not improve the activity of the combination GATETA [38]. In a subsequently work, the dose o GAT in the combination GAT-ETA was evaluated. Mice treated with 100 mg/Kg of GAT and 25 mg/Kg of ETA had about 10 CFU/lung after 12 weeks, whereas in mice treated with 300 mg/Kg of GAT and 25 mg/Kg of ETA no bacteria could be cultivated [41]. These results suggested that ETA can be a promising single agent for the use in combination with GAT. Due to the important results in animal models, GAT is nowadays, in advanced clinical trials. 3.2.3 Clinical Trials In 2008, results of phase II clinical trial carried out by Gatifloxacin for TB Study Team (OFLOTUB) were published [42,43]. In this study, EMB was replaced by GAT, MXF or OFX in the standard regimen. MXF promoted a high reduction of colony count during the early phase, whereas during the late phase MXF and GAT led to a significant acceleration of bacillary elimination. Both substances improved the sterilizing activity of the treatment and could be considered important candidates to shorten the TB treatment regimen. Actually, a Phase III clinical trial with GAT is being conduced in several African countries [31]. The main goal of this study is to develop a 4 month treatment regimen combining GAT with others anti-TB drugs. The most important results obtained using GAT with others anti-TB drugs are summarized in Table 4.
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Table 4. Gatifloxacin Combined with Other Anti-TB Drugs Tests
Drugs
Results
in vitro
GAT + EMB
Decreased GAT activity [23].
in mice
GAT + EMB
Kept the activity unchanged [38].
RIF + GAT
Improvement in the activity of GAT Similar activity to that of INH + RIF [40].
INH + GAT + RIF
Improvement in the activity of GAT. Similar activity to that of INH + RIF [38].
GAT + ETA
Improvement in the activity of GAT [38].
GAT + ETA + PZA
Similar activity to that of GAT + ETA [38].
GAT + ETA + PZA + EMB
Similar activity to that of GAT + ETA [38].
GAT + INH + PZA + RIF
Significant acceleration of bacillary elimination during the late phase [42,43]
in humans
3.2.4 Gatifloxacin synthesis GAT is synthesized from a common intermediary of MXF, the 1-cyclopropyl-6,7difluoro-1,4-dihydro-8-methoxy-4-oxo-3-quinoline (5). Substitution of the fluorine atom at the C-7 position of 1 by 2-methylpiperazine (11) yields GAT (Scheme 3). The synthesis of 5 is described in MXF section (Scheme 1 and 2). O F
CO2H
F
DMSO 55-70oC 24 h CH3
N OCH3 5
NH
Gatifloxacin 84%
HN 11
Scheme 3. GAT synthesis.
3.3 Diarylquinoline TMC207 The diaryquinoline TMC207 (Fig. 4) was developed by the researcher Koen Andries and coworkers at Johnson & Johnson Pharmaceutical Research & Development. They have synthesized several compounds of diarylquinolines series and tested them for inhibition of multiple-cycle growth of M. smegmatis by a whole-cell assay. This test was chosen due to its ability to concomitantly assess multiple targets. In this context, they were found 20 potent derivatives (MIC below 0.5 g/mL), among them the most active R207910 (now called TMC207), exhibiting promising in vitro activities with MIC values ranging from 0.03 to 0.12 g/mL.
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CH3
OH
N
Br
CH3 N
OCH3
TMC 207
Fig. (4). Structure of TMC207.
3.3.1 Synthesis TMC207 is a quinoline derivative, which possess into its structure two chiral centers (R,S). The synthesis of this compound was carried out in four steps producing a mixture of four isomers. Firstly, 4-bromo aniline 12 was reacted with 3-phenylpropionyl chloride 13 in the presence of triethylamine and methylene chloride. The obtained product 14 was submitted to a Vilsmeier-Haack formylation followed by a cyclization using POCl3 in DMF to produce the quinoline 15. After that, a methoxy group was introduced by using MeONa in MeOH to lead the compound 16. The next step was based on the coupling reaction between 16 and 17 with diisopropylamine lithium (LDA) to produce 18 (TMC207) as a mixture of 4 diastereoisomers, which were purified by column chromatography, obtaining two pure fractions diastereoisomers (R,R and S,S) and (R,S and S,R), being separated by chiral HPLC (Scheme 4). O NH2 + Br 12
H N Cl Et3N CH2Cl2
Br POCl3 DMF
O
Br
N
14
13
CH3 O
CH3
OH
MeONa MeOH
N CH3 Br
N
Br
CH3 N
OCH3
LDA THF
18
+ N 17
4 Diastereomers (R, R), (S, S), (R, S) and (S, R) Column Chromatography
(R, R) (S, S)
(R, S) (S, R)
Chiral HPLC
Cl
15
TMC 207 (R, S)
Scheme 4. Synthetic route for preparation of the diarylquinoline TMC 207.
OCH3 16
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3.3.2 Mechanism of Action This substance will become the first TB drug with a new mechanism of action in 40 years. To determine its mechanism Andries et al [44] performed a comparative genomic analysis of mutants of M. tuberculosis and M. smegmatis resistant to TMC207, which demonstrated that the only gene affected in the mutants encodes aptE, suggesting that TMC207 inhibits subunit c (the proton pump) of mycobacterial ATP synthase. This enzyme uses the transmembrane proton-motive force to generate ATP for the mycobacteria and the subunit c forming a membrane-spanning oligomer, which is essential for this proton transport. Higher organisms also use this enzyme as supply cells with the bulk of their ATP via oxidative phosphorilation, which occurs in the mitochondria of eukaryotic cells. Therefore, the selectivity evaluation of TMC207 towards mycobacterial ATP synthase compared to mitochondrial ATP synthase is a relevant issue to investigate the toxicity of this compound. The lack of selectivity leads to mitochondrial toxicity, which is related to the development of several adverse events promoted by drugs, such as pancreatitis, peripheral neuropathy and cardial or skeletal myopathies. Hence, Haagsma and coworkers [45] studied this selectivity and concluded that TMC207 specifically inhibits mycobacterial ATP synthase. In addition, they showed that the human mitochondrial ATP synthase displayed much lower sensitivity for TMC207 than the mycobacterial enzyme, which indicates that this compound is very specific and unlikely to induce target-based toxicity in mammalian cells. Another important characteristic of TMC207 is that it promotes a disturbance on ATP homeostasis in dormant mycobacteria. Koul and coworkers [46] demonstrated that when the mycobacteria is latent, it still possess residual ATP synthase enzymatic activity. This activity is blocked by little concentrations of TMC207, including a decrease of ATP levels, which causes a significant bactericidal effect. They also verified an increase of susceptibility of dormant mycobacteria toward TMC207 as compared to actively growing bacteria. These results suggest that ATP synthase is an excellent target to treat latent TB infections and TMC207 could be an attractive candidate for this purpose. 3.3.3 Antimycobacterial Spectrum of TMC207 A study about the in vitro antimycobacterial spectrum of TMC207 verified that there is indeed no difference among the MICs of DS (drug-susceptible) and MDR strains. Furthermore, this substance exhibited a strong inhibitory effect against the majority of NTM (nontuberculous mycobacteria) species tested and it shows also promising results against MAC (M. avium complex) infections (MICs 0.25g/mL). Due to the broad antimycobacterial spectrum of TMC207, it could be considered an interesting candidate to treat other mycobacterial infections [47]. 3.3.4 Drug Combinations The combination of TMC207 with other anti-TB drugs in murine models has been performed by many research groups. The first study was reported by Andries et al [44], where they demonstrated that when TMC207 was administrated (12.5 and 25mg/Kg) in infected rats, it was significantly more active than INH (25 mg/Kg), which is the most powerful anti-TB drug. Moreover, when TMC207 was administrated as monotherapy, it was at least as active as the standard therapy [triple drug combination: RIF, INH and PYZ]. It is important to be mentioned that when added to this combination, TMC207 promoted a greater decrease in bacterial load in the lungs (relative to the standard RIF-INH-PYZ
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treatment regimen) by 2 log units after 1 month of therapy and by a further 1 log unit after 2 months of therapy. Other combinations were tested through the substitution of each first-line drug of the standard therapy by TMC207 and the results demonstrated that the activity of each new combination was improved when compared with RIF-INH-PYZ regimen, mainly after 1 month of treatment. Additionally, after 2 months of treatment with TMC207-INHPYZ and TMC207-RIF-PYZ, the lungs of all animals were culture-negative. However, the differences between the bactericidal activities of the combinations TMC207-INH-RIF, TMC207-INH-PYZ, and TMC207-RIF-PYZ were not significant. The bactericidal activity obtained by the RIF-INH-PYZ combination after 2 months of therapy was comparable to the combinations TMC207-INH-PYZ and TMC207-RIF-PYZ after just 1 month of therapy. Nevertheless, Ibrahim et al [48] demonstrated an important synergistic interaction between TMC207 and PYZ in murine model. It was observed that after only 2 months of treatment this combination led to complete eradication of the bacilli in mice lungs. In addition, it was verified that the addition of RIF, INH or MXF to the TMC207-PYZ regimen could not further increase the activity after 2 months of treatment. Nevertheless, the addition of a third drug in this combination could provide potential benefits, for instance to increase the sterilizing activity by introduction of RIF or MXF in the regimen. For example, the administration of PYZ in humans can not prevent the selection of mutants resistant to the companion drug suggesting that the introduction of a third drug in this proposed combination could be necessary. Another important combination studied was TMC207 and RIF, because there is a pharmacokinetic interaction between these drugs, which is responsible by 50% reduction in the level of TMC207 exposure in humans. Due to this problem, Lounis and coworkers [49] have studied the impact of the interaction of TMC207 with RIF on the standard treatment (RIF-INH-PYZ). This study verified that TMC207 has significant activity even when its exposure is reduced by 50% and also when it is added to a strong background. The treatment of MDR-TB is also an important problem related to TB global control. Currently, WHO recommends the use of a regimen including AMK, ETA, MXF and PYZ to MDR-TB treatment. In order to verify the influence of TMC207 in the efficacy of this regimen, infected rats were treated five times per week with TMC207 alone or various combinations of TMC207 with the second-line drugs AMK, PYZ, MXF and ETA. All TMC207-containing regimens were significantly more active than the non- TMC207containing regimens after 1 month of therapy. When TMC207 was combined with secondline drugs, the combinations were more active than the currently recommended regimen of MDR-TB (AMK-ETA-MXF-PYZ), and culture negativity of both lungs and spleen were reached after 2 months of treatment in almost every case. These results can be considered very promising because they indicate a hope for shortening MDR-TB treatment duration (9 months to 2 or 3 months) [50]. 3.3.5 Clinical Studies Preclinical safety assessments supported the administration of TMC207 to humans. Hence, Andries and coworkers [44] studied the pharmacokinetics, safety and tolerability of TMC207 in health male adults in a double-blind, randomized, placebo-controlled design. The results showed that TMC207 has a good oral absorption, long plasma half-life, high tissue penetration and long tissue half-life, which are important characteristics for treatment of chronic infections. Furthermore, they also demonstrated that TMC207 has a good tolerability and the adverse effects were mild or moderate in severity.
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After that, Rustomjee and co-workers [51] reported the results of a phase IIa clinical tests, which were verified the pharmacokinetics, safety, tolerability, and extended early bactericidal activities of three different daily oral doses (25mg, 100mg and 400mg) of TMC207 administered as monotherapy over a treatment period of 7 days in treatment-naive patients with sputum smear-positive pulmonary TB. They demonstrated that the administration of 400mg TMC207 promotes a relevant bactericidal activity from day 4 onward and it was comparable to those of INH and RIF over the same period. In Table 5, there is a summary of pharmacokinetics data of TMC207 after oral administration at 400mg dose. Furthermore, Diacon and coworkers [52] reported the results of a phase IIa clinical tests, which were assigned 47 patients with newly diagnosed, smearpositive pulmonary infection caused by MDR-TB. This patients receive either TMC207 (400 mg daily for 2 weeks, followed by 200 mg three times a week for 6 weeks or placebo in combination with a standard second-line anti-TB regimen (KAN, OFX, ETA, PYZ and CYC or terizidone). They observed that the rates of conversion to a negative culture were 48% in the TMC207 group (10 of 21 patients) and 9% in the placebo group. Furthermore, the rates of negative smears for acid-fast bacilli at week 4 were 57% for the placebo group and 77% for the TMC207 group, and at week 8 were 68% for the placebo group and 84% for the TMC207 group. Moreover, the most adverse events were mild to moderate, and only nausea occurred significantly more frequently among patients in the TMC207 group than among patients in the placebo group (26% vs. 4%). These results confirmed the safety and efficacy of TMC207 for MDR-TB treatment. Table 5. Pharmacokinetics of TMC207 after Oral Administration at 400mg a Parameterb
Value
n (Day 7)
12
Ch (ng/ml)
1,530 ± 438.0
Cmin (ng/ml)
1,448 ± 437.4
Cmax (ng/ml)
5,502 ± 2,965
tmax (h)
4.00 (2.05-6.02)
AUC0-24 (ng . h/ml)
6,4750 ± 20,700
Css.av (ng/ml)
2,696 ± 865.4
FI (%)
144.3 ± 46.22
a
All doses q.d. for 7 days. Data are expressed as mean ± SD, for tmax: median (range). b n, number of subjects with values; Cøh, plasma concentration at hour zero, i.e., predose; Cmin, minimum concentration of drug in serum; Cmax, maximum concentration of drug in serum; tmax, time to maximum concentration of drug in serum; AUC0–24, area under the concentration-time curve, 0 to 24 h; Css,av, average steady-state plasma concentration; FI, fluctuation index.
3.4 PA-824 Bicyclic nitroimidazofurans was originally investigated for cancer treatment [53], but were found to have antimycobacterial activity. The lead compound of this series CGI 17341 (Fig. 5) was mutagenic and futher investigations were discouraging [54]. In 2000, in spite of these results, Baker and co-workers [55-57] prepared a series of 328 substances on the basis
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of the structure of CGI 17341 (Fig. 5). Among these, over 100 compounds possessed relevant antitubercular activity, with no unfavorable mutagenic features. The structureactivity relationship (SAR) of this class is summarized in Fig. (6). Therefore, they chose PA-824 (Fig. 5) as a lead compound, because this substance exhibited significant in vitro and in vivo activity (see above). In 2002, due to the promising results, TB Alliance and Chiron Corporation signed a license agreement that gave TB Alliance rights to develop this molecule and its analogues. Chiron pledged to make the technology available royalty-free in endemic countries [58]. O2N
N
O
N
O2N
O
N
N
O (PA-824)
(CGI 17341)
OCF3
Fig. (5). Structures of the nitroimidazoles CGI 17341 and PA-824.
O2N
N
O S enantiomers only active N X
X= O, urea, carbamate or carbonate
R
Substituents in ortho and para or in meta positions
R Substituents in para position. They should be large or slightly eletronegative
Modifications that positively contributes to biological activity Modifications that negatively contributes to biological activity
Fig. (6). SAR of nitroimidazo[2,1-b]oxazine series (adapted from reference [59])
This substance has many attractive characteristics, such as its activity in vitro against all tested drug-resistant clinical isolates, its activity as both, a potent bactericidal and a sterilizing agent in mice and its novel mechanism of action, which is responsible for avoiding cross-resistance to current anti-TB drugs. All of these characteristics will be detailed in the further sections. 3.4.1 Mechanism of Action The mechanism of action of PA-824 is very complex. Stover and co-workers observed that the nitroimidazopyrans (NAP) inhibit the synthesis of proteins and mycolic acids. However, these effects can not explain the cell killing under non-replicating conditions, because theses processes are not extensively occurring under anaerobic conditions [57]. Therefore, Singh and co-workers have found that PA-824 is converted into three main metabolites by Rv3547, a deazaflavin-dependent nitroreductase (Dnd). The major
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metabolite is the des-nitroimidazole (des-nitro), which is directly related to the anaerobic killing of M. tuberculosis by generation of reactive nitrogen species, such as nitric oxide. These species react with cytochromes and cytochrome c oxidase leading to interference in the electron flow, interruption the coupling of respiration to reduction of oxygen and, consequently, the cell killing [60]. 3.4.2 In vitro Studies Lenaerts and co-workers [61] reported that PA-824, when tested in vitro against a wide collection of MDR clinical isolates retrieved internationally, it was found to be highly active (MIC < 1g/mL) against all strains tested. Furthermore, this substance was assessed against M. tuberculosis isolates grown under conditions of transient oxygen depletion and PA-824 showed a strong activity against these persistent bacteria in a dose-dependent manner. The observed activity was slightly less than those of GAT and MXF, but it was very similar to that of metronidazole (MET), which is an antibiotic that possess activity against M. tuberculosis isolates that survive under anaerobic conditions [62]. In addition, the efficacy of PA-824 was evaluated in a short-course mouse infection model, which demonstrated that this substance has an action comparable to those of INH, RIF and MXF after nine oral treatments at 50, 100 and 300 mg/Kg of body weight. Moreover, in a long-term treatment with PA-824 at 100mg/Kg the bacterial load was reduced to bellow 500CFU in the lungs and spleen. When the activity of PA-824 was compared to other single drug regimens tested (INH at 25mg/Kg, RIF at 10mg/Kg, GAT at 100mg/Kg and MXF at 100mg/Kg ) no difference could be observed. It is important to be mentioned that due to the poor solubility of PA-824 in aqueous solution, the used doses in these experiments were formulated in cyclodextrin/lecitin (CM2). 3.4.3 In vivo Studies Using Murine Model of TB Tyagi and co-workers [63] studied the bactericidal activity of PA-824 in a murine model of TB. After determine the MIC of this substance (0.125 g/mL), they established the minimal effective dose (MED) and the minimal bacterial dose (MBD), which were 12.5 and 100 mg/Kg of body weight/day, respectively. Afterwards, an experiment was performed to characterize the bactericidal activity of PA-824 during the initial phase of therapy. They discovered that when PA-824 was administrated as monotherapy at the MBD, it exhibited similar bacterial activity to that of the equipotent dose of INH in humans during the 2-month initial phase of treatment. In addition, when administrated in combination with INH, PA824 prevented the selection of INH-resistant mutants. Another study was performed to measure the activity of PA-824 during the continuation phase of treatment (sterilizing activity), which was verified that this substance also demonstrated activity on persistent bacilli that, survived after the intensive initial phase (2 months with INH, RIF and PYZ). 3.4.4 Combinations of PA-824 with First-Line Drugs in vivo Nuermberger et al [64] studied the incorporation of PA-824 to the standard 6-month treatment regimen (INH, RIF and PYZ) with the aim of determining if it is an alternative way that could reduce the duration of treatment. However, when PA-824 was added to the standard regimen it was not able to improve its sterilizing activity and, consequently, to decrease the period of treatment. Furthermore, the substitution of PA-824 for INH promotes an extensively lower lung CFU counts after 2 months of treatment and a more quick culturenegative conversion compared to standard treatment. Nevertheless, there was no difference in the proportion of mice relapsing after completing 6 months of therapy. Moreover, they
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observed that no other PA-824- containing regimen tested was superior to the standard regimen. The authors tried to explain this result by some issues, such as the lack of additive activity of PA-824 when combined with the drugs in the standard regimen, the limited sterilizing activity of PA-824 and the antagonism between PA-824 and one or more drugs in the RIF-INH-PYZ regimen. Nevertheless, PA-824 has the potential to contribute to entirely novel regimens when combined with other new investigational drugs or second-line drugs and it could be important in combinations for MDR-TB treatment. To determine the applicability of introduction of PA-824 in other regimens, Nuermberger et al [65] propose that the introduction of this substance into the RIF-MXFPYZ therapy could improve it. This regimen is more active than the standard treatment and promotes a reduction of the period of treatment by up to month (from 6 to 4 months) [24, 25]. The authors hypothesized that the addition of PA-824 would permit a further reduction in the treatment duration from 4 to 3 months. However, the results showed that the incorporation of PA-824 to the experimental 4-month RIF-MXF-PYZ regimen did not allow the shortening of the treatment duration to 3 months. Nevertheless, they observed that the combination of PA-824, MXF and PYZ was at least as effective as RIF-MXF-PYZ in reducing organ CFU counts but may be somewhat less effective in keep a durable culturenegative state after treatment. In addition, PA-824-MXF-PYZ regimen cured mice more quickly than the first-line regimen (RIF, INH and PYZ). Another important information about PA-824 is that it substitutes relatively well RIF. This fact is highly relevant because RIF is a potent metabolic inducer of many compounds, thus PA-824-containing regimens may provide attractive alternatives to RIF-containing combinations in evaluations of new drug candidates metabolized by P450 enzymes, such as TMC207. Tasneen and co-workers have investigated the potential benefits of substitution of PA824 for INH in the standard regimen, because this drug has a proven antagonistic activity on the activity of RIF-PYZ in a murine model [49]. They found that the addition of PA-824 at 12.5 and 25 mg/kg/day into RIF-PYZ did not increase the activity of these drugs, but the addition of PA-824 at 50 and 100 mg/kg/day increased the activity in a dose-dependent manner. Therefore, they verified that the combination of RIF, PA-824 (100 mg/kg) and PYZ led to all mice culture negative after 2 months of treatment and free of relapse after 4 months of treatment, while some mice receiving the standard regimen remained culture positive and 15% relapsed after completing 4 months of treatment. This experiment also showed that PA-824 and PYZ exhibited synergistic activity that was equivalent to that combination of first-line drugs [66]. 3.4.5 Phase I Studies In 2009, Ginsberg and co-workers [67] reported the phase I study of PA-824 where they evaluated the safety, tolerability and pharmacokinetics of this substance. This study was divided in two stages: a single-dose and a multiple-dose (up to 7 days of daily dosing). In both cases, PA-824 was readily absorbed, bioavailable and well tolerated. The pharmacokinetics parameters indicated oral bioavailability and a half-life consistent with a once-per-day dose–regimen. Furthermore, no significant or serious adverse events were observed in the 58 subjects dosed with up to 1.000 mg PA-824 for up to 7 days (the multidosing was halted at 5 days at 1.000 mg due to increases in serum creatinine). Furthermore, serum creatinine elevation has been shown to be unrelated to human safety when directly examined in a renal effects study.
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With the aim to verify if the observed effect of PA-824 on circulating creatinine is of clinical significance relative to renal function, Ginsberg and co-workers [68] conducted a phase I clinical study to assess the renal effects of PA-824 (compared to those of a placebo) at doses of 800 and 1.000 mg per day for 8 days in normal volunteers by determining of changes on the levels of serum creatinine (SC), glomerular filtration rate (GFR), effective renal plasma flow (ERPF), filtration fraction (FF), creatinine clearance (CrCl), extraglomerular creatinine excretion (EGCE), blood urea nitrogen (BUN), and uric acid (UA). The results demonstrated that at a dose of up to 1.000 mg/day for 8 days, PA-824 was safe and well tolerated in healthy volunteers with no significant impact on renal physiology. 3.5 OPC-67683 OPC-67683 (Fig. 7) is another promising anti-TB compound that was found through the optimization of CGI 17341 (Fig. 5), which was the lead compound of the bicyclic nitroimidazofurans series [54]. Sasaki and co-workers [69] at Otsuka Pharmaceutical Development & Commercialization, Inc. (OPDC) synthesized a series of 6-Nitro-2,3dihydroimidazo[2,1-b]oxazoles being the structure-activity relationship (SAR) of this class summarized in Fig. (7). Hydrophilic group improves bioavailability
Lipophilic phenoxy
Ring directely attached to hetero atom
Hetero atom eliminates mutagenicity Me x H
O
N
O N OPC-67683
para substituents F3CO
S is less active than R
O N
NO2
CGI-17341 core
CF3, F, Cl
Subtitle: modifications that positively contributes to biological activity modifications that negatively contributes to biological activity important issues to mutagenicity or pharacokinetics parameters
Fig. (7). Structure of OPC-67683 and SAR of 6-Nitro-2,3-dihydroimidazo[2,1-b]oxazoles series.
3.5.1 Synthesis Essentially, OPC-64683 was prepared by coupling the key intermediate (R)-form of epoxide 24 with a specific phenol 28, followed by ring closure in the presence of sodium hydride in DMF. The key intermediate 24 was prepared in four steps. Firstly, the nitroimidazole 19 was reacted with the epoxide 20 in Et3N and AcOEt to produce the
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compound 21, which was de-esterificated using MeOH and K2CO3 to afford the diol 22. This compound was reacted with MsCl in pyridine to furnish the mesylate 23, which was converted into the epoxide 24 by using DBU in AcOEt. The preparation of the phenol was performed in two steps. Initially, 2-(4-bromophenoxy) tetrahydropyran 25 was reacted with the 4-phenoxypiperidine 26 by the Buchwald palladiumcatalyzed amination method to give 27. Then, this derivative was deprotected with pyridinium p-toluenesulfonate in ethanol to afford the desired phenol 28 (Scheme 5) [70]. NH
O
O
O
+ O2N
N
Cl
Et3N
O
N
AcOEt
19
20
O2N
N
OH
O2N
N
OMs OH
DBU
Cl
O
N
AcOEt
O2N
23
N
+
N
MeOH O2N
Cl 21
NO2
N
K2CO3
O
HO
OH OH
MsCl Py
Cl
N
22
NO2
O
N
NaH DMF
Cl
OPC-67683
28
24
OCF3 Pyridinium p-toluene sulfonate, EtOH
O
O O 25
Br
+ HN
O
Pd(OAc)2
O
N
O
Rac BINAP
26 OCF3
Cs2CO3 toluene
27 OCF3
Scheme 5. Synthetic route for preparation of OPC-67683.
3.5.2 Mechanism of Action Matsumoto and co-workers [71] evaluated the inhibitory activity against mycolic acid biosynthesis and the results showed that OPC-67683 inhibits the synthesis of methoxy and keto-mycolic acids, but not the synthesis of –mycolic acids while INH inhibits all mycolic acid subclasses. Similarly to PA-824, OPC-67683 also needs metabolic activation by M.tuberculosis to produce its anti-TB activity. Through the isolation of OPC-67683-resistant strains, they observed that these strains did not metabolize the compound. Thus, they confirmed a mutation in the Rv3547 gene among these strains, which shows that Rv3574 is the key enzyme involved in the activation of OPC-68673. Another study indicated that the major metabolite of OPC-67683 was identified as a non-active desnitro-imidazooxazole. This information suggests that Rv3547 has a reduction potency of the nitro group and that an intermediate between OPC-67683 and desnitroimidazooxazole could be the active form. Furthermore, in an experiment with radioactive OPC-67683, they found that in standard strains, 20% of the radioactive substances were not recovered at the end of the exposure while in OPC-67683-resistant strains 100% of radioactivity was recovered. This data suggests that a radical intermediate that appears as the intermediate for the metabolism of a nitro residue covalently binds to the target molecule. This hypothesis could explain the strong post-antibiotic effect observed with OPC-67683 against intracellular mycobacterium,
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which was demonstrated in an experiment that examined the killing activity intracellular TB in macrophages. This study is an important information about the capacity of this drug to be effective against latent TB. 3.5.3 In vitro Studies OPC-67683 was assessed in vitro against both drug-susceptible and drug-resistant standard or clinically isolated strains. This substance exhibits MIC ranging from 0.006 to 0.012 g/mL, being most potent than RIF, INH, EMB, streptomicyn, CGI-17341 and PA824. This experiment also proved that OPC-67683 does not show cross-resistance to any currently used anti-TB drug [71]. 3.5.4 In vivo Studies Using Mouse Model of TB In this model, OPC-67683 exhibited the most potent anti-tubercular activity in comparison with the standard anti-TB drugs. The viable bacterial counts in the lung were clearly reduced dose-dependently this substance at 0.313 mg/kg and higher. Moreover, OPC-67683 was so effective in a TB model established using immunodeficient mice than using standard mice. These data are an important evidence that this substance could be helpful in the treatment of TB patients co-infected with HIV/AIDS [71]. 3.5.5 Combinations of OPC-67683 with First-Line Drugs In vitro studies demonstrated that OPC-67683 did not exhibit antagonistic effects in any of tested combinations and also produced partial or total synergistic effects when combined with RIF and EMB. When combined in vivo with RIF and PYZ (2 months) followed by combination with RIF for more two months, OPC-67683 produced a rapid reduction in bacterial load over the first 3 months. Considering that, this substance could be effective in shortening the clinical treatment duration [71]. 3.5.6 In vitro Metabolism in Human and Animal Liver Microsomes This experiment is very important because many drugs interactions are caused by inhibition of drug-metabolizing enzymes, such as Cytochromes P450 (CYPs). An opportune example is RIF, which induces CYP3A4 enzymes that lead to a reduction in the bioavaibility of the drug itself as well as other CYP-intermediate drugs, including protesase inhibitors that are essential for HIV/AIDS treatment. The results demonstrated that OPC67683 did not exhibit inductive, stimulatory or inhibitory effects on CYP enzyme activities at its expected therapeutic concentration. Thus, OPC-67683 would not be expected to cause clinically significant interactions with other CYP-metabolized drugs, such as RIF. These results, together with data supporting non-compromised anti-TB activity inimmunodeficient animals, suggest that OPC-67683 could be useful in treating TB patients who are also coinfected with HIV/AIDS. 3.5.7 Clinical Trials A phase II trial to evaluate the safety, efficacy and pharmacokinetics of four oral doses of OPC-67683 in patients with uncomplicated, smear-positive, pulmonary tuberculosis was completed in 2007 and appear to have been successful. Currently, a placebo-controlled,
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phase 2 trials to evaluate OPC-67683 in patients with pulmonary sputum culture-positive, multidrug-resistant TB are in progress [31]. 3.6 SQ-109 SQ-109 (Fig. 8) is an EMB analog developed at Sequella Inc. in collaboration at NIH/NIAID. Protopopova and co-workers [72] have synthesized a library of 63 238 compounds based on the pharmacophore of EMB (1,2-ethylenediamine) by solid-phase synthesis using an acylation-reduction sequence that is compatible with high-throughput screening. After evaluation of the in vitro activity against Mycobacterium tuberculosis, they have identified 26 substances with activity that equals or exceeds that of EMB (MIC = 7 M). Thus, another study was performed to identify a candidate drug for clinical development through a set of tests in vitro and in vivo, such as determination of MIC for M. tuberculosis H37Rv, cytotoxicity, intracellular antimycobacterial activity, permeability evaluation and in vivo efficacy testing. Among all tested substances, the compound SQ-109 was identified as the most potent, which had a MIC of 0.7–1.56 mM, a selectivity index (SI) of 16.7and demonstrated activity against drug-resistant strains of M. tuberculosis. Another important information is that SQ-109 reduced the intracellular M. tuberculosis burden by 99% at its MIC (1.56 mM). Furthermore, this compound did not verify toxicity to mice. Preliminary in vivo studies showed that SQ-109 had high activity, mainly in lungs, and it was effective to treat TB infection in mice at 1 mg/kg (EMB at 100 mg/kg) [73]. Pharmacophore of ethambutol: 1,2-ethylenediamine
CH3
CH3
H3C
N H
H N Bulky core: adamantyl
Isoprenyl units
Fig. (8). Structure and SAR of SQ-109.
3.6.1 Pharmacodynamics and Pharmacokinetics Data Jia and co-workers [74] studied the pharmacodynamics and pharmacokinetics of SQ109. Firstly, they confirmed the antimicrobial activity of SQ-109 in vitro and in vivo and compared to isoniazid INH and EMB. These tests showed that SQ-109 has potency and efficacy similar to INH in inhibiting intracellular M. tuberculosis, but superior to EMB. When administrated in vivo via oral to the mice for 28 days (0.1–25 mg kg-1 day-1), SQ-109 promotes a dose-dependent reductions of mycobacterial load in both spleen and lung comparable to that of EMB administered at 100 mg kg-1 day-1, but was less potent than INH at 25 mg kg-1 day-1. The study of monitoring of drug levels in mouse tissues demonstrated
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that lungs and spleen contained the highest concentration of SQ-109, at least 10 times above its MIC. The pharmacokinetic profile of SQ-109 in mice following a single administration was summarized in Table 6. The main relevant information about these data is the low oral bioavailability (4%), which could be explained by its poor oral absorption, high first-pass effect and its high dissociation constants from blood proteins. However, SQ-109 displayed a large volume of distribution into various tissues; for instance, in the targeted tissue concentrations of SQ-109 were at least 120-fold higher than that in plasma. Table 6. Pharmacokinetic Parameters (mean ± s.e.m.) of SQ109 in Mice Route Parameter Intravenous ( i.v.)
Oral (p.o.)
Dose (mg kg-1)
3
25
AUC0 (ng . h/ml)
792 ± 369
254 ± 184
a
0.07± 0.051
---
T1/2 (h)b
0.43 ± 0.35
---
3.5 ± 6.6
5.2 ± 1.1
Cmax (ng/ml)
1038 ± 93
135 ±10
Tmax (h)
---
0.31± 0.06
Vdss (ml kg )
11826±14878
---
Bioavailability (%)
---
4
T1/2 (h)
T1/2el (h)
c
-1
a b c
Half-life of the distribution phase. Half-life of the initial elimination phase. Half-life of the terminal elimination phase.
To explain these pharmacokinetic data, Jia and co-worker [75] also characterized the interspecies absorption, distribution, metabolism and elimination (ADME) profile of SQ109. They observed that after oral administration of [14C]SQ-109 to rats the highest level of radioactivity was in the liver, followed by the lung, spleen and kidney. After that, they determined the main metabolites in the human liver microsomes as products of oxidation, epoxidation and N-dealkylation of SQ109. Another relevant information is that SQ-109 is predominantly metabolized by CYP2D6 and CYP2C19. These facts evidenced that the rapid liver microsomal metabolism of SQ-109 is the key to the significant observed first-pass effect. 3.6.2 Combination with First-Line Drugs in Mouse Model of Chronic TB Chen and co-workers [76] have determined interactions of SQ-109 with other anti-TB drugs (INH, RIF, PYZ, EMB and Streptomycin) in vitro. They were found that no antagonism was observed with any two-drug combination tested. Furthermore, SQ-109 did not show any positive interaction (additive or synergistic) with either EMB or PYZ. However, SQ-109 showed an additive effect with Streptomycin and a synergistic effect with INH and RIF. Due to the intensity of the observed synergistic interaction in the combination
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of SQ-109-RIF, they also studied the effect of this interaction in RIF-resistant strains of M. tuberculosis (H37rv) and they demonstrated that these results suggested that this synergy between SQ-109 and RIF was specific for this combination of drugs and that the enhanced drug interactions inhibited M. tuberculosis functions in both susceptible and resistantstrains. Nikonenko and co-workers [77] compared the efficacy of TB therapy with INH-RIFPYZ and INH-RIF-PYZ-EMB where SQ-109 was added or replaced by EMB in the more standard chronic mouse model of TB. They demonstrated that the combination of SQ109INH-RIF-PYZ afforded a new and very effective anti-TB intensive phase treatment regimen that achieved a better and faster rate of mycobacterial kill than the therapeutic regimen INHRIF-EMB-PYZ. 3.6.3 Mechanism of Action The precise target(s) for SQ-109 activity is not yet known, but it affects the synthesis of M. tuberculosis cell walls. Protapopova et al postulated that SQ-109 and EMB have different mechanism of action or activating mechanism, because SQ-109 possess activity against ETA-resistant strains and it shows differences of behavior in genearray studies. [77, 73]. Another evidence of this difference was reported by Jia and co-workers [78]. Through a proteomic approach comparing the effects of 24-hour drug on Mycobacterium tuberculosis H37Rv, they demonstrated that SQ-109 did not affect EmbA or EmbB, the target of EMB. 3.7 LL-3858 The story of LL-3858 (Fig. 9) started with the pyrrole BM-212 (Fig. 9) described in the 1900´s by Deidda and co-workers [79] and Biava and co-workers [80], which evaluated a series of pyrroles with promising antimicrobial activity. Few years later, in 2004 at Lupin Ltd were synthesized a new series of this class with improved anti-TB activity. In this study, they identified the lead compound called LL-3858. This compound possess activity against drug-susceptible (MIC= 0.12-0.025 mg/ml) and drug-resistant strains of M. tuberculosis. It also showed in vitro synergistic activity with RIF. Furthermore, LL-3858 probably has a novel mechanism of action, but this mechanism has not yet been established. N Ph
N HN
CF3 N
N
N CH 3 N
O LL-3858
CH3
BM-212
N
Fig. (9). Structure of pyrrole LL-3858 and BM-212.
3.7.1 In vivo Studies In vivo studies demonstrated that when combined with the first-line drugs, LL-3858 sterilized the lungs and spleens of infected animals in a shorter time than the standard regimen. In addition, when administrated alone, LL-3858 it is more effective than INH.
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3.7.2 Phase I Trial A Phase I trial was performed in healthy adult male volunteers. This study showed that LL-3858 is better absorbed under fasting conditions and it seemed to be safe and well tolerated in doses bellow 600mg. 4. CURRENT AND FUTURE DEVELOPMENTS The worldwide problem caused by TB and the lack of new drugs in the market makes it imperative to have new drugs to fight efficiently against the rapid spread of multi-drug resistant TB strain against all major anti-tuberculosis drugs in the market. In this context, there is an urgent need for TB drugs with fewer toxic side effects, improved pharmacokinetics properties, extensive and potent activity against Gram-positive and Gram-negative bacteria, including resistant strains and drugs able to reduce the total duration of treatment. REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16]
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Drug Effects on Drug Targets: Inhibition of Enzymes by Neuroleptics, Antimycotics, Antibiotics and Other Drugs on Human Pathogenic Amoebas and their Anti-Proliferative Effects Raúl N. Ondarza*,&,‡,# #
Department of Biochemistry, Faculty of Medicine, National Autonomous University of Mexico (UNAM), University City, Mexico 04510, Mexico, &Center of Research on Infectious Diseases, National Institute of Public Health, Cuernavaca, Morelos, Mexico 62508, and ‡ Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California, San Diego (UCSD), La Jolla, CA 92093-0204, USA Abstract: This paper reviews the inhibition of various enzymes by neuroleptics, anti-mycotics, antibiotics and other drugs on three species of human pathogenic amoebas, mainly Entamoeba histolytica, Acanthamoeba polyphaga and Naegleria fowleri, and their antiproliferative effects. A recent patent registered by Philip relates to the combination of an antibacterial formulation and antifungal agent for producing a therapeutically effective quantity of an antimicrobial that is suitable for suppressing or treating fungal growth. The rationale behind this patent focused on essential and valid targets with a description of the main pathogenic characteristics of these amoebas. The study of new targets, such as trypanothione and trypanothione reductase, and the drug effects of selected agents were arranged into six main groups: A) Inhibition of disulfide reducing enzymes by neuroleptics, antimycotics and antibiotics; B) Comparative evaluation of the efficacies of several drugs with antiproliferative activities; C) Inhibition of the enzymes for the synthesis of trypanothione, such as ornithine decarboxylase, spermidine synthase and trypanothione synthetase; D) Inhibition of the glycolytic enzyme PPidependent phosphofructokinase (PFK) from Entamoeba and Naegleria by pyrophosphate analogues, different from the host enzyme; E) Inhibition of enzymes secreted by these parasites to invade the human host, for example cysteine proteinases; and F) Inhibition of encystment pathways and cyst-wall assembly proteins.
Keywords: Drug effects, neuroleptics, antimycotics, antibiotics, antiproliferative effects, Entamoeba histolytica, Acanthamoeba polyphaga, Naegleria fowleri. INTRODUCTION The development of new antiparasitic drugs is becoming more demanding every day since existing drugs either have too many side effects, or they tend to lose effectiveness due *Corresponding author: E-mail: [email protected]
Atta-ur-Rahman / M. Iqbal Choudhary (Eds.) All rights reserved – © 2010 Bentham Science Publishers.
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to resistant parasitic strains [1,2]. Consequently, one of the most promising approaches in developing candidate drugs is to find new targets that are present in the genome of the parasites but absent in the human host genome [3]. Comparing the metabolic pathways of parasites and their hosts helps to identify new, potential drug targets [4], and such groups and pathways considered useful for comparison include outer-membrane proteins, proteins assembling the vesicular with the plasmatic membrane, permeases and enzymes of intermediary metabolism. The search for potential “drug targets” that derive from human pathogens depends on essentiality and validity, in that the “target” must be essential for the growth, replication and viability of the pathogen. An example would be cysteine proteinases, which are attractive potential targets because they are essential for pathogenesis [5]. Further research is required to characterize the cellular functions of these gene products and validate them as targets. Some of the enzymes studied here, such as trypanothione reductase [6] and PPi-dependent phospho fructokinase [7], which are unique to Entamoeba histolytica and Naegleria fowleri and not present in the human host, could be used as potential drug targets. A recent patent registered by Goyal Neena relates to a process in the prokaryotic system for heterologous expression and large scale production of trypanothione reductase, a functionally active enzyme of Leishmania donovani [8]. It is assumed that proteins encoded by essential genes are valid targets, and in this context Barrett et al. [9] discussed the validity and process of identification, reaching the following tentative conclusion: “if the gene cannot be deleted from any life-cycle stage, it means that is an essential gene, and so is a good drug target”. GENERAL CHARACTERISTICS OF ENTAMOEBA HISTOLYTICA, ACANTHAMOEBA POLYPHAGA AND NAEGLERIA FOWLERI Entamoeba histolytica is a protozoan considered to be the world’s second leading cause of death after malaria [10]. Recently, the genome of this atypical eukaryote was published [11] as was the genome of Trichomonas vaginalis [12]. E. histolytica reveals a variety of metabolic deficiencies [11], which include the reduction or elimination of most mitochondrial metabolic pathways. E. histolytica uses oxidative stress enzymes, generally associated with anaerobic prokaryotes and bacterial-like fermentation enzymes, but it lacks proteins from the tricarboxylic acid cycle and mitochondrial electron transport chain. In fact, E. histolytica has been re-classified as two genetically distinct intestinal parasites: one pathogenic and one benign. The name E. histolytica has been retained for the parasite that causes invasive intestinal and extra-intestinal amoebiasis, while the name Entamoeba dispar has been given for the non-pathogenic intestinal commensal organism that is visually indistinguishable from E. histolytica [13]. There are several species of Acanthamoeba a free-living amoebae that can cause Granulomatous Amebic Encephalitis (GAE), a chronic progressive disease of the central nervous system, as well as amoebic keratitis, a chronic eye infection. The recent increase in the incidence of GAE is due in part to infection in patients with acquired immune deficiency syndrome (AIDS), while increases in cases of amoebic keratitis in healthy individuals has been linked to the increased use of contact lenses [14, 15]. In the natural environment, very few Acanthamoeba are actually pathogenic to humans but differentiation between pathogenic and non-pathogenic species is difficult to achieve
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[16]. Many attempts have been made to differentiate the two and several properties have been identified as potential identifying factors such as the ability to grow at temperatures similar to those found in mammals. In addition, classification of Acanthamoeba has been problematic since it has been based on morphological features only, but it is possible to obtain better results using genetic techniques based on 18S rRNA sequence analysis [17]. Naegleria fowleri is a free-living amoeba that can be pathogenic to humans producing a Primary Acute Meningo-encephalitis (PAM) that causes death of the host within a few days of the first symptoms being detected. It is not common for N. fowleri to be pathogenic but it is an important amoeba because diagnosis can be difficult and PAM usually is rapidly fatal [18]. The first case of meningo-encephalitis in the United States produced by N. fowleri was reported by Butt [19] who coined the term PAM. Although N. fowleri is just one of several species in the genus Naegleria, to date, it remains the only known Naegleria species to produce this human disease. This amoeba is a facultative pathogen capable of living many generations without infecting a host. Infection begins via the nasal mucosa and olfactory nerves, crossing the cribriform plate and gaining access to the central nervous system. Once in the brain, extensive inflammation, hemorrhage, and necrosis occurs leading to death within 3 to 7 days [20]. Naegleria fowleri infections [21] have occurred in children and young adults who have had recent exposure to swimming or diving in warm fresh water. Due to its thermophilic nature, N. fowleri is capable of surviving in waterways contaminated by thermal discharges from power plants, waste water sewage, heated swimming pools, and even hot springs. DRUGS EFFECTS A) Inhibition of Disulfide Reducing Enzymes by Neuroleptics, Antimycotics and Antibiotics Prior to 1978, it was accepted that glutathione (GSH) and its dependent enzyme glutathione reductase were the only normal components of eukaryotes and prokaryotes maintaining a reducing environment within the cell. However, some time later Fahey et al. [22] reported that some Gram-positive bacteria do not produce glutathione, and Ondarza et al. [23] established that Arqueobacteria and strict anaerobe bacteria do not have glutathione reductase. Fahey et al. [24] then showed that the human parasite Entamoeba histolytica also lacks this system and in January 1993 Ondarza et al. decided to initiate studies on new thiol compounds and disulfide reducing enzymes from E. histolytica. In 1985, Fairlamb et al. [25] were studying trypanosomatids and found a new Gsp (glutathione-spermidine) conjugate together with the related enzyme trypanothione reductase. The new conjugate was given the common name of trypanothione [N1,N8bis(glutathionyl)spermidine] [26]. T(S)2 + NADPH + H+ T(SH)2 + NADP+ TR
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While most organisms utilize glutathione within their cells to maintain an intracellular reducing environment, Leishmania and the trypanosomes uniquely utilize trypanothione as the antioxidant molecule. In the case of Entamoeba histolytica and Naegleria fowleri, trypanothione reductase (TR) also maintains the required levels of reduced trypanothione by catalyzing the NADPHdependent reduction of its substrate, trypanothione disulfide. It has been possible to detect the presence of the precursor Gsp (glutathionylspermidine) and trypanothione in E. histolytica by HPLC using the fluorescent bimane reagent that is specific for thiol compounds [27]. Moreover, the results published by Fahey et al. [22] confirmed the inability of Entamoeba to synthesize glutathione. In addition we showed that when grown in a glutathione-depleted medium, this parasite was unable to synthesize a compound identified by HPLC as trypanothione, and although the amoeba could still grow in this environment it did so at a slower rate than that of the control rate [28]. Using MALDI-TOF (matrix-assisted laser-desorption ionization- time-of-flight) Mass Spectrometry, the thiol-bimane compound isolated and purified from E. histolytica, [29] and later from N. fowleri trophozoites, [30] has been shown to correspond to the characteristic monoprotonated ion of trypanothione-(bimane)2. Meanwhile, the amplified PCR product obtained from amoebic DNA corresponds to a previous sequence analysis of the trypanothione reductase enzyme from Trypanosoma cruzi, and more generally to the NADPH-dependent disulfide reductases [6]. Furthermore, a partially purified extract from Entamoeba histolytica, which had no GSSG reductase activity, showed the presence of a NADPH-dependent TR activity [6]. In terms of a preliminary discourse, current in vitro studies with partially purified extracts from Naegleria fowleri have also detected clear TR and GR activities in the presence of NADPH and their respective substrates [30]. The presence of the trypanothione/trypanothione reductase system in E. histolytica and N. fowleri opens up the possibility for using trypanothione synthetase and disulfide reductase as new “drug targets” for rationally designed drugs, and to eliminate the parasite without affecting the human host. The analysis of the molecular structure of enzymes that can be inhibited by rationally designed compounds has already been applied to trypanothione reductase, which can be found in trypanosomatids such as T. cruzi, T. brucei, and T. congolense, as well as in various species of Leishmania [31]. Trypanothione reductase is an essential component of the anti-oxidant defenses of parasitic Trypanosomes and Leishmania, and differs markedly from the equivalent human host enzyme, glutathione reductase (hGR), in the binding site for disulfide substrate. A patent registered by Hjort described a protein disulphide isomerase variant with increased reducing properties when compared with the wild-type protein, which was capable of being expressed extracellularly in a high yield [32]. Molecular modeling of this region suggested that certain tricyclic compounds might bind selectively to trypanothione reductase without inhibiting host glutathione reductase. This was confirmed by Benson et al. [33] who tested 30 phenothiazine and tricyclic antidepressants, of which clomipramine was found to be the most potent.
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The relevant structural aspects of the target have been compared with the homologous structure found in the mammalian hosts to indicate the molecular basis by which selective toxicity is likely to be achieved. TR and hGR are mutually exclusive for their respective substrates [34]. Despite playing identical roles, the two major factors accounting for the selectivity of inhibitors against the respective enzyme are charge and size. In 1999, Douglas et al. [35] showed that the tricyclic neuroleptics clomipramine and chlorpromazine have lethal effects on Leishmania donovani and L. major, and their studies indicated that the phenothiazine inhibitors of trypanothione reductase are potential candidates for anti-trypanosomal and anti-leishmanial drugs. Since Entamoeba histolytica does not have the capacity to form glutathione and lacks glutathione reductase, but incorporates glutathione from the culture medium to initiate a trypanothione metabolism similar to that of trypanosomatids, studies on the inhibitory effect of five phenothiazine- and five tricyclic-derived compounds * on the reduced thiols of this human parasite have been conducted [36]. Similar to the effects observed with the trypanosomatids, the Entamoeba histolytica trophozoites were also found to be susceptible to phenothiazine and tricyclic derivatives when incubated at a concentration of 100 μM for 24 h [36]. NMe2
R N
CF3
N
Cl
S Phenothiazine nucleus
Table l.
Clomipramine
Effects of Nine Drugs on Thiol Compounds from Acanthamoeba polyphaga Cys-SH
GSH
X-SH
Normal
0.85 nmol
0.87 nmol
2.8 nmol
Trifluoperazine
(0.0 nmol)
(0.0 nmol)
(0.0 nmol)
100 %
100 %
100 %
(0.0 nmol)
(0.0 nmol)
(0.12 nmol)
100 %
100 %
96 %
(1.1 nmol)
(1.7 nmol)
(1.3 nmol)
29%
95%
53%
(0.0 nmol)
(0.30 nmol)
(0.53 nmol)
100 %
66%
81 %
Chlorpromazine
Amphotericin
Ketoconazole
*The following phenothiazine derivatives were synthesized by O. Faruk Khan Prof. K.T. Douglas’ laboratory at Manchester University, UK: OFK001; OFK006; OFK008; OFK027, and OFK043. Five commercially available tricyclic compounds, chlorpromazine HCl, clomipramine HCl, doxycycline succinate, triflupromazine HCl, and diphenydramine HCl, were also used.
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(Table 1) Contd…..
Miconazole
Rifampicin
Pentamidine
Mepacrine
Metronidazole
Cys-SH
GSH
X-SH
(0.0 nmol)
(0.66 nmol)
(0.30 nmol)
100 %
24%
89 %
(2.1 nmol)
(2.4 nmol)
(1 nmol)
147%
175%
64 %
(0.36 nmol)
(0.60 nmol)
(0.32 nmol)
58 %
31 %
89 %
(1.32 nmol)
(2 nmol)
(1.2 nmol)
56 %
130 %
57 %
(0.63 nmol)
(0.72 nmol)
(2.4 nmol)
26 %
18 %
14%
Note: The effects produced by each drug are measured at a concentration of 32 μg/ml for 24 h starting after 72 h of culture. The amounts are expressed in nmol / 1 X 10 6 trophozoites. [See Ref. 38].
When the HPLC elution pattern corresponding to T. cruzi grown under normal conditions is compared with the elution patterns for T. cruzi treated with OFK006 and clomipramine, there is a complete disappearance of T(SH)2 in both cases. The same effect was obtained with E. histolytica, except that OFK 006 did not have as great an inhibitory effect when compared with clomipramine, which totally inhibited the formation of dihydrotrypanothione. All the other compounds studied (OFKs and chlorpromazine, doxycycline succinate, triflupromazine, and diphenydramine) had the same inhibitory effect on T(SH)2. With regard to Acanthamoeba polyphaga, another free living human amoeba pathogen, previous studies [37] have shown the presence of a trypanothione-like compound (compound X) but its identity still needs to be ascertained. Studies have also been carried out showing the in vitro effects of the neuroleptic agents, chlorpromazine and trifluoperazine, the antimycotics, amphotericin B, ketoconazole and miconazole, and the antibiotics, pentamidine, rifampicin, mepacrine and metronidazole, on the NADPH-dependent disulfide reducing enzymes, cystine reductase (CysR), glutathione reductase (GR) trypanothione reductase (TR), and on a putative disulfide reductase for compound X in Acanthamoeba polyphaga derived from the human pathogens A. polyphaga and Naegleria fowleri [38]. Considering the effects against A. polyphaga, all nine drugs showed the capacity to inhibit the putative disulfide reductase (substrate = X-SH) from the trophozoites at a concentration of 32 g/ml over a 24 h incubation period as follows: the neuroleptics trifluoperazine (100%) and chlorpromazine (96%), the antimycotics miconazole (89%) ketoconazole (81%) and amphotericin B (53%) and the antibiotics pentamidine (89%), rifampicin (64%), mepacrine (57%) and metronidazole (14%). Only six of the nine drugs simultaneously inhibit CysR, GR and the putative disulfide reductase (see Table 1). In the case of N. fowleri, (see Table 2) the most potent inhibitors of trypanothione reductase were amphotericin B and miconazole which showed a 100% inhibition at a
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concentration of 32ug/ml over a 24 h incubation period followed by the neuroleptics trifluoperazine (92%) and chlorpromazine (80%) and the antibiotic mepacrine (70%). All these compounds also inhibited CysR and GR from the trophozoites apart from mepacrine, which inhibited CysR and TR only. Ketoconazole, rifampicin (which does not affect CysR), pentamidine and metronidazole, had opposite effects, since they did not inhibit but increased the amount of the three thiols. Table 2.
Effects of Nine Drugs on Thiol Compounds from Naegleria fowleri Cys-SH
GSH
Trypanothione
Normal
0.12 nmol
0.13 nmol
1.1 nmol
Trifluoperazine
(0.0 nmol) 100%
(0.0 nmol) 100%
(0.08 nmol) 92%
Chlorpromazine
(0.0 nmol) 100%
(0.0 nmol) 100%
(0.22 nmol) 80%
Amphotericin
(0.0 nmol) 100%
(0.0 nmol) 100%
(0.0 nmol) 100%
Ketoconazole
(0.15 nmol) 25%
(0.18 nmol) 38%
(1.3 nmol) 18%
Miconazole
(0.0 nmol) 100%
(0.0 nmol) 100%
(0.0 nmol) 100%
Rifampicin
(0.12 nmol) 0.0%
(0.14 nmol) 7%
(1.7 nmol) 55%
Pentamidine
(0.13 nmol) 8%
(0.30 nmol) 130%
(1.54 nmol) 40%
Mepacrine
(0.0 nmol) 100%
(0.21 nmol) 61%
(0.32 nmol) 70%
Metronidazole
(0.61 nmol) 400%
(0.51 nmol) 292%
(1.85 nmol) 68%
Note: The effects for each drug are measured at a concentration of 32 μg/ml over 24 h starting after 36 h of culture. The amounts are expressed in nmol/ 1 106 trophozoites. [See Ref. 38].
B) Comparative Evaluation of the Efficacies of Various Drugs with Antiproliferative Activities The presence of trypanothione in Entamoeba histolytica enables the inhibitory effect of various phenothiazine and tricyclic derivatives on this human parasite to be studied [39]. Drugs like clomipramine (KD002), the most potent in vitro inhibitor of trypanothione reductase, tested at 25 M after 24 h of culture under aerobic conditions, causes a substantial decrease in the number of E. histolytica HK9 trophozoites, from approx. 15106 to 5.37106 cells, and at 100 M to 0.8106 cells. A clear inhibitory effect on cell proliferation can also be demonstrated with metronidazole (used clinically against amoebiasis). Under similar experimental conditions other tricyclic and phenothiazine derivatives (OFKs), have an inhibitory effect between 16 to 95%. For comparison, similar results are obtained using clomipramine and a phenothiazine derivative (OFK006) with Trypanosoma cruzi and Crithidia luciliae. Experiments comparing two E. histolytica strains show that normal cell proliferation under anaerobiosis is higher in strain HK9 than in HM1, which is highly virulent, but metronidazole and clomipramine are less effective against HM1. Two other drugs tested, diphenydramine (KD005) and a phenothiazine derivative (OFK008), also have significant but lower inhibitory effects on both strains [39].
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The inhibitory activity on cell proliferation and the lytic effects on this human parasite by the tricyclic compounds clomipramine, chlorpromazine and others, as well as by the phenothiazine derivatives, suggest that they could be considered as potential anti-amoebic agents. Kolarich et al. [40] have found by two-dimensional gel electrophoresis and mass spectrometry, five protein targets from the soluble proteome of metronidazole-treated E. histolytica cells. Of about 1,500 proteins visualized, only five formed covalent adducts with metronidazole metabolites, including thioredoxin, thioredoxin reductase, superoxide dismutase, purine nucleoside phosphorylase, and a previously unknown protein. In addition to these proteins targets, small thiol molecules, including cysteine, formed adducts with metronidazole. Supplementation with cysteine allowed the cells to survive otherwise lethal metronidazole concentrations. Taken together, their work reveals a new area of molecular interactions of activated metronidazole with cellular components. With regard to Acanthamoeba, there are several species that can infect humans; the species implicated are Acanthamoeba cultbersoni, A. polyphaga and A. castellanii amongst others. Infection due to Acanthamoeba spp. is frequently present in immune deficient patients and the chronically sick [41]. Currently, the medical treatment is cumbersome due to the parasite‘s resistance to treatment, however, satisfactory results have been seen in some patients through the use of combined neomycin and propamidine isethionate [41]. Since an increase in the number of cases has occurred worldwide, these protozoa have become important as human disease agents [42]. Antimicrobial therapy for these infections is generally empirical, and patient recovery is often problematic. For example, encephalitis and other infections caused by Acanthamoeba have been treated, more or less successfully, with antimicrobial combinations including sterol-targeting azoles (clotrimazole, miconazole, ketoconazole, fluconazole, itraconazole) pentamidine isethionate, 5-fluorocytosine, and sulfadiazine. In the case of N. fowleri, although highly sensitive to the antifungal agent amphotericin B, delay in diagnosis and the fulminant nature of the disease result in few survivors [42]. A recent patent by Roberts, Craig William is based on compounds that modulate the shikimate pathway and/or a separate pathway branching from the shikimate pathway in members of the amoebida order [43]. The use of drug combinations ensures that at least one of the drugs may be effective against the amoebae. This is particularly true for Acanthamoeba keratitis that causes a nonopportunistic infection of the cornea, which responds well to treatment with chlorhexidine gluconate and polyhexamethylene biguanide in combination with propamidine isethionate (Brolene), hexamidine (Desomodine), or neomycin [44]. As with other infectious diseases, recovery is dependent not only on antimicrobial therapy, but also on the patient's immune status, the infective dose and virulence of the amoebic strain, and the timing of diagnosis and drug therapy initiation. In 1992, Schuster and Jacob [45] studied the magainins, a group of naturally occurring (and synthetic) channel-forming membrane-active peptides isolated from the skin of the African clawed frog Xenopus laevis [46], and showed them to be active in vitro against a clinical isolate of Acanthamoeba polyphaga. A recent patent by Dr. Borazjani, consider around ophthalmic solutions such as eye drops and contact lens solutions that include a polycationic material to enhance antimicrobial efficacy against protozoans, such as Acanthamoeba [47]. Two magainins tested extensively exhibited minimal inhibitory and amebicidal values for magainin MSI-103 and magainin MSI-94. However, both
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amoebistatic and amoebicidal activities are enhanced by combining the magainins with other marginally effective antimicrobial agents. These combinations act against both the trophic and cystic stages of the Acanthamoeba life cycle and show promise as potential antimicrobial agents in the treatment of amebic keratitis. Other studies by Mattana et al. [48] demonstrated the in vitro effectiveness of the macrolide, rokitamycin, and the phenothiazine compound, chlorpromazine, against Acanthamoeba castellanii. Growth curve evaluations revealed that both drugs inhibit trophozoite growth in dose- and time-dependent ways. The effects of both drugs when used at the MICs at which 100% of isolates are inhibited were amoebistatic, but at higher doses they were amoebicidal, as well as cysticidal. In addition, experiments showed that when rokitamycin was associated with chlorpromazine or amphotericin B, rokitamycin enhanced their activities. Furthermore, low doses of rokitamycin and chlorpromazine, alone or in combination, blocked the cytopathic effect of A. castellanii against WKD* cells derived from the human cornea [48]. These results may have important significance in the development of new anti-Acanthamoeba compounds.*(Woringer-Kolopp Disease). Recently, Schuster et al. [49] carried out in vitro tests of the anticancer agent miltefosine and the antifungal drug voriconazole against Acanthamoeba spp. and Naegleria fowleri. In these experiments, Acanthamoeba spp. recovered from exposure to the 2 week drug effects from 40 μM dose of miltefosine, but it did not recover from an 80 μM dose. Attempts to define the minimal inhibitory (MIC) and amoebicidal concentrations (MAC) for Acanthamoeba more specifically were difficult due to the persistence of non-proliferating trophic amebas in the medium. For N. fowleri, 40 and 55 μM were the MIC and MAC, respectively, with no trophic amoebas seen at the MAC [49]. Voriconazole has shown strong inhibitory effects on Acanthamoeba spp. and N. fowleri at all drug concentrations up to 40 μg/ml when tests were carried out on trophic amoebas. It seems clear that miltefosine and voriconazole are potentially useful drugs for the treatment of free-living amoebic infections, although sensitivities differ between genera, species, and strains. Martín-Navarro et al. [50] propose that chlorhexidine, rather than polyhexamethylene biguanide (PHMB), or other biguanides, should be considered for inclusion in contact lens maintenance solutions, especially in light of the fact that PHMB has been reported to be cytotoxic to human corneal cells. Additionally, these authors indicate that the concentration of chlorhexidine or other biguanides, should be re-evaluated as the maintenance solutions were shown to be ineffective against the strains isolated during their study with high pathogenic potential. It is well known that Acanthamoebae can cause infections of several organs, including eye, skin, lung and brain but except for Acanthamoeba keratitis, these infections are linked to immunodeficient patients. Moreover, their treatment is problematic, due to the lack of sufficiently effective and easily manageable drugs. So, in view of these facts, Walochnik et al. [51] followed an study and discovered that miltefosine (hexadecylphosphocholine) is highly active against Acanthamoeba spp. in vitro, and is suitable for the topical treatment of Acanthamoeba infections. These authors conclude that since Miltefosine has been approved for oral and topical treatment of leishmaniasis, may also be a promising candidate for the topical treatment of Acanthamoeba infections.
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According to Henriquez et al. [52] Tubulin is an essential structural element of the cytoskeleton of eukaryotic cells, where it plays a central role in chromosomal segregation, organelle movement, and cellular motility. However, despite the highly conserved nature of -tubulin and -tubulin across the phyla, organisms present diverse degrees of susceptibility and resistance to the different groups of antimicrotubule agents. For instance, some protozoans, including apicomplexans like Toxoplasma gondii, are susceptible to dinitroanilines, while others, such as Trypanosoma cruzi, are resistant. Similarly, there is considerable variation in susceptibility of protozoans to paclitaxel, as exemplified by Leishmania spp. and T. gondii, and in the case of few protozoa, such as Giardia lamblia, are susceptible to benzimidazoles, a class of drug used to treat helminth infections. This is the reason that Tubulin has been exploited as a target for antineoplastics, antifungal, and antiprotozoal compounds. However, this group of research [52] has demonstrated that in the case of Acanthamoeba spp., these protozoa are resistant to five antimicrotubule compounds, unlike any other eukaryote studied so far and its resistance correlates with critical amino acid differences within the inhibitor binding sites of the tubulin heterodimers that influence tertiary structure or alter inhibitor-docking regions, responsible for determining resistance to antitubulins. Due to the fact that Amoebic keratitis is difficult to treat, without total efficacy in some patients because of cysts are less susceptible than trophozoites, S. Bouyer et al. [53] investigated the in vitro effectiveness of caspofungin, a new antifungal, against three species of Acanthamoeba: A. castellanii, A. polyphaga and A. culbertsoni. (Caspofungin is an echinocandin, a synthetically modified lipopeptide which inhibits the synthesis of ß-Dglucan in fungal cell walls). According to their results, the trophozoites of the three tested species were susceptible in vitro to caspofungin at a concentration of 250 mg/L (206 M) and cysticidal at a concentration of 500 mg/L (412 M) against A. castellanii and A. culbertsoni. However still it will be necessary to conduct further studies in animal models to verify the absence of toxicity of caspofungin at higher concentrations and to confirm its efficacy on Acanthamoeba keratitis in vivo. The authors concluded that this new antimicrobial agent could be promising to treat, alone or in combination with another anti-amoebic drug, against Acanthamoeba keratitis. As already documented, Primary Amoebic Meningoencephalitis (PAM) is an emerging disease with a rapidly fatal outcome and only eight reports of cured cases have appeared in the medical literature (up to the year 2005). One of the success stories was achieved by Vargas-Zepeda et al. [54] who treated a 10-year-old boy that developed PAM caused by Naegleria fowleri 1 week after swimming in an irrigation canal. He was admitted to the hospital after 9 h with a severe headache, vomiting, fever, ataxic gait, mild confusion, and evident seizures. Trophozoites were identified in the cerebrospinal fluid (CSF). Treatment with intravenous (i.v.) dexamethasone, amphotericin B, fluconazole, and oral rifampicin was initiated and it was concluded following recovery by the boy that early treatment of PAM using this i.v. combination can offer some hope for curing for this devastating disease. In our laboratory, [55] using reproducible conditions in vitro, a study was embarked upon to obtain a comparative evaluation of the efficacies of several tricyclic neuroleptics, antimycotics and antibiotics with antiproliferative activities against Acanthamoeba polyphaga and Naegleria fowleri trophozoites.
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An overview of the inhibitory concentration (IC50) for the nine drugs studied against A. polyphaga trophozoites expressed in μg/mL and on a molar basis appear in the following Table 3. In general, the neuroleptics, antimycotics and antibiotics, except rifampicin, offered significant inhibitory activity against this pathogenic free-living amoeba. In the case of N. fowleri, the most effective drugs expressed as (IC50) were as follows: the antimycotics Ketoconazole and Amphotericin B, followed by Trifluoperazine, Mepacrine, Chlorpromazine, Miconazole and Metronidazole. The least effective were Rifampicin and Pentamidine. The most potent growth inhibitors (MIC100) against N. fowleri were the antimycotics Amphotericin B and Ketoconazole and the neuroleptic Trifluoperazine. It is clear that there are major differences between the two amoebas in their susceptibility to some of the drugs. The inhibitory concentrations (IC50) of the nine drugs tested against Naegleria fowleri trophozoites expressed in μg/mL and on a molar basis appear in Table 4. The drugs with the minimal inhibitory concentration (MIC) values could be considered alone or in combination as potential anti-amoebic agents for the treatment of diseases produced by these amoebas. New positive results have been obtained by Kim et al. [56] that evaluated the in vitro and in vivo efficacies of amphotericin B, miltefosine and chlorpromazine against pathogenic N. fowleri. The results showed that the growth of the amoeba was effectively inhibited by treatment with these three drugs but especially chlorpromazine had the best therapeutic activity for the treatment of PAM than amphotericin B. Another study done by Soltow and Brenner [57] has been done combinig amphotericin B and azithromycin in vitro and in vivo with a mouse model of PAM. For the in vitro studies, they found that amphotericin B and azithromycin were synergistic at all three of the fixed combination ratios and in the mouse model, a combination of amphotericin B and azithromycin protected 100% of the mice, whereas amphotericin B alone protected only 27% and azithromycin alone protected 40%. This study indicates that amphotericin B and azithromycin act synergistically against the Lee strain of N. fowleri, suggesting that the combined use of these agents may be beneficial in treating PAM. A different approach has been followed by Wang, Band and Kopachik [58] that used the microtubule-disrupting herbicide trifluralin (TFL) to inhibit the growth and flagellate transformation of Naegleria fowleri and N. gruberi. They found that Naegleria shows a high sensitivity to TFL like trypanosomatids and propose that TFL is potentially useful for the study of flagellate transformation, especially since colchicine does not effect Naegleria and may be useful in the treatment of primary amoebic meningoencephalitis. Three strains of the genus Naegleria, isolated from patients who had died of primary amebic meningoencephalitis infection, were investigated for its sensitivity to antifungal drugs: amphotericin B, ketoconazole, fluconazole and itraconazole by T. Junnuan [59]. They found, that ketoconazole was slightly more effective than amphotericin B because its action is directed to the permeability of the amebic membrane, but In the case of the amebae which were more resistant to fluconazole it was due to the action of the cytochrome P450 multienzyme and in the effect on blocking cytochrome P450-dependent chitin synthesis, to itraconazole. The authors concluded that amphotericin B and ketoconazole remain the main drugs with proven activity against pathogenic Naegleria spp.
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According to Alisky [60] primary amebic meningoencephalitis (PAM) by Naegleria fowleri, has a very high mortality rate, probably exceeding 95% and few people have survived after getting intravenous and intrathecal amphotericin, variably coupled with other agents that include dexamethasone, diflucan, chloramphenicol and rifampin, but even with prompt initiation of therapy, still is a very unsuccesfull battle. So He suggests that instilling medications intranasally, intravenously and intrathecally would target the primary reservoir of infection and its common sites of spread. For instance, survival could be improved by combining intrathecal, intranasal and intravenous amphotericin, diflucan and rifampin, with adyuvant intravenous chloramphenicol, muramyl dipeptide, azithromycin, minocycline and linezolid; intramuscular trifluoperazine; intranasal Cry1C protoxin and intrathecal antiNaegleria immune globulin and dexamethasone. Intrathecal dexamethasone should attenuate cerebral edema, a primary cause of death in PAM. The author concludes that azithromycin and minocycline appear to have synergy with amphotericin in killing N. fowleri in animal models, and the other agents, which also showed efficacy, should also be additive or synergistic as well. In synthesis this would be an approach PAM, like in the chemotherapy for tuberculosis and cancer, with multidrug therapy to assure complete eradication. C) Inhibition of Enzymes for the Synthesis of Spermidine and Trypanothione The polyamines spermidine and spermine are present in a variety of eukaryotes and as positively charged amines interact with DNA. It may be that these polyamines exert some of their effects through this interaction in the regulation of apoptosis, cellular proliferation, and progression through the cell cycle [61]. In the particular case of the trypanosomatids and amoebas like E. histolytica and N. fowleri, spermidine is essential for the synthesis of trypanothione. In Acanthamoeba polyphaga there is no trypanothione [37] however, S-Adenosyl-L-methionine decarboxylase (AdoMetDC) [62] has been purified from Acanthamoeba castellanii and recently, Shon and Nam [63] also demonstrated the presence of an ornithine decarboxylase activity that can be inhibited by a chitosan oligosaccharide in this same amoeba. Table 3.
Acanthamoeba polyphaga Neuroleptics
Antimycotics
Drug
Trifluoperazine
Chlorpromazine
Amphotericin B
Ketoconazole
Miconazole
IC50
0.25 μg/mL (0.52 μM)
0.25 μg/mL (0.7 μM)
8 μg/mL (8.6 μM)
1.5 μg/mL (2.8 μM)
2 μg/mL (4.2 μM)
Antibiotics Drug
Rifampicin
Pentamidine
Mepacrine
Metronidazole
IC50
>32 μg/mL (>38.8 μM)
2 μg/mL (3.4 μM)
0.7 μg/mL (1.5 μM)
3 μg/mL (17.5 μM)
[Ref. 50]. Ondarza, R.N., Iturbe, A., Hernandez, E. in vitro antiproliferative effects of neuroleptics antimycotics and antibiotics on the human pathogens Acanthamoeba polyphaga and Naegleria fowleri. Arch Med Res 2006 Aug; 37(6): 723-9.
Ornithine is derived from the amino acid arginine as part of the urea cycle and its enzyme ornithine decarboxylase (ODC) produces putrescine. Amino-propyl groups from
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decarboxylated adenosyl methionine (SAM) are progressively added to putrescine to produce spermidine and then spermine. Studies with difluoro methyl ornithine (DFMO), a specific and irreversible inhibitor of ornithine decarboxylase, have been conducted by Gillin et al. [64] on Giardia lamblia, Entamoeba histolytica and Trichomonas vaginalis. Table 4.
Naegleria fowleri Neuroleptics
Antimycotics
Drug
Trifluoperazine
Chlorpromazine
Amphotericin B
Ketoconazole
Miconazole
IC50
1 μg/mL (2 μM)
2 μg/mL (5.6 μM)
1.5 μg/mL (1.6 μM)
0.25 μg/mL (0.23 μM)
8 μg/mL (16.7 μM)
Antibiotics Drug
Rifampicin
Pentamidine
Mepacrine
Metronidazole
IC50
>32 μg/mL (>38.8 μM)
>32 μg/mL (>54 μM)
2 μg/mL (4.2 μM)
8 μg/mL (46.7 μM)
[Ref. 50] Ondarza, R.N., Iturbe, A., Hernandez, E. In vitro antiproliferative effects of neuroleptics antimycotics and antibiotics on the human pathogens Acanthamoeba polyphaga and Naegleria fowleri. Arch Med Res 2006 Aug; 37(6): 723-729.
Growth of G. lamblia is inhibited by DFMO at concentrations greater than or equal to 1.25mM but inhibition is reversed when spermidine is previously added. However, the growth of E. histolytica and T. vaginalis is not inhibited even at higher concentrations (by 20mM DFMO). These studies indicate that polyamine biosynthesis from ornithine is required for G. lamblia growth. Following purification from trophozoites of the parasite protozoan E. histolytica, the same ODC enzyme was studied by Arteaga-Nieto et al. [65] and was found to be insensitive to inhibition by -difluoro methyl ornithine (DFMO). This suggests that the enzyme may not be a suitable drug target, at least as an anti-parasitic drug in this case. Jhingran et al. [66] claimed to have cloned, expressed, and characterized for the first time, the putative ODC-like sequence (Ornithine Decarboxylase) from E. histolytica, a rate limiting enzyme in the polyamine biosynthesis pathway. Computer modeling revealed that three of the critical residues required for binding of Difluoromethylornithine (DFMO) to the ODC enzyme are substituted in E. histolytica, resulting in the likely loss of interactions between the enzyme and DFMO. These authors think that the expression of this protein, will facilitate the studies of its structural and functional aspects which could prove to be an important anti-amoebic target. Other studies [67] have demonstrated that the proliferation of Acanthamoeba castellanii (Neff strain) in either a broth medium or a defined medium can be arrested by alphamonofluoro methyl dehydro ornithine (-MFMOme), -difluoro methyl ornithine (DFMO), and (R,R')--methyl--acetylenic putrescine (MAP), three specific inhibitors of ornithine decarboxylase. Although all three inhibit the amoeba enzyme, -MFMOme is the most effective inhibitor of multiplication. Growth inhibition is reversed by adding polyamines, and DFMO caused encystment when supplemented with CaCl2 or MgSO4.
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On the other hand, studies by Kishore and Shukla [68] have shown that the multiplication of A. culbertsoni in peptone medium is not inhibited by -difluoromethyl ornithine (DMFO) while a partial and transient inhibition of cell multiplication was observed using 10-20 mM DFMO in a proteose peptone, yeast extract, or glucose (PYG) medium. However, ornithine decarboxylase (ODC) activity in the cells and cell free extracts was strongly inhibited by DFMO. In the case of E. histolytica, the presence of trypanothione synthetase activity (TryS) in vitro has been demonstrated using partly purified extracts incubated with glutathione, spermidine and ATP in the presence of Mg2+ [69]. The thiol products were detected by HPLC and compared with commercial Gsp and trypanothione standards. In Crithidia fasciculate, the synthetase activity can be related to two enzymes, [70] but in T. cruzi [71] T. brucei [72, 73] and Leishmania major [74] only a single enzyme was found [75]. Recently however, the TryS of C. fasciculata, like that of Trypanosoma species, has been reported to catalyze the entire synthesis of trypanothione, whereas its glutathionylspermidine synthetase appears to be specialized for Gsp synthesis [76]. It seems clear that the trypanothione synthetase in N. fowleri [30], as well as in E. histolytica [58], could also be a suitable drug target. With regard to the above, Stuhlmann et al. [77] patented the N5-substituted benzo [2,3] azepino [4,5-b] indol-6-one compound as a pharmaceutically active agent performing as a trypanothione synthetase inhibitor for prevention and/or treatment of diseases caused by parasites associated with trypanothione, such as African trypanosomiasis or sleeping sickness, South American trypanosomiasis, Kala-Azar, visceral leishmaniasis, cutaneous leishmaniasis (CL), Chagas disease, trichomoniasis, giardiasis, lamblia dysentery, amoebiasis, primary amebic meningoencephalitis (PAM) and keratitis. D) Inhibition of PPi-Dependent Phosphofructokinase (PFK) by Pyrophosphate Analogues Bruchhaus et al. [78] amplified a genomic DNA fragment using oligonucleotide primers derived from regions highly conserved in prokaryotic and eukaryotic phosphofructokinase sequences. They used it to isolate cDNA and genomic clones coding for PPi-dependent phosphofructokinase (PPi-PFK) of Entamoeba histolytica. The purified recombinant protein was found to be enzymically active and the Km values for PPi and fructose 6-phosphate of the native and the recombinant PPi-PFKs were nearly identical. Various bisphosphonates (synthetic pyrophosphate analogues) were also tested and found to be competitive inhibitors for amoeba PPi-PFK activity and amoebic growth. The best inhibitors were 3-[N-(2-phenylthioethyl)-N-methylamino]-1-hydroxypropylidene1, 1-bisphosphonate (CGP48048) and 2-(imidazol-1-yl)-1-hydroxy-ethylidene-1, and 1bisphosphonate (zoledronate; CGP 42446), with Ki values of 50 M. One of the Bisphosphonates (risedronate) was inhibitory at a concentration of 10 M showing them to be potential therapeutic agents for the treatment of amoebiasis. Orozco has patented the vaccine composition, which is used to control amoebiasis and which is based on the 112KDA surface protein from Entamoeba histolytica [79]. For the purpose of designing drugs to treat parasitic infections, Byington et al. [80] constructed a PPi-PFK model from E. histolytica based on the three-dimensional structure of the ATP-dependent PFK from Bacillus stearothermophilus. The model was used to predict the binding of pyrophosphate and selected bis-phosphonates to the enzyme. These
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drugs were tested against E. histolytica and showed that they inhibited the growth of amoebae in vitro. It may be possible in the future to make use of this inhibitory capacity in the treatment of serious parasitic infections. Of the PPi-dependent enzymes, phosphofructokinase (PFK) has been studied most extensively and its sequence data has been shown to be homologous to the ATP-dependent phosphofructokinase (ATP-PFK) [81]. The PPi-PFK from Entamoeba histolytica, as well as a number of other parasitic protozoa, utilizes PPi as the phosphoryl donor instead of ATP. The study by Chi and Kemp [81] specifically demonstrated the presence of a latent nucleotide binding site in the PPi-PFK of E. histolytica and proposed that the ancestral PFK was an ATP-dependent enzyme and that PPi -PFKs are a later evolving adaptation. The PPi -PFK of E. histolytica displays a million fold preference for inorganic pyrophosphate (PPi) over ATP, but after the introduction of a single mutation by sitedirected mutagenesis, it changes its preference from PPi to ATP. In the case of ATP-dependent phospho fructokinase (PFK), this enzyme is a key regulatory enzyme in glycolysis, in part because of its irreversible nature. The PPi-dependent enzyme differs significantly from ATP-dependent phosphofructokinase found in humans and is the rate-limiting glycolytic enzyme found in the amitochondrial parasitic protists Entamoeba histolytica, Giardia lamblia, Toxoplasma gondii, Trichomonas vaginalis and Naegleria fowleri, and as such represents an important drug target. The PPi-dependent enzyme was first isolated, partially purified and characterized in 1974 by Reeves’ laboratory [82]. The reaction catalyzed was: Fructose 6-phosphate + PPi fructose-1,6 - bisphosphate + Pi PFK Some time later, the genomic sequence for a PPi-PFK from E. histolytica was reported and in a further study it was purified to near-homogeneity [83]. The sequences for the gene and cDNA for this PPi-PFK were determined and the properties of the expressed gene were shown to be identical with those of the enzyme in extracts of axenically grown E. histolytica. Considering that the synthesis of ATP in the human parasite E. histolytica is carried out solely by the glycolytic pathway, the biochemical characterization of recombinant glycolytic enzymes and flux control analysis were reported by Saavedra et al. [84]. Their work characterized 10 recombinant enzymes, established the kinetic constants at optimal and physiological pH values, analyzed the effect of activators and inhibitors, and investigated the storage stability and oligomeric state. The analysis suggested that among the 10 enzymes studied, phospho-glycerate mutase (PGAM) and pyruvate phosphate dikinase (PPDK) exhibited the highest flux control coefficient values at physiological pH. According to Stephen et al. [85] pyruvate phosphate dikinase (PPDK) is the key enzyme essential for the glycolytic pathway in most common and perilous parasite Entamoeba histolytica and inhibiting the function of this enzyme could control the wide spread of intestinal infections caused by Entamoeba histolytica in humans. With this objective, they modeled three dimensional structure of the PPDK protein. The Virtual screening was carried out using the genetic docking algorithm GOLD and a consensus scoring function X-Score to substantiate the prediction. The small molecule libraries (ChemDivision database, Diversity
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dataset, Kinase inhibitor database) were used for screening process. Along with the high scoring results, the interaction studies provided promising ligands for future experimental screening to inhibit the function of PPDK in Entamoeba histolytica and the phylogeny study to assess the possibility of using the proposed ligands as inhibitors in related pathogens. Turning to Naegleria fowleri, the enzyme PPi-dependent phosphofructokinase (PPiPFK) was detected by Mertens et al. [86] and later purified to near homogeneity. Sizeexclusion chromatography [87] revealed the existence of two forms: a large one, approximately 180 kDa, (presumably a tetramer), which was active, and a smaller one, approximately 45 kDa (presumably the monomer), which was inactive, but could be reactivated and converted into the large form by incubation in the presence of AMP. Following phylogenetic analyses of biochemically characterized PPi-PFKs, Siebers et al. [88] grouped the enzymes into three monophyletic clusters: PFK group I that represented only classical ATP-PFKs from Bacteria and Eucarya; PFK group II that contained only PPiPFKs from the genus Propionibacterium, plants and amitochondriate protists; and PFK group III that consisted of PFKs with either cosubstrate specificity, i.e., the PPi-dependent enzymes from T. tenax and Amycolatopsis methanolica and the ATP-PFK from Streptomyces coelicolor. E) Inhibition of Cysteine Proteinases Some of the main drug targets that can be identified in the human parasites E. histolytica, A. polyphaga and N. fowleri are the peptidases that form two groups of enzymes: the endopeptidases and the exopeptidases, which cleave peptide bonds at points within the protein and remove amino acids sequentially from either the N or C-terminus. The term proteinase is used as a synonym word for endo-peptidase and four mechanistic classes are recognized by the International Union of Biochemistry and Molecular Biology, which are: serine proteinases; cysteine proteinases; aspartic proteinases and the metallo proteinases. The amebic cysteine proteinases are inhibited by the cysteine proteinase-specific inhibitor isolated from Aspergillus japonicum, a broad inhibitor of cysteine proteinases, Ltrans-epoxysuccinyl-leucylamido-(4-guanidino)butane (E-64) but not by the serine proteinase-specific inhibitor phenylmethylsulfonyl fluoride [89]. New generations of cysteine proteinase-specific inhibitors, including diazomethanes, vinyl sulfones, and synthetic peptide inhibitors, have been active in the micromolar to nanomolar range against T. cruzi [90], as well as other parasites. Cysteine proteinases are putative virulence factors for which specific genes appear to be present or over expressed in E. histolytica, and absent or unexpressed in non-invasive E. dispar [91]. Compared with E. dispar, invasive E. histolytica strains release significantly greater amounts of active proteases, both intracellular and extracellularly. According to Que and Reed [91], there are a number of roles that cysteine proteinases play in the infection and invasion by the pathogenic ameba E. histolytica, which include: aiding attachment by degrading mucus and debris overlying the intestinal mucosa; aiding penetration of host tissue by digesting the extracellular matrix; aiding dissemination to produce metastatic lesions; degrading host proteins to circumvent the immune response; and activating host cell proteolytic cascades such as complement.
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Since cysteine proteinases are attractive potential targets for the treatment of amebiasis, because they are essential to pathogenesis, the availability of recombinant enzymes and the detailed study of their kinesis, should allow inhibitors and substrates with high potency and specificity to be designed [91]. According to Bruchhaus et al. [92], E. histolytica contains 20 cysteine proteinase genes, of which only a small subset are expressed during in vitro cultivation. Therefore, it is likely that at least some of these enzymes are required for infection of the human host or for completion of the parasite’s life cycle. The activity of elastase was first reported in the free-living amoebae Naegleria and Acanthamoeba [93], although protease activity had been identified prior to its involvement in pathogenesis. Collagenase activity was later discovered in a culture supernatant in which Acanthamoeba had been cultured, but not in cysts [94]. All of the Acanthamoeba spp. that have so far been isolated and tested contain a proteinase that is active against collagen, which is present in the growth medium presumably having been secreted by amoebae [95]. The fact that all Acanthamoeba strain seems to secrete proteinases whether or not they happen to be pathogenic raises the question of why Acanthamoeba (and other amoeba) secrete proteinases. Whatever the purpose in protease secretion, it is certain that the proteases do have the potential to degrade cornea once an infection of Acanthamoeba has been established, and it is believed that the proteolytic activity is a key fact in Acanthamoeba keratitis. Aldape et al. [96] characterized a secreted histolytic cysteine proteinase from Naegleria fowleri. They hypothesized that the protease released by the parasite contributed to tissue destruction and facilitated host invasion. Analysis of N. fowleri cultures revealed a major 30-kDa proteinase with substrate and inhibitor specificity consistent with cysteine proteinases. Amino-terminal amino acid sequence of the purified enzyme showed it to be a thiol protease with homology to cathepsin L. It catalyzed the in vitro degradation of the extracellular matrix and had a cytopathic effect on mammalian cells. The cytopathic effect was inhibited by Z-Phe-Ala-fluoromethyl ketone, an irreversible cysteine proteinase inhibitor. The results indicated that N. fowleri secretes a cysteine proteinase with the capacity to destroy host tissue. Naegleria gruberi, a non-pathogenic species, expresses a similar proteinase but, unlike its pathogenic relative, is not thermo tolerant to temperatures above 30°C. In addition to this proteinase, N. fowleri possesses a phospholipase [97] and poreforming peptides, [98] all of which have been implicated in the pathogenic process. F) Inhibition of Encystment Pathways and Cyst-Wall Assembly Proteins Resistance to antiprotozoal drugs is likely to increase and efforts to find new targets for chemotherapy must be continued. In the case of cyst-forming pathogenic protozoa, one such target might be encystment pathways and cyst-wall assembly [99]. It is known that Giardia and Entamoeba contain unique proteins and polysacharides in cyst walls, which differ from those of their hosts and thus make them potential targets. Enough evidence exists to study them with some of the antifungal drugs, especially those that target mannoproteins and chitin and glucan synthesis, similar to those found in fungi [99].
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In this sense, Villagomez et al. [100] measured chitinase activity in extracts of Entamoeba invadens as a function of time of encystation in axenic conditions using a 4methylumbelliferyl--D-cellotrioside (4-MU(Ch)3) substrate and found that Allosamidin strongly inhibited hydrolysis of the substrate by the amebic chitinase in vitro. When added to the axenic medium, the drug markedly retarded encystment though it was partially recovered after longer periods of incubation. In fact, the antiproliferative effects of the antimycotics amphotericin B, ketoconazole and miconazole against the free living pathogenic amoebas A. polyphaga and N. fowleri have been demonstrated in vitro. The study revealed amebistatic effects for all the drugs against the two pathogens and in the case of N. fowleri, amphotericin B and ketoconazole were the most effective [50]. CURRENT AND FUTURE DEVELOPMENTS The main results of drugs studied on Entamoeba histolytica, Acanthamoeba polyphaga and Naegleria fowleri are as follows: 1)
Similar to the effect observed in the trypanosomatids, Entamoeba histolytica trophozoites are susceptible to phenothiazine and tricyclic derivatives. All the other compounds studied (OFK001, OFK008, OFK027, OFK043, chlorpromazine, doxycycline succinate, triflupromazine, and diphenydramine) had the same inhibitory effect on T(SH)2. There are also inhibitory effects in vitro by the neuroleptics, antimycotics and antibiotics on the NADPH-dependent disulfide reducing enzymes cystine reductase (CysR), glutathione reductase (GR), and trypanothione reductase (TR) from the human pathogens A. polyphaga and Naegleria fowleri. In terms of A. polyphaga, all drugs studied had the capacity to inhibit the putative disulfide reductase from the trophozoites. The drugs concerned were: trifluoperazine and chlorpromazine, miconazole, ketoconazole and amphotericin B, and the antibiotics pentamidine, rifampicin, mepacrine and metronidazole. In the case of N. fowleri, the most potent inhibitors of trypanothione reductase are amphotericin B and miconazole, followed by trifluoperazine and chlorpromazine, and the antibiotic mepacrine. Ketoconazole, rifampicin, pentamidine and metronidazole have opposite effects since they do not inhibit but increase the amount of the three thiols. The results obtained with phenothiazine and tricyclic-derived compounds clearly indicate that they can inhibit E. histolytica, A. polyphaga and N. fowleri trophozoitereduced thiol compounds, suggesting that they are potential antiamebic chemicals.
2)
Several Neuroleptics, Antimycotics, Antibiotics and various other drugs have been found to have antiproliferative activities against E. histolytica, A. polyphaga and N. fowleri trophozoites. Drugs like clomipramine, tested under aerobic conditions, cause a substantial decrease in the number of E. histolytica HK9 trophozoites. A clear inhibitory effect on cell proliferation could also be demonstrated with metronidazole (used clinically against amoebiasis). A recent patent by Zhou suggests that the peptide based compounds will display anti-non-inflammatory properties and inhibition of cell proliferation [101].
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With relation to Acanthamoeba spp. (frequently present in immune deficient patients and the chronically sick), satisfactory results have been seen in some patients through the use of combined neomycin and propamidine isethionate. Antimicrobial therapy for these infections is generally empirical and patient recovery often problematic, for instance encephalitis and other infections caused by Acanthamoeba have been treated, more or less successfully, with antimicrobial combinations including sterol-targeting azoles (clotrimazole, miconazole, ketoconazole, fluconazole and itraconazole), pentamidine isethionate, 5fluorocytosine and sulfadiazine. In the case of N. fowleri, although highly sensitive to the antifungal agent amphotericin B, delay in diagnosis and the fulminant nature of the disease results in few survivors. The use of drug combinations is particularly true in Acanthamoeba keratitis, a nonopportunistic infection of the cornea, which responds well to treatment with chlorhexidine gluconate and polyhexamethylene biguanide, in combination with propamidine isothionate (Brolene), hexamidine (Desomodine), or neomycin. Other studies with magainins have shown to be active in vitro against a clinical isolate of A. polyphaga. Two magainins tested extensively have minimal inhibitory and minimal amebicidal values and the amebicidal activities are enhanced by combining them with other marginally effective antimicrobial agents. These combinations have activity against both trophic and cystic stages in the Acanthamoeba life cycle and show promise as antimicrobial agents in the treatment of amebic keratitis. The in vitro effectiveness of the macrolide rokitamycin and the phenothiazine compound chlorpromazine against A. castellanii has also been demonstrated. Experiments show that when rokitamycin was associated with chlorpromazine or amphotericin B, rokitamycin enhanced their activities. These results may have important significance in the development of new anti-Acanthamoeba compounds. The anticancer agent miltefosine and the antifungal drug voriconazole have been tested in vitro against Acanthamoeba spp. and N. fowleri. Voriconazole has strong inhibitory effects on Acanthamoeba spp. and N. fowleri at all drug concentrations tested. All testing were carried out on trophic amoebas; in conclusion miltefosine and voriconazole are potential, useful drugs for treating free-living amebic infections, though sensitivities differ between genera, species and strains. The inhibitory activity on cell proliferation and lytic effects on these human parasites by tricyclic compounds, as well as by phenothiazine derivatives and other drugs, indicate that they can be considered potential anti-amoebic agents. 3)
The enzymes for the synthesis of trypanothione, all present in the trypanosomatids and in the amoebae E. histolytica and N. fowleri, are putative drug targets although some studies with alpha-difluoro methyl ornithine (DFMO) indicate that in the case of E. histolytica, growth is not inhibited even at higher concentrations. According to other researchers, the enzyme ornithine decarboxylase (ODC) after being purified from E. histolytica trophozoites is also largely insensitive to inhibition by alphadifluoro methyl ornithine (DFMO); it is concluded that the enzyme may not be a suitable target, at least for this anti-parasitic drug.
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However, in another study of A. castellanii (Neff strain), it was found that the proliferation of this amoeba can be arrested by three specific inhibitors of ornithine decarboxylase: alpha-monofluoro methyl dehydro ornithine (-MFMOme), difluoro methyl ornithine (DFMO) and (R,R')--methyl--acetylenic putre-scine (MAP). Although the three drugs inhibit the amoebic enzyme, delta-MFMOme is the most effective inhibitor of multiplication, although inhibition is reversed by adding polyamines. 4)
Various bisphosphonates (synthetic pyrophosphate analogues) were found to be competitive inhibitors for Entamoeba and Naegleria PPi-PFK activity and amoebic growth. The best inhibitors were 3-[N-(2-phenylthioethyl)-N methylamino]-1hydroxypropylidene -1,1-bisphosphonate (CGP 48084) and zoledronate, with Ki values of 50 microM. One of the bisphosphonates (risedronate) was inhibitory at a concentration of 10 microM; bisphosphonates are therefore potential therapeutic agents for the treatment of amoebiasis.
5)
The amoebic cysteine proteinases can be inhibited by the specific cysteine proteinase inhibitor L-trans -epoxysuccinyl-leucylamido-(4-guanidino) butane (E-64) but not by the serine proteinase-specific inhibitor phenylmethylsulfonyl fluoride. New generations of cysteine proteinase-specific inhibitors, including diazomethanes, vinyl sulfones, and synthetic peptide inhibitors, have also been found to be active in the micromolar to nanomolar range against E. histolytica, as well as against other parasites. The purified cysteine proteinase enzyme from N. fowleri has been shown to be a thiol protease with homology to cathepsin L. It catalyzes the in vitro degradation of the extracellular matrix and has a cytopathic effect on mammalian cells. The cytopathic effect is inhibited by Z-Phe-Ala-fluoromethyl ketone, an irreversible cysteine protease inhibitor.
6)
The encystment pathways and cyst-wall assembly proteins from Entamoeba cyst walls that contain unique proteins and polysaccharides different from those of their hosts, make them potential targets for a variety of chemotherapeutic attacks. Enough evidence exists in this aspect for Giardia and Entamoeba that it seems prudent to treat them with some of the antifungal drugs, especially those that target mannoproteins, chitin synthesis, and beta glucan synthesis. Some researchers have measured chitinase activity in extracts of Entamoeba invadens and found that allosamidin strongly inhibits amoebic chitinase in vitro, but when added to axenic medium, the drug markedly retarded encystment, though it partially recovered following longer periods of incubation. The drugs described here and used in vitro against Entamoeba, Acanthamoeba and Naegleria, promise to be good candidates for further studies with experimental animals in vivo. It is now known that E. histolytica and N. fowleri secrete proteinases, have a PPidependent phospho-fructokinase different to the enzyme from the host, and contain an essential and valid trypanothione/trypanothione reductase system, as well as other new “drug targets”. It is believed that these amoebae need to be studied with lead drugs rationally designed against these parasites.
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In particular, Acanthamoeba shares the presence of cysteine proteases and the enzymes for the synthesis of spermidine with the other two amoebae. The use of drug combinations with synergistic effects also promises to be a good strategy to combat these human pathogenic amoebas. ACKNOWLEDGEMENTS I am indebted to Professor W. Fenical (Director of the Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, UCSD) who gave me the facilities to spend a leave of absence in his laboratories, where part of this study was completed. I would also like to thank Dr Khurshid Zaman from Bentham Science Publishers Ltd. who kindly advised me on some of the topics contained in this manuscript and to Mr Ian Shepherd from England, GB who thoroughly revised the English version. I acknowledge also the support from Consejo Nacional de Ciencia y Tecnologìa (CONACyT) and from the Dirección General de Asuntos del Personal Académico (UNAM). REFERENCES [1] [2] [3]
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Macrophage Inflammatory Protein 1 and CCR5 as Potential Therapeutic Targets for HIV Infection and Acquired Immunodeficiency Syndrome Tsuyoshi Kasama*, Ryo Takahashi, Michihito Sato and Kuninobu Wakabayashi Division of Rheumatology, Department of Medicine, Showa University School of Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8666, Japan Abstract: Chemokines play key roles in inflammatory and immune responses mediated by their respective target cell populations. For instance, release of chemokines from inflammatory cells is a crucial step in the recruitment of cells needed to establish local inflammatory responses (e.g. rheumatoid arthritis). Moreover, recent advances in our understanding of the pathogenesis of human immunodeficiency virus (HIV) infection have revealed that chemokines and chemokine receptors are crucially involved in the molecular mechanism of HIV entry into target cells. Studies have shown that the chemokine receptor CCR5 serves as a critical coreceptor during the viral entry stage of HIV infection, while its ligands macrophage inflammatory protein (MIP)-1α and β and RANTES act as endogenous inhibitors of HIV infection. This makes chemokine/chemokine receptor systems an attractive potential target for the development highly specific drugs with which to improve in the management of HIV. In this review, I will discuss the latest developments in the research on chemokine/chemokine receptor systems, especially MIP-1 and CCR5, with a particular focus on their role in the mechanism of HIV infection and on the development of effective therapies against acquired immunodeficiency syndrome (AIDS).
Keywords: MIP-1, RANTES, CCR5, HIV, AIDS, chemokine, TAK779, AK602, TAK652, SCH-C, SCH532706, SCH-D, GSK 873140, UK-427, 857, E913, PRO140, HGS004. INTRODUCTION The interval between initial infection with human immunodeficiency virus (HIV)-1 and depletion of CD4 lymphocytes and subsequent progression to acquired immunodeficiency syndrome (AIDS) varies among HIV-infected individuals. But amongst the multitude of factors that govern the natural history and pathogenesis of HIV-1, elements that are dependent on both viral and host factors and their interactions are thought to be especially important determinants of disease progression and outcome [1, 2]. Over the past decade, there have been extraordinary advancements in our understanding of the mechanism of HIV entry into cells. Although the importance of the CD4 molecule as a receptor for HIV-1 entry *Corresponding author: Tel: +81-33784-8942; Fax: +81-33784-8946; E-mail: [email protected]
Atta-ur-Rahman / M. Iqbal Choudhary (Eds.) All rights reserved – © 2010 Bentham Science Publishers.
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is well recognized, several lines of evidence suggest that additional receptors also play significant roles. For instance, chemokine receptors have been identified as principal coreceptors for both T-tropic and M-tropic HIV-1 isolates [3-8]. What’s more, infection with HIV often results in dysregulation of the chemokine profile, thereby diminishing the protective effect of some chemokines act as potent natural inhibitors of HIV infection [9]. This makes chemokines and their receptors excellent targets for the development of highly specific drugs that may be of benefit in the management of HIV. The two chemokine receptors that appear to be the most widely utilized as coreceptors for HIV entry are CC chemokine receptor 5 (CCR5), which is the receptor for macrophage inflammatory protein (MIP)-1α and β, as well as regulated on activation normal T cells expressed and secreted (RANTES), and CXCR4, which is the receptor for stromal cellderived factor (SDF)-1. In this review, we will focus first on the biology of MIP-1 and CCR5, then on their involvement in the mechanism of HIV infection and, finally, on the development of effective therapies against AIDS. MIP-1 Chemokine Family Overview Amongst the chemokine superfamily is a recently identified group of molecules that share substantial structural homology – in particular four conserved cysteine residues [10, 11]. Of those, the CXC family of chemokines, in which the first two cysteines are separated by another amino acid residue [e.g., IL-8 (also known as CXCL8), GROα (CXCL1), expression of PMN activating protein-78 (ENA-78; CXCL5), interferon (IFN)-inducible protein 10 (IP-10; CXCL10), monokine induced by interferon-gamma (Mig; CXCL9), and interferon-inducible T cell a chemoattractant (I-TAC; CXCL11)], induces chemotaxis in polymorphonuclear neutrophils (PMNs) and T-cells. On the other hand, the CC chemokine family, in which the first two cysteine residues are in juxtaposition [e.g. MIP-1 (CCL3), macrophage chemoattractant protein (MCP)-1 (CCL2) and RANTES (CCL5)], induces chemotaxis in monocytes and subpopulations of T cells. More recently, two other minor groups (the C and CX3C chemokine families) have also been identified. The members of all these families show considerable homology in their amino acid sequences and often possess overlapping chemoattractant specificities. All of these molecules exert their effects through interaction with chemokine-specific receptors on the surfaces of target cells [10, 12]. Expression and Regulation of MIP-1 in Inflammation and Viral Infection MIP-1 is a member of the CC chemokine family and is a lipopolysaccharide (LPS)inducible, heparin-binding polypeptide first isolated from LPS-treated murine RAW 264.7 cells [13-15]. Native MIP-1 is comprised of two 8-kDa polypeptides, MIP-1α and MIP-1β, which share 68% amino acid identity[16]. In addition, a high degree of nucleotide sequence similarity (69%) was found between the murine MIP-1 cDNA and two human cDNAs, GOS19 and LD78, which were cloned from stimulated lymphocytes [17, 18]. LD78 and GOS19 are thus presumed to be human counterparts of murine MIP-1. Although macrophages and monocytes are often thought of as the major sources of MIP-1, other cell types, including fibroblasts, endothelial cells, keratinocytes and PMNs, are also capable of expressing both MIP-1 mRNA and protein in response to various inflammatory stimuli [19, 20]. As described below, enhanced expression of both MIP-1 and RANTES has been detected in HIV-1-infected macrophages and T cells [21, 22].
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MIP-1 possesses a variety of biological activities. In neutrophils, for instance, MIP-1 induces both chemokinesis and hydrogen peroxide production [13]. It also serves as a prostaglandin-independent endogenous pyrogen [23], stimulates secretion of TNF, IL-1α and IL-6 from murine peritoneal macrophages [24], and induces chemotaxis in basophils and histamine release from mast cells [25]. MIP-1 also is known to play important roles in numerous pathological conditions, including rheumatoid arthritis, pulmonary fibrosis and multiple sclerosis [26-30], as well as viral infections. For example, Cook et al. reported that influenza virus-infected mutant mice lacking MIP-1α showed less inflammation and slower clearance of virus than wild-type mice [31]. With respect to the pathophysiology of HIV, CD4+ T cells and macrophages infected with M-tropic strains of HIV-1 secrete significant amounts of MIP-1α and β and RANTES [32, 33]. On the other hand, MIP-1 and RANTES appear to exert an inhibitory effect on the activity of HIV released by CD8+ T cells [9, 34]. Notably, in that regard, greater expression of MIP-1 by either peripheral CD8+ T cells or mononuclear cells was detected in asymptomatic individuals with HIV infection than in uninfected donors or patients in whom HIV had progressed to AIDS [35, 36]. It may be, therefore, that MIP-1 plays an important role in the shift or progression from latent HIV infection to the clinical stages of AIDS. CCR5 AND ITS ROLE IN HIV INFECTION CCR5, the specific receptor for MIP-1α and β and RANTES, is a member of the Gprotein chemokine receptor (GPCR) superfamily and shares 55% amino acid identity with CCR1 [37, 38]. Important breakthroughs that occurred in 1995-1996 were the discoveries that CCR5 and CXCR4 (the receptor for SDF-1) respectively served as coreceptors for entry of M-tropic (R5) and T-tropic (X4) HIV strains [3, 39-41], and that dual-tropic HIV-1 isolates can use either CCR5 or CXCR4 as an entry cofactor [5]. HIV-1 enters its target cells through direct fusion of the viral and target cell membranes (Fig. 1). The external HIV envelope glycoprotein gp120 sequentially interacts with two cellular receptor molecules, the CD4 glycoprotein and a chemokine receptor (CCR5 or CXCR4), leading to the activation of the fusogenic domain of the transmembrane viral glycoprotein gp41, which results in formation of a hairpin configuration and, in turn, triggers fusion between the viral and cellular membranes [42, 43]. The importance of the role played by CCR5 in HIV infection was confirmed by the finding that downregulation of its expression in macrophages was correlated with a reduction in virus entry and replication [44], and humans homozygous for an inactive mutant CCR5 allele show a reduced risk of infection by HIV-1 [45, 46]. This CCR5 mutation generates a nonfunctional receptor that does not support membrane fusion or infection by M-trophic or dual-tropic HIV-1 strains. Paxton et al. also reported that individuals carrying this mutation show significantly higher levels of CCR5 ligands [34]. Collectively then, the findings summarized so far demonstrate the importance of the MIP-1/CCR5 system for virus entry and replication, which should make this system an important new therapeutic target for the treatment of HIV/AIDS. It is also noteworthy that Philpott et al. [47] demonstrated that shifts to CCR5 from CXCR4 in coreceptor usage by HIV may be indicative of the clinical efficacy of an antiretroviral therapy. Furthermore, to make use of this phenomenon in a clinical setting, those investigators recently developed a new diagnostic method for assessing the suitability of antiretroviral treatment regimens by analyzing the shifts in coreceptor usage between CCR5 and CXCR4 in HIV-infected patients [48].
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Fig. (1). Schematic diagram illustrating the stepwise process of HIV-1 entry into target cells. Following interaction of gp120 with CD4 glycoprotein, a structural conformational change occurs in the viral envelope complex, resulting in the formation of CCR5-binding determinants. The interaction of gp120 with CCR5 leads a second conformational change, this time in the viral glycoprotein gp41, which triggers fusion of the viral and cellular membranes.
INHIBITORS TARGETING THE INTERACTION CHEMOKINE/CHEMOKINE RECEPTOR SYSTEM
OF
HIV-1
AND
THE
1. Chemokines or Small Peptide Antagonists Although MIP-1, RANTES and their various derivatives may be potent natural inhibitors of HIV in vitro [49, 50], the risk of inducing adverse inflammatory effects or causing dysregulation of the homeostatic chemokine system represents a potential limitation to their clinical use. However, Lusso et al. reported that small synthetic peptides corresponding to the N-terminal loop of RANTES not only inhibited HIV-1 infection and fusion, but also acted as a RANTES antagonist in chemotaxis assays [51]. This suggests that small synthetic peptides with structures that enable them to interfere with or modify chemokine-chemokine receptor interaction could be useful therapeutic agents in the future treatment of AIDS. 2. Nonpeptide Small-Molecule Antagonists Alternatively, a number of nonpeptide chemokine receptor antagonists and other small molecules that inhibit HIV-1 infection also have been described. These include a number of small-molecule chemokine antagonists that act at the CCR5 coreceptor to inhibit HIV entry (TAK779, AK602 ,TAK652, SCH-C, SCH532706, SCH-D, GSK873140, UK-427,857 and E913) as well as new types of fusion inhibitors that act via the same mechanism as enfuvirtide (e.g., T1249). The simplified structures of these molecules are shown in Fig. (2). TAK779 A group at Takeda Chemical has discovered numerous small-molecule CCR5 antagonists using high-throughput screening with RANTES- and CCR5-transfected Chinese hamster ovarian cells. Among these compounds, TAK-779 was found to be a highly potent and selective CCR5 antagonist with an IC50 of 1.4 nM in binding assays [52, 53]. On the other hand, there appears to be some donor-dependent variability in the anti-HIV-1 activity of TAK-779 [54].
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Fig. (2). Chemical structures of various CCR5 antagonists. The structures of some compounds such as TAK652, SCH532706, GSK873140, and UK-427,857 have not been made public as of this writing.
AK602 In 2004, Maeda et al. reported a new CCR5 antagonist, AK602 [55]. In vitro, this compound potently inhibited the binding of both MIP-1α and gp120 to CCR5. Moreover, analysis of its pharmacokinetics in healthy subjects showed that high levels of AK602 persist in serum 2 hours and 12 hours after administration, and they were well tolerated. TAK 652 Recent reports have shown that another new CCR5 antagonist, TAK-652, selectively inhibits entry and replication of six R5 HIV-1 clinical isolates in vitro, including those containing NRTI and PI-resistant mutants [56, 57]. Moreover, a single oral administration (25 to 100 mg) of TAK-652 solution was found to be safe and well tolerated with good oral absorption, and Tremblay et al. reported favorable antiviral interactions between TAK-652 and ZDV, 3TC, EFV, IDV and enfuvirtide [58].
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SCH-C This orally bioavailable compound selectively inhibits HIV-1 infection mediated by CCR5, but not by CXCR4, in U-87 astroglioma cells [59]. SCH-C has broad and potent antiviral activity in vitro, with mean IC50s ranging between 0.4 and 9 nM. SCH532706 SCH532706 is a novel small molecule CCRS antagonist with high in vitro potency (mean 90% inhibitory concentration [IC90] 0.15-7.0 nM) against diverse HIV type-1 (HIV1) isolates. Clinical study by Pett et al. showed that ten days of dosing with SCH532706 coadministered with ritonavir demonstrated potent antiretroviral activity and well tolerated [60]. SCH-D The same group that described SCH-C also described another potent CCR5 antagonist, SCH-D [61], has now been named Vicriviroc [62], which was safe and well tolerated at all dose levels tested; no severe or serious adverse events were reported. In addition, Scurmann et al. showed that SCH-D elicited a dose-related decrease in viral load, with mean reductions of 1.08, 1.56 and 1.62 log10 units from baseline in the 10, 25 and 50 mg BID dose groups, respectively [63]. The percentages of patients with a >1.0 Log10 unit reduction in viral load were 55%, 69% and 81% in the 10, 25 and 50 mg BID dose groups, respectively. GSK 873140 Lalezari et al. conducted a Phase I study of GSK 873140 [64]. At a dose of 600 mg BID, GSK 873140 reduced viral load by 1.6 log10 units in 8 patients. Although the half life of this compound in serum is relatively short, clinical studies showed that the antiviral benefits persist even after the drug is no longer detectable in serum. UK-427,857 This piperidine-based CCR5 antagonist, which exhibits potent anti-HIV activity in vivo, has now been named Maraviroc [65, 66]. Maraviroc was approved for marketing by the Food and Drug Administration (FDA) in 2007, for use in combination with other antiretroviral agents in the treatment of HAART-experienced adult patients whose HIV infection is resistant to multiple classes of antiretroviral drugs [67]. Analyses from these two studies, MOTIVATE trial[67] and MERIT trial[68] revealed that twice-daily maraviroc decreased HIV-1 RNA by 1.84 log copies/mL, compared with 0.78 log copy/mL with placebo. Forty-six percent of subjects attained an HIV-1 RNA concentration of <50 copies/mL, compared with only 17% with placebo. E913 A novel low-molecular-weight spirodiketopiperazine derivative, E913, specifically inhibited both the binding of MIP-1α to CCR5 and MIP-1α-evoked cellular calcium mobilization, resulting in potent inhibition of replication of laboratory and primary R5 HIV1 strains, as well as various multidrug-resistant monocyte/macrophage tropic strains [64]. In vitro, this inhibitory effect of E913 was synergistically augmented by combination with a CXCR4 antagonist, AMD-3100.
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3. Anti-CCR5 Antibodies PRO140 Recent in vitro analyses have led to speculation that anti-CCR5 antibodies may exert beneficial and/or protective effects in HIV therapy [69, 70]. In that regard, Trkola et al. have described a murine monoclonal antibody (PRO140) against a complex epitope spanning multiple extracellular domains of CCR5 that does not affect CCR5’s chemokine receptor activity [71, 72]. In vitro, low nanomolar concentrations of PRO140 inhibit infection of primary peripheral blood mononuclear cells by all CCR5-using R5 viruses and inhibited entry by many subtypes of R5 virus into various target cells. Lehner et al. also reported that anti-CCR5 monoclonal antibodies enhanced the in vitro inhibitory effects of suboptimal concentrations of MIP-1α, MIP-1β or RANTES, while the presence of these CCR5 ligands enhanced the inhibitory effect of suboptimal titers of anti-CCR5 antibodies on HIV [73]. These results demonstrate that anti-CCR5 monoclonal antibodies, such as PRO140, may serve as potent inhibitors of HIV-1 infection. Indeed, recent patent applications have focused upon this action. For instance, in their applications, Combadiere et al. reported establishing stable, nonhuman cell lines that coexpress human CD4 and CCR5, and which may serve as valuable tools for continuing research on HIV infection; they also proposed that anti-CCR5 antibodies, CCR5 variants and CCR5-binding agents capable of blocking membrane fusion between HIV and target cells represent potential anti-HIV therapeutics for M-tropic strains of HIV [74-76]. And as a technical advance, Li et al. have applied for a patent on methods to generate a variety of antibodies against human GPCR (e.g., CCR5) polypeptides, the polypeptides themselves and the DNA encoding the polypeptides [77]. HGS004 HGS004 is a fully human IgG4 monoclonal antibody that specifically binds to the second extracellular loop of CCR5, thereby inhibiting HIV envelope–dependent cell-cell fusion and blocking viral entry [78]. This antibody is also a potent inhibitor of chemokine (MIP-1α, MIP-β, and RANTES) binding to the receptor, and does not induce signaling or mediate cytotoxicity in human cells. Furthermore, in vitro studies with currently approved agents representing each of the antiretroviral drug classes; nucleoside reverse-transcriptase inhibitors (zidovudine and lamivudine), nonnucleoside reverse-transcriptase inhibitors (efavirenz), protease inhibitors (indinavir), and fusion inhibitors (enfuvirtide), demonstrated that HGS004 acts synergistically with all currently approved classes of antiretroviral agents [79]. CURRENT AND FUTURE DEVELOPMENTS Chemokines are intricately involved in processes related to homeostasis, development, inflammation and viral infection, especially HIV infection. It has therefore become an issue of considerable importance to determine as specifically as possible the correlation between each clinical feature of HIV infection and the patterns of chemokine receptor usage and chemokine regulation. Despite the challenge inherent in such a complex investigation, the knowledge derived from ongoing studies of the involvement of chemokine biology in the pathogenesis of HIV may make a crucial contribution to much needed improvements in the clinical management of HIV/AIDS, as well as to new therapeutic strategies that increase the efficacy of the actually used highly active anti-retroviral therapy (HAART) regimens. Moreover, new insights into the molecular basis underlying the regulatory effects of
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mutations and polymorphisms among chemokine/chemokine receptors in HIV infection may bring us additional new targets for therapeutic intervention. ABBREVIATIONS MIP-1
=
Macrophage inflammatory protein-1
HIV
=
Human immunodeficiency virus
AIDS
=
Acquired immunodeficiency syndrome
CCR5
=
CC chemokine receptor 5
RANTES
=
Regulated on activation normal T cells expressed and secreted
CXCR4
=
CXC chemokine receptor 4.
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Small-Molecule Inhibitors of Raf for Treatment of Malignant Diseases Li Li1, Shuhong Wu2, Wei Guo2 and Bingliang Fang*,2 1 2
Department of Laboratory Medicine, First Hospital of Shanghai, Shanghai, China; Department of Thoracic and Cardiovascular Surgery, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA Abstract: Ras/Raf/MEK/ERK pathway is a critical downstream signal transduction cascade of most growth factor receptors and is pivotal in oncogenesis, tumor cell malignancy, viral infection, neuronal degeneration, and lymphocyte activation. A number of gain-of-function mutations of Raf genes have been detected in various cancer cells. Moreover, increased MEK/ERK activities were also found in various tumor tissues. Consequently, Raf/MEK/ERK pathway as an anticancer target has been intensively investigated. Numerous small-molecule Raf/MEK/ERK-inhibiting compounds have been reported in the literature and in patent applications. One of Raf inhibitors, the urea derivative Bay 43-9006 (Sorafenib), has recently been approved for treatment of kidney cancer and is under clinical trials for treatment of other cancers. Several Raf inhibitors or mutant B-Raf-selective inhibitors (RAF265 and PLX4032) were also in various stages of clinical trials for cancer treatment. Those inhibitors have also been evaluated preclinically for treatment of viral infection, neuronal degeneration, and inflammatory disease. Thus, small molecule inhibitors of Raf/MEK/ERK pathway may have broad applications in addition to cancer therapy.
Keywords: Raf-1, A-Raf, B-Raf, anticancer agents, small molecule, virus infection, Ras, MAP kinases. INTRODUCTION The Ras/Raf/MEK/Erk pathway is a critical downstream signal transduction cascade of most growth factor receptors and is critical for cell survival, growth, differentiation and transformation [1,2]. As important as it is in the proliferation and survival of many tumor cells [3,4], the Ras/Raf/MEK/ERK pathway has attracted much attention in cancer therapy [5-7]. Raf proteins are serine/threonine kinases and are the key components of the Rasmediated Raf/MEK/ERK signaling pathway. Humans and other vertebrates have three Raf proteins, A-Raf, B-Raf, and C-Raf (Raf-1), which are encoded by their corresponding genes [8,9]. Sharing common conserved regions, the Raf proteins carry out similar but indispensable functions in development. They can all be activated by Ras on the binding of extra-cellular ligands, such as growth factors, cytokines, and hormones, to their cell surface receptors. Activated Raf can all phosphorylate and activate the dual-specificity protein kinase mitogen-activated protein kinase kinase (MEK), which in turn phosphorylates both *Corresponding author: Tel: (713) 563-9147; Fax: (713) 794-4669; E-mail: [email protected] Atta-ur-Rahman / M. Iqbal Choudhary (Eds.) All rights reserved – © 2010 Bentham Science Publishers.
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tyrosine (Tyr-185) and threonine (Thr-183) residues of extracellular signal-regulated kinases (ERK) proteins [10]. Humans and other mammals have two MEK proteins, MEK1 and MEK2, which are encoded by their corresponding genes, are expressed ubiquitously, and activate ERK through its dual-specificity protein kinase activity [10]. ERK also has two isoforms, ERK1 and ERK2, encoded by their corresponding genes [11,12]. Activated ERK is then transferred to the nucleus, where it activates transcriptional factors such as Elk-1. Because of its critical roles in the proliferation and survival of tumor cells [3,13], the Ras/Raf/MEK/ERK pathway has attracted much attention in cancer therapy [5-7]. Small molecules that interrupt this pathway have been intensively investigated as pharmaceutical agents against cancer. One of Raf inhibitors, the urea derivative Bay 43-9006 (Sorafenib), has been approved for treatment of kidney cancer [14]. Several other Raf inhibitors or BRaf-selective inhibitors were also under clinical trials for cancer treatment. For example, RAF265 and PLX4032 are currently under clinical trials for treatment of advanced metastatic melanoma. Promising results with disease stabilization have been observed in a small group of patients with melanoma. Evidences also suggested that modulation of the biologic functions of this pathway might also be used to treat viral infection, neuronal degeneration, and inflammatory disease. REGULATION OF RAF/MEK/ERK PATHWAY Regulation of Raf activities involves complex processes, including the phosphorylation and subcellular localization of Raf proteins and their interaction with other proteins. Recruitment of Raf from the cytosol to the membrane by activated Ras represents an important step in Raf regulation because it changes the phosphorylation status and activates Raf. Numerous studies have delineated the regulation of C-Raf phosphorylation. Several serine residues in C-Raf can be phosphorylated by protein kinase A or B and thus suppress C-Raf activity [15-19]. On the other hand, Ras and protein phosphatase 2A can cooperate to dephosphorylate C-Raf to some extent and thereby re-activate it [20]. Once activated, Raf turns on the MEK/ERK cascade as described above. Activated C-Raf can also be translocated to the mitochondria and there execute antiapoptosis signaling by interacting with proteins regulating apoptosis [21,22]. For examples, Raf proteins interact with 14-3-3, [23-25], c-Flip [26], cytokeratine K8/K18 [26,27], apoptosis signal-regulating kinase 1 [28] BAG-1 [29], and Bad [30-32]. Thus, Raf may exert an antiapoptotic effect by interacting with various molecules in apoptosis pathways. Various mutations of the B-Raf gene have been identified in human cancers, including 60-70% of malignant melanomas, 36-50% of thyroid cancers, 5-22% of colorectal cancers, 30% of serous ovarian cancers, and lower percentages of other cancers [33]. Several studies have demonstrated that mutated B-Raf proteins have elevated kinase activity and transform NIH 3T3 embryonic mouse cells into tumorigenic cells [33,34]. Wan et al analyzed 22 BRAF mutants and found 18 of them had elevated kinase activity which activates ERK in vivo directly [34]. Interestingly, three mutants that had reduced kinase activity towards MEK in vitro but activated C-RAF in vivo, leading to MEK/ERK activation in cells [34]. In vitro studies have also demonstrated that constitutively active form of MEK is sufficient for cellular transformation, leading to highly tumorigenic cell lines [35,36]. Evidence also demonstrated that activation of MEK/ERK has been implicated in development of a broad range of human tumors [37,38]. For examples, enhanced activation of MEK/ERK has been detected in about 30-60% of primary lung cancers [37,39-42]. Elevated levels of phosphoryated ERK in primary tumors is observed in both smokers and non-smokers [39],
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is associated with hyperactivation rather than overexpression of ERK, and is associated with poor survival [40]. Moreover, C-Raf is a pivotal regulator of endothelial cell survival during angiogenesis [43,44]. Ablation of C-Raf renders cells hypersensitive to apoptosis despite normal regulation of extracellular signal-regulated kinases [45]. In fact, many proteins critical in cell survival/apoptosis pathways are regulated by phosphorylation through Raf/MEK/ERK pathway, including forkhead transcriptional factors [46], TSC2/mTOR complex [47], Bad [48], caspase-9 [49] cyclin D1 [50]. INHIBITION OF THE RAS/RAF/MEK/ERK PATHWAY Because Raf is activated by Ras and in turn activates MEK/ERK, agents that inhibit Ras, MEK, or ERK may induce biologic functions similar to those of Raf inhibitors. Similarly, agents that modulate activities of other kinases, such as protein kinase A, AKT, and SRC, may dramatically affect Raf functions. However, because all of those molecules interact with many other target proteins that have diverse biologic functions or are involved in different pathways, agents targeted at different molecules of the Ras/Raf/MEK/ERK cascade may have their own safety and efficacy profiles. In addition, the clinical application of these agents will depend on their specificity, potency, and chemical and pharmacologic properties [51-54]. Because of numerous publications on the small molecule inhibitors of the Ras/Raf/ERK/ERK pathway, this review will focus on small molecule inhibitors of Raf. Readers interested in inhibitors to other proteins in this pathway are referred to other recent reviews [55-57]. Raf can be inhibited specifically by a variety of agents, including dominant negative genes, antisense oligonucleotides, small interfering RNA, peptides, intracellular antibodies, and small-molecule compounds. Here we focus on some small-molecule compounds that inhibit Raf. USE OF SMALL-MOLECULE UREA DERIVATIVES IN CANCER THERAPY Urea derivatives are one of several major groups of compounds that inhibit Raf, mainly C-Raf (compounds 1-5). Except for one patent on malonamide derivatives that was filed by Merck Patent [58], all patent applicants have been filed by Bayer AG [59-63]. One of the compounds, Bay 43-9006 (marketed as Sorafenib or Nexavar, compound 1) that is jointly developed by Bayer and Onyx Parmaceuticals, is in extensive clinical trials. Initially, 200,000 compounds were screened against recombinant C-Raf kinase, and a 3-thienyl urea compound was identified as the most potent inhibitor of C-Raf in biochemical assays (IC50 = 17 μM) [64]. A subsequent intensive structure-activity relationship study on more than a thousand urea derivatives led to identification of Bay 43-9006 as the compound that inhibited C-Raf with an IC50 = 12 nM in the biochemical assays [64]. In cultured cancer cells, the IC50 for inhibition of Raf-dependent MEK activation and tumor cell proliferation were in single-digit micromolar values. For example, Bay 43-9006 inhibited HCT 116 human colon cancer cell proliferation at IC50 = 4.6 μM [65]. Bay 43-9006 also inhibited the growth of SKOV3, a human ovarian cancer cell line that contains wild-type Ras but has a constitutive activation of the Ras/Raf/MEK/ERK pathway because of overexpression of epidermal growth factor and Her-2 receptors [65]. BAY 43-9006 has also been shown to be active against several receptor tyrosine kinases involved in neovascularization and tumor progression, including vascular endothelial growth factor receptors (VEGFRs) 2 and 3, platelet-derived growth factor receptor beta, Flt-3, and c-KIT [66]. Thus, an activated Ras oncogene is not necessary for Bay 43-9006-mediated antitumor activity.
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O Cl
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Fig. (1). Urea derivatives. 1, Bay 43-9006 (Sorafenib or Nexavar).
Pharmacokinetic analyses in rodents showed that orally administered Bay 43-9006 was rapidly absorbed and had a terminal half-life of 3.2-4.2 hours [65]. Plasma concentrations increased proportionally with dose and exceeded 50 μM at 100 mg/kg. Preclinical studies on the HCT 116 xenograft tumor mouse model showed that daily administration of Bay 439006 (10-300 mg/kg) dose-dependently inhibited tumor growth by up to 70% [65]. Phase I/II clinical trials showed that Bay 43-9006 has favorable safety profile. The compound was administered orally at doses up to 800 mg twice daily [5,67]. Diarrhea and
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skin toxicity were dose-limiting toxicities at 600-800 mg. The maximum tolerated continuous dose was 400 mg twice daily [67]. At this dose, Bay 43-9006 reached a mean plasma level of 20 μM and had a terminal half-life of 36 hours [5]. Combination therapy of Bay 43-9006 and other chemotherapeutic agents, such as doxorubicin and irinotecan, has also been reported in clinical trials [68-70]. The clinical trials in renal cancer patients have so far shown significantly longer progression-free survival in patients who received BAY 43-9006 than among those who received placebo. This compound was approved treatment of advanced renal cancer in the United States and European Union in 2006 and marketed as Nexavar or sorafenib tosylate. A phase III study of Bay 43-9006 in patients with advanced liver cancer [14,71] and phase II-III studies evaluating BAY 43-9006 plus carboplatin and paclitaxel to treat patients with advanced metastatic melanoma, thyroid cancer and lung cancer [72,73] have also been reported. Bay 43-9006 is also being evaluated in single-agent phase II-III clinical trials in breast, and other cancers and in phase II-III clinical trials studying this agent plus a range of standard chemotherapeutic agents. USE OF OTHER SMALL-MOLECULE RAF INHIBITORS IN CANCER THERAPY PLX4720 (6), a 7-azaindole derivative developed by Plexxikon, Inc., specifically inhibits B-Raf V600E mutant with an IC50 of 13 nM [74]. Compared with a broad spectrum of other kinases, PLX4720 preferentially inhibits the V600E mutant B-Raf and induce cytotoxic effects exclusively in cells bearing the V600E allele. PLX4720 effectively Inhibits ERK phosphorylation in B-Raf(V600E)-bearing tumor cell lines and suppressed tumor growth of B-Raf(V600E)-dependent tumor xenograft, without evidence of toxicity [74]. Plexxikon Inc announced recently that Phase I clinical study on PLX4032, an oral active and highly selective drug that targets the BRAFV600E cancer-causing mutation, showed objective responses in metastatic melanoma patients (www.medicalnewstoday.com/ articles/152309.php). In patients whose cancer harbors this mutation and who were treated with therapeutic doses of PLX4032, tumor shrinkage and extended progression-free survival have been observed. Larger clinical trials with this compound are planned to start later in 2009. Starting from a urea compound of Raf inhibitors, investigators in Norvatis Institutes for Biomedical Research developed a series of arylaminobenzimidazoles compounds (7) and evaluated their structural-activity relationship in Raf kinase inhibition as well as in vitro and in vivo properties [75]. Most of them have their IC50 for B-Raf and C-Raf at a few nM ranges. Some of them have favorable in vivo properties, including complete oral bioavailability. Moreover, testing on tumor-bearing animals showed that one those compounds has significant antitumor activities in HT29 (B-Raf V600E mutant) human colorectal xenofraft model [75]. Based on information provided on Norvatis website (www.novartisoncology.com), Norvatis is currently performing clinical trials on a compound named RAF265, which is a potent and selective inhibitor of all three Raf isoforms, including mutant B-Raf. In addition, RAF265 has antiangiogenic activity through inhibition of VEGFR-2. This compound is now investigated in Phase I clinical trials in malignant melanoma. Investigators in UK Center for Cancer Therapeutics also developed a series of pyridoimidazolone (8) compounds with potent activities against mutant B-Raf [76-78]. Compounds with an IC50 of 1 nM for purified V600E-BRAF and nanomolar activity in cells have been identified through activity-relationship analysis and optimization [76]. On the other hand, a series of pyrazole-based inhibitors with potent activity and selectivity for B-
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Raf is currently under development by Genentech Inc [79,80]. One of such compound named GDC-0879 (9), which has IC50 of 0.13 nM for purified B-Raf V600E enzyme and 63 nM for MALME-3M cell line [80]. Analysis on the relationship between GDC-0879 plasma concentrations and tumor growth inhibition in A375 melanoma and Colo205 colon cancer xenografts showed that plasma concentrations required for tumor stasis and inhibition of phosphorylated MEK in those tumor models is about 3 μM - 4.5 μM. Moreover, a threshold of >40% pMEK1 inhibition is required for tumor growth inhibition, and a minimum of approximately 60% pMEK1 inhibition is required for stasis in A375 xenografts treated with GDC-0879 [79]. Compound ZM-336372 (10), which was patented by AstraZeneca, was found to be a potent and specific inhibitor of Raf isoforms in vitro, and of 20 other protein kinases tested, only p38 was inhibited by ZM-336372 [81]. However, ZM-336372 did not prevent growth factor- or phorbol ester-induced activation of MKK1 or ERK, and it did not reverse the phenotype of Ras- or Raf-transformed cell lines. Exposure of cells to ZM336372 induced a 100-fold activation of C-Raf, suggesting that Raf may suppress its own activation by a novel feedback loop such that inhibition is always counterbalanced by reactivation [81]. Another B-Raf inhibitor under evaluation by AstraZeneca is AZ628, with IC50 for wide type B-Raf 2.14 μM and mutant B-Raf 196 nM [82]. A substantial numbers of cancer cell lines from melanoma, thyroid and colorectal cancers with B-Raf mutations were sensitive to this compound [82]. However, elevated C-Raf activity may convey resistance to AZ628 [83]. Investigators in Glaxo Wellcome also reported that some benzylidene oxindole derivatives (11) were C-Raf kinase inhibitors that induced kinase enzyme inhibition at low nanomolar concentrations [84,85]. In addition to directly inhibiting Raf function, some compounds very effectively suppress Raf expression. For example, SU5416, a potent and selective inhibitor of VEGFR tyrosine kinase activity developed by Sugen [86,87], blocked cisplatin-induced increases in AP-1 expression, JNK activity, and C-Raf protein level [88]. SU5416 is currently being investigated in clinical trials for treatment of renal cell carcinoma, melanoma, and leukemia. [89-91]. Another example is tyrphostin AG 879, a tyrosine kinase inhibitor also developed by Sugen, that is specific for ErbB2 and VEGFR and markedly inhibited expression of the C-Raf gene [92]. Inhibition of Raf protein expression will ultimately lead to suppression of its biologic function. Interestingly, BAY 43-9006 has been reported to strongly suppress several receptor tyrosine kinases, including VEGFR [66], which suggests that Raf inhibitors and VEGFR inhibitors may have broader targets or cross activities. In fact, the patents filed by Bayer AG for urea derivatives describe their use as VEGF inhibitors for VEGF-mediated diseases such as angiogenesis disorders [59-62]. USE OF SMALL-MOLECULE RAF INHIBITORS IN TREATING OTHER CONDITIONS Although small-molecule Raf inhibitors have been extensively investigated as possible cancer therapeutic agents, we believe that they may also be used to treat other conditions because in addition to oncogenesis, the Ras/Raf/MEK/ERK pathway involves many important biologic functions, including virus infection, neuronal degeneration, and lymphocyte activation. For example, binding of human immunodeficiency virus type 1 (HIV-1) virions to CD4 receptors stimulated the association between Lck and C-Raf and resulted in the activation of C-Raf in a Ras-independent manner [93,94]. This mode of Raf activation may promote HIV replication in lymphocytes because the functional consequences of active Raf in T
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lymphocytes include activation of NF-B, transactivation of the HIV long terminal repeat, and synthesis and release of HIV particles [95]. In addition, Raf and Raf-induced VEGF expression promoted Kaposi sarcoma-associated herpesvirus infection and the pathogenesis of Kaposi sarcoma [96,97]. Furthermore, the integrity of the C-Raf/MEK signal transduction cascade appears to be essential for hepatitis B virus gene expression [98]. The Raf/MEK pathway was activated by human papillomavirus [99] and hepatitis C virus core protein [100] and blockage of this pathway impeded propagation of influenza virus and bornavirus [101,102]. Thus, we hypothesize that compounds that block the Ras/Raf/Mek/ Erk signal tranduction pathway may be used to treat viral infections. In fact, a patent application filed by Ludwig and Pleschka used inhibitors of the Raf/MEK/ERK signal cascade to treat DNA and RNA viral infections [103]. More specifically, MEK inhibitors, such as U0126, PD98059, and PD184352, blocked the replication of influenza A virus and bornavirus in cultured cells. H N
N
F R2
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Fig. (2). Non-Urea Compounds. 6, PLX4720; 7, arylaminobenzimidazoles; 8, pyridoimidazolone; 9, GDC-0879; 10; ZM-336372; 11, benzylidene oxindole derivatives.
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Raf inhibitors, especially B-Raf inhibitors, have also been useful in the treatment of or as prophylaxis for disorders associated with neuronal degeneration resulting from ischemic events, including cerebral ischemia after cardiac arrest, stroke and multi-infarct dementia, and cerebral ischemic events resulting from surgery or during childbirth [104]. For example, 4-(4-chloro-3-hydroxyphenyl)-2-phenyl-5-(4-pyridyl)-1H-imidazole protected against oxygen-glucose deprivation-induced neuronal cell death in organotypic hippocampal culture (IC50 = 100 nM) [104]. The Ras/Raf/MEK/ERK pathway is important in the activation and proliferation of many inflammatory cells, and the use of Raf kinase inhibitors to treat inflammatory diseases, such as rheumatoid arthritis, osteoarthritis, rheumatoid spondylitis, and gouty arthritis, has been described in a patent application by Astex Technology [105]. Activation of the Raf/MEK/ERK pathway is required for B- and T-cell clonal expansion and generation of effector populations [106-109]. Suppressing this pathway may suppress the immune response in organ or tissue transplantation. CURRENT AND FUTURE DEVELOPMENTS The identification of Raf genes two decades ago [110-112] aroused much interest in Raf as a therapeutic target. Substantial effort has been directed toward developing Raf inhibitors, mostly for cancer treatment. In addition to Raf-inhibiting small compounds that have been evaluated preclinically and clinically, Raf-inhibiting antisense oligonucleotides have also been used in clinical trials for treatment of cancers [113]. At present, clinical evaluations of Raf inhibitors have mostly been cancer therapy. However, because of the roles of the Ras/Raf/MEK/ERK pathway in viral infection, neuronal degeneration, and lymphocyte activation, Raf inhibitors may have clinical applications that go beyond cancer therapy. The success of Raf inhibitors as therapeutic agents will depend on the chemical, biologic, pharmacologic, and toxicologic properties of each compound. The presence of three Raf homologues in mammals and their similar but non-redundant biologic functions may complicate the evaluation of Raf inhibitors. The lethality of Raf knockouts also suggests that profound inhibition of Raf function may induce toxicity, although clinical trials with Bay 43-9003 have shown that this agent can provide clinical benefit without obvious toxicity, indicating that Raf inhibitors might be well tolerated and their use can be explored with substantial safety margins. Nevertheless, recent development in mutant B-Raf selective inhibitors may minimize toxicity to normal cells, thereby improving the safety profile of those inhibitors. However, because the Ras/Raf/MEK/ERK signal transduction pathway interacts with a variety of other molecular targets and pathways, it is expected that changes in other signaling pathway may dramatically affect the sensitivity of cancer cells to those inhibitors. Therefore, combining Raf inhibitors with other therapeutic agents may be a useful approach to treating various diseases and infections. ACKNOWLEDGEMENT Grant support: National Cancer Institute grants RO1 CA 092487 (to B. Fang).
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Tigecycline: A New Treatment Choice Against Acinetobacter baumannii Virginia Bosó-Ribelles*,1, Eva Romá-Sánchez1, Jorge Carmena2, Cristina Cáceres2 and Daniel Bautista3 1
Pharmacy Department, Hospital La Fe, Valencia, Spain, 2Infectious Diseases Department, Hospital Dr. Peset, Valencia, Spain, 3Preventive Medicine Department, Hospital Dr. Peset, Valencia, Spain Abstract: Acinetobacter baumannii (AB) is a gram-negative organism that has emerged recently as a major cause of nosocomial infections, because of the extent of its antimicrobial resistance and its persistence in the hospital environment, where intensive care units are the place of greatest risk for acquiring AB. There is no treatment of choice for AB and it’s treatment is based on clinical experience and in vitro susceptibility testing. Also, nowadays Acinetobacter resistance to carbapenems is common and isolates resistant to colistin and polymyxin B have been reported. Tigecycline, the 9-tert-butyl-glycylamido derivative of minocycline, exhibits a broad-spectrum of activity against numerous pathogens, including AB and several reports place it among the antimicrobials with lower MIC for AB. Tigecycline overcomes the two major mechanisms of resistance to tetracyclines (ribosomal protection and efflux), but tigecycline resistance emerging during therapy has been reported. Tigecycline efficacy has been demonstrated in clinical studies in skin and skin structure infections and in complicated intra-abdominal infections but, although it seems a good alternative for the treatment of AB infections, there is little evidence about its use in these cases and more clinical experience and adequate trials are needed. The present review shows the recent patents related to treatment by tigecycline in different AB infections.
Keywords: Acinetobacter baumanii, tigecycline, glycilcyclines, multiresistance, nosocomial infection. ACINETOBACTER TREATMENT
BAUMANNII:
EPIDEMIO-LOGICAL
FEATURES
AND
Acinetobacter baumannii (AB) is a gram-negative organism that has emerged recently as a major cause of nosocomial infections, because of the extent of its antimicrobial resistance and its persistence in the hospital environment [1]. AB is ubiquitous, living in soil and water. The organism can survive for extended periods in the environment and tolerates both *Corresponding author: E-mail: [email protected] Atta-ur-Rahman / M. Iqbal Choudhary (Eds.) All rights reserved – © 2010 Bentham Science Publishers.
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wet and dry conditions [2], making nosocomial transmission extremely difficult to control. This opportunistic pathogen may cause severe clinical disease with a high mortality [3]. The factors of poor prognosis include: patient severity, septic shock, disseminated intravascular coagulation, and inappropriate antimicrobial treatment. The latter is the most changeable, although the possibilities of improving the empirical treatment are reduced in parallel to the increase of AB resistance. The epidemiology of AB infection is often complex, with the coexistence of epidemic and endemic infections, the latter of which often is favored by the selection pressure of antimicrobials [1, 4]. The transition from colonization to infection is determined by the performance of invasive procedures that alter the normal barriers of a particular host. Crosscontamination plays an important role in epidemics caused by AB [5], and may occur despite implementation of stringent infection control measures [6]. Tolerance to desiccation contributes to the maintenance of Acinetobacter in the environment. This provides enough opportunities for contamination of patients and staff [7]. It may be present in the hands of health care workers and hospital devices [4]. Resistance to currently used disinfectants is probably not a major factor in the epidemic spread of AB. However, even minor deviations from the recommended procedures leading to decreased concentrations or exposure times may facilitate nosocomial cross-transmission [8]. Although combating AB infections is important in all hospital services, the intensive care unit (ICU) is the place of greatest risk for acquiring AB, since the incidence is much higher than in the rest of the hospital [3]. In the ICU, large number of patients and health personnel confluence, more invasive diagnostic and therapeutic procedures per patient are performed, and broad-spectrum antimicrobial agents are used against multi drug resistant microorganisms. Recent trends indicate increasing antimicrobial resistance of Acinetobacter isolates worldwide [9-13]. The ability to acquire multidrug resistance can be due to either the acquisition of genetic elements carrying multiple resistant determinants or mutations affecting the expression of porins and/or efflux pumps, which can affect unrelated antimicrobial agents [14]. Unfortunately, the accumulation of multiple mechanisms of resistance leads to the development of multiply resistant or even "panresistant" strains [15, 16]. There is no treatment of choice for AB because there are no comparative clinical studies. The treatment is based on clinical experience and in vitro susceptibility testing [12]. Generally, imipenem was the most active agent against AB. However, carbapenem resistance in AB is common because of porin or penicillin-binding protein modifications. [17]. Likewise, Acinetobacter isolates resistant to colistin and polymyxin B have also been reported [18, 19]. Furthermore, toxicity of these antibiotics complicates the choice of appropriate therapy. Thus, new antimicrobials or synergistic combinations of antimicrobials are needed for the treatment of infections caused by multidrug resistant AB. A NEW THERAPEUTIC CHOICE FOR AB: TIGECYCLINE Pharmalogical and Pharmacokinetic Aspects Tigecycline (TBG-MNO or GAR 936) is the fist antibiotic in the glycylcycline family, synthetic analogs of tetracycline that emerge from the integration of radical tbutylgylcilamido position 9 of minocycline (Fig. 1). It is a structured modification which betters its spectrum and enables a better profile of anti-resistance [20-23].
Tigecycline
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N(CH3)2
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N(CH3)2 OH NH2
OH
O
OH
OH
O
O
Minocycline N(CH3)2
N(CH3)2 OH
H H3C H3C
N CH3
O NH2
N H
OH
O
OH
OH
O
O
Tigecycline
Fig. (1). Chemical structure of tigecycline.
Just like tetracyclines, it inhibits its protein translation by reversibly binding to the subunit 30S of the ribosome in bacteria, which impedes the unification of amino acids and its subsequent prolongation in peptic chains [22, 24-26]. Glycylcyclines bind 5 times more effectively than tetracyclines [27], allowing it to influence in its ability to overcome its resistance to tetracycline based on ribosome protection [23]. Even more so it seems that the interaction of tigecycline with ribosome is different from that of tetracyclines [28, 29]. It has been approved by the FDA (Food and Drug Administration) and EMEA (The European Medicines Agency) in the treatment of complicated skin and skin structure infections (cSSSIs) as well as complicated intraabdominal infections (cIAI). Due to its low oral bioavailability, it is only administrated intravenously (IV) [24, 30]; with an initial dose of 100 mg followed by a dose 50mg every 12 hours (5 to 14 days). It binds to plasma proteins (range of 71-89%) and is amply distributed to the tissues [24]. Tigecycline has a large volume of distribution (7-9 L/kg) [24, 26, 30] and circulates principally as an unaltered drug [30]. It is estimated that 20% of the administered dose is metabolised before its excretion and the main metabolites are a glucuronide, a N-acetyl and an epimer. Globally, the primary route of elimination is through biliary excretion of the unaltered drug, while glucuronidation and renal excretion are secondary routes (the renal clearance is approximately 13% of total clearance) [24]. Fifty-nine per cent of the dose is eliminated by biliary/ fecal excretion and 33% is by renal excretion (22% as an unaltered drug). The average clearance is 0.2 to 0.3 L/h/kg and the half life is 42 hours [24, 26]. It doesn’t interact with other drugs because of hepatic microsomal metabolism. It interacts with the warfarin diminishing its clearance although international normalized ratio (INR) alterations have not been observed [31]. The interaction of digoxin has also been studied without finding any significant interactions in the concomitant administration of oral digoxin and tigecycline in healthy volunteers [22, 32].
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ANTIBACTERIAL ACTIVITY, EFFICACY AND SECURITY Tigecycline is a broad-spectrum antibiotic with in vitro activity against a great variety of pathologic microorganisms including among others, methicillin-resistant S. aureus, microorganisms producers of extended spectrum beta-lactamases (ESBLs) and Acinetobacter spp. It is generally considered a bacteriostatic agent, although it has bactericidal activity against S. pneumonia, H. influenza and N. gonorrhoea [33, 34]. Acinetobacter spp and S. maltophilia are the non-fermentative gram-negatives bacillus with the lowest tigecycline MIC. In most published studies MIC90 values for Acinetobacter spp of 2mg/l [35-39] are given, although in some studies 8mg/l (90% sensitivity) is reached [40, 41]. The Tigecycline Evaluation and Surveillance Trial program found major differences in the susceptibility of AB depending on the geographical distribution in the USA, with as many as 29.3% of the analyzed samples being resistant to 3 or more drugs [39]. Tigecycline was the antibiotic with the lowest MIC (amongst amikacin, ampicillin, amoxicillinclavulanic, ceftazidime, ceftriaxone, cefepime, imipenem, levofloxacin, minocycline and piperacillin-tazobactam). In another study carried out in Spain, it was observed that tigecycline, after polymyxin B, was the drug with the highest activity against AB ahead of sulbactam, levofloxacion, ampicillin-sulbactam, imipenem, cefepime, piperacillin-tazobactam and gentamicin [41]. Tigecycline avoids the two principal mechanisms of resistance against tetracyclines: the protection of ribosomes and the active efflux of the drug from the inside of the bacteria cell [22, 24, 42]. It seems to be because of the steric impediment conferred by the position 9 substituent [22, 23, 28]. It eludes the exiting efflux pumps TET (A-E) [43, 44] which cause most cases of acquired resistance to tetracycline and minocycline in enterobacterias and Acinetobacter spp. Also, resistance mechanisms such as beta-lactamases (BLs) (including ESBL), metallo-beta-lactamases (MBLs) target modifications, macrolide expulsion pumps or topoisomerase alterations do not affect the tigecycline, and crossed resistance with other antibiotics has not been observed [22-24]. Therefore, it is a potential option in the treatment of microorganisms producers of ESBL [45]. Aside from all this, tigecycline is not exempt from the development of resistance, possibly from mutations of the exiting efflux pumps [42]. According to the results of the TEST program (4247 samples) approximately 5% of the analysed samples had CMI>2mg/l [46]. However, in a study carried out in Israel with 82 clinical analyses of multi-resistant AB only 22% were sensitive to tigecycline and 95% of the strains that were not sensitive to imipenem were also not to tigecycline [47]. Cases of AB bacteriemia in patients undergoing treatment with tigecycline for other reasons have been published, possibly due to the low serum concentration of the drug [48-50]. Tigecycline has demonstrated its efficacy in diverse clinical trials designed to determine the noninferiority to an active comparative agent. The trials published up to date were taken out in hospitalized adults with cSSSIs (an open trial in phase II [51] and two randomized, controlled, double blind, phase III trials [52, 53]) and in patients with cIAI (an open trial in phase II [54] and two randomized, controlled, double blind, phase III trials [25, 55]). In the cSSSI clinical trials, patients randomly received tigecycline (initial dose of 100mg followed by 50mg every 12 hours) or a combination of vancomycin and aztreonam IV during a maximum of 14 days. The main efficacy criteria was the clinical response in the evaluation visit in the clinical modified population (c-mITT, including patients included the
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intent-to-treat population who received at least one dose of the drug and who had clinical evidence of a cSSSI by meeting the minimal disease criteria) and the clinically evaluable population (CE, including patients in the c-mITT who did not have P. aeruginosa as a sole baseline isolate, received no concomitant antibiotic after their first dose of study medication, and had an assessment of cure or failure at the test-of-cure visit). The analysis set of results from both trials were published, which is shown in Table 1 [56]. cIAI patients randomly received tigecycline or imipenem-cilastatin during 5 to 14 days. It was stratified depending on APACHE II. The main efficacy criteria was the clinical response in the evaluation visit in the microbiological-modified population (m-mITT, consisting of patients in the intent-to-treat population who received at least 1 dose of study drug –mITT- and had clinical evidence of a complicated intraabdominal infection, by meeting the minimal disease criteria, and had a confirmed baseline isolate) and in the microbiologically evaluable (ME) population. The combined clinical analysis of both results is shown in Table 1 [57]. Generally, tigecycline is a well tolerated drug. Its most frequent adverse effects in clinical studies were gastrointestinal [22, 24, 52, 53, 55]: nausea (33.5% of the tigecycline group vs. 19.9% of the control group), vomiting (22.3% vs. 13.4%) and diarrhoea (12.9% vs. 11.9%). The nausea and vomiting episodes occurred during the 1st and 2nd day of treatment, in most cases, they were of slight -moderate intensity and reverted when treatment was suspended [22, 25, 58]. In respect to serious adverse affects, infections (6.7% vs. 4.6%) and sepsis/shock septic (1.5% vs. 0.5%) [24] should be pointed out. The FDA classified its safeness as D during pregnancy. There are no studies in humans but tigecycline, just like tetracyclines, can produce discoloration and harm to the dental enamel and a subnormal delay in the ossification in foetuses exposed in the last half of gestation. In children under 8 years it can have the same effects because of its tissue distribution due to the high replacement of calcium and the formation of complex calcium quelantes. Its use is also unadvisable during breast feeding [24, 26, 58]. Several studies describing the use of tigecycline in Gram-negative MDR infections are currently published. In a prospective open-label and non-comparative study of tigecycline in the treatment of patients with selected serious infections due to resistant Gram-negative organisms including Enterobacter species, AB and K. pneumoniae (the most commonly isolated resistant pathogens was AB (47%)) the clinical cure rate was 72.2% and the microbiological eradication rate was 66.7% [59]. A retrospective study compared the use of tigecycline in the treatment of infections due to MDR AB and K. pneumoniae (n=45) as monotherapy or in combination (co-administered antimicrobial(s) were resistant in vitro or had been clinically and microbiologically failing). Successful clinical outcome was 81.8% vs. 78.3%, respectively [60]. Tigecycline (MIC90≤2 mcg/mL) use in MDR AB infections involving 24 patients resulted in an overall mortality rate of 16.7% [61]. On the other hand, a case-series study including infections from MDR AB (n=29, MIC90≥4 mcg/mL) resulted in very low clinical and microbiological response rates (28% and 44%, respectively) [62]. Gordon et al. found a poor correlation between microbiological and clinical outcomes in a retrospective study including 34 patients receiving tigecycline for MDR AB. 68% had a positive clinical outcome and the overall mortality was 41% [63].
Tigecycline iv 100mg, 1st dose; 50 mg / 12h (5-14 days)
Babinchak et al., 2005 (57) R, DB, C, MC (n=1.658)
Clinical responseb - c-mITTc (n=1.057) - CEd (n= 833) Microbiologic erradication Clinical success rates by baseline diagnosis Soft tissue Abcesses Ulcers Burns Clinical responseb - c-mITT (n=1.601)c - CE (n=1.382)d - ME (n=1.025)f - m-mITT (n=1.262)e MIcrobiologic erradication Clinical success rates by baseline diagnosis Complicated appendicitis Complicated cholecystitis Intra-abdominal abscess Perforation of the intestines Complicated diverticulitis Gastric and abdominal perforations Peritonitis Concomitant bacteremia
Imipenem / cilastatina 500mg/500mg / 6h (5-14 days)
Outcomes
Vancomycin iv 1gr / 12h + Aztreonam iva 2gr / 12h (14 days)
Control group
82,0% (n=800) 87,1% (n=697) 86,2% (n=513) 81,5% (n=631) 86,2% (n=513)
89,3% (n=262) 94,6 % 77,8% 72,5% 71,4% 92,0% 90,0% 80,0%
88,2% (n=263) 97,1% 78,4% 74,5% 71,9% 92,0% 88,9% 82,5%
87,3% 91,4% 82,6% 100,0%
86,3% 87,1% 80,0% 100,0%
79,8% (n=801) 86,7% (n=685) 86,1% (n=512) 80,2% (n=631) 86,1% (n=512)
81,9% (n=519) 88,6% (n=411) 86,2%
Control group
79,7% (n=538) 86,5% (n=422) 82,1%
Tigecycline group
-1,1% (-27,4 a 23,8) 2,5% (-16,0 a 19,6)
-1,1% (6,8 a 4,6) 2,5% (-6,4 a 11,4) 0,7% (-17,0 a 18,8) 2,0% (-17,0 a 21,8) 0,4% (-22,1 a 21,7) 0,0% (-20,6 a 20,6)
-2,2% (-6,2 a 1,8) -0,4% (-4,1 a 3,3) 0,0% (-4,5 a 4,4) -1,3% (-5,8 a 3,2) 0,0% (-4,5 a 4,4)
-0,9% (-7,1 a 5,2) -4,3 (-13,2 a 4,5) -2,6% (-25 a 22,4) 0% (-37,1 a 37,1)
-2,1% (-7,1 a 2,8) -2,1% (-6,8 a 2,7)
Absolute risk reduction (CI 95%)
Results
<0,001 <0,001 <0,001 <0,001 <0,001
<0,001 <0,001 <0,001
Test for Non inferiority, P value
0,2851 0,9003 1,0000 0,6167 1,0000
0,4183 0,4133
p
Tigecycline: Nausea (24,4%); Vomiting (19,2%); Diarrhea (13,8%) Control: Nausea (19%); Vomiting (14,3%); Diarrhea (13,2%)
Tigecycline: Nausea (34,5%); Vomiting (19,6%) Control: Skin and appendagesa (19,3%); Cardiovascular system (14,7%)
Complications
R:randomized; DB: double blind; C: controlled; MC: multicentric a Aztreonam could be discontinued after 48 h, according to the investigator’s clinical judgment. b Primary efficacy end point: clinical response within the CE and c-mITT populations at the test-ofcure visit (12 to 92 days after last dose). Patients were considered to have a clinical cure if the patients had resolution of signs and symptoms such that no further antibiotic therapy was required. c c-mITT: clinical modified population, including patients included the intent-to-treat population who received at least one dose of the drug and who had clinical evidence of a cSSSI by meeting the minimal disease criteria. d CE: clinically evaluable population, including patients in the c-mITT who did not have P. aeruginosa as a sole baseline isolate, received no concomitant antibiotic after their first dose of study medication, and had an assessment of cure or failure at the test-of-cure visit. e m-mITT: microbiological-modified population, includes patients in the intent-to-treat population who received at least 1 dose of study drug (mITT) and had clinical evidence of a complicated intraabdominal infection, by meeting the minimal disease criteria, and had a confirmed baseline isolate. f ME: microbiologically evaluable population.
Tigrcycline iv 100mg, 1st dose; 50 mg / 12h + Placebo iv (14 days)
Tigeciclyne group
Treatment
Phase III Clinical Trials Results in Complicated Skin and Skin Structure Infections and Complicated Intra-Abdominal Infections
Ellis-Grosse et al., 2005 (56) R, DB, C, MC (n=1.153)
Author, year
Table 1.
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Other studies describing the use of tigecycline in gram negative MDR infection, including AB, have been published describing good results in clinical response and microbiological eradication. However most of the patients received concomitant therapy with other active drugs such as imipenem or colistin [64, 65]. The interest of tigecycline derives especially from the progressive increase in the number of multidrug resistan AB isolates. When referring to microbiological efficacy it should be kept in mind that even though the aforementioned clinical studies demonstrate tigecycline’s noninferiority to active comparators, measuring eradicating rates and MICs, for AB it is not measured since it isn’t a microorganism that is implicated in these pathologies. Therefore, further conveniently designed studies are necessary to determine in which position to place tigecycline in respect to other antibiotics like colistin, whose profile has more serious adverse affects. Tigecycline is a viable alternative in treating infections involving AB in the absence of concomitant bacteraemia, however with the possibility of acquired resistance, an antibiogram should be done before administrating tigecycline [66]. CURRENT AND FUTURE DEVELOPMENTS Only two indications of tigecycline have been approved by FDA and EMEA: skin and skin structure infections and complicated intra-abdominal infections. Hunter et al. described the devices and implants of different drug combinations having anti-scarring properties [67]. Shah et al. describe the treatment of gastrointestinal infections by the oral composition of tigecycline [68]. Clinical trials in patients with pneumonia have been carried out (not published) and there are different studies still in progress in diabetic foot, catheter infection, biliar cirrhosis or human bone. Indeed, recently a patent was awarded for the treatment of infection in bone, bone marrow, joint or surrounding tissue with tigecycline (BR0413770A). Tatapudy et al. deals with the method of delivering antibiotics in respiratory disorders [69]. Also a phase 3, open-label, noncomparative study of tigecycline for the treatment of infections due to resistant gram-negative organisms such as AB has been completed, but not yet published. Testa et al. described in the patent, the treatment of osteomyelitis and septic arthritis by the combination of rifampin and tigecycline or by tigecycline alone [70]. Although in vitro studies are encouraging and a few reports and case series published show that tigecycline is likely to be useful in high risk patients vulnerable to multi-resistant pathogens, there is still little evidence about the use of tigecycline in AB infections. Despite growing clinical experience in this field, adequate trials in this type of patients are needed in order to determine efficacy and position tigecycline versus other drugs, like colistin, traditionally used in these cases; and also because acquired resistance to tigecycline is a possibility that has not to be forgotten. REFERENCES [1] [2]
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Fluit AC, Florijn A, Verhoef J, Milatovic D. Presence of tetracycline resistance determinants and susceptibility to tigecycline and minocycline. Antimicrob Agents Chemother 2005; 49: 1636-1638. Petersen PJ, Jacobus NV, Weiss WJ, Sum PE, Testa RT. In vitro and in vivo Antibacterial activities of a novel glycylcycline, the 9-t-butylglycylamido derivative of minocycline (gar-936). Antimicrob Agents Chemother 1999; 43: 738-744. Hope R, Warner M, Potz NA, Fagan EJ, James D, Livermore DM. Activity of tigecycline against ESBLproducing and AmpC-hyperproducing Enterobacteriaceae from south-east England. J Antimicrob Chemother 2006; 58: 1312-1314. Tigecycline Evaluation and Surveillance Trial (TEST). Available from: http://www.testsurveillance. com/home.php [Accessed: l Oct 2007]. Navon-Venezia S, Leavitt A, Carmeli Y. High tigecycline resistance in multidrug-resistant Acinetobacter baumannii. J Antimicrob Chemother 2007; 59: 772-774. Peleg AY, Potoski BA, Rea R, et al. Acinetobacter baumannii bloodstream infection while receiving tigecycline: a cautionary report. J Antimicrob Chemother 2007; 59: 128-131. Sader HS, Jones RN, Stilwell MG, Dowzicky MJ, Fritsche TR. Tigecycline activity tested against 26,474 bloodstream infection isolates: a collection from 6 continents. Diagn Microbiol Infect Dis 2005; 52: 181186. Yang K, Guglielmo BJ. Diagnosis and treatment of extended-spectrum and AmpC beta-lactamaseproducing organisms. Ann Pharmacother 2007; 41: 1427-1435. Postier RG, Green SL, Klein SR, et al. Results of a multicenter, randomized, open-label efficacy and safety study of two doses of tigecycline for complicated skin and skin-structure infections in hospitalized patients. Clin Ther 2004; 26: 704-714. Breedt J, Teras J, Gardovskis J, et al. Safety and efficacy of tigecycline in treatment of skin and skin structure infections: results of a double-blind phase 3 comparison study with vancomycin-aztreonam. Antimicrob Agents Chemother 2005; 49: 4658-4666. 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Cefepime and its Role in Pediatric Infections Sukhbir K. Shahid* Consultant Pediatrician and Neonatologist, Mumbai-400 077, India Abstract: Cefepime is a semi-synthetic fourth generation cephalosporin with broader Gram-positive and excellent Gram-negative bacterial coverage. Its extended anti-microbial activity and infrequent tendency to develop resistance makes it popular for treatment of infections due to multi-drug resistant organisms. It has good efficacy against -lactamase and ESBL (extended spectrum lactamase)-secreting pathogens, and it has shown great promise in the management of children with severe and nosocomial infections. It possesses superior bactericidal action compared to other cephalosporins and is a cheaper and safe alternative to the carbapenems. It is well-tolerated but needs dose adjustments in newborns, and in children with renal insufficiency. Cefepime is a valuable antibiotic but it should be used judiciously as unnecessary, improper and prolonged use may lead to emergence of cefepime-insensitive bacteria and risk of drop in the efficacy of cefepime. Various recent patents of cefepime have been launched which deal with improvements in its preparation, and with its combinations with -lactamase inhibitors and newer antibiotics such as linezolid. These developments may further augment the usefulness of cefepime in pediatric infections.
Keywords: Cefepime, pediatric infections, child, efficacy, safety, antibiotic, resistant, nosocomial, -lactam, cost-effective. INTRODUCTION Multi-drug resistant infections, whether community- or hospital-acquired, have shown an alarming rise in the last few years [1-3]. They cause considerable morbidity and mortality, and have enormous economic impact due to need for hospitalization and expensive, higher dose, parenteral antibiotics [4, 5]. Prompt initiation of appropriate empiric antibiotic therapy in these cases can cut down on fatalities and hospital costs [5-7]. However, inadequate treatment selection may lead to clinical failure and possible development of bacterial resistance [8, 9]. Cephalosporins, alone or in combination with other agents, at present are widely used for treatment of these infections. They are broad-spectrum antibiotics which are traditionally grouped by their anti-microbial activity into four generations [10]. Cefepime (BMY-28142) is a newer semi-synthetic fourth-generation cephalosporin which has excellent activity against both Gram-positive as well as Gram-negative organisms. It is a potent anti-staphylococcal and anti-pseudomonal antibiotic and acts on lactamase-producing organisms [11-14]. *Corresponding author: Tel: 0091-9869036606; E-mail: [email protected], [email protected] Atta-ur-Rahman / M. Iqbal Choudhary (Eds.) All rights reserved – © 2010 Bentham Science Publishers.
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CHEMISTRY Cephalosporins are basically derived from cephalosporin C, a natural antibiotic obtained from Sardinian sewage moulds [15]. They have a beta-lactam ring as their major structural component, and their anti-bacterial activity varies, depending on substitutions at the 3 and 7 positions of the cephalosporin nucleus [10]. The first-generation cephalosporins are active against most Gram-positive pathogens (except Listeria) and act on a limited scale against some Gram-negative organisms, while second-generation cephalosporins have increased activity against Gram-negative organisms. The third-generation cephalosporins have extended potency against Gram-negative bacteria but are generally less active against susceptible Gram-positive bacteria such as Staphylococci. Fourth-generation cephalosporins which includes cefepime has broader anti-bacterial activity including against Gram-positive organisms and the notorious enterobacteriaceae. Cefepime [Compound (1), C19H24N6O5S2) is marketed as lyophilized cefepime hydrochloride for parenteral use [16]. Chemically, cefepime hydrochloride (MAXIPIME® BristolMeyers-Squibb Company) is 1-[[(6R, 7R)-7-[2-(2-amino-4-thiazolyl)-glyoxylamido] -2carboxy-8-oxo-5-thia-1-azabi-cyclo [4.2.0] oct-2-en-3-yl] methyl]-1-methylpyrroli-dinium chloride, 72-(Z)-(O-methyloxime), monohydrochloride, monohydrate, which corres-ponds to the structural formula shown in Fig. (1) [17]: O N
H H N
N CIH
H2N
O S
H S
Cl N+
N
OH2
O HO
O
Fig. (1). Chemical structure of cefepime.
It is a white to pale yellow powder and is highly soluble in water. Injections for intramuscular and intravenous use are supplied as sterile, dry mixture of cefepime hydrochloride and L-arginine in strengths of 0.5, 1 and 2 g. It is not degradable by -lacatamases. It is essentially prepared by treating sulphate obtained in the synthesis with an ion exchange resin to release the pure zwitterion from which the desired dihydrochloride salt is prepared [16, 18-20]. Since this method is far from straightforward, research into simpler and more productive methods of preparation of purer cefepime is ongoing [21-28]. SPECTRUM OF ACTIVITY Cefepime is bactericidal with similar Gram-negative but broader Gram-positive coverage compared to ceftazidime. It can inactivate Gram-positive organisms such as Staphylococcus aureus, Streptococcus pyogenes and Streptococcus pneumoniae. Multi-drug resistant streptococcus pneumonia remains sensitive to cefepime. Escherichia coli, Haemophilus influenzae, Pseudomonas aeruginosa, Moraxella catarrhalis, Morganella morganii, Proteus mirabilis are susceptible to cefepime. Strains of Acinetobacter, Citrobacter, Enterobacter, Klebsiella, Providencia, and Serratia are also sensitive to cefepime. Indeed, virtually all of the chromosomal -lactamase and majority of ESBL (extended spectrum lactamase)-producing strains of Gram-negative bacteria are still susceptible to cefepime [17, 29-31]. The in vitro activity of cefepime against Enterobacteriaceae includes those resistant to ceftazidime, cefotaxime, cefoperazone and aminoglycosides [32]. Those organisms in the
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family Enterobacteriaceae that have demonstrated less susceptibility to cefepime include Citrobacter freundii, Enterobacter cloacae, and Serratia marcescens. Cefepime has shown very limited activity against Enterococcus faecalis, P. cepacia, P. fluorescens, Xanthomonas maltophilia, Listeria monocytogenes, Bacteroides fragilis [33]. It does not act on methicillin-resistant Staphylococcus aureus or enterococcus [34, 35]. Most strains of Clostridium difficile remain resistant to cefepime. Being a weak inducer of lactamase, emergence of bacterial resistance with cefepime is infrequent [36-38]. MECHANISM OF ACTION Cefepime acts by inhibition of bacterial cell wall synthesis. Cefepime zwitterion has a net neutral charge that allows it to penetrate the outer membrane of Gram-negative bacteria faster than third generation cephalosporins. This penetration is enhanced by its N-methylpyrrolidine moiety. It is unaltered by chromosomal -lactamases because of lower affinity of the enzymes for cefepime when compared with third generation cephalosporins. It has only mild -lactamase induction capacity and has higher affinity for penicillin-binding proteins [39, 40]. PHARMACOKINETICS Cefepime displays a linear dose-dependent pharmacokinetic profile in children. It has a wide distribution in body tissues and fluids. The serum cefepime levels are well above the mean inhibitory concentration level for most of the time and its concentration in sputum, blister fluid, peritoneal fluid and bile is good. It does cross the inflamed blood brain barrier. Clinical trial carried out on 88 patients aged 2 months to 16 years revealed that after a single intravenous dosing, the volume of distribution at steady state of cefepime was 0.31 liter/kg (0.33 to 0.40) and total body clearance 3.1 ml/min/kg (1.43 to 4.01). It has an elimination half-life of about 2 hours and is primarily excreted unchanged via the kidneys with urinary recovery of around 72%. Bio-availability after intramuscular dose is 82.3% [41-44]. Elimination of cefepime is hampered in newborns due to their immature renal function. Hence, dose or frequency adjustment is required in newborns [45]. Cefepime can also be administered as continuous infusion with similar efficacy and the advantage of total reduced dose [46, 47]. DOSING Cefepime is available as sterile lyophilized powder with L-arginine (MAXIPIME® , Bristol-Meyers-Squibb Company). L-arginine is added to control the pH of the constituted solution at 4.0-6.0. It is used at doses of 50 mg/kg/dose 12 or 8 hourly in children (maximum of 6 g/day). In newborns, doses of 30 mg/kg/dose 12 hourly or 50 mg/kg every 24 hours suffice. At these doses, effective and non-toxic serum cefepime levels are reached [45]. Dose adjustment is required in renal insufficiency when the creatinine clearance is less than 60 ml/min [29]. However, liver-impaired individuals need no alteration in cefepime dosages. CLINICAL USES Confirmed Indications of Cefepime Cefepime has been approved for treatment of pneumonia, urinary tract infections including pyelonephritis, uncomplicated skin and soft tissue infections, intra-abdominal
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infections and as empiric therapy for febrile neutropenic patients in children above 2 months of age and adults. Recent studies have revealed the safety of cefepime in infants less than 2 months of age as well [45, 48]. The efficacy and safety of cefepime in children has been demonstrated in numerous comparative and non-comparative trials. Its twice-a-day dosage schedule and enhanced activity against enterobacteriaceae and Gram-positive organisms puts it into an advantageous position. It is a front line agent when infection with Enterobacter is known or suspected. It has low cross-resistance with other cephalosporins, and comparable clinical and slightly better microbiological cure compared to ceftazidime. It has low propensity for selection of resistant mutants and low potential for induction of bacterial resistance. Cefepime is as effective and safe as other cephalosporins in lower respiratory tract infections and serious urinary tract infections in children [49, 50]. Cefepime, alone or in combination, is useful in the treatment of febrile neutropenia and this treatment is less costly compared to monotherapy with carbapenem [51, 52]. Cefepime is safe and therapeutically equivalent to cefotaxime and ceftriaxone for management of bacterial meningitis in children; however, at present, insufficient clinical data exists to support the use of cefepime in treatment of Haemophilus influenzae type b infection in children. An alternative agent with demonstrated clinical efficacy should be used in suspected or documented meningitis due to this pathogen [53]. It is a good alternative to ceftazidime for nosocomial infections, specially hospital-acquired pneumonia. Limited Studies/Reports on Cefepime Recent studies have also revealed the efficacy and safety of cefepime in late-onset VAP in infants, in bacterial exacerbations of cystic fibrosis and for prophylaxis in neurosurgical patients with EVD in situ [48, 54, 55]. Cefepime has successfully treated acalculous Salmonella paratyphi B cholecystitis in a child with leukemia [56]. Cefepime was also used to treat Enterobacter cloacae ventriculitis with good outcome [57]. Cefepime therapy also tends to minimize on hospital days, blood component requirements and such supportive therapy [48]. Hence, it is a cost-effective modality for management of notorious infections. Comparison of Cefepime Microbiological Activity with other Cephalosporins In the study by Kurchavov et al. 92% of the bacterial isolates were susceptible to cefepime, whereas only 70-75% of them were sensitive to the other cephalosporins [58]. Cefepime seems to have better microbiological clearance compared to ceftazidime, specially with E.coli and polymicrobial infection [48]. Cefepime Monotherapy vs. Combination with other Antibiotic Combination of cefepime with other antibiotic is supposed to enhance its antibacterial activity. This synergy is however poorly supported. In a recent meta-analysis, no benefit was found for cefepime combination therapy over monotherapy in terms of mortality or prevention of resistant germs. In 74 mechanically-ventilated patients, cefepime monotherapy was compared with cefepime in combination of either amikacin or levofloxacin. No clinical or biological benefit was elicited with combination therapy when compared with cefepime monotherapy [59].
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ADVERSE EFFECTS Cefepime has a tolerability profile similar to that of other parenteral cephalosporins. Cefepime is relatively safe drug with no nephrotoxicity. Commonly reported side effects are related to skin and gastrointestinal system. Rash, phlebitis and pruritus have been noticed in minority of cases. Some patients have complained of loose motions, nausea and vomiting with cefepime use. Headache, transient leucopenia, neutropenia, agranulocytosis and thrombocytosis have been noticeable in few patients on cefepime. In renal-impaired children, risk of encephalopathy, seizures and myoclonus increases even at adjusted dosages. Minor changes in liver enzymes, renal function, serum phosphorus level and PT/PTT may be seen. Cefepime is contraindicated in children with known hypersensitivity to cephalosporins. Immediate IgE type of hypersensitivity reaction may be seen in them. On the whole, however, cefepime is well-tolerated by all pediatric age groups and is equally safe when compared to the other cephalosporins [29]. There was a suspicion in between that cefepime may be linked with higher risk of death. A meta-analysis performed by Yahav D et al. in 2007 revealed that cefepime use had a higher all-cause mortality compared with the other -lactam antibiotics [60]. However, US FDA reviewed this study and conducted additional analyses based on 88 trials and patientlevel data and determined that available data did not indicate a higher rate of death in cefepime-treated patients. Hence cefepime remains a safe and appropriate therapy for its approved indications [61]. CURRENT AND FUTURE DEVELOPMENTS Various new roles of cefepime are being considered. Addition of -lactamase inhibitor, such as clavulanate, tazobactum or sulbactum to cefepime tends to widen its anti-bacterial spectra and makes it an attractive alternative to carbapenems against ESBL-producers [62,63]. Cefepime can also be combined with newer metallo--lactamase inhibitor for enhanced antimicrobial coverage [64]. A formulation comprising cefepime, tazobactum and linezolid has been suggested and tried as empiric therapy in critical infections due to ESBLproducing organisms and MRSA, VRSA, E. faecalis and Bacteroides [65]. In this formulation, cefepime is the active ingredient and tazobactum is an enzyme inhibitor which acts to enhance cefepime’s anti-bacterial activity by inhibiting the beta-lactamases. Linezolid, an oxazolidinone, is a new class of antibacterial which has good activity against anaerobes, MRSA and VRSA. This formulation may prove useful for nosocomial infections specially in units where antibiotic resistance is an increasing problem. Though cefepime monotherapy is beneficial in many infections at present, the increased emergence and spread of multi-drug resistant pathogens coupled with relative dearth of new antibiotic development makes the situation precarious for treatment of critical and drugresistant infections. Hence, continuing focuses on employing rational antibiotic usage, on appropriate infection control measures and on research targeted at development of inhibitors with broader enzyme inhibitory activity and strategies to combat bacterial resistance are simultaneously warranted. To summarize, cefepime with/without inhibitors offers unique advantages for antimicrobial therapy in children. It is safe and is a useful alternative to ceftazidime and other broad spectrum cephalosporins for serious and nosocomial infections. But emphasis on rational usage of this antibiotic is required in order to preserve its usefulness for several more years.
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Cefepime: pharmacokinetics and clinical response in patients with cystic fibrosis. Am J Dis Child 1992; 146: 797-802. Blumer JL, Reed MD, Knupp C. Review of the pharmacokinetics of cefepime in children. Pediatr Infect Dis J 2001; 20 (3): 337-342. Barbhaiya RH, Forgue ST, Gleason CR, et al. Safety, tolerance, and pharmacokinetic evaluation of cefepime after administration of single intravenous doses. Antimicrob Agents Chemother 1990; 34: 118122. Rhoney DH, Tam VH, Parker D, McKinnon PS, Coplin WM. Disposition of cefepime in the central nervous system of patients with external ventricular drains. Pharmacotherapy 2003; 23(3): 310-314. Capparelli E, Hochwald C, Rasmussen M, Parham A, Bradley J, Moya F. Population pharmacokinetics of cefepime in the neonate. Antimicrob Agents Chemother 2005; 49 (7): 2760-2766. Sprauten PF, Beringer PM, Stan GL, Synold TW, Gill MA. Stability and antibacterial activity of cefepime during continuous infusion. Antimicrob Agents Chemother 2003; 47 (6): 1991-1994. Emmanuel B, Dominique B, Frederic D, et al. Steady-state plasma and intrapulmonary concentrations of cefepime administered in continuous infusion in critically ill patients with severe nosocomial pneumonia. Crit Care Med 2003; 31(8): 2102-2106. Shahid SK. Efficacy and safety of cefepime in late-onset ventilator-associated pneumonia in infants: a pilot randomized controlled study. Ann Trop Med Parasitol 2008; 102 (1): 63-71. Bradley JS, Antonio A. Empiric use of cefepime in the treatment of lower respiratory infections in children. Pediatr Infect Dis J 2001; 20 (3): 343-349. Antonio A, Bradley JS. Empiric use of cefepime in the treatment of serious urinary tract infections in children. Pediatr Infect Dis J 2001; 20 (3): 350-355. Corapçioglu F, Sarper N. Cefepime versus ceftazidime + amikacin as empirical therapy for febrile neutropenia in children with cancer: a prospective randomized trial of the treatment efficacy and cost. Pediatr Hematol Oncol 2005; 22 (1): 59-70. Agaoglu L, Devecioglu O, Anak S. Cost-effectiveness of cefepime + netilmicin or ceftazidime + amikacin or meropenem monotherapy in febrile neutropenic children with malignancy in Turkey. J Chemother 2001; 13 (3): 281-287. Xavier SL, Miguel O. Cefepime in the empiric treatment of meningitis in children. Pediatr Infect Dis J 2001; 20 (3): 356-361. Robinson CA, Kuhn RJ, Craigmyle LJ, Anstead MI, Kanga JF. Susceptibility of Pseudomonas aeruginosa to cefepime versus ceftazidime in patients with cystic fibrosis. Pharmacotherapy 2001; 21(11):1320-1324. Wong GK, Poon WS, Lyon D, Wai S. Cefepime vs. ampicillin/sulbactam and aztreonam as antibiotic prophylaxis in neurosurgical patients with external ventricular drain: result of a prospective randomized controlled clinical trial. J Clin Pharm Ther 2006; 31(3):231-235. Erduran E, Arslan MK, Dereci S. Acalculous cholecystitis caused by Salmonella paratyphi B infection in a child with acute pre-B-cell lymphoblastic leukemia. Pediatr Hematol Oncol 1999; 16 (5): 473-476. Barnes BJ, Wiederhold NP, Micek ST, Polish LB, Ritchie DJ. Enterobacter cloacae ventriculitis successfully treated with cefepime and gentamicin: case report and review of the literature. Pharmacotherapy 2003; 23(4): 537-542. Kurchavov VA, Beloborodova NV, Biriukov AV, Vostrikova TIu, Rogatina EL, Krutskikh EN. The comparative activity of cefepime and other current antibiotics against microorganisms isolated from patients in pediatric intensive therapy units. Antibiot Khimioter 1999; 44 (11): 23-30. Damas P, Garweg C, Monchi M, et al. Combination therapy versus monotherapy: a randomised pilot study on the evolution of inflammatory parameters after ventilator associated pneumonia. Crit Care 2006; 10 (2): R52. Yahav D, Paul M., Fraser A., Sarid N., Leibovici l. Efficacy and safety of cefepime: a systematic review and metanalysis. Lancet Infect Dis. 2007; 7(5): 338-348 US FDA. Information for Health Care Professionals: Cefepime (marketed as maxipime). FDA Alert. 17/6/09. Available from: http://www.fda.gov/drugs/drug safety/postmarket drug safety information for
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Antibacterial Properties of Organosulfur AntiInfectives: A Review of Patent Literature 1999-2009 Monika I. Konaklieva*,1 and Balbina J. Plotkin2 1
2
Department of Chemistry, American University, 4400 Massachusetts Avenue, NW, Washington, DC 20016-8014, USA
Department of Microbiology and Immunology, CCOM, Midwestern University, 555 31 St., Downers Grove, IL 60515, USA Abstract: A wide variety of organosulfur compounds that exhibit antibacterial properties are being patented. Functionally, organosulfur groups can act as metal chelators, powerful nucleophiles, or electrophiles depending on the local reaction environment. In this review of patent literature from 1999-2009, the reliance of these compounds on the reaction of the sulfur moiety with its biological target(s) will be discussed with regards to activity, specificity, and antimicrobial spectrum.
INTRODUCTION The utility of sulfur-containing compounds functioning as antimicrobials has been the focus of several patent applications from the years 1999-2009. The principle focus of the majority of these patents is the utility of a component of garlic (Allium sativum), allicin, and related compounds as antimicrobials. There are a variety of patents proposing the antiinfective functionality of other thio-containing compounds such as thioethers, as well as the bioactivity of methylthiolated -lactams having unusual modes of action, e.g., interacting with cellular thiols and thiol-containing -lactams as inhibitors of both serine- and metallo -lactamase. Garlic has been known for centuries to have antimicrobial properties. The biologically active components of garlic appear to be mainly those compounds containing sulfur. The principal antimicrobial component, allicin, is a diallyl thiosulfinate 1 (Fig. 1) [1, 2], which is also responsible for garlic’s aroma. Intact garlic cloves contain the precursor of allicin, i.e., alliin ((+) (S)-allyl-L-cysteine sulfoxide 2) (Fig. 1) which is converted to allicin upon the crushing or cutting of the garlic clove. The enzyme responsible for converting alliin to allicin is a C-S lyase (also termed allicin lyase or alliinase), which is kept compartmentally segregated from alliin in the garlic clove. Therefore, only when the clove is damaged by cutting or crushing are the alliinase and alliin brought together and allicin formed. Allicin is a relatively unstable compound, having a half-life of 16 hours at 23o C. The mechanism of action of allicin was initially proposed to be due to its reaction with cysteine [1] to form disulfides and react with free thiol groups essential for bacterial proliferation. Subsequently, allicin was shown to be a very potent inhibitor of thiol-containing enzymes [3] which is supported by experimental evidence and is now the commonly accepted *Corresponding author: Tel: (202) 885-1777; Fax: (202) 885-1752; E-mail: [email protected] Atta-ur-Rahman / M. Iqbal Choudhary (Eds.) All rights reserved – © 2010 Bentham Science Publishers.
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mechanism of action [4]. Allicin upon isolation is spontaneously degraded to two major components: Ajoene (4,5,9-trithiadodeca-1,6,11-triene-9-oxide) 3 (Fig. 1) and diallyltrisulfide (DATS) 4 (Fig. 1). Extensive research has shown that these two allicin derivatives also have biological activity [5]. DATS has been marketed for the treatment of human bacterial, fungal and parasitic infections in China since 1981 under the name DASUANSU. However, despite the potential utility of these compounds as antimicrobial agents [6], a cost effective, eco-friendly straight forward synthesis has been difficult to achieve. Such an approach for the synthesis of diorganotrisulfides and particularly DATS is presented in one of the patents [7]. The synthesis of diorganosulfides is achieved by reacting of an organic halide (alkyl, allyl or benzyl) with tin (II) chloride, catalytic copper (II) halide and elemental -rhombic sulfur in a mixture of organic solvents. This one-pot synthetic approach allows for preparation of DATS in two steps with 50% yield of the pure product. O S
O
NH2
S
S 1
COOH 2
O S
S
S S 4
3 S
S S 5
S
S 6
Fig. (1). Sulfur-containing compounds from garlic.
Allicin, cysteine and related organosulfur compounds from garlic such as diallyl disulfide 5 and diallyltrisulfide 6 (Fig. 1) can also act as immuno modulators to prevent/treat disease occurrence, in addition to having a variety of intrinsic antimicrobial activities. A method of non-enzymatic formation of allicin utilizing the localized generation of H2O2 by immune system cells capable of respiratory burst, e.g. neutrophiles and macrophages, is described in another invention [8]. The intent of this method is to simultaneously increase the antimicrobial effect of allicin while reducing the cytotoxicity to the host. It has been experimentally determined that the rate of allicin production from H2O2 and an alliumrelated organosulfur compound is a non-linear function of the local H2O2 concentration; there is additional enhanced localization of allicin production from the allium-related organosulfur compound. The authors also show that allicin can be enzymatically formed by cytochrome P-450 in the liver, which provides a more uniform in situ concentration over time than the more conventional approach of using the enzyme aliinase to form allicin [8]. Different ways to formulate allium-related compounds with advantages over existing dietary supplements and medicinal product are also presented [8]. Corollary uses of garlic and its components as antibacterials have been the addition of garlic extract, allicin and related sulfur-containing compounds to blood products [9], e.g. whole blood, red blood cells, white blood cells, platelets, and serum, to inhibit selected microorganisms, and thus increase their current shelf life (platelets < 5 days). This addition of the garlic derived compounds extended the platelet storage life by at least 20%. Thus, this invention allows for increasing the time for storage of blood products [9].
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Preparations of novel sulfenate esters and thiosulfinate esters that induce expression of metabolic enzymes, particularly those of Phase II enzymes, such as glutathione-Stransferase, DT-diaphorase and Ferritin H have been also reported in the patent literature. Phase II enzymes are responsible for the detoxification of reactive electrophilic and nucleophilic metabolites [10]. For example, glutathione S-transferases catalyze the reaction of glutathione, a tripeptide, with electrophiles, such as epoxides, alkyl and aryl halides and , -unsaturated ketones, thus making them less toxic and carcinogenic and more easily excreted from the body. Sulfenate esters and five membered ring thiosulfinate esters may be used in humans who have been or may be exposed to certain cancer causing compounds. In addition, sulfenate esters and five membered ring thiosulfinate esters compounds have been shown to be effective in displacement of a zinc ion from retroviral zinc finger nucleocapsid proteins of the HIV, thus inhibiting the HIV replication [10]. O R
O
S O
H3C
O
S
7
R = (4-OMe)phenyl, (4-tBu)phenyl, cyclohexenyl
S O
H3CO
O
O S O O
H3CO 8 O
9
10
O
Fig. (2). Sulfenate esters having activity against HIV.
The zinc finger containing nucleocapsid proteins of retroviruses appears to be relatively stable, thus it is a particularly attractive target for drug action since HIV-1 exhibits a high mutation rate to drug resistance [11Ref. Cohen, 1997, patent December 10, 1999]. In addition to the described disulfides [12 Ref. Rice and Turpin, 1996, patent December 10, 1999] already reported in the literature, the authors of this patent describe the utility of sulfur-containing esters 7- 10 (Fig. 2) as novel compounds for targeting HIV nucleocapsid containing the zinc finger [10]. Synthetic routes for the preparation of these sulfanate and thiosulfenate esters have been described. Microbial colonization of surfaces, such as catheters, is associated with adverse patient outcomes. Thioethers (poly- or oligo-thioethers) 11 (Fig. 3) have been utilized in coating surfaces leading to a reduced protein and bacterial adhesion to these surfaces [13]. The thioether block in the multiblock polymers of propylene sulfide and ethylene glycol, e.g., poly(propylene sulfide)-poly(ethylene glycol) copolymers, may serve several functions. In certain of these polymers the blocks are designed to be very hydrophobic, therefore allowing strong adsorption to hydrophobic surfaces with high stability in polar solvents (water or alcohols). Poly- or oligo-thioethers may include multiple sulfur atoms along their backbone chains, which are typically hydrophobic, in contrast to homologous polyethers. The coatings with these thioethers typically reduce adsorption, or cell adhesion, or both on a surface. The surface coatings may also include a biologically active moiety, such as an organic compound, including a given antibiotic, a nucleic acid, a protein, an enzyme substrate, an enzyme inhibitor, or an antibody, which can aid in the effect of reducing in protein adsorption or bacterial cell adhesion relative to an uncoated surface.
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p
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O O
PEGn
PEGp
O PPSm
O
O n
S S
m
S
11
Fig. (3). Thioether polymers used for coating surfaces.
N-Sulfenated monocyclic -lactams 12 (Fig. 4) are another class of sulfur-containing antibacterial compounds recently discovered which [14], have anti-methicillin resistant Staphylococcus aureus (MRSA) activity. Despite the presence of a -lactam ring these compounds, unlike the rest of the -lactam-containing class of antibiotics, do not act as cell wall biosynthesis inhibitors. It is believed that these -lactams have an unusual mode of action for compounds having a -lactam ring, where the N-S functionality may interact with cellular thiols in a same fashion as disulfide [15], i.e., intracellular thiol attacks the sulfur atom to form a mixed disulfide which in turn inhibits bacterial growth. These thiolated lactams demonstrate a narrow range of antibacterial activity – mainly against the staphylococci, including MRSA, S. epidermis and Bacillus anthracis, the causative agent of anthrax [16].
R1
O
R2 N
O
S
12
R1 = Me, Ac, SO2Me, SO2Ph CH3
R2 =Ph,
Cl F
O NO2 O
N
SR R = different alkyl groups
R2 13
R1
Fig. (4). N-thiolated -lactams and N-thiolated oxazolidinones as antibacterial agents for MRSA.
Testing of N-alkylthio -lactams 12 [16-19] and N-alkylthio-2-oxazolidinones 13 [20] (Fig.4) demonstrated that they had narrow spectrum bacteriostatic activity against MRSA and B. anthracis [21]. The structural requirements of N-alkylthio -lactams and N-alkylthio2-oxazolidinones substantially differ from the structure-activity profiles of -lactams and 2oxazolidinones in clinical usage. For these compounds the presence of a thioalkyl moiety has been demonstrated to be essential for antibacterial activity. Of these compounds, antibacterial activity correlates with those compounds with highly lipophilic residues on the heterocyclic ring and sulfur side chain. Recently, the mode of action of the N-methylthio lactams have been determined; they act as inhibitors of type II fatty acid biosynthesis in S.
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aureus through an initial transfer of the N-alkylthio moiety onto a coenzyme-A (CoASH) [22], which is present in large quantities in the cytoplasm and comprises the redox buffer system for these bacteria. It is postulated that sulfenylation of coenzyme A produces an alkyl-CoA disulfide (CoASSR) responsible for inhibiting lipid biosynthesis (Scheme 1). R1
O
R2
R1
CoASH N O
S
O
R2 + CoASSR NH
R
O
Scheme 1. Sulfenylation of coenzyme A by N-thiolated -lactams.
There has been considerable interest in development of -ketoacyl-ACP synthetase III (FabH) inhibitors as antibacterial agents [23, 24]. Fatty acid biosynthesis is an essential universal process for the generation and maintenance of cell membranes. It is carried out by two related but distinct fatty acid synthetase (FAS) systems that are present in prokaryotes and mammals. One system present exclusively in mammals utilizes a large multifunctional protein, with one or two polypeptides (type I FAS). While FAS system (type II FAS), which is present in prokaryotes, protozoa and plants, is composed of discrete monofunctional enzymes, with activities that correspond to the domains of the type I single-chain FAS [2527]. The key role of the FabH in type II FAS, and the unique differences between bacterial and mammalian FAS pathways make this enzyme an excellent target for selective inhibition of bacterial growth. FabHs from different bacterial species display different substrate specificities for the acyl-CoA primer, e.g. the Escherichia coli enzyme is specific for acetylCoA while Mycobacterium tuberculosis (Mtb) enzyme is specific for lauroyl-CoA. Recent studies on the effects of mixed alkyl-CoA disulfides (CoASSR) showed that they covalently modify the fatty acid biosynthesis protein, FabH in Mtb and that these inhibitory effects are highly dependent on the nature of the alkyl side chain R [28]. The most active inhibitor of Mtb and E. coli FabH enzymes is disulfide 14 (Fig. 5); these compounds exert their activity by “capping” an active-site cysteinethiol by sylfenylation, thus destroying the catalytic function of the enzyme. O O
S
S
OH
14
Fig. (5). FabH inhibitor disulfide 14.
X-ray crystallography of E. coli FabH shows the methylthio group from methyl-CoA disulfide covalently bound to the active-site cysteine (112Cys). Further, the catalytic activity inactivated by the methylthilation of FabH could not be regenerated by dialysis; however, treatment with dithiothreitol (DTT) quickly restored full catalytic function. This suggests that formation of the cysteine-alkylated FabH is irreversible under buffered aqueous conditions, but is reversible when a thiol is added, by restoring the catalytic form of the FabH enzyme via thiol-disulfide exchange. Unfortunately, this activity did not translate to in situ activity. When Staphyloccocus aureus was incubated with mixed disulfides CoASSR, bacterial growth was unaffected. This deficiency in activity is attributed to the lack of cytoplasmic
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target acquisition due to the multiply charged CoA moiety’s inability to successfully traverse the cell membrane. This finding led to the realization N-thiolated -lactams and Nthiolated oxazolidinones have the necessary cell permeability and sulfenation capabilities, to generate CoASSR adducts intracellularly. Since the -lactam ring alone does not appear to have roles beyond that of a carrier of the sulfur moiety across the bacterial cell membrane and being able to serve as a good leaving group during nucleophilic attack on sulfur, synthesis of lipophilic disulfides have been envisioned (Scheme 1) as a new means of delivering electrophilic sulfur species effectively into the bacterial cell [29]. In nature, there are disulfides and trisulfides compounds that have anti-infective properties [30] (Fig. 1). In many cases the biological targets and mode of action of these compounds are not known; however, there are distinct similarities between N-thiolated lactams/N-thiolated oxazolidinones and allicin. Both the lactams and allicin strongly inhibit fatty acid biosynthesis [31-34], and partially inhibit protein and nucleic acid biosynthesis [35]. In mammalian cells the mixed alkyl disulfides function as inhibitors of redox buffers, in this case as a means to restore normal cellular function. As described in two recent patent applications, their functionality is attributed to their high reactivity toward thioredoxin [36, 37]. Interestingly, both N-thiolated -lactams/N-thiolated oxazolidinones and allicin exhibit their highest level of activity against bacteria, e.g. S. aureus and B. anthracis that express low levels of gluthatione and relatively high levels of CoA. Allicin is reported to react with sulfhydryl residues of proteins including RNA polymerase, thioredoxin reductase, and alcohol dehydrogenase, and reversibly inhibit acetyl-CoA synthetases [38, 39]. Of the prepared unsymmetrical aryl methyl disulfides [40] (Fig. 6), p-nitrophenyl analogue 22 showed the strongest in vitro activity against S. aureus, with zones of inhibition comparable to that of some N-thiolated -lactams. Changing the position of the nitro group on the phenyl ring to ortho, (24, Fig. 7) diminishes the activity somewhat, while the disulfide with meta-nitro group (23, Fig. 7), has similar activity to the p-nitro isomer. SSCH3 F
SSCH3
SSCH3
SSCH3 OH
H3C
15
16
F
17 OCH3CH3
CH3 SSCH3
SSCH3 N
AcHN
19
18
H2N 20
SSCH3
21
O2N
SSCH3
22
Fig. (6). Mixed methyl-aryl disulfides.
The greater bioactivity of the nitrophenyl disulfides against S. aureus and B. anthracis, as compared to other aryl-substituted analogues, suggests that the nucleophilic scission of the S-S bond is enhanced by strong electron deficiency on the S-centers. Additionally, branching of the alkyl chain of the alkyl-aryl disulfides appears more important for the bioactivity of these compounds than the length of the alkyl chain, i.e. the isopropyl 24c, 22c and sec-butyl 22d (Fig. 7). Furthermore, these mixed alkyl-aryl disulfides, as well as N-
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thiolated lactams (Fig. 2), have been proven to be inhibitors of purified E. coli FabH enzyme, in the absence of coenzyme A. SSR
O2N
22 a-f
SSR
SSR
23 a-f
NO2
24 a-f
NO2 a. R = methyl, b. R = ethyl. c. R = isopropyl, d. R = sec-butyl. e. R = n-propyl. f. R = n-butyl
Fig. (7). The most active alkyl-aryl disulfides - nitrophenyl disulfides.
These findings provide an indication that both mixed alkyl-aryl disulfides and Nthiolated -lactams directly inhibit the FabH enzymes in vitro. However, once inside bacterial cells these two groups of compounds appear to indirectly inhibit the FabH catalysis by inhibiting the CoA mixed disulfides, which in turn impairs the FabH function. Heterosubstituted N-thiolated -lactams [41] (Fig. 8) and S-heterosubstituted disulfides of type RRXSSXR (X= O, N, or S) [42] (Fig. 9) have also shown antibacterial properties against bacterial infections caused by the staphylococci, including MRSA. The activity (μg/ml) of many of the S-heterosubstituted disulfides against MRSA are at least equal to, and in certain cases more than that of Penicillin G. The mechanism of action of Sheterosubstituted disulfides is thought to be similar to that of previously reported Nthiolated -lactams (N-alkylthio -lactams), based on structural and chemical similarities. N-thiolated -lactams (N-alkylthio -lactams) have been shown to create alkyl-CoA disulfides through a thiol-disulfide exchange within the cytoplasm ultimately inhibiting type Il fatty acid synthesis. Br
Br
Cl O
O
Me S R
S R
N
N Me
O
N
RS
RS
O
S
O
O
Me
N O
S
O
Me
S R
N
N S
O
Me
O
O
O
Cl 25
26
Br
27
Fig. (8). Heterosubstituted N-thiolated -lactams.
These compounds may also be characterized as essentially "stable" analogues of natural disulfides obtained from garlic and onions, which are fatty acid biosynthesis inhibitors. The potential utility of these compounds is high given their mode of action, i.e., inhibition of
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fatty acid biosynthesis. These compounds have reported activity against Francisella tularensis, Staphylococcus aureus including MRSA, B. anthracis and fungi. Ph Me R N
S
Ph
O
S
N R Me
S
Ph
O R Et
S
O
Me
30
S
Me S N
Et
Et Me
Ph
Ph
28
R Me
Et
Ph
Ph
N S Me
S
Ph
29
S
S Me
S
O R Et Me
31
Et N H
S
S
H N
Et Ph
32
O
Me
S
S
33
O
Et Ph
Fig. (9). Representatives of antibacterial S-heterosubstituted disulfides.
The most common form of bacterial resistance to the -lactam class of antibiotics is the production of one or more types of -lactamases. More than 300 -lactamases have been characterized and are divided into four classes A-D. Classes A, C, and D are serine enzymes, while class B -lactamases are zinc metalloproteases. Although strains expressing class A are currently the most clinically relevant, increasing numbers of bacterial strains are developing resistance by production of class B, C, and D enzymes. Recently, new inhibitors that inactivate both metallo- and serine -lactamases [43] have been described. These inhibitors 33 (Fig. 10) are penicillin-based to ensure recognition by both classes of lactamases, but they also incorporate a sulfur-containing ligand, i.e., thiol to inhibit the metallo--lactamases. These new compounds display in vitro synergy with piperacillin against -lactamase-producing bacteria, including Pseudomonas aeruginosa. ROCHN b
H S 5 1 6 2 7 N4 3
O CO2H penicillins SH H (O)n S
HS
N
N
O 33a
H (O)n S
CO2H
O 33b
CO2H
n = 0, 2
Fig. (10). Penicillin-derived inhibitors of both serine- and metallo--lactamases.
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In conclusion, organic compounds that contain sulfur cover an extraordinary range of chemical structures and reactivities. For millennia, sulfur-containing natural product extracts from the edible representatives of Liliaceae, including garlic, which contains the thiosulfinate allicin, have been used in folk medicine as anti-infectives. The likely mode of action of allicin is reaction of the thiosulfinate with a microbial thiol to produce mixed disulfide. In general, except for their sometimes pungent odor and chemical instability, most of these antimicrobially active organosulfur compounds display few side effects. FUTURE DIRECTIONS There now exists for virtually every class of human infection representative examples of organosulfur anti-infective agents. Given this era of drug resistance, in which there is an ever increasing demand for new antibiotics, organosulfur compounds may provide alternate avenues for new therapies. It appears that many of the compounds being developed are based on the thiol, sulfide and thiosulfinate containing compounds varying from small molecules, such as -lactams, to polymers. Most of the sulfur-containing compounds where the biological activity is due to participation of sulfur will continue to be designed due to abundance of thiol-bearing enzymes, e.g., thioredoxin or glutaredoxin, in a large majority of life forms. REFERENCES [1] [2] [3] [4] [5]
[6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19]
Cavallito CJ, Bailey JH. Allicin, the antibacterial principle of Allium sativum. I. Isolation, physical properties and antibacterial action. J Am Chem Soc 1944; 66:1950-1951. Cavallito CJ, Bailey JH. Allicin, the antibacterial principle of Allium sativum. II. Determination of the chemical structure. J Am Chem Soc 1944; 66:1952-1954. Wills ED. Enzyme inhibition by allicin, the active principle of garlic. Biochem J 1956; 63:514-520. Rabinkov A, Miron T, Konstantinovski L, Wilchek M, Mirelman D, Weiner L. The mode of action of allicin. Trapping of radicals and interaction with thiol containing proteins. Biochim Biophys Acta 1998; 1379: 233-244. Takada N, Matsuda T, Otoshi T, et al. Enhancement by organosulfur compounds from garlic and onions of diethylnitrosamine-induced glutathione S-transferase positive foci in the rat liver. Cancer Res 1994; 54: 2895-2899. Lun ZR, Burri C, Menziger M, Kaminsky R. Antiparasitic activity of diallyl trisulfide (Dasuansu) on human and animal pathogenic protozoa (Trypanosoma sp., Entamoeba histolytica and Giardia lamblia) in vitro. Ann Soc Belg Med Trop 1994; 74: 51-59. Pradipta, S., Sujit, R.: US20020198410 (2002). Ott, D. M.: US20040235946 (2004). Goodrich, L. L.: US20030077264 (2003). Welker, M. E., Torti, S. V., Torti, F. M., Townsend, A. J., Pietsch, E., Hurley, A. L.: US:20010605 (2001). Cohen J. The media's love affair with AIDS research: hope vs. hype. Science 1997; 275: 298-299. Turpin JA, Terpening SJ, Schaeffer CA, et al. Inhibitors of human immunodeficiency virus type 1 zinc fingers prevent normal processing of gag precursors and result in the release of noninfectious virus particles. J Virol 1996; 70: 6180-6189. Hubbel, J.A., Bearinger, J.P., Napoli, A., Textor, M., Tirelli, N.: US20030133963 (2003). Turos, E.: US20030191108 (2003). Long TE, Turos E. N-thiolated -lactams. Curr Med Chem – Anti-Infec Agents 2002; 1:251-268. Turos E, Long TE, Heldreth B, et al. N-Thiolated -lactams: a new family of anti-Bacillus agents. Bioorg Med Chem Lett 2006; 16: 2084-2090. Turos E, Konaklieva MI, Ren RX-F, et al. N-thiolated bicyclic and monocyclic -lactams. Tetrahedron 2000; 56: 5571-5578. Long TE, Turos E, Konaklieva MI, et al. Effect of aryl ring fluorination on the antibacterial properties of C4 aryl-substituted N-methylthio -lactams. Bioorg Med Chem 2003; 11: 1859-1863. Turos E, Long TE, Konaklieva MI, et al. N-Thiolated -Lactams: novel antibacterial agents for methicillin-Resistant Staphylococcus aureus. Bioorg Med Chem Lett 2002; 12: 2229-2231.
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[21] [22]
[23] [24] [25] [26] [27] [28]
[29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43]
Konaklieva and Plotkin
Mishra RK, Revell KD, Coates CM, Turos E, Dickey S, Lim DV. N-thiolated 2-oxazolidinones: a new family of antibacterial agents for methicillin-resistant Staphylococcus aureus and Bacillus anthracis. Bioorg Med Chem Lett 2006; 16: 2081-2083. Turos, E., Mishra, R.K.: US2006252809 (2006). Revell KD, Heldreth B, Long TE, Jang S, Turos E. N-thiolated -lactams: studies on the mode of action and identification of a primary cellular target in Staphylococcus aureus. Bioorg Med Chem 2007; 15: 2453-2467. Marrakchi H, Zhang Y-M, Rock CO. Mechanistic diversity and regulation of Type II fatty acid synthesis. Biochem Soc Trans 2002; 30: 1050-1055. Campbell JW, Cronan JE. Bacterial fatty acid biosynthesis: targets for antibacterial drug discovery. Annu Rev Microbiol 2001; 55: 305-332. Clough RC, Matthis M, Barnum SR, Jaworski JG. Purification and characterization of 3-ketoacyl-acyl carrier protein synthase III from spinach. A condensing enzyme utilizing acetyl-coenzyme A to initiate fatty acid synthesis. J Biol Chem 1992; 267: 20992-20998. Rock CO, Cronan JE. Escherichia coli as a model for the regulation of dissociable (type II) fatty acid biosynthesis Biochim Biophys Acta 1996; 1302: 1-16. Surolia N, Surolia A. Triclosan offers protection against blood stages of malaria by inhibiting enol-ACP reductase of Plasmodium falciparum. Nat Med 2001, 7: 167-173. Wright HT, Reynolds KA. Crystal structure of a substrate complex of mycobacterium tuberculosis ketoacyl-acyl carrier protein synthase iii (fabh) with lauroyl-coenzyme a. J Mol Biol 2005; 346:13131321. Turos E, Revell K D, Ramaraju P, et al. Unsymmetric aryl-alkyl disulfide growth inhibitors of methicillinresistant Staphyloccocus aureus and Bacillus anthracis. Bioorg Med Chem 2008; 16: 6501-6508. Heldreth B, Turos E. Microbiological properties and modes of action of organosulfur-based antiinfectives. Curr Med Chem Anti-Infect Agents 2005; 4: 295-315. Adetumbi M, Javor GT, Lau BH. Allium sativum (garlic) inhibits lipid synthesis by Candida albicans. Antimicrob Agents Chemother 1986; 30: 499-501. Ghannoum MA. Studies on the anticandidal mode of action of Allium sativum (garlic). J Gen Microbiol 1988; 134: 2917-2924. Mayeux PR, Agrawal KC, Tou JS, et al. The pharmacological effects of allicin, a constituent of garlic oil. Agents Actions 1988: 25: 182-190. Augusti KT, Matthew PT. Lipid lowering effect of allicin (diallyl disulfide oxide) on long term feeding to normal rats. Experentia 1974; 30: 468-470. Feldberg RS, Chang SC, Kotik AN, et al. In vitro mechanism of inhibition of bacterial cell growth by allicin Antimicrob Agents Chemother 1988; 32: 1763-1768. Kirkpatrick, D.L.: US20030176512 (2003). Kirkpatrick, D.L., Powis, G.: US20040116496 (2004). Focke M, Feld A, Lichentlaler HK. Allicin, a naturally occurring antibiotic from garlic, specifically inhibits acetyl-CoA synthetase FEBS Lett 1990; 261: 106-108. Ozolin ON, Uteshev TA, Kim IA, Deev AA, Kamzolova SG. Specific modification of the alpha-subunit of Escherichia coli Rna polymerase by monomercuric derivative of fluorescein mercuric acetate. Mol Biol (Mosk) 1990; 24: 1057-1066. Turos, E., Revell, K.D.: US2008182815 (2008). Turos, E., Rmaraju, P.: US2008033278(2008). Turos, E., Rmaraju, P.: US2009055677(2009). Buynak, J.D., Chen, H.:WO 2003087105 (2003).
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A Review of the Carbapenems in Clinical Use and Clinical Trials Tze Shien Lo*,1, Justin M. Welch2, Augusto M. Alonto3 and Eileen Anne R. Vicaldo-Alonto4 1
Section of Infectious Diseases, Veterans Affairs Medical Center, Fargo, ND, USA 2
3
4
Pharmacy Department, Veterans Affairs Medical Center, Fargo, ND, USA
Section of Infectious Diseases, MeritCare Medical Center, Fargo, ND, USA
Department of Internal Medicine, University of North Dakota School of Medicine & Health Sciences, Fargo, ND, USA Abstract: Despite alarming data showing the ever increasing number of bacteria becoming resistant to different classes of antibiotics through various mechanisms, the carbapenems remain a unique class of antibiotics that possess the broadest spectrum against Gram-positive, Gram-negative, aerobic and anaerobic organisms. However, bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa, carry mechanisms that can inactivate the carbapenems. This article gives a review of the carbapenems that are currently in clinical use as well as discusses the new carbapenems that are in clinical trials. These new carbapenems show promising potential to overcome the resistance against the presently existing carbapenems. The present article shows the recent patents using carbapenems as an effective antibiotic.
Keywords: Carbapenems, new agents, resistant organisms, nosocomial infection, broad spectrum. INTRODUCTION The carbapenems are anti-microbial agents that belong to the -lactam family. They possess a broad spectrum of bactericidal activity against both Gram-positive and Gramnegative bacteria. Along with the other members of the -lactam group, they inhibit the transpeptidation step of bacterial cell wall synthesis by binding to penicillin binding proteins (PBPs). They are also stable against -lactamase hydrolysis[1, 2]. The carbapenems are derived from thienamycin, a naturally occurring extract of Streptomyces cattleya. Thienamycin was discovered to have a broad spectrum of activity and was stable against -lactamase. However, thienamycin is chemically unstable. Researchers developed more chemically stable carbapenems resulting in another class of agents: the synthetic carbapenems[3]. *Corresponding author: Tel: (701) 232-3241; Fax: (701) 237-2491; E-mail: [email protected] Atta-ur-Rahman / M. Iqbal Choudhary (Eds.) All rights reserved – © 2010 Bentham Science Publishers.
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The unique chemical structure of carbapenems is important for their activity and stability. Carbapenems differ from penicillins and cephalosporins due to the replacement of the sulphur atom with a carbon atom at position 1 and the presence of an unsaturated bond between C2 and C3 at its 5-membered -ring structure. The trans configuration of hydroxyethyl at position 6 renders it more stable against -lactamase hydrolysis compared to penicillins and cephalosporins which possess a cis configuration [1, 2]. Among Gram-positive cocci, carbapenems have good activity against Staphylococcus and Streptococcus spp. They are inactive against Enterococcus faecium, methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus epidermidis (MRSE). The carbapenems possess good activity against many Gram-negative bacteria and anaerobic organisms[4]. The carbapenems are widely utilized in hospitals due to their extensive activity and costeffectiveness. They are very useful agents in polymicrobial infections due to their effect both on Gram-positive and Gram-negative bacteria. They are used as monotherapy or in combination therapy in central nervous system, lower respiratory tract, skin and soft tissue, urinary tract, joint, muscle, obstetric, gynecologic and abdominal infections. They are also good agents in managing febrile neutropenia and complications due to cystic fibrosis [1]. This review article aims to discuss the chemistry, mechanisms of action, mechanisms of resistance, microbiology, pharmacokinetics, pharmacodynamics, adverse effects and clinical indications of the four Food and Drug Administration (FDA)-approved drugs in the United States – imipenem, meropenem, ertapenem and doripenem. We will also discuss the carbapenems undergoing clinical trials. The compositions patented by Romesberg et al. are useful in enhancing the sensitivity of both drug-resistant and drug-sensitive microorganisms and cells[5]. Coleman and Han showed the antibiotic compositions comprised of a targeting moiety covalently linked to an antibiotic moiety[6]. CHEMISTRY The carbapenems are semi-synthetic beta-lactam antibiotics that differ from penicillins by substituting a carbon atom at position 1 and an unsaturated bond between C-2 and C-3 of the thiazolidine ring structure. The 6-trans-hydroxyethyl group on carbapenems is responsible for the stability to beta-lactamase activity [1, 7] (Fig. 1). HO
HO H
H H
CH3 S
N H
N O
CH3
H
O
CH3 S
N
NH
N
O
Imipenem
H
H
Meropenem OH
CH3
H O
CH3 N
OH
O
OH NH
H CH3
H3C
S
O
CH3
O
O
HO
NH
OH
OH
CH3
N H
O
S
N
NH
O
O Ertapenem
Fig. (1). Chemical structure of the four FDA-approved carbapenems.
OH
O S
NH
O Doripenem
NH2
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Imipenem is metabolized by human renal dehydropeptidase I (DHP-1) which requires the co-administration of cilastatin, a DHP-1 inhibitor to maintain antimicrobial activity in urine and to extend its half-life. The addition of a methyl group on the carbapenem nucleus decreases the susceptibility to DHP-1 degradation and is found on meropenem, ertapenem and doripenem [1, 8, 9]. These three carbopenems are more stable than imipenem and do not require co-administration with a DHP-1 inhibitor. Differing side chains on each carbapenem leads to some differences in side effects, pharmacokinetics and spectrum. The dimethylcarbamylpyrrolidinethiol side chain on meropenem may be responsible for the reduced incidence of seizure activity compared to imipenem and enhanced activity against Gram-negative organisms [1, 7]. A metasubstituted benzoic acid group gives ertapenem a longer half-life than other carbapenems. Doripenem has a sulfamoylaminoethyl-pyrrolidinylthio side chain that increases its activity against Gram-negative microorganisms [1, 9]. Christensen et al. showed the formula, preparation and antibiotic use of carbapenems [10]. Choi et al. showed that beta-methyl carbapenem compounds are useful in the treatment of bacterial infections and explained the methods for treating such infections using such compounds and/or compositions [11]. MECHANISM OF ACTION Carbapenems exert their bactericidal activity by binding to penicillin-binding proteins (PBPs) and interrupting the synthesis of the bacterial cell wall. The difference in binding affinities of various carbapenems for specific PBPs is believed to be responsible for some of the differences in antimicrobial activity [1, 2, 8, 12-17]. The affinities of each carbapenem for specific PBPs vary by species as well as by strain of bacteria. While some general conclusions can be made, caution is advised in interpreting PBP studies that assess a single strain[7]. Imipenem binds with highest affinity to PBP-2, then –1a and –1b and has weak affinity for PBP-3. Meropenem and ertapenem also have high affinity for PBP-2, then PBP-3 and then –1a and –1b [18, 19]. Doripenem binds to PBP-2, PBP-3 and PBP-4 [17]. MICROBIOLOGY Carbapenems have a very broad spectrum and are highly active against most aerobic Gram-positive and Gram-negative microorganisms and anaerobes. Table 1 summarizes the spectrum of activity of major pathogens. Their spectrum includes staphylococci, streptococci, certain enterococci, Neisseria spp., Enterobacteriaceae, as well as Pseudomonas Table 1. Spectrum of Activity Imipenem
Meropenem
Ertapenem
Doripenem
Staphylococcus aureus (MSSA)
Susceptible
Susceptible
Susceptible
Susceptible
Staphyococcus aureus (MRSA)
Resistant
Resistant
Resistant
Resistant
Gram Positives:
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(Table 1) Contd…..
Imipenem
Meropenem
Ertapenem
Doripenem
Streptococcus pneumoniae
Susceptible
Susceptible
Susceptible (excluding penicillin-resistant)
Susceptible
Streptococcus pyogenes and viridans group
Susceptible
Susceptible
Susceptible
Susceptible
Enterococcus faecalis
Susceptible (excluding VRE)
Susceptible (excluding VRE)
Resistant
Susceptible (excluding VRE)
Enterococcus faecium
Resistant
Resistant
Resistant
Not reported
Enterobacter
Susceptible
Susceptible
Susceptible
Susceptible
Acinetobacter
Susceptible
Susceptible
Resistant
Susceptible
Neiseria
Susceptible
Susceptible
Susceptible
Not reported
Pseudomonas aeruginosa
Susceptible
Susceptible
Resistant
Susceptible
Gram Negatives:
Modified from Nicolau DP, 2008 [12].
aeruginosa and Acinetobacter spp. Carbapenems are inactive against E. faecium, vancomycin-resistant Enterococcus faecalis, MRSA, MRSE, Burkholderia cepacia and Stenotrophomonas maltophilia. In general, meropenem is slightly more active against Gram-negative pathogens than imipenem, which is slightly more active against Grampositive pathogens [2, 12, 13, 20]. Ertapenem has similar coverage of most aerobic Grampositive and negatives and anaerobes as the other carbapenems, but is not active against Pseudomonas aeruginoas, Acinetobacter spp. and enterococci. In general, doripenem has better activity against Gram-positive organisms than meropenem and is more potent against Gram-negative organisms than imipenem. Doripenem appears to have improved in vitro activity against P. aeruginosa and Acinetobacter spp. and is the most potent of the carbapenems against penicillin-resistant streptococci [1, 9]. Mor et al. discussed the novel class of antimicrobial polymeric agents which are designed to exert antimicrobial activity[21]. MECHANISMS OF RESISTANCE 1. Alteration in Penicillin-Binding Proteins (PBPs) All carbapenems manifest their bacterial activity by binding covalently to PBP. However, some Gram-positive cocci possess PBPs that render weak affinity to -lactam antibiotics including the carbapenems. For example, PBP 2a of MRSA [22] and PBP 5 of Enterococcus faecium [23] are poorly bound to carbapenems, thus, generating resistance of these two bacteria to the carbapenems.
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Gram-negative bacteria such as Proteus mirabilis, Pseudomonas aeruginosa, and Acinetobacter baumannii were shown to be associated with resistance to carbapenems by decreasing their PBPs affinities to the carbapenems [24]. 2. -lactamases Some bacteria produce -lactamases to inactivate -lactam antibiotics by opening the lactam ring of the antibiotics. The two -lactamses produced by some Gram-negative bacilli that can inactivate most of the -lactam antibiotics are the extended-spectrum -lactamase (ESBL) and the AmpC -lactamase. The two most common Gram-negative bacilli that produce ESBL are Escherichia coli and Klesiella species. ESBLs are generally plasmid mediated. In contract, AmpC -lactamses are chromosomally mediated and are produced by Gram-negative bacilli, such as Enterobacter cloacae, Citrobacter freundii, Serratia marcescens and Pseudomonas aeruginosa[25, 26]. Gram-negative bacteria such as Escherichia coli and Klebsiella pneumoniae, whether they are the wild types or extended-spectrum -lactamases (ESBL) producing isolates, were observed to be 100% sensitive to imipenem, meropenem, doripenem and ertapenem by the National Committee for Clinical Laboratory Standards (NCCLS) 2005 interpretive criteria. The minimum inhibitory concentration MIC90 for ertapenem was found to be higher than the other three carbapenems for the ESBL isolates [27, 28]. Ge and co-workers made similar observations that imipenem, meropenem, ertapenem and doripenem have good in vitro activity against Enterobacter spp. and Serratia marcescens regardless of whether they are AmpC -lactamase-producing isolates or wildtype isolates, although ertapenem showed greater increase in the MIC compared to the other three carbapenems [29]. Therefore, ESBL or AmpC -lactamase producing Gram-negative bacteria are generally susceptible to all the carbapenems including ertapenem [18]. Jacoby et al. reported that in order for K. pneumoniae to become resistant to a carbapenem, a combination of ESBL and membrane permeability defects is needed [30]. Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Burkholderia cepacia are Gram-negative bacteria that can produce metallo--lactamases which rapidly hydrolyze all carbapenems and markedly deprive their activities against these bacteria [25, 31-33]. Kaeaeriaeinen et al. modified the metallo-beta-lactamase to reduce amino terminal heterogeneity in a recombinant DNA production system[34]. Mansour et al. showed that certain tricyclic 6-alkylidene penems act as inhibitors of class-D enzymes and ssLactamases hydrolyze ss-lactam antibiotics, and serve as the primary cause of bacterial resistance[35]. The invention of Balkovec et al. which substituted succinic acid metallobeta-lactamase inhibitors is useful in treating bacterial infections in animals or humans[36]. 3. Impermeability Carbapenems can usually penetrate the OprD porins of Gram-negative bacteria including Pseudomonas aeruginosa because of their low molecular weight and zwitterionic structures [37]. However, ertapenem is generally considered not clinically useful compared to other carbapenems in treating infections caused by P. aeruginosa because this bacterium can exhibit resistance to ertapenem by virtue of a combination of alteration in permeability of the cell membrane and an increased activity in the membrane-associated efflux pump[31]. Some P. aeruginosa exhibit resistance to carbapenems through a combination of reduction of OprD porin expression and increased expression of ampC -lactamase [31].
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PHARMACOKINETICS Table 2 summarizes the major pharmacokinetic properties of the commercially available carbapenems. All carbapenems are formulated to be given intravenously. The maximum plasma concentration (Cmax) of imipenem/cilastatin after a 500 mg dose infused over 20 minutes ranged from 21 to 58 mcg/ml and declined to below 1 mcg/ml at 4 to 6 hours [14]. After a 500 mg dose of meropenem infused over 30 minutes, Cmax ranged from 14 to 26 mcg/ml and took 6 hours to decline to approximately 1 mcg/ml [15]. When a single 1 gram dose of ertapenem infused over 30 minutes is given, the mean Cmax was 155 mcg/ml [16]. The mean Cmax of doripenem after a single 1 hour infusion of a 500 mg dose was 23.0 mcg/ml [17]. Table 2. Pharmacokinetic Properties of Carbapenems in Healthy Subjects[2, 14-17, 19]
T(h)
% Excreted Unchanged in Urine
Renal Dose Adjust
~1
70 (w/cilastatin)
Yes
~1
70
Yes
572.1
~4
38
Yes
36.3
~1
70
Yes
IV Dose (g)
Cmax(mcg/ml)
AUC(mcg*hr/ml)
0.5
21-58
42.2
1
69.9
94.38
0.5
14-26
27.2-32.4
1
61.6
77.5
Ertapenem
1
155
Doripenem
0.5
23.0
Drug
Imipenem
Meropenem
Distribution Carbapenems are widely distributed. They penetrate into the majority of body tissues and fluids [8]. Imipenem penetrates well into cerebrospinal fluid, lung, pleural, peritoneal, bone and skin tissues with mean levels that exceed MICs for most susceptible bacteria [8, 14]. Meropenem also penetrates several body fluids and tissues including the cerebrospinal fluid and reaches levels that exceed MICs for most susceptible bacteria [8, 15]. Doripenem penetrates intra-abdominal and urinary tract tissues sufficiently to match or exceed MICs for most susceptible bacteria [17]. Metabolism and Elimination When administered alone, imipenem is metabolized by dehydropeptidase (DHP-1) in the kidneys reducing its level in the urine. Therefore, imipenem must be given with cilastatin, a dehydropeptidase inhibitor, which prevents renal metabolism to improve efficacy and to prevent potential nephrotoxicity [1, 14, 19]. Meropenem, ertapenem and doripenem are more resistant to DHP-1 [1, 13, 19]. Ertapenem is metabolized by hydrolysis into an inactive ring-opened derivative [16]. Doripenem is metabolized by dehydropeptidase-1 to an inactive ring-opened metabolite
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[17]. In vitro studies in human liver microsomes show that ertapenem and doripenem do not interact with the cytochrome P450 system [16, 17]. Imipenem, meropenem, ertapenem and doripenem are primarily eliminated by the kidneys [14-17]. Therefore, all available carbapenems require dose adjustment for renal insufficiency [14-17]. Ertapenem has the longest half-life of the available carbapenems at 4 hours and is highly protein bound [19]. Together these pharmacokinetic parameters allow ertapenem to be dosed once daily [16, 19]. Imipenem, meropenem and doripenem have halflives around 1 hour and, therefore, require dosing intervals of every 6 to 8 hours [14,15,17]. Imipenem, meropenem, ertapenem and doripenem are removed by hemodialysis [14-17]. PHARMACODYNAMICS Carbapenems are time-dependent (concentration-independent) and, therefore, the most important variable of efficacy is that the plasma concentration is maintained above the MIC for at least 30-40% of the dosing interval [9, 12, 19]. A post antibiotic effect has been reported against some Gram-negative pathogens, (Enterobacteriaceae, P. aeruginosa, and B. fragilis) as well as E. faecalis and S. aureus with the carbapenems [2, 8, 19]. ADVERSE EFFECTS Carbapenems are generally well tolerated. The incidence of adverse reactions of the four carbapenems is shown in Table 3. Table 3. Drug-Related Adverse Reactions Reported as Percentage for the Respective Carbapenem
Adverse event
Imipenem/cilastatin (n=1723)[14]
Meropenem (n=5026)[38]
Ertapenem (n=1152)[39]
Doripenem (n=477)[17]
Nausea/vomiting
3.5
1.4
4.7
12
Diarrhea
1.8
2.3
5.6
11
Thrombophlebitis
3.1
0.8
1.0
8
Injection site complications
1.4
1.1
3.2
N.O.
Rash
0.9
1.4
1.1
5
Pruritus
0.3
0.4
1.5
3
Seizure
0.4
0.8
*N.O.
*N.O.
Headache
<0.2
0.4
2.3
4
n = total number of patients in respective source study. N.O.= not observed. * Both ertapenem and doripenem were not found to cause seizure in respective studies; however, there was literature suggestive of seizures associated with ertapenem [40-43]. So far, no human cases of doripenem-induced seizure has been reported in the literature.
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Among the above four carbapenems, imipenem/cilastatin has the most epileptogenic potential. The risk factors for imipenem-induced seizure are advanced age, renal dysfunction, over-dose of imipenem, and preexisting central nervous system diseases such as stroke, encephalitis/meningitis, brain tumor, or other brain pathology [44]. DRUG INTERACTIONS Drug interactions with the four carbapenems are shown in Table 4. Table 4. Drug Interactions with Carbapenems [14-17, 45]
Imipenem
Cyclosporine
Ganciclovir
TyphoidVaccine
Probenecid
Valproicacid
Meropenem
Ertapenem Doripenem indicates increase in level or effects/side effects of the carbapenem. indicates increase in the level or effects/side effects of the drug in row 1. indicates decrease in the level or effects/side effects of the drug in row 1.
CLINICAL INDICATIONS Imipenem, meropenem and doripenem exhibit broad-spectrum activity against Grampositive, Gram-negative, aerobes and anaerobes; except for MRSA, MRSE, Enterococcus faecium, vancomycin-resistant Enterococcus faecalis, Burkholderia cepacia, Stenotrophomonas maltophilia, Corynebacterium jeikeium, and Legionella spp.[12, 13]. Ertapenem has a similar spectrum to the above-mentioned carbapenems, but it exhibits poor activity against P. aeruginosa, Acinetobacter calcoaceticus-baumannii complex and Enterococcus spp. Ertapenem’s long half-life allows it to be administered once a day. Thus, ertapenem can be used to treat outpatients with mild to moderated infections such as skin and skin structure infections, community-acquired pneumonia or complicated urinary tract infections, including pyelonephritis [18, 46]. Odink et al. demonstrated the use of carbapenems in prevention and treatment of pulmonary infections [47]. The FDA-approved clinical indications are shown in the following Table 5. Table 5. Indications for Carbapenems That Are FDA-Approved[14-17, 45] Indication
Imipenem
Meropenem
Ertapenem
Doripenem
Intra-abdominal infections
Skin and skin structure infections
Urinary tract infections
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(Table 5) Contd…..
Indication
Imipenem
Lower respiratory tract infections
Bone/joint infections
Endocarditis
Obstetric/gynecological infections
Sepsis
Polymicrobic infection
Meropenem
Ertapenem
Doripenem
Pediatric meningitis Acute pelvic infection
Diabetic foot infection
Community-acquired pneumonia
denotes FDA-approved indication.
USE OF CARBAPENEMS AND DEVELOPMENT OF RESISTANCE Because the carbapenems have the broadest antibacterial spectrum, there is a concern that abuse or overuse of this class of antibiotic will lead to development of resistance. Indeed, Lepper et al. and Carmeli et al. demonstrated an increase in consumption of imipenem not only led to an increase in resistance of Pseudomonas aeruginosa to imipenem, it also contributed to an increase in resistance to other antibiotics of the same bacterium[48, 49]. Another alarming sign is an increasing resistance rates of Acinetobacter baumannii to the carbapenems. A citywide surveillance done in New York City in 2000-2001 found that only one-third of the A. baumannii isolates were sensitive to meropenem[50]. The MYSTIC (Meropenem Yearly Susceptibility Test Information Collection) Program antimicrobial surveillance study conducted at 40 European centers in 2006 reported an increase in the number of carbapenem-resistant A. baumannii isolates compared to previous years [51]. The increase in carbapenem resistance is not limited to a particular geographic region and Table 6 demonstrates a trend of increased resistance of Pseudomonas aeruginosa in Table 6. Trends in Antimicrobial Resistant Pseudomonas aeruginosa Isolates in Various Regions of the World from 1997-1999 (from SENTRY Antimicrobial Surveillance Program Data)[52] Asia-Pacific
Canada
1998
1999
1997
1998
Imipenem
10
13.8
17
18
Meropenem
8.5
12.2
7.6
8.4
Europe 1999
1997
1998
8
10.7
5.1
10.2
Latin America
United States
1999
1997
1998
1999
1997
1998
1999
21
28.4
23
23.3
25.7
12
14.8
19.1
14.4
26.2
17
20.3
23.4
7.6
9.2
9.1
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different regions of the world [52]. Thus, the increase and prevalence of resistant isolates warrants judicious use of carbapenems. CURRENT AND FUTURE DEVELOPMENTS Carbapenems have been very useful in clinical practice. Their effectiveness against a broad spectrum of Gram-negative and Gram-positive infections has been proven. Studies are progressing to the development of newer and more advanced drugs in the carbapenem family. Carbapenems that can be administered orally are also currently being researched. Other molecules are also being introduced that are more stable and have more widespread antibacterial activity. This section aims to discuss the latest developments on three of the newest carbapenem drugs in pharmacologic research – tebipenem, tomopenem, and razupenem. Presently undergoing Phase II clinical trials in Japan is an oral agent tebipenem/ tebipenem pivoxil [1, 53, 54]. This agent exhibited good anti-microbial activity and is recommended as a potential therapy for respiratory, urinary tract and otolaryngological infections. Tebipenem also exhibited good stability against hydrolysis by dehydropeptidase1. A recent study tested the in vitro activity of tebipenem against penicillin-nonsusceptible Streptococcus pneumoniae. Samples were collected from a pediatric population with the following conditions: respiratory tract infection, acute otitis media, sepsis and meningitis. Out of the 202 clinical isolates, 34 were penicillin-susceptible, 96 were penicillinintermediate and 72 were penicillin-resistant. It was suggested that tebipenem may be a potential therapy for multi-drug resistant Streptococcus pneumoniae [54]. Another clinical investigation demonstrated the antimicrobial activity of tebipenem. This study examined the antimicrobial actions of several antibiotic agents against Streptococcus pneumoniae and Haemophilus influenzae. Tebipenem was compared to penicillin G, ampicillin, cefotaxime, ceftriaxone, panipenem, meropenem, vancomycin, cefditoren, cefcapene, cefteram and faropenem against Streptococcus pneumoniae while another group of various antibiotic drugs was utilized against Haemophilus influenzae. It was determined that among the oral agents tested, tebipenem had the highest activity against Streptococcus pneumoniae [55]. A double-blind intergroup comparative study investigated the efficacy, safety, and PKPD analysis of tebipenem pivoxil for the treatment of otolaryngological infections in adults to establish the recommended clinical dosage. The drug was tested against agents causative for otolaryngological infections – Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Moraxella catarrhalis and Haemophilus influenzae. This study suggests high clinical usefulness of tebipenem pivoxil for the treatment of otolaryngological infections in adults [56]. Another new drug undergoing clinical investigation is the injectable carbapenem, tomopenem (formerly RO49084631/CS-023). It possesses a broader spectrum of activity compared to its counterparts being effective against Gram-positive and Gram-negative bacteria, MRSA and Pseudomonas aeruginosa. Tomopenem has the same mechanism of action as other carbapenems, inhibiting cell wall synthesis. It is stable against dehydropeptidase-1 hydrolysis and has a longer elimination half-life due to its decreased rate of renal tubular secretion [57]. These qualities render it a clinically efficient drug for possible future use.
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Tomopenem stability was tested against against Class A (Sme-1 and KPC-3), Class B (IMP-1, VIM-2 and VIM-6) and Class D (OXA-23) carbapenemases. Through spectrophotometry, the stability of tomopenem to the purified carbapenemases was assayed and compared to other carbapenems, which include imipenem, panipenem, meropenem, biapenem and doripenem. Tomopenem had superior stability against carbapenemase breakdown compared to imipenem and meropenem [58]. Three investigations were conducted which demonstrated the efficacy of tomopenem against Gram-positive, Gram-negative and anaerobic bacteria. The first was an in vitro study on the antibacterial effect of tomopenem against seven strains of Staphylococcus aureus including methicillin-sensitive Staphylococcus aureus (MSSA) and MRSA. The study suggested that tomopenem has similar pharmacodynamic effects as the other carbapenems, but can also be clinically useful against MRSA [59]. One study determined the in vitro activity of tomopenem against recent clinical isolates of Gram-positive and Gram-negative bacterial pathogens in Japan. There were 1,275 clinical isolates from 23 species that were collected between the year 2005 and 2006. Tomopenem demonstrated the greatest activity compared to the carbapenems tested against clinically significant, drug-resistant pathogens except for Enterococcus spp. and Acinetobacter spp. It was concluded that tomopenem exhibited good in vitro activity against recent clinical isolates of Gram-positive and Gram-negative pathogens[60]. Another study was geared towards exhibiting the in vitro activity of tomopenem against anaerobic bacteria. Tomopenem was compared to other carbapenems and demonstrated broad activity against 63 reference species. The investigation suggests that the activity of tomopenem was potent and comparable to those of meropenem and doripenem and more potent than that of panipenem [61]. Another carbapenem undergoing clinical investigation is razupenem (formerly PZ-601). Razupenem is an intravenous novel carbapenem with a wide spectrum of antimicrobial activity, which includes multidrug-resistant Gram-positive (MRSA and Enterococcus faecium) and extended-spectrum -lactamase producing Gram-negative bacteria. A study was done to determine the safety and multiple dose pharmacokinetics of razupenem in healthy male volunteers. In this randomized, three-cohort (6 razupenem, 2 placebo) study, a total of 24 male subjects were given twice daily infusion of razupenem (600 mg, 1,000 mg, 1,500 mg) or placebo for 7 days. The study assessed the presence of adverse events, laboratory, ECGs, and plasma and urinary levels of razupenem or its ring-opened metabolite. It was shown that razupenem did not cause any serious adverse events. There were neither laboratory nor ECG findings that created clinical concerns. The most common side effects were infusion site and gastrointestinal disorders [62]. A recent study determined the activity of razupenem against Enterobacteriaceae with defined resistance mechanisms. This study demonstrated that razupenem has good activity against producers of extended-spectrum -lactamases. However, the activity of razupenem was negatively affected by AmpC enzymes and carbapenemases [63]. The penems are a class of -lactam worth mentioning. The penems are structurally similar to the carbapenems except, instead of a –CH2– in position 1 of the five-membered ring, a sulfur atom is in that position. They are derived as a hybrid of penam (penicillin) and cepham (cephalosporin) nuclei[64]. The penem that has been actively developed is faropenem. Both forms of faropenem are orally-active. The sodium salt, which is faropenem sodium, has been marketed in Japan since 1997[65]. Currently under phase 3 clinical
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investigation is the prodrug form, which is faropenem medoxomil (also known as faropenem daloxate). Both forms have been shown to have excellent activity against Grampositive, Gram-negative and anaerobic organisms[1, 66-68]. A new drug application (NDA) was filed in the US for approval of faropenem medoxomil for the following indications: acute bacterial sinusitis, com-munity-acquired pneumonia, acute exacerbations of chronic bronchitis and uncomplicated skin and skin structure infections. However, it was considered “non-approvable” by the US FDA in October 2006 [69]. CONCLUSION Carbapenems have proven their use and importance in clinical practice. This class of antimicrobials possesses excellent activity against Gram-positive, Gram-negative, and anaerobic organisms. Currently, none of the available agents on the market are active against MRSA. It is very promising that there are agents under investigation that may be active against MRSA. These investigational agents, if approved and released, will make therapy for polymicrobial, hospital acquired infections, more convenient and potentially less toxic. Research and development of other oral agents, which can be utilized as possible stepdown therapy will also make treatment for polymicrobial infections easier and more effective. We are looking forward to future developments in carbapenem therapy. As these agents are discovered, studied and undergo improvement, the medical community can now expect more highly effective, broader spectrum, and more tolerable carbapenems available for clinical use. ACKNOWLEDGEMENTS We thank Renae Fjeldheim, Pharm D for her assistance with preparation of this manuscript. All contributing authors have no conflict of interest or significant relationships to disclose. REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10]
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Saidel-Odes L, Borer A, Riesenberg K, Smolyakov R, Schlaeffer F. History of cerebrovascular events: a relative contraindication to ertapenem treatment. Clin Infect Dis 2006; 43(2): 262-263. Seto AH, Song JC, Guest SS. Ertapenem-associated seizures in a peritoneal dialysis patient. Ann Pharmacother 2005; 39(2): 352-356. Calandra G, Lydick E, Carrigan J, Weiss L, Guess H. Factors predisposing to seizures in seriously ill infected patients receiving antibiotics: experience with imipenem/cilastatin. Am J Med 1988; 84(5): 911918. Faulds D, Heel RC. Ganciclovir. A review of its antiviral activity, pharmacokinetic properties and therapeutic efficacy in cytomegalovirus infections. Drugs 1990; 39(4): 597-638. Shah PM, Isaacs RD. Ertapenem, the first of a new group of carbapenems. J Antimicrob Chemother 2003; 52(4): 538-542. Odink D.A., Truex P.F., Ge Y.: US20050065141 (2005). Lepper PM, Grusa E, Reichl H, Hogel J, Trautmann M. Consumption of imipenem correlates with betalactam resistance in Pseudomonas aeruginosa. Antimicrob Agents Chemother 2002; 46(9): 2920-2925. Carmeli Y, Troillet N, Eliopoulos GM, Samore MH. Emergence of antibiotic-resistant Pseudomonas aeruginosa: comparison of risks associated with different antipseudomonal agents. Antimicrob Agents Chemother 1999; 43(6): 1379-1382. Quale J, Bratu S, Landman D, Heddurshetti R. Molecular epidemiology and mechanisms of carbapenem resistance in Acinetobacter baumannii endemic in New York city. Clin Infect Dis 2003; 37(2): 214-220. Turner PJ. Meropenem activity against European isolates: report on the MYSTIC (Meropenem Yearly Susceptibility Test Information Collection) 2006 results. Diagn Microbiol Infect Dis 2008; 60(2): 185-192. Gales AC, Jones RN, Turnidge J, Rennie R, Ramphal R. Characterization of Pseudomonas aeruginosa isolates: occurrence rates, antimicrobial susceptibility patterns, and molecular typing in the global SENTRY Antimicrobial Surveillance Program, 1997-1999. Clin Infect Dis 2001; 32 (Suppl 2): S146S155. Wang Y, Bolos J, Serradell N. Tebipenem pivoxil/tebipenem. Drugs Future 2006; 31(8): 676. Kobayashi R, Konomi M, Hasegawa K, et al. In vitro activity of tebipenem, a new oral carbapenem antibiotic, against penicillin-nonsusceptible Streptococcus pneumoniae. Antimicrob Agents Chemother 2005; 49(3): 889-894. Sakata H. Relationship between protein binding and antimicrobial activities of antibiotics against Streptococcus pneumoniae and Haemophilus influenzae science links Japan [Online], [Date Accessed: 2008 March 7]: URL: http://sciencelinks.jp/jeast/article/200623/000020062306A0919819.php Baba S, Yamanaka N, Suzuki K, et al. Clinical efficacy, safety and PK-PD analysis of tebipenem pivoxil on a phase II clinical trial in otolaryngological infections. Jpn J Antibiot 2009; 62(2): 155-177. Revill P, Serradell N, Bolos J. Tomopenem. Drugs Future 2007; 32(1): 37. Sugihara K, Ishii Y, Tateda K, Yamaguchi K. Carbapenemase Stability of CS-023 (RO4908463) and Other Carbapenems. 47th ICAAC, Chicago, IL, USA September 17-20 (2007). Macgowan AP, Bowker KE, Noel AR. The pharmacodynamics of the antibacterial effect and emergence of resistance to Tomopenem (formerly RO4908463/CS-023) in an in vitro pharmacokinetic model of Staphylococcus aureus infection Antimicrob Agents Chemother [Online], [Date Accessed: 2008 Jan 28]: URL: http://aac.asm.org/cgi/content/abstract/AAC.01153-07v1 Sugihara K, Tateda K, Shibuya R, et al. In vitro activity of CS-023 (RO4908463) against recent clinical isolates of gram-positive and gram-negative pathogens in Japan. 47th Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, USA September (2007). Tanaka K, Mikamo H, Nakao K, et al. In vitro activity of tomopenem (CS-023/RO4908463) against anaerobic bacteria. Antimicrob Agents Chemother 2009; 53(1): 319-322. Young CL, Rusca A, Di Stefano AFD, et al. Safety and multiple dose pharmocokinetics of intravenous PZ-601 (SMP-601): a novel carbapenem. 47th Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, USA September (2007). Livermore DM, Mushtaq S, Warner M. Activity of the anti-MRSA carbapenem razupenem (PTZ601) against Enterobacteriaceae with defined resistance mechanisms. J Antimicrob Chemother 2009; 64(2): 330-335. Bryskier A. In: Antimicrobial agents - Antibacterials and antifungals. Washington DC: ASM Press 2005; 319-335. Yokota T, Azagami S, Abe T, et al. Efficacy and safety of faropenem in pediatric patients with bacterial infectious diseases [Japanese]. Jpn J Antibiot 2008; 61(6): 366-378. Gettig JP, Crank CW, Philbrick AH. Faropenem medoxomil. Ann Pharmacother 2008; 42(1): 80-90.
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Dalhoff A, Thomson CJ. The art of fusion: from penams and cephems to penems. Chemotherapy 2003; 49(3): 105-120. Hamilton-Miller JM. Chemical and microbiologic aspects of penems, a distinct class of beta-lactams: focus on faropenem. Pharmacotherapy 2003; 23(11): 1497-1507. [No Authors Listed]. Faropenem medoxomil: A0026, BAY 56-6854, BAY 566854, faropenem daloxate, SUN 208, SUN A0026. Drugs R D 2008; 9(2): 115-124.
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Anti-Infective Quinone Derivatives of Recent Patents Junko Koyama* Faculty of Pharmaceutical Sciences, Kobe Pharmaceutical University, Higashinada-ku, Kobe 658-8558, Japan Abstract: Quinones are important naturally occurring pigments widely distributed in nature and are well known to demonstrate various physiological activities as antimicrobial and anticancer compounds. This review will focus on the preparation, therapeutic application, and administration of several benzoquinones, naphthoquinones, and anthraquinones having anti-infective, e.g. antiviral and antibacterial activities, in recent patents.
Keywords: Benzoquinone, naphthoquinone, anthraquinone, anti-infective, antiviral activity, antibacterial activity, antimalarial activity, patent. INTRODUCTION Infectious diseases have not yet been overcome. The worldwide death toll is 52 million people a year according to the estimate by WHO, and a third of them, 17 million people, die from infectious diseases. It is reported that most infections around the world are the diarrhea syndrome and 4 billion patients reported every year, those who are infected with malaria amount to 500 million, and pneumonia attacks 400 million. Considering that the earth’s population is 6 billion, such infections are a threat to human. The viruses related to hepatitis, such as HEV and HCV, and the new infectious diseases, such as the Ebola virus, Legionella pneumophila, and human immunodeficiency virus (HIV), have been discovered in the past 20 years. Furthermore, the appearance of a multidrug-resistant strain by antibacterial medicine use is a big factor. One of the more significant achievements of the 20th century has been the discovery and commercial development of numerous therapeutic agents that now provide reliably effective treatments for many infectious diseases that had previously caused extensive mortality, morbidity, and fear. The search for suitable drugs effective for the cure of human diseases is a continuing process. A number of methods can be used to perform susceptibility tests with antibacterial agents in a clinical laboratory setting or for research purpose, such as assessing the activities of new antimicrobial agents. These methods include the broth microdilution, disk diffusion, antibiotic gradient (epsilometer-test), and automated-instrument methods. For the antibacterial agents often used, quinone compounds are tetracyclines, new quinolones, anthraciclines, and mitomycins. More than 2000 naturally occurring quinones, for example - anthraquinones, naphthoquinones, and benzoquinones (Fig. 1), are now known and widely distributed in nature as pigments and as intermediates in cellular respiration and photosynthesis [1, 2]. Some quinones have important roles in the biochemistry of energy production and serve as vital links during electron transport. Most *Corresponding Author: Tel: +81-441-7549; Fax: +81-441-7550; E-mail: [email protected] Atta-ur-Rahman / M. Iqbal Choudhary (Eds.) All rights reserved – © 2010 Bentham Science Publishers.
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quinones, which are aromatic compounds present in bacteria and eukaryotes [3], are often involved in electron transport and include, ubiquinones and menaquinones [4]. They provide a defense role as a result of their effectiveness at inhibiting the growth of bacteria, fungi, or parasites [5-8]. Therefore, a number of them have various physiological activities as antimicrobial and anticancer compounds [9]. The mechanism of toxicity is still under investigation, but two theories dominate the literature [10], with some quinones proposed to exhibit one or both mechanisms. Redox cycling is the concept in which compounds catalytically cycle and generate oxidative radicals, such as hydrogen peroxide and superoxide, which then damage the cell. Alkylation is when quinones are activated inside cells and become covalently attached to proteins, DNA, or other targets. The most important reaction of quinones as far as biology is concerned is their reversible reduction to the corresponding hydroquinone (Fig. 2). O
O
O
O
O
O
(1) anthraquinone
(3) 1,4-benzoquinone
(2) 1,4-naphthoquinone
Fig. (1). Structures of quinones. O
NAD(P)H + H+
NAD(P)+
O
O•
O2
O2– •
H2O2
O-
Fig. (2). Redox cycling of NADH or NAD(P)H and quinone.
This study covers the preparation, the therapeutic application, and the administration of several benzoquinones, naphthoquinones, and anthraquinones having anti-infective, e.g., antiviral and antibacterial effects, in recent patents (2000-2009). The quinone compounds in 22 recent patents are classified into four categories based on targets; antibacterial, antiviral, antifungal, and antiprotozoal application, and reviewed.
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ANTIBACTERIAL APPLICATION Bacteria are very small, relatively simple, single-celled organisms whose genetic material is not enclosed in a special nuclear membrane. Two types of chemotherapeutic agents are synthetic drugs and antibiotics. In this section, 15 patents are classified into 1) antimycobacterial, 2) antichlamydial and/or anti-methicillin-resistant Staphylococcus aureus (MRSA), and 3) antibiotic. 1) Antimycobacterial Naphthoquinone Derivatives and their Use in the Treatment and Control of Tuberculosis [11] This invention relates to the use of the naphthoquinone derivative for the treatment and control of tuberculosis caused by Mycobacterium tuberculosis. Tuberculosis (TB) remains a major global public health problem. Nearly 2 million people died of TB, with a global case fatality rate of 23% but reaching > 50% in some African countries due to high rates of coexisting HIV infection. Man infected with HIV are very susceptible to TB [12]. If control of tuberculosis is not further strengthened, WHO estimates that between 2000 and 2020, nearly one billion people will be newly infected, 200 million people will become sick, another 35 million people will die from TB. Mycobacteria are aerobic, non-endspore-forming, nonmotile, slightly curved or straight rod. These bacteria are relatively resistant to normal staining procedures. Once stained, however, mycobacteria are not easily decolorized, even with acid-alcohol and are therefore classified as acid-fast. This characteristic reflects the unusual composition of the cell wall which contains mycolic acids together with free lipids. Treatment options of tuberculosis are limited, the drugs have significant side effect, and no new antibiotics have been developed against any mycobacteria since the 1970s. The present treatment regimes for TB are based on multidrug therapy with usually 3 or 4 antituberculosis drugs. However, the problem of multidrug resistant tubercle bacilli is emerging for various drugs. The need for new antituberculosis agents is urgent due to the increasing resistance of mycobacteria to the classic antituberculosis drugs. It is essential to have new antituberculosis agents, preferably those that can readily and simply be produced from some local sources. Twenty South Africa medicinal plants used to treat pulmonary diseases were screened for activity against the drug-resistant and sensitive strains of M. tuberculosis. A preliminary screening of acetone and a water plant extract, against a drug-sensitive strain of M. tuberculosis, H37Rv, was carried out using the agar plate method. The minimal inhibitory concentration (MIC) for the extract of Croton pseudopulchellus, Ekebergia capensis, Euclea natalensis, Nidorella anomala and Polygala myrtifolia was 0.1 mg/ml against the H37Rv using the radiometric method. The extracts of Chenopodium ambrosiodes, Ekebergia capensis, Euclea natalensis, Helichrysum melanacme, Nidorella anomala and Polygala myrtifolia were active against the resistant strain at 0.1 mg/ml. In this invention, the naphthoquinone derivatives of Formulas A and B have been found to be particularly effective against Mycobacterium tuberculosis [13]. (Fig. 3) In particular diospyrin (4) and methyljuglone (5) have been found to inhibit several antibiotic resistant as well as antibiotic susceptible strains of M. tuberculosis. Diospyrin and methyljuglone [1416] were isolated from E. natalensis and other species in this genus. A combination
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treatment of diospyrin and methyljuglone, which may be more effective than a single drug treatment of the two naphthoquinones, is also being considered. The oral administration of diospyrin or methyljuglone in an appropriate pharmaceutical composition with suitable diluents and carriers will typically be used to treat or control tuberculosis. R
O R
O
R1
(4) diospyrin: R= OH, R1= CH3 O R1 O [Formula A]
R
O (5) methyljuglone: R= OH, R1= CH3
R1 O [Formula B]
Fig. (3). Structures of naphthoquinones.
There are some papers about the mechanism such as extrusion pumps, cell penetration, and redox cycling, of naphthoquinone [17-20]. However, the mechanism is still under investigation. 2) Antichlamydial and/or Anti-MRSA Antichlamydial Agent [21] This invention relates to obtaining an antichlamydial agent which shows an antimicrobial effect, e.g., detergency, disinfection, infection prophylactic effect or the like on chlamydia using bamboo extract. Many problems exist during the treatment of chlamydia, including widely spread urogenital infections [22]. Chlamydia, which involves intracellular parasites, is hardly accessible to the majority of existing compounds. The chlamydia genus that is a pathogenic microorganism is neither a bacillus nor a virus from 1974, and is independently classified. As for chlamydia, three kinds (C. trachomatis, C. pneumoniae, and C. psittaci) are known. Chlamydia is bimorphic which is a fancy way of saying that they come in 2 stages: the elementary body (EB) and the reticular body (RB). The EB is the only infectious stage of the chlamydial developmental cycle. It functions as a tough "spore-like" body whose
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purpose is to permit chlamydial survival in the non-supportive (to chlamydiae) environment outside the host cell. They have either no or a very small amounts of peptidoglycan. Chlamydia is treated with antibiotics as doxycyclin and not penicillin. It is not an antichlamydia agent that improves the infection prevention to EB, though the antibiotic such as the tetracycline derivatives used for a treatment after infecting chlamydia. In this invention, the antichlamydial agent is obtained from the extract of bamboo, e.g., Phyllostachys pubescens [23] or the like, and an alcohol or ether as an extraction raw material and an extracting solvent, respectively, as the active ingredient. The infection preventive effect is due to inhibit the protein synthesis catalyzed ATP. This antichlamydial agent contains benzoquinone in the extract, improves the infection prevention of somatic cell to EB, and provide the certain antibacterial activity by kill out RB in the cell. The more specific antimicrobial effect is exhibited when the extract contains liquid benzoquinone in a formation amount of 0.2 - 0.5%. Anti-Drug Resistant Strain Agents and Antichlamydia Agents [24] The invention relates to novel antibacterial agents for drug-resistant bacteria and antichlamydia agents which comprise highly active furanonaphthoquinone derivatives as effective compounds. In recent years, MRSA has been seriously considered as the casual bacterium of hospital infection. Since this MRSA is a multiple drug resistant bacterium for a variety of antibiotics, there is a limitation to the drugs that may be effectively used as therapeutic agents and not antibacterial agents which show a stronger antibiotic activity against MRSA than against MSSA (sensitive bacterium). Accordingly, the antibacterial agents for drug-resistant bacteria with a high antibacterial activity have been desired. The Chlamydiae differ from the other main order of intracellular bacteria, the Rickettsiales, in their characteristic dimorphic growth cycle. Chlamydia, which is known to be the casual bacterium of parrot fever, comes from infected pet birds and infections caused by sexual intercourse, urethritis, cervicitis, lymphogranuloma, etc. Antibiotics such as macrolide derivatives and tetracycline derivatives have been used for its treatments. Since recent new drugs have a broad spectra, the acquisition of resistance to other bacteria against drugs has become a problem. Therefore, the development of novel highly specific antichlamydia agents of which the action mechanism is different from that of the drugs known so far, is anticipated. This invention provides antibacterial agents for drug-resistant bacteria and antichlamydia agents comprising, as the effective component, the furanonaphthoquinone derivative (Fig. 4 wherein each R may be the same or different representing any one of the following a) to e)). These various compounds may be produced by a variety of known methods of chemical synthesis [25] or by methods such as the extraction of naphthoquinones as natural products. For example, 8-hydroxy-2-methylnaphtho[2,3-b]furan-4,9-dione (6, FNQ13) was tested for 14 strains of MRSA and 12 strains of MSSA as the control, and six fluconazoleresistant Candida albicans (FRCA) and fluconazole-sensitive Candida albicans (FSCA) as the control. It was found that the MIC of MRSA was 5.36 μg/ml, and MSSA 11.98 μg/ml, indicating that FNQ13 exhibits a stronger antimicrobial activity against MRSA. FNQ13 exhibits the same degree of antibacterial activity against the FRCA of Candida albicans as that against FSCA. Furthermore, antibiotics by concomitant use with a low concentration of FNQ13 decreases the resistance of MRSA against antibiotics such as ampicilin, cefaclor, vancomycin, etc., from 1/2 to 1/4.
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R
O
R
O R
R R
R1
299
a) b) c) d) e)
R= -R' R= -OR' R= -COR' R= -OCOR' R= -SR'
R
O
O O R
(6) FNQ13: R= CH3, R1= OH (7) FNQ3: R= CH3, R1= H
O
Fig. (4). Structures of furanonaphthoquinones.
2-Methylnaphtho[2,3-b]furan-4,9-dione (7, FNQ3) and 2-hydroxymethylnaphtho[2,3b]furan-4,9-dione inhibited the growth of all the tested strains of chlamydia at a low concentration of 0.25 to 1.0 μg/ml. In addition, as for the toxicity to human cells, the concentration that caused 100% necrosis in human cancer cells is about 5 μg/ml, while that for normal human cells is about 20 μg/ml [26]. Each compound of the present invention, which may be used as the effective component in antibacterial agents against drug-resistant strains, are advantageous in that it exhibits a drug efficacy as an anti-cancer agent, but shows no toxicity to normal cells and causes no side effects, at effective concentrations as antibacterial agents. When A549 cells were treated with 7, reactive oxygen species was produced in mitochondria and carcinoma cells compared to normal cells damaged as 10-fold [27]. The same reaction may be occurred in the bacteria. Antibacterial Compound Against VRE and/or MRSA [28 - 30] This invention relates to the specific quinone methide compounds having a strong antibacterial activity against vancomycin-resistant Enterococcus (VRE) and MRSA, and the synthetic methods of these new compounds (Fig. 5). In recent years, infections caused by bacteria resistant to multiple antibiotics have been significant problems. Especially, MRSA and VRE are spreading to medical institutions all over the world. It was found that totarol, abietane diterpene, had a strong antibacterial activity for MRSA by searching for a new antibacterial compound [31, 32]. Abietane diterpenes are widely distributed in nature with various biological activities, e.g., antivirus [33, 34], antibacterial [34-36], antimalarial [37], antioxidant [38], and antitumor activities [39, 40]. The inventors reported the synthesis of oxidized abietane diterpene derivatives and their antibacterial activities against MRSA and VRE, and that the quinone methide compound showed a very potent activity against both MRSA and VRE [41, 42]. In this invention, the compounds having a more potent antibacterial activity against MRSA and VRE were synthesized using a previously reported synthetic route [41, 42] or a new method suitable for the mass production in liquid phase [29]. From the previously reported results, it was suggested that the C ring structure of this compound has a greater influence than the structure of the A or B ring against the antibacterial activity. Therefore, even if the structure
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O
OH X1
O
X3
X2
X4
O
X5
A C
B R1
H
Y3
Y1 Y2
R2
H
Y5
Y4 R3
Y6 (10)
(9)
(8)
H
OH
OH
O
Br
O
O
(11)
(13)
(12)
OH Br
OH Br
Br
O (14)
(15)
Fig. (5). Structures of abietane diterpene compounds.
of the A or B ring of the compound shown in the figure is somewhat different, it is thought that the compound which has the same partial structure as the C ring has the same antibacterial activity. For example, the synthetic route of compounds, 11-15 were reported. The antibacterial activities against MRSA and VRE of the synthesized abietane diterpenes and their derivatives were evaluated. The test organisms, three strains of MRSA (MRSA664, MRSA-730 and MRSA-996) and three strains of VRE (VanA, VanB and VanC) were obtained from the Department of Acterial and Blood Products, National Institute of Infection Disease of Japan. MIC of the test compounds are listed in (Table 1). In Table, compounds, 12, 13, and 15 showed potent antibacterial activity. At present, antibacterial mechanism is not clear. Many strains of MRSA possess efflux pumps such as the specific TetK and MsrA transporters which export certain tetracyclines and macrolide, and the multidrug resistance proteins NorA and QacA which confer
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resistance to a wide range of structurally unrelated antibiotics. The activity is likely to be related to the inhibition of pumps [43]. Table 1.
Minimum Inhibitory Concentrations (MIC) of Abietane Derivatives Strain
Compound
MIC (μg/mL) VRE
MRSA
VanA
VanB
VanC
996
730
664
11
64
64
128
64
64
64
12
-
-
-
2
2
4
13
4
4
4
4
4
4
14
16
32
16
32
64
64
15
8
8
16
8
8
8
vancomycin
256
128
16
2
2
2
Naphthoquinone Derivative Compound [44, 45] The purpose of this invention is to provide the following: (1) a new naphthoquinone derivative that can be used as an agent such as a cell cycle inhibitor, anti-bacterial agent, cell cycle progression inhibitor, cell proliferation inhibitor, anti-bacterial agent, imipenem O
R4
R2
R5
O
Formula I (17) R1=Cl, R2= OCH3, R3= R4= R5= H
CH3 (18) R1= H (19) R1= CH3
O
R3
R1 R2
OH
H3CO
O
O
OR1
O
R3
R1
OR1 OH
O 6
7
H3CO
O (16) chloroquinocin: R1= Cl, R2= OH R3= OH, 6C C7 (20) R1= Cl, R2= OH, R3= OH, 6C C7 (21) R1= H, R2= OCH3, R3= OCH3, 6C C7 (22) R1= H, R2= OCH3, R3= OCH3, 6C C7
Fig. (6). Structures of naphthoquinone derivatives.
OCOCH3 O (23) R1= H (24) R1= CH3
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activity promoter, or bacterial cell proliferation inhibitor, (2) a pharmaceutical composition containing this compound as an active component that can be used for cell cycle progression inhibition, cell proliferation inhibition, bacterial inhibition, imipenem activity promotion and bacterial cell proliferation inhibition, and (3) an activity promoting agent and an activity promoting method for this compound. These naphthoquinone derivatives (Formula I) (Fig. 6) were prepared by a variety of established methods [46, 47]. Chloroquinocin (16), a naphthoquinone that was isolated from Streptomyces sp. LL-A9227 culture broth, shows a moderate inhibitory activity against Gram-positive bacteria, including MRSA. Chloquinochin showed weak activity for the Gram-negative bacteria and no activity for the yeast. Yeast Candida albicans Among the extensive efforts to acquire highly potent agents, the inhibitory potential of 16 against Gram-positive bacteria makes it an interesting and significant lead for this purpose. In addition to being a chlorine substituent, chloroquinocin has a novel pyranonaphthoquinone framework. The various derivatives that have this structurally unique formula were prepared and examined for their anti-bacterial activity against a Gram- positive bacterium, such as the MRSA, and Gram-negative bacteria. Imipenem promotes the activity of naphthoquinone derivatives and their pharmacologically permissible salts. Furthermore, the anti-MRSA activity is increased when these compounds are combined with imipenem (Table 2). These naphthoquinone derivatives and a pharmaceutical composition containing the naphthoquinones as an active component effectively inhibit bacterial infection and disease resulting from bacterial growth. Table 2.
Anti-MRSA Activity* of Naphthoquinones Compound
Compound only (10 μg/ml)
+ Imipenem (10 μg/mL)
17
-
8
18
11
15
19
9
12
20
11
15
21
21
22
22
9
18
23
12
21
24
7
13
*inhibitory zone (mm): paper-disk method.
3) Antibiotic New Anthraquinone Derivatives [48] This invention relate to the new bianthraquinone derivatives, for example, 2,2´, 4,4´,5,5´,8-heptahydroxy-7,7´-dimethoxy[1,1´-bianthracene]-9,9´,10,10´-tetrone (25, YM 187781) and 2,2´,4,4´,5,5´-hexahydroxy-7,7´-dimethoxy [1,1´-bianthracene]-9,9´,10,10´tetrone (26, YM187787) were obtained by culturing a strain belonging to the genus
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Verticillium (A Hyphomycetes Verticillium lecanii Q57371) that have an antibacterial activity toward various bacteria (Fig. 7). OH
O
R
O
OH
H3CO
OH O
H3CO
OH
OH
O
OH
(25) YM187781: R= OH (26) YM187787: R= H
Fig. (7). Structures of bianthraquinones.
Conventionally, there are the -lactam antibiotics, such as penicillin and cephalosporin, macrolide antibiotics, such as erythromycin, and aminoglycoside antibiotics, such as kanamycin, which the microbes produce. These inventors describe new antibacterial compounds, a manufacturing process with a microbe having the ability to produce a new compound, and a new microbe. As a result of a detailed study, they discovered a microbe which has the ability to produce a compound having an antibacterial activity, and they identified it with the Hyphomycetes Verticillium lecanii Q57371 strain which belonged to the genus Verticillium that was separated from soil collected on Yaku island in Kagoshima, Japan [49]. In order to manufacture new antibacterial compounds, a cultivation is started by inoculating the Verticillium lecanii Q57371 strain into the culture medium containing the source of nutrition of the Verticillium lecanii Q57371 strain and growing it aerobically. The cultivation method performed by the same production method used for general antibiotics. These antibacterial compounds react with acids and form inorganic salts or organic salts. The active compounds described here may be formulated for addition to a pharmaceutical carrier in accordance with known techniques [50]. Rifamycin Derivatives [51 - 57] Rifamycins are natural products with potent anti-microbial activity. The rifamycin antibiotic from Streptomyces mediterranei was discovered in late 1957 by Piero Sensi [58], and subsequently rifampicin was synthesized in 1965 [59] and introduced into medical care in 1968. Rifampicin is still the most important agent in the treatment of tuberculosis, leprosy and diseases caused by nontuberculous mycobacteria. Rifamycins are distinguished by a unique chemical structure, particularly an aromatic chromophore with an ansa chain [60] and by their specific anti-bacterial action that selectively blocks the -subunit of the DDRP enzyme (DNA-dependent RNA polymerase), which controls the rebuilding process of bacterial peptides [61,62]. However, rifamycins can be enzymatically inactivated by a
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recently described class of rifamycin-specific ADP-ribosyl transferases [63]. In addition, the therapeutic applications of naturally occurring rifamycins are limited due to their poor oral bioavailability, weak activity against Gram-negative pathogens and low distribution in infected tissues, although they have high activity towards a broad spectrum of Grampositive bacteria and mycobacteria. Because of these unusual properties, the rifamycin derivatives are of great pharmaceutical interest. Therefore, new biologically active semisynthetic rifamycins are being extensively studied, and significant efforts have been made to identify semi-synthetic rifamycin derivatives to address these deficiencies. As a result, many semi-synthetic rifamycin derivatives with improved spectrum and pharmacological profiles have been identified. Zhenkun’s group was granted patents on the rifamycin derivatives (Fig. 8) sequentially in 2005-2006 [51-57]. These invitations relate to rifamycin derivatives that have antiH3COCO
RO
R5O 25
H3CO
25 OH
OH
OH 1
OH
H3CO
OH
OH
O OH 1
OH NH
O
O
O
OH
O
OH
OH
OH 1
30
NH
O NH
2 3
H
4
11
OH R6
O
2 3 O
25
H3CO
A
X
4
11
2 3 O
O
R3 OR2
rifamycin
WO200507940
X
OH
O
R4
A
N
4
11
WO200507941 R1 N
RO 25 H3CO
25 OH
OH
OH
OH 1
H3CO
O
O
OH NH X3 O
O
Y
N O
NH
OH
OH
OH
OH 1
11
N
X11
O
US20050203076
O
Y
L11
O NH 2
X
4 N
L11
OH 1
25 H3CO
O
2 3
L3
4
11
RO OH
OH
O
2 3
O
R2
RO
O
Y
Z
X11
X
4
11
US20050256096
US20050203085 RO
R 25
H3CO
25 OH OH
H3CO
OH OH 1
OH
OH
OH
OH 1
O NH 2 3
O
O
Y1
O
Y2
O
4
11 O
(CH2)m
Fig. (8). Structures of refamycin derivatives.
OH
F L N
O
(CH2)n COOH
Z US20050277633
NH 2 3
X
4 N
11
O
US20060019985
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microbial activities, their compositions, and the associated methods that can be used to treat and prevent microbial infections. The compounds associated with the current invention have improved anti-microbial and anti-bacterial activity against resistant pathogens. In particular, the rifamycin derivative in the current invention is a rifamycin moiety that is covalently linked to a linker through the C-3 carbon of the rifamycin moiety, and the linker, in turn, is covalently linked to a therapeutic moiety, an anti-bacterial agent, or pharmacophore in WO200507940 [51]. In WO200507941 [52], the rifamycin derivative in the current invention is a rifamycin moiety covalently bound to a linker through an iminomethylenyl group (-CH=N-) at the C-3 carbon of the rifamycin moiety, and the linker, in turn, is covalently linked to a quinolone moiety. In US20050203076 [53], the invention comprises rifamycin derivatives, in which the natural rifamycin C-11 keto group is converted to the C11oxime group. In US20050203085 [54], one aspect of the current invention comprises 3,4cyclo-11-deoxy-11-oxyiminorifamycins, in which the C-11 keto group of the natural rifamycin, rifabutin, rifalazil, rifaximin, rifamycin P origin, and their derivatives is converted to a C-11 oxime group. One of the major problems associated with the rifamycin class of anti-microbial agents is the rapid development of microbial resistance. The compounds in the US20050256096 invention [55] are designed to address rifamycin resistance by covalently attaching another functional group to the C-25 position of the rifamycin scaffold, which provides an additional interaction with RNA polymerase or an additional enzyme target. The compounds of the US20050277633 invention [56] are structurally distinct because they contain a 6-membered heterocycle fused to the 3,4position of rifamycin and have a spirosystem in their structure. The compounds of the invention in US20060019985 [57] contain a series of rifamycins that are chemically designed to address drug resistance by chemically linking molecules derived from hybridization of rifamycin and 4H-4-oxoquinolizine carboxylic acid. These compounds have potent anti-bacterial pharmacophores joined together through a stable bivalent linker. The inventive rifamycin derivatives are active against drug-resistant microorganisms, and microorganisms have a reduced frequency of developing mutational resistance to these derivatives (Table 3). Another aspect of the current inventions is a method for treating a microbial infection in a subject, which is any species in the animal kingdom but more specifically refers to humans and animals. The method involves administering an effective amount of one or more compounds from the present invention to the microbe-infected subject. The compounds of the current invention are effective against drug-resistant microbes and particularly rifamycin-resistant microbes. Table 3. Antimicrobial Activity of Selected Compounds Antomicrobial Activity (MIC, μg/mL) Organism
Rifam
Cipro
1
2
3
4
5
6
7
Staphylococcus aureus ATCC29213
RifS
0.008
0.25
0.0040.5
0.0081
0.0040.5
0.00050.125
0.0021.95
0.0080.032
0.008 -2.0
Staphylococcus aureus ATCC29213 rpoBH418Y
RifR
8
0.25
0.0616
0.0616
0.12516
<0.06>62.5
2.0->64
0.97731.25
0.06 ->64
Staphylococcus aureus ATCC29213 rpoBD417Y
RifR
>64
0.25
0.0632
0.06>64
0.25-32
4.0->62.5
0.98>64
16->250
0.06 -32
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Antomicrobial Activity (MIC, μg/mL) Organism
Rifam
Cipro
1
2
3
4
5
6
7
Staphylococcus aureus EN1252aa gyrAS84L grlAS80F
cipR
0.004
8
0.0040.5
0.0080.125
0.0040.5
0.00050.125
-
-
-
Staphylococcus epidermidis ATCC12228
RifS
0.03
0.125
0.0080.063
0.0080.1
0.00050.063
0.00050.125
0.00010.8
0.000130.031
0.004 -2.0
Staphylococcus pneumoniae ATCC6303
RifS
0.61
1
0.0080.125
0.0080.06
0.00020.125
<0.000060.031
0.0000625.5
0.000250.063
0.002 -0.25
Staphylococcus pyogenes ATCC19615
RifS
0.013
0.5
0.0080.063
0.0080.03
0.00050.063
<0.000060.063
0.0022.0
0.0020.063
0.002 -0.25
Enterococcus faecalis ATCC29212
RifS
1
0.5
2-32
0.24>64
2-32
0.5-31.25
0.25-62
0.0658.0
0.06 ->64
Haemophilus unfluenzae ATCC10211
RifS
0.24
0.008
0.1252
0.1254
0.03-2
0.125-8
0.06>51
0.1258.0
0.008 ->64
Escherichia coli ATCC25922
RifS
16
0.03
0.1258
1->64
0.125-8
4.0->62.5
3.9->64
2.0>62.5
0.03 ->64
Mycobacterium smegmatis ATCC700084
RifS
64
0.125
0.5-64
0.25>64
0.5-64
4.0->62.5
0.063-31
1->64
-
a For strain MT1222 see: Ince & Hooper, Anti6bicrobial agenta and chemotherapy, 2000, 44, 3344-50. rifam: rifampin, cipro: ciprofloxacin, 1: WO2005070940, 2: WO2005070941, 3: US20050203076, 4: US20050203085, 5: US20050256096, 6: US20050277633, 7: US20060019985.
ANTIVIRAL APPLICATION Viruses contain a single type of nucleic acid, either DNA or RNA surrounding by a protein coat and are obligatory intracellular parasites. They multiply by using the host cell’s synthesizing machinery to cause the synthesis of specialized elements that can transfer the viral nucleic acid to other cells. In this section, 4 patents are classified into two categories; 1) HIV and 2) others. 1) HIV Anti-Viral Multi-Quinone Compounds and Regiospecific Synthesis Thereof [64] The present invention relates to a method of the regiospecific synthesis of multi-quinone compounds and to novel biquinones and trimeric quinones, those that have antiviral activity and can be used to treat viral infections, particularly HIV infections. Acquired immune deficiency syndrome (AIDS) is a fatal disease caused by HIV that afflicts millions of people worldwide. Many current commercially available drugs used to treat HIV act by inhibiting either the enzymes reverse transcriptase or protease. The use of combinations or cocktails of these two classes of drugs has enabled a great number of HIV-
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infected individuals to keep the virus in check and remain alive. However, there are some patients who cannot respond to multi-drug therapy, and the side effects of several drugs can be also serious. Since HIV came to gain resistance to existing drugs, there is a pressing need to discover new anti-HIV medicines. Conocurvone, being a trimeric naphthoquinone, was isolated from a plant of the genus Conospermum, commonly known as the western Australian smoke bush by Boyd et al. [6567]. The Boyd patents disclose that conocurvone had been found to inhibit the growth and replication of viruses, and particularly retroviruses such as HIV, and synthesized trimeric naphthoquinones through the acid-coupling or base-coupling of 2,3-deoxy-1,4-naphthoquinone with two other naphthoquinone monomers. The monomeric and dimeric naphthoquinones were both found to be devoid of antiviral activity in the Boyd patents. Conocurvone and other trimeric quinones may possess a completely novel mechanism of HIV-inhibition by acting against integrase and the fusion of HIV to CD4 T-lymphocytes [65]. The synthetic method that is regiospecific and produces a good yield is thus needed [68, 69]. The quinone includes various quinone derivatives including benzoquinones and naphthoquinones. The multiquinone compounds can include identical quinone monomers or two or more different quinone monomers, such as a biquinone having a benzoquinone monomer bonded to a naphthoquinone monomer. The first quinone includes at least two directing groups at the C-2 position of the first quinone and a second directing group at C-3. The first directing group is selected from a group consisting of a halogen, and a nonhalogen, and the second directing group is selected from a group consisting of a halogen and non-halogen. Using the first directing group that is different from the second directing group allows for the efficient regiospecific bonding of the hydroxyquinone anion obtained by reacting a hydroxyquinone in the presence of a base, such as potassium hydroxide or cesium carbonate to the first quinone. The reaction can occur between any hydroxyquinone anion and any first quinone in a solution containing cesium carbonate and acetonitrile in an inert atmosphere at room temperature in about six to seven days (Scheme 1). O
O R1
A
OR3
+ R2
Cs2CO3 CH3CN, rt 6-7 days
O
HO O
R3
R1
B O (27)
O O (28)
R2
B O
(29)
Scheme 1.
In one embodiment of this invention, the representative biquinone can be further reacted in the presence of a base and or a chemical reagent to substitute the hydroxyl group for any chemical group. The biquinone can also be further reacted with a nucleophile. The nucleophile can substitute for the other in the first and second directing group. The nucleophile, for example, can be an amine analog or a second hydroxyquinone anion. Reacting the biquinone with the second hydroxyquinone anion results in a trimeric quinone in a polar aprotic solvent at about 60°C in about 1-3 days (Scheme 2). One aspect of this invention relates to a method for treating a viral infection demonstrating through in vitro antiviral assays [70]. Multi-quinone compounds of the present invention have been shown to inhibit retroviruses, particularly the human immunodeficiency
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virus, including different strains of HIV-1. The malti-quinone compounds may be formulated into various compositions for use in therapeutic antiviral treatment compositions. Antiviral compositions of this invention include one or more antiviral multi-quinones of this invention, as well as a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers and methods of administration are well-known to those skilled in the art. Inhibition of Carbohydrates Metabolism by Quinone Compounds [71] The present invention relates to an optically pure enantiomer of a synthetically prepared avarol. The enantiomers of avarol and derivatives are demonstrated to be potent and selective inhibitors of -glucosidase and -mannosidase. The inhibition of these two enzymes is useful for a variety of assays and probes, and offers particular utility in the treatment of retroviral infection-associated syndromes, such as AIDS. O Y
R1
O R2 R3
O O-
R1
R3 R1
O
R2
HO
R2 O
(31)
or
O
R2 O
(28)
(32)
O X
R1
O
O Y
O
O O-
O
or crown ether, 1-3 days
O
O
Y
(30)
R1
O R2 R3
O O
O
R3 HO O
(33)
Scheme 2.
Studies conducted with simple achiral quinone have suggested that their toxic activity can be attributed not only to their ability to undergo redox cycling but also to their potential binding and alkylation of nucleic acids and proteins [72]. Given the facile conversion of hydroquinones to quinones under aerobic conditions [73, 74], it stands to reason that chiral substituents on a hydroquinone nucleus might impart a degree of selectivity to the interaction between the respective quinone and asymmetric cellular components such as nucleic acids and highly organized proteins. Glycosyl hydrolases [75] are important enzymes that catalyze the hydrolysis of glycosidic bonds in polysaccharides and glycoproteins. The ability to inhibit the biosynthetic pathways to carbohydrate and carbohydrate-protein conjugates is significant in the study of cellular and extracellular events and in the development of antiviral [76], antidiabetic [77], and antitumor [78]
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chemotherapeutic strategies. All the currently approved drugs target one of two key retrovial enzymes, reverse transcriptase or protease, which are essential for replication and survival of the virus. Another promising strategy indirectly targets the initial association and recognition event between the HIV virus and the host cell. The CD4 surface protein has been shown to be a specific cellular receptor for HIV [79, 80]. The inhibitors of certain glycosidases having a profound effect on both the cell surface expression and function and topology of glycoproteins [81], are potential candidates for the therapeutic treatment of HIV infection. In 1974, avarol and avarone, having various biological effects [82, 83], were isolated from the marine sponge Dysidea avara by Minale et al. [84] (Fig. 9).
H
H
OH
HO
OH
HO
(34) (+)-avarol
(35) (-)-avarol
H
H
O
O
O
O (36) (+)-avarone
(37) (-)-avarone
Fig. (9). Structures of avarol and avarone.
In this invention, optically pure enantiomers of avarol were synthetically prepared. A survey of the potential inhibitory effect by avarol against twelve glycisidases was performed according to general method [85], and the avarols proved to be selective, potent inhibitors of -glucosidase and -mannosidase. The selective inhibition of -glucosidase (Type IV, brewer’s yeasy, EC 3.2.1.20) and -mannosidase (hack bean, EC 3.2.1.24) was observed
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with virtually no inhibitory activity against the other assayed enzymes. The value of Ki (9.5 and 25 μM) of (-)-avarol and natural ()-avarol were obtained from Lineweaver-Burk analyses [86]. Interestingly, the unnatural isomer (-)-avarol was significantly more active than the naturally occurring enantiomer in both cases. The IC50 for the unnatural (-)-avarol was 7.6 μM and the natural ( )-isomer was greater than 20 μM. The magnitude of inhibition of -glucosidase (yeast) by avarol is comparable to that exhibited by deoxynojirimycin (Ki=23 μM, yeast -glucosidase) and the castanospermine derivative (Ki=1.27 μM, cellular -glucosidase) which are currently under investigation as potential anti-HIV drugs. Avarol and its derivatives [87, 88] and avarone [89] had their pharmaceutical compositions described and used as AIDS agents. New potent anti-HIV agents may be prepared by incorporating into avarol some of the salient chemical functionality inherent to several known glycosidase inhibitors while ideally retaining the documented low toxicity of both avarol and avarone. 2) Others Heterocyclic Quinones as Pharmaceutical Agents [90] This invention relates to synthetic methods for the preparation of pyrrolylquinones and indolylquinones, the compounds so prepared, and uses thereof in the treatment of disease, viral infections, neurodegenerative disease, and proliferative disease. A large class of natural products derived from Aspergillus fungi is based upon the dihydroxy-bis-indolylquinone unit that is prenylated in various ways and sometimes Omethylated, and are called asterriquinones. They have the ability to activate the insulin receptor, the TrkA nerve growth factor (neutrophin) receptor [91, 92], and antitumor activity. They have further developed a synthetic version identified as “compound 38” [9196] (Fig. 10). O HO
Ph
OH O N
Compound (38)
Fig. (10). Structure of compound (38).
A first aspect of the present invention is the compound of Formula II and an acidcatalyzed method of producing a compound of Formula II (Fig. 11) by reacting a substituted or unsubstituted 2,5-dichloro-1,4-benzoquinone with at least one pyrrole in a polar organic solvent, for example, tetrahydrofuran, and in the presence of an acid, such as HCl, H2SO4, AcOH or a mixture to produce a first intermediate, and then reacting the first intermediate with an oxidization agent, such as dichlorodicyanobenzoquinone, Ag2CO3, or a mixture to produce the said compound of Formula II. The method may further include reacting Formula II with an alkali metal hydroxide to produce a compound of the compound 39 (R1=R3 =OH in Fig. 11).
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R1 O
R2
R7
O R3
R6
N
R4
R5 Formula II Compound (39): R1=R3= OH
Fig. (11). Structure of Formula II.
A second aspect of the invention is the compound of the Formula III and an acidcatalyzed method of producing these compounds (Fig. 12). O
R1
R9
R2
R8 R4
R3
O
N
R7 R6
R5 Formula III
Fig. (12). Structure of Formura III.
A further aspect of this invention is a method of treating a viral infection, a proliferative disease, and neurodegenerative disease. The method includes the administration of compounds of Formulas II and III. The active compounds described here may be formulated for administration in a pharmaceutical carrier in accordance with known techniques [97]. The administration routes of these active compounds are in pharmaceutically acceptable carriers for oral, rectal, topical, buccal, parenteral, intramuscular, intradermal, intra-venous, and transdermal administration. The preferred routes of parenteral administration include intrathecal injection, including directly into the tumor, and intraventricular injection into a ventricle of the brain. There are compara-tively fewer antivirals than there are antibiotics. Since viruses engage in much of their infective activity by hijacking a cell’s machinery and essentially directing the cell to manufacture virus particles, agents with an antiviral effect may additionally inhibit cellular functions in non-infected cells. For example, iododeoxyuridine, one of the first antiviral agents, depresses the DNA synthesis by inhibiting the incorporation of thymidine. The present invention describes use against several families of viruses, both in traditional antiviral targets and in families that currently have no antiviral medication. In one embodiment of the invention, compounds of the invention are effective in treating an infection by the viruses of the family Poxviridae, such as variola and vaccinia. Compounds included in this invention may have utility against Filoviridae, such as Ebola and Marburg, Hepadnaviridae, such as Hepatitis B,
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Herpesviridae, such as HSV-1, and Retroviridae. Tested compounds of this invention inhibited phosphatase Cdc25B. Cysteine Protease Inhibitors [98] The present invention relates to the compounds having one of the structures represented by 117 formulas, quinone and quinone analogs (examples in Fig. 13) useful for pharmaceutical preparations which inhibit cysteine proteases, in particular, the caspases and 3C-cysteine proteases. The cystein protease inhibitor is used for treatment of viral diseases, neurodegenerative diseases, and inflammatory diseases. Q1
O Z2 Z1
A
O
O
R2
Q2
R2
R1
Q3
R1
R2
Z2
R1 R17
O
Q4 (40)
Z1
(41)
Q1
(42) R18
Q1 O
Q2
R2
Q3
R1
R2
Q3
R1 Q4
R16 R17
O
Q2 Q3 Q4
R17
O
(44)
(43)
R18
O
Q2
O
Q1
R16
O
Q1 R2
Q2
R1
Q3
O
R2 O Q4
O (46)
(45) Q2
Q10
Q1
Q9
O Q3
Q8
N Q4
O O Q5 (47)
Fig. (13). Structures of quinone analogs.
Q6
O
Q7
R1
Anti-Infective Quinone Derivatives
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Cysteine proteases are a major family of peptide-bond-cleaving hydrolases isolated from viruses, bacterial protozoa, plants, mammals and fungi, wherein the thiol group of the cysteine residue serves as a nucleophile in the catalytic process. A variety of physiological disorders or diseases have been attributed to the presence of excessive or insufficient levels of cysteine proteases. The caspases (one family of cysteine proteases) are involved in the biochemical pathway that mediates apoptosis. Apoptosis is one method by which multicellular organisms eliminate unwanted cells. Normally, apoptosis is a means for regulating the cell number, facilitating morphogenesis, and eliminating harmful, abnormal or nonessential cells. Inappropriate apoptosis has been implicated in a number of diseases. Modulators of apoptosis are a potential target for therapeutics for these diseases. Inhibitors of caspases have been shown to be useful for the treatment of diseases in which excessive apoptsis occurs, such as Alzheimer, Perkinson, etc., and enhancers of caspases in which insufficient apoptosis occurs, such as cancer, viral infections and certain autoimmune diseases [99-102]. Recognized as important proteins in the maturation of the picornaviral life cycle, the 3C and 2A proteases have been a prime target for extensive structural and mechanistic investigations during the past few years [103]. While a variety of compounds have been identified to treat viral diseases by reacting with certain 3C protease or 3C protease-like proteins, which are essential for viral replication and the activity of various proteins [104-106]. Several chemical compounds useful as inhibitors of cysteine proteases, in particular, caspases and 3C cysteine proteases have been found. These inhibitors can be used in in vitro applications as well as pharmaceutical preparations. ANTIFUNGAL APPLICATION Fungi includes yeasts, molds, and freshly fungi (mushrooms). Yeasts are unicellular and molds are multicellular filamentous organisms. Cell type of fungi is eucaryotic with welldefined nuclear membrane. Cell membranes contain sterols and cell walls contain glucans, mannans, and chitin. Of the more than 100,000 species of fungi, only about 100 are pathogenic for humans and other animals. Benzonaphthacenequinone Derivative [107] This invention relates to a novel compound that is prepared by covalently bonding a benzonaphthacenequinone having an antimicrobial activity to a specific organic compound, thus providing the antimicrobial activity of the benzonaphthacenequinone, and increasing the solubility in water to attain the efficient and selective transportation of medicines with high safety and reduced side-effects. During the screening for microbial products as lead compounds for the treatment of mycoses, benanomicin was isolated from Actinomadura sp. by Gomi [108] and pradimicin was from Actinomadura hibisca by Oki [109]. Benanomicin and pradimicin, termed Mannose-Binding Quinone glycosides (MBQ) [110], are composed of a polyketide-derived benzo[a]naphthacenequinone aglycon, a D-amino acid and monosaccharide residue. MBQ recognizes D-mannosides and binds to the yeast cell surface [111, 112] in the presence of calcium ions. This binding is essential for MBQ to exert its fungicidal action. MBQ has an ideal profile for an antifungal agent, with high selectivity, fungicidal activity, low toxicity, and broad spectrum. Although this development has been withdrawn due to side-effects, the MBQ derivative is believed to be one of the most promising candidates. In this invention, a new compound is a benzonaphthacenequinone covalently bonded to a polyether or glycosaminoglycan represented by the formula (R1 is H, lower alkyl, lower
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hydroxyalkyl; R2 is hydroxy group, amino group, mono- or di(lower alkyl)amino; R3 is H, nonsulfated or sulfated D-xylosyl, D-glucosyl; R4 is H, lower alkyl, D-xylosyl), typically monodecyloxy-tetraethylene glycolbenanomicin A. The compound is prepared, for example, by protecting the hydroxyl groups, then allowing the carboxyl group to react with the amino groups or hydroxyl groups to form the acid amide bonds or ester bonds. Also claimed are pharmaceutical compounds containing the derivatives or their salts (Fig. 14). For example, 8.7 mg of benanomicin in DMF was treated with 29.0 mg of aminohexaethylene glycol methyl ether in the presence of dicyclohexylcarbodiimide to produce 8.5 mg of the amide product. The benanomicin A polyethylene glycol derivative showed a dose-dependant antifungal activity against Candida albicans 3143, and the activity was enhanced in the presence of calcium. R1 CONHCHCOX HO OH
O R4
CH3
O
OH
OH
O
O
O
CH3
HO R3O
(48)
R2
Fig. (14). Structures of benzonaphthacenequinone derivative.
The derivative of this invention can be used in the medical treatment of various diseases (infective desease, HIV, cancer, etc.) of mammals including humans. ANTIPROTOZOAL APPLICATION Protozoans are one-celled, eucaryotic organisms that belong to the Protista. All protozoans live in areas with a large supply of water. Protozoans are mostly aerobic heterotrophs and classified by their means of locomotion: the Sarcidina move by amoeboid motion; the Mastigophora use flagella for motility; the Sporozoa lack a means of locomotion and are obligate parasites; the Ciliata possess cilia. Atovaquone Derivatives [113] Pharmaceutical combination of 2-[4-(4-chlorophenyl) cyclohexyl]-3-hydroxy-1,4naphthoquinone and proguanil and pharmaceutical preparation: The present invention relates to the synergistic combination of 2-[4-(4-chlorophenyl)cyclohexyl]-3-hydroxy-1,4naphthoquinone (atovaquone) and proguanil which have anti-parasitic activity. The compound 2-[4-(4-chlorophenyl)cyclohexyl]-3-hydroxy-1,4-naphthoquinone (atovaquone) has previously been disclosed, for example in EP123238 [114] which relates to the 2-substituted 3-hydroxy-1,4-napnthoquinones of Formula IV having antiprotozoal activity. Specifically, compounds of Formula IV wherein n is zero are said to be active against the human malaria parasite Plasmodium falciparum and also against Eimeria
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species. Among the compounds specifically named is atovaquone having the formula in which n is zero, R1 is hydrogen and R2 is the 4-chlorophenyl group (Fig. 15). Proguanil is a well-known drug for prophylaxis, but not for the treatment of malaria. It is one of the safest antimalarial drugs. However, a resistance of P. falciparum to proguanil has appeared, particularly in southeast Asia. In order to combat drug resistance, it is becoming standard practice to use combinations of more than one antimalarial, either simultaneously or sequentially. However, many such combinations are antagonistic, resulting in a reduced effectiveness. In this invention, it has been found that potentiation of the antiparasitic and antimalarial activities is achieved by combining, either concomitantly or sequentially, atovaquone and proguanil. Atovaquone inhibits the electron transport system in the mitochondria of parasites, thereby blocking nucleic acid synthesis and inhibiting replication [115]. Proguanil also acts against the asexual erythrocytic stage of the parasite by selectively inhibiting plasmodial dihydrofolate reductase. But it significantly enhanced the ability of atovaquone to cause collapse of the mitochondrial membrane potential when used in combination [116]. The present invention provides a method for the treatment and/or prophylaxis of a protozoal parasitic infection such as malaria and toxoplasmosis, and an infection caused by P. carinii in mammals including humans, which comprises administering an effective amount of atovaquone or a physiologically acceptable salt thereof and concomitantly or sequentially administering an effective amount of proguanil. The pharmaceutical combination of proguanil and atovaquone is present in a weight ratio ranging from 1:1 to 1:3. Preferred are the pharmaceutical preparations containing 50 mg to 3 g of each of the agents, more preferably 500 mg of atovaquone and 200 mg of proguanil. O
R1 (CH2)n
Formula IV
R2 OH O O Cl
(49) atovaquone OH O
Fig. (15). Structures of atovaquone derivatives.
Naphthoquinone Derivatives [117] This invention relates to naphthoquinone derivatives isolated from solid cultures from two species in the palaeotropical plant families Dioncophyllaceae and Ancistrocladaceae. It further relates to the methods of their production and their use as antileishmanial pharmaceuticals. Leishmaniasis is a widespread parasitic disease caused by protozoan parasites of the genus Leishmania [118]. This disease occurs in two major forms: cutaneous (CL) and visceral leishmaniasis (VL). The most severe form, VL, known as kala-azar, is nearly
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always fatal if untreated. Leishmaniasis is endemic in tropical and subtropical areas in both the Old and New Worlds. It is estimated that about 1.5 million new CL cases and 0.5 million VL cases occur annually, and over 350 million people are at risk of infection [119]. Over 20 species of Leishmania have been identified. VL is mainly caused by L. donovani in which the parasite spreads from the site of inoculation to multiply as an amastigote in macrophages in the spleen, liver, lymph nodes, and bone marrow. In CL, the disease is normally localized to the site infection within dermal macrophages. Typically, papules develop at the site of infection, enlarge to a nodule, and progress to ulcerated lesions, which last for less than a year. Pentavalent antimonials, e.g. sodium stibogluconate and meglumine antimoniate, have been used for over 50 years as the first-line parenteral therapy for all types of leishmaniases. However, these drugs require long courses of parenteral administration, have variable efficacies with potential harmful effects, and are associated with increasingly efficacy due to drug resistance. Hence, it is highly important to develop more effective and less toxic drugs for the treatement of leishmaniasis. The search for new bioactive compounds often starts with the plant kingdom with traditional folk remedies and leads to a number of additional antiparasitic agents in the pharmacopoea. A phytochemical analysis of tropical lianas of the families Dioncophyllaceae and Ancistrocladaceae led to the discovery of a number of novel natural products exhibiting a broad spectrum of anti-infection activities [120-123]. The anti-protozoal activity of hydroxynaphthoquinones was reported over 10 years ago [124]. Caltivating these tropical lianas under modified conditions and establishing callus cultures from sterile plant parts expose the plants to chemical and physical stress [125], thereby causing the formation of secondary metabolites. For example, two 1,4-naphthoquinones plumbagin and droserone were detected in callus cultures of Triphyophyllum peltatum Airy Shaw (Dioncophyllaceae) and Ancistrocladus abbreviates Airy Shaw (Ancistrocladaceae) [126, 127]. Many naphthoquinones have been tested against L. donovani, and other species both in vitro and in vivo as oral, subcutaneous, and topical administrations. The object of the present invention is to provide further bioactive substaces that can be used to treat of infectious diseases (Table 4) and tumors, as well as the method for their productions. These goals are met by a compound with the general formula V that can be Table 4. Activities against Leishmania Donovani
Compound
Inhibition of Cell Growth (%) (c=4.8 μg/mL)
Inhibition of Cell Growth (%) (c=0.8 μg/mL)
50
80.3
49.6
51
87.6
79.2
52
31.7
11.4
53
30.5
19.6
54
28.6
0.3
miltefosine (control)
53 (c=0.22 μg/mL)
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used to treat and/or prevent tumoral and/or infectious diseases. (Fig. 16) The aims of the present invention are further solved using a compound preparation method that involves cultivating callus cultures of tropical lians of the families Dioncophyllaceae and Ancistrocladaceae as well as other related families under specifically optimized conditions, isolating at least one defined compound, and further comprising the synthetic derivatives of the isolated compound(s). The invention has also provided general methods for the chemical synthesis of compounds that have the general formula V. Currently, the anti-leishmanial mechanism is not clear. Diospyrin, which has the significant anti-leishmanial activity as a bisnaphthoquinone, was found to specifically inhibit the type I DNA topoisomerase enzyme of Leishmania donovani [128]. DNA topoisomerases are considered to be important therapeutic targets for the retional design of anti-protozoal drug. The proposed compounds may inhibit the electronic transport system as well as atovaquone or the DNA topoisomerase enzyme as well as diospyrin. The proposed mode of action for hydroxynaphthoquinones involves the ability of the drug to form free radicals during the interaction with the parasite’s respiratory chain. OR2
OH
O
R3O
OR1
O
RO
OH CH3
CH3
R4 R5
O
O
(50) R= sugar (51) R= H
Formula V
OR1
OCH3 O H3CO
OH CH3 OH
O (52)
O
O
OH CH3
O R2
O
(53) R1= H, R2= OH (54) R1= CH3, R2= H
Fig. (16). Structures of naphthoquinone derivatives.
CURRENT AND FUTURE DEVELOPMENTS Quinone compounds are intermediates in many pathways of gene regulation, enzyme protein induction, feedback control, and waste product elimination in addition to the role as substrates and products in metabolism. Quinones play a pivotal role in energy metabolism, many other key processes, and even in chemotherapy where redox cycling drugs are utilized. However, the molecular mechanisms involved in quinone cytotoxicity and pharmaceutical activity are still mostly unknown. Their widespread use as antibiotics, antiparasitic agents, antitumor agents, and a variety of other agents makes it imperative to understand their effects on cellular function. Until this is clarified, it is not possible to use a rational approach to search for or design more effective quinone agents with less side-
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effects, and the current approach of random screening and analog development will continue. Malarone (atovaquone and proguanil) as antimalarial agent and the bamboo extract containing benzoquinone as antichlamidial detergent in this review will be approved for use because of their commercial clinical use and low toxicity. The fate of new other compounds will be decided in clinical trials. It is unclear that research will yield the next breakthrough discovery, but it is certain that therapeutic advances will continue to happen. REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20]
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Targets and Patented Drugs for Chemotherapy of Chagas Disease Vilma G. Duschak*, 1 and Alicia S. Couto2 1
Instituto Nacional de Parasitología “Dr. Mario Fatala Chabén”, ANLIS-Malbrán, Ministerio de Salud. Av. Paseo Colon 568 (1063), Buenos Aires, Argentina; 2CIHIDECAR (CONICET) Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, CP 1428, Argentina Abstract: Chagas disease or American Trypanosomiasis, a parasitic infection typically spread by triatomine bugs, affects millions of people throughout Latin America. Current chemotherapy based on the nitroaromatic compounds, benznidazole and nifurtimox provides unsatisfactory results and suffers from considerable side effects and low efficacy. Therefore, there is an urgent need for new drugs to treat this neglected disease. Over the last two decades, new advances and understanding in the biology and the biochemistry of Trypanosoma cruzi have allowed the identification of multiple targets for Chagas´ disease chemotherapy. This chapter summarizes antichagasic agents obtained based on i) target metabolic biochemical pathways or parasite specific enzymes, ii) natural products and its derivatives, iii) design and synthesis of lead compounds. Related patents filed and issued from 2000 to early 2009 are also discussed. Most of them claimed inhibitors on specific parasite targets such as cysteine proteinase, sterol biosynthesis, protein farnesyltransferase, etc. Particularly, those related with cysteine proteinase inhibitors were the most represented. Natural products also displayed many anti-T cruzi lead compounds. In addition, a few patents claiming natural or synthetic compounds with antichagasic activity, disclosed no specific target. However, only a small proportion of all these patents displayed specific data of biological trypanocidal activity.
Keywords: Chagas disease, Trypanosoma cruzi, drug targets, natural and synthetic inhibitor compounds.
INTRODUCTION Tropical parasitic diseases are produced by different eukaryotic protozoa. Among them, trypanosomes are known to be responsible for sickness presenting quite different clinical manifestations, geographical distribution, life cycle and insect vectors [1]. Chagas disease, also known as American Trypanosomiasis is one of the most serious protozoan diseases which occurs throughout Latin America, particularly in South America. Its etiological agent is Trypanosoma cruzi (T. cruzi), a flagellate protozoan, which is transmitted to humans and *Corresponding author: Tel: (+5411) 4331-4010/19; Fax: (+5411) 4331-7142; E-mail: [email protected] Atta-ur-Rahman / M. Iqbal Choudhary (Eds.) All rights reserved – © 2010 Bentham Science Publishers.
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other mammals mostly by hematophagous insects of the Family Reduviidae, subfamily Triatominae. T. cruzi has a complex life cycle, with proliferative stages in the vector (epimastigotes) and the vertebrate (intracellular amastigotes), as well as non-proliferative infectious stages (trypomastigotes) in both hosts. The World Health Organization has estimated that some 16-18 million people are infected throughout the American continent, and that more than 100 million are at risk [2]. The disease has also emerged as a public health problem in the United States of America and Europe [3, 4]. Despite Chagas disease transmission has been eliminated in several countries by control of the Triatomine vector using insecticide spraying and serological screening of blood donors [5, 6], the disease continues to be endemic in large areas of Latin America. The disease is characterized by three clinical phases: acute, indeterminate and chronic. In the acute phase, a local inflammatory lesion appears at the site where metacyclic trypomastigotes enter, and the parasite spreads throughout the host organism. The indeterminate phase comprises a period that may last 10-20 years between the acute and chronic phases and is generally symptomless. On the contrary, the chronic phase is characterized by the presence of myocarditis and/or pathological disturbances in the peripheral nervous and gastrointestinal systems. Thirty to forty per cent of chronic infected individuals develop cardiac abnormalities and as many as 10 % develop digestive tract disease [7]. Recently, night blindness was investigated as new clinical symptom in patients with chronic Chagas' disease and retinal dysfunction was associated to anti-Trypanosoma cruzi antibodies that cross-react with rhodopsin [8]. Two mechanisms were proposed for pathogenesis in the chronic phase: inflammatory reactivity due to the persistence of the parasite inside the host tissues and induction of auto-immune responses targeted in infected tissues. Both events would indicate that the elimination of T. cruzi from infected patients would lead to arrest the evolution of the disease [9]. Diagnosis of Chagas disease has been performed by the traditional direct detection of the parasite in blood during the acute phase or by serodiagnosis. DNA amplification using the polymerase chain reaction (PCR) as well as single or mixtures of recombinant antigens used for serodiagnosis, are currently available tools to evidence the presence of the parasite [10, 11]. In addition, the use of chimerical synthetic peptides containing antigenic sequences of immunodominant regions of T. cruzi as coating antigens have shown to be useful for the immunodiagnosis of this disease [12]. To date, there are no prophylactic drugs to prevent infection with T. cruzi. Moreover, current chemotherapy of Chagas disease based on the nitroaromatic compound benznidazole is questionable because it provides unsatisfactory results, suffers from considerable side effects and is effective only for recent (acute, congenital or experimental) infections and its utility during the chronic phase of Chagas disease is controversial. However, the use of this compound may pose a lesser risk to heart function than nifurtimox when any cardiopathy is present [13, 14]. Taking into account that nifurtimox and benznidazol are far from the requirements to consider them ideal as trypanocidal drugs (very safe, very effective, very stable and inexpensive) in addition to the fact that in the last decade trials with allopurinol showed poor results [15], the search for new compounds with anti-T. cruzi activity, with low toxicities and increased efficacies during the indeterminate and chronic phases, continues. The identification of new antichagasic agents may be based not only on rational drug design and natural products screening [16], but also taking advantage of compounds already in use against other human diseases, which have already passed several of the clinical trials necessary for the development of any new drug. Thus, there is an urgent need to identify specific enzymes and metabolic pathways in the parasite useful as potential targets for drug
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development. However, in spite of the urgency of the matter, pharmaceutical industry has restricted investment in research and development in this disease [17]. In addition, health innovation networks to help developing countries address neglected diseases were created [18]. On the other hand, as a result of the parasite genome sequencing project, available since 2005 [19] the possibility of identifying new specific pathways and novel drug targets in the near future is opened. Over the last two decades, new advances and understanding in the biology and the biochemistry of T. cruzi has allowed the identification of multiple targets for Chagas disease chemotherapy. The main promising targets for antiparasitic agents involve proteinases (particularly cysteine proteases), sterols and isoprenoids biosynthetic pathways and thioldependent redox metabolism. In addition, polyamine metabolism and transport pathways, enzymes of the glycolytic and pentose phosphate biosynthetic pathways, lipidic (alkyllysophospholipids, glycosphingolipids) and purine salvage pathways, have also been intensively studied. Moreover some organelles functions including DNA modulation in nucleus and kinetoplast involving topoisomerases as well as the exchanger Na+/H+ mechanism from acidocalcisomes are also considered promising targets for antiparasitic drugs. Among them, particularly those that target the validated biochemical pathways of the parasite including cysteine proteinase inhibitors (CPIs) and inhibitors able to block ergosterol biosynthesis are currently in the pipeline. In summary, the aim of this review is to present a whole view including patents and recent advances on antichagasic agents obtained from different sources. Data were divided into three major sections: Part I will include targets and most patents referring to specific drug targets. Part II will refer to natural compounds and their derivatives as chemotherapeutic agents. Part III will include designed and synthesized parasiticidal drugs. Metabolic pathways or specific enzymes used as targets and patents will also be discussed. The search strategy for patent literature claiming for trypanocidal activity against T. cruzi was performed through the Delphion Research intellectual property network including international and US patent search database (2000-early 2009).
DEVELOPMENT OF NOVEL DRUGS AGAINST CHAGAS DISEASE PART I. DRUG TARGETS AND LEAD COMPOUNDS T. cruzi Metabolism-Targets Trypanosomes diverged very early from the common eukaryotic lineage, probably due to independent evolution of Kinetoplastida, one of the oldest lineages of protozoa [20]. Thus, they have several unusual biochemical pathways which differ in numerous aspects from that of mammalian cells. This fact may provide selective targets for drug development, particularly, rational design of metabolic pathway inhibitors or for specific enzymes chosen as drug targets. However, it is worth mentioning that a simple difference between host and parasite is not sufficient to consider a compound as a drug target. Target validation is an essential step in any rational approach to chemotherapy. The usual method to verify that an enzyme is essential for an organism is based on the use of a highly specific inhibitor, but such compounds are not always available, so genetic approaches such as knock-out mutants or the inducible depletion of the specific mRNA by RNA interference, are now widely in use [21, 22]. Moreover, although an enzyme has proven to be essential, it may not constitute
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necessarily a drug target. In fact, a high protein abundance of the target enzyme will difficult the maintenance of the high drug concentration required for the binding of a reversible inhibitor within the cell. On the other hand, a rapid de novo synthesis of the essential enzyme would overcome the effect of an irreversible inhibitor.
1- PROTEINASES Proteinases play multiple roles in disease pathogenesis. They have been involved in host invasion, in the migration of the parasite through tissue barriers, in the degradation of haemoglobin and other blood proteins, in immune evasion as well as in activation of the inflammation process. The multiple roles suggested for several of the T. cruzi proteolytic enzymes make them attractive potential targets for the development of new drugs against Chagas disease [23]. The complete sequence from T. cruzi clone CL Brener genome has allowed the prediction of seventy cysteine proteinases (CPs), forty serine peptidases (SPs, none of them belonging to the chymotrypsin family), about two hundred and fifty metallopeptidases (MPs, most of them presenting homology to leishmanolysin), twenty five threonine peptidases with high homology to proteasome subunits, and only two aspartic peptidases which do not belong to the pepsin family [19].
1A-CYSTEINE PROTEINASES CPs regulate host-parasite interaction being involved in modulation of a variety of pathobiological effects including nutrient uptake, immune evasion and host tissues degradation. The specific inhibition of these enzymes by immunoprophylaxis or chemotherapy may potentially impair the survival mechanisms of the parasite. Therefore, CPs are promising targets for vaccines or chemotherapy. Cruzipain (Cz), also known as cruzain or GP57/51 [24-26], the most abundant member of the papain C1 family of CPs of the parasite, is expressed as a complex mixture of isoforms by the major developmental stages of the parasite and present microheterogeneities [27]. Although the bulk of the enzyme is lysosomal, it is also present in an epimastigotespecific pre-lysosomal organelle called 'reservosome'. In addition, some plasma membranebound isoforms [28] and Cz forms released into the medium [29] have been reported. The T. cruzi enzyme consists of a catalytic domain with high homology to cathepsins S and L and a particular C-terminal domain (C-T) which is absent in all other CPs of the C1 families described so far [30]. The enzyme is an immunodominant antigen in human chronic Chagas disease and seems to be important in the host/parasite relationship, it was associated with virulence [31], the interaction between plasma membrane-bound isoforms with alphamacroglobulins was reported [32] and the humoral immune response to Cz appeared to be related with the severity of chronic Chagas disease [33]. Since membrane bound isoforms of Cz were detected, and sialylation is a surface reaction in T. cruzi, it was interesting to identify the presence of sialic acid in the C-terminal domain of cruzipain. In addition, Nacetyl-D-glucosamine in O-glycosidic linkages has also been determined. These findings might contribute to elucidate the migratory route followed by Cz [34]. Interestingly, we have reported for the first time the presence of sulfated oligosaccharides in this glycoprotein [35]. These structures are main targets for immune responses and are involved in tissue damage in mice immunized in absence of infection [36]. Finally, all the aspects related to the major CP of T. cruzi studied so far including recent advances as proteinase, antigen and glycoprotein were recently reviewed [37].
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The addition of fluoromethyl ketone as CPI to infected mammalian cells showed that the enzyme (Cz) is essential for replication of the intracellular parasite and the differential susceptibility of parasite versus host CPs to these inhibitors suggested that T. cruzi major CP might represent a potential lead target for new chemotherapy of Chagas disease [38]. However, the possibility that some other, minor and highly specific, CPs may be involved in the inhibition of the parasite life cycle, should not be discarded. Recently, we have reported a novel CP present during T. cruzi metacyclogenesis [29]. In addition, the presence of a group of atypical Cz molecules which do not bind to ConA-Sepharose columns (NACrI), that represent a minor sub-class with a different oligosaccharide pattern and different preference of chromogenic substrates, was also studied [39]. The advances in the study of the structure and specificity of Cz, including the obtainment of the crystal structure bound to various inhibitors [40, 41], favored the development of new and more specific inhibitors. However, not only the presence of minor CPs but also atypical responses to inhibitors of other classes of proteinases should be taken into account. In fact, it was described that the SP oligopeptidase B was strongly inhibited by the CP inhibitor Z-Phe-Arg-fluoromethylketone [42] in a similar way to the atypical SP oligopeptidase B from Trypanosoma brucei [43]. CP INHIBITORS (CPIs) Studies were performed with synthetic peptidyl and non peptidyl inhibitors. Among peptidic compounds, the following groups of irreversible or reversible inhibitors can be mentioned (Table 1): Table 1. Chemical Structures of Peptide-Based Cysteine Proteinase Inhibitors (CPIs). Representative Compounds A. Irreversible O HN
NH
O
O
Leu-Val-Gly-CHN2
O
N H
N S
O
H Peptidyl diazomethane [44]
S
H N
O
O
O
H N
S O
O O
Peptidyl ketone based [45]
O
O
N
N
N
O
N H
O
O
O
H N
S
H N
NH H N
O n Peptidyl vinyl sulphone [46-53]
O
H
N O
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(Table 1) Contd…. B. Reversible OH
O
OH
N N H O
O N H
BnO
H N
O OMe
O
O
O H N
Bis-arylacylhydrazides [54] S
O N H O
O
i-Pr Ketone based (cyclic structures) [56, 41-57]
N O
O
O
H N
N
S
N
O
O
NH O N O Br
O
O
O N
O Azepanone based [68-70]
Nitrile based [71-75]
I1-IRREVERSIBLE PEPTIDIC INHIBITORS a- Peptidyl Diazomethane Inhibitors The interaction between Cz and biotin-labeled peptidyl diazomethane inhibitors showed a strong reaction when the inhibitor included a spacer arm containing part of the sequence of known proteic inhibitor, cystatin, at difference with the mammalian counterparts, probably due to differences in the topologies of the binding site [44]. b- Peptidyl Ketone Based Inhibitors The design and synthesis of a variety of peptidyl fluoromethylketones, potent irreversible inhibitors of Cz, revealed that dipeptidyl alpha', beta’-epoxy ketones resulted more effective inhibitors of Cz than E-64c. In addition, D-Phe- and D-Tyr containing epoxysuccinate derivatives from the peptidyl-epoxysuccinate E-64, selective irreversible inhibitor of CP, obtained by substituting the L-Leu residue of this compound, showed to be potent irreversible inhibitors of Cz but they were little effective against T. cruzi in cell cultures [45].
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c- Peptidyl Vinyl/Allyl Sulphone Inhibitors The potential toxicity associated to the use of the known irreversible inhibitors led to the screening of compounds including vinyl sulphones. The first report of trypanocidal activity involved to these compounds. It was demonstrated by in vivo assays that vinyl sulphone derivatized dipeptides were able to effectively rescue the mice from an acute lethal inoculation of T. cruzi reducing the number of parasites in blood of infected animals significantly although chronic phase was not evaluated [46]. In particular, the vinyl sulphones morpholinourea-FhF-vinyl sulphone phenyl (MFhFVSPh) and morpholinourea-FhF-fluoromethylketone arrested growth of the epimastigotes and caused parasite death, probably due to accumulation of the enzyme in the Golgi [47]. The fact that these compounds inhibit Cz allowed to identify it as a promising therapeutic target in the treatment of Chagas disease [48]. Besides, a second generation of new potent N-alkoxyvinylsulfonamide inhibitors of Cz has been developed. One of them, named inhibitor 13 resulted to be highly effective against T. cruzi trypomastigotes in a tissue culture assay [49]. In addition, the novel dipeptidyl allyl sulphones were determined to be more potent than the dipeptidyl vinyl sulfones [50]. Two patents related to these compounds (Table 6) were disclosed by Georgia Tech Research Corporation, providing peptidyl allyl sulfone compositions for inhibiting proteases, methods for synthesizing the compositions, and methods of using the disclosed protease inhibitors either in vivo or in vitro [51]. The structure/activity relationship (SAR) based design has evolved focusing on irreversible compounds, most of which rely particularly on covalent attachment to the active enzyme thiol group, to minimize the potential toxicity associated to the use of reversible inhibitors. Among vinyl sulphones, Doyle et al., 2007 [52] have reported that the dipeptidic inhibitor N-methyl-Pip-F-homo F-vinyl-sulfonylurea phenyl (K11777) is in late-stage preclinical development and have studied the course of infection in immunodeficient and normal mice infected with T. cruzi, finding that immunodeficient mice treated with this dipeptidic inhibitor rescued them from lethal Chagas infection. The immunodeficient mice treated with the inhibitor had increased survival, negative PCR, and normal tissues by histopathological examination. On the other hand, vinyl sulphonecontaining macrocycles were synthesized via olefin ring-closing metathesis to evaluate conformationally constrained inhibitors. Unfortunately, they resulted substantially less active as inhibitors of cruzain and other CPs compared to the acyclic vinyl sulphone K11777 [53].
I2-REVERSIBLE PEPTIDIC INHIBITORS d- Bis-arylacylhydrazides, Aryl Ureas Some reversible inhibitors, have been designed based on the known structure of the active site of Cz, and synthesized including a family of bis-arylacylhydrazides [54] and some aryl ureas [55] as new class of CPIs. e- Ketone Based Inhibitors Among potent ketone based peptides, some of them reversible against Cz by formation of hemithioacetal complexes with CPs, inhibiting the enzyme in the nM range, have been developed by using solid-phase parallel synthesis [56]. Crystal structures of these reversible ketone-based inhibitors of Cz were studied [41]. Choe and co-workers synthesized a novel series of alpha-ketoamide-, alpha-ketoacid-, alpha-ketoester-, and aldehyde-based inhibitors
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of Cz. Some of them displayed picomolar potency in in vitro assays and three inhibitors representing different alpha-keto-based inhibitor scaffolds demonstrated anti-trypanosomal activity in cell culture [57]. A search for patents showed that scientist from Glaxo Smith Kline Corp designed a series of cyclic ketone compounds as protease inhibitors which form a hemithioacetal with the cys 25 residue and retain reliable oral bioavailability and improved pharmacokinetics. However, these compounds have not been tested against Cz [58]. In order to address the epimerization problem in the ketone based inhibitors, Medivir UK Ltd, Genzyme Corp have reported the synthesis of a series of substituted amides and 2 acylamide-bicyclic ketone derivatives as inhibitors of CPs and its potential use in infectious diseases including Chagas disease. In the first patent dealing with substituted amides [59], a tetrahydropyran-3-one derivative was used as cathepsin S inhibitor but no biological data were presented. Similarly, Incenta have also designed a series of peptide mimics 2-acylamino bicyclic ketone derivatives including tetrahydrofuran-3-one derivatives which claimed to be more potent inhibitors of Cz than those of the previous series mentioned. In addition, Amura also patented inhibitors of Cz and other CPs [60-63]. Similarly, Amura disclosed a series of pirrole compounds, with activity on Cz and also cathepsins K, S and L, useful for the in vivo therapeutic treatment of diseases in which participation of a CP is implicated [64] and other peptide based CPIs, claimed by Corvas International Inc as useful antiparasitic agents, were tested as effective against Cz (IC50 values lower than 50 nM), but no specific biological data are available [65]. Amura Therapeutics Ltd have also patented some amide molecules that inhibit Cz more effectively than they inhibit mammalian CPs, such as bovine cathepsin S, human cathepsins L and K [66] as well as inhibitor compounds of Cz and other CPs useful as therapeutic agents for Chagas disease, or for validating therapeutic target compounds [67], (Table 6). f- Azepanone Based Inhibitors In their search for cathepsin K inhibitors, Smith Kline Beecham Corp published several patents describing the synthesis and use of peptidomimetics based on azepine or thiazepane [68, 69]. These compounds were tested as cathepsin K inhibitors and claimed to be useful against different parasitic diseases including trypanosomiasis. However, only two patents reported biological data [68-70] and only the latter [70] disclosed the inhibition by 4aminoazepan-3 one derivatives of seven parasitic proteases including Cz in the analysis. 43 out of about 222 compounds tested, showed Ki values lower than 5 nM against Cz. The most potent CPIs against Cz were the 1-(pyridine 2-ylsulfonyl) azepan-3 one derivatives [70], (Table 6). g- Nitrile Based Inhibitors Novartis has patented a series of novel peptidic heteroaryl nitrile derivatives as therapeutic agents [71] for the treatment of osteoporosis and several parasitic diseases, one of them with Ki value of about 50 nM for human cathepsin K. The patent assessed that the compound would be useful in the prevention and treatment of several parasitic diseases including Chagas disease. The Combio Company [72] has recently disclosed a series of novel alpha-amino-carbonitrile-derived inhibitors of human dipeptidyl peptidase and cathepsin B, H and L, claiming that can be used for Chagas disease. However, data with regard to their efficacy against parasitic diseases have not been reported. Boehringer Ingelheim Pharmaceuticals, Inc disclosed 404 novel nitrile compounds claiming they were
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useful as reversible inhibitors for treatment of diseases mediated by CPs, particularly cathepsin K and S and a variety of pathological conditions exacerbated by these proteases but no detailed experimental results were shown [73,74]. Ten compounds were assayed against Cz with Ki values ranging from 0.09 to 20 M [75]. Although the specific claim of these patents, biological data for these nitrile based inhibitors (including some of them nonpeptidic) regarding their efficacy on parasitic diseases are also absent (Table 6).
II-NON PEPTIDIC INHIBITORS Structure activity relationships (SAR) for non peptidic inhibitors of Cz based on different scaffolds were reported, including the following (Table 2): Table 2.
Chemical Structures of Non Peptidic-Based Cysteine Proteinase Inhibitors (CPIs) Representative Compounds
a- Thiosemicarbazones R Cl
H N
N
H N
Br
NH2
NH2
N S
Cl
S [76- 85]
b-Aminoacylthiazolidones S N S N N H H
CH3
CH3
S N
S
N H
O CH3 [86, 87]
Cl
N
O
H
c- Ethenylbenzofuroxan derivatives
O2N N
F
O
N
S
O
O O
N
[88]
N
d-Inhibitors with cysteine protease mechanism-based N NH
N
F
O O
N
F
N NH
N
O
F N
F
N [89]
O S
N
Bu
Bu
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(Table 2) Contd…..
e- Oxorhenium (v) and Palladium (II) metal complexes S O S Re N S
O
N
S S
Re
Cl
S
[90]
a-Thiosemicarbazones Among non-peptidic inhibitors those based on the thiosemicarbazone lead were reported as active Cz inhibitor at the nM range; many of them, of small size and low cost showed trypanocidal activity against intracellular amastigotes in vivo making them attractive candidates for drug development [76-79]. The appearance of parasite populations resistant to some of these inhibitors was reported. A phenotypically stable cell line of T. cruzi (R-Dm28) displayed increased resistance to the irreversible CPI Z-(SBz)Cys-Phe-CHN 2, which preferentially inactivates cathepsin L-like enzymes suggesting that this fact could represent a possible limitation of CPs as targets for chemotherapy [80]. However, further assays with non-stable cell lines, showed that the phenotype was reversed upon removal of the inhibitor from the culture media [81]. On the other hand, the Chem Bridge data base was used for virtual screening to identify novel drug-like non-peptidic inhibitors of parasitic CPs. Several non-peptidic inhibitor compounds were able to avoid protease hydrolysis in living systems, retaining in vivo activity as well as selectivity [82]. Recently, it was reported that the treatment of dogs with K177, inhibitor of Cz, abrogated myocardial damage by T. cruzi, as documented by histopathological lesion scores and serum troponin I levels [83]. The design of lead optimization libraries of thiosemicarbazone inhibitors was performed. The screening of some of these compounds on different CPs and on their respective parasites showed that they were able to kill several species of protozoan parasites through the inhibition of CPs as well as other novel targets [84]. Among the active CPIs tested, several inhibited proliferation of cultures of T. brucei potently but only a modest activity was observed in inhibition of T. cruzi growth [79]. In the years 2005 and 2009, Reagents of the University of California presented two patents related to thiosemicarbazone and semicarbazone inhibitors of CPs and methods of using such compounds to prevent and treat protozoan infections such as trypanosomiasis, malaria and Leishmaniasis [85], (Table 6). b- Aminoacylthiazolidones A novel series of thiosemicarbazone and aminoacyl thiazolidones derivatives were also synthesized. Some of them were able to inhibit T. cruzi growth in non-cytotoxic concentrations to mammalian cells [86]. In vitro studies performed with aryl-4-oxothiazolylhydrazone derivatives against T. cruzi have shown to be very active at non-cytotoxic concentrations in in vitro assays with mammalian cells and showed potency comparable with reference drugs [87].
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c- 5-Ethenylbenzofuroxan Derivatives These compounds were developed and studied as antiproliferative T. cruzi agents displaying remarkable in vitro activities against different strains and were able to reduce the parasite loads of animals with fully established T. cruzi infections [88]. Recently, by introducing additional binding interactions in the S3 pocket of cruzain, optimized substrates were converted to inhibitors by the introduction of CP mechanism-based pharmacophores. One of them showed to be reversible even after the incorporation of the vinyl sulphone pharmacophore which is well documented as irreversible cruzain peptidic inhibitor. d- Inhibitors with CP Mechanism-Based Another, a previously unexplored beta-chloro vinyl sulphone pharmacophore led to the development of potent irreversible acyl- and aryl-oxymethyl ketone cruzain inhibitors. Among these inhibitors, 2, 3, 5, 6-tetrafluorophenoxymethyl ketone was identified as one of the most potent inhibitors against this enzyme describing its capacity to eradicate the parasite from mammalian cell cultures completely [89]. e- Oxorhenium (V) and Paladium (II) Metal Complexes The activity of gold (III), and palladium (II) cyclometallated complexes, and oxorhenium (V) complexes against mammalian and parasitic CPs was investigated. Six complexes were tested against the parasite CPs, cruzain from T. cruzi, and CPB from L. major; the most potent inhibitors were two rhenium complexes. The compounds were also evaluated in assays for parasite growth. Preliminary results showed that two oxorhenium (V) compounds and the palladium compound 11 inhibited T. cruzi intracellular growth suggesting that metal complexes targeted at parasite CPs showing promise for the treatment of both Chagas disease and Leishmaniasis [90].
MINOR CPS The presence of cathepsin B-like CPs in T. cruzi was demonstrated but it is not still known neither how many different enzymes of this type are present or their possible functions. Among them, a 30 kDa cathepsin B-like enzyme has been described [80, 91]. On the other hand, the presence of a novel CP, TcCPmet, secreted by metacyclic trypomastigotes was reported. This novel CP showed a different elution pattern on ConA-Sepharose than Cz and was not recognized by anti-Cz serum. In addition, TcCPmet was able to hydrolyse the same chromogenic peptides as Cz at optimal alkaline pH values, although with a different order of effectiveness. The results obtained strongly suggest a different nature between TcCPmet and Cz [29]. Although there is no data still available, these minor CPs may constitute new targets for the development of novel inhibitors. Regarding the results obtained so far with this type of drugs on animal models, an effective chemotherapy of the American Trypanosomiasis based on CPIs seems to be possible in the near future.
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1B-SERINE PEPTIDASES (SPs) Oligopeptidase B is a member of the prolyl oligopeptidase family involved in Ca2+ signaling during mammalian cell invasion [92,42]. A secreted prolyl endopeptidase (Tc80), with collagenolytic activity, was also purified and partially characterized from T. cruzi [93]. The inhibition pattern and its ability to hydrolyze peptide bonds at the carboxyl side of Pro residues suggested that the enzyme is a prolyl endopeptidase also belonging to the S9ASP family, but distinct from the oligopeptidase B. Selective inhibitors of the enzyme have been synthesized [94, 95], with Ki values in a low nM range, and shown to be able to block the entry of the parasite into the host cells [96]. This SP looks, therefore, as a new very promising target for the development of new drugs against Chagas disease. Other putative SPs have also been described [23]. Recently, a secreted 75 kDa T. cruzi serine oligopeptidase was purified and the subcellular localization was restricted to intracellular structures, including the flagellar pocket, plasma membrane and cytoplasmic vesicles resembling reservosomes [97].
SERINE PROTEINASE INHIBITORS Synthetic prolylprolylisoxazoles and prolylprolylisoxazolines, potent inhibitors of human and trypanosome prolyloligopeptidase (POP), were shown to inhibit T. cruzi and T. brucei in vitro systems with ED50 in the low M range [98]. Novel inhibitors were assayed with rPOP Tc80, and the most efficient ones presented values of inhibition coefficient Ki lower than 1.52 nM. Infective parasites treated with these specific POP Tc80 inhibitors attached to the surface of mammalian host cells, but were incapable of infecting them [99].
1C-METALLOPROTEINASES Enzymes with homology to the gp63 of Leishmania spp. are also present in T. cruzi [23, 100]. Studies related with metalloproteinases inhibitors have not been still reported in T. cruzi.
1D- ASPARTYL PROTEINASES Once that the presence of two aspartyl proteinases was predicted after the obtention of the complete sequence from T. cruzi genome [19], two aspartyl peptidase activities, cruzipsin-I (CZP-I) and cruzipsin-II (CZP-II), were identified and isolated from T. cruzi epimastigotes [101]. Similarly to metalloproteinases, there are no studies on aspartyl proteinase inhibitors reported in T. cruzi so far.
1E-THREONINE PROTEINASES (PROTEASOME) Proteasomes are intracellular complexes that control protein degradation in organisms ranging from Archaebacteria to mammals. In protozoan parasites, the proteasome is involved in cell differentiation and replication, and could therefore be a promising therapeutic target [102]. In T. cruzi, the presence of proteasome with properties similar to those of other eukaryotes was reported [103] and its inhibition by lactacystin blocks some differentiation steps in the life cycle of the parasite. However, clasto-lactacystin, an inactive
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analogue of lactacystin, and cell-permeant peptide aldehyde inhibitors of T. cruzi CPs did not show effect. The use of proteasome inhibitors determined the accumulation of ubiquitinated proteins and showed that cytoskeleton proteins associated with the flagellum are targets of the ubiquitin-proteasome pathway [104]. It was suggested that inhibition of the ubiquitin-proteasome pathway with lactacystin in T. cruzi epimastigotes block parasite growth, does not block adhesion, but disrupts cell division and affects factors triggering differentiation [105]. Although several parasite proteasome subunits have been cloned and sequenced showing homology to the corresponding subunits from other eukaryotic proteasomes [23], at difference with other kinetoplastida, no studies about proteasome specific inhibitors are still available for T. cruzi. Nereus Pharmaceuticals, Inc. [106] presented a patent claiming the use of analogue compounds of salinosporamide A, a bacterial marine natural product, as proteasome inhibitor for the treatment of neoplasm, inflammation and microbial infection. This heterocyclic compound was able to inhibit proteasome activity with an IC50 value of 11.8 nM. However, despite the well known potential of proteasome inhibitors against trypanosomes in vitro [23, 107], no biological data of anti-trypanosomal activity was disclosed.
2-ERGOSTEROL BIOSYNTHESIS PATHWAY The sterols are essential structural components of cellular membranes serving as precursors of steroid hormones and vitamin D in mammals as well as modulators of growth and development in unicellular organisms [108, 109]. Trypanosomatids contain sterols in plasma, inner mitochondrial and glycosomal membranes [110]. Depletion of sterol end products causes Trypanosomal cell death as a result of membrane disruption, especially in the exponentially dividing stages of the parasite [111, 112]. The finding that the main sterol in T. cruzi metabolism is ergosterol instead of cholesterol unlike human hosts triggered an intensive search for the identification and potential effect of inhibitors of ergosterol biosynthesis (EBIs) [113]. The singularity of this pathway in kinetoplastid parasites, the strict requirement of T. cruzi for specific endogenous sterols for cell viability and growth, similarly to fungi and yeast, and the susceptibility to sterol biosynthesis inhibitors (EBI) in vitro [114-117] and in vivo [114, 116-119] have shown sterol biosynthesis pathway as a promising target for drug therapy against T. cruzi [120]. Among potential drug target enzymes of sterol biosynthesis for treatment of Chagas disease can be mentioned the following enzymes from this metabolic pathway (Fig. 1). 2A- Sterol C14 -demethylase Sterol C14 -demethylases are essential enzymes in sterol biosynthesis in eukaryotes and drug targets in antifungal therapy. These enzymes catalyze oxidative removal of the C14 -methyl group from postsqualene sterol precursors (Fig. 1). They are found in Trypanosomatids. It was early reported that even with only 22-33% amino acid identity across the biological kingdoms the orthologous enzymes from bacteria to mammals preserve strict catalytic region- and stereo-specificity and have a very limited range of substrates [121]. The sterol C14 -demethylase from T. cruzi (TcCYP51) was found to be catalytically closely related to animal/fungi-like CYP51 and prefers C4-dimethylsterols. By contrast, the
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Fig. (1). Enzymes of the Ergosterol biosynthetic pathway as drug targets. The scheme shows the chemical structures and names of the major intermediates of the ergosterol biosynthesis. Enzymes are shown in blue italics and drug classes that act on them are shown in red squares.
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ortholog from T. brucei, similarly to plant CYP51 requires C4-monomethylated sterol substrates. The substrate preferences of these enzymes imply differences in the postsqualene portion of sterol biosynthesis in different trypanosomes. The phyla specific residue can be used to predict preferred substrates of new CYP51 sequences and subsequently for the development of new artificial substrate analogues, which might serve as highly specific inhibitors. It is worth noting that the CYP51 family is very special as its members preserve strict functional conservation in enzyme activity in all biological kingdoms. As mentioned above, amino acid identity across the kingdoms as low as 25-30%, they all catalyze essentially the same three-step reaction of oxidative removal of the 14/*alpha*/-methyl group from the lanostane frame. This reaction is the required step in sterol biosynthesis of pathogenic microbes and it was shown that specific inhibition of protozoan CYP51 can potentially provide treatment for human trypanosomiases [122]. On the other hand, the effects of sterol biosynthesis inhibitors (simvistatin, zaragosic acid, terbinafine, ketoconazole, and others) on the regulation of different sterol biosynthesis genes and their protein products, demonstrating that T. cruzi can specifically regulate sterol C14demethylase gene expression were reported [123].
I- AZOLE INHIBITORS The azole drugs (ketoconazole, itraconazole, Table 3), target the lanosterol C14-demethylase enzyme in the ergosterol biosynthesis pathway causing the accumulation of 14 -methylsterols and decreasing production of ergosterol. It was reported that the triazole derivatives, inhibitors of fungal P-450-dependant C14- -sterol demethylase, posaconazole, (SCH56592, Schering-Plough Research Institute), D0870 (Astra-Zeneca Pharmaceuticals), and TAK-187 (Takeda Chemical Company) are capable of inducing parasitological cure in murine models of both acute and chronic Chagas’ disease with no toxic side effects to the hosts [114, 118, 119, 124]. Compounds such as itraconazole and fluconazole markedly reduced or prevented chronic phase symptoms [125]. D0870, D (+) isomer of fluconazole, displayed a striking inhibitory activity in vivo, both in acute and chronic models, leading to unprecedented percentages of parasitological cure [118]. Albaconazole (UR-9825; Uriach & Company, Barcelona, Spain) resulted one of the most potent EBIs tested against this organism [87, 126]. Among triazole derivatives of probed antifungal activity, ketoconazole, failed to eradicate T. cruzi from experimentally infected animals or human patients [113, 116] whereas ravuconazole resulted one of the most advanced candidates for clinical trials for a new, rationally developed trypanocidal activity in vivo and in vitro [124]. However, the use of such compounds as chemotherapeutic agents was questioned due to the cross resistance between ketoconazole, miconazole and itraconazole revealed in in vitro experiments, in addition to the induction of resistance of T. cruzi to some azoles [127]. Interestingly, a series of peptidomimetic disubstituted imidazoles resulted highly effective against T. cruzi. The compounds were administered orally to mice with acute T. cruzi infection and caused significant decrease in parasitemia leading to 100% survival [128]. Moreover, Tipifarnib (R115777), an inhibitor of human protein farnesyltransferase (PFT), is shown to be a highly potent inhibitor of T. cruzi growth (ED50: 4 nM). Surprisingly, this was attributed to the inhibition of the mentioned CYP51, the cytochrome P450 sterol C14-demethylase [129]. Moreover, when three sets of CYP51 inhibitors were tested in vitro and in Trypanosomal cells including azoles, non-azole compounds (50% T. cruzi cell growth inhibition at 5 M) and substrate analogs of the 14/*alpha*/-demethylase reaction, the compound 32-methylene
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cyclopropyl lanost-7-enol exhibited selectivity toward T. cruzi with 50% cell growth inhibition at 3 M [130]. Recently, the antiarrhythmic compound amiodarone, frequently prescribed for the symptomatic treatment of Chagas disease patients, was reported to have direct activity against T. cruzi, both in vitro and in vivo, and that it acts synergistically with posaconazole [131] and with itraconazole for the treatment of chronic Chagas disease [132]. These results open up the possibility of novel combination chemotherapy approaches for the treatment of Chagas disease using currently approved drugs [133]. A recent report showed that dialkyl imidazoles structurally simpler than posaconazole, tested as inhibitors of T. cruzi lanosterol14alpha-demethylase (L14DM), displayed potency for killing T. cruzi amastigotes in vitro with values of EC50 in the nM range. Two compounds given after establishment of parasite infection by using a mouse model of acute Chagas disease reduced parasitemia in the blood to undetectable levels. These dialkyl imidazoles, substantially less expensive to produce than posaconazole are proposed as appropriate for further development toward an antiChagas disease clinical candidate [134]. 2B-Oxidosqualene Cyclase or Lanosterol Synthase (OSC) OSC is a key enzyme in sterol biosynthesis, which converts 2, 3-oxidosqualene to the tetracyclic product, lanosterol (Fig. 1). The synthesis of lanosterol is an essential step in the production of mature sterols. In yeast and higher eukaryotes (including humans), OSC directly catalyzes the synthesis of lanosterol from 2, 3-oxidosqualene by a complex cyclization-rearrangement reaction involving the formation of a total of six new carboncarbon bonds by a single enzyme. The fact that OSCs from Trypanosomes and animals use different catalytic motifs could lead to the development of specific inhibitors for this enzyme [135]. II- NON AZOLE INHIBITORS Among this type of inhibitors (Table 3), the following can be mentioned: Table 3. Inhibitors of Ergosterol Metabolism 1-Azole inhibitors -sterol C14- demethylase [121-134] N
N
N OH N CH2 C CH2 N
N
H3C
N
O CH2 Cl
O N
C N
OCH2
O Cl
Ketoconazole O H O F
F
N
N
R N
N
F
N
F
Fluconazole
CH3 O Me HO N N S N N S OH H N N N F Me N
N Posaconazole
F
TAK-187
OCH2CF2CF2H
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(Table 3) Contd….. N
O
N
N
N
N
N F
N
Cl
N
CH3CH2 CH
Albaconazole
F
OCH2
N
O Cl
O
Itraconazole
H3C N
OH
N
N
N
N N
CN
S
F
N
N
N
CH3 N
CH2 Cl
O
O
O N
HN
H2N N
F Ravuconazole compounds
HN
OH
O
S
Peptidomimetic Imidazol
Cl Cl Tipifarnib
2-Non-Azole Inhibitors - Squalene epoxidase [140, 141]
-oxidosqualene cyclase[135-139] (CH2) n CH3
N
S+ N+
BF4-
N+
-phenylthio based
-pyridinium ion based O
-allylamine based
- squalene synthase [142-148] SCN
O -thiocyanate derivatives
O
H3CO
O
N
OH
O
HO
CO2H CO2H
HO O
N
-quinuclidine based
OH zaragosic acid
HO2C
R HO
O O O
O CH3-CH2-CH-C
H OH H
CH2 CH2
O 24-SMT [149] - azasterol
OH H3C
H CH3
H3C HMGCoA reductase [150] - mevinolin
HO
OH
O O
OH
OH
OH
OH
OH
O H
COONa CH3
H3C O No enzyme target assigned [151] - polyene based
H3C O
OH H2N
OH
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Pyridinium Ion Based Inhibitors N-Alkyl- and N-prenylpyridinium ions showed to be potent and specific inhibitors on C. albicans OSC and to exhibit antifungal activity [136]. Besides, it was reported that these compounds have potent activities against T. cruzi and inhibit sterol biosynthesis in these organisms. The anti-trypanosomal activities from specific non-azole inhibitors including the lead compound N-(4E, 8E)-5, 9, 13-trimethyl-4, 8, 12-tetradecatrien-1-ylpyridinium and a series of compounds designed to inhibit OSC, were tested against mammalian-stages and 12 of them resulted highly active in the nM range against trypomastigotes [137]. Phenylthiovinyl Derivatives By using a recombinant T. cruzi OSC expressed in yeast, 19 inhibitors: aza, methylidene, vinyl sulfide, and conjugated vinyl sulfide derivatives of oxidosqualene and squalene, were tested. Many inhibitors of control OSC showed comparable IC50 for T. cruzi OSC, but some phenylthiovinyl derivatives showed to be 10-100 times more effective on the T. cruzi enzyme than on the control enzymes [138]. Buckner et al. presented a patent claiming that OSC inhibitors could be used to treat fungal, bacterial and parasite infections including Trypanosomatids based on the drug induced blockade of sterol biosynthesis (University of Utah Research Foundation) [139], (Table 6). Five promising compounds were described with in vitro growth inhibitory effects against T. cruzi and L. mexicana with IC50 values in the nM range and antiparasitic activity confirmed in a murine model of Chagas disease. 2C- Squalene Epoxidase This enzyme catalyzes the conversion of squalene to (3S) 2, 3-oxidosqualene (Fig. 1). It was described in vertebrates as a nonmetallic, flavoprotein monooxygenase and is also considered as potential target for the design of therapeutic agents to be used against different pathogen organisms [140]. Allylamine Based Inhibitors It is known that among antifungal drugs, the allylamine terbinafine (Table 3) inhibits squalene epoxidase in the sterol biosynthesis pathway and was shown to be synergistic with ketoconazole against cultures of T. cruzi [141]. 2D-Squalene Synthase (SQS) SQS catalyzes a head-to-head reductive dimerization of two molecules of farnesyl pyrophosphate (FPP) in a two-step reaction to form squalene (Fig. 1), the first step in sterol biosynthesis. This enzyme is currently under intense study as a possible target for cholesterol-lowering agents in and has been recently shown as a promising target for antiparasitic chemotherapy [142, 143].
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Thiocyanate Derivatives The 4-Phenoxyphenoxyethyl thiocyanate (Table 3) resulted to be an effective and potent agent against epimastigote proliferation and produced the accumulation of low molecular weight metabolites from mevalonate to squalene [144]. Searching for new chemotherapeutic and chemoprophylactic agents, some aryloxyethyl thiocyanate derivatives, structurally related to 4-phenoxyphenoxyethyl thiocyanate were designed, synthesized, and evaluated. Some of these drugs proved to be effective growth inhibitors of T. cruzi with values comparable with those presented by ketokonazole, others proved to be potent inhibitors of epimastigotes multiplication, and one of them was reported to be an effective antichagasic agent with prospective as a lead drug for further in vivo studies [145]. The growth inhibition of T. cruzi epimastigotes induced by 4-phenoxyphenoxyethyl thiocyanate (WC-9) was associated with a reduction in the content of the parasite's endogenous sterols due to a specific blockade of their de novo synthesis at the level of squalene synthase [146]. Quinuclidine Based Inhibitors Among the synthesized quinuclidine inhibitors (Table 3), 3-(biphenyl-4-yl)-3-hydroxyquinuclidine (BPQ-OH) showed to be a powerful non-competitive inhibitor of T. cruzi SQS, with a Ki value in the nM range. This compound was able to eradicate intracellular T. cruzi amastigotes from culture Vero cells with no side effects on host cells [142, 111]. In addition, the compounds E5700 and ER-119884 were found to be potent noncompetitive or mixed-type inhibitors of T. cruzi SQS with Ki values in the low nanomolar to subnanomolar range. In vivo studies indicated that E5700 by oral administration is capable of providing complete protection against acute Chagas’ disease [143]. In vitro and in vivo activities of these two novel quinuclidine SQS inhibitors are currently under development by Eisai Company Ltd. (Ibaraki, Japan) as cholesterol- and triglyceride-lowering agents in humans [147]. Recently, some biphenylquinuclidine derivatives were evaluated as inhibitors of SQS in order to explore their potential in the treatment of the parasitic diseases such as Leishmaniasis and Chagas disease. The compounds were screened against a recombinant Leishmanial SQS, against L. mexicana promastigotes, and T. cruzi intracellular amastigotes. Compounds that inhibited the enzyme also reduced the levels of steroids and caused growth inhibition of L. mexicana promastigotes [148]. 2E-Delta 24(25)-Methyltransferase (24-SMT) This enzyme is essential for the biosynthesis of ergosterol, but not required for the biosynthesis of cholesterol (Fig. 1). A series of potential transition state analogues of 24SMT were designed, synthesized and evaluated against recombinant L. major 24-SMT and the parasites L. donovani and T. cruzi in vitro. Some of the compounds (Table 3) showed inhibition of the recombinant (L. major) 24-SMT and inhibited parasite growth. Others, although did not show enzyme inhibition, presented anti-parasitic activity against T. cruzi [149]. 2F-3-Hydroxy-3-methyl-glutaryl-coenzymeA (HMGCoA) Reductase The antiproliferative effects of mevinolin (Table 3), an inhibitor of HMGCoA, were tested on T. cruzi both in vitro and in vivo (Fig. 1). In addition, its ability to potentiate the action of specific EBIs, such as ketoconazole and terbinafine was evaluated. A synergic action against the proliferative stages of T. cruzi of combined EBIs suggested that mevinolin
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combined with azoles, such as ketoconazole, could be used in the treatment of human Chagas disease [150]. Finally, among other antifungal drugs, different Amphotericin B-lipid formulations which associates with ergosterol to disrupt the integrity of the cell membrane were also tested in in vitro and in vivo assays against experimental T. cruzi infections showing potent anti-T. cruzi activities [151]. Despite the enzymes of this biosynthetic pathway are showing growing indications, this is not reflected in the number of disclosed patents. Only the synthesis of 5-amino-1-benzylimidazole derivatives, inhibitors of the C-14 -demethylase, with antibacterial, antifungal and anti-trypanosomal activity was presented by scientist of the Yale University. The compounds were tested on intracellular amastigotes; they were non toxic to the cells and showed a remarkable IC50 from M to pM values. The authors analyzed in vivo assays in mice and suggested that a phenylbenzylimidazole moiety is responsible for the inhibition of the enzyme and consequent antiparasitic activity. However, no data of enzymatic inhibition is presented [152-154], (Table 6).
3-BIOSYNTHESIS OF POLYISOPRENOIDS 3A- Farnesylpyrophosphate Synthase (FPPS) In pathogenic protozoa, farnesylpyrophosphate synthase (FPPS) is the enzyme responsible for the formation of farnesylpyrophosphate that marks the branching point in the synthesis of a variety of sterols and other essential isoprenoids. In T. cruzi, the gene TcFPPS that codifies for this enzyme was cloned, sequenced, expressed and characterized as an essential enzyme for parasite survival. Enzymes from the isoprenoid pathway have been assigned to different compartments in eukaryotes, including Trypanosomatids. T. cruzi FPPS localizes to the cytoplasm of both T. cruzi and T. brucei, and is not present in other organelles such as the mitochondria and glycosomes [155]. Farnesylpyrophosphate Synthase Inhibitors The above mentioned pathways can be blocked by bisphosphonates (Table 4), metabolically inert inorganic PP analogues that inhibit FPPS [156]. The recombinant enzyme was inhibited by the nitrogen-containing bisphosphonates risedronate and pamidronate causing the latter a decrease of parasitemia in infected mice and inhibiting the in vitro intracellular replication of amastigotes [113]. By contrast, the non-nitrogen-containing bisphosphonate etidronate did not affect parasite growth [156]. Risedronate inhibited the proliferation of epimastigotes and sterol biosynthesis at a pre-squalene level as shown by sterols analysis in treated parasites, associating these results with the inhibition of FPPS, turning out as a promising lead compound for the development of new drugs against T. cruzi [157-159]. The treatment of human bone resorption disorders currently involves bisphosphonatecontaining drugs which due to their potential innocuousness are good candidates to control tropical diseases. Some fatty acids-derived bisphosphonate compounds resulted potent inhibitors of the proliferation of T. cruzi intracellular amastigotes at low M level, but none of them was effective against epimastigotes [160,161]. The drug accumulation in parasite acidocalcisomes seems to be responsible for the selective action displayed by bisphophonate compounds against T. cruzi [162]. FPPS condenses the diphosphates of C5 alcohols to form
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C10 and C15 diphosphates (geranyl and farnesyl). The analysis of the structures of the T. cruzi FPPS alone and in complexes with substrates and inhibitors revealed that after binding the enzyme undergoes conformational changes facilitating the enzyme to bind a bisphosphonate inhibitor. Structural studies as well as molecular dynamics may lead to the design of new, more potent anti-trypanosomal bisphosphonates [163, 164].
Table 4.
Isoprenoid Metabolism Inhibitors
-farnesyl PP synthase [155-164] Bisphosphonates
O
O
OH P
H2N-(CH2)2
OH OH
P
OH
OH P
OH OH
P O
OH OH
O
OH
H2N-(CH2)3
P O
Risedronate
Alendronate
Pamidronate
N
OH
OH OH
P O
OH OH
-farnesyl transferase [163-171] Benzophenone based
NO2
O
H2N
N H O
H N
O
O
3B- Protein Farnesyltransferase (PFT) This enzyme catalyzes the transfer of a farnesyl residue from farnesylpyrophosphate to the thiol of a cysteine side chain of proteins which carry at the C-terminus the so called CaaX-sequence. The attachment of polyisoprenoids to specific proteins, protein prenilation, is involved in signal transduction and anchorage of protein to cell membranes. Prenilation was demonstrated in Trypanosomatids, [165, 166] and PFT of both T. cruzi and T. brucei were cloned, finding differences with its mammalian counterpart. These facts validated the use of PFT as trypanocidal chemotherapeutic target [166].
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Farnesyl Transferase Inhibitors PFT inhibitors are known as potent antitumoral drugs in experimental animals, some of them assayed in treatment of human cancer [167]. The development of PFT inhibitors is clearly directed towards the so-called non-thiol farnesyltransferase inhibitors (Table 4) because of adverse drug effects connected to free thiols. Recently, several farnesyltransferase non-thiol inhibitors based on the benzophenone scaffold were assayed in vitro and in vivo with T. cruzi. The common structural feature of all inhibitors resulted to be an amino function which can be protonated. R-phenylalanine and N-propylPiperazinyl derivatives showed the best in vitro activity with IC50 values in the nM range. These inhibitors showed no cytotoxicity to cells. When tested in vivo, the survival rates of infected animals were 60 to 80 % at day 115 post infection [168]. As mentioned above in section 2A, the PFT inhibitor tipifarnib, now in phase III anticancer clinical trials, was previously found to kill T. cruzi by blocking sterol 14 alpha-demethylase. Rational modification was performed developing tipifarnib analogues that display reduced affinity for human PFT to reduce toxicity while increasing affinity for the mentioned parasite demethylase against T. cruzi and resulted efficacious in a mouse model of acute Chagas disease [169]. The use of PFT inhibitors, such as the natural antibiotic manumycin A and other synthetic cyclic hexenone compounds, to treat parasitic diseases was patented by Mark Field from the Imperial College of Sciences, Technology and Medicine (UK) [170] but no description of the synthesis procedure was included. Schering Corp recently disclosed 21 PFT inhibitors based on Piperazine or Piperidine scaffold for the treatment of T. brucei infection [171]. The compounds were claimed to inhibit PFT in a M range and in vivo inhibition of the parasite ranged between 0.2-10 M. However, no patents specifically related with PFT inhibitors acting on T. cruzi have been reported yet (Table 6). 3C-Protein Geranylgeranyltransferase Type I (PGGT-I) Similarly to PFT, PGGT occurs in many eukaryotic cells and consists of two subunits, a common alpha subunit and a distinct beta subunit. A putative protein that consists of 401 amino acids with approximately 20% amino acid sequence identity to the PGGT-I beta of other species was identified in the gene database of T. cruzi. Recombinant T. cruzi PGGT-I ortholog was cloned and characterized showing geranylgeranyltransferase activity with distinct specificity toward the C-terminal CaaX motif of protein substrates compared to that of the mammalian PGGT-I and T. cruzi PFT. Several candidates for T. cruzi PGGT-I or PFT substrates containing the C-terminal CaaX motif were also found in the T. cruzi gene database. However, only one out of five of the peptide tested, a peptide of a Ras-like protein ending with CVLL was selectively geranylgeranylated by T. cruzi while the others were specific substrates for T. cruzi PFT but not for PGGT-I. On the other hand, the mRNA and protein of the T. cruzi PGGT-I beta ortholog were detected in three stages of parasite development. In addition, it was shown that cytosol fractions from trypomastigotes and epimastigotes contained 100-fold lower levels of PGGT-I activity compared with PFT activity [172]. Protein Geranylgeranyltransferase Type I Inhibitors Although the CaaX mimetics, known as PGGT-I inhibitors showed very low potency against T. cruzi PGGT-I compared to the mammalian enzyme, it was suggested as potential target to develop selective inhibitors against the parasite enzyme [172].
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4-THIOL-DEPENDENT REDOX METABOLISM Biosynthesis of Trypanothione Trypanosomatids present a unique thiol-dependant redox metabolism, which is based on trypanothione (a low molecular weight thiol-polyamine conjugate, N1, N8-bis(glutationyl) spermidine T(SH)2), exclusively found in parasitic protozoa of the order Kinetoplastida and specific enzymes including a trypanothione reductase (TR) in replacement of the ubiquitous glutathione reductase (GR) [173]. The sensitivity of Trypanosomatids towards oxidative stress and the absence of trypanothione in the mammalian host validate the enzymes of the trypanothione metabolism as drug-target molecules. Trypanothione Reductase (TR) TR is a key enzyme of the parasite antioxidant defense, and is essential for all trypanosomatids studied so far. It is an NADPH-dependent flavoprotein that maintains trypanothione in its reduced form and able to be oxidized by trypanothione oxidase, leading to reduction of free radicals levels and contributing to the maintenance of an intracellular reducing environment. X ray crystallography studies solved the three-dimensional structure of the purified TR in free form, in complex with substrates and in the presence of inhibitors [174]. The differences on the substrate specificity found between TR and the mammalian counterpart determined that TR had been widely used as a target for rational drug design against trypanosomiasis [175].
TRYPANOTHIONE REDUCTASE INHIBITORS A great proportion of trypanocidal agents are involved in the trypanothione metabolism [176]. Among them, a lot of them are inhibitors of T. cruzi TR (Table 5).
I-IRREVERSIBLE INHIBITORS a- Subversive Substrates or Sabotage Inhibitors Sabotage inhibitors are molecules that convert an antioxidative disulfide reductase into a prooxidative enzyme. Typical subversive substrates are reduced in single-electron steps to the respective radicals which then react with molecular oxygen to yield superoxide anion radicals, enhancing the effect of oxidative stress. Among the compounds capable to act as subversive substrates of TR and other flavoenzymes are nitrofurans and naphthoquinones [177, 178]. These compounds can be reduced by a variety of cellular reductases triggering the production of oxygen radicals, followed by the consumption of thiol species. When the acting reductase is TR, the subversive process may take place avoiding the regeneration of T (SH)2 [178]. On the basis of the redox properties, nitrofuran compounds, resulted moderate inhibitors of TR and GR and some of them, namely nifuroxime and nifuroxazide were no substrates for GR and proved to be better inhibitors of T. cruzi in culture as compared to nifurtimox. Among some promising nitrocompounds reported, Chinifur, a bactericidal nitrofuran derivative, is an inhibitor and subversive substrate of TR, but it interacts weakly to some structurally related antioxidant enzymes [179]. However, a series of nitroderivatives
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(nitrofurazones and nitrothienyl analogues), were not found to be significantly better inhibitors of T. cruzi in vitro growth [180]. Table 5. Trypanothione Reductase Inhibitors. Representative Compounds I-Irreversible Inhibitors a- Subversive substrates or sabotage inhibitors [177-188]
b-Nitrosoureas [189]
O O
O
O N H
4
O
OH
Cl
O N
N H
N H
OH
O
c- Ajoene [190]
Cl
R
O
N H
S O
NH
N
4
N
S
N Pt2+
S d- Organo-metallic complexes[191-194]
H
N
CH3
II-Reversible Inhibitors b- Aminodiphenylsulfides [202-203]
a- Tricyclic compounds [98-201]
O
N
O H N
S
O
NH O
Br
N H H N
N
12
S
O O
O Cl
N
Br
HN
NH
N
N N
N c-Polyamine derivatives [204-206]
d- Bisbenzylisoquinoline alkaloids derivatives [207] OCH3 O N
NH
N
NH
H3C
N
O O
N H
H O OCH3
CH3
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(Table 5) Contd…. f- Nitrofuryl [210, 211] S
e-Quaternary arylalkylammonium phenothiazines [208, 209] O N O2N
O
N H
OR
Cl
N
Me2 N+ R h- Analogues of Dethiotrypanothione [214]
g- Natural products scaffolds [213]
H Cbz N O
N R2N
H N
N R1
O
N H CH2
O HN NH2+
CH2
CH3 H3CO
O
N R1
H N
S Cbz N H
HN O
O
Naphthoquinones group is composed of very reactive molecules capable to undergo redox cycling, present in all aerobic cells, which display multiple applications in medicine. Some of them, menadione, plumbagin, and lapachol showed notable trypanocidal activities but interacted with TR as well as human GR [178]. With respect to parasite infections, some naphthoquinone derivatives, both synthetic and obtained from natural sources, have been assayed as trypanocidal agents [181-183]. With the aim to obtain trypanocidal compounds with specificity for T. cruzi TR, a series of menadione, plumbagin, and juglone derivatives have been synthesized. The most potent derivatives contained two 1, 4-naphthoquinone moieties linked by a polyamine spacer. It was reported that the inhibition of TR alone is not sufficient for a significant trypanocidal activity but the combination of both inhibition of T(SH)2 reduction and redox cycling would render the parasite more susceptible to the harmful effects of free radical species [178]. The trypanocidal activity of new synthesized naphthoimidazoles from beta-lapachone with an aromatic moiety linked to the imidazole ring using phenylic and heterocyclic aldehydes was assayed finding no correlation between biological activity and the structure of the phenylic series [184]. In addition, several oxyranes structurally related to -lapachone, nor- -lapachone, -lapachone, and 4methoxy-1,2-naphthoquinone showed similar trypanocidal activity to –lapachone although less cytotoxicity than the corresponding naphthoquinones [185]. Finally, four new naphthofuranquinones were evaluated for trypanocidal activity in assays with T. cruzi trypomastigotes. The IC50 values for these compounds were between 157 and 640 M, while those for crystal violet were about 540 M. The trypanocidal activity of the new naphthofuranquinones bearing redox properties reinforces a rational approach in the chemotherapy of Chagas' disease [186]. Recently, taking into account that methylene blue has trypanocidal activity, the interaction of this phenothiazine drug was tested with a number of specific molecules of the parasite antioxidant metabolism, disulfide reductases and its thiol products, finding inhibition of T. cruzi trypanothione reductase,
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serving as a significant subversive substrate of this enzyme [187]. Furthermore, the diaryl sulfide-based inhibitors of TR were also investigated as subversive substrates with antitrypanosomal properties [188]. b- Nitrosoureas The fact that substrate accumulation cannot overcome inhibition constitutes an advantage for using covalent inhibitors. Among them, the drug carmustine is an irreversible inhibitor of TR; however, it also inactivates human GR [189]. c- Ajoene ((E,Z)-4,5,9-trithiadodeca-1,6,11-triene-9-oxide), the spontaneous degradation product of allicin, a major sulfur garlic-derived natural compound, is known for its antifungal, antiviral, antiTrypanosomal, and antimalarial activity and is a covalent inhibitor and subversive substrate of both human GR and T. cruzi TR. A crystal structure of GR inhibited by (E)-ajoene revealed a mixed disulfide between the active site Cys58 and a specific moiety of ajoene. The interactions between the flavoenzymes and ajoene are expected to increase the oxidative stress of the respective cell. The antiparasitic and cytostatic actions of ajoene may at least in part be due to the multiple effects on key enzymes of the antioxidant thiol metabolism [190]. d- Organ-Metallic Complexes Platinum II organometallic complexes extensively used in therapy of cancer are also irreversible ligands of T. cruzi TR but not of human GR. They display trypanocidal activity both in vivo and in vitro assays [191, 192]. It was also reported that complexation of known antiparasitic drugs such as ketoconazol with ruthenium II or III and rhodium II enhances the activity of the parental drugs overcoming primary and secondary drug resistance [193]. The evaluation of synthesized copper (II) and gold (I) clotrimazole and ketoconazole complexes against T. cruzi growth exhibited significantly higher inhibitory activity than their respective parental compounds [194]. A patent from Isis Innovation Ltd claimed that some (2, 2 ’6 ’2 ’’terpyridine) platinum II complexes resulted useful as antitumoral and antiprotozoal agents [195], (Table 6). About 40 complexes were synthesized and characterized including pyridine-2-thiolate-(4-chloro-2, 2’, 6’, 2’’terpyridine) platinum (II), which inhibit the reduced form of the TR, are active against tumoral cell lines and on T. cruzi and other Trypanosomatids. Unsaturated Mannich bases irreversibly inactivated TR from T. cruzi and structural studies revealed a divinyl ketone as the active compound responsible for the enzyme inactivation. It was proposed that the interaction of these compounds with both trypanothione and TR could account for their potent trypanocidal effect reported against T. brucei [196]. Sixteen novel palladium (II) complexes with bioactive nitrofuran-containing thiosemicarbazones as ligands were synthesized. Most complexes showed higher in vitro growth inhibition activity against T. cruzi than nifurtimox. The complexes showed strong DNA binding; however the main trypanocidal mechanism of action seems to be due to the production of oxidative stress as a result of their bioreduction and extensive redox cycling. Moreover, the complexes were found to be irreversible inhibitors of TR [197].
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Table 6. Patent Protected Drug Targets, Natural and Synthetic Trypanocidal Compounds Target
Part I
CPs
Part III
Patent Number*
CP inhibitors
2001: WO0195911 [58] 2002: WO0240462 [59]; WO02057246 [60]; WO02057248 [61]; WO02057249A1[61]; WO02057270A1 [61]; CA2436462AA [61]; WO00217924 [70]; WO02100849 [73]; 2003: EP1362052A1[62]; NO20033220[62]; WO0248097A1 [65]; WO0248097B1/C2 [65] WO03053331 [68]; WO03103574 [69]; WO03104257 [69]; WO03097593 [69]; WO03097664 [75] 2004: CN1486320A [63]; MX3006224A [63]; ZA0305259A [63]; NZ0526913A [63]; WO04007501A1 [64]; WO04020441A1, WO04110988A1 [72] 2005: 6958358 [66]; US6897240 [85]; US7521427 [51] 2006: US6982263 [74] 2009: US7521427 [51]
OSC inhibitors C14 demethylase
2000: WO0076316A1 [139] 2003: WO03006012A1, CA 2453396AA [152-153] 2004: BR0211098A [154]
Synthesis of Poliisoprenoids
PFT inh (in T. brucei)
2001: WO00105384A3 [170] 2003: US03134846A1 [171]
Redox metabolism
TR inh.
2000: WO0050431A1 [195]
DNA nucleotide synthesis
DHFR inh.
2001: WO0153276A1 [282]; WO0114401A1 [284]
Acidocalcisome nucleus
Exch. Na+/H+ inh DNA binder antimitotic drugs topoisomerase II
2000: US6114393 [309] 2002: WO02057224 [297] 2003: WO03090678 [300] 2005: US6967205 [293]; US6906076 [307]
Sialic acid transference
Neuraminidase/ sialidase inh
1999: WO9906369A1 [329] 2000: US6114386 [330]
Natural compound and its derivatives
2003: WO03000272A1 [433]; WO 03080600A1 [435] 2004: WO04067514A1 [430]; WO 04065349 [431]; WO04050092A1/B1 [434] 2007: US7521569 [437]; 2008: US7317114 [437] 2009:US7521569 [437];
Synthetic compounds
2000: BR 09805381A [387]; WO0032201A2 [488] 2004: WO04062590 [486]; WO04080390 [489] US7504501 [491] 2008: US7429540 [490] 2009: US7504501 [491]; US7476686 [492]
Ergosterol Biosynthesis
Part II
Inhibitor
nd
nd
*The first two letters in the Patent number corresponds to PCT (Patent Corporation Treaty) contracting states: BR, Brazil; CA, Canada; CN, China; EP, Europe; MX, Mexico; NO, Norway; US, United States of America; WO, World Intellectual Property Organization; ZA, South Africa. CP, cysteine proteinase; OSC, oxidosqualene cyclase; PFT, protein farnesyl transferase; TR, trypanothione reductase; DHFR, dihydrofolate reductase; nd, non determined.
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II-REVERSIBLE INHIBITORS a- Tricyclic Compounds The structure of tricyclic neuroleptic compounds showed to be a promising class of TR inhibitors [198]. Phenothiazines and related compounds are tricyclic drugs with different biological activities. These drugs also exert trypanocidal effects upon epimastigote and trypomastigote forms: anticalmodulin action (clomipramine); disruption of mitochondria (trifluopherazine and thioridazine); serious cell membrane disorganization (prometazine). Moreover, clomipramine and thioridazine were also effective in treatment of mice with experimental Chagas disease [199]. Clomipramine, a tricyclic antidepressant drug with antiTR and anti-calmodulin effects, was used for treating mice infected with trypomastigotes. 70 % of the mice survived for more than 2 years demonstrating that clomipramine could be a promising trypanocidal agent for the treatment of Chagas' disease [200]. Mepacrine, the acridine derivative that prevents the transmission of Chagas disease by blood transfusion, similar to phenothiazines, is a reversible competitive inhibitor of TR but not of GR. The coupling of mepacrine to the active site of T. cruzi TR allowed the obtention of a crystallographic TR-inhibitor complex [201]. b- Aminodiphenylsulfides Some compounds of the series of 2-amino diphenylsulfides, with lower neuroleptic activity than phenothiazines, were potent inhibitors of TR [202]. To avoid the disadvantages of the neuroleptic activity of phenothiazines, some compounds of the series of 2-amino diphenylsulfides, were synthesized resulting potent inhibitors of TR and showing that the active site of TR easily accommodates extremely bulky ligands [202, 203]. c- Polyamine Derivatives Several potent spermidine and spermine-based inhibitor compounds have been synthesized. In many cases, the spermine derivatives were significantly more effective than the corresponding spermidines [204]. Screening of a library of spermidine-peptide conjugates revealed that N 1, N 1, N 4, N 8, N 12-penta (3-phenylpropyl) spermine was the most effective competitive inhibitor of T. cruzi TR. The compounds of this series were strong trypanocides but a clear correlation between enzyme inhibition and antiparasitic activity was not observed. Several polyamine derivatives were prepared and found to be potent competitive inhibitors of T. cruzi TR. The most effective inhibitor studied was compound 12 with a Ki value of 0.151 M [205]. The antihypertensive agent Kukoamine A, a natural spermine derivative from the root bark of Lycium chinense, is a mixed-type inhibitor of TR. Kukoamine showed no significant inhibition of human GR providing thus a novel selective drug lead [206]. d- Bisbenzylisoquinoline Alkaloids These compounds were also studied in their capacity of inhibiting TR of T. cruzi, finding that daphnoline and cepharanthine showed to be TR inhibitors. Daphnoline led to a significant decrease in parasitemia as well as an increase in parasitological cure rate in comparison
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with Benznidazol-treated acute infected mice and in 70 % of the treated chronic mice no parasite was detected [207]. e- Quaternary Arylalkylammonium Phenothiazines Substituted benzyl [3-(2-chloro-phenothiazine-10-yl)propyl] dimethylammonium salts were synthesized to introduce a permanent positive charge into inhibitor molecules. These compounds were linear competitive inhibitors against trypanothionedisulfide. The strongest inhibitor of this series rendered a Ki value of 0.12 M, approximately 2 orders of magnitude more inhibitory than the parent chlorpromazine [208]. Quaternization of the nitrogen atom of 2-amino-4-chlorophenyl phenyl sulfide analogues of chlorpromazine improved T. cruzi TR inhibition approximately 40-fold with a linear competitive Ki value in the M range. The quaternized analogues of the 2-chlorophenyl phenyl sulfides had strong antitrypanosomal and antiLeishmanial activity in vitro [209]. f- Nitrofuryl Derivatives New 5-nitrofuryl derivatives were synthesized and tested as anti-T. cruzi agents finding that more than 75 % of the prepared derivatives showed higher activity than nifurtimox [210]. The design of 5-nitrofuryl derivative compounds combining in the same molecule the recognized 5-nitrofuryl group, an oxidative stress promoter, and lateral chains that could interact with biomolecules such as TR showed to be very active against the epimastigote forms of the parasite in comparison with the reference drug nifurtimox [211]. Two structurally new types of inhibitors of TR but not of GR were studied: the antimicrobial chlorhexidine {1, 1'-hexamethylenebis [5-(4-chlorophenyl) biguanide]}, a linear competitive inhibitor and a Piperidine derivative acting as mixed inhibitor. Although these compounds did not exert an improved inhibitory potency compared to chlorhexidine, the change from competitive to mixed-type inhibition resulted advantageous, since substrate accumulation does not overcome inhibition [212]. g-Natural Product Scaffolds In the search for TR inhibitors, natural product scaffolds were used as leads. Thus, the harmaline, 10-thiaisoalloxazine, and aspidospermine frameworks were identified as the basis of inhibitors of T. cruzi TR. Two new heterocyclic compounds showed moderately strong, linear competitive inhibition with K(i) values in the mM range Aspidospermine inhibited T. cruzi TR but none of the compounds tested inhibited glutathione reductase [213]. h- Dethiotrypanothione Analogues Synthesis and activity of dethiotrypanothione and analogues as inhibitors of T. cruzi TR was also performed. The synthesis of these macrocycles feature ring-closing olefin metathesis (RCM) reactions rendered a Derivative number 4 as the most potent inhibitor obtained with a Ki=16 M [214]. Other enzymes of the trypanothione metabolism, such as trypanothione synthetase without counterparts in the mammalian host, could be also mentioned as potential drug targets [174]. Particularly, TcAPX, a plant-like ascorbate-dependent hemoperoxidase was
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reported in T. cruzi. This enzyme belongs to the oxidative defense system of the parasite and is involved in the reduction of the parasite-specific thiol trypanothione by ascorbate in a process that involves non-enzymatic interaction. The absence of this redox pathway in the human host may be therapeutically exploitable [215].
GLUTATHIONE CYCLE A gene codifying for a novel T. cruzi protein containing the glutaredoxin (Grx) pattern CXXC was cloned. TcGrx, the recombinant protein, showed homology to glutathione-Stransferases (GSTs) and was recognized by a serum anti-recombinant TcGrx in parasite lysates. It was confirmed that it is a thiol containing NADPH dependent reductase and binding assays suggested that it might use another thiol different from GCS as substrate1 . TcGrx could be a putative target for the design of specific inhibitors with antiparasitic properties. Phosphinopeptides Structurally Related to Glutathione In addition, a series of phosphinopeptides structurally related to glutathione was designned, synthesized, and evaluated as T cruzi-antiproliferative agents. Two of them resulted potent growth inhibitors against amastigote forms [216]. Phosphonate and Phosphinate Analogues of Glutathionyl-Spermidine These compounds were previously shown to be potent inhibitors of glutathionylspermidine synthetase (GspS) from E. coli, are equally potent against GspS from C. fasciculata (CfGspS). The phosphinate analogue inhibited recombinant trypanothione synthetase from C. fasciculata, L. major, T. cruzi and T. brucei with K(i)(app) values 20-40-fold greater than that of CfGspS. This phosphinate analogue remains the most potent enzyme inhibitor identified to date, and represents a good starting point for drug discovery for trypanosomiasis and Leishmaniasis [217].
5-GLYOXALASE SYSTEM The glyoxalase system, comprising the metalloenzymes glyoxalase I (GLO1) and glyoxalase II (GLO2), is an almost ubiquitous metabolic pathway involved in the detoxification of highly reactive aldehydes such as the glycolytic byproduct methylglyoxal to dlactate, using glutathione as a cofactor. Recent studies in Trypanosomatids have revealed a unique dependence upon the Trypanosomatid thiol trypanothione as a cofactor suggesting that the trypanothione-dependent glyoxalase system may be an attractive target for rational drug design against the Trypanosomatid parasites. Cloning, expression and kinetic characterization of glyoxalase I from T. cruzi was performed. T. cruzi glyoxalase I isomerised hemithio-acetal adducts of trypanothione more than 2400 times more efficiently than glutathione adducts, with the methylglyoxal adducts 2-3-fold better substrates than the 1
García GA, Garavaglia PA, Esteva MI, Duschak VG, Ruiz AM. Identification, characteri-zation and purification of a putative thiol containing NADPH dependent reductase from Trypanosoma cruzi Reunión de Protozoología y Enfermedades Parasitarias, Rosario, Santa Fe, Argentina (2004).
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equivalent phenylglyoxal adducts. However, glutathionylspermidine hemithioacetal adducts were most efficiently isomerised [218]. A recent comparative study of methylglyoxal metabolism in Trypanosomatids was reported pointing out major differences between this metabolism in T. brucei in comparison with those found in L. major and T. cruzi [219]. S-4-Bromobenzylglutathionylspermidine This glutathionylspermidine-based inhibitor was found to be a potent linear competitive inhibitor of the T. cruzi enzyme with a K(i) near to 5 M. Prediction algorithms, combined with subcellular fractionation, suggested that T. cruzi glyoxalase I localizes not only to the cytosol but also the mitochondria of T. cruzi epimastigotes. The contrasting substrate specificities of human and Trypanosomatid glyoxalase enzymes suggest that the glyoxalase system might be an attractive target for anti-trypanosomal chemotherapy [218].
6-GLYCOLYSIS It is known that T. cruzi amastigotes possibly derive its energy entirely from glycolysis, that is the reason why the inhibition of glycolytic enzymes of trypanosomes may be considered attractive targets for the development of anti-T. cruzi drugs [220]. 6A- Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) The structural differences found between the glycosomal GAPDH (gGAPDH) in comparison with that of the mammalian counterpart led to the development of specific inhibitors [221]. Adenosine was found to be a very poor inhibitor, however the addition of substituents to the 2' position of ribose and the N6-position of adenosine led to a series of disubstituted nucleosides, finding that the adenosine derivative [N6-(1-naphthalenemethyl)2'-(3-chlorobenzamido)adenosine] inhibited the proliferation of amastigotes without effect on the corresponding GAPDH human enzyme. A tight binding competitive inhibitor of an enzyme in the glycolytic pathway has been suggested to block the energy production in Trypanosomatids [222, 223]. Besides, flavonoids from the fruits of Neoraputia magnifica were isolated, and among these compounds 3', 4', 5', 5, 7- pentamethoxyflavone resulted to be the most active over flavones and pyrano chalcones displaying inhibitory effect against the GAPDH of the parasite [224]. Studies performed on T. cruzi and T. brucei gGAPDHs showed that despite the high homology between the two trypanomatid enzymes (> 95%), some specific interactions identified could be useful to design selective irreversible inhibitors against T. cruzi gGAPDH [225]. 3-Piperonylcoumarins Based on the structures of previously identified natural products, these coumarin derivatives were designed as inhibitors of gGAPDH from T. cruzi. The molecules could be clustered in different groups according to the chemical substitutions regarding the biological activity, finding that the most active synthesized derivatives contained heterocyclic rings at position 6. Molecular modeling studies by docking suggested a different binding mode for these derivatives, when compared to natural chalepin [226].
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Cis and Trans-Methylpluviatolides The trypanocidal structure-activity relationship for racemic mixtures of cis and transmethylpluviatolides was evaluated in vitro by using trypomastigote forms of T. cruzi and the enzymatic assay of T. cruzi gGAPDH. The mixture of the trans stereoisomers displayed trypanocidal activity with IC50 value in the molar range. Only the (-) enantiomer was active against the parasite. Despite being inactive the (+) enantiomer acted as an antagonistic competitor. In addition, at the evaluated concentrations trans-methylpluviatolide displayed low toxicity, and neither inhibited gGAPDH activity nor hindered peroxide and NO production [227]. "Bi-Substrate" Analogues When a series of "bi-substrate" analogues were synthesized as potential inhibitors of the GAPDH, only one lead compound could be identified capable to inhibit the enzyme from T. cruzi with good affinity and 50-fold high specificity [228]. Anacardic Acids, Glucosylxanthon and Flavonoid Derivatives The combination of structure and ligand-based virtual screening techniques allowed the identification of seven natural products, including anacardic acids, flavonoid derivatives, and one glucosylxanthon as novel inhibitors of T. cruzi GAPDH. The structural diversity of this series of promising natural products showed to be of special interest in drug design, and might be useful in future medicinal chemistry efforts aimed at the development of new GAPDH inhibitors with increased potency [229]. Additionally, the inhibitory effects of a library of natural and synthetic anacardic acid derivatives against this target enzyme were evaluated. The most potent inhibitors, 6-n-pentadecyl- and 6-n-dodecylsalicilic acids, showed IC50 values of 28 and 55 μM, respectively. The effects of these compounds on the T. cruzi GAPDH-catalyzed reaction showed non competitive inhibition with respect to both substrate and cofactor [230]. 6B- Hexose-Phosphorylating Enzymes Glucose, an essential substrate for T. cruzi, is intracellularly phosphorylated to glucose 6-phosphate. It is well known that hexokinase is the first enzyme involved in glycolysis in most organisms. An hexokinase responsible for this phosphorylation has been characterized. In T. cruzi, unlike the human enzyme, it presents an unusual inhibition by inorganic diphosphate (PPi) [231]. In addition, an ATP-dependent glucokinase in T. cruzi exhibiting a ten-fold lower substrate affinity compared to the hooknose was further identified. Both enzymes, which belong to very different groups of the same family, are located inside glycosomes, the peroxisome-like organelles of Kinetoplastida that are known to contain the first seven glycolytic steps as well as enzymes of the oxidative branch of the penthose phosphate pathway. Glucokinase genes, found in the genome databases of T. cruzi and L. major, were cloned and sequenced. Their expression resulted in the synthesis of soluble and active enzymes, named TcGlcK and LmjGlcK, with a molecular mass of 43 kDa and 46 kDa, respectively. The enzymes were purified, and values of their kinetic parameters determined. It was found that no inhibition was exerted by glucose-6-phosphate. Similarly, no inhibition by inorganic pyrophosphate was found in contrast to previous observations made for the T. cruzi and L. mexicana hooknoses. Multiple sequence comparisons, as well as kinetic properties, support the notion that these Trypanosomatid enzymes belong to group
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A of the hexokinases, in which they, according to a phylogenetic analysis, form a separate cluster [232]. The crystal structure of T. cruzi glucokinase revealed features determining oligomerization and anomer specificity of hexose-phosphorylating enzymes. Mass spectrometry analysis was used to confirm the existence of TcGlcK monomeric and dimeric states. In contrast to hexokinases, which show a moderate preference for the alpha anomer of glucose, the electron density showed the d-glucose bound in the beta configuration in the T. cruzi glucokinase. Kinetic assays with alpha and beta-d-glucose further confirmed a moderate preference of the T. cruzi glucokinase for the beta anomer. Structural comparison of the glucokinase and hexokinases allowed the identification of a possible mechanism for anomer selectivity in these hexose-phosphorylating enzymes. The fact that T. cruzi hexokinase and glucokinase show preference for distinct anomers suggests that in T. cruzi these kinases are not directly competing for the same substrate and are probably both present because they exert distinct physiological functions [233]. -Bisphosphonates It was recently reported that bisphosphonates, non-hydrolysable analogues of PPi, are potent inhibitors of T. cruzi hexokinase (TcHK). The most active compound against T. cruzi hexokinase was found to have a 2.2 M IC50 versus intracellular amastigote forms showing selective activity against the parasite [241]. A kinetic analysis of the effects of three bisphosphonates on homogeneous TcHK, as well as on the enzyme in purified intact glycolsome, on glucose consumption by intact and digitonin-permeabilized T. cruzi epimastigotes, and on the growth of such cells in liver-infusion tryptose medium was performed. These compounds resulted several orders of magnitude more active than PP(i) as non-competitive or mixed inhibitors of TcHK, blocked the use of glucose by the epimastigotes, and did not affect the sterol composition of the treated cells, indicating that they did not act as inhibitors of farnesyl diphosphate synthase and suggesting that these novel bisphosphonates act primarily as specific inhibitors of TcHK and might represent a novel class of selective antiT. cruzi agents [234].
7-PENTOSE PHOSPHATE PATHWAY Recent results regarding the pentose phosphate pathway (PPP) have been reported in T. cruzi. All the enzymes of the PPP are present in the four major developmental stages of the parasite [235]. The seven enzymes of the pathway were cloned and expressed in E. coli as active proteins. Glucose 6-phosphate dehydrogenase (6PGDH), which controls glucose flux through the pathway by its response to the NADP/NADPH ratio, is encoded by a number of genes per haploid genome. The kinetic parameters from a recombinant form of T. cruzi 6PGDH showed to be identical to the values reported for 6PGDHs from mammals, however Km for NADP was significantly lower than the value reported for the human enzyme, and closer to that for the T. brucei enzyme, suggesting that inhibitors of the T. brucei 6PGDH might also be successful for the chemotherapy of Chagas disease. The enzyme shows a similar behavior to the redox regulated G6PDHs from chloroplasts and cyanobacteria in addition to a considerable G6PDH increase in metacyclic trypomastigotes under oxidative stress conditions, suggesting that the enzyme might play a prominent role in the defense mechanisms of the parasite against oxidative stress becoming an important target for chemotherapy [236].
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The genes encoding 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase (6PGD), transaldolase and transketolase are present in the CL Brener clone as a single copy per haploid genome. Although 6-phosphogluconate dehydrogenase is very unstable, it was stabilized introducing two salt bridges by site-directed mutagenesis. Ribose-5-phosphate isomerase belongs to Type B; genes encoding Type A enzymes, present in mammals, are absent. Ribulose-5-phosphate epimerase is encoded by two genes. The enzymes of the pathway have a major cytosolic component, although several of them have secondary glycosomal localization and also minor localizations in other organelles [237]. In T. cruzi, this functional pentose phosphate pathway is probably essential for protection against oxidative stress and also for ribose 5-phosphate (R5P) production for nucleotide synthesis. The haploid genome of the CL Brener clone of the parasite contains one gene coding for a Type B ribose 5-phosphate isomerase (Rpi), but genes encoding Type A Rpis, most frequent in eukaryotes, seem to be absent. The recombinant RpiB catalyzes the isomerization of R5P to Ru5P (ribulose 5-phosphate) with Km values of 4 mM (R5P) and 1.4 mM (Ru5P). 4phospho-D-erythronohydroxamic acid, an analogue to the reaction intermediate when the Rpi acts via a mechanism involving the formation of a 1,2-cis-enediol, was capable to inhibit the enzyme competitively, with an IC50 value of 0.7 mM and a Ki of 1.2 mM. The mechanism of the Rpi reaction was studied by site-directed mutagenesis. Moreover, in the absence of RpiBs in the genomes of higher animals also signs this enzyme a possible target for chemotherapy of Chagas disease [238]. On the other hand, T. cruzi trypanothione-dependent antioxidant system must have a current supply of NADPH, provided by G6PD and 6PGD, enzymes of the pentose pathway, to work properly. In this sense, different T. cruzi strains, Tulahuen 2 and Y, were studied regarding growth rate, cytosolic tryparedoxin peroxidase (TcCPX) concentration and pentose phosphate pathway dehydrogenases activities. TcCPX concentration, resistance to H2O2, growth index and G6PD activity values were higher in Tul 2 than in the Y strain. The different patterns of G6PD and 6PGD activities observed among strains along the growth curve and when cells were challenged with H2O2 reinforce the heterogeneity within T. cruzi populations as well as the importance of G6PD in protecting the parasite against reactive oxygen species [239].
8- ARGININE KINASE Vertebrates, including human, use creatine kinase for the storage of ATP in the form of phosphocreatine, capable to maintain ATP homeostasis during muscle contraction. A few years ago, it was reported that T. cruzi and T. brucei, possess an alternative pathway which uses arginine kinase as the catalyst for arginine phosphorilation to produce the analogous phosphagen, phosphoarginine. Phosphagens, posphoarginine and phosphocreatine, play a critical role as energy reserve because the high-energy phosphate is ready to be transferred to adenosine diphosphate ADP when the production of ATP is required. In addition, the molecular and biochemical characterization of arginine kinases in trypanosomes have been reported [240]. This pathway is also widespread through the invertebrate phylum, including a great variety of phosphagens other than arginine, but not creatine. Creatine kinase and arginine kinase are homologous proteins belonging to the family of guanidino kinases, conserved proteins with phosphotransferase activity. There is a close relationship between the energy requirements within the cell and the activity of guanidino kinases. Particularly, in T. cruzi, it has been suggested that the action of arginine kinase acquires relevance during the vertebrate stage of the parasite life cycle, due to variations in
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the insect feeding condition. Thus, during bursts of cellular activity or under starvation stress conditions, phosphoarginine results a rapid source of energy allowing the parasite to adaptate either to environmental changes or stress conditions [241]. Studies in parasites overexpressing arginine kinase showed significantly increased survival capability during hydrogen peroxide exposure suggesting the participation of arginine kinase in oxidative stress response systems [242]. Besides, crystal structure was reported [243]. In addition, recent subcellular localization assays showed that the digitonin extraction pattern of arginine kinase differed from those obtained for reservosomes, glycosomes and mitochondrial markers, and resulted similar to the cytosolic marker. However, immunofluorescence analysis revealed that although arginine kinase is localized mainly in unknown punctuated structures, previously observed in many cytosolic proteins of Trypanosomatids, and also in the cytosol, it did not co-localize with any of the subcellular markers [244]. Moreover, some reports showed that arginine kinase inhibition resulted in parasite growth inhibition in culture. Arginine kinase was also inhibited by the arginine analogs agmatine, canavanine, nitroarginine and homoarginine. Among them, canavanine turned out to be a potent inhibitor of arginine kinase. The trypanocidal action of green tea catechins against two different developmental stages of T. cruzi was demonstrated. In addition, recombinant T. cruzi arginine kinase was inhibited by the polyphenols catechin, gallate or gallocatechin gallate [245]. However, patents have related to these compounds with catechins compounds in the last years relate these compounds with anticancer activity. It is worth mentioning that amino acid metabolic routes as possible therapeutic targets against Chagas disease have been properly reviewed by Silber et al., 2005 [246].
9- PROLINE RACEMASE This enzyme catalyzes the interconversion of L- and D-proline enantiomers and was originally found in the bacterium Clostridium sticklandii, it contains cysteine residues in the active site and does not require co-factors or other known coenzymes. The first eukaryotic amino acid (proline) racemase was identified in T. cruzi and is encoded by two paralogous genes per parasite haploid genome, TcPRACA and TcPRACB that give rise, respectively, to secreted and intracellular protein isoforms. Interestingly, the secreted form of proline racemase is a potent host B-cell mitogen supporting parasite evasion of specific immune responses. Functional intracellular or secreted versions of the enzyme exhibit distinct kinetic properties that might be relevant for their relative catalytic efficiency. Studies with an enzyme-specific inhibitor and abrogation of enzymatic activity by site-directed mutagenesis of the active site Cys330 residue encouraged the potential of proline racemase as a new target for drug development against Chagas disease [247]. On the other hand, overexpression of TcPRAC led to an increase in parasite differentiation into infective forms and in its subsequent penetration into host cells. In addition, parasite viability was impaired in functional knock-down parasites emphasizing the fact that TcPRAC is considered a potential target for drug design as well as for immunomodulation of parasite-induced B-cell polyclonal activation [248]. The enzyme is a homodimer, with each monomer folded in two symmetric alpha/beta subunits separated by a deep crevice. The crystal structure of TcPRAC in complex with a transition-state analog, pyrrole-2-carboxylic acid, revealed the presence of one reaction center per monomer, with two Cys residues optimally located to perform acid/base catalysis through a carbanion stabilization mechanism. It was shown that mutation of the catalytic
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Cys residues abolishes the enzymatic activity while preserving the mitogenic properties of the protein. By contrast, inhibitor binding promotes the closure of the interdomain crevice abrogating B cell proliferation, suggesting that the mitogenic properties of TcPRAC depend on the exposure of transient epitopes in the ligand-free enzyme [249] Recently, the relative contribution of TcPRAC to D-proline availability and its further assembly into peptides was estimated through the use of wild-type parasites and parasites over-expressing TcPRAC genes suggesting that D-proline-bearing peptides, similarly to the mucopeptide layer of bacterial cell wall, might be of benefit to T. cruzi by providing resistance against host proteolytic mechanisms [250]. A patent related with the identification and characterization of racemase, in particular proline racemase was disclosed by Institute Pasteur, including definition of protein signatures, as well as a test for detecting D-amino acid and for screening molecules capable of inhibiting the activity of the enzyme. In addition, it relates to methods and kits for detecting racemases using the nucleic acid molecules of the invention, as well as the peptides consisting of the motifs and antibodies to these peptides but no specific inhibitors for the chemotherapy of the trypanosomiasis are included [251].
10- PROTEIN KINASES Protein kinases (PK) were presented as promising drug targets for a number of human and animal diseases including trypanosomiasis and Leishmaniasis. Genome sequences of the three human-infective Trypanosomatid protozoa, L. major, T. brucei and T. cruzi have been completed, thus defining the eukaryotic protein kinases or kinome for each parasite representing one third of the human complement. Kinome analysis will allow exploiting differences between parasite and mammalian protein kinases to develop novel anti-parasitic chemotherapeutic agents [252]. On the other hand, cyclic AMP-protein kinase A (PKA) signalling is important for the growth and differentiation of T. cruzi. In this sense, recent immunofluorescence assays suggested that PKA can associate with the plasma membrane of trypomastigotes, finding that the PKA regulatory subunit was capable to interact with several P-type ATPases, which might play a role in anchoring PKA to the plasma membrane in T. cruzi [253]. Moreover, a possible correlation between T. cruzi metacyclogenesis induced by oleic acid (OA) and the activation of a particular PKC isoenzyme was investigated by using the specific PKC inhibitors Ro 32-0432 and Rottlerin. These compounds were capable to abrogate both epimastigote differentiation and membrane translocation of PKC beta, gamma, and delta supporting a key role for classical and novel PKC isoenzymes in the signalling pathways involved in T. cruzi metacyclogenesis induced by OA [254]. Protein Kinase Inhibitors To evaluate PKs as drug target, three PK inhibitors: staurosporine (serine/threonine kinase inhibitor), genistein (tyrosine kinase inhibitor), and wortmannin (phosphatidylinositol 3' (PI3) kinase inhibitor) were tested on the growth and ultrastructure of T. cruzi epimastigotes and the effect of these drugs on intracellular amastigotes were evaluated. Wortmannin inhibited parasite growth at the lowest concentrations. However, staurosporine was the most effective after 24 h treatment and genistein caused the stronger inhibition during the whole treatment (60-70 % inhibition) whereas wortmannin showed the lower IC50 in the mM range. In addition, these PK inhibitors showed strong ultrastructural effects on the epimastigotes, they did not interfere neither with the division of intracellular amastigotes nor with their differentiation to trypomastigotes. However, as trypanosomes
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have kinomes that contain a large set of protein kinases and phosphatases, PKs should not be disregarded as an important target for chemotherapy of Chagas disease [255]. The effect of 1-O-hexadecylphosphocoline (Miltefosine), a very active compound against T. cruzi, was evaluated on the protein kinase C (PKC) as well as Na (+)-ATPase activities present in the plasma membrane of the parasite. The drug inhibited the parasite PKC activity through a Na(+)-ATPase-independent way indicating that miltefosine inhibits T. cruzi growth through, at least in part, by inhibition of both PKC and Na(+)-ATPase activities [256].
11- POLYAMINE METABOLISM AND TRANSPORT PATHWAYS In parasitic protozoa, polyamine metabolism and transport pathways comprise valuable targets for chemotherapy. Polyamines are involved in multiple functions inside the cell: in chromatin condensation, in stabilization of tRNA´s structure, in DNA conformational transitions, in neurotransmission modulation and post-translational modification of proteins [257]. In T. cruzi, the polyamine spermidine forms a part of trypanothione, essential member of the dithiol redox metabolism, contributing to the maintenance of an intracellular reducing environment. Polyamines are essential requirements for parasite cell growth and differentiation and polyamine metabolism has attracted considerable attention as a chemotherapeutic target in parasite infections. Although ornithine decarboxylase (ODC) is a key enzyme of the polyamine biosynthesis pathway usually inhibited by the rationally designed drug difluormethylornithine, it has not been detected in any stage of T. cruzi´s life cycle and T. cruzi is not affected by difluormethylornithine. However, T. cruzi was found to be susceptible to a compound related to ODC, difluoromethylarginine (DFMA), which is supposed to inhibit arginine decarboxylase (ADC) but ADC activity in T. cruzi was only found in the trypomastigote form although at almost undetectable levels [258]. On the other hand, taking into account that T. cruzi cannot synthesize putrescine, but uptakes it from the extracellular milieu, the putrescine analogue 1,4 -diamino-2-butanone (DAB) inhibited T. cruzi epimastigotes' in vitro proliferation and produced remarkable signs of oxidative stress such as mitochondrial destruction and cell architecture disorganization. In addition, thiobarbituric-acid-reactive substances were measured to assess lipid peroxidation. A dose-dependent response was found indicating that putrescine uptake by this diamine auxotrophic parasite might be important for epimastigote axenic growth and cellular organization [259]. A patent related with polyamine transport inhibitors from Laval University, Canada includes design, synthesis and therapeutic use of a variety of novel inhibitors of polyamine transport to prevent poliamines salvage in tumoral cells [260]. However, the importance of polyamines in cell survival as well as the complete knowledge of the synthetic pathways in T. cruzi still needs further investigation. Taking into account that T cruzi genome contains neither ODC nor ADC genes, transformation with a recombinant plasmid bearing the complete coding region of C. fasciculata ODC gene was performed and, the transgenic parasites were able to synthesize putrescine and simultaneously became susceptible to alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC. The emergence of DFMO-resistant T. cruzi after one-step selection of ODC-transformed parasites cultivated in the presence of high levels of the drug were reported in parasites transfected with ODC gene [261].
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Polyamine Biosynthetic Enzyme Inhibitors Since the targeting of the enzymes of the poliamine pathway might provide novel therapy approach to inhibit the deoxyhypusine hydroxylase and in order to identify new lead compounds, Piperidines were produced and biologically evaluated [262]. Considering that the enzymes involved in spermidine synthesis and utilization are promising targets for drug development to therapy of African sleeping sickness, Chagas' disease, and Leishmaniasis different inhibitors were tested [263].
12-PURINE SALVAGE PATHWAY AND NUCLEOTIDE SYNTHESIS Whereas in mammals nucleotides are synthesized both de novo and salvaged from recycled purine bases, most parasites are obligate purine auxotrophs, it means that they must salvage purines from their host and they have developed systems to transport, internalize and metabolize the required substrates: the components of nucleic acids and ATP. Accordingly, T. cruzi depends on the scavenging of exogenous purines for nucleotide synthesis. Among Trypanosomatid enzymes involved in the scavenging of purines from the host can be mentioned: 12A- Purine(Hipoxantine/Guanine)-Phosphoribosyltransferase (HGPRT) The HGPRT catalyzes the transfer of a phosphoribosyl moiety on the nucleobase hypoxanthine or guanine converting purine bases to ribonucleotides and is responsible for the initiation in the parasite of the metabolism of certain cytotoxic purine base analogues, such as allopurinol. Thus, either inhibitors or substrates of HGPRT are good targets for effective and selective chemotherapeutic agents. The hgprt genes from T. cruzi and other pathogenic Trypanosomatids have been cloned, sequenced and overexpressed in E. coli, and the recombinant proteins have all been purified and characterized [264]. It was reported that the purine (3'-azido-3’deoxyinosine, 3'-deoxyadenosine) and pyrimidine (3'-azido- 3'-deoxythymidine) analogues inhibited the proliferation of amastigotes in culture cell lines [265]. Allopurinol (4-hidroxy-pyrazol-(3,4d)-pyrimidine) has been used in humans for the treatment of gout and it is transformed in vertebrates in oxypurinol, a potent inhibitor of xanthine oxidase (XO). In Trypanosomatids, deficient in XO, the compound acts as a purine analogue and is incorporated via HGRPT into DNA disrupting the synthesis of RNA and proteins. Allopurinol was shown to be active in murine models of acute Chagas disease with differences in susceptibilities among T. cruzi strains [266] however, there are some conflictting reports related to its efficiency in humans. The drug did not show in vivo activity due to low incorporation in vertebrate stages of T. cruzi and probably to inadequate pharmacokinetic properties. Purine analogues were assayed for their interaction with the HGPRTs from T. cruzi and its human counterpart and some of them showed affinity for the Trypanosomal enzyme [267]. A structure-based docking method identified several potential inhibitors of the Trypanosomal HRPT. Among them, three compounds (2,4,7-trinitro-9fluorenyl-idenemalononitrite, 3-(2-fluorophenyl)-5-(phenoxy)-1,2,4-triazolo(4,3-C)-quinazoline and 3,5-diphenyl-4´-methyl-2-nitrobiphenyl) showed trypanostatic activity in cell culture (against intracellular amastigotes) and one [6-(2,2-dichloro-aceta-mido)chrysene] was a potent inhibitor of the enzyme [268]. Wenck and co-workers (2004) stated the difficulty in designing a mechanism-based inhibitor of the Trypanosomal HPRT that would only inhibit the human cognate enzyme based on kinetic parameter analysis [269].
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12B-Dihydrofolate Reductase (DHFR) Dihydrofolate reductase and thymidylate synthase are two widespread enzymes involved in DNA nucleotide synthesis. Both constitute a bifunctional protein present in different species of protozoa which has been successfully used as a drug target in chemotherapy of cancer, malaria and infectious diseases. The gene coding for the DHFR domain from T. cruzi was cloned and expressed [270]. Several derivatives of methotrexate, inhibitor of the human enzyme, were designed and synthesized using a structure-based approach, and some of them showed higher selectivity for the parasite enzyme than for the human counterpart. Another group of compounds were designed, synthesized and screened as inhibitors of DHFR of Trypanosomatids, showing weak activity in in vitro assays with intracellular amastigotes of T. cruzi [271]. On the other hand, with the aim to generate a library of selective lead inhibitors for further development as antiparasitic agents, a structure-based three-dimensional quantitative structure-activity relationship (3D-QSAR) approach was used to predict the biochemical activity for inhibitors of T. cruzi dihydrofolate reductase-thymidylate synthase (DHFR-TS). Crystal structures of complexes of the enzyme with eight different inhibitors of the DHFR activity together with the structure in the substrate-free state (DHFR domain) were used to validate and refine docking poses of ligands that constitute likely active conformations.3D-QSAR models were obtained for T. cruzi DHFR-TS and human DHFR that show a very good agreement between experimental and predicted enzyme inhibition data [272]. Recently, in order to gain a detailed understanding of the structure-function relationship of the bifunctional enzyme, dihydrofolate reductase-thymidylate synthase (DHFR-TS), the three-dimensional structure of this protein in complex with various ligands was studied. The crystal structures of T. cruzi DHFR-TS with three different compositions of the DHFR domain were reported: the folate-free state, the complex with the lipophilic antifolate trimetrexate (TMQ) and the complex with the classical antifolate methotrexate (MTX). The DHFR active site of the T. cruzi enzyme showed subtle differences compared with its human counterpart. These differences may be exploited for the development of antifolate-based therapeutic agents for the treatment of T. cruzi infection [273] Among dihydrofolate reductase inhibitors can be mentioned: a- 2, 4 –Diaminopyrimidines It was shown that 5-benzyl-2, 4-diaminopyrimidines are selective inhibitors of the Trypanosomal as well as Leishmanial enzymes. Various compounds with alkyl/aryl substitution on the 6-position of the pyrimidine ring were prepared and evaluated against both the recombinant enzymes and the intact organisms finding that the presence of a substituent did not enhance the inhibitor activity neither against the enzyme nor intact parasites in comparison with unsubstituted compounds [274]. On the other hand, the synthesis of 4'-substituted and 3', 4'-disubstituted 5-benzyl-2, 4-diaminopyrimidines was performed and these compounds were then assayed against the recombinant parasite and human DHFRs. Some of the compounds showed good activity against T. cruzi in in vitro assays. A molecular modeling showed that those compounds which bound within the enzyme pocket of Trypanosomatid enzymes presented the highest selectivity [275].
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b- 2,4-Diaminoquinazolines A series of 2, 4-diaminoquinazolines were designed, synthesized and evaluated as inhibitors of dihydrofolate reductase of different Trypanosomatids. Some of these compounds showed potent activity against T. cruzi [276]. c- Antifolate Drugs Recently, the lipophilic trimetrexate (TMQ), a FDA-approved drug for the treatment of Pneumocystis carinii infection in AIDS patients, showed to be a potent inhibitor of T. cruzi DHFR activity and was also highly effective in killing T. cruzi trypo and amastigotes. Unluckily, TMQ also showed to be a good inhibitor of human enzyme [277]. 12C-Pteridine Reductase (PTR) Many important cellular functions require reduced pteridines. Trypanosomatids unlike their mammalian host are pteridine auxotrophs and salvage the precursor pteridines from the host and reduce them to the respective biologically active tetrahydro forms using parasite enzymes which may serve as drug targets. The enzyme pteridine reductase 1 (PTR1), only found in Trypanosomatids and plant pathogens, was first related with reduction of unconjugated pteridins. However, it also catalyzes the reduction of folate to dihydrofolate and tetrahydrofolate mediating in the salvage of oxidized pteridines showing a lower sensitivity to methotrexate than DHFR, interfering in the effectiveness of antifolate drugs targeting DHFR [278]. In addition, pteridine reductase 2 (PTR2), which can only reduce dihydropterin and dihydrofolate substrates but not oxidized pteridines was identified and expressed in T. cruzi [279, 280]. A docking study was recently performed on a set of pteridine analogues at the active site of PTR2 and better results than that of methotrexate, were obtained for the assayed compounds [280]. Recently, the crystal structure of an inhibitor (methotrexate) and a substrate (dihydrofolate)-complex of this enzyme was performed [281]. Isis Innovation Ltd, 2001 described triazine derivatives as useful novel DHFR inhibitors claiming that these compounds were useful for parasitic infections including Chagas disease [282]. Most of the recently disclosed patents on purine analogues are related to antiviral and /or anticancer activity [283], only a few claim their effects on parasitic diseases. Among ATP analogues, Bottaro et al., claimed that nucleoside pirophosphate and triphosphate analogues, were useful against infectious diseases caused by some protozoans including Chagas disease. However no experimental evidences were given [284], (Table 6). A rapidscreening strategy using a folate-based library with structure-based design was used to identify inhibitors of L. major and T. cruzi PTR1. Assays were carried out against folatedependent enzymes including PTR1, dihydrofolate reductase (DHFR), and thymidylate synthase. Affinity profiling determined selectivity and specificity of a series of quinoxaline and 2,4-diaminopteridine derivatives, and nine compounds showed greater activity against parasite enzymes compared with human enzymes. Biological evaluation of selected inhibitors was performed against the extracellular forms of T. cruzi and L. major, both wildtype and overexpressing PTR1 lines, as a model for PTR1-driven antifolate drug resistance and the intracellular form of T. cruzi. An additive profile was observed when PTR1 inhibitors were used in combination with known DHFR inhibitors, and a reduction in toxicity of treatment was observed with respect to administration of a DHFR inhibitor alone.
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The combination of antifolates targeting two enzymes proved to be successful in the development of novel antiparasitic drugs [285]. 12D- Dihydroorotate Dehydrogenase (DHOD) In T. cruzi, the fourth enzyme of the pathway catalyzing production of orotate from dihydroorotate markedly differs from the human enzyme. Searching for potent inhibitors against T. cruzi DHOD activity, a number of methanolic extracts prepared from green, brown, and red algae were tested. T. cruzi DHOD activity was inhibited by the extracts from two brown algae, Fucus evanescens and Pelvetia babingtonii. In addition, these extracts were effective against the protozoan infection and proliferation in mammalian cells [286] and a recombinant enzyme form, Tc DHOD, was recently crystallized complexed to orotate [287], opening the possibility for future inhibitors design. Genetic studies have shown that this enzyme is essential for T. cruzi survival, validating the idea that it can be considered an attractive target for the development of antichagasic drugs. Thus, a detailed analysis of its crystal structure has allowed suggesting potential sites to be further exploited for the design of highly specific inhibitors through the technology of structure-based drug design [288]. Furthermore, structural analysis of T. cruzi DHOD complexed with substrates and products has allowed investigating atomic resolution insights into mechanisms of dihydroorotate oxidation and fumarate reduction [289]. By combination between a rapid screening strategy using a folate-based library and a structure-based design, assays were carried out against folate-dependent enzymes including and dihydrofolate reductase (DHFR), and thymidilate synthase. A series of quinoxaline and 2, 4 diaminopteridine derivatives showed higher activity against parasite enzymes compared with human enzymes. An additive profile was observed when PTR1 inhibitors were used in combination with known DHFR inhibitors, and a reduction in toxicity of treatment was observed with respect to administration of a DHFR inhibitor alone. The combination of antifolates targeting two enzymes was proposed as high potential for such an approach in the development of previously non described antiparasitic drugs [290].
13-ORGANELLES AS TARGETS 13-1-Nucleus, Kinetoplast and DNA Modulation DNA topoisomerases are essential enzymes for nucleic acid biosynthesis and cell survival which modify the topology of DNA. In kinetoplastids, topoisomerases are involved in the metabolism of both nuclear and mitochondrial (kinetoplast) DNA. DNA topoisomerases from parasites have been the focus of molecular and cellular biology studies and have been also considered as target for antiparasitic chemotherapy, particularly, topoisomerase II, required for kinetoplast replication. Several inhibitors of bacterial DNA topoisomerase II showed to be effective against T. cruzi, producing damage to kinetoplast and/or the nucleus of epimastigotes and inhibiting both proliferation and differentiation processes, suggesting that both organelles could be the targets of the drugs [291]. On the other hand, complex II (succinate: ubiquinone reductase) often plays a pivotal role in adaptation of parasites in host organisms and could be a potential target for new drugs. Complex II from T. cruzi was studied finding that it is composed of six hydrophilic (SDH1, SDH2N, SDH2C, and SDH5SDH7) and six hydrophobic (SDH3, SDH4, and SDH8-SDH11) nucleus-encoded subunits. Orthologous genes for each subunit were identified in T. brucei and L. major. A detailed
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study showed unusual unique features in Trypanosomatids that make Complex II a target for new chemotherapeutic agents [292]. -Quinolone Derivatives T. cruzi is particularly sensitive to quinolone derivatives probably through DNA topoisomerase II inhibition. Quinolone derivatives were patented by New Pharma Research Sweden AB as useful agents in treatment of bacterial and parasitic diseases including those caused by Trypanosomatids but no specific data for the claimed compound were reported [293], (Table 6). Currently, these compounds are patented as antibacterial compounds and especially suitable for treatment of coccidiosis. On the other hand, Camptothecin, an antitumoral drug and a well-characterized inhibitor of eukaryotic DNA topoisomerase I, caused disruption of nuclear and mitochondrial DNA in T. cruzi [294]. -Dicationic Guanidine and Reverse Amidine Derivatives Among DNA modulating agents, described as promising agents for the treatment of African trypanosomiasis, twenty dicationic molecules containing either diguanidino or reversed amidine cationic groups were tested in vitro versus T. cruzi. The most active compounds belong to the reversed amidine series and six exhibited IC50 values of less than 1 M [295, 296]. Scientist from University of North Carolina at Chapel Hill synthesized dicationic reversed amidines such as novel 2, 5-bisalkyl (or aryl) imino aminophenyl furans and thiophenes, compounds with strong DNA binding affinities and a patent claimed them useful for mycobacterial, fungal and protozoal infections including Trypanosoma cruzi [297], (Table 6). Aromatic diamidines are DNA minor groove-binding ligands that display antimicrobial activity against fungi, bacteria and protozoa. The effects of a diarylthiophene diamidine, DB1362, were tested on amastigotes and bloodstream trypomastigotes of T. cruzi showing a potent in vitro activity against both forms at dosis that did not exhibit citotoxicity [298]. Later, studies on the activity of four such diamidines (DB811, DB889, DB786, DB702) and a closely related diguanidine (DB711) against bloodstream trypomastigotes as well as intracellular amastigotes of T. cruzi in vitro and toxicity assays of these compounds against mammalian cells in vitro were performed. Most of the diamidines compounds exerted high anti-parasitic activity and low toxicity to the mammalian cells suggesting that the compounds merit in vivo studies [299]. -Dinitroaniline Sulfonamide Derivatives These antimitotic compounds with activity against tubulin were disclosed by scientist from Ohio State University as useful for the treatment of diseases caused by parasitic protozoa, particularly Leishmaniasis. Despite their good in vitro activity, these compounds failed to cure parasite infected mice. The putative toxicity of compounds with nitroaromatic groups remains to be addressed [300], (Table 6). Vanadium Mixed-Ligand Complexes Four novel mixed-vanadyl ligand complexes, [V(IV)O(L(2)-2H)(L(1))], including a bidentate polypyridyl DNA intercalator (L(1), and a tridentate salycylaldehide semicarbazone derivative (L(2) as ligand were synthesized, characterized and evaluated, being as active on epimastigotes of T. cruzi as nifurtimox. DNA was evaluated as potential parasite
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target and data obtained by electrophoretic analysis suggest that the mechanism of action of these complexes could include DNA interactions [301]. Diphenylamine Derivatives The pursuit of small molecules that bind to DNA has led to the discovery of selective and potent anti-trypanosomal agents, specifically 4,4'-bis(imidazolinylamino)- and 4,4'bis(guanidine)diphenylamine compounds, CD27 and CD25, respectively. Anti-Trypanosomal properties of these compounds have been characterized. To detail the nature of the interaction of these compounds with DNA, the crystal structure was analyzed, suggesting the basis for understanding the mechanism of anti-trypanosomal activity of these symmetric diphenylamine compounds [302]. Bisbenzimidazol Derivatives Novel bisbenzimidazol derivatives characterized by 3, 4-ethylenedioxy-extension of thiophene core, revealed pronounced affinity and strong thermal stabilization effect toward ds-DNA. Compounds 4-6 showed moderate to strong antiproliferative effect towards a panel of carcinoma cell lines. Among them, compound 5 was capable to inhibit the growth of T. cruzi epimastigotes [303]. The inhibition of trypanosome growth was caused by the specific interaction of typical ligands (benzimidazoles, colchicine and vinblastine) with trypanosome tubulin. Then, in kinetoplastids, tubulin has been proposed as a potential target [304]. Selective lead compounds against kinetoplastid tubulin have been identified and have been suggested as starting point for the development of new drug candidates against these parasites [305]. Ribavirin (1,2,4-Triazole-3-carboxamide Riboside) It is a well-known antiviral drug and has also been reported to inhibit human S-adenosylL-homocysteine hydrolase (Hs-SAHH), that catalyzes the conversion of S-adenosyl-Lhomocysteine to adenosine and homocysteine. The drug is structurally similar to adenosine, produces time-dependent inactivation of Hs-SAHH and T. cruzi SAHH (Tc-SAHH). Ribavirin binds to the adenosine-binding site of the two SAHHs and reduces the NAD (+) cofactor to NADH. The reversible binding step of ribavirin to Hs-SAHH and Tc-SAHH has similar K (I) values but the slow inactivation step is 5-fold faster with Tc-SAHH. Thus, ribavirin might provide a structural lead for design of more selective inhibitors of Tc-SAHH as potential anti-parasitic drugs [306]. Among patents related with compounds and methods of use to treat infectious diseases, scientist from Bradley Cytokine Pharmasciences, Inc, describe some of them, used to target specific nuclear localization, signal blocking importation of specific proteins or molecular complex into the nucleus of a cell claiming their use for treatment or prevention of infectious diseases, such as parasitic and viral diseases [307], (Table 6). 13-2-Acidocalcisomes and Exchanger Na +/H+ Mechanism The storage of calcium in specialized acidic organelles, termed acidocalcisomes constitutes another unusual feature of T. cruzi, in comparison with mammalian cells. These structures are involved in polyphosphate and calcium storage as well as in adaptation to
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environmental oxidative stress [308]. 3,5 dibutylhydroxitolueno blocks Ca2+ release via the acidocalcisomal exchanger Na+/H+. Then, the acidocalcisomal exchanger Na+/H + is a mechanism involved in Ca2+ and pH homeostasis exclusive of this organism to be potentially used as drug target. -Guanidine Derivative Compounds Hoechst Marion Russel Deutchland CmbH claimed the use of Na+/H + exchange inhibitors for the treatment of protozoal infections including Chagas disease. However, these compounds were described but neither synthesis nor characterization was shown [309], (Table 6). --lapachone-Derived Naphthoimidazoles Among 45 semi-synthetic derivatives of naphthoquinones isolated from Tabebuia sp, naphthoimidazole N1 resulted one of the most active compounds against T. cruzi trypomastigotes. The effect of N1 against the proliferative forms of T. cruzi suggested that in epimastigotes, reservosomes, mitochondrion, and nucleus contain N1 targets. In trypomastigotes, in which reservosomes are absent, the organelles affected by the compound were also the mitochondrion and nucleus, as well as acidocalcisomes, in which the decrease in electron density could be due to the use of polyphosphate as an alternative energy supply [310]. 13-3 Membrane Components, Contractile Vacuole Complex and Osmoregulation Searching for novel drug targets, among parasite membrane components, transport proteins for nutrients and metabolites of the parasite-host interface are getting into focus. Genes coding for aquaporin water and solute channels have been identified in the protozoan genomes. Six protozoan aquaporins have been cloned and functionally characterized. Amino acid compositions of the individual pore entries were compared and permeability properties attributed to specific protein features. Furthermore, possible physiological roles in osmotic protection and metabolism were assigned to aquaporins. The presence of TcAQP, corresponding to an aquaporin gene from T. cruzi, was reported in acidocalcisomes and contractile vacuole complex of the parasite [311]. The potential of protozoan aquaporins for use as a target or entry pathway for chemotherapeutic compounds was recently reviewed by Beitz and co-workers [312]. Moreover, a contractile vacuole complex is involved in osmoregulation in T. cruzi. A microtubule- and cyclic AMP-mediated fusion of acidocalcisomes to the contractile vacuole complex in T. cruzi results in translocation of aquaporin and the resulting water movement which, in addition to swelling of acidocalcisomes, is responsible for the volume reversal not accounted for by efflux of osmolytes. Polyphosphate hydrolysis occurs during hyposmotic stress, probably increasing the osmotic pressure of the contractile vacuole and facilitating water movement [313]. A subset of transporters that are essential for parasite viability could serve as targets for novel drug therapies by identifying compounds that interfere with their uptake functions [314]. A T. cruzi phosphatidylinositol 3-kinase (TcVps34) plays a prominent role in vital processes for T. cruzi survival such as osmoregulation, acidification, and vesicular trafficking [315]. In addition, the cloning, expression, purification, and characterization of the T. cruzi exopolyphosphatase (TcPPX) were reported. TcPPX differs from most exopolyphosphatases in its preference for short-chain polyphosphate (poly P). Heterologous expression
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of TcPPX in E. coli produced a functional enzyme which was dramatically inhibited by low concentrations of Zn2+, high concentrations of basic amino acids (lysine and arginine), and heparin. TcPPX is a processive enzyme and does not hydrolyze ATP, pyrophosphate, or pnitrophenyl phosphate, although it hydrolyzes guanosine 5'-tetraphosphate very efficiently. Overexpression of TcPPX resulted in a dramatic decrease in total short-chain poly P and partial decrease in long-chain poly P, accompanied by a delayed regulatory volume decrease after hyposmotic stress supporting the role of poly P in T. cruzi osmoregulation [316]. Parasite membrane contains ecto-enzymes whose active site faces the external medium rather than the citoplasm. Recently, Cr-ATP (chromium (III) adenosine 5'-triphosphate complex) was shown as a new inhibitor of ecto-ATPases of Trypanosomatids as a tool for a better understanding of properties and role of ecto-ATPases in the biology of parasites. DIDS (4, 4 diisothiocyanatostilbene 2,2' disulfonic acid), suramin and ADP were also effective as inhibitors. Only ADP presented no additive inhibition with Cr-ATP. The pattern of partial inhibition by Cr-ATP was observed for the ecto-ATPase activities of L. amazonensis, T. cruzi and T. rangeli. Cr-ATP emerges as a new inhibitor of ecto-ATPases and as a tool for a better understanding of properties and role of ecto-ATPases in the biology of parasites [317]. On the other hand, inositol is the precursor for most T. cruzi surface molecules, including phosphoinositides, glycosylinositolphospholipids and glycosylphosphatidylinositol anchors. As the parasite is an inositol auxotroph, the inositol transport system might be a potential target for new trypanocide drugs, as some of its properties are different from its mammalian counterpart. The modulation exerted by effectors of PKA and PKC on this transport system to comply with the parasite physiology was studied concluding that the myo-inositol transport system in T. cruzi epimastigotes is inhibited by PKA and stimulated by PKC effectors [318]. 13-4 Glycosome and Vitamin C Synthesis It was demonstrated that both T. brucei and T. cruzi have the capacity to synthesize vitamin C and the reaction occurs in a unique single-membrane organelle of the parasite, the glycosome. Tacking into account that the capacity to synthesize vitamin C (ascorbate) is widespread in eukaryotes but is absent from humans, this aspect constitutes another potential chemotherapeutic drug target [319]. Different studies pointed to the localization of a solanesyl-diphosphate synthase, TcSPPS from T. cruzi in glycosomes. Taking into account that ubiquitine is has a central role in energy production and in reoxidation of reduction equivalents, TcSPPS is proposed to be promising as a new chemotherapeutic target [320]. 13-5 Mitochondrion Natural quinones isolated from Brazilian flora and its derivatives were tested as alternative chemotherapeutic agents against T. cruzi. Three naphtofuranquinones were synthesized and showed to be active against trypo and epimastigote forms. Ultrastructural analysis of treated epimastigotes and trypomastigotes indicated a potent effect of the naphtofuranquinones on the parasite mitochondrion, which appeared drastically swollen and with a washed-out matrix profile. In addition, naphtofuranquinones produced a collapse in the mitochondrial membrane potential, decreased specifically mitochondrial complex I-III activity, and in parallel to the reduction in succinate induced oxygen consumption indicating an association between trypanocidal action of these compounds and mitochondrial
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dysfunction leading to increased reactive oxygen species generation and parasite death [321]. A series of over a hundred furoxans, alkylnitrates and related compounds were tested as anti-trypanosomal agents in in vitro assays. Among the studied compounds, derivative 4 emerged as lead compound inhibiting trypo and amastigote forms of T. cruzi, attributing the effect to action on mitochondrial dehydrogenases [322]. In the search for new therapeutic agents for Chagas disease, extracts obtained from the Brazilian plant Pterodon pubescens were screened. Oleaginous ethanolic extract of P. pubescens seeds and its fractions as well as geranylgeraniol (GG-OH), the sole component of the hexane fraction were tested for trypanocidal activity. Fraction 2 and GG-OH showed similar potency on blood trypomastigotes and GG-OH inhibited the proliferation of intracellular amastigotes, at concentrations which do not affect the mammalian host cell. Ultrastructural studies pointed to mitochondrion of both epimastigotes and of trypomastigotes, an organelle that plays a central role in apoptosis, as the major suggested target of GG-OH [323]. (2E)-N-(1,3-benzothiazol-2-yl)-3-(2,5-dimethoxyphenyl)-2-propenamide (CAD-1) The preparation and in vitro evaluation of cinnamic acid as potential anti-protozoan agent showed that 0.05 mM CAD-1 induced 58 % of T. cruzi epimastigotes death; mainly by apoptosis. The diminution in the transmembrane mitochondrial electrical potential together with the increase in the intracellular generation/accumulation of reactive oxygen species, suggest the parasites mitochondria as the main target for CAD-1-induced death. The concentration of 0.05 mM CAD-1 is not low enough to consider it as a potent trypanocidal agent. However, the novel mechanism that induces T. cruzi death, together with the novelty of its chemical structure, pointed out CAD-1 as a head group compound that could serve as a template to obtain new, more potent anti-Chagas disease agents [324].
14- SIALIC ACIDS TRANSFERENCE Trypanosomes are unable to synthesize sialic acids but can scavenge them from its mammalian hosts by using a unique neuraminidase with trans-sialidase activity able to transfer sialic acid molecules from host glycoconjugates to mucin-like acceptors present in the parasite surface membrane. In addition, the action of this particular developmentally regulated trans-sialidase (TS) seems to be essential for T. cruzi survival and cell invasion in the host [325, 326]. Then, TS inhibitors are also considered potential trypanocidal therapeutic agents. The X-ray structure of TcTS and TcTS in complex with substrates and sialidase inhibitors has been published. A significant number of amino acid residues are conserved within the active site of TcTS that are common to all known sialidases, reflecting a strong evolutionary link to other microrganisms. However, critical amino acid residue differences between mammalian sialidases and the parasite trans-sialidase provide a basis for an explanation of the particular glycotransfer enzymatic activity of TcTS [327]. A recent report describes some target synthetic sialylmimetics-cyclohexenephosphonate monoester compounds displaying promising inhibitory properties when tested with parasitic or bacterial sialidases [328]. Among patented compounds, novel N-substituted Piperidines were disclosed by Horenstein and Parr from the University of Florida, claiming that these compounds with neuraminidase inhibitory activity could be used for the treatment of bacterial, viral and parasitic infections including diseases caused by trypanosomes [329]. In addition, scientist from University of Alabama disclosed inhibitor and methods of treating
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and preventing bacterial or Trypanosomal infection using a bacterial sialidase inhibitor [330], (Table 6). However, in both cases no data on their activity against the parasite enzymes were presented.
15-BIOSYNTHESIS OF LIPIDS 15-A-Alkyl-Lysophospholipids (ALPs) Another group of promising compounds active against proliferation and differentiation of T. cruzi, in vitro and in vivo are alkyl-lysophospholipids (ALPs). These synthetic analogues of lisophospholipids designed as potential immunomodulators have been developed as antitumoral and antileukaemial agents [331]. Although the mechanism related with antiparasitic activity is still not known, the anti-T. cruzi activity of ALPs has been related with a selective blockade of phosphatidyl-choline (PC) biosynthesis in the parasite involving the transmethylation pathway, in contrast with the situation in the vertebrate host, where the CDP-choline pathway is predominant. These ALPs present good oral activity and low toxicity [332]. In addition, lysophospholipid analogues (LPAs) originally developed as anti-cancer agents, have also shown significant activity against Leishmania spp. and T. cruzi, both in vitro and in vivo. Miltefosine was registered in 2002 for the oral treatment of visceral Leishmaniasis. LPAs interfere with lipid synthesis in T. cruzi and cancer cells, but the activity is about >20-fold higher against the parasite [333]. It was reported that LPAs present antiproliferative synergy with ketoconazole against both epimastigotes and intracellular amastigotes of T. cruzi. Whereas edelfosine or ketoconazole alone induced morphological alterations in the plasma membrane and reservosomes of the parasites, combinated also led to severe mitochondrial damage, formation of autophagic structures and multinucleate, possibly by interference with lipid metabolism [334]. Recently, the LPA edelfosine was also tested on trypomastigotes. LPAs induced alterations in the plasma membrane of the three developmental stages of the parasite and in the mitochondria in epimastigotes suggesting that these organelles are potential targets of these analogues [335]. LPAs interfere in the lipid biosynthesis in epimastigotes altering the amount of phospholipids and sterols, and consequently the physical properties of the membrane [336]. A series of analogues of the naturally occurring antibiotic thiolactomycin (TLM) has been evaluated against P. falciparum proliferation taking into account that TLM is an inhibitor of Type II fatty acid synthase but not of Type I fatty acid synthase in mammals. A number of the analogues showed inhibition equal to or greater than TLM and some of them showed activity when assayed against the parasitic protozoa, T. cruzi and T. brucei [337]. 15-B- Glycosphingolipids (GSLs) Lipid metabolism has also been attracting a lot of attention with respect to basic biology and applications for chemotherapeutic purposes. Although glycosphingolipids (GSLs) are ubiquitous in eukaryotic cells, very little is known about their role in parasites. The presence of an active Glucosylceramide synthase (GCS) in the intraerythrocytic stages of P. falciparum has been demonstrated [338]. Taking into account that glucosylceramide is a pivotal precursor of numerous GSLs, the special features presented by this enzyme compared with the mammalian counterpart signal GCS as a potential target [339]. In T. cruzi, different GCS inhibitors were tested as antiproliferative agents in culture and
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bloodstream forms. PPMP produced 79 to 95.5 % of parasite lysis in in vitro assays. In vivo assays in infected mice are under development with PPMP as well as with some citostatic drugs involved in the alteration of this GSL pathway2.
PART II. DRUGS DERIVED FROM NATURAL SOURCES The use of natural products for the treatment of protozoal infections is well known and has been early documented. Several recent works report the investigation of trypanocidal activity of a wide variety of crude natural extracts or compounds isolated, particularly of vegetal origin as well as semi-synthetic analogues. Among them different groups can be found in the literature:
1- ANTI-MICROTUBULE AGENTS Microtubules play fundamental roles in eukaryotic cells. The antimicrotubule drug taxol, obtained from the bark of Taxus brevifolia as well as its synthetic derivatives, employed in cancer chemotherapy, also interferes with the proliferation of Crithidia fasciculata and T. cruzi, leading to morphological alterations, interruption of nuclear division and cytokinesis, and inhibitory effect on endocytosis of proteins by epimastigotes [340, 341]. On the other hand, the antimicrotubule agents vinblastine and vincristine, alkaloids obtained from Vinca rosea showed selective and reversible effects inhibiting both nuclear division and cytokinesis thus interfering with epimastigotes proliferation [342]. 2- ALKALOIDS A variety of alkaloids have been tested against epimastigotes of T. cruzi. The activity of apomorphine [343] as well as the activity of -carboline alkaloids on nifurtimox resistant parasites [344] was associated to the inhibition of respiratory chain. Besides, some glycolalkaloids including -chaconine and -solamargine as well as some aglycones (demissidine, solanidine, etc) were tested against epimastigotes, bloodstream and metacyclic trypomastigotes, showing higher activity than ketoconazole [345]. Five new bisbenzylisoquinoline derivatives were isolated from the stem bark of Guatteria boliviana, among them, funiferine, antioquine and guatteboline were active against trypomastigotes [346]. In addition, trypanocidal effects of the natural alkaloid Piperine were evaluated and twelve synthetic derivatives were tested against epimastigote and amastigote forms of T. cruzi, pointing out Piperidine as a suitable template for the development of new drugs with trypanocidal activity [347]. Recently, five out of 64 diterpenoid alkaloids tested, were active on T. cruzi epimastigotes: atisinium chloride and 13-oxocardiopetamine were potent T. cruzi epimas-tigote growth inhibitors with activity levels similar to that of benznidazole. In vitro assays showed that these compounds reduced metacyclic forms capacity of invasion to mammalian cell, their intracellular replications and their transformation into trypomastigotes, with no toxicity to the host cell suggesting that these alkaloids are structural leads of clinically active compounds against T. cruzi [348]. The antiparasitic effects of the canthinones compounds canthin-6-one, 5-methoxycanthin-6-one, canthin-6-one N-oxide, as well as that of the total alkaloids of Zanthoxylum 2
Duschak, VG; Landoni, M; Garabaglia, P.; Esteva, MI; Couto, AS. Glucosylceramide synthase as target for new antiparasitic drugs. Kinetoplastid Diseases, Dakar, Senegal, Africa (2006).
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chiloperone stem bark, were examined in Balb/c mice infected either acutely or chronically with T. cruzi. In the case of acute infection, parasiteamia was significantly reduced following oral treatment with canthin-6-one. Moreover, the total alkaloids of Z. chiloperone stem bark led to high levels of parasitological clearance. Seventy days post-infection, the serological response in the acute model was significantly different between oral canthin-6one and benznidazole-treated mice. Chronic model of the disease showed that both canthin6-one and the alkaloidal extract at the above dosage induced 80-100% animal survival compared to untreated controls. Thus, canthin-6-one exhibited trypanocidal activity in vivo in the mouse model of acute or chronic infection. It was suggested that a long-term oral treatment with this natural product of very low toxicity could prove advantageous compared to the current chemotherapy of Chagas disease [349].
3- STILBENOIDS Isonotholaenic acid, a natural dihydrostilbenoid and some synthetic series of related heterocyclic compounds were tested on cultures of epimastigote and trypomastigote forms of T. cruzi, finding that some of these compounds showed activity similar to benznidazol against epimastigotes, and others were more active against trypomastigotes than the reference drug gentian violet [350].
4- GANGLIOSIDES Ganglioside treatment of acute infected mice determined long-term survival and clearance of parasites from the bloodstream and organs, producing additional complete prevention of clinical manifestations of the infection, and progression into the chronic stages of the disease, for at least 18 months post-infection. It was suggested that the effect of gangliosides could be due to inhibition of phospholipase A2 enzymes, which are involved in membrane destabilization interfering parasite penetration into the host cells. However, the fact that these compounds had no toxic effect on the parasite turned non probable this hypothesis, considering that the in vivo effect could be due to modulation of the host immune system [351]. Exogenous gangliosides, therapeutic agent in experimental Chagas disease, produced biochemical and structural modifications in axenic cultured treated epimastigotes as well as in trypomastigotes altering lipid order, inhibiting membrane enzymes, shifting the parasite energy source from glucose to amino acids and ending on a structural transformation which signals parasite cell death [352].
5- SNAKE VENOM AND AMPHIBIAN SKIN SECRETIONS Proteins and peptides from snake venoms have also been considered as novel drug candidates, showing effective activities. Venom from three different snake species was tested in vitro against T. cruzi. Epimastigotes proliferation was inhibited by Venom from Cerastes cerastes and Naja haje at levels similar to benznidazol. Venom from C. cerastes was also active against trypomastigotes [353]. In terrestrial ecosystems, amphibians present a unique efficient skin secretion system with a variety of glands which produce a myriad of potent bioactive compounds such as peptides, alkaloids, biogenic amines and lipids. Bufadienolides are cardioactive steroids from animals and plants that have also been reported to possess antimicrobial activities.
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Two steroids, telocinobufagin and hellebrigenin, demonstrated activity against L. chagasi promastigotes, but only the latter was active against T. cruzi trypomastigotes. This novel biological effect of R. jimi steroids could be used as a template for the design of new therapeutics against Leishmaniasis and American Trypanosomiasis [354].
6- JUVENILE HORMONE AND ANALOGUES The juvenile hormone-III and the analogues methoprene and fenoxycarb inhibited macromolecular biosynthesis and growth of epimastigotes [355]. Some analogues showed lytic activity on blood trypomastigotes and reduced the parasitemia and mortality levels in infected mice in a moderate degree [356]. Sulphur-containing derivatives structurally related to fenoxycarb showed to be potent growth inhibitors against the intracellular form of the parasite [357]. On the other hand, whereas in vitro experiments showed that methoprene cause cellular death of T. cruzi, this compound failed to clear bloodstream trypomastigotes in in vivo experiments but a decrease of parasitemia levels of infected mice was observed, suggesting that this compound might serve as an effective agent to sterilize blood for transfusions [358].
7- FLAVONOIDS AND PROPOLIS Antiplasmodial, leishmanicidal and anti-trypanosomal activities of eight natural biflavonoids were estimated in vitro on the respective parasites. Among them, ginkgetin and isoginkgetin showed the best anti-trypanosomal activity with low IC50 values in the M range [359]. In addition, the strong antimicrobial activity of propolis, the natural resin produced by honey bees is associated mainly with flavonoids and also with derivatives of hydroxycinnamic acid. In T. cruzi, the effect of different types of propolis was evaluated, finding in vitro activity against epimastigotes, trypomastigotes and intracellular amastigotes but no effect was observed on the course of acute infection [360]. In the last years, four derivatives of hydroxycinnamic acid isolated from a Brazilian propolis were assayed against trypomastigotes showing lower activity than crystal violet [361]. In addition, two ethanolic Bulgarian propolis extracts with a high content of flavonoids presented strong inhibitory activity against T. cruzi proliferative epimastigotes, but were more susceptible than trypomastigotes [362]. Multivariate analysis was applied to evaluate the efficiency of different extracts of a Brazilian propolis from Apis mellifera finding different degrees of trypanocidal activity [363]. It was also reported that the treatment of T. cruzi-infected mice with ethanolic extracts of Bulgarian propolis interferes with the basic properties of immune cells promoting changes in the immune response [364]. A sensitive technique that takes advantage of ((3H)thymidine uptake by dividing Trypanosomatids has been adjusted for quantification of the parasiticidal effect of natural products, finding that the flavonoids hispidulin and santin, obtained from the Argentine medicinal plants Ambrosia tenuifolia and Eupatorium buniifolium, respectively, showed trypanocidal and leishmanicidal activities. The IC50 values obtained on epi and trypomastigote forms of T. cruzi in addition to the absence of citotoxicity on lymphoid cells makes hispidulin and santin potential lead compounds for the development of new natural drugs [365]. The dichloromethane extract of Cassia fistula fruits (Leguminosae) led to the isolation of the active isoflavone biochanin A, identified by spectroscopic methods. This compound showed effectivity against promastigotes of L.(L.) chagasi. Additionally, presented an anti-T. cruzi activity, resulting in an
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EC50 value of 18.32 g/ml and a 2.4-fold more effectiveness than benznidazole. Thus, contributing with novel antiprotozoal compounds for future drug design studies [366].
8- NATURAL NAPHTHOQUINONES Among natural naphthoquinones present in plants, bioactive compounds known are lapachol, which was isolated from the heartwood of Tabebuia sp and and -lapachone, both obtained as contaminants in the process of lapachol isolation. Lapachol derivatives were assayed against infective trypomastigote blood forms of T. cruzi and the triacetoxy derivative of reduced lapachol showed relevant trypanocidal activity [181]. - lapachone showed trypanocidal activity against epimastigotes, which was associated to generation of free radicals and inhibition of nucleic acids and protein synthesis [182]. Other quinone compounds isolated from natural products were assayed against T. cruzi and showed trypanocidal activity including trihydroxylated anthraquinone purpurin, obtained from the roots of Rubia tinctorum (Rubiaceae) [367]; the 1,4-naphthoquinone 2,3,3-trimethyl-2-3dihydronaphtho[2,3-b]furan-4,9-quinone isolated from Calceolaria sessilis, [368]; and the polyprenylated benzoquinone 7-epiclusianone, isolated from Rheedia gardneriana (Clusiacease). The latter was active in vitro against trypomastigote, but showed no effect on experimentally infected mice [369]. Recently, the epoxy-alpha-Lap, an oxyran derivative of alpha-lapachone, which presents a low toxicity profile and a high inhibitory activity against different strains of T. cruzi was pointed as a potential candidate for Chagas disease chemotherapy [370].
9- CYCLOSPORIN ANALOGUES Cyclophilin and FK506-binding protein families, known as immunophilins, include the major binding proteins of certain immunosuppressive drugs: cyclophilins for the cyclic peptide cyclosporin A and FK506-binding proteins for the macrolactones FK506 and rapamycin. Tacking into account the antiparasitic activities of cyclosporins, macrolactones and non-immunosuppressive derivatives of these compounds, immunophilins may mediate drug action and/or may themselves represent potential antiparasitic drug targets [371]. In T. cruzi, cyclosporin A (CsA) nonimmunosuppressive analogues were evaluated against the parasite and on a parasite cyclophilin named TcCyP19. Among them, two out of eight CsA analogues (H-7-94 and F-7-62), showed the best anti-parasitic effects on epimastigote proliferation, trypomastigote lysis and inhibition of trypomastigote infection in vitro assays in comparison to CsA control suggesting that this ciclophilin might be involved in the trypanocidal effects [372]. Further In vivo and in vitro parasiticidal effect of CsA analogues showed the most efficient anti-T. cruzi effect with H-7-94, F-7-62 and MeVal-4 CsA derivative suggesting that this effect could be due to inhibition of the peptidyl prolyl cistrans isomerase activity on the T. cruzi recombinant cyclophilins tested and considering to these compounds as promissory parasiticidal drugs worthy of further studies [373].
10- CRUDE PLANT EXTRACTS AND ITS COMPONENTS Plants contribute with several anti-trypanosomal compounds derived mainly from their secondary metabolism. In vitro screenings of plant extracts testing the antiprotozoal activity from different plant families have been performed. Preliminary studies on Bolivian
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medicinal plants evidenced that some of the extracts showed activity against epimastigotes of different strains of T. cruzi [374]. Similarly, extracts from several plants used in Guatemala for the treatment of protozoal infections showed high activity against trypomastigotes. Among them, Neurolaena lobata showed in vitro and in vivo trypanocidal activity [375]. When 79 total extracts obtained from Asteraceae, Araceae, Moraceae, Solanaceae, Rhamnaceae, Zingiberaceae, Leguminosae and Sapotaceae were tested on different parasite models, only nine of them showed trypanocidal activity [376]. The evaluation of trypanocidal activity against trypomastigotes of crude plant extracts of different species of Rutaceae showed that eight out of 32 were significantly actives, being the most active the one obtained from the stems of Pilocarpus spicatus [377]. Besides, crude ethanolic extracts and several fractions obtained by solvent partition of 13 plants from Brazilian Rain Forest were tested for trypanocidal activity with promising in vitro activity against different forms of the parasite. Particularly, activity was observed in both dichloromethane and hexane fractions of Polygala sabulosa and P. paniculata [378]. In addition, extracts obtained from C. podantha and M. arenosa showed high percentages of growth inhibition of epimastigote forms from T. cruzi [379]. Moreover, among selected plants, Casearia sylvestris var. lingua was the most active against both T. cruzi and L. donovani and extracts of Annona crassiflora, Duguetia furfuracea, and Casearia sylvestris var. lingua were active with IC50 values between 0.3-10 g/ml against amastigotes of T. cruzi [380]. On the other hand, a variety of organic crude extracts obtained from 65 Mexican medicinal plants was screened for trypanocidal activity, the methanolic extract of seeds of Persea americana (avocado), six 1,2,4-trihydroxyheptadecane derivatives and two 1,2,4trihydroxy-nonadecane derivatives, isolated from the active fractions showed a moderate activity against epimastigotes and trypomastigotes [381]. Finally, organic and aqueous extracts from 12 Argentine medicinal plants were tested for their in vitro trypanocidal activity on epimastigote forms from T. cruzi. Among the selected species, the organic extracts of Ambrosia scabra, Ambrosia tenuifolia, Baccharis spicata, Eupatorium buniifolium, Lippia integrifolia, Mulinum spinosum and Satureja parvifolia, and the aqueous extracts of E. buniifolium, L. integrifolia, M. spinosum and S. parvifolia showed trypanocidal activity with a percentage of growth inhibition higher than 70 % at a concentration of 100 g/ml [382]. A lot of plant extract components were isolated and also tested for trypanocidal activity. Among them can be mentioned acetogenins from the seeds of Annona glauca (glaucanisin, annonacin A, squamocin and annonacin) which showed activity against trypomastigotes [383], or those extracted from the stem barks of Rollinia emarginata showing in vitro leishmanicidal and trypanocidal properties [384]; cryptofolione derivatives isolated from Cryptocarya alba fruits, were actives against trypomastigotes, but with moderate cytotoxicity for both amastigotes and macrophages, indicating little selectivity for T. cruzi [385]; among antibiotic macrolides, megalomicin, produced by Micromonospora megalomicea, showed potent activity against epimastigotes and intracellular amastigotes at lower concentrations than those that interfere with the mammalian organelle [386] while some polyene macrolides produced by genetically modified Streptomyces appeared to be especially potent and selective trypanocidal compounds [387] and among lignans, methylpluviatolide extracted from the leaves of Zanthoxyllum naranjillo (Rutaceae) which was tested both in vitro and in vivo assays against different strain of T. cruzi resulted highly effective [388] or eupomatenoid-5, a neolignan dihydrobenzofuranic compound isolated from leaves of Piper regnellii var. pallescens which showed antiprotozoal activity against the epimastigote proliferative stages and intracellular amastigote forms of T. cruzi produced ultrastructural
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alterations [389, 390]. Five chromenes isolated from Piper gaudichaudianum and P. aduncum and seven derivatives were assayed in vitro against epimastigote forms of T. cruzi, showing most of them potent trypanocidal activity. The most active compound, [(2S)methyl-2-methyl-8-(3''-methylbut-2''-enyl)-2-(4'-methylpent-3'-enyl)-2H-chromene-6-carboxylate], was almost four times more potent than benznidazole (the positive control) and showed an IC50 of 2.82 M [391]. In the search for antiparasitic agents, extracts from Piper glabratum and P. acutifolium were analyzed, affording nine new benzoic acid derivatives. Their structures were elucidated on the basis of spectroscopic data and the compounds were evaluated in vitro against the promastigote forms of Leishmania spp., T. cruzi, and P. falciparum. Among the evaluated compounds, methyl 3,4-dihydroxy-5-(2-hydroxy-3methylbutenyl)benzoate, methyl 4-hydroxy-3-(2-hydroxy-3-methyl-3-butenyl)benzoate, and methyl 3,4-dihydroxy-5-(3-methyl-2-butenyl) benzoate showed significant trypanocidal activity [392]. Recently, prenylated benzoic acid derivatives from the leaves of Piper heterophyllum and P. aduncum also displayed antiparasitic activity. Among the tested ones, 3-[(2E,6E,10E)-11-carboxy-3,7,15-trimethyl- 2,6,10,14-hexadecatetraenyl)-4,5-dihydroxybenzoic acid and 4-hydroxy-3-(3-methyl-1-oxo-2-butenyl)-5-(3-methyl-2-butenyl)benzoic acid showed moderate antiplasmodial and trypanocidal activities, respectively [393]. In vitro trypanocidal activity of prenylated hydroquinone and benzoic acid derivatives isolated from Piper crassinervium was demonstrated against epimastigote forms of the parasite [394]. Hexanic, methanolic, and hydroalcoholic extracts, and 34 isolated compounds from Vitex polygama Cham and Siphoneugena densiflora were screened for their trypanocidal effects on bloodstream forms of T. cruzi. Their enzymatic inhibitory activities on glicosomal gGAPDH and TR enzymes from T. cruzi were tested. Polar extracts and some of the tested compounds have shown good results in comparison to positive controls of the bioassays [395]. A screening performed in the extracts of two trees from the American tropical rain forests, Calophyllum brasiliense and Mammea americana, showed high trypanocidal activity. Several mammea-type coumarins, triterpenoids and biflavonoids were isolated from the leaves of C. brasiliense and tested in vitro against epimastigotes and trypomastigotes of T. cruzi. Several active coumarins were also tested against normal human lymphocytes in vitro, which showed that mammea-type coumarins were not toxic and could be a valuable source of trypanocidal compounds [396]. Crude extracts and fractions from leaves and stems of Peperomia obtusifolia were evaluated in vitro against epimastigote forms of T. cruzi. The most active extracts afforded seven known compounds, including three chromanes, two furofuran lignans and two flavone C-diglycosides. The most active compounds were the chromanes peperobtusin A and 3,4-dihydro-5-hydroxy-2,7-dimethyl-8-(2''-methyl2''-butenyl)-2-(4'-methyl-1',3'-pentadienyl)-2 H-1-benzopyran-6-carboxylic acid, evidencing Trypanosomal activity in addition to unspecific citotoxicity of chromanes from P. species [397]. Trypanocidal activity was studied on methanolic extracts and tannin compounds from the stem bark of Anogeissus leiocarpus and Terminalia avicennoides [398], on a new pterocarpan and other secondary metabolites of plants from Northeastern Brazil flora. [399], presenting some of them significant activity, without revealing serious toxicity. On the other hand, several flavonoid glycosides from a Turkish plant were tested in mouse models showing a moderate activity against T. cruzi, and only chrysin dimethylether and 3hydroxydaidzein had IC50s lower than 5.0 g/ml. In addition, it was reported that 7,8 dihydroxyflavone and quercetin appear to ameliorate parasitic infections in mouse models resulting potent and effective antiprotozoal agents [400]. In addition, a comparative study on the anti-trypanosomal activity of the isolated triterpenoids and sterols and some related
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compounds from the leaves of Strychnos spinosa and related compounds has indicated that the presence of an oxygenated function at C-28 or an oxygenated side chain at C-17 seems to be important for the anti-trypanosomal activity of triterpenoids and sterols, respectively [401]. Terpenes A variety of terpene compounds were isolated from different plants as follows: a- Diterpenes Some kaurane diterpenes, isolated from the aerial parts of Wedelia paludosa (Asteraceae), showed activity in in vitro assays against trypomastigotes [402]; among diterpenoids isolated from Azorella compacta, the products azorellanol and mulin-11,13dien-20-oico acid were active against amastigotes and the cytotoxicity to mammalian cells was lower than that of nifurtimox [403]. Besides, two new norditerpen aldehydes and five known diterpenes from the fruits of Vitex trifolia also showed in vitro trypanocidal activity with minimum lethal concentrations against epimastigotes in the M range [404]. In addition, komaroviquinone, a potent trypanocidal diterpene, was reduced by T. cruzi old yellow enzyme (TcOYE) to its semiquinone radical. The reductase activity in trypanosome lysates was completely immunoabsorbed by anti-TcOYE antibody. It was suggested that the fact that TcOYE is expressed throughout the T. cruzi life cycle, turns komaroviquinone in an interesting candidate for developing new antichagasic drugs [405]. On the other hand, the oleoresin from Pinus oocarpa was fractionated yielding two diterpenes, pimaric acid and dehydroabietic acid among other compounds, which were tested in vitro against epimastigotes of T. cruzi resulting primaric acid as well as the sesquiterpene longifolene and the oleoresin the most active compounds, being as active as the reference compound nifurtimox [406]. A novel icetexane diterpene, 5-epi-icetexone (ICTX) from Salvia gilliessi resulted active against epimastigotes from different T. cruzi strains [407]. A chloroform extract from roots of Craniolaria annua provided six new C-11 unsubstituted abietane diterpenoids and two known compounds, ferruginol and stigmasterol. Among them, abietanes 1, 1A, 3-5 and ferruginol showed cytotoxic effects against trypomastigote and epimastigote forms of T. cruzi and against fibroblastic Vero cells [408]. b- Triterpenes Crude extracts and fractions of Bertholletia excelsa stem barks were tested for trypanocidal activity. In vitro assays performed with the acetonic and methanolic extracts showed significant activity against trypomastigote forms since in the concentration of 500 g/ml, the parasites were reduced in 100 % and 90.3 % respectively, whereas a triterpene betulinic acid pure isolated from an hexane extract presented 75.4 % [409]. In addition, some bioactive constituents were obtained from an ethanolic extract of Dracocephalum subcapitatum including five flavonoids, calycopterin, xanthomicrol, isokaempferide, luteolin and apigenin, together with five terpenoids, oleanolic acid, ursolic acid, geranial, neral and limonene-10-al. Among them, citral and limonene-10-al were the most effective components against epimastigotes of T. cruzi [410]. Triterpene acids were isolated from methylene chloride extracts of the Miconia sellowiana and M. ligustroides species and their activities against the trypomastigote blood forms of T. cruzi were evaluated. The in vitro assays showed that ursolic acid and oleanoic acid were the most active showing IC50 values in the
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M range. In vivo assays showed that ursolic acid and its salt derivative produced the most significant reduction in parasitemia (75.7 % and 70.4 %, respectively) increasing the survival time for all the treated mice [411]. Different extracts and some fractions obtained from stem bark of Ampelozizyphus amazonicus, commonly used as antimalarial, antiinflammatory as an antidote to snake venom, and were also investigated for trypanocidal activity due to the presence of pentacyclic triterpenes resulting against T. cruzi. Fractions containing mainly betulin, lupenone, 3beta-hydroxylup-20(29)-ene-27, 28-dioic acid, and 2alpha, 3beta-dihydroxylup-20(29)-ene-27, 28-dioic acid showed more activity than crude extracts. Thus, A. amazonicus showed to be a potential source of bioactive compounds that exhibited in vitro parasite lysis against trypomastigote forms of T. cruzi at concentrations >100g/ml [412]. A study showed that Cedrella fissilis is a promising source of active compounds for the control of Chagas disease. Among them, 15 crude extracts and 14 compounds (limonoids and triterpenes) as well as the isolation of 25 known compounds (6 limonoids, 12 triterpenes, 1 sesquiterpene, 5 steroids, and 1 flavonoid) showed trypanocidal activity. Moreover, the inhibitory activity found for odoratol was considered potentially useful as an alternative for the chemoprophylactic gentian violet [413]. c- Sesquiterpenes The sesquiterpene lactone dehydroleucodine affects the growth of cultured epimastigotes of T. cruzi, resulting lethal for the parasites at the higher concentrations tested [414]. By contrast, the sesquiterpene lactones: helenalin and some structurally related derivatives showed anti-trypanosomal activity towards both T. cruzi and T. brucei. Helenalin was the most active compound in the series with IC50 values in the M range [415]. New assays on trypanocidal effect of sesquiterpene lactones including helenalin and mexicanin on cultured epimastigotes was analyzed concluding that both are deleterious for T. cruzi epimastigotes and that their mechanism of action is different from that of the related lactone, dehydroleucodine [416]. Besides, the ethyl acetate extract from leaves plus inflorescences of Lychnophora salicifolia showed significant trypanocidal activity against trypomastigote forms of T. cruzi, which was due to the flavonoid quercetin-7, 3', 4'-trimethyl ether and the sesquiterpenoid lychnopholic acid [417]. In addition, new sesquiterpene hydroperoxides with trypanocidal activity from Pogostemon cablin were also described by Kiuchi and coworkers [418]. Moreover, chemical constituents of L. pohlii, crude extracts from leaves plus inflorescences of L. pohlii and the active sesquiterpene lactones lychnopholide, centratherin, goyazensolides, caffeic acid as well as the isolated flavonoids luteolin and vicenin-2 were analyzed for trypanocidal activity [419]. The trypanocidal sesquiterpene lactone eremantholide C isolated from L. trichocarpha Spreng gave five new oxide derivatives, which were evaluated against Y and CL strains of T. cruzi. All of them were inactive against the Y strain. Compounds 2 and 5 displayed 100% activity on the CL strain while compounds 4 and 6 were partially active on the CL strain [420]. Two bioactive sesquiterpene lactones which were isolated from the organic extract of Ambrosia tenuifolia Sprengel (Asteraceae), were identified as psilostachyin and peruvin and showed significant trypanocidal and leishmanicidal activities. Both compounds showed a marked in vitro trypanocidal activity against T. cruzi epimastigotes with IC50 values of less than 2 g/ml. Psilostachyin exerted a significant in vitro activity against the trypomastigote forms of T. cruzi (IC50, 0.76 g/ml) and was selected for in vivo testing. Psilostachyin-treated mice had a survival of 100% and lower parasitemia values than control mice. Both compounds also presented high selectivity for Leishmania spp suggesting that psilostachyin and peruvin could be considered potential candidates for the development of new antiprotozoal agents against Chagas disease and
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Leishmaniasis [421]. Recently, the in vitro antiprotozoal activity of three irregular, linear sesquiterpene lactones recently isolated from Greek Anthemis auriculata, namely anthecotulide, 4-hydroxyanthecotulide and 4-acetoxyanthecotulide was evaluated. All compounds showed potent trypanocidal and leishmanicidal activity. 4-Hydroxyanthecotulide appeared to be the most active compound against all parasites tested, whereas 4-acetoxyanthecotulide was the least active. All three metabolites possessed toxicity on mammalian cells, which might limit their use as antiprotozoal agents [422].
11- MACROPHYTES, MARINE SPONGE AND ALGAE In the marine ecosystem ecological pressures, such as competition for space and predation, may have favored several invertebrate organisms to select unique metabolites with an assortment of astonishing biological activities. Some Turkish freshwater macrophytes and marine macroalgae were assayed for their in vitro antiprotozoal activity. Whereas all crude extracts displayed appreciable trypanocidal activity on different Trypanosomatids and plasmodes none of the extracts was active against T. cruzi [423]. The marine sponge (Agelas sp.) metabolite agelasine D, as well as other agelasine analogs and related structures were screened for inhibitory activity against P. falciparum, L. infantum, T. brucei and T. cruzi, as well as for toxicity against fibroblast cells. Two compounds displayed IC50 <1 g /ml against T. cruzi in combination with relatively low toxicity against MRC-5 fibroblast cells [424].
12-FUNGI AND SOIL MICROORGANISMS The fungus Lentinus strigosus was selected in a screen for inhibitory activity on T. cruzi TR. The crude extract of L. strigosus was able to completely inhibit TR at 20 g /ml. Two triquinane sesquiterpenoids (dihydrohypnophilin and hypnophilin), in addition to two panepoxydol derivatives (neopanepoxydol and panepoxydone), were isolated using a bioassay-guided fractionation protocol. Hypnophilin and panepoxydone displayed inhibitory activity on TR while the other two compounds were inactive. The activity of hypnophilin was confirmed using intracellular amastigote forms of T. cruzi. It was suggested that the ability of hypnophilin to kill the intracellular forms of T. cruzi while modulating human PBMC proliferation could make this terpenoid to be considered a promising prototype for the development of new chemotherapeutical agents for Chagas disease [425]. The organic extract of the culture of the endophytic fungus Alternaria sp, isolated from the plant Trixis vauthieri DC, was able to inhibit TR by 99%, when tested at 20 g/ ml. Fractionation of the extract identified altenusin, a biphenyl derivative inhibitory activity in the TR assay opening new perspectives for the design of more effective derivatives that could serve as drug leads for new chemotherapeutic agents to treat trypanosomiasis and Leishmaniasis [426]. Basidiomes and fermentation broth extracts from a high number of Basidiomycota fungi were screened in a bioassay panel including the enzyme TR from T. cruzi as well as amastigote forms of L. amazonensis, in addition to human cancer cells lines and peripheral blood mononuclear cells. Among all the results obtained, thirty-four extracts inhibited the activity of the TR. This screening aimed to show the potential of Basidiomycota fungi as sources of bioactive natural products that might be developed into new therapeutic agents for cancer and neglected diseases such as trypanosomiasis and Leishmaniasis [427]. On the other hand, a screening program to discover new trypanocidal compounds from soil microrganisms as well as from the antibiotics library of the Kitasato Institute for Life
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Sciences, showed two microbial metabolites, KS-505a and alazopeptin which exhibit moderate anti-trypanosomal characteristics after studies on in vitro and in vivo anti-trypanosomal activities and cytotoxicities of both compounds, compared with some commonly-used antitrypanosomal drugs [428]. Similarly, three peptide antibiotics, leucinostatin (A and B), alamethicin I and tsushimycin exhibited potent or moderate anti-trypanosomal activity [429]. PATENTS RELATED TO NATURAL COMPOUNDS Among chemotherapeutic drugs derived from natural products, 8 patents claimed the use of natural extracts or synthetic natural products derivatives against Chagas disease (Table 6). Quinoline Derivatives A series of 3, 3-dimethyl-8-oxo-isoquinoline derivatives from natural naphtylisoquinoline alkaloids, originally isolated from African plants, were synthesized. These compounds claimed as useful antiviral and antitumoral agents as well as for treatment of neurodegenerative diseases showed in vitro activity on T. cruzi infected L6-cells with values ranging from 10 to 50 M. One of them displayed selectivity towards the parasite but with citotoxicity on the cells at concentrations higher than 440 M [430]. Similar claims were also described for 1-phenyl-2-aminomethyl naphthalene derivatives. Among them, a sulfonate derivative showed selectivity and an IC50 value in the M range [431]. Moreover, antidesmone, an isoquinoline alkaloid isolated from different species of Euphorbiacea and Ancistroealaines A and B, two new bioactive naphthylisoquinolines, and related naphthoic acids which were isolated from Ancistrocladus ealaensis, exhibited in vitro activity against L. donovani and T. cruzi [432]. Some of these tetrahydroisoquinoline derivatives were disclosed as useful for treating tropical diseases, especially Leishmaniasis, trypanosomiasis or Chagas disease showing in vitro activity against T. cruzi with high IC50 values ranging from 0.02 to 30 M [433]. Derivatives from Plant Extracts The use of canthin 6-one and its derivatives, extracted from Zanthoxylum chiloperone, were disclosed for treatment of T. cruzi infection. This natural compound was assayed in chronic and acute mouse models resulting in both cases more effective than benznidazole. Mice treated with this drug resulted parasite free and protected from death [434]. The lignans obtained from leaves of Zanthoxylum naranjillo such as cubebin, or methylpluviatolide showed trypanocidal activity [388]. The trypanocidal effect of six lignan lactones, (-)cubebin, (-)-O-methyl cubebin, (-)-O-benzyl cubebin, (-)-6, 6’-dinitrohinokinin, (-)hinokinin and dimethoxymorelensin were evaluated in vitro and in vivo. The compounds with higher anti-epimastigote activity were screened against intracellular amastigote of T. cruzi. Among these, (-)-hinokinin was selected to be assayed in vivo. It was observed that three of these compounds showed higher trypanocidal activity against epimastigote forms of T. cruzi. Five of these compounds were also evaluated against intracellular amastigote forms of T. cruzi, with (-)-hinokinin displaying similar activity to benznidazole. In vivo assays showed significant reduction of parasitaemia after administration of this compound in mice infected [389]. A patent was presented by Fundation Pesquiza do Estado de Sao Paulo claiming that these lignans and semisynthetic dibenzylbutyrolactonic derivatives were
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useful for the treatment and prophylaxis of Chagas’ disease. Assays performed with cubebin with blood trypomastigotes showed IC50 values ranging from about 2 to 270 M. Some of them displayed in vitro total inhibitory activity as well as were claimed to show chemoprophylactic ability into healthy mice [435]. Recently, five cubebin derivative compounds were evaluated on in vitro assays against free amastigotes finding that hinokinin was the most active compound (IC50 = 0.7 M) [436]. A novel recent presentation of Fundação De Amparo À Pesquisa Do Estado de São Paulo, Sao Paulo, Brazil included the process to obtain to obtain lignans, especially to obtain cubebin and methylpluviatolide from leaves of Zanthoxylum naranjillo or Piper cubeba as well as semi-synthetic derivatives of cubebin, dibenzylbutyrolactonic lignans, such as: hinokinin, o-acetyl cubebin, o-methyl cubebin, 6, 6'-dinitrohinokinin and o-dimethylethylamine cubebin, synthetic derivatives from lignans bearing anti-Chagas chemoprophylactic and therapeutical activities, were patented [437].
PART III. DESIGN AND SYNTHESIS OF NEW ANTI T. CRUZI DRUGS The literature describes rationally developed drugs, some of them active against different parasitic forms as potential new candidates for the treatment of the Chagas' disease [438]. Recent advances include:
BENZOTROPOLONE DERIVATIVES This new synthetic series of compounds bearing an endocyclic hydrazine moiety were evaluated as potential anti-protozoan agents showing limited or no in vitro activity against L. donovani, P. falciparum and T. brucei rhodesiense. However, several of these compounds were active against T. cruzi in the M range, comparable to that of benznidazole [439].
HETEROAROMATIC COMPOUNDS A 2000-compound chemical library was screened using a recombinant T. cruzi expressing beta-galactosidase strain. Three classes of compounds were selected for their high activity against T. cruzi and low toxicity to host cells in vitro. PCH1, NT1 and CX1 (IC(50): 54, 190 and 23 nM, respectively, each presenting a different mechanism of action on intracellular proliferation of T. cruzi amastigotes, providing new candidate molecules for the development of treatments against Chagas disease and Leishmaniasis [440]. NITROIMIDAZOLE DERIVATIVES New 1, 3, 4-thiadiazole-2-arylhydrazone derivatives of nitroimidazole or phenyl series were synthesized. The evaluation of the activity against bloodstream trypomastigote forms of T. cruzi allowed the identification of brazilizone A, a new potent trypanomicide compound which present an IC50/24 h=5.3 M [441]. BENZO[G]PHTHALAZINE DERIVATIVES A new series of 1,4-bis(alkylamino)benzo[g]phthalazines 1- 4 containing the biologically significant imidazole ring were synthesized, finding when tested a remarkable in vitro
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antiparasitic activity against T. cruzi epimastigotes, in particular in compound 2. Interestingly, inhibition of iron superoxide dismutase (Fe-SOD) resulted efficient, whereas effect on human Cu/Zn-SOD have resulted negligible [442].
5-NITRO INDAZOLE DERIVATIVES A series of 5-nitroindazoles with good antiprotozoal activities, against T. cruzi epimastigotes and Trichomonas vaginalis, were formerly identified. Most of them have shown very low unspecific toxicity on macrophage cell lines. These compounds were tested on T. cruzi bloodstream trypomastigotes and different species of Leishmania promastigotes. Derivatives 1, 2, 7 and 8 displayed remarkable trypanocidal activity (>80% lysis) equivalent to gentian violet. An oxidative stress-mediated mechanism of action was confirmed for derivatives 1, 10 and 12 on T. cruzi epimastigotes. Supported by the in vitro activities, derivatives 1 and 2 were submitted to in vivo assays using an acute model of Chagas disease. None of the animals treated with derivatives 1 and 2 died, unlike the untreated control and benznidazole groups [443]. The development of new indazole derivatives was performed to study structural requirements for adequate anti-T. cruzi activity finding that a butylaminopentyl substituent located in the position 1 of indazole ring affords good activity, but N-oxidation of omega-tertiary amino moiety yields completely inactive compounds. Similarly, the substituent at position 3 of indazole ring affects drastically the in vitro activity. On the other hand, electrochemical studies showed that the trypanocidal 5-nitroindazole derivatives yielded nitro-anion radical via one-electron process at physiological pH. This electrochemical behavior and ESR spectroscopic studies with the T. cruzi microsomal fraction showed that 5-nitroindazole derivatives suffer bio-reduction without reactive oxygen species generation [444].
IMIDAZOLIDINE DERIVATIVES Imidazolines can be considered ethylenediamine/carbonyl precursors that interfere with the biosynthesis of polyamines into the parasite. Then, imidazolidine derivatives were studied as anti-T. cruzi agents. Some of the derivatives were found to have high and selective activity against the proliferative stages of the parasite, with IC50 values against the epimastigote form in the low molar range as the reference drug nifurtimox. It was proposed that these derivatives affect the mitochondrial integrity according to the excreted end-products found in the NMR studies. The QSAR studies indicated that the bioactivities are correlated with the lipophilicities. Thus, a new and relevant bioactivity was described for imidazolidines supporting further in vivo studies of some of these imidazolidine derivatives [445].
NITROFURAZONE DERIVATIVES Nitrofurazone (NF) and its derivative, hydroxymethylnitrofurazone (NFOH), have shown antichagasic activity due to TR inhibitory activity. In addition to this activity, in vitro cruzain inhibition tests were performed for both compounds, showing IC50 values for both compounds in the μM range. Moreover, AM1 semi-empirical molecular modelling studies corroborated the observed cruzain inhibitory activity [446].
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HETEROCYCLIC CATIONIC MOLECULES Among aromatic diamidines, furamidine (DB75) and its phenyl-substituted analogue (DB569) were tested. DB569 displayed higher trypanocidal activity compared to furamidine and also had higher ability to induce apoptosis-like death in treated parasites [447, 448]. Taking into account the broad-spectrum antimicrobial activity of the aromatic dicationic compounds, a study focused on the activity of four such diamidines (DB811, DB889, DB786, DB702) and a closely related diguanidine (DB711) against bloodstream trypomastigotes as well as intracellular amastigotes of T. cruzi in vitro. Most of the diamidines compounds tested exerted high anti- T. cruzi activity and low toxicity to the mammalian cells suggesting that reversed diamidines merit in vivo studies [449]. In addition, several different heterocyclic cationic compounds including diamidines (DB1195, DB1196 and DB1345), a monoamidine (DB824), an arylimidamide (DB613A) and a guanylhydrazone (DB1080) against amastigotes and bloodstream trypomastigotes of T. cruzi. All compounds exerted, at low-molar doses, a trypanocidal effect upon both intracellular parasites and bloodstream trypomastigotes of T. cruzi. A potential application in the prophylaxis of banked blood was proposed for the compounds DB613A and DB1196, because their trypanocidal effects were not affected by plasma constituents. In addition, potency and selectivity of DB613A, towards intracellular parasites, confirmed the promising activity of arylimidamides against this parasite [450].
MELAMINE-BASED NITROHETEROCYCLES Various nitro heterocycles compounds were tested on different Trypanosomatids. Some of them showed significant activity in vitro against T. cruzi [451] and others on T. brucei spp [452].
QUINONE AND NAPHTHOQUINONE DERIVATIVES A set of 25 quinone compounds with anti-trypanosomal activity was studied by using the density functional theory (DFT) method. Two of them were predicted as active against T. cruzi [453]. New naphthoquinone derivatives were synthesized and assayed against bloodstream trypomastigote forms of T. cruzi. Five substituted ortho-naphthofuranquinones, a non-substituted para-naphthofuranquinone, a new oxyrane and an azide were prepared from nor-lapachol and a new non-substituted para-naphthofuranquinone from alpha-lapachone. Five substituted ortho-naphthofuranquinones recently designed as cytotoxic, were also evaluated. The compounds were rationalized based on hybrid drugs. The most active compounds against T. cruzi were the ortho naphthofuranquinones derivatives [454]. In addition, [1,2,3]-triazole derivatives of nor-beta-lapachone were synthesized and assayed against the infective bloodstream trypomastigote forms of T. cruzi, resulting all the naphthoquinoidal [1,2,3]-triazole derivatives more active than the original quinones, showing IC50 values in the range of 17 to 359 M, emerging as new lead compounds for the chemotherapy of Chagas disease [455]. Derivatives of natural quinones with biological activities, such as lapachol, alpha- and beta-lapachones, have been synthesized and their trypanocidal activity evaluated in vitro in T. cruzi. All tested compounds inhibited epimastigote growth and trypomastigote viability. Several compounds showed similar or higher activity as compared with nifurtimox and benznidazole. It was shown that anti-T. cruzi activity of the alphalapachone derivatives can be increased by the replacement of the benzene ring by a pyridine moiety. Free radical production and consequently oxidative stress through redox cycling or
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production of electrophilic metabolites are the potential biological mechanism of action for these synthetic quinones [456]. A recent screening of 65 derivatives of natural quinones using bloodstream trypomastigotes of T. cruzi, the 3 naphthoimidazoles derived from betalapachone (N1, N2 and N3) were selected as the most active. Studies on the mode of action of naphthoimidazoles showed mitochondrion, reservosomes and DNA as their main targets. These compounds promote different death phenotypes in T. cruzi, resulting autophagy the predominant one [457].
THIOSEMICARBAZONE DERIVATIVES A series of new derivatives were designed combining in the same molecule the thiosemicarbazone function recently described as a potent Cz-inhibitor moiety and the recognized 5-nitrofuryl group, an oxidative stress promoter. Some of the derivatives were found to be very active against epimastigotes, being 1.5-1.7-fold more active than nifurtimox [458]. In order to get insight into the bioreductive mode of action of antitrypanosomal 5-nitrofuryl containing thiosemicarbazones, electron spin resonance spectra of radicals generated in T. cruzi by bioreduction were analyzed finding three different pattern of signals with the different compounds tested in accordance with the changes in the T. cruzi-oxygen uptake promoted by these compounds [459].
QUINOXALINE DERIVATIVES In vitro assays of some synthetic compounds presented similar inhibitor growth activity than nifurtimox. Among them, 13, a quinoxaline N, N'-dioxide derivative, and the reduced derivatives 19 and 20 were the most cytotoxic compounds against the protozoan [460]. Novel quinoxaline-N-acylhydrazon derivatives, planned as cruzain inhibitor candidates were designed, synthesized, studied by docking analysis and tested for trypanocidal activity. Two salicylaldehyde N-acylhydrazones presented IC50 values of the same magnitude order than the standard drug nifurtimox when tested in vitro against epimastigote forms of T. cruzi and resulted non-toxic at the highest assayed doses in assays with macrophages [461]. On the other hand, when a series of novel quinazoline-type compounds were designed as inhibitors of the parasite specific enzyme TR, and their biological activities were evaluated, some of them inhibited TR, showed selectivity for TR over human glutathione reductase, and inhibited parasite growth in vitro, suggesting that the quinazoline framework is a privileged structure that can be purposely modified to design novel TR inhibitors. In this sense, the use of privileged motifs might emerge as an innovative approach to antiparasitic lead candidates [462]. BENZOFUROXAN DERIVATIVES A series of new benzo [1, 2-c] 1, 2, 5-oxadiazole N-oxide derivatives as antitrypanosomal compounds were generated. In vitro activity of these compounds was tested against T. cruzi. The most effective derivatives showed IC50 of the same order as that of the reference drug [463, 464]. Hybrid compounds containing hydrazones and benzofuroxan pharmacophores were designed as potential T. cruzi-enzyme inhibitors. Some of the synthesized derivatives were moderate inhibitors of cysteinyl site enzymes of T. cruzi, Cz and TR. The mechanism of action of the trypanocidal effect was assigned to the oxidative stress into the parasite [465]. New benzofuroxans were developed and studied as antiproliferative T. cruzi
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agents. Compounds displayed remarkable in vitro activities against different parasite strains. The most active derivatives, the vinylsulfinyl- and vinylsulfonyl-containing benzofuroxans, showed to with glutathione in a redox pathway and showed good in vivo activities when they were studied in an acute murine model of Chagas disease [466]. 2-PROPEN-1-AMINE DERIVATIVES The cis and trans isomers of the unsubstituted and bromo-2-propen-1-amine derivatives were evaluated in vitro and in vivo assays on T. cruzi. It was suggested that these derivatives should inhibit the enzyme squalene synthase of the parasite ergosterol biosynthesis pathway [467].
RING-CONTRACTED AMANTADINE ANALOGS The synthesis of several (3-noradamantyl)amines, [(3-noradamantyl)methyl]amines, (3,7-dimethyl-1-bisnoradamantyl)amines, and [(3,7-dimethyl-1-bisnoradamantyl)methyl] amines were evaluated against a wide range of viruses. Several of the polycyclic amines tested showed an interesting activity as NMDA receptor antagonists and a rimantadine analogue displayed significant trypanocidal activity. Moreover, to further characterize the pharmacology of these compounds, their effects on dopamine uptake were also assessed [468]. Synthesis and pharmacological evaluation as NMDA receptor antagonists of several (2-oxaadamant-1-yl) amines was performed. Several of them were more active than amantadine, but none was more potent than memantine. Two of the derivatives showed a significant level of trypanocidal activity [469]. N-OXIDE DERIVATIVES 3-Cyano-2-(4-iodophenyl)-2H-indazole N1-oxide among a series of synthesized N-oxide derivatives exhibited interesting antichagasic and leishmanicidal activity in some of the parasitic strains evaluated [470]. In addition, three series of benzimidazole N-oxide derivatives were developed and were examined for their activity against Trypanosomatid parasites in in vitro and in vivo assays (T. cruzi and Leishmania spp). Among them, the series of 2H-benzimidazole 1, 3-dioxides displayed remarkable in vitro activities against both parasites resulting selective toward both Trypanosomatid parasites [471]. NIFUROXAZIDE (NX) ANALOGUES In vitro anti-T. cruzi activity assays of a set of novel 5-nitro-heterocyclic compounds such as 5-nitro-2-furfuryliden and 5-nitro-2-theniliden derivatives were performed. The majority of the tested derivatives showed increased anti-T. cruzi activity in comparison with the reference drug, benznidazole. Additionally, the 5-nitro-2-furfuryliden derivatives presented better pharmacological profile than the 5-nitro-2-theniliden analogues [472].
GONIOTHALAMIN ANALOGUES Sixteen 5, 6-dihydro-2H-pyran-2-ones were evaluated in an in vitro assay against trypomastigotes forms of T. cruzi. The relevant structural features for the trypanocidal activity of these goniothalamin analogues against T. cruzi were established by a structure-activity
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relationship study (SAR), finding that the non-natural form of goniothalamin was threefold more potent than the natural styryl lactones. Some analogues were identified as potent compounds against T. cruzi with IC50 values in the mM range and significant low toxicities [473].
BETA CARBOLINE-3 CARBOXILATES / CARBOXAMIDES Several beta carboline compounds were evaluated for in vitro trypanocidal activity against T. cruzi. Beta-carboline derivative 4 showed good activity against the different forms of the parasite with a dose dependant inhibitory effect, low toxicity and a selective index 30 times higher to the parasite than for mammalian cells. A comparative study of the trypanocidal activity of the nitrophenyl-tetrahydro-beta-carbolines derivatives and benznidazole using theoretical calculations and cyclic voltammetry was performed. To this aim, the cis and trans isomers of methyl 1-(m-nitro) phenyl and 1-(p-nitro) phenyl-1,2,3,4tetrahydro-9H-beta-carboline-3-carboxylates were synthesized and evaluated in vitro against epimastigote forms of T. cruzi. Among all of the evaluated tetrahydro-beta-carboline derivatives, the compound trans-methyl 1-(m-nitro)phenyl-1,2,3,4-9H-tetrahydro-beta-carboline3-carboxy-late (3b) was found to exhibit significant trypanocidal activity (IC50=22.2 M) [474, 475].
AZAHETEROCYCLIC ANALOGS OF MEGAZOL Design, synthesis and trypanocidal evaluation of new azaheterocyclic derivatives was performed. These compounds were designed as megazol (1) analogs based on bioisosterism tools and were synthesized to investigate the possible pharmacophoric contribution of the 1,2,4-triazole nucleus, the position of the heterocyclic nucleus and presence of the nitro group, to the activity against the bloodstream trypomastigote forms of T. cruzi. Compound 6, a nitro derivative obtained by substitution of a thiadiazole by a triazole ring and by moving the nitro group from C-5 position, to the C-4 position resulted the most potent one [476]
CYCLOANALOGUES OF SPHINGOSINE 2-aminocyclohexanol, 1,2-cyclohexanediamine derivatives and other related cycloanalogues of sphingosine were synthesized and assayed in vitro against Leishmania spp. and T. cruzi, resulting most of these compounds potent parasiticides, with IC50 values in the M or lower range and potencies higher than those of pentamidine and benznidazol [477].
3,7-BIS(DIALKYLAMINO)PHENOXAZINIUM SALTS These salts were synthesized and evaluated for in vitro activities against P. falciparum, T. cruzi, T. brucei rhodesiense, and L. donovani. Notably, the compounds showed potent antiprotozoal activities, especially against P. falciparum and T. cruzi. High selective indices and good activities were found in the compounds with alkyl side chains containing less than three carbons in length [478].
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BENZIMIDAZOLE DERIVATIVES The development of analytical methodologies by UV spectrophotometry and HPLC allowed the characterization of five nitroarylbenzimidazole derivatives with activity against T. cruzi. The five compounds presented an inhibitory effect on the epimastigote form growth at 1-100 M concentration range. Additionally, cyclic voltammetric data revealed that the nitroarylbenzimidazole derivatives might sustain their effects on growth and oxygen uptake on T. cruzi epimastigotes [479].
N-PHENYLPYRAZOLE BENZYLIDENE-CARBOHYDRAZIDES Synthesis, in vitro trypanocidal evaluation, cytotoxicity assays, and molecular modelling and SAR/QSAR studies of a new series of N-phenylpyrazole benzylidene-carbohydrazides was performed. The halogen-benzylidene-carbohydrazide presented the lowest potency whereas 6l showed the most promising profile with low toxicity. The best equation from the 4D-QSAR analysis (Model 1) was able to explain 85% of the activity variability. The QSAR graphical representation revealed that bulky X-substituents decreased the potency whereas hydrophobic and hydrogen bond acceptor Y-substituents increased it [480].
N-QUINOLIN-8-YL-ARYL SULFONAMIDES Twelve N-quinolin-8-yl-arylsulfonamides and tested in vitro for trypanocidal and leishmanicidal activities against both extra and intracellular forms [481].
N-ALLYL AND N-PROPYL OXAMATES The trypanocidal activity of N-allyl (NAOx) and N-propyl (NPOx) oxamates and that of the ethyl esters of N-allyl (Et-NAOx) and N-propyl (Et-NPOx) oxamates were tested in vitro on cultured epimastigotes and in vivo in murine trypanosomiasis using five different T. cruzi strains. NAOx and NPOx did not penetrate intact epimastigotes. Thus, no trypanocidal effect was observed with these oxamates. Whereas the ethyl esters (Et-NAOx and EtNPOx), exhibited in vitro and in vivo trypanocidal activity on all the T. cruzi strains tested at difference with control treated with reference drugs showing that only three of the five tested T. cruzi strains were affected by in vitro and in vivo trypanocidal activity of these compounds [482].
1H-PYRAZOLO[3,4-B]PYRIDINE DERIVATIVES The synthesis, the in vitro biological evaluation, and the SAR results of 1H-pyrazolo [3, 4-b] pyridine derivatives as new antichagasic agent series was reported. The presence of fluorine, hydroxyl or nitro group at Y position resulted in at least one or two promising compounds in each set of derivatives. The SAR study showed that trypanocidal activity observed depends on both geometric and stereoelectronic parameters (MEP and frontier molecular orbitals HOMO and LUMO). The Osiris program was used for calculating and comparing the fragment based drug-likeness of the most active derivative with current toxic antichagasic drugs [483].
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Finally, in the search for new anti-trypanosomal compounds, computational approaches were described. A novel non-stochastic quadratic fingerprints-based approach was satisfactorily applied for virtual evaluation. The anti-trypanosomal activity of a series of 10 already synthesized compounds was in silico predicted as well as in vitro and in vivo explored against T. cruzi. The model was able to predict correctly the behavior of these compounds in 90 % of the cases [484]. A new ligand-based approach applying non-stochastic linear fingerprints to the identification of potential antichagasic compounds was recently introduced. A few compounds with trypanocidal activity against epimastigote forms of T. cruzi were predicted with a confidence of 95 % [485].
PATENTS RELATED WITH SCREENED SYNTHETIC COMPOUNDS Among them, the synthesis and in vitro activity of different bicyclic carbohydrates as antiprotozoal bioactive for the treatment of parasitic diseases, such as Leishmaniasis and trypanosomiasis was disclosed by Kemin Pharma Europe [486]. Besides, scientist from the Universidade Estadual de Campinas in Brazil described the synthesis and in vitro activity against the different stage forms of T. cruzi, among other parasites from a series of 4bromophenil metanona and 2-propen-1-amine derivatives, claiming that among them a furanyl derivate showed a considerable IC50 value (9.5 M) [487]. In addition, Hollis-Eden Pharmaceuticals, Inc, claimed the use of 17-ketosteroid compounds and derivatives, metabolites and precursors in the treatment of Malaria and African and American trypanosomiasis or to ameliorate or reduce one or more symptoms associated with a Plasmodium or Trypanosoma infection [488]. Later, Merck and Co, Inc presented a series of novel synthesized imidazopyridine compounds and N-oxide derivatives and claimed to be useful in the treatment and prevention of protozoan diseases including Trypanosomiasis Americana among other parasitic diseases but no relevant biological data were included [489, 490]. Medial Limited, Duluth, GA, United States of America also patented antiprotozoal imidazopyridine compounds pharmaceutically acceptable salts, or N-oxides thereof, described as useful for the treatment and prevention of protozoal diseases in mammals and birds. In addition to anticoccidial agent activity, treatment and prevention of mammalian protozoal diseases, such as, for example, toxoplasmosis, malaria, African trypanosomiasis, Chagas disease, and opportunistic infections comprise administering the compound alone, or in combination with one or more antiprotozoal agents [491] (Table 6). Finally, Achillion Pharmaceuticals, Inc. disclosed a series of substituted aryl thioureas and related compounds as inhibitors of viral and infectious diseases [492]. Moreover, invention directed to alpha ketoamide and haloalkyl containing compounds as CPIs were presented in different patents in 2009 in particular, towards cathepsins B, K, L, F, and S and are therefore useful in treating diseases mediated by these proteases but not specifically directed towards protozoan CPs.
CURRENT AND FUTURE DEVELOPMENTS The drugs available for the treatment of the Trypanosomiasis Americana are not satisfactory; they present toxic side effects and are expensive. Moreover, there is currently no drug effective once the disease has progressed to the chronic stage. In addition, these drugs are not dispensed in pediatric version, which complicates the treatment of children. The disease affects 16-18 million people in the Americas, particularly in South America, only in Argentina about two million people are infected and it is estimated that from 5000 to 10000
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people die every year. In addition, the disease has emerged as a public health problem in the United States of America and Europe. Therefore, there is an urgent need to solve this problem. However, it is well-known that pharmaceutical industry has restricted investment in research and development of diseases affecting primarily poor populations in low-income developing countries [17]. The identification of new antichagasic agents may be based not only on rational drug design and synthetic or natural products screening [16], but also taking advantage of compounds already in use against other human diseases which have already passed several of the clinical trials necessary for the development of any new drug. The current state of knowledge of parasite biochemistry has favored the development of new chemotherapeutic approaches based on newly validated biochemical targets. Multiple metabolic pathways and specific enzymes useful for the development of targeted trypanocidal drugs have been investigated. In addition, as a result of the parasite genome sequencing project, available since 2005 [19] the possibility of identifying new specific pathways and novel drug targets in the last years has increased. As it has been shown, the biology of the parasite has been intensively studied and a large number of compounds have emerged, however, despite all the new information available, a true applicable drug has not been identified so far. Thus, a huge effort of the global research community is needed, gathered to sustainable financial resources, in order to translate the basic scientific knowledge into a number of selected drug candidates in the pipeline. In fact, only some cysteine proteinase inhibitors and ergosterol biosynthesis inhibitors are currently in the pipeline. A search through the patent literature during the last decade involving parasiticidal activity against T. cruzi was performed including target-based drugs, natural products and its derivatives and new synthetic compounds as well as old ones rediscovered as novel drugs against Chagas disease. Most patents found are related with specific target-based drugs, and some of them that claim compounds useful for the treatment of human diseases such as various cancers, bone diseases or antiviral activity also report possible trypanocidal activity. Among them, can be mentioned those related with cysteine protease inhibitors, purine analogues, organometalic complexes. Others disclose compounds with specific protozoan, parasitic or trypanocidal activity as the main claim, including Chagas disease [282, 297, 309]. Only some of them are related with targets in pipeline (CPIs, sterol biosynthesis inhibitors). It is worth mentioning that despite the abrupt increase of knowledge about the parasite biochemistry, this is not reflected in the number of disclosed patents, furthermore only a few number of the analyzed patents showed specific data of biological anti T-cruzi activity. Whereas most patents found are related with parasitic targets disclosing interesting in vitro activity against T. cruzi, only a few important in vivo results were reported. In particular, those inhibitors based on the drug induced blockade of specific enzymes involved in sterol biosynthesis specially, C-14 demethylase [152] and OSC [139] can be mentioned. In the last years, patents related with CPIs were the most represented among those claiming potential chemotherapeutic agents against T. cruzi, involving azapanone based inhibitors [68-70] peptidyl allyl sulfone compounds for inhibiting proteases [51] or thiosemicarbazone and semicarbazone inhibitors [85]. Among patents related with natural compounds, two of them showed interesting results. Some cubebin derivatives showed total in vitro inhibitory activity [435]. In addition, cantin6-one derivatives showed not only an important inhibitory activity but also showed
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interesting in vivo results [434] suggesting that screening of natural products as well as libraries of synthetic compounds against T. cruzi should allow the discovery of new trypanocidal lead compounds. Moreover, structural studies as well as molecular dynamics leading to the development of more potent antichagasic drugs based on specific molecule analogues should help to find Trypanosoma cruzi specific chemotherapeutic agents. On the other hand, multiple libraries of synthetic compounds are under evaluation and some of them were patented as possible anti-trypanosomal agents. However, no clinical successful results have been shown yet. Hence, as suggested, a pragmatic approach for the rapid development of new anti-T.cruzi chemotherapy would be based on the clinical assessment of drug combination with existing trypanocides [493]. In fact, synergistic effects between an anthyarrithmic compound commonly prescribed for the symptomatic treatment of the disease with azole drugs have been reported [131] opening the possibility of novel approaches including combination of current approved drugs for the treatment of the disease. Under the light of the results obtained so far, despite the multiple efforts done, currently there are near absence of adequate therapeutics for curing patients with chronic Chagas disease and no drugs are available in clinical trial for Chagas disease. Therefore, additional effort to develop better drugs needs to be a priority. Unluckily, Chagas disease still remains a challenge for effective chemotherapy.
ACKNOWLEDGEMENTS This work was supported by grants from CONICET; ANPCyT; UBA and INP, ANLISMalbrán, Ministerio de Salud de la Nación, Argentina. V.G.D. and A.S.C. are Members of the National Research Council (CONICET) from Argentina.
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Recent Patents on Development of Nucleic Acid-Based Antiviral Drugs against Seasonal and Pandemic Influenza Virus Infections Edward G. Saravolac1 and Jonathan P. Wong*,2 1
2
Formulation Technology Consulting, 3 Essex St., Footscray, Victoria, 3011 Australia
Defence R&D Canada - Suffield, Biotechnology Section, P.O. Box 4000 Main Station, Medicine Hat, Alberta, T1A 8K6 Canada Abstract: Influenza viruses are etiological agents of deadly flu that continue to pose global health threats, and have caused global pandemics that killed millions of people worldwide. The global crisis involving the avian H5N1 and more recently porcine H1N1 influenza both provide compelling reasons for accelerate fast track development of novel antiviral drugs against the potential pandemic virus. The availability of neuraminidase inhibitors such as oseltamivir (tamiflu) improves our ability to defend against influenza viruses, but the incidences of tamiflu-resistance are on the rapid rise. Nucleic acid-based antiviral drugs are promising classes of experimental antiviral drugs that have been shown in preclinical studies to be effective against seasonal and avian influenza viruses. The potency and versatility of these drugs make them potential candidates to be used in seasonal and pandemic influenza scenarios. Here we review recent patent activity in the development of nucleic acid based drugs directed at influenza. The review will assess the recent patents, research and development of antisense oligonucleotides, immunomodulating RNA and the most rapidly developing area, the exploitation of small interfering RNA for the prevention and treatment of influenza infection.
Keywords: Patents, influenza infection, nucleic acid-based drugs, antisense, small interfering RNA. INTRODUCTION Influenza is a leading cause of human mortality and morbidity worldwide, and is responsible for resulting in loss of billions of dollars in health care costs. As a leading cause of death and mortality, influenza is responsible for over 36,000 deaths per year in the US and over 500,000 worldwide and is thus highly desirable target for siRNA therapy [1, 2]. Influenza’s most potent threat is its potential to cause deadly global pandemics. The 191819 Spanish influenza pandemic caused the loss of over 50 million human lives [3] and highlighted the vulnerability of humans to pandemic influenza viruses. In recent history, the emergence of new strains of influenza in the form of avian H5N1 influenza [4] and the more *
Corresponding author: Tel: +1 403 544 4689; Fax: +1 403 544 3388; E-mail: [email protected] Atta-ur-Rahman / M. Iqbal Choudhary (Eds.) All rights reserved – © 2010 Bentham Science Publishers.
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recently, pandemic H1N1 swine flu strain has caused global crises provided testament to the challenge of defending against a deadly virus that is unpredictable and ever changing. Table 1.
Current Anti-Influenza Pharmaceutical Development Activities
Company
Anti Influenza Drug
Type
Phase
Representative Patent/Application
AVI Biopharma
NeuGene™ (AVI 60010)
Antisense (Morpholino)
Phase I
20070004661* [7]
Replicor Inc.
Rep9
PS - oligonucleotide
Preclinical
20050196382 [8]
Coley Pharmaceutical Group
CpG7909 (Promune™) CpG10101 (Actilon withdrawn)
Oligonucleotide TLR-9 Agonists
PhaseI PhaseII (withdrawn)
20050256073 [9]
Hemispherx Biopharma
Ampligen™ Mismatched dsRNA Poly A: Poly U
poly(I): poly(C12U)
Preclincial Preclinical
IRX Therapeutics
MIMP (5 methyl inosine monophosphate)
APC and T-cell stimulator
Preclinical
Multicell Technologies
MCT-465
dsRNA
Preclinical
20050222060 [14]
Nastech (MDRNA)
G00101
siRNA
Preclinical
20040242518 [15]
siRNA
Preclinical
20060293271 [16] 20060293272 [17] 20060217337 [18]
Sirna Therapeutics
20060035859* [10] 20070224219 [11] US5614504 [12] 20050148538 [13]
Alnylam
ALN-Flu01
siRNA
Preclinical
US7579451 [19]
Protiva
ProFlu™
siRNA
Preclinical
20050064595*[20]
BioDelivery Sci
Bioral™ siRNA
siRNA
Preclinical
20050013855 [21]
* Patents rejected by the USPTO or abandoned by the authors.
Vaccination with trivalent influenza vaccine is effective in reducing the impact of the annual spread of seasonal influenza, although its prophylactic effectiveness can be significantly impacted by strain matching with circulating strains, strains used for vaccine production, and by virus mutations. Given the ability of influenza virus to undergo constant antigenic change, there is a compelling requirement to develop alternative prophylactic countermeasures to protect against seasonal and pandemic influenza. Antiviral therapy is used clinically to reduce the duration and severity of influenza, and stockpiling of antiviral drugs is an important component of many influenza pandemic preparedness plans developed by many western nations [5]. Currently there is a limited arsenal of antiviral compounds which includes M2 ion channel inhibitors (amantadine, rimantidine) and neuraminidase inhibitors (perimivir, oseltamivir and zanamivir). However, analysis of recent avian H5N1
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isolates from infected patients revealed that these virus isolates are completely resistant to rimantidine and amantidine and are increasingly resistant to oseltamivir (tamiflu) [6]. In view of the steady increase in influenza viruses developing drug-resistance, and the global threat of a looming influenza pandemic, the requirement to develop novel antiviral drugs that are less likely to give rise to drug resistance becomes more urgent. Rapid advances in viral genomics and gene-based drug design demonstrate that antisense oligonucleotides, siRNAs, ribozymes and DNAzymes are versatile in their mechanisms of action, can inhibit viral replication at the molecular level in the early phase of infection, and can be custom designed to match antigenic shifts, mutations or recombination in the virus. The pharmaceutical industry, academic research and defense departments have been quick to recognize the potential value of nucleic acid based antiviral agents. An outline of the current development activities (Table 1) [7-21] reveals that indeed several newly established companies are employing a wide range of nucleic acid technological strategies to combat the threat of influenza. Advances in this area may be attributed to a keen appreciation of both public health and bio-defense considerations driving increased funding and R&D efforts in this area. This has resulted in a corresponding accumulation of intellectual property relating to the designs and development of nucleic acid-based anti-influenza drugs. This review will survey the recent and significant patents on designs and applications of nucleic acid-based drugs, and will provide an overview on their prophylactic and/or therapeutic applications against influenza virus infections A survey of the most recent and significant patents on nucleic acid-based drugs, reflecting the rapid advances made in this subject area, is shown in Table 2 [7-43]. This overview of patent activity reveals a diverse range of nucleic acid-based drug designs and strategies. These novel or improved drug designs can be broadly classified into 4 major classes: antisense oligonucleotides, immunomodulating nucleic acids (CpG oligonucleotides and ds RNA), catalytic nucleic acids (ribozymes and DNAzymes) and small interfering RNA (siRNA). Upon reviewing these patents and patent applications, it is particularly significant that most of these drug candidates have been used to demonstrate anti-influenza activity in either established tissue culture cell lines and/or animal infection model systems. However any review of these gene-based strategies is severely limited by the relative paucity of strong clinical data. The diversity inherent in exploiting the influenza gene sequence yields both enormous flexibility to this area of antiviral treatment and in the same measure adds complexity to their pharmaceutical development particularly in terms of modes of delivery, toxicology and potential non-specific activity. Nevertheless, current patents reveal the state-of-the-art in nucleic acid based approaches for anti-influenza drug development, their modes of action, current status of development and potential applications of each of these approaches directed against influenza are outlined summarized here. A) ANTISENSE OLIGONUCLEOTIDES Antisense oligonucleotides have been developed as a means of gene blockade by specifically hybridizing to target mRNA sequences [44]. Antisense oligonucleotides can be designed to bind to coding region of virus mRNA thereby interfering viral protein synthesis, or bind to the promoter region or initiation codon thus stopping the initiation of viral protein translation. In addition, the binding of antisense oligos to mRNA forms duplexes recognized by the cellular enzyme RNAse H, which in turn cleaves the viral mRNA. These antisense effects result in the silencing of viral protein expression and inhibition of viral replication. The most successful antiviral application of this technology is the marketed
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phosphorothioate (PS) oligonucleotide Fomiversin which is an antisense directed against cytomegalovirus (CMV) infections in the human eye [45]. Table 2.
Recent Anti-Influenza Patents and Patent Applications
Patent
Author
Title
Assignee
Publication Date
Antisense Oligonucleotides 5,194,428 [22]
Agrawal et al.
Inhibition of influenza virus replication by oligonucleotide phosphorothioates
Mt Sinai School of Medicine
Mar 16, 1993
5,580,767 [23]
Cowert et al.
Inhibition of influenza viruses by antisense oligonucleotides
Isis Pharmaceuticals
Dec 3, 1996
5,637,573 [24]
Agrawal et al.
Influenza virus replication inhibiting analogues and their pharmaceutical compositions
Authors
Jun 10, 1997
6,326,487 [25]
Peyman et al.
3 Modified oligonucleotide derivatives
Aventis Pharma Deutchland
Dec 4, 2001
6,495,675 [26]
Takaku et al.
Pharmaceutical composition for treating for preventing influenza, and novel capped oligonucleotide
Chiba Institute of Technology, China
Dec 17, 2002
6,683,167 [27]
Metelev et al.
Hybrid oligonucleotide phosphorothioates
University of Massachusetts
Jan 27, 2004
7,045,609 [28]
Metelev et al.
Hybrid oligonucleotide phosphorothioates
University of Massachusetts
May 16, 2006
20070004661*[7]
Stein et al.
Antisense antiviral compound and method for treating influenza viral infection
AVI Biopharma
Jan 4, 2007
Wong et al.
Therapy of respiratory influenza virus infection using free and liposome-encapsulated ribonucleotides
Defence R&D Canada - Suffield
April 8, 2003
Ribozyme Pharmaceuticals
Jul 10, 2001
6,544,958 [29]
Ribozymes 6,258,585 [30]
Draper
Method and reagent for inhibiting influenza virus replication
Immunomodulatory/ Non-Complementary Nucleic Acids 5,614,504 [12]
Hadden et al.
Method of making inosine monophosphate derivatives and immunopotenitating uses thereof
The University of South Florida
Apr 21, 1995
US7491706 [31]
Yu et al.
The artificial CPG single strand deoxidation oligonucleotide and its antiviral uses
Changchun Huapu Biotechnology Co.
Feb 17, 2009
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(Table 2) Contd….. Patent
Author
Title
Assignee
Publication Date
20050148538 [13]
Hadden et al.
Adjuvant formulations for bacterial and virus vaccines and method for making same
IMP Therapeutics
Jul 7, 2005
20050196382 [8]
Vaillant et al.
Antiviral oligonucleotides targeting viral families
Replicor Inc.
Sept 8, 2005
20050222060*[14]
Bot et al.
Composition and methods to initiate or enhance antibody and majorhistocompatability class I or class II-restricted T-cell responses by using immunomodulatory, noncoding RNA motifs
20050256073 [9]
Lipford et al.
Immunostimulatory viral RNA oligonucleotides
Coley Pharmaceuticals
Nov 17, 2005
6,468,558 [32]
Wong et al
Liposome encapsulated poly ICLC
Defence R&D Canada
Oct 22, 2002
6,506,559 [33]
Fire et al.
Genetic inhibition by doublestranded RNA
Carnegie Institute of Washington
Jan 14, 2003
20060035859* [10]
Carter et al.
Treating severe and acute viral infections
Hemespherx Biopharma
Feb 16, 2006
20070224219[11]
Carter et al.
dsRNA as influenza virus vaccine adjuvants or immunostimulants
Hemepsherx Biopharma
Sept 27, 2007
20040242518* [15]
Chen et al.
Influenza Therapeutic
M.I.T.
Dec 2, 2004
EP1647595 [34]
Berkhout et al.
Nucleic acids against viruses in particular HIV
Universiteit van Amsterdam
Oct 15, 2004
20050013855* [21]
GouldFogerite et al
Cochleate compositions directed against expression of proteins
BioDelivery Sciences
Jan 20, 2005
20050058982* [35]
Han et al.
Modified small interfering RNA molecules and methods of use
Chiron Corporation
Mar 17, 2005
20050064595*[20]
MacLachlan et al.
Lipid encapsulated interfering RNA
Protiva Biotherapeutics Inc.
Mar 24, 2005
US 7,297,786[36]
McCray et al.
RNA interference in respiratory epithelial cells
University of Iowa
Apr 20, 2006
20060217337*[18]
McSwiggen et al.
RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (SINA)
Sirna Therapeutics Inc
Sept 28, 2006
20060282921 [37]
Lam et al .*
Transgenic plant-derived siRNAs for suppression of influenza virus in mammalian cells
Astral Inc. (Multicell Technologies)
Oct 6, 2005
dsRNA
siRNA
Dec 14, 2006
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(Table 2) Contd…. Patent
Author
Title
Assignee
Publication Date
US7199109 [38]
Pal et al.
Potent inhibition of influenza virus by specifically designed short interfering RNA
Cal Poly Pomona Foundation
Apr 30, 2007
US7288531 [39]
Pal et al.
Potent inhibition of influenza virus by specifically designed short interfering RNA
Cal Poly Pomona Foundation
Oct 30, 2007
US7304042 [40]
Pal et al.
Potent inhibition of influenza virus by specifically designed short interfering RNA
Cal Poly Pomona Foundation
Dec 4, 2007
20070099858 [41]
Jadhav et al.*
RNA interference mediated of inhibition of influenza virus gene expression using short interfering nucleic acid (SINA)
Sirna Therapeutics Inc
April 3, 2007
20070218122 [42]
MacLachlan et al.
siRNA silencing of influenza virus gene expression
Protiva Biotherapeutics Inc
Sept 20, 2007
20080279920 [43]
Tang et al.
Compositions for treating respiratory viral infections and their use
Intradigm Corporation
Nov 13, 2008
20060293271[16]
McSwiggen et al.
RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (SINA)
Sirna Therapeutics Inc
Dec 28, 2006
20060293272*[17]
McSwiggen et al.
RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (SINA)
Sirna Therapeutics Inc
Dec 28, 2006
* Patents rejected by the USPTO or abandoned by the authors.
Several antisense oligonucleotides have been designed for antiviral applications against influenza virus infection [46]. These antisense oligonucleotides were directed against the translation initiation codons in the PB2 and PA genes that encode for the influenza virus RNA polymerase, and were found to be effective in the inhibition of both PB2 and PA gene expression in cultured cells. Treatment of influenza A virus-infected mice with PB2 antisense encapsulated in cationic liposomes significantly prolonged overall survival rates, reduced lung virus loads and pulmonary consolidation [46]. However, the PS oligonucleotides are attributed to non-specific effects and are susceptible to nuclease degradation in vivo and in vitro [44]. A number of oligonucleotide chemical modifications have been introduced, most specifically here, the phosphorodiamidate morpholino oligonucleotides (PMO) [47]. The uncharged backbone is attributed with improved uptake into target host cells and is reported to lead to greatly reduced non-antisense effects (Fig. 1). Recently Ge et al. characterized the antiviral activity of an influenza specific morpholino oligonucleotides - conjugated to an alanine rich peptide
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(to aid in cellular uptake) - designed to base pair with FLUAV RNA sequences that are highly conserved across viral subtypes and considered critical to the FLUAV biologicalcycle [48]. Several PMO were highly efficacious, and two PMO targeted to the AUG translation start site region of PB1 to the 3′-terminal region of nucleoprotein viral genome proved to be potent against several other FLUAV strains [48], including A/WSN/33 (H1N1), A/Memphis/8/88 (H3N2), A/Eq/Miami/63 (H3N8), A/Eq/Prague/56 (H7N7), and the highly pathogenic A/Thailand/1(KAN-1)/04 (H5N1). The novel mopholino oligonucleotides have also recently been found to be similarly effective against several strains of the Dengue fever virus [49] and have recently been tested in clinical trials for antitumor applications [50]. The anti-influenza drug AVI60010 NeuGene antisense oligonucleotide is currently being developed by AVI Biopharma. AVI60010 antisense has been demonstrated in vitro to inhibit multiple types of influenza including the highly pathogenic H5N5 avian influenza virus [48]. Preclinical studies and an investigational new drug (IND) submission were planned for this product.
Fig. (1). Structural representation of the peptide conjugated phosphorodiamidate morpholino antisense structure. Adapted from ref. [38].
Single-stranded RNA has also been investigated for use as an anti-influenza agent. A recent patent by Wong et al. [29] described synthetic ribonucleotide oligonucleotides (RNO) which were designed to suppress the gene expression of heamagglutinin protein, a surface spike protein of influenza A responsible for virus attachment to target host cells. The single- stranded RNO 15-mers were evaluated in murine influenza models and were demonstrated to be effective in both prophylaxis and treatment of the disease and could be effective delivered as naked RNO or encapsulated in liposomes [29]. The sequences that were active in protecting and treating mice against influenza virus were found to be either sense or antisense strands while control random sequence lacked anti-influenza activity. More recent work suggested that ssRNA from viral sources may result in the production of high levels of type-1 interferon (IFN-α/β). ssRNA-stimulated interferon production is
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mediated either in the cytoplasm or the endosome. In the cytoplasm, this effect is mediated through RIG-1 (RNA helicase enzymes retinoic acid–inducible gene 1), an element of the innate immune system which recognizes 5′-phosphorylated ssRNA [51]. Such an effect has also been seen with transfection of cells with siRNA [52]. In the endosome the TLR-7 receptor mediates the production of interferon upon interaction with ssRNA [53]. Further work will be required to determine whether antisense or interferon mediated effects are responsible for the anti-influenza effects observed with these RNOs. In addition to antisense oligonucleotides, anti-influenza oligonucleotide drug products that do not rely on sequence complementarity are also being developed [54, 55]. Replicor is developing randomer PS development of Rep9, an anti-influenza randomer PS-oligonucleotide which has been reported to be effective against H5N1 influenza (A/Vietnam/ 120I3/04) and which prevents the spread of influenza when administered as aerosols to the lungs of mice. It has been demonstrated for some time that polyanionic compounds such as polysulfones, sulfated polysaccharides and phosphorothioate modified nucleotides inhibit the fusion of viruses to the host cell surface [55]. This process was further characterized by investigators who demonstrated that long chain phosphorothioate oligonucleotide randomers act as anti-viral agents [56]. Using HIV-1 as a model virus they demonstrated that PS oligomers of optimal lengths (~40mer) blocked viral fusion by a mechanism involving blocking gp41 six helix bundle formation. As gp41 represents the type I fusion protein the data suggests that the other viruses which employ type I proteins including, influenza, ebola and coronavirus could be susceptible to this form of PS-randomer antiviral activity. B) CATALYTIC DNA AND RNA (DNAZYMES AND RIBOZYMES) Catalytic DNA and RNA, (DNAzymes and ribozymes respectively), like morpholino oligonucleotides, have RNAase H independent mechanisms of action [57]. The thermodynamic energy of hybridization of these oligonucleotides drives a catalytic core to cleave the RNA of the target site resulting in gene blockade. DNAzymes are entirely synthetic and require modified nucleotides (3′ inversion, PS, etc.) for stability against nucleases in the binding sequences flanking the phosphodiester “catalytic core” nucleotides (Fig. 2). Ribozymes may be transcribed in situ from a plasmid or retrovirus, such as those targeted against HIV or are also designed and made synthetically [58]. DNAzymes have
Fig. (2). Schematic representation of the structure of a DNAzyme binding to target RNA. Adapted from ref. [51].
been described that are effective against influenza A viruses. DNAzyme drug candidates were designed and characterized thar are capable of cleaving the sequence at the AUC initiation codon of the PB2 gene in influenza A [59, 60]. Cell culture studies revealed that these DNAzymes were effective in reducing levels of influenza levels by 99% in MDCK
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cells [59, 60]. Using a different strategy, Lazareve et al. [61] constructed cell lines that endogenously expressed ribozymes targeted against a region of the influenza PB1 gene conserved across several strains. These cell lines were demonstrated to have substantial resistance to influenza infection reducing the virus levels up to 94% versus. While both ribozymes and DNAzymes have shown promise in as therapeutic agents in experimental models, a great deal of preclinical work will be required to show promise to support further clinical study DNAzyme [62]. C) IMMUNOMODULATORY NUCLEIC ACIDS CpG Oligonucleotides Amongst the non-hybridization dependant effect of PS oligonucleotides is the CpG activation of B cells via the Toll-like receptor 9 (TLR-9). The extent of activation is dependant up on the sequence context around the CpG motifs of non-methylated DNA [63]. Much effort has been expended designing CpG containing oligonucleotides as immunomodulators and adjuvants. Immunomodulating oligonucleotides form a second class of antiviral compounds. Coley Pharmaceutical group (now part of Pfizer) had developed a range of oligonucleotide TLR-9 receptor agonists such as CpG 7909 (Promune™) and CpG10101 (Actilon). CpG 7909 was demonstrated in early (Phase Ib) trials to enhance the efficacy of fluvarix influenza vaccine and has shown promise in as a late phase (II and III) anticancer adjuvant [64]. However, early pre-clinical and clinical success does not guarantee a clinical success. Such is the case for Actilon (CpG10101) where shortly after fast tracking by the FDA, clinical trials were cancelled due to insufficient evidence of efficacy due to poor Phase II results against HCV. CpG oligonucleotides have also been shown to induce immunological responses to protect experimental animals against multiple lethal dose challenge with influenza A virus [65]. Wong et al. demonstrated that liposome encapsulation could be used for the intranasal delivery of CpG oligos in a mouse influenza model. These pre-clinical studies demonstrated that 5 µg of naked oligonucleotide given 5 days prior to infection offered protection to 50% of mice infected with 10 LD50 influenza A. Liposome encapsulation of the CpG oligos increased the survival rate to 80% [65]. Double Stranded RNA (dsRNA) Double stranded (ds) RNA has long been known to be a strong mediator of a nonspecific immune response acting as a TLR-3 agonist resulting in stimulation of interferon-α, -β and -γ production [66, 67]. Several examples of dsRNA immunomodulation employed for the treatment and prophylaxis against influenza, including have been reported [68-70]. Poly ICLC is a synthetic double-stranded polyriboinosinic-polyribocytidylic acid (poly IC) stabilized with poly-L-lysine and carboxymethyl cellulose (LC). When poly ICLC was encapsulated and/or complexed to liposomes, the duration in window of protection against influenza infection, and the safety profile were enhanced in mice [69, 70]. Preclinical studies in mice have demonstrated that protection against seasonal influenza virus could last up to 21 days [70, 71]. Recent studies have shown that liposomal poly ICLC was found to be effective in the protection of mice against lethal challenge of avian influenza A/H5N1/chicken/Henan strain [70]. A related nucleic acid-based immunostimulator, ampligen ®, which is a mismatched dsRNA poly (I): poly (C12U) developed by Hemispherx Biopharma Inc. Ampligen has been
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used in antiviral applications. As with poly IC, this dsRNA stimulates the 2′-5′ oligoadenylate synthetase/RNase L pathway for viral RNA destruction. Published work on its effectiveness has only been reported for coxsackie B3 virus [71], Nipah virus [72] and in clinical trials for HIV [73]. Methyl inosine monophosphate (MIMP) is another immunomodulating nucleic acid drug that has been demonstrated to have anti-influenza activity during development by IRX Therapeutics [74]. Recent studies, (IRX Therapeutics) have questioned the effectiveness of this compound [75]. In the earlier studies, MIMP was protective against aerosol delivered mouse adapted influenza A in a strain of outbred mice. However, when the MIMP was tested in a mouse adapted influenza A inoculated in an inbred mouse strain (BALB/c) by intranasal administration, the drug was shown to be ineffective. The authors correctly concluded that care must be taken to consider the age, strain and routes of administration when extrapolating data from preclinical in vivo models of viral disease. D) siRNA Small interfering RNA (siRNA) is one of the most active areas in nucleic acid research. The 2006 Nobel Prize winning research on genetic interference observed first in nematode Caenorhabditis elegans by Fire and Mello [76] led soon after to its characterization in mammalian cells [77]. Here double stranded RNA (dsRNA) directs sequence specific degradation of messenger RNA (mRNA). The process involves the cutting of small (~25 mer) dsRNA from large dsRNA (Fig. 3). This is part of the naturally occurring process used by these eukaryotic cells as defense mechanisms against viruses and transposons. The discovery that these siRNA could mediate sequence-specific gene silencing effect had generated much excitement in the biotechnology sector in that these siRNA molecules can either be synthetically produced in large scale, or can be expressed by ribozyme or lentiviral vector expression systems [78]. siRNA are well suited to be used as antiviral agents. Since its discovery, siRNA technology platform has been successfully used to treat and/or prevent viral diseases including hepatitis C [79], HIV [80] and influenza [81]. More recently, the emergence of pandemic bird and swine flu variants of the virus has added urgency to the discovery that siRNA is effective in vitro against a range of influenza strains including the pandemic, bird flu H5N1 and other seasonal variants [82, 83]. siRNA have also been designed to target the nucleocapsid protein (NP) as well as polymerase (PA) RNAs of influenza A virus [84, 85]. These siRNA were found to suppress viral mRNA, virion and complementary RNA levels in cell culture and chicken embryos [86]. In a mouse study, treatment of influenza-infected mice with siRNAs specific for NP and PA protected mice against lethal virus challenge and caused significant reduction of virus titers in the lungs. The protection was specific and was not mediated by an interferon response. Furthermore, this specific siRNA treatment was later found to be effective against the highly pathogenic avian influenza viruses of both H5 and H7 subtypes [87]. In another study in mice, siRNAs specific against conserved regions of NP and PA, found to be very effective to prevent or treat influenza virus infection. In this study, the antiviral activity of siRNAs were similar whether given intravenously when complexed with polycation carrier, or transcribed from a DNA expression vector [88]. A series of 3 patents for an antiviral siRNA for influenza were awarded to Pal et al. [3840]. The patents claim conserved (sense and antisense) sequences in the influenza nucleoprotein (NP), providing protection against the A, B and C variants of the virus as well
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Fig. (3). Overview of intracellular siRNA production. A. Representation of the cellular gene silencing process. Naturally occurring RNAi is initiated by the dsRNA-specific endonuclease, called Dicer, which processively cleaves long dsRNA into double-stranded fragments between 21 and 25 nucleotides long, termed short interfering RNA (siRNA) [68]. siRNAs are then incorporated into a protein complex (RNA Induced Silencing Complex or RISC) that recognizes and cleaves target mRNAs resulting in Translational Gene Silencing (TGS) on the genome or Post Translational Gene Silencing (PTGS) at the mRNA level. B. Schematic structure of siRNA. Adapted from ref. [40].
as the H5N1 strain. In studies described in the patent, siRNA is nasally delivered (aqueous mist) using an expression vector either via a transient plasmid or AAV (Adeno-associated virus) vector resulting in a permanent integration of the siRNA in the host chromosome. In prophylactic murine models, Pal et al. pretreated mice with either cationic polymercomplexed plasmid or AAV containing the anti-NP siRNA. Administration of the plasmid vector yielded a dose-responsive resistance to morbidity (weight loss) and mortality against
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a lethal challenge with influenza. Persistent resistance to escalating (4-fold) lethal influenza challenges up to 24 days post-treatment was observed in mice pre-administered the AAV vector coding the anti-NP siRNA. The remaining influenza-specific patent applications extant in the public domain describe attempts to target a range sites on the influenza genome. Chen et al. [15] submitted a broadly based patent application describing siRNA, shRNA and RNAi vectors targeted against various sequences with the influenza NP. PA, PB1, PB2 and M genes. To date the patent claims have yet to be allowed. The application of Lam et al. [37] described a novel approach where siRNA targeted to the NS1 gene of the Influenza A H1N1 strain was produced in vivo and isolated from the leaves of transgenic tobacco plants. The siRNA thus produced demonstrated to be functional in suppressing viral replication in mammalian cell lines. Jadhav et al. [41] submitted an application which described processes for the design and preparation of a range of RNAi (siRNA, shRNA, RNAi, etc.) targeted against a range of conserved influenza A gene sequences. Interesting features of the design were the description of a range of multifunctional (bi-functional, tri-functional and dendrameric) RNAi structures. The patent claims were largely rejected and the application was subsequently abandoned. McSwiggen et al. [89] also described synthetic siRNA targeted against influenza NP and PA gene sequences. The polyethyleneimine (PEI) - complexed siRNA sequences were evaluated both in vitro and in vivo in a murine influenza challenge model. Increased survival and reduced viral lung titre resulted from pretreatment with the combined NP and PA targeted siRNA. However claims from this application were rejected upon examination due to the discovery of existing prior art. Two more recent patent applications relating to siRNA targeted to influenza have yet to have their claims tested. MacLachlan et al. [42] designed modified synthetic siRNA targeted to influenza NP and PA genes delivered transiently using a stabilized lipid-nucleic acid particle (SNALP) formulation. 2′-O-methyl- modified nucleotides used in the synthetic siRNA reduced the induction of interferon in the in vitro and murine models. The readout for the prophylactic study was to determine viral replication as a function of haemagglutinin (HA) levels 48 h after administration of the virus. HA levels were 40% lower in stable nucleic acid lipid particles (SNALP)-formulated siRNA treated mice than with the PBS control - suggesting suppressed viral replication in vivo [90]. Tang et al. [43] claim in their patent application, sequences in the genomes of both influenza A H1N5 strain and respiratory syncytial virus (RSV). In vitro studies indicated that synthetic siRNA from the H1N5 genome NP1 and M2-1 were the most potent at inhibiting viral replication in vitro. An interesting variation on the use of siRNA is the recent development of siDNA [91]. Molling’s patent claims that siDNA consists of a homologous antisense strand and a second strand partially complementary to the antisense strand with both strands being held together with a linker (e.g. 4 thymidines). It is suggested that the siDNA is superior to siRNA because it is more stable, and forms more stable hybrids with the target mRNA. The originality of this DNA containing construct await patent testing and further pharmaceutical development. The efficacy of siRNA against influenza or any other virus will of course have to await rigorous pre-clincial and clinical investigation. Recently, MDRNA (formerly Nastech) reported (unpublished) in vivo efficacy for seasonal influenza with their in lead siRNA antiinfluenza candidate MDR-03030 directed against the virus [92]. The NIH has also recently supported the development of this work with a 5 year grant suggesting the course of development may show promise [93]. Indeed in the recent past companies such as Protiva (Tekmira), Sirna Therapeutics (Ribozyme Therapeutics), Senesco Technologies and
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BioDelivery Sci had initiated preclinical studies with siRNA directed against influenza. Each of these companies has reported the development of not only siRNA as a drug but development is also driven by the introduction of enabling delivery systems and nucleotide chemistries to potentiate these novel therapeutics. Substantial reports of in vivo efficacy remain however pending. Recent Advances in Delivery of Antiviral Nucleic Acids Nucleic acid-based antiviral agents are extremely versatile in their antiviral mechanisms of action. Whether these drugs exert their anti-influenza activity through induction of broadspectrum antiviral immune responses (ds RNA, CpG oligos), or inhibition of gene expression and viral replication at the molecular level (antisense, siRNA), the delivery of these antiviral agents to the sites of virus infection is one of the greatest challenges. Drug delivery systems are of paramount importance in their therapeutic applications of nucleic acid-based drugs as these delivery vehicles enhance the transport of these highly charged macromolecules across cell membranes, as well as protecting them against nuclease degradation in the body for both local (regional) and systemic applications. Such systems can include cationic polymers or lipids, particles, liposomes, viral vectors, peptides and chemical modifications. Drug delivery systems such as liposomes and nanoparticles are effective in targeting nucleic acid-based drugs to the site of viral replication, thereby avoiding potential toxicity to non-infected organs. The use of liposomes to deliver dsRNA poly ICLC has been shown to enhance antiviral efficacies against influenza virus infection, as well as reducing adverse drug effects in the body [69, 70]. In addition to carrier technologies, extensive advances have been made in the chemical modifications of the nucleic acids that make up the synthetic oligonucleiotide drugs. These modifications serve to not only protect the nucleic acids from in vitro degradation but also aid in enhancing the specificity of their antiviral activity [94]. Viral vectors such as adenovirus, lentivirus and adenoassociated virus are also very effective and commonly used in the delivery of nucleic acid-based drugs, and they permit expression of these nucleic acid-based drugs at the transfection site. For a comprehensive reviews of the recent developments in the area of delivery of nucleic acid-based antiviral agents, particularly for anti-influenza applications, readers are encouraged to refer to these reference review articles [95-97]. CURRENT AND FUTURE DEVELOPMENTS In light of growing drug-resistance of seasonal and avian influenza viruses to antiviral drugs, and the increasing global threat of a potential avian H5N1 influenza pandemic, the need to fast track development of new antiviral drugs to combat influenza has never been more urgent. Nucleic acid-based antiviral agents may have a significant role to play as novel weapons to the existing arsenal of existing antiviral drugs against seasonal or avian influenza viruses. This survey of the recent patents on nucleic acid-based antiviral agents reveals that these drugs are versatile in their mode of action in that they can be designed to elicit protective and broad-spectrum antiviral immune responses, interfere with viral replication, suppress gene expression of key viral proteins, or cleave viral mRNAs. The potency and versatility of these drugs make them potential candidates for used in seasonal or pandemic influenza situations. The fundamental issue with this class of drug is whether the promising efficacy seen in preclinical studies in animals can be fully translated in human patients.
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Comparing the potential of these nucleic acid approaches would depend upon the strategy for use. Drugs based on immunomodulation such as poly ICLC or CpG could be ideal where the strategy requires non-specificity, where the strain or species of the virus is less important. Such a strategy could be important in forming a rapid first line of defense, particularly for rapidly emerging serotypes about which very little is known. Alternatively the use of modified antisense or siRNA may be preferred if specificity and potentially reduced side-effects is desired. Overall, once beyond the investigators bench, development of such drugs is complicated. Predicting success is difficult to predict because so few nucleic acid based drugs have made the transition from the bench to the clinic and ultimately the market. Indeed as with developing any novel product, issues such as manufacturability, toxicity, scalability and stability are all important for bringing a new therapeutic entity to clinical and commercial use. Furthermore, it will be quite likely that both the nucleic acid drug and the accompanying (and previously untried) delivery system will have to be clinically evaluated. For example despite its potential potency and specificity against influenza gene targets, siRNA is particularly unstable and would require a delivery system to be effective in vivo. Such a delivery system may be fraught with stability, toxicity and cost issues of its own. Thus would such a drug be cost-effective enough to be made widely available for prevention and treatment of pandemic influenza? Such an analysis may prove telling for each class of nucleic acid-based drug. Ultimately however, clinical efficacy will be the most important factor in judging the success of any nucleic acid-based drug as an anti-influenza agent. Thus it is important to guard against unrealistic expectations from nucleic acid-based drugs. As with conventional pharmaceutical therapeutics many of these drugs have failed at various stages of clinical trials. As these drugs are entering the various phases of clinical studies against various cancer and infectious diseases, preliminary results from a small number of studies appear to indicate that these drugs are relatively safe and well tolerated in patients [98]. Nevertheless, the therapeutic usefulness of the nucleic acid-based antiviral agents against influenza infection will need to be determined in phase II and III studies in humans. It is to be hoped that amongst the many approaches and targets available, clinical success will further guide ongoing development and innovation. REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14]
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Author Index
Author Index to Volume 1 Alonto, A.M. .............................................. 279 Aneja, R. ...................................................... 49 Arabshahi, M. .............................................. 49
Lee, V.J. ......................................................138 Li, L. ...........................................................238 Lo, T.S. .......................................................279
Bautista, D. ................................................ 251 Bosó-Ribelles, V. ...................................... 251 Bughani, U. .................................................. 49 Burza, M.A. ............................................... 124
Massi, E. .........................................................1 Morini, S. ....................................................124
Cáceres, C. ................................................. 251 Campo, S.M.A. .......................................... 124 Carmena, J. ................................................ 251 Chandra, R. .................................................. 49 Chiarella, P. ................................................... 1 Couto, A.S. ................................................. 323 Cristofari, F. ............................................... 124 de Lima Ferreira, M. ................................. 176 De Robertis, M. ............................................. 1 de Souza, M.V.N. ...................................... 176 Duschak, V.G. ........................................... 323 El-Khatib, W.F. ........................................... 70 Fang, B. ...................................................... 238 Fazio, V.M. .................................................... 1 Gonçalves, R.S.B. ...................................... 176 Guo, W. ...................................................... 238 Harris, F. ...................................................... 17 Hassan, C. .................................................. 124 Joshi, H. ........................................................ 49 Kalman, D. ................................................... 49 Kasama, T. ................................................. 227 Konaklieva, M.I. ........................................ 269 Koyama, J. ................................................. 294
Nadal, I. ......................................................107 Noreddin, A.M. ............................................70 Ondarza, R.N. .............................................202 Pagès, J.-M. ................................................138 Phoenix, D.A. ...............................................17 Plotkin, B.J. ................................................269 Ridola, L. ....................................................124 Romá-Sánchez, E. ......................................251 Sánchez, E. .................................................107 Sanz, Y. .......................................................107 Saravolac, E.G. ...........................................409 Sato, M. .......................................................227 Shahid, S.K. ................................................261 Signori, E. .......................................................1 Takahashi, R. ..............................................227 Tong, T.R. ....................................................83 Van Bambeke, F. ........................................138 Vangapandu, S.N. ........................................49 Vicaldo-Alonto, E.A.R. .............................279 Wakabayashi, K. ........................................227 Welch, J.M. ................................................279 Wong, J.P. ..................................................409 Wu, S. .........................................................238 Zullo, A. ......................................................124
