A D V A N C E S IN
Pharmacology VOLUME 4
ADVANCES IN PHARMACOLOGY ADVISORY BOARD
D. BOVET Istituto Superiore d i Sanitci Rome, Italy B. B. BRODIE National Heart Institute Bethesda, Maryland
J. F. DANIELLI Department of Biochemical Pharmacology School of Pharmacy State University of N e w York at Buffalo Buffalo, N e w York
J. H. BURN Oxford University Oxford, England
R. DOMENJOZ Pharmakologisches Institut Universitat Bonn Bonn, Germany
A. CARLSSON Department of Pharmacology University of Gothenburg Gothenburg, Sweden
B. N. HALPERN Dhpartement de Me'decim Eqe'rimentale CollBge de France Paris, France
K. K. CHEN Lilly Research Laboratories Indianapolis, Indiana
A. D. WELCH Department of Pharmacology Yale University Medical School New Haven, Connecticut
ADVANCES IN
Pharrnacology EDITED BY
SlLVlO GARATTlNl
PARKHURST A. SHORE
Zstituto di Ricerche Farmacologiche "Mario Negri" Milano, Italy
Department of Pharmacology The University of Texas Southwestern Medical School Dallas, Texas
VOLUME 4 1966
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COPYRIQHTO 1966,
BY
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CONTRIBUTORS TO VQLUME 4 RAYMOND L. CAHEN,Pharmacology Department, Pfizer-Clin-Research Center, Amboise (Indre et Loire), France R. DOMENJOZ, Znstitute of Pharmacolgy, Rheinische Friedrich-Wilhelms Universitat, Bonn, Germany
ERVING. ERDOS,Department of Pharmacology, University of Oklahoma School of Medicine, Oklahoma City, Oklahoma JAMESR. GILLETTE,Laboratory of Chemical Pharmacology, National Heart Institute, National Institutes of Health, Bethesda, Maryland ALEXANDER B. GUTMAN,Department of Medicine, The Mount Sinai Hospital, New York, New York
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CONTENTS CONTRIBUTORS . .
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Hypotensive Peptides: Bradykinin, KalMin, and Eledoisin
ERVING. ERD& I. Introduction . . . . . . . . . 11. Releasing Enzymes (Kininogenases) . . . . 111. Kininogen (Kallidinogen, Bradykininogen) . . 1V. Kinins (Bradykinin, Kallidin, Met-Lys-Bradykinin) V. Eledoisin . . . . . . . . . . VI. Conclusions . . . . . . . . . References . . . . . . . . . .
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1 4 19 21 64 72 74
Uricosuric Drugs, with Special Reference to Probenecid and Sulfinpyrazone
ALEXANDER B. GUTMAN I. Uricosuric Activity Defined . . . . . . . . 11. Physiological Basis for Use of Uricosuric Drugs . . . 111. Nature of Uricosuric Response in Normal and Gouty Man;
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Species-Dependency . . . . . . . . . . . . IV. The Search for a Suitable Uricosuric Agent . . . . . . . V. The Akylsulfonamidobenzoic and N-Alkylsulfamylbenzoic Acids . . VI. The Pyrarolidinediones . . . . . . . . . . . . VII. Incidental Compounds Possessing Uricosuric Properties : Phenolsulfonphthalein, Mersalyl, Iodopyracet, Corticotropin and Adrenocortical Steroids, Coumarins and Indandiones, Chlorprothixene, Acetohexamide, Ethyl-pchlorophenoxyisobutyrate.The Paradoxical Action of Benzothiadiazines . . . . . . . . . . . . . . References . . . . . . . . . . . . . . .
96 9!4 107 117
132 134
Synthetic Anti-Inflammatory Drugs: Concepts of Their Mode of Action
R. DOMENJOZ I. Introduction . . . . . . . . . . . . . 11. Former Interpretations of the Effects of Antipyretic/Non-Narcotic
Analgesic Drugs . . . . . . . . . . 111. The Pituitary-Adrenal Axis and Drug-Induced Inhibition of Inflammation . . . . . . . . . . . vii
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143 144
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CONTENTS
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IV. The Inflammatory Focus as Site of Action of Anti-Inflammatory Drugs 171 V. Summary . . . . . . . . . . . . . . . 204 References . . . . . . . . . . . . . . . 204
Biochemistry of Drug Oxidation and Reduction by Enzymes in Hepatic Endoplasmic Reticulum
JAMES R . GILLETTE I. Introduction . . . . . . . . . . . . . I1. Oxidation of Foreign Compounds by Enzymes in Hepatic Endoplasmic Reticulum . . . . . . . . . . . I11. Reduction of Foreign Compounds by Enzymes in Hepatic Endoplasmic Reticulum . . . . . . . . . . . IV. Mechanisms of Oxidation and Reduction by Enzymes in Hepatic Endoplasmic Reticulum . . . . . . . . . . . V . Factors Which Limit! Drug Metaholism in Living Animals . . References . . . . . . . . . . . . . .
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234 254 255
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Experimenta I a nd CIinicaI Chemot eratog enesis
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RAYMOND L CAHEN I. Introduction . . . . . . . . . . . I1. General Survey . . . . . . . . . . . I11. General Principles . . . . . . . . . . IV. Experimental Conditions . . . . . . . . V. Experimental Techniques . . . . . . . . VI . Teratogenic Drugs . . . . . . . . . . V I I . Nature and Mechanism of Action of Teratogenic Drugs . VIII . Conclusions . . . . . . . . . . . Glossary . . . . . . . . . . . . References . . . . . . . . . . . .
AUTHORINDEX . . SUBJECTINDEX . .
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263 264 266 269 278 291 320 333 334 334
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Hypotensive Peptides: Bradykinin, Kallidin, and Eledoisin* ERVING. ERDOS Department of Pharmacology, University of Oklahoma School of Medicine, Oklahoma City, Oklahoiiza
I. Introduction . , . . . 11. Releasing Enzymes (Kininogenases) A. Kallikrein . . . . . B. Trypsin . , . . . C. Plasmin . . . . . D. HagemanFactor . . . E. Permeability Factor . . . F. Snakevenoms . . .
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G. Bact,erial Enzymes . . . . , H. Pepsin. . . . . . . . 111. Kininogen (Kallidinogen, Bradykininogen) . . IV. Kinins (Bradykinin, Kallidin, Met-Lys-Bradykinin) A. Structure . . . . . . . . . B. Physiology and Pharmacology . . . . C. Metabolism of Kinins , . . . . . D. Pathology . . . . . . . . E. Other Sources of Kinins . . . . . . V. Eledoisin . . . . . . . . , . A. Structure and Metabolism . . . . . B. Pharmacology . . , . . . . . VI. Conclusions . . . . . . . . References . . . . . . . . .
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1 4 4 14 14 15 16 17 18 19 19 21 22 26 45 56 62 64 64 67 72 74
I. Introduction
This review deals with hypotensive peptides and with eiizynies which liberate them. Two groups of peptides are included: one belongs to kinins, the bradykinin-kallidin-type peptides, the other consists of eledoisin and its derivatives. The cardiovascular effect is but one of the coninion features between these groups. Although eledoisin-type peptides have riot yet been found in the mammalian body, these inaterials have been tested frequently on experimental animals and clinical subjects. The precursor of bradykinin and kallidin occurs in abundance in blood plasma. Some of the studies described here were supported in part by Grants H E 04592 and NB 05196 from the National Institutes of Health, U.S.P.H.S. and by a Wellcome Research Travel Grant from the Wellcome Trust, London. 1
2
ERVIN G . ERDOS
Some other peptides, although they lower the blood pressure, are not mentioned here. The discussion of oxytocin, substance P, or gastrin would go far beyond the frame of this article, and the major actions of these substances are not related to the cardiovascular response they may elicit. Systematic investigation of kallikrein started in 1925 and led to the discovery of kallidin. Studies with another enzyme, trypsin, resulted in the finding of bradykinin in 1949. In the same year the first description of eledoisin appeared in the literature. Very likely some of these discoveries were accidental. That may have contributed to the negative response they received first (Frey, 1963). Although 24 years passed between the discovery of kallikrein and eledoisin, within a 2-year period, between 1960 and 1962, bradykinin, kallidin, and eledoisin were synthesized. No doubt the developments in methods for separating natural products, for establishing the structure of peptides, and for peptide synthesis made all these rapid advances possible. When the chemistry of the peptides stopped, semantics took over the field. This latter science seems to have fewer limitations than chemistry, with the result that we have now a large number of names for a few bradykinin-type peptides (Fig. 1). The term “kallidin” was used for Structure of kinins
H-Arg1-Pro*-Pros-Gly4-Phe 6-Ser 6-ProT-Phe8-Argg-OH Bradykinin (Kallidin I, kallidin-9, kinin-9, nonapeptide) H-Lys 1-Arga-ProS-Pro4-Gly’-Phe6-SerT-Pro8-PheB-Arg1o-OH Kallidin (Kallidin 11, lysyl-bradykinin, kinin-10, decapeptide) H-Met 1-Lysz-ArgS-Pro4-Pro6-Gly6-Phe7-Sera-ProB-Phe ‘O-Arg”-OH
Methionyl-lysyl-bradykinin (Methionyl-kallidin, kinin 11, undecapeptide, hendecapeptide)
FIG.1. Structure of kinins.
the substance released by kallikreins and “bradykinin” for the active material released by trypsin or snake venoms. The two peptides were considered to be identical by those few who believed in them. Schachter and Thain in 1954 called a similar factor in wasp venom “venom kinin” or simply “kinin.” Later a group of English pharmacologists applied the name to all bradykinin-like peptides derived from plasma proteins, including
HYPOTENSIVE PEPTIDES
3
bradykinin and kallidin. The word kinin as used here refers to either bradykinin, kallidin, methionyl-lysyl-bradykinin (met-lys-bradykinin) (Fig. l), or to other analogs. In the absence of an agreement on terminology a generic name such as kinin is useful to describe any one of these peptides, especially since it is quite difficult to distinguish among the naturally occurring peptides by pharmacological methods. Serious objections can be raised against using this terminology. For example, kinins are entirely different tJypes of compounds in botany; the name “plasma kinin” was coined first to characterize a lipid in blood (Laki, 1943). For the purpose of this review, however, the term kinin is used to describe bradykinin-type peptides. In line with this reasoning-if not further characterized-we call kininogeriases enzymes which liberate kinins, kininases enzymes which inactivate kinins, and kininogen the precursor of kinins. A simplified version of the release and inactivation of kinins is shown in Fig. 2. For example, kallikreins (kininogenase) occur in plasma and in tissues Simplified scheme of release and inactivation of kinins Activator Inhibitor (e. g., hexadimethrine)
P
Preenzyme (e. g . , kallikreinogen)
Kininogenase (e. g . , kallikrein)
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P Inhibitor
(e. g . , kallikrein inhibitor, Trasylol)
Kininogen
t
Kinin
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Kininase (e.g., carboxypeptidase N )
Inactive split product
Inhibitor (e. g.,
chelating agents, heavy metals)
FIG.2. Simplified mechanism of the release of kinin.
4
ERVIN G . ERDOS
as inactive zymogcn, kallikreinogen (preenzyme). The active kallikrein is an enzyme which releases the active peptide (kinin) from the substrate (kininogen). The liberated peptide is broken down by a carboxypeptidase and other enzymes in the blood and tissues (kininase) and the split product is inactive (Fig. 2). II. Releasing Enzymes ( Kininogenases)
A. KALLIKREIN
If the number of occasions of rediscovery (with different names) were indicative of popularity for a substance, kallikrein would be among the most popular. The volume of information published about this enzyme under real or assumed names is considerable. Abelous found a hypotensive principle in the urine in 1909 (see Abelous and Bardier, 1909), and Petroff (1925) discovered one in the pancreatic juice. Frey (1926) characterized the substance in urine and, in collaboration with his associates Kraut and Werle, quickly discovered similar agents in blood plasma, pancreas, and other tissues (Frey and Kraut, 1926; Kraut et al., 1928, 1930; Frey, 1931). Frey believed that all these substances were identical. Since highest concentration was found in the pancreas, Frey (Kraut et al., 1930; Frey, 1931) named them kallikrein from the Greek word for pancreas, lcallilcreas. In the nineteen twenties and thirties numerous properties of kallikreins were described by various research groups (Frey and Kraut, 1926; Kraut et al., 1928, 1930; Elliot and Nuzum, 1931; Krayer and Ruhl, 1931; Felix, 1934; Bischoff and Elliot, 1935). I n addition to kallikrein, plasma and many tissues contain proteolytic inhibitors that reversibly inactivate kallikrein (Kraut et al., 1928, 1930). Werle et al. discovered in 1937 that kallikrein is an enzyme which acts by releasing an active principle from plasma proteins (Fig. 3). This principle was named substance D K (Darmkontrahierende Substanz) (Werle and Grunz, 1939). It is known now as kallidin (Werle and Berek, 1948). Currently lcallikreins may be defined as a group of closely related enzymes, which release a kinin from the plasma protein, kininogen. The activity of kallikreins is measured by their ability t o lower the blood pressure. One Frey unit (U) equals the drop in the arterial blood pressure of a dog caused by the intravenous injection of 5 ml of pooled human urine. Other methods of kallikrein determination are based on reduction of perfusion pressure in the perfused hind limb, increase in capillary permeability in laboratory animals, in vitro release of kinins, or ester hydrolysis caused by the enzyme. The first and the last methods are frequently used (Frey et al., 1950; Trautschold and Werle, 1963). Hog pancreatic kallikrein contracts smooth muscle preparations such as
HYPOTENSIVE PEPTIDES
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FIG.3. Liberation of kallidin by kallikrein in vitro. Isolated surviving guinea pig colon. (1) Dog submaxillary gland kallikrein; (2) kallikrein incubated with 0.8 ml human serum for 1 minute; (3) Same as (2), but incubated for 5 minutes (Werle et al., 1937).
the isolated dog or cat gut (Frey et al., 1950). Since kininogen was not found in these organs, the release of kallidiri would be an unlikely explanation for this action (Werle, 1963). The major sources for kallikreins in the body are blood plasma, glandular tissues, and urine. They occur abundantly in the pancreas, in the parotid and submaxillary glands, in the intestinal wall, in feces, in duodenal juice (Frey et al., 1950), and to a lesser degree in the kidney (Werle, 1955). The kallikrein content of various fluids and tissues varies a great deal. For example, the human pancreas contains 4.5 U/gni, pancreatic fistula fluid 0.5-6 U/ml, ldood serum 2 U/ml, and urine 0.2 U/m1 (Frey and Werle, 1933). Swine pancreas is a very rich source of a kallikrein; the content can range from 40 to 90 U/gm (Kraut et al., 1930; Frcy et nl., 1950; Kraut and Korbel, 1957). Bird pancreas contains ornithokallikrein. This differs froin niaiiinialian kallikreins in that it lowers the blood pressurc of the chicken, but is inactive in nianinials (Werle and Hurter, 1936). Mammalian pancreatic kallikrein, on the other hand, is inactive in birds. Aiiiong inaniinals, species difference can influence the action of kallikrein (Werle et al., 1937; Fasciolo and Halvorsen, 1964). For example, kallikrein of the guinea pig salivary gland does not release kallidin from guinea pig plasma (Schachter, 1960). The pancreas stores kallikrein mainly as an inactive zyinogen, kallikreinogen (Werle and Urhahn, 1940; Werle, 1955). This can be activated by trypsin or by duodenal niucosa. Fetal pancreas contains kallikrein, but fetal subinandibular gland usually does not (Werle, 1960). Other glands such as sublingual, submaxillary, or parotid gland are good sources for active kallikrein (Werle and von Roden, 1936; Frey et al., 1950). Species
6
ERVIN G . ERDOS
differences are quite significant here; for example, rat submandibular gland contains 3000 U/gm of kallikrein (Werle, 1960), cat submaxillary gland has over 100 U/gm, while the human gland has only about 1-3 U/gm (Werle and von Roden, 1936). The release of kallikrein from the pancreas and from the submandibular gland is associated with the exocrine and not the endocrine functions of the glands (Werle, 1960; Forell, 1960). Meat diet, for example, increases pancreatic and urinary kallikrein excretion (Forell, 1960; Vogel et al., 1962a). The average excretion of kallikrein in the human is 210 U during a 24hour period (Werle and Korsten, 1938). Carnivora, such as lion, fox, dog, rat, and swine, have high kallikrein levels in the urine (Vogel et al., 1962a). The horse excretes very little urinary kallikrein (Frey et al., 1950). Blood plasma apparently has two types of kallikreins, both of them in inactive form (Frey et al., 1950). One is activated by treating the serum with acetone (Kraut et al., 1933), the other with papain or acid (Kraut et al., 1928). The activation (for example, by lowering the pH) was attributed to the dissociation of the enzyme-inhibitor complex. But apparently kallikrein occurs in blood as a precursor, kallikreinogen ; trypsin can activate this preenzyme (Werle et al., 1955; Forell, 1955). Trypsin (1 mg) can release 3 4 U of kallikrein from 1 ml serum (Werle, 1960). Numerous processes and components of blood can liberate the active enzyme from kallikreinogen (Sections II,C,D, and E). Plasma kallikrein is different from glandular or urinary kallikrein ; it releases mainly bradykinin (Webster and Pierce, 1963; Habermann and Blennemann, 1964b), while the other kallikreins liberate chiefly kallidin. Acidification of plasma activates a n endogenous enzyme (or enzymes) which, depending on the circumstances, releases either bradykinin (Habermann and Okon, 1961; Webster and Pierce, 1963) or met-lys-bradykinin (Elliott et al., 1963; Elliott and Lewis, 1965). The origin of blood kallikrein is not clear. It does not come from the pancreas, but it may originate from intestinal wall via the lymphatic system or from the liver. Blood kallikreinogen can decrease after hepatectomy, CCI, adniinistration (Werle et al., 1963), or in hepatitis (Forell, 1957); it increases after adrenocorticotropic hormone (ACTH) or cortisone administration (Forell, 1957). Kallikrein in the intestinal wall also does not, originate from the pancreas; it is more concentrated in the colon than elsewhere. Carnivora have more kallikrein in the intestine than other animals (Werle et al., 1963). Gut usually contains more kallikreinogen than kallikrein. The highest kallikreinogen content was found in the cat colon, about 6-15 U/gm (Werle, 1960).
HYPOTENSIVE PEPTIDES
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The occurrence of quantities of kallikrein in the intestinal wall might be of importance in pathological conditions such as shock. In experimental endotoxin shock, where changes in the splanchnic circulation have been implicated (Lillehei et al., 1964), a hypotensive agent (maybe kallikrein) was found in the circulation (Kobold et al., 1964) and the kallidinogen level decreased in plasma (Meyer and Werle, 1964). Thus, it is possible that damage to the intestinal wall or extensive changes in its circulation might release kallikrein which in turn may contribute to circulatory collapse. Another condition in which kallikrein release may cause pathological symptoms was described in patients with carcinoid tumor (Oates et al., 1964). I n some of these individuals, large quantities of kallikrein were found in tumor metastatic to the liver. The cutaneous flushes observed in these Fersons were ascribed to the release of kinins by the increased amounts of circulatory kallikrein. It is also possible that epinephrine would contribute to the liberation of kallikrein from the tumors. The cutaneous flushes in this disease somewhat resemble the symptoms which follow intravenous injections of kallikrein (Reeke and Werle, 1935), or the symptoms found in pancreatitis where the liberation of active kallikrein in the circulating blood is assumed to be of importance (Forell, 1961). Pancreatic kallikreinogen can be activated by various pathological conditions (Section IV,D). For example, necrosis of the pancreas (Forell, 1955), alloxan administration (Forell and Dobovicnik, 1960), or autocatalytic activation of trypsin may lead to the conversion of kallikreinogen to kallikrein in the pancreas and in the serum and to the release of active peptides (Werle et al., 1958). The activation of kallikrein in pancreatitis is considered by some to be an important contributing factor to the circulatory collapse in this disease (Section II,A,2). Recently the presence of active proteolytic enzymes (Forell and Dobovicnik, 1964) in the pancreas during experimental pancreatitis has been questioned (I. T. Beck et al., 1962) although active proteolytic enzymes were found in some in vitro experiments in the homogenized pancreas (Nagel and Willig, 1964; Nagel et at., 1965; Schon et al., 1963). Probably this activity is not caused by trypsin. Various nephrotoxins enhance the excretion of kallikreinogen in rat urine (Werle and Vogel, 1960; Dobovicnik and Forell, 1961) and decrease that of active kallikrein. Tubular damage seems to be involved in this process. Patients with nephrogenic hypertension (Frey el al., 1950) and with Addison’s disease or hypophysial deficiency (Vogel et al., 1962b) excrete less kallikrein. In rats hypophysectomy lowers the amount of urinary kallikrein (Vogel et al., 1962b). Removal of the pancreas probably does not affect urinary kallikrein (Frey et al., 1930; Beraldo e l al., 1956). Kallikrein has been purified from various sources. Kraut et al., prepared
8
ERVIN G. ERD6S
the first purified kallikrein in 1932. Of this material, 0.05 pg/kg caused a significant drop in the blood pressure of a dog. The currently used methods of purification include, among others, cellulose column chromatography and Sephadex filtration (Moriya et al., 1963; Werle and Trautschold, 1963). The purest swine pancreatic kallikrein preparation contains 588 U/mg, according to Werlc and Trautschold (1963); 800 U/mg, according to Habermann (1963a); or 530 U/mg, according to Moriya et al. (1963). Currentfly even more active hog pancreas kallikrein preparations are available (E. Werle, unpublished results, 1965) (Fig. 4). The best hog
I
1 MINUTE
1I 1
FIG.4. Effect of intraarterial injection of purified hog pancreatic kallikrein on the perfusion pressure in the dog; 10.5-kg dog, autoperfused hind limb preparation. (1) 4 ng/kg kallikrein (Erdos, unpublished results, 1965).
submaxillary kallikrein preparation has 510 U/mg (Werle and Trautschold, 1963)) and human urinary kallikrein 430 U/mg (Moriya et al., 1963), human salivary kallikrein 200 U/mg (Moriya, 1965). E. S. Prado et al. (1962) extracted kallikrein from horse urine, and obtained a 330-fold purification. The molecular weight of swine pancreatic kallikrein was calculated as
HYPOTENSIVE PEPTIDES
9
33,000 (Moriya, 1959), 24,000 (Habermann, 1962a, 1963a), or 48,000 (Kraut, cf. Werle, 1955 p. 22). Human urinary kallikrein has a molecular weight of 40,500; human pancreatic kallikrein, 31,200 (Moriya et al., 1963). Even highly purified kallikrein preparations show two components in the electrophoresis (Habermann, 1963a). Serum kallikrein is quite unstable beyond the 100 U/mg purification levels. Pure hog pancreatic kallikreiri coiitains about 15 (Moriya, 1959) or 16 (Werle and Trautschold, 1963) amino acids. Injections of hog pancreatic kallikrein produce antibodies in the rabbit which are inactive against serum kallikrein, suggesting additional diffcreiices between the kallikreins from different sources (Habermann, 19G2b). Kallikrein which was inhibited by DFP (diiosopropyl fluorophosphate) still induced formation of antibodies. Antibodies against human urinary kallikrein inhibited human pancreatic kallikrein as well, but not dog urinary or hog pancreatic kallikrein. These experinleiits also point out the importance of species differences in the structure of this enzyiue (Webster el nl., 1963). All three types of kallikreiiis have a pH optinium of about 8.5 with ester substrate; their activities decreased when the pH was lowered toward neutrality (Webster and Pierce, 1961). Although the main function of kallikrein is the release of active peptides, it also has an esterase activity which is maintained throughout purification procedures (Contzen et al., 1959; Werle and Kaufmann-Boetsch, 1959,1960; Habermann, 1959; Webster and Pierce, 1961). The esterase activity provides a convenient method for assaying the enzyme either by spectrophotometric techniques (Trautschold and Werle, 1961, 1963; Webster and Pierce, 1961) or by titrating the acid liberated during the hydrolysis of the ester bonds (Trautschold and Werle, 19Sl). The relationship between lowering the blood pressure and the hydrolysis of ester bonds varies according to the source of kallikrein. The value of the esterase activity could be converted t o biological activity units by niultiplying it with a factor which ranges from 0.8 to 8.8, depending on the origin of kallikrein (Trautschold and Werle, 1963). The substrates used were methyl or ethyl esters of arginine. Unfortunately, these substrates are not specific for kallikrein. Arginine esters are hydrolyzed by trypsin and other tryptic enzymes. In addition, a crude guinea pig kallikrein preparation was capable of hydrolyzing lysine esters (Davies and Lowe, 1963). A partially purified hog pancreatic kallikrein preparation also splits a substrate of chyniotrypsin, a tyrosine ester (Webster and Pierce, 1961). Some of these substrates inhibit, probably competitively, the release of kinin by kallikrein (Werle and KaufmannBoetsch, 1960; Webster and Pierce, 1961). Since the kiniriogenase preparations tested were not able to hydrolyze amides, the conclusion was drawn
10
ERVIN G . ERDOS
that the release of kinin is related to the breaking of ester bonds (Rocha e Silva, 1960; Elliott, 1963). It should be pointed out, however, that tryptic enzymes are not true esterases, and the more rapid hydrolysis of esters compared to the corresponding amide probably reflects the lower stability of the ester bond. Enzymes such as trypsin hydrolyze ester substrates 60 times faster (Schwert et al., 1948; Elliott, 1963), and snake venom enzymes (Habermann, 1961) several hundred times faster than the corresponding amide. In addition, benzoyl-L-arginine amide, although not hydrolyzed by the kallikrein preparations a t the concentrations used, inhibits the release of kallidin by kallikrein (Webster and Pierce, 1961). Kallikrein has been in clinical use for several decades (Frey, 1931). The preparation called Padutin is produced from hog pancreas and contains 10 U, or recently, 40 U per ampule. The use of kallikrein has been indicated mainly in circulatory diseases, especially when the peripheral blood flow has keen impaired (Frey et al., 1953; Golenhofen et al., 1958; Lund, 1958). The clinical use is based on the experimental observations that kallikrein increases the blood flow in the extremities, in the skin, and in the coronary artery, and elsewhere. The long-lasting Padutin injections are called DepotPadutin and are administered intramuscularly. The exact mechanism of the action of this latter preparation is not yet clear. Additional studies are needed to explain how a material so strongly inhibited by blood serum can be absorbed in small doses and exert its effect in various parts of the body.
1. Ka’likrein Inhibitors Kallikrein is inhibited by numerous agents; some of them are proteins or peptides which occur in blood and in various tissues including glandular tissues, lymph glands, and lung (Frey et al., 1950). Other inhibitors originate, from plants or are fairly simple organic compounds (Table I ; see Section IV,B,4). The tissue and blood serum inhibitors were assumed to be basic peptides which combine with the acidic kallikrein, probably via ionic bonds (Werle and Daumer, 1940). Acid treatment of serum dissociates the kallikrein serum inhibitor complex (Kraut et al., 1928). The activity of the kallikrein inactivator (inhibitor) has been measured in biological units; 1 U of inactivator completely inhibits 1 U of kallikrein at pH 8 and 37°C in 2 hours (Frey et al., 1950). The affinity of kallikreins to inhibitors depends on the source of the enzyme. This property is helpful in differentiating pancreatic kallikrein from serum kallikrein or from trypsin. Serum kallikrein is the only kallikrein which is sensitive t o soybean trypsin inhibitor (Werle and Maier, 1952a; Werle and Kaufmann-Boetsch, 1960; Webster and Pierce, 1961). Ovomucoid trypsin inhibitor blocks only the papain-activated serum kallikrein, but not the acetone-activated one
11
HYPOTENSIVE PEPTIDES
TABLE I INHIBITORS OF KALLIKREIN~ Inhibitor
Source of kallikrein Inhibition
DFP
O,O-Bis-(2-chloroethyl)-O(2-dichlorovinyl) phosphate Antipyretics, non-narcotic analgesics
1, 2
1 1,2
+ + + + + -
Soybean trypsin inhibitor
1 only
Plasma inhibitor
1; 2, 3
Pancreatic trypsin inhibitor
1, 2, 3
Kallikrein inhibitors from various tissues
1 , 2, 3
Ester substrates of kallikrein
1, 2, 3
Potato inhibitor
1, 2, 3
Plasmin inhibitor from plasma* Heparin
1
1
+ + + + + + + + + + + + + + +
Reference Habermann (1960, 1961); Wehster and Pierce (1961) Webster and Pierce (1960). Guth (1960); Northover and Subramanian (1961); Hebhorn and Shaw (1963); Spector and Willoughby (1962,) Werle and Maier (1952a); Werle and Kaufmann-Boetsch (1959, 1960); Webster arid Pierce (1960) Kraut et al. (1928); Trautschold and Riidel (1963) Webster and Pierce (1960, 1961) Frey et al. (1950); Werle and Appel (1959); Trautschold and Riidel (1963) Werle and Kaufmann-Boetsch (1960) ; Wehster and Pierce (1961) Werle and Maier (1952b); Werle et al. (1951) Webster and Pierce (1960) McConnell et al. (1965)
Key: 1, plasma; 2, pancreas; 3, urine; f, inhibition; -, no inhibition. Recently obtained pure plasmin inhibitor preparations do not inhibit kallikrein (I. Trautschold, unpublished observation, 1965). 0
b
(Werle and Maier, 1952a). Inactivators from the parotid or lymph gland (Kraut et al., 1933, 1934) inhibit urinary and pancreatic kallikrein, but not serum kallikrein (Werle and Maier, 1952a). This statement was modified later, and inhibitors from various tissues were described to inhibit all kallikreins (Trautschold and Rudel, 1963). The approximate ratio of concentrations of soybean, pancreatic, and ovomucoid trypsin inhibitors used to inhibit plasma kallikrein was 1 :310 : 100,000 (Webster and Pierce, 1960). Ovoniucoid trypsin inhibitor was fairly ineffective even a t that high concentration level. An inhibitor of many
12
EHVIN G. ERDOS
hydrolytic enzymes, DFP, inactivates kallikrein as well (Haberniann, 1960, 1961). As mentioned elsewhere, substrates of kallikrein can prevent the release of kallidin from kallidinogen by the enzyme (Webster and Pierce, 196l), possibly by competitive inhibition. 2. Trasylol
Studies with various kallikrein inhibitors (Frey et al., 1950; Werle and Maier, 1952a,b; Werle and Appel, 1959) led to the development of a commercially available product, Trasylol (Bayer 128). This inhibitor was first purified from the parotid gland of cattle (Kraut et al., 1934; Kraut et al., 1963). It is now manufactured from bovine lung (Werle, 196413; Kraut and Bhargava, 1964). Trasylol is a polypeptide of known amino acid content with a molecular weight of about 11,000 (Kraut et al., 1960, 1963) for the dimer. The true molecular weight is 6,500 (Anderer and Hornle, 1965). I n addition to kallikrein it also inhibits trypsin and chymotrypsin (Werle et al., 1952; Kraut and Korbel-Enlthardt, 1958; Kraut and Bhargava, 1963), the conversion of plasminogen to plasmin, and to a certain degree the digestion of fibrin by plasmin (Steichele and Herschlein, 1961; Kraut and Bhargava, 1963; Berghoff and Glatael, 1963; Blix, 1964; Marx et al., 1959, 1963; E. Deutsch arid Marschner, 1963; Marx, 1963; E. Beck et al., 1963); 0.15 pg of the purest preparations inhibits 1 U of kallikrein (Kraut et al., 1963). It has been suggested that the peptide is identical with the pancreatic trypsin inhibitor of Kunitz. The crystallized kallikrein inhibitortrypsin complex is similar to the crystallized trypsin inhibitor-trypsin complex (Kraut et al., 1963). The amino acid composition of the two inhibitors seems to be identical (Anderer, 1965). The effective in vitro use of proteolytic inhibitors against trypsin and kallikreiri suggested in vivo experiniental and t,herapeutic applications for Trasylol. The inhibitor is stable in blood; it is not attacked by enzymes there (Werle and Trautschold, 1961). It disappears very rapidly from the circulation in nian or laboratory animals (Trautschold et al., 1964). The half-life of the agent is about 1 hour in clinical subjects after infusion and 10-15 minutes after single injection. In the rat the kidney removes the peptide and slowly metabolizes it, as shown with tritium-treated material. Therapeutic use of Trasylol is aimed a t the first place against conditions which are considered to lead to the activation of the zymogens of proteolyt]ic enzymes in the pancreas, to the activation of kallikrein, and to result in the release of kinins. Although it is debatable whether or not in experimental pancreatitis active trypsiri can be found in the rat or in the dog (Creutzfeldt et al., 1964; Trautschold, 1964; Forell and Dobovicnik,
HYPOTENSIVE PEPTIDES
13
1964; Creutzfeldt et al., 1965) or the zymogen content can be depleted, many reports are quite favorable toward the clinical use of Trasylol. The use of a trypsin inhibitor in hemorrhagic pancreatitis in dogs was first attempted by Rush and Clifton (1952). Their favorable results with soybean trypsin inhibitor were not confirmed by Hoffman et al. (1953). I n the dog, Trasylol seemed to have a protective effect against experimental pancreatitis or pancreatic necrosis (Herinann and Knowles, 1962; Hoferichter et al., 1962; Mallet-Guy et al., 1961; Thal et al., 1963; McCutcheon and Race, 1963; McHardy et al., 1963; Nemir et al., 1963; Smith et al., 1963; Grozinger et al., 1964). The suggesl ion was made, however, that the beneficial effects of Trasylol are not related to the inhibition of trypsin (I. T. Beck et al., 1962, 1965) but to inhibiting active kallikrein (Trautschold and Rudel, 1963; Trautschold et al., 1964; Creutzfeldt et al., 1965). Some authors had mainly negative results to report on the use of Trasylol in experimental pancreatitis (I. T. Beck et al., 1965; Creutzfeldt et al., 1965; Schutt et al., 1965). One particularly disturbing report (Cliffton and Agostino, 1964) indicated that Trasylol increased the incidence of lung metastases in rats bearing malignant tumors. This phenomenon was attributed to the inhibition of plasmin. I n addition to experimental pancreatitis, Trasylol was also used to block the Shwartzman phenomenon in rabbits. Like another trypsin inhibitor (Chryssanthou and Antopol, 1961), Trasylol inhibited the development of local Shwartzman reaction (Halpern, 1964). Pretreatment with Trasylal decreased the systemic effects of endotoxin in dog (Meyer arid Werle, 1964; Massion and Erdos, 1965). The kallikrein inhibitor gave controversial results when used to combat the aftereffects of burns in small animals (Koslowski et al., 1963; Allgower, 1962; Veragut, 1962). Some of the conflicting results in animal experiments might be explained by too low doses of the inhibitor. In clinical use the initially applied 10,000-U dose of Trasylol was increased to 1,000,000 U (Trautschold et al., 1964). I n man Trasylol lowered the trypsin content of human pancreatic fistula (Nehrbauer, 1959). The clinical use of Trasylol has been indicated in peritonitis (Forell, 1963), in parotitis (Lorbek, 196l), in pancreatitis (Werle et al., 1958; Asang, 1960; Frey, 1962), in chemotherapy of malignant tuniors (Rolle, 1964), and in spontaneous fibrinolysis (Schmutzler and Beck, 1962; Steichele and Herschlein, 1962). Kazmers (1964) and Maurer (1964) described the beneficial effect of prophylactic treatment of postoperative pancreatitis with Trasylol. The latter author, after surveying a
14
ERVIN G. ERDOS
large number of clinical cases, concluded that for effective administration Trasylol should be used prophylactically.
B. TRYPSIN Intravenous injection of trypsin lowers the blood pressure of laboratory animals (Rocha e Silva, 1940b). This effect was attributed first to the release of histamine. It was observed later, however, that the administration of a n antihistamine does not protect the animal against trypsin ;thus, in addition to histamine, another substance was assumed to be released by this enzyme (Wells et al., 1946). Trethewie (1942) has described such a slow-reacting substance which contributed to the response of smooth muscles to trypsin. Independent of the discovery of kallidin, Rocha e Silva et al. (1949) found the in vitro release of bradykinin by trypsin. It was established much later that less than 1/10 of the kinin released by trypsin is kallidin (Webster and Pierce, 1963; Habermann and Blennemann, 1964b). The effects of the in vivo injection of trypsin rest upon the release of bradykinin and upon the activation of kallikrein (Werle et al., 1955; Werle, 1963). Since plasma kallikrein releases bradykinin, this peptide is the end product of both of the actions of trypsin. Injected kallikrein, however, acts on the circulation in several thousand times lower concentration than trypsin (Werle, 1953). This might be attributed to the high trypsin inhibitor level of plasma.
C. PLASMIN In his first publication dealing with bradykinin, Rocha e Silva stated that chloroform treatment of plasma results in release of the peptide (see Rocha e Silva et aZ., 1949). He attributed this to the activation of plasmin in blood. Beraldo (1950) incubated plasmin with plasma globulin and observed the release of a smooth muscle-stimulating substance, probably bradykinin. Soybean trypsin inhibitor blocked this liberation of the peptide. Schachter (1956) attributed the kinin release to the activation of plasma kallikrein by plasmin. The precursors of the two enzymes in blood are different since numerous properties distinguish kallikreinogen from plasminogen (ltebster and Innerfield, 1965). Lewis (1958) claimed that human plasmin preparations have a proteolytic and a plasma kinin-forming activity which run parallel. Later the theory was advanced (Horton and Lewis, 1959) that, in the body, two types of enzymes can release kinins: one acts rapidly and is not inhibited by proteolytic inhibitors (e.g., soybean trypsin inhibitor). The other acts slowly and is inhibited by these agents (Lewis, 1959). Presumably the quicker enzyme would be identical with kallikrein, the slower one with plasmin. In the light of recent experiments done by others, this distinction no longer holds true (Eisen, 1963). For example, numerous proteolytic inhibitors, are known which can inhibit kallikrein
15
HYPOTENSIVE PEPTIDES
from various sources (Webster and Pierce, 1960). I n addition, no correlation between the fibrinolytic activity and kinin release by plasmin was found (Bhoola et al., 1960). Webster and Pierce (1960) have shown that plasmin is not involved in the acetone activation of plasma kallikrein; plasmin does not destroy kallidinogen. According to Eisen (1963) only a small fraction of the kinin available in plasma is released by plasmin (Back and Steger, 1965). Vogt goes even further and suggests that plasmin fornis kinins only through the activation of plasma liallilireinogeri (Vogt, 1964). It is also unlikely that a direct in vivo kinin release would be an important factor in the hypotensive effect of plasmin (Back et al., 1963a). Conversely, intravenous injection of bradykinin or kallidin increases fibrinolysis in dog plasma (Holemans, 1965).
D. HrZGEMAN FACTOR The relationship between blood coagulation and peptide release has been extensively studied by Margolis and others. The observations of Schachter (1956) and Armstrong et al. (1957) that dilution or contact with glass releases from plasnia a substance which contracts smooth niuscle preparations and causes pain led Margolis to explore the rolc of the Hageman factor in peptide release. The Hagenian factor is a plasma globulin which is missing from the blood of patients with the Hageman trait (Ratnoff and Colopy, 1955; Margolis, 1959). This is a congenital condition which is characterized by a grossly prolonged clotting time in zitro in the absence of any hemorrhagic symptoms. Hageman factor appears to be responsible for the initiation of a series of reactions when plasma is exposed to foreign surfaces such as glass, some of the fatty acids (Margolis, 1962), or AlzO, (Margolis, 1963). Acetone and chloroform can also activate the factor. Patients aEicted with the Hageman trait may also be lacking a precursor of kallikrein (Bhoola et al., 1960; Webstcr and Ratnoff, 1961). According to Margolis (1963) during the in vifro release of kinins the glass-activated Hagernan factor activates a component A of blood plasma, which in turn releases an active kinin from the substrate (coniponent B). The scheme of activation is shown in the accompanying diagram: Foreign surface I
1
Hagemai factor,Comprnt
Activated HF
A
Activated component A
_ _ _ _ +Component
B ---+ Kinin
16
ERVIN G. ERDOS
Component A may be kallikreinogen (Margolis, 1963; Margolis and Bishop, 1963). The activated component A would be identical with kallikrein and with a plasma permeability factor. Component B would represent approximately one third of the total kininogen in blood. This part of the kinin precursor would be more readily available for the endogenous enzyme than the rest of the substrate. This scheme of activation might be even more complex in the light of recent experiments done with purified Hageman factor. Here Hageman factor acted as a n enzyme which released PF (permeability factor) (Ratnoff and Miles, 1964). Presumably this PP in turn releases kallikrein from kallikreinogen (Becker and Kagen, 1964) (see Section II,A and E). Heparin accelerates plasma kinin formation (Amstrong and Stewart, 1962). Consequently, heparin antagonists such a s protanine sulfate and hexadimethrine bromide antagonize this induced kinin formation. The suggested mechanism of this antagonism depends on interference with the activation of Hageman factor on glass and other foreign surfaces (Eisen, 1964).
E. PERMEABILITY FACTOR A factor was found in guinea pig blood seruin which increased capillary permeability when injected intraderrnally to guinea pigs (Mackay et ab., 1953). The characteristics of this protein in various animals and its relation to the plasma kallikrein system has been studied extensively by a number of investigators. Recent studies indicate that blood plasma contains two permeability factors. One of them niight be identical with kallikrein; the other is a different protein. The latter factor would mediate the activation of kallikrein. Permeability factors appear when serum is diluted or stands undiluted for several days in the refrigerator. These factors were named accordingly PF/Dil (permeability factor) and PF/Age (Mackay et al., 1953; P. B. Stewart and Bliss, 1957). PF/Age makes up less than 1% of the total PF available in guinea pig seruin (Miles and Wilhelm, 1955). [The abbreviation PF refers to PF/Dil (Miles, 1964).] Serum of inan (P. B. Stewart and Bliss, 1957), rabbit (Pashkina, 1956), rat (Spector, 1958), and other animals contain PF, but it is iiiost active in the guinea pig (Miles, 1961). During the course of these investigations it has been noticed that numerous properties of PF resemble those of plasma kallikrein. PF is inhibited by soybean trypsin inhibitor (Miles and Wilhelm, 1955; Elder and Wilhelm, 1958) or DFP, but not by ovoniucoid trypsin inhibitor (Becker et al., 1959). I n vivo its effects are blocked by salicylate (Mill et al., 1958; Spector and Willoughby, 1959). Normal blood contains an inhibitor of PF which inhibits kallikrein and C’-1-esterase as well (Donaldson and Rosen, 1964; Becker
HYPOTENSIVE PEPTIDES
17
and Kageri, 1964). The IT-inhihitor coniplcx can dissociate upon dilution in glass. Administrat ion of t iirpcntine releases I’F in rat pleural exudate which is activated by niitochondria (Spector, 1958). This material also liberates a bradykinin-like, slow-cont ract ing suhstance (Spector and Willoughby, 1962b). The PI’ lowers the blood pressure of guinea pigs (Mackay et al., 1953). The hypotensive effects arid the ~ienncabilitychanges caused by PF are closely correlated (Wilhelm el al., 1955). The pcrineability factor occurs in blood in the form of a precursor in sonic species (Miles and Wilhelm, 1960); dilution or fractionation activates this protein. It has an enzyniic function aiid hydrolyzes sonic esters of aiiiino acids (Miles, 1961; Kagen, 1964). Two persons with angioneurotic edema were described to have much higher than nornial PE’ values. In atlditioii to plasina, guinea pig lyniph also contains a PF (Miles aiid Wilhelin, 1060). In laboratory animals, shock, radiation, or bactereniia fails to change thcl equilihriuin between the PF precursor and inhibitor systeiii. The pennc~al)ilityfactor is not very effective in inducing leucocyte migration (Spector a i d Willoughby, 1964). Human plasma (Kagen et al., 1963) rontaiiis two 1’Fs. One of them, similar to kallikrein, niigratcs with the 7-globulin; the other PF behaves like a 0-globulin. Only the PF in the -y-globulin was considered to be identical with kallikrein. It can he recalled here, however, that two kallikreins have been described to exist in blood plasma (Frey el al., 1950). The two human PFs scparatecl (Becker and Kagrn, 1964) were not tested on the blood pressure of aninialb. This test could provide some additional inforniation on the characteristics of the factors. Human serum kallikrein and PI? are distinguishable by their electrophoret ic niobilities, chromatographic properties, sediiiieiitatiaii in the ultracentrifuge, and by the release of kinin from heated substrate (Becker and Kagen, 1964). The results of these studies are conipatible with the view that the true PF niediates release of kiniri by activating ciiclogeiious kallikrein (Miles, 1964).
F.
SNARE
VENOMS
Snake venoms in general provide a rich source of various enzymes. One of them releases bradyltinin from bradykiniiiogen. This releasing enzyme probably is riot identical with the enzynies or factors in the verionis which inactivate bradykiiiin, accelerate the coagulation of blood, hydrolyze proteins such as casein or henioglobin, or kill the aninials. It is also possible that this kiniiiogenase is different from the peptidase that can convert met-lys-bradykinin to bradykiiiin (Hahermann and Blenneinann, 1964b). Some of the esterase activity of the venoms, however, has not separated from the kininogenase.
18
ERVIN G. ERDOS
The release of bradykinin by Bothrops jararacu venom was noted by Rocha e Silva and his colleagues (1949). The observation that in vitro incubation of the venom with dog plasma liberated a smooth musclecontracting principle stimulated a great deal of subsequent research on this peptide. Some snake venoms, however, were inactive. This problem has been studied extensively by H. F. Deutsch and Diniz (1955). They have noticed that the release of the peptide and the inactivation of the peptide are caused by different enzymes in the venoms (Hamberg and Deutsch, 1958.) The ratio of these two components varies from venom to venom. Venoms of Agkistrodon contortix (copperhead) or A . piscivorus (water moccasin), for example, release bradykinin, but they do not inactivate the peptide. Agkistrodon contortix venom (Deutsch and Diniz, 1955) has the highest releasing enzyme content, Crotolus atrox (rattlesnake) has the most active kininase. Thrombin-like and proteolytic enzymes also appear in the venoms independently (Deutsch and Diniz, 1955). The liberation of the kinin by snake venom kininogenase was not blocked by soybean trypsin inhibitor (Hamberg and Rocha e Silva, 1957), but DFP prevented it (Habermann, 1961). Treatment with DFP, however, did not change the toxicity of the venom. Ester and amide substrates of the enzyme can inhibit the kinin release. The kininogenase in the Bothrops jararaca venom seems to be resistant to boiling for a few minutes (Hamberg and Roche e Silva, 1957). This venom released more bradykinin from purified bradykininogen than did trypsin, although the venom hydrolyzed arginine ester slower than did trypsin (Henriques et al., 1962). Fractionation studies with Bothrops venom also indicated that the kininase, protease, and thrombin-like activities can be separated (Holtz and Raudonat, 1956; Henriques et al., 1960). A kininogenase which had no effect on blood clotting and hydrolyzed arginine esters very little (Sato et ad., 1965) was purified from Agkistrodon halys blomhojii venom. The enzyme was inhibited by the kallikrein inhibitor, Trasylol (Suzuki et al., 196513). The purified kinin which is liberated when bovine plasma is incubated with snake venom has the full potency of the peptide from other sources (Zuber and Jaques, 1960; Jaques and Meier, 1960). Chemically it is identical with bradykinin (Hamberg et al., 1961). Some experiments suggested the possibility that A . contortix venom might be able to liberate kallidin in addition to bradykinin (Webster and Pierce, 1963).
G. BACTERIAL ENZYMES A cysteine activated proteinase of Clostridium histolyticum (clostripaine) releases a kinin from bovine bradykininogen. The source of substrate was bovine plasma globulins precipitated with ammonium sulfate or bovine
HYPOTENSIVE PEPTIDES
19
Cohn fraction IV-4 (J. L. Prado et al., 1956). The release of kinin was not attributed to activation of an endogenous kallikrein by this enzyme, because soybean trypsin inhibitor did not block the formation of the peptide (J. L. Prado and Prado, 1962). Another bacterial proteinase from Bacillus subtilis N (Nagarse) liberated a kinin from horse plasma kininogen. Nagarse had practically no effect on arginine esters. In contrast to soine other kininogenases it did not hydrolyze p-toluenesulfonyl-L-arginine methyl ester or benzoyl-L-arginine ethyl ester to any appreciable extent (J. L. Prado et al., 1964; Prado, 1964). Interestingly, Nagarse liberates only a hypertensive material, probably arigiotensin 11, from human plasma fraction IV-4 (Huggins et al., 1964). H. PEPSIN Pepsin digestion of serum releases among others an oxytocic substance, pepsitocin (Croxatto, 1955; Croxatto and Barnafi, 1960). The material has been purified from bovine globulin (Turba and Hetzel, 1954). The possibility exists that the peptide may derive from bradykininogen. It may be identical with one of the smaller active fractions of bradykininogen (Habermann and Schuck, 1964) obtained after peptic digestion (Greenbauni and Hosoda, 1963b; Werle and Hochstrasser, 1963; Greenbaum et al., 1965). I l l . Kininogen (Kallidinogen, Bradykininogen)
The precursor of bradykinin and kallidin in blood plasma is called kininogen. Very likely bradykininogen (Rocha e Silva et al., 1949) and kallidinogen (Effkemann and Werle, 1941; Werle and Berek, 1948) are identical proteins (Werle and Berek, 1950). Various enzymes can release either bradykinin or kallidin from the same precursor. Investigators working on the characterization of this protein encountered certain problems. Crude kininogen preparations may contain, among others, an enzyme which inactivates kinins and a proteolytic inhibitor which inhibits kininogenases. The methods of preparation ainied to destroy these two factors usually include steps which involve either heating the protein, or using acid pH in the solution, or the combination of both. Some of the difficulties stemming from this treatment are caused by the spoilt aneous activation of plasma kininogenase contamination at acidic pH (Frey et al., 1950). This phenomenon has been attributed to the dissociation of the kallikreininhibitor complex. Subsequently, there is a LLspontaneous”appearance of kinin and the exhaustion of available precursor. Although the kallikreiri contamination in the kininogen preparation is destroyed by boiling, heat denaturation of the protein renders it less sensitive to kaIlikrein (Werle and Preisser, 1956) or snake venom (Rocha e Silva, 1963b), and more
20
ERVIN G. ERDOS
readily available to trypsin (Haberniann, 1963a). Using denatured protein as a precursor of kinins makes the task of estimating the changes in kininogen level in blood plasma in various conditions difficult. The problem of obtaining a high yield of kinin was partially overcome by using inhibitors of the kinin-destroying enzymes. These include cysteine (Werle and Grunz, 1939; Van Arman, 1955), phenanthroline (Erdos, 1962, 1963a; Vogt, 1964), or EDTA (Armstrong et al., 1955; Erdos, 1962, 1963a; Aniundsen et al., 1963). Werle et al. (1937) prepared kininogen by heating serum for 3 hours at' 56°C t o destroy both a kallikrein inhibitor and a kininase. Horton achieved the sairie result by lowering the p H of dog plasma to 2 for a short time (1959a). The destruction of the kinin inactivator by acidification has already been noticed by Werle et al. (1937). The kininogen level varies in the various niamnials. Rats have the lowest and ox the highest levels, a difference of 7-fold. Other plasma samples from various laboratory animals ranged between these two (Diniz and Carvalho, 1963). Human plasma contains a relatively high level of kininogen (Werle and Hambuechen, 1943). The first concentration of kininogen was achieved by Rocha e Silva et al. (1949) who precipitated bradykininogen from half-saturated animonium sulfate solution. Van Arman found (1952, 1955) the kinin precursor in plasnia globulins, and in Cohn fractions IV-1, IV-4, and IV-6. More recently kiniriogen has been purified from plasma by means of chromatographic techniques. Horse serum kininogen was purified 170-fold with respect to snake venom and only 43-fold when trypsin was used as a releasing enzyme (Henriques et al., 1962) (the experimental conditions used here allowed only the ineasurenieiit of instantaneously released kinin). Webster and Pierce (1963) reported a 150-fold concentration of human kininogen. Greenbauni and Hosoda (1963a) concentrated bovine kininogen 30-fold; Werle and Hochstrasser (1963), 60-fold; Suzuki et al. (1965a) obtained an approximately 40-fold purification over the starting material which was an aninioniuin sulfate precipitate of bovine plasma. Pepsin can break down kiriinogen to even smaller units (Greenbaum and Hosoda, 1963b; Werle and Hochstrasser, 1963; Greenbaum et al., 1965). Some of the products of the peptic digestion are biologically active in that they contract smooth muscle (Greenbauni and Hosoda, 196313; Greenbauni et al., 1965) (see Section 11,H). Using Cohn fraction IV-6 of bovine plasma as his starting material, Haberniann (196313) obtained a highly purified bradykiriin precursor which released 20 kg bradykinin per iiig protein (Habernann et al., 1963). T. Suzuki (unpublished, 1965) had comparable results. The kiniiiogeii is probably an a-glycoprotein of a molecular weight of 48,000 (Habennann, 1963b; Haberniarin et al., 1963). According to
HYPOTENSIVE PEPTIDES
21
Habermann,* the bradykinin molecule would be held in a peptide chain the following way:
Met-Lys-ilrg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Ser-Val-Glu(NH2) Changes in kininogen level in human blood plasma were also scrutinized by various authors. Arnistrong et al. (1960) estimated that up to 95% of the available kininogen can be depleted in the blood of women during labor. Martinez et al. (1962) observed only a 25% drop. The difference in the values might be due to the different techniques used (Armstrong and Stewart, 1960). While Armstrong et al. (1960) used native plasma, the Brazilian group boiled the kiniiiogen in acetic acid. It is possible that Arnistrong et al. assayed the decrease in the readily available kiniiiogen [the so-called cornponent B of Margolis (1963)], while Martinez et al. used a denatured substrate. Another source of discrepancy niighh have been the difference in temperature during the handling of plasma. At lower temperature the plasma kininogen is depleted very rapidly (Arnistrong et al., 1965). A decrease in the kininogen level in blood during delivery was also observed by Periti et al. (1962). The newborn has an wen lower value than the niother (Battaglia et al., 1963). Generally the arterial blood contains more kininogen than the venous blood (Sicuteri et al., 1963a). In addition t o blood plasma, kininogen has been found in ascites (Frey et al., 1950), in colostrum (Werle, 1960; Guth, 1959), in bone marrow (Werle and Preisser, 1956), and in lymph (Schachter, 1960), but not in the cellular elements of blood. Bradykininogen niay originate from the liver, since CCI, treatment orhepatectomy decreases its level in rabbits (Diniz and Carvalho, 1963). Lower than normal values were also observed in patients suffering from cirrhosis of the liver (Sicuteri et nl., l962a). Kallikrein infusion (Werle, 1955; Webster and Clark, 1959) or fibrinolytic therapy (Salmon and Lecomte, 1963) can deplete the availahlc kininogen in the blood. The kininogen was also lower in the blood of rabbits in anaphylactic shock (Lecomte, 1961; Diniz and Carvalho, 1963) or in dogs in eiidotoxin shock (Meyer and Werle, 1964), but ncphrcctomy in dogs had no effect (Fasciolo, 1962). IV. Kinins ( Bradykinin, Kallidin, Met-Lys-Bradykinin )
Bradykinin has the strange distinction among hypotensive peptides iii that it was synthesized before the final amino acid sequence of the natural product was established. Retween 1960 and 1964 more than 70 analogs of . kinins had been prepared (Schrodcr and Hempel, 1964), but only a limited * See note added in proof a t the end of this chapter.
22
ERVIN G . ERDOS
number of conclusions could be drawn from these studies. I n this respect bradykinin may be at a disadvantage to vasopressin or oxytocin (Boissonnas et al., 1961). The analogs of these peptides have been studied more extensively because they have been available for a longer period and more specific assay systems have been developed for them than for bradykinin. The shorter derivatives of some other peptides such as ACTH (Hofmann et al., 1962) or eledoisin (Sturmer et al., 1964; Schroder and Lubke, 1964) remain biologically effective, but only the longer analogs of bradykinin are active. The deca- and hendeca- and even the dodeca- and tridecapeptide kinins are quite potent (Schroder, 1965) while the shorter octapeptide (boguskinin) is less active by orders of magnitude.
A. STRUCTURE In the early purification attempts, good use was made of the solubility of kinins in organic solvents which precipitate contaminating proteins. Kallidin is soluble in trichloroacetic acid and in 96% ethanol (Frey et al., 1950). Bradykinin is soluble in glacial acetic acid and hot methyl alcohol (E. S. Prado et al., 1950). The first significant purification of bradykinin was achieved by Andrade and Rocha e Silva in 1956. They purified their material by using cellulose and ion exchange column chromatography. The best preparation contained 1 U per 200 ng (80 ng of pure bradykinin equals 1 U). Later Elliott et al. (1960a,b, 1961) used acid-treated ox plasma as a starting material. Bradykinin was liberated by trypsin from kininogen and purified on CM-cellulose columns with the help of volatile buffers. A magnesium contamination was removed with EDTA. In another purification process snake venom was the liberating enzyme (Zuber and Jaques, 1960). The amino acid sequence established by these workers (Elliott et al., 1960d; Zuber and Jaques, 1960) omitted Pro7 from the polypeptide structure. Three different groups of peptide chemists synthesized the suggested octapeptide, named “boguskinin” (Boissonnas et al., 1960a; Nicolaides et al., 1960; Schwyzer et al., 1960) which was inactive. Boissonnas and his colleagues (1960b), however, after considering the possibility of several sources of error, synthesized a number of peptides. One of them was a nonapeptide, with proline in the seventh position; it turned out to be the true bradykinin (Boissonnas, 1963). Shortly thereafter Elliott et al. independently revised (1960b) the structure of bradykinin suggested by them and included Pro7 in the peptide chain. Bradykinin obtained from human plasma protein (Hamberg, 1962a,b) or even from amphibian skin (Anastasi et al., 1965) has the same structure as the bovine variety. Kallidin was purified (see Fig. 5) by Pierce and Webster (1961) and almost simultaneously by Werle et al. (1961). Pierce and Webster used acidified human plasma and urinary kallikrein as starting material ; Werle
23
HYPOTENSIVE PEPTIDES 12,
I
I
I
,
I
I
I
I
,
400
I
A
1
300
!
-j200
j
4100 1
I 6 G O
:
g
2 '5 C
0
1 0 20 30 40 50 60 70 80 90 Effluent volume (ml)
FIG.5. Purification of kallidin from outdated human plasma. The peptide was released by human urinary kallikrein. A. Sephadex G-25 chromatography a t 4" of the pH 8.2 eluate from XE-64. Sample: 200 mg from 4 liters of plasma, in 2 ml water. Column: 1.7 X 72 cm. Eluent: 0.002 M HCl. Fraction volume: 1 ml. B. CM-cellulose chromatography at 4" of the product from second Sephadex chromatogram. Sample: 26 mg from 20 liters of plasma, in 0.5 mlO.01 M HCOONH4, p H 5.0. Column: 0.67 x 23 em. Eluents: 80 ml 0.01 M-0.5 M HCOONH, and from pH 5.0-7.5. Volume of mixing chamber: 125 ml. Fraction volume: 2 ml (Pierce and Webster, 1961).
et al. released kallidin from bovine plasina with subinaxillary kallikrein. The former authors obtained a significant aiiiouiit of bradykinin in addition to kallidin, but this niight have been due l o the acidification of plasma. Werle et al. (1961) found only 10% bradykinin after purifying kallidin. The purification procedure of Pierce and Webster consisted of the sequential use of adsorption on Amberlite XE-64 ion exchange resin, Sephadex filtration, and CM-cellulose chroniatography (see Fig. 5). Very shortly after the announceinent of the structure two laboratories synthesized kallidin (Nicolaides et al., 1961; Pless et al., 1962). This peptide is different from bradykinin by having ail additional N-terminal lysine (Fig. 1). Acid treatment of plasma activates endogenous enzymes which release active peptides. One of them was found to be identical with bradykinin (Haberinann and Okon, 1961; Hamberg, 196213); the other is niet-lysbradykinin, a hendecapeptide (Elliott et al., 1963; Schroder, 1964a; Merrifield, 1964; Elliott and Lewis, 1965). The reason for obtaining two different
24
ERVIN G. ERDOS
peptides may have been due to the difference in the methods of treating the substrates. Haberniaiin and Okon (1961) acidified the substrate only for 10 minutes, while Elliott and Lewis (1965) dialyzed the ammonium sulfate precipitate of plasma for 3 days against 0.01 N HCl. The activity of met-lys-bradykinin, in some systems approaches, in some others surpasses, bradyltinin (see Table 11).
Analogs Studies with the synthetic derivatives of kinins yielded information about structure-activit,y relationship, but the lack of knowledge about the receptor sites makes the interpretation of the experimental data difficult. Aniong the nuniber of kallidin and bradykinin analogs available, only a few are equally or more active than the parent compound. A fluro-Phe8 derivative is more active than bradykinin; however, substitution of Pheg of kallidin with fluorophenylalaiiine did not yield a more potent agent (Nicolaides et al., 1963b,c). When Ser6was replaced with Gly6 (Bodanszky et al., 1963a)b; Schroder and Hempel, 1964), the hypotensive effect of the peptide actually increased in several species (Rubin et al., 1963; Erdos et al., 1963a). A cautious interpretation of these data suggests the alcoholic OH of serine to be of no great importance (Bodanszky et al., 1963b), although replacement of L-serine with D-serine decreases the activity (De Wald et al., 1963). D-Arginine in the first or ninth position virtually abolished the hypotensive effect of bradykiriin (Nicolaides et al., 1965). Substituting the N-(Nicolaides et al., 1963a) or C-terminal arginine (Wunsch et ul., 1964; Schroder and Hempel, 1964) with other basic amino acids such as lysine, citrulline (Bodanszky et al., 196311; Ondetti, 1963; Schroder, 19640), or ornithirie (Schroder arid Hempel, 1964; Schroder et al., 1964) leads to a big loss in the effect of the bradykinin, but Argl-kallidin remained active (Schroder and Henipel, 1964). Among the other kallidin derivatives the biological effects of Glyl-kallidin, or Phel-kallidin are similar to those of kallidiri (Schroder arid Hempel, 1964). Removal of either N- or C-terminal arginine or shortening the peptide chain of bradykiiiin renders the peptide virtually inactive (Nicolaides et al., 1963~). The structure-activity relationship was studied also by the technique of systematically replacing the amino acid links in bradykinin with alanine. Only one of these derivatives, Ala3,was fully active (Fig. 6). Ala6 had 1/10 of the original activity arid the other analogs were even less potent (Schroder and Hempel, 1964; Schroder, 196413). Reversing the sequence of bradykinin (Vogler et al., 1962; Bodanszky et al., 1963b; Lande, 1962) yields inactive “retrobradykinin.” Another bradykinin where Args does not have a free carboxyl group, bradykinin-
HYPOTENSIVE PEPTIDES
25
amide, is also inactive (K. Vogler, unpublished observations, 1963; Erdos et al., 1963a; Pieri and Marrazzi, 1964). As nientioned above, shortening the peptide by deleting Pro7 results in the loss of biological activity (see Section IV,A).
FIG.6. Biological activities of the alanine analogs of bradykinin. Effect of bradykinin rabbit blood pressure and on isolated smooth muscles = 1. (Schroder and Hempel, 1964). 011
The various biological effects of kinins do not always change in a parallel manner in their derivatives; for example, the replacement of amino acids in some analogs of bradykinin decreases the bronchoconstrictor effect much more than the hypotensive activity of the pcptidcs (Nicolaides et al., 1963~). The fact that Lysg-braclykinin is much less active in most species than the parcnt compound (Schroder, 1964c) hut is more hypotensive than bradykinin in the guinea pig (Erdos and Cano unpublished observation, 1965) illustrates the difficulties encountered in the determination of the potency of peptide analogs. A variety of kinin derivatives can dilate the coronary artery of the isolated guinea pig heart. Their potency in other assay systems can vary by orders of magnitude, but maxinium difference here was about threefold. For example, Alal-bradykinin retained 34% of the activity of bradykinin on the coronary arteries, but in geiieral its biological effect was 1/1000 of that of bradykinin. Schroder and Henipel (1964) after surveying studies done in their laboratory and elsewhere concluded that Pro7 and Phe* are the most essential amino acids in the structure of bradykinin. Pro3and Sere
26
ERVIN G. ERDOS
can be replaced with a partial loss or even with no loss in activity. Substitution of Argl or Argg leads to a complete or incomplete loss of the biological effects of the peptide.
B. PHYSIOLOGY AND PHARMACOLOGY Although kiniris have a variety of actions in vivo and in vitro (Erdos, 196313; Habermann, 1963c) none of these properties is unique but is shared with some other pharmacologically active agent. The distinction of bradykinin rests upon the abundant presence of its precursor in blood plasma (and possibly in extracellular fluid) and upon the high releasing enzyme content of blood and tissues. In addition, bradykinin is active in some assay systems a t a concentration lower than other substances which occur in the body. For example, in contracting the isolated rabbit or guinea pig intestine or in increasing capillary permeability (Carr and Wilhelm, 1964) the peptide is much more effective than histamine (Trautschold and Rudel, 1963). The fact that large amounts of the peptides are available in the body contributes to the importance of kinins. Potentially mammalian plasma contains 2 to 14 pg/ml bradykinh (Margolis and Bishop, 1963; Diniz and Carvalho, 1963). This means that a healthy man might have 4 to 11 mg bradykinin in inactive form in every liter of circulating blood plasma. 1. Smooth Muscles and Assay Kinins were first characterized by using crude kallidin and crude bradykinin preparations as sources of peptides. The value of these observations can be appreciated even more if we consider that 0.2-0.5 U (0.2-0.5 mg) of crude bradykinin was used (Prado et al., 1950) to contract the isolated guinea pig ileum. Since 1 U is equivalent to 80 ng of synthetic bradykinin (Collier et al., 1960) the crude pool of bradykinin must have contained over 99.9% impurities, thus less active material than a laboratory reagent would have as contamination. Kinins are mainly hypotensive; they contract isolated smooth muscles but relax rat duodenum or hen’s rectal caecum. They increase capillary permeability and cause pain (Schachter, 1964). Kinins are frequently assayed on isolated smooth muscle preparations. The rat uterus in estrus is the most sensitive organ. Bradykinin is a t least 10 times less active on the isolated guinea pig ileum (see Table 11).Bradykinin causes the characteristically slow contraction of the guinea pig intestine, which inspired its name. This stimulation of smooth muscles is not blocked by specific compounds which would eliminate the effect of acetylcholine, histamine, or serotonin. Bradykinin relaxes the isolated
HYPOTENSWE PEPTIDES
27
rahhit intestine lwiefly; this is followed by contraction. The relaxation is not caused by release of catecholaniiiics (Turker et nl., 1964). Isolated rat duodenuni or hen’s rectal caecum are also relaxed by bradykinin. Bradykinin inhibited in situ intestinal motility in dog (Levy, 1963) ant1 cat (Turker ef nl., 1964). Diphcnhydraniine arid atropine blocked this effect in the (log as did pretreatinent with reserpine in the cat. Although bradykiniii is oxytocic on a number of isolated uterus preparations, it has only a slight effect 011 tlie uterus in situ. In wonien no oxytocic effect was seen after the intravenous administrat ion of bradykinin (Berde and Saameli, 1961). The peptide also failed to increase the pressure in the lactating mammary gland of the rabbit (Berde arid Cerletti, 1961) or to contract the uterus of this animal in si2u in reasonably low coricentration (Fregnan and Glasser, 1964). Bradykinin, however, blocks the spontaneous or induced corit raetions of the isolated human myonic%rial strip (Landesman et al., 1963). Intraarterial injections into the perfused hind leg of the dog is also a very sensitive method for assaying the peptides (Fasciolo et al., 1958; Nicolaides et nl., 1963c; Bergamaschi and Glasser, 1964; McCarthy et al., 1965). The ratio of activities among bradykinin, kalliclin, and met-lys-bradykinin varies. It would be tempting to look for similarities between kinins and angiotensins, since both peptides are liberated by enzymes from plasma globuliiis. The longer angiotensin analog, arigiotensin I, has to be converted I o angiotensin I1 before becoming active. Kallidin and met-lys-bradykinin, on the othcr hand, have definite actions of their own. Kallidiri in some in vitro bioassay can be even more active than bradykinin. The possibility of the in. t1iz)o conversion of some of the available kallidin to bradykinin, however, exists in some animals (Webster and Pierce, 1963; Erdoa et al., 19631)). The use of synthetic bradykinin and kallidin instead of the crude material was not the answer to all the problems. Dilute solutions of the peptitles are quite unstable. This instability is caused at least in part by losing the active material by adsorption on glass. Various techniques are recommended for avoiding this apparent decrease of activity. Using acidwashed glassware which has not been exposed to synthetic detergents (Gladner et al., 1963), 0.25% casein solution as solvent (Webster and Gilniore, 1965), or small amount of p-toluenesulfonic acid (Greenbauni et al., 1965) could be helpful in preventing the loss of material. Commercial preparations of bradykinin may contain chlorobutanol as a preservative (Collier, 1965). This solvent can interfere with the bioassay of the peptide in systems such as the isolated hen rectal caecum (Hamberg, 1964).
COMPARISON Test In vitro Rat uterus Guinea pig ileum Rabbit large intestine Rabbit duodenum Rabbit uterus Hen rectal caecum Rat duodenum In vivo; i n situ Guinea pig bronchial muscle Cat uterus Rabbit uterus
Bradykinin
OF THE
TABLE I1 RELATIVE ACTIVITIES OF KININSAND
Kallidin
Met-lys-bk
ELEDOISINn
Eledoisin
Threshold dose of bradykinin
Referenceb
1 ; contraction 0 . 6 ; contraction 0.3; contraction
0.03 ng/ml
1,2,3,5,9,13
1 ; contraction 1; contraction
1 ng/ml -lOOng/ml 1n g / d - 0 . 2 ng/ml
1 , 2 , S,5,8,9,1S 9 1, 3 , 9 2, 9 2, s, 9 2,3,6,7,13
1 ; contraction 1; eontraction
1 ; relaxation 1; relaxation
0.001-0.002; cont.raction 0.3; contraction 0.1-0.3; contraction 5 ; contraction 13; contraction 2; contraction contraction 200; contraction 2; relaxation contraction 0.5; relaxation 0.25; relaxation contraction
0 . 1 ng/ml
B
4
? 1; contraction 0.3; contraction 0.25; contraction
10; contraction
1 ; contraction 1 ; contraction
15; contraction 200; contraction (irregular) 50-100; decrease 5-10; increase
Dog blood pressure (iv) 1; decrease Dog hind leg blood 1 ; increase flow (ia) Dog blood pressure 1; decrease (unrestrained; iv) Cat blood pressure (iv) 1; decrease Diuresis rat 1; antidiuretic Rabbit blood pressure (iv) 1; decrease Rat, blood pressure (iv) 1 ; decrease
decrease >1; increase
(Decrease) (Increase)
1 ; decrease
l(r20; decrease
0.6; decrease 2.8; antidiuretic 1.9; decrease 2-3; decrease 3.3; decrease 1; decrease
8; decrease 4-50; decrease 10; decrease
0 . 2 Mdkg
1, 2 , 4 , 6 , 1 s
M
u 2 2
0 . 2 pg/kg 1 , 2, 7, 10 0 . 0 1 ng/kg 4, 7 , 1 2 11
0 . 5 pg/kg
1, 2, 3, 10
s
0.05 pg/kg 1 , 2 , 3 , 6 , 1 0 , 1 3 0.2-1.0 1 , 2 , s , 1s pdkg
0: u,
Guinea pig blood pressure (iv) Chicken blood pressure (iv)
Guinea pig capillary permeability (id)
1 ; decrease 1; initial decrease then increase 1 ; increase
0 . 7 ; decrease
1 ; increase
1 ; increase
0.2r g / k
1 , 2,
initial decrease then increase
40/80 @g/kg
1, 2, 10
1 ; increase
1 ng
1,2,3,6,13
Bradykinin = 1.
* References:
1. Konzett and Sturmer (1960a,b)
8. Stiirmer and Berde (1963a,b) S. Sturmer and Berde (1963~)
4. Nicolaides et al. (1963~) 6. Schroder and Hempel (1964) 6. Elliott et al. (1963)
4,
10; decrease
7. Bergamaschi and Gldsser (1963, 1964) 8. Habermann and Blennemann (1964b) 9. Erspamer and Erspamer (1962) 10. Erspamer and Glaesser (1963) 11. Olmsted and Page (1962) 12. McCarthy et al. (1965) 13. Elliott and Lewis (1965)
10
30
ERVIN G . ERDOS
2. Tachyphylaxis The development of tachyphylaxis to kinins depends on the bioassay employed and on the rate of administration of the peptides. Kinins can be added to a bath containing isolated guinea pig ileum or rat uterus every few minutes without any decrease in the sensitivity of the test organ. In experiments measuring the blood pressure of dogs, kinins can be injected every 5 or 10 minutes, while in rabbits (Konzett and Sturmer, 1960b; Erdos et al., 1963b) or in rats (Parratt, 1964a) it' is inore advisable to keep the injections 15 minutes apart. Bradykinin is not tachyphylactic in causing visceral pain in dogs (Guzman et al., 1964)) but is strongly tachyphylactic on the human blister base even when applied at, l-hour intervals (Horton, 1963). Some other actions of kinins, especially on the respiration, are also followed by diminishing response upon repeated administration. Tachyphylaxis was observed during the administration of bradykinin and kallidin in the pulmonary artery of the rabbit (Hauge et al., 1964) or in the perfused dog lung (Waaler, 1961). Another assay system also dealing with the lung registers the bronchoconstrictor effect of kinins in guinea pigs. This preparation also beconies refractory to bradykinin (Collier et al., 1960; Bisset and Lewis, 1962; Collier, 1965). Intracarotid injection of kinins stiniulates respiration in guinea pig and rabbit. This effect cannot be repeated within an hour (Gjuris et al., 1964b). Asthmatic patients who were usually very sensitive to bradykinin became resistant upon repeated administration (Stresemann, 1963). Tachyphylaxis was also reported in man during the intravenous infusion of bradykinin when some effects on the circulation were measured (Feruglio et al., 1963). Bradykinin stimulates the superior cervical sympathetic ganglion of the cat and close arterial injection causes vasodilation in the submaxillary gland. Tachyphylaxis was observed in these preparations as well (Lewis and Reit, 1965; Bhoola et al., 1965). Successive injections of sheep or human urinary kallikrein resulted in diminished hypotensive response in the dog. Such desensitization in dogs blocked the response to plasmin as well (Back et al., 1963b). A seemingly tachyphylactic effect to kallikrein was observed by Werle (1955). After the infusion of sufficient amount of kallikrein to exhaust the kallidinogen level in blood, the dog became resistant to both kallidin and kallikrein. 3. Central Nervous System
The effects of bradykinin on the central nervous system have not been definitely established; the interpretations of the experimental data are somewhat controversial. Large doses of crude bradykinin (1mg/gm) injected subcutaneously to
HYPOTENSIVE PEPTIDES
31
mice did not influence experimentally induced catatonia or tremor (Zetler, 1956). Intraveiitricular administration of crude bradykinin via the Feldberg-Sherwood cannula caused tranquilization in cats (Rocha e Silva, 1960; Rocha e Silva et al., 1960). Capek (1963) injected 50 mg of crude bradykinin intraventricularly to cats. The aniiiial responded with excitation followed by depression. Others (Lewis, 1963a; Norton, 1963; Heath, 1963) had only negative results to report with pure or synthetic bradykinin. Rocha e Silva (1963a) attributed the differences in results to the rapid destruction of the peptide. According to him more consistent effects were observed when the animals were pretreated with BAL (2,3-diniercaptopropanol) or cysteine. Relatively very little radioactive material entered the brain, however, during the intravenous infusion of labeled bradykinin to rats (Bunipus et al., 1964). I n cross-circulation experiments (Buckley et al., 1963) dogs showed either direct or indirect stimulation of the central parasympathetic system by bradykinin. Intracarotid injection of bradykinin affects the respiration (see Section IV,8). It has been indicated that this might be a consequeiice of the cerebral vasodilator activity of the peptide (Rocha e Silva, 1963a). No such interrclatioiiship was seen when the synaptic inhibition of the transcallosal pathways by bradykiniii were studied (Pieri and Marazzi, 1964). No correlation was found either between the iieurogenic and musculotropic activities of various bradykinin analogs (Krivoy et al., 1963). 4. Permeability
It has been known since the last century that breakdown products of tissues can increase capillary permeability (Miles and Wilhelni, 1960); thus, capillaries which under nornial conditions are not permeable to larger molecules such as proteins can be affected to let the material pass through the walls of these vessels into surrounding tissues. Many peptides with unrelated structure can cause these changes (Feldberg, 1956; Wilhelin, 1962). Leukotaxine (Menkin, 1956), products of peptic, tryptic, and papain digestion of proteins (Duthie and Chain, 1939), and soiiie basic peptides (Frimmer, 1964) can be mentioned here. Kallikrein was shown a long time ago to be an in1portant factor in increasing capillary pernieability (Christensen, 1939; Rocha e Silva, 1940a) ; bradykinin and kallidin are among the most potent agents in this respcct (Schachter, 1963a). For example, bradykinin is 15 times more active than histamine when compared on a molar basis (Elliott el al., 1960~). The methods of measuring capillary permeability changes in the skin include either the use of a vital dye (Miles and Miles, 1952),labeled colloidal
32
ERVIN G. ERDOS
gold (Frinimer, 196l), or P31-labeled albumin (Ascheim et al., 1963). The leakage of vital dye in the peritoneal cavity of the mice can also be followed (Northover, 1963). In addition to direct lesions which can be caused by the injection of kallikrein or kinins, various processes and interrelated factors in blood, for example, dilution (Schachter, 1960), permeability factor (Miles, 1964), or Hageman factor (Margolis, 1963), may act indirectly by activating kallikrein and releasing kinins (Sections II,D and E). Although kinins can increase capillary permeability, they cannot reproduce all the phases of inflammation. Their action may resemble the first phase of phlogistic process, but neither kinins nor histaniine can be made responsible for the second, delayed increase in pernieability (Miles, 1964). Bradykinin acts on the capillary wall after topical administration (Witte et al., 196l), but kinins are relatively ineffective in promoting migration of leucocytes (Spector and Willoughby, 1964). They induce this phenomenon only in relatively high concentration (1-100 pg/ml) (Lewis, 1961, 1962). The claim that topical application of bradykinin causes the sticking of leucocytes in the venules of the mesentery (Lewis, 1962) has not been confirmed (Zweifach, 1964). Sticking of leucocytes has been observed, however, in the rabbit ear chamber (Graham et al., 1965). Bradykinin was more active than histanline in promoting phagocytosis by leucocytes in vitro (Ludany et al., 1964). In another type of experiment, bradykinin did not raise t,he permeability of the brain stein of guinea pigs (Berkinshaw-Smith et al., 1962). Among the enzymes which are either directly or indirectly involved in the release of kinins, plasinin is at least 1000 times (Bhoola et al., 1960) and trypsin 300 times less effective (Frimmer, 1961) than kallikrein in permeability tests. Experiments showing a relatively low activity of kallikrein (Miles and Wilhelm, 1960; Wilhelm, 1962; Rocha e Silva, 1964) were obtained with commercial, and probably very crude preparations. The species difference also may have contributed to the impression of low activity. Kallikrein is a potent permeability factor. I n addition to the skin, it increases the capillary permeability in the splanchnic circulation in larger doses (Frey et al., 1950). Of the other kininogenases, the secretion of guinea pig coagulating gland (Freund et al., 1958) increases the capillary pernieability in the guinea pig skin (Bhoola et al., 1962a). Intradermal injection of bradykinin in man causes a wheal and a shortlasting flare (A. Herxheimer and Schachter, 1959; Schachter, 1963a; Witte et al., 1961; Mitchell and Krell, 1964). In animal experiments the lesion caused by bradykinin or kallidin can be distinguished readily from that caused by histamine. The delay in the appearance of the circulating dye a t the site of the injection is shorter after the administration of kinin, the form of the lesion and the dose-response curve is different from that of
HYPOTENSIVE PEPTIDES
33
hist anline. Preliminary electron niicroscopic observations showed that bradykinin causes a marked discontinuity in the endothelium in the capillaries of the guinea pig skin (Schachter, 1963a). Another iiiechanisni whereby bradykinin can increase the capillary permeability in the rat paw was suggested by Rowley (1964). According to his experiment A, bradykiniri increases the pressure in the proximal venules which leads to edema forination. Bradykinin and kallidin are about equally potent in increasing capillary permeability, but another kinin, wasp venom kinin, might be even more active (Schachter, 1963b). Two bradykiniri derivatives, the hexa- and the octapeptides, were 30 to 100 times less effective than the parent compound (Burckhardt, 1962). Edema forniation in the rat paw, which follows the adiiiinistration of bradykinin, can be blocked by some antiphlogistic agents (Lisin and Leclercq, 1963), but this type of drug was ineffective in the rabbit skin (Friinnier and Iirych, 1963). A iiuniher of other agents also gave negative results against bradykiiiin in the rat skin (Bonaccorsi et al., 1963). Soybean trypsin inhibitor (Bhoola et al., 1960) and salicylates (Spector and Willoughby, 1962b, 1963) can block the effect of kallikrein, although they do not influence the action of bradykinin (Collier and Shorley, 1960). I n the guinea pig, however, pretreatment with carboxypeptidase B reduces the effect of intradernial bradykinin (Erdos et al., 1963a). Intravenous injection of plasniin or urokinase to guinea pigs yielded siiiiilar results (Copley and Tsuluca, 1962, 1963b). 5. Pain Bradykinin is ariiong the most potent pain-producing agents. It is active in low concentration, although this property is shared with substances such as substance P, acetylcholine, histamine, or serotonin. Since pain is a subjective event, it is difficult to measure it, except in human experiments. Electrophysiological studies do not provide independent objective data which substitute for subjective estiniation of pain, according to Keele and Ariiistrong (1964). Kinins may be involved in causing pain in several ways. Bradykinin or a siniilar peptide is released during painful stiniulation; kiniiis occur in certain animal venoms (see Section IV,E,3). In addition, under painful condit ions tissues can be inore sensitive to bradykinin, and, finally, bradykinin has been shown to act on nervous elements which are supposed to be pain receptors in the viscera and elsewhere. Arnistrong et al. (1953, 1954) have shown that blister fluid, plasma stored in glass, synovial fluid from the knee joint of arthritic patients, and ascites fluid contain a pain-producing factor, which is probably identical
34
ERVIN Q. ERDOS
with bradykinin (1957). A similar substance was found in pleural fluid a s well (Galletti et al., 1960). According to Ostfeld et al. (1957), blister fluid contributes to headache when injected subcutaneously into the scalp. Most inflammatory exudates develop kinin readily only when they come in contact with glass. Possibly these fluids do not contain active kinins, but a kinin is released from kininogen when glass activates a kininogenase in the system (Keele and Armstrong, 1964). On the other hand, a material, which was probably active kinin, was found in the peritoneal cavity of mice during a “squirming” response (Whittle, 1964). Hot water treatment of the skin can also release a bradykinin-like substance subcutaneously. When anesthesia was suggested to patients in hypnosis, the peptide release was blocked (Chapman et al., 1959) (this certainly would be the first instance when hypnosis or another type of persuasion would hold back a kinin release). After subcutaneous perfusion of the forearm in the zone of the axon reflex caused by histamine, the effluent contains a kinin (Chapman et al., 1959). The kinin released from the zone of the axon reflex was named neurokinin. Bioassay and behavior toward various blocking agents distinguished it from bradykinin, according to Ramos et al. (1963a,b,c), but some other investigators disagree (Keele and Armstrong, 1964). The release of kinin in the forearm was not attributed by Chapnian to the function of sweat glands (R. H. Fox and Hilton, 1958), or to permeability changes (Hilton, 1963; Chapman and Goodell, 1964). The stimulation of the distal portion of the transsected dorsal route also releases kinin in man (Chapman et al., 1961). Application of bradykinin on a blister base in human skin (0.1 pg/rnl) (Elliott et al., 1960c; Keele and Armstrong, 1964) or intraarterial injection of bradykinin causes pain (R. H. Fox et al., 1961; Burch and De Pasquale, 1962). Intracarotid injection of the peptide increases migraine pain in patients (Sicuteri et al., 196313). Ischemia, in general, greatly enhances the pain in humans following the injection of bradykinin in the artery (Sicuteri et al., 1964b). Intradermal injection of bradykinin also causes burning pain in humans (Harpman and Allen, 1959; Cormia and Dougherty, 1960; Witte et al., 1961; Mitchell and Krell, 1964). Kallikrein causes severe itching when administered intradermally (Cormia and Dougherty, 1960). Kallidin is a somewhat less active pain-producing agent than bradykinin in man. The peptides were compared by injecting them in the dorsal vein of the hand following sensitization with serotonin (Sicuteri et al., 1965). No evidence for tachyphylaxis was seen in these latter experiments. Bradykinin also causes conditions which are considered to be painful to various animals. In dogs, Lim and his associates (Guzman et al., 1962, 1964; Lim et al., 1964) measured the vocalization which follows the intraarterial
HYPOTENSIVE PEPTIDES
35
injection of bradykinin. The peptide is assumed to act on the paravascular nerve fibers which are unniyelinated. Various narcotic and non-narcotic analgesics and aniphetainine block this action in dogs (Lim et al., 1964) and similar painful conditions in mice (Emele and Shananiaii, 1963) and guinea pigs (Collier and Lee, 1963). In the dog, non-narcotic analgesics block the response to bradykinin peripherally, while narcotic analgesics and amphetamine block the visceral pain-producing effect of the peptide centrally (Lini et al., 1964). The visceral pain caused by bradykinin is short lasting arid does not lead to tachyphylaxis (Guznian et al., 1964), while application of bradykinin to the exposed skin is strongly tachyphylactic (Elliott et al., 1960c; Horton, 1963). 6. Cardiovascular E j e c t s The effects of synthetic kinins on the circulation resemble the circulatory changes which follow the administration of kallikrein. Intravenous injection of synthetic peptides, however, cannot produce all the signs induced by kallikrein. Kallikreiri can have a longer lasting effect on the circulation than do kinins (e.g., kallidin) (Werle and Grunz, 1939), apparently because a portion of the injected kinins is rapidly inactivated in blood before reaching the site of action (Kroneberg and Stoepel, 1963). Injection of kallikrein lowers thc systemic blood pressure of man aiid animal, increases the heart rate, and causes vasodilatation (Frey et al., 1950). Various vascular beds react differently to kallikrein. It dilates the coronary arteries (Fig. 7) (Hochrein and Keller, 1931; Krayer aiid Ruhl,
Time (min)
FIG.7. Kallikrein increases the coronary blood flow in the dog heart-lung preparation in four experiments. KU = units of kallikrein. (Krayer and Ruhl, 1931).
36
ERVIN G. ERDOS
1931; Elliot and Nuzum, 1931; Felix, 1934), increases blood flow of the skin (Werle and Multhaupt, 1937), muscles (Zipf and Giese, 1933), and brain (Schneider and Springoruni, 1939). The observation that kallikrein lowers the peripheral resistance in ariinials led to its therapeutic application. Kallikrein, however, increases the pulnioriary arterial pressures of cat and dog (Frey and Kraut, 1928; Krayer and Ruhl, 1931; Frey et al., 1950). Injection of trypsin also lowers the blood pressure; this effect is due in part t o activation of kallikrein in the blood plasma (Werle et al., 1955). Most vascular areas react to the intravenous or intraarterial administration of kinins with vasodilatation which is not blocked by atropine or antihistamines. In ii1animals, kinins transiently lower both the systolic and diastolic components of the systeniic arterial pressures. The rabbit is most sensitive to intravenous injection of bradykinin; about 0.05 pg/kg is the threshold dose (Konzett and Sturiner, 1960b). Other animals also react to the administration of peptides with transient hypotension (Table 11). Pretreatment with an iiihibitor of the enzymic metabolism of kinins modifies the degree and the duration of hypotensive response considerably (Erdos and Wohler, 1963a,b). Unanesthetized dogs seem to be less sensitive to the injection of kinins than anesthetized animals (Olmsted and Page, 1962; Kroiieberg and Stoepel, 1963). Bradykinin causes marked dilatations of various vascular trees and lowers the total and regional peripheral resistance (Page and Olmsted, 1961; Rowe et al., 1963; Gersnieyer and Spitzbarth, 1961; Concioli el al., 1961; Carpi and Corrado, 1961). Among the most sensitive blood vessels which react with vasodilatation to bradykinin are the coronary arteries of the isolated guinea pig heart (Antonio and Rocha e Silva, 1962), the superior mesenteric arteries of dog (Chou et al., 1965), and the blood vessels in the hind limb of the dog. The threshold dose of bradykinin injected into the fenioral artery is about 0.01 ng/kg (Bergamaschi and Gliisser, 1964). In the superior mesenteric circulation, locally infused bradykinin was about 50 times more potent than histamine or acetylcholine. With kallidin the ratio was about 1 :36 (Chou et al., 1965). Some blood vessels, however, have been described to react to bradykinin with vasoconstriction; for example, veins in the rabbit ear (Guth et QZ., 1963), or in the rat paw (Rowley, 1964). Burch and De Pasquale (1962) in their investigations with digital rheoplethysniograph suggested a selective constriction of A-V anastomoses in man, but this interpretation of their results was questioned by some (Paldino et al., 1962; Kontos et al., 1964a). Bradykinin contracts the spiral strips of isolated sheep coronary artery (Kovalcik, 1962) or ox carotid artery (Kobold and Thal, 1963).
HYPOTENSIVE PEPTIDES
37
I n experimental aninials bradykinin or kallidin adniinistered in various ways increases nionientarily the cardiac output, heart rate (Page and Olnisted, 1961; Olnistcd and Page, 1962), coronary blood flow (Maxwell et al., 1962; Rowe et al., 1963; Berganiaschi and Glasser, 1963), oxygen consumption (Afonso et al., 1962), and systemic venous return (Nakano, 1965b). Bradykinin and kallidin given intravenously increase pulmonary arterial pressure of man and animal (Carlier, 1963; Gersnieyer and Spitzbarth, 1961; Klupp and Konzett, 1963, 1965; J. M. Bishop et al., 1963; Hauge et al., 1964). Pulmonary vessels of various aninials are constricted by bradykinin and kallidin (Leconite arid Troquet, 1960; Moog aiid Fischer, 1964; Greeff and Moog, 1964; Klupp and Konzett, 1965). The ratio of activities of bradykinin and kallidin on the circulation varies from species to species (see Table 11). For example, kallidin is iiiore active than bradykiniii in the dog (Webster and Pierce, 1963), rat, and rabbit (Sturnier and Berde, 1963c), arid less active in the cat (Sturiiier and Berde, 1963~)and in the guinea pig (Nicolaides et al., 1 9 6 3 ~ )Met-lys. bradykiriin is twice as active as bradykiniri on the blood pressure of the rabbit (Schroder, 1964a). Lys-lys-bradykinin and lys-lys-lys-bradykinin were described to be 8 to 10 tiines more hypotensive in the rabbit than bradykinin (Schroder, 1965). In rats with a systemic blood pressure of about 60 niiii Hg or less (nephrectoniy, pentolinium treatnient) bradykinin raises the blood pressure. This rise is caused probably by the release of epiriephriiie (Croxatto and Belmar, 1961; Croxatto et a/., 1962; Parrat, 1964a). Large doses of bradykinin lower the blood pressure of the chicken but slightly; this is followed by a nioderate rise (Konzett and Sturiiier, 1960b). In reserpinized dogs the hypotensive effect of bradykinin was grealer, but basically the heinodynamic action of the peptide was the same as in nornial aninials. Catecholaniincs are probably released duriug hypot ensiori as a hoiiicostatic niechariisni (Rowe et al., 1963). It can be recalled here, however, that a vasopressor peptide, angiotensin, was about 50 to 100 tiriles more effective than bradykiiiin in releasing catecholamines froiii the suprarenal niedulla of the cat (Feldberg aiid Lewis, 1964). The liypotensive effect of bradykinin in the cat was increased by pretreatment with reserpine or synipatholytic drugs (Rocha e Silva et nl., 1960). Ilexamethoniuni had the same effect in rats (Lloyd, 1962). According to Roclia e Silva (1963b), potentiation of bradykiiiin action by drugs such as phenoxyberizamine or dichloroisoprotereno1 cannot be explained adequately a t present. The direct effect of kinins on the heart muscle has also been considered by sonic authors. Bradykiniii in the dog papillary muscle decreased the
38
ERVIN G . ERDOS
duration of Purkinje action potential (Vick et al., 1965)) but this activity can be attributed to the chlorobutanol solvent used. Bradykinin stimulated the metabolic heat production in the heart of laboratory animals, while kallidin did not (Parratt, 196413). A cardiac-stimulating action in the rat has also been suggested (Rosas et al., 1965). On the other hand, bradykinin in low concentrations did not affect the myocardial contractile force of isolated guinea pig atria (Nakano, 1965b), but a n increase was reported a t 0.1 pg/inl level by Heeg and Meng (1965). I n human subjects, bradykinin causes a short lasting drop in the systemic blood pressure; the primary target is the peripheral circulation (Broghamnier and Wernitsch, 1962, 1963). In some pathological conditions the peripheral vessels do not react with vasodilatation to bradykinin after intravenous injection or intraarterial infusion (Broghamnier and Wernitsch, 1962; Konzett et al., 1964). Intravenous or intraarterial infusion of bradykinin enhances the blood flow in the leg or arm. (Ehringer et al., 1961; R. H. Fox et ul., 1961; Javett and Coffman, 1962; Coffman and Javett, 1963; Kontos et al., 196413). Bradykinin infusion increases the cardiac output and heart rate of man (Kontos et ul., 1964a; De Freitas et al., 1964; J. M. Bishop et al., 1963) and decreases the splanchnic resistance (Feruglio et aZ., 1963, 1964). Siniilar to kallikrein (Frey et al., 1935; Reeke and Werle, 1935)) bradykinin causes intracranial vasodilatation and increases the pressure in the cerebrospinal fluid (Sicuteri et d,1962b, 1963e). The role of peptides in functional vasodilatation has been debated for decades. It has been shown that the stimulation of the chorda tympani releases a vasodepressor from the salivary gland (Feldberg and Guiniarais, 1935). The material in the saliva was found to be similar to the pancreatic depressor substance (Guiniarais, 1936), namely, to kallikrein (Werle and von Roden, 1936, 1939; Ungar and Parrot, 1936). The proposed mechanism of action stipulates that stimulation of the nerve releases kallikrein from the gland and causes vasodilatation. Lately functional vasodilatation in various secreting glandular cells was connected to the release of a kallikreinlike enzyme, which would liberate a kiiiin in the interstitial fluid (Hilton, 1962). This was also shown in the submandibular salivary gland by stimulating the chorda or by infusion of acet,ylcholine, epinephrine, or norepinephrine (Hilton and Lewis, 1956) (if the releasing enzyme originating from the glands is indeed identical with kallikrein, then the authors were dealing with kallidin in these experiments). Human sweat glands can also forin kinins; this would play a role in the periglandular vasodilatation. Active vasodilatation in human forearm skin which accompanies heating was attributed to bradykinin released by sweat gland activity (R. H. Fox and Hilton, 1958). Some of the observations of Chapman e l al. (1960), however,
HYPOTENSIVE PEPTIDES
39
argue against this interpretation. Muscular work or thermal vasodilatation did iiot increase the level of kinin in venous blood (Carretero et al., 1965). Glandular activity does not seem to be connected with kinin release in all species. According to Schachter (1964), his results are iiot consistent with the view that kallikrein iiiediates all functional vasodilatation in the salivary gland. For example, the guinea pig has some glands which do not release kiiiins from guinea pig plasma. Chorda-lingual stiniulation of the subiiiaxillary gland of cats, where the content in kallikrein has been depleted, still causes vasodilatation (Beilenson et al., 1965). 7. Kidney
Kinins can be diuretic or aiitidiuret,ic in laboratory animals. The effect is determined by the mode of administration of the peptides and by the selcctioii of the animal. The antidiuretic effect is probably due to the release of vasopressin, and the diuresis is caused by the increased blood flow in the kidney. In addition, bradykinin was diuretic in clinical subjects. It has been observed that intraperitoneal injections of the urinary peptide, substance 2 (urinary kinin), causes antidiuresis in rats (Werle and Erdiis, 1954; Gonies, 1959). Bradykinin is also antidiuretic in dog (iv) (Barac, 1957) or rat (Stiirmer and Berde, 1963~).Kallidin was more active in the latter experiments. This antidiuretic activity is not due to direct action on the kidney, but it is caused by the release of vasopressin froin the posterior pituitary gland by bradykinin (Rocha e Silva, Jr., and Malnic, 1964). The intravenous or intraarterial injection of bradykinin to cats (Barer, 1963) increases the renal blood flow. Both kallidin and bradykinin (iv) increase the renal voluiiie of cats (Stunner arid Rerde, 1963~).Intravenous injection of kallikrein to dogs (Szakhll, 1932) or infusion of lcallidin (1-3 pg/minute) directly into the renal artery enhances the flow of urine. The concentration of sodium and chloride increases in urine. The excretion of potassium and total solutes is moderately elevated after the administration of kalliclin (Webster and Gilniore, 1964). Infusion of bradykinin yielded similar results (Heidenreich el al., 1963; Barraclough and Mills, 1965). Intravenous infusion (0.5-1 pg/kg/niinute) of bradykinin to dogs had an antidiuretic effect in the experiments of Heidenreich et al. (1964), while, in agreement with the previous findings, infusion of bradykinin or kallidin in the renal artery caused diuresis (Heidenreich et al., 1964). Bradykinin infusion (i.v., 0.15-0.30 pg/kg/minute) in man (Mertz, 1963, 1964) increases the renal blood flow, lowers renal vascular resistance, and is diuretic. This effect was similar in healthy individuals and in patients suffering from kidney disease.
40
ERVIN G . ERDOS
8. Respiration Kinins affect the respiration of man and animal. In contrast, kallikrein only moderately influences the respiration in connection with the hypotension it causes (Frey and Kraut, 1928; Krayer and Ruhl, 1931; Frey et al., 1950; Gjuris et al., 196413). Intravenous injection of bradykinin causes tachypnea in guinea pigs which can be blocked by vagotomy. Larger doses of bradykinin (24 bg/kg) and kallidin (16 pg/kg) induce respiratory arrest in guinea pig and rabbit. This effect on the respiration seems to be of central origin because intracarotid administration of the peptide is more effective than intravenous injection (Gjuris et al., 1964a,b). The stiniulation of the respiration in the cat by intraarterial injection of bradykinin was not completely abolished by vagotomy or by destruction of the innervation of the carotid sinus (Rocha e Silva et al., 1960). Intravenous injection of Gly6 derivative of bradykinin stimulates the respiration of cats and guinea pigs more than the parent conipound (Erdos et al., 1963a). Bradykinin and kallidin administered in vitro or in vivo are strongly bronchoconstrictor in the guinea pig (Collier et al., 1959; Collier et al., 1960; Bhoola et al., 196213;Moog and Fischer, 1964; Greeff and Moog, 1965). This bronchoconstriction induces resistance to inflation in the lung which is not affected by cutting the vagus or destroying the spinal cord (Collier et al., 1960; Berry and Collier, 1964). X-ray studies showed that bradykinin acts on the respiratory bronchioli (Jankala and Virtama, 1963). Smooth muscle fibers in the pleura may also contribute to the effects (Gjuris and Westermann, 1965; Collier, 1965). Kallidin has about 1/3 of the activity of bradykinin on the guinea pig bronchus (Bhoola et al., 1962b). The dose of bradykinin here is above 0.5 fig/kg i.v. (Collier, 196313) (Fig. 8). Rabbits also react to the administration of bradykinin with bronchoconstriction. The smooth muscles of the brcnchi of other animals are less scnsitive to bradykinin (Bhoola et al., 1962b). Bradykinin is ineffective when given in aerosol to guinea pigs or healthy wen. Asthmatic patients, however, are very sensitive to bradykinin aerosol (Melon and Lecomte, 1962), especially after consuming hard liquor (Schnaps) (H. Herxheimer and Streseniann, 1961, 1963; Stresemann, 1963). Some of these patients reacted to the peptide with an exceptionally violent attack. 9. Interaction with Other Agents Kinins or kallikrein can act either synergistically with some other pharmacologically active agents, or they can antagonize them (Section I1,C). This will depend to a cert,ain extent on the bioassay employed for
HYPOTENSIVE ’ PEPTIDES
41
FIG.8. Resistance to inflation of guinea pig lungs in vivo. Action of SRS-A and bradykinin and their antagonism by acetylsalicylic acid after pithing the spinal cord and crushing the sympathetic cervical nerves and vagi. Guinea pig 700 gm;time (upper signal), 10 seconds; H, 2pg of histamine; S, 80 U of SRS-A; B, 1 and 4 pg of bradykinin; Asp, 2 mg/kg acetylsalicylic acid. Doses were given at 5-minute intervals. Ordiriate: Overflow volume. (Berry and Collier, 1964.)
the particular test. In addition, there are more complex relationships, whereby neurohorinones such as acetylcholine can induce the release of kinins or epinephrine niay activate proteases, which in turn libearte a kinin. Shortly after the discovery of kallikrein, it was found that injected kallikrein can abolish the pressor effect of epinephrine (Elliot and Nuzuni, 1931; Sivo and Dobozy, 1934). Later it was noted that kallikrein can also block the vasoconstriction caused by norepinephrine in the skin (Papenberg and Hensel, 1959). Epinephrine antagonizes the action of kallikrein on the isolated cat intestine, dog intestine, or cat uterus (Werle and Flosdorf, 1938). The antagonistic effect of kallikrein against vasoactive agents, including BaClz and histamine, has been studied on the perfused hind leg of the rabbit, on the Langendorff’s heart preparation, and on smooth niuscle preparations (Tripod and Meier, 1958). Epinephrine (Feldberg and Guimarais, 1935), as well as norepinephrine or acetylcholine (Hilton, 1962), can release a kininogenase from the salivary gland which is probably kallikrein (Section IV,B,6). In various pathological processes the release of epinephrine niay activate proteases, with the subsequent liberation of kinins (Rocha e Silva, 1963a). An interaction between bradykinin and epinephrine was observed by Oates et al. (1964) in carcinoid patients; during the flushes induced by injected epinephrine, the level of a kinin increased in blood. Di Mattei (1962) indicated that bradykinin is the mediator of a type of pulmonary edenia in the rabbit which can be caused experinlentally with epinephrine. On the other hand, injection of bradykinin leads to the release of epinephrine (Croxatto and Belmar, 1961; Lecomte et al., 1961). For example,
42
ERVIN G. ERDOS
injections of bradykinin or kallidin in the coeliac artery of the cat liberated epinephrine from the adrenal glands (Feldberg and Lewis, 1964). Bradykinin also releases catecholamines from the inferior mesenteric ganglion and adrenal medulla of dog (Muscholl and Vogt, 1964; Vogt, 1965). The peptides stimulated the sympathetic, superior cervical ganglion of the cat (Lewis and Reit, 1965). Bradykinin was less effective on the adrenal gland of cat than angiotensin. The release of catecholamines by peptides depends on the presence of calcium ions (Poisner and Douglas, 1965). Bradykinin, being a potent vasodepressor, can abolish the hypertensive effect of vasopressors such as angiotensin or vasopressin (Konzett, 1963). Bradykinin is synergistic with antiotensin on the guinea pig ileum or rat uterus, but has an opposite effect on rat duodenum (Rocha e Silva, 1963~). Local administration of dopa and dopamine reduces the action of bradykinin or kallikrein on the capillary permeability of the skin in rats (Willoughby and Spector, 1964). In the guinea pig, intravenously injected bradykinin partially blocks the effects of histamine on the capillaries (Copley and Tsuluca, 1963a). Intravenous injection of histamine or the histamine releaser 48/80 increases the kinin-forming activity in the lymph of dogs (Edery and Lewis, 1963). Pain-producing animal venoms, for example, hornet venom, contain a mixture of substances such as histamine, serotonin, acetylcholine, and a kinin, hornet kinin (Bhoola et al., 1961). This is an almost ideally effective combination of pain-producing agents (Keele and Arnistrong, 1964) (at least as far as the hornet is concerned). Experimental application of serotonin a,lsosensitizes man to the algogenic effects of bradykinin and kallidin (Sicuteri et al., 1965). 10. Blocking A gents In contrast to acetylcholine, histamine, or serotonin, no organic cornpounds of relatively low molecular weight have been found which would specifically block the action of kinins in a wide range of tests. Although some compounds block the effects of the peptides, they are not selective. They inhibit the activity of other agents a t the same concentration as well (Section IV,B,5). Some other blocking agents prevent the action of bradykinin in a concentration lower than that of other substances, but only in connection with bronchoconstriction in guinea pig (Collier, 1963a). Finally, pretreatment with carboxypeptidase B abolishes the in vivo action of bradykinin and kallidin, because this enzyme metabolizes the peptides very rapidly (Erdos, 1962; Erdos et al., 1963a,b). Collier and Shorley (1960) found that aspirin (2 mg/kg. i.v.) and other analgesic, antipyretic drugs blocked the bronchoconstriction caused by bradykinin (see Table 111). Aspirin was inactive against histamine at the
43
HYPOTENSIVE PEPTIDES
TABLE I11 AGENTSWHICHBLOCKTHE EFFECTS OF BRADYKININ Blocking agent
Effects
Aspirin, mefenamic acid, flufenamic acid
Guinea pig bronchoconstriction
Analgesic antipyretic drugs
Rabbit circulation Rat circulation Constriction of guinea pig pulmonary vessels
Anti-inflammatory substances Corticosteroids Plasmin, proteolytic enzymes Urine extract Phenothiazines Dicyclomine Cyproheptadine, imipramine, tremaril Pancreatic carboxypeptidase B
Respiration in guinea pigs Edema of rat paw Capillary permeability, human skin Guinea pig skin permeability Guinea pig ileum and skin Guinea pig ileum in vitro Guinea pig ileum in vitro Guinea pig ileum in vitro
Guinea pig skin permeability Blood pressure Leus-OAcThre-Leu8methyl Rat uterus in vilro ester analog of bradykinin Nociceptive responses in Codeine guinea pigs and mice Visceral pain, peripheral Non-narcotic analgesics block, dog; mice Visceral pain, central Narcotic analgesics, block; dog amphetamine
References Collier and Shorley (1963) Collier (196313) Berry and Collier (1964) Lecomte (1999) Lecomte and Troquet (1962) Klupp and Konzett (1963, 1965) Greeff and Moog (1964) Gjuris et al. (1964a,b) Lisin and Leclercq (1963) Frank et al. (1964) Copley and Tsuluca (1962, 1963a,b) KovBcs and Melville (1963) Mariani (1961) McGrath et al. (1964) Rocha e Silva and Leme (1963, 1965) Erdos et al. (1963a,b) Erdos and Yang (1965) Webster et al. (1965) Stewart and Woolley (1964)
Collier and Lee (1963) Emele and Shanaman (1963) Collier el al. (1964) Lim et al. (1964) Lim et al. (1964)
concentration used (see Fig. 8). Other non-narcotic analgesics were also effective, roughly in the same order as they protected the skin against erythema caused by UV-irradiation. Except for the isolated trachea of the guinea pig (Collier, 1963a), aspirin did not show this specific effect in other animals or in other organs (Bhoola et al., 1962b). It has been also postulated that bradykinin may act on several types of receptors, and that nonnarcotic analgesics and antipyretic drugs would coinpete with the peptide for one of them in the guinea pig (Collier and Shorley, 1963). In addition to bradykinin, aspirin antagonized the bronchoconstrictor effect of kallidin, wasp venom kinin (Bhoola et al., 1962b), bradykinin analogs (Collier,
44
ERVIN G. ERDOS
1963b), and SRS-A (a slow-reacting substance produced in anaphylaxis) (Fig. 8) (Berry and Collier, 1964). Among the newer anti-inflammatory agents mefenamic and flufenaniic acids acted similarly to aspirin. Phenylbutazone and, in large doses, sodium salicylate block some of the effects of the peptides on the circulation. The former compound prevents the drop in systemic blood pressure caused by bradykinin in the rabbit (Lecomte, 1959) and in the rat (Lecomte and Troquet, 1962). Both of these drugs act on the pial circulation of the guinea pig (Concioli et al., 1962). l'ret,reatnient with aspirin or phenylbutazone blocks the apnea or tachypnea caused by the injection of bradykinin or kallidin to guinea pigs (Gjuris et al., 1964a,b). Administration of plasmin, urokinase, or other proteolytic enzymes block the permeabilit
Several types of compounds can potentiate the effect of kinins in vivo or in vitro. The agents which act in vivo are inhibitors of the enzymic nietabolism of bradykinin, or are sympatholytic or ganglion-blocking compounds. (See Sections IV,B,6 and IV,C.) The mechanism of in vitro potentiation has not yet been explained satisfactorily. Cysteine (Lewis, 1960; Picarelli et al., 1962), BAL, or thioglycolic acid (Ferreira and Rocha e Silva, 1962) increase the size of contraction induced by bradykinin on the isolated guinea pig ileum. Since these compounds are among the inhibitors of the enzymic destruction of bradykinin (Ferreira and Rocha e Silva, 1962; Erdos and Wohler, 1963b), it has been suggested that the increase in the effect of kinins is due to blocking of the inactivation of the
HYPOTENSIVE PEPTIDES
45
peptide by the isolated tissues. However, bradykiiiiri is destroyed by the guinea pig ileum too slowly in the experiments of Picarelli et al. (1962) to explain the rapid action of a compound such as cysteine. Tetraethylaninioniuiii enhances the effect of bradykiniti on the isolated guinea pig ileuin (Khairallah and Page, 1961) and on the rat uterus (Capek and Knesslova, 1959). Low concentrations of guanethidine, dibenamine, and phenoxybenzamine acted similarly on the guinea pig ileum (Rocha c Silva and Leme, 1963). Thrombin liberates two peptides during the conversion of fibrinogen to fibrin. One of them, peptide B, greatly increases the effect of bradykinin on the isolated rat uterus (Gladner el al., 1963). The effect persists for a while even after washing the organ (Osbahr et al., 1964). It does not seem to depend on the integrity of the peptide chain or on the presence of the C-terminal arginine of peptide B. The individual amino acid components of the peptide, on the other hand, are inactive. The action of this naturally occurring peptide might be of possible physiological importance. Adding proteins such as trypsin, chymotrypsin, or chyniotrypsinogen to the isolated organ bath increased the sensitivity of guinea pig ileum or rat uterus to bradykinin (Edery, 1964, 1965). Venom of Bothrops jararaca contains a n alcohol-soluble factor which potentiates both the in vitro and the in vivo effects of bradykinin (Ferreira, 1965).
C. METABOLISM OF KININS Kinins are rapidly inactivated in blood plasma. This honieostatic mechanism is probably a major factor in determining the duration of their action, although not the only one the body has at its disposal. In some species the very rapid removal of the peptides from the circulation by the tissues might, be of importance. Compared to other pharmacologically active peptides, bradykinin and kallidin are inactivated fastest by nornial blood plasma. The observation of this inactivation was inade simultaneously with the discovery of kinins. Later, it was established that the liberation of kiriin and the inactivation are independent, processes frequently occurring in the same system in vitro. 1. Blood and Tissues
Werle found that after the release of an active principle (DK or kallidin) by kallikrein, blood, probably enzymically, inactivates this agent (see Werle et al., 1937; Werle and Grunz, 1939) (Fig. 3). Rocha e Silva (1951) had similar experience with bradykinin. Guinea pig serum was especially active in this respect (Werle et al., 1937; Werle and Hambuechen, 1943; Schachter, 1960; Klett, 1962).
46
ERVIN G . ERDOS
Plasma fractions IV-1 and IV-4, which contain the precursor of bradykinin (bradykininogen), also inactivate kinins (Van Arman, 1952, 1955; Erdos and Sloane, 1962). Plasma destroyed the urinary peptide, substance 2, as well (Werle and Erdos, 1954). Until bradykinin and kallidin became available in synthetic form, little was known about the mechanism of inactivation beyond a few isolated observations. For example, cysteine preserved the activity of both kallidin (Werle and Grunz, 1939) and bradykinin (Van Arman, 1955; Rocha e Silva, 1955). EDTA prolonged the existence of a pain-producing substance (bradykinin) in plasma (Armstrong et aZ., 1955). In 1961 Erdos characterized the enzyme in hunian plasma fraction IV-1 which inactivates bradykinin and probably kallidin. It is a carboxypeptidase which cleaves the C-terminal arginine of the peptides. The removal of the Argg of bradykinin was shown by the sequential use of paper chromatography and electrophoresis. The inactivation of bradykinin by the enzyme was inhibited by various chelating agents such as phenanthroline or EDTA (Table IV) and accelerated by cobalt. A number of inhibitors of the pancreatic carboxypeptidase B (Folk, 1956), for example, eamino-n-caproic acid, inhibit the plasma enzyme. Depending on the source of blood, there are some differences in the inhibition pattern of the enzyme. Plasmas of many animals contain a carboxypeptidase which seems to be similar to the human variety (Erdos et al., 1963b), but the enzyme in blood plasma is different from the pancreatic carboxypeptidase B (Erdos and Sloane, 1962; Erdos et al., 1964). The suggestion was made to name the enzyme in human plasma fraction IV-1 (Erdos, 1962) which inactivates bradykinin carboxypeptidase N. This name conforms more with the terininology of the biochemist than the previously used name kininase, since proteolytic enzymes are usually bond specific, and should not be named after a whole peptide chain (Dixon and Webb, 1964). Human red blood cells are also very active in destroying bradykinin. Combined biological and chemical studies indicated that this inactivation is probably caused by a prolidase (imidopeptidase) in the red blood cells (Table VI). The enzyme in erythrocytes was inhibited by agents which do not block the plasma carboxypeptidase (Erdos et al., 196313; Amundsen and Nustad, 1964). During the hydrolysis of bradykinin by hemolyzed erythrocytes, the released arginine (probably Arg') is converted to ornithine by the arginase present in the cells (Erdos et al., 1963b). In order to find a convenient chemical, spectrophotometric assay system for the carboxypeptidase in blood the hydrolysis of various short peptide substrate was measured. Among the substrates of pancreatic carboxypeptidase B, hippuryl-L-lysine was hydrolyzed by carboxypeptidase N, or
HYPOTENSIVE PEPTIDES
47
by a closely related carboxypeptidase, much faster than hippuryl-Largiriine or hippuryl-L-ornithine (Erdos et al., 1964). Hippuryl-L-lysine was cleaved a t a rate of 0.77 pmole/minute/ml in normal human plasma with cobalt chloride added (Erdos et al., 1965). Later peptide fragments related to the C-terminal end of bradykinin were employed in enzymic assay; for example, acetyl-L-phenylalanyl-Larginine. Hippuryl-L-lysine is split in the blood sera or plasma of various animals, including mamnials, birds, aniphibia, and fish. The human plasma enzyme, which hydrolyzed hippuryl-L-lysine, bradykinin, and the related shorter peptides, was purified about 80-fold. This purification procedure, which consisted of aninionium sulfate precipitation of the enzyme, followed by DEAE-cellulose and DEAE-Sephadex chromatography, suggested the possibility of bradykinin being the substrate of several related enzymes in blood (Erdos et al., 1964). Aguinea pig kininase, 25 times concentrated, was obtained by means of chromatography on hydroxylapatite column by N e t t (1962). The activity of the plasma enzyme ceases a t an acid pH (Werle et al., 1937; Horton, 1959a). Edery and Lewis (1962); Lewis, (1963b) pointed out the importance of the low activity of the enzyme below pH 7. According t,o these authors, the releasing enzyme would still liberate kinins rapidly at a pH where the destruction of bradykinin might be slowed down by the slightly acid milieu. In inflammation, the lowered pH of the tissues may lead to the accumulation of the peptide and aggravation of the existing symptoms. This attractive theory has to be confirmed in a more comprehensive system. The enzymic hydrolysis of bradykinin was iiieasured here in a phosphate buffer. The effect of pH on kinin hydrolysis in this medium depends very much on the chloride ion concentration (Aarsen and Kemp, 1963). In general, the pH curve of a rnetallopeptidase such as carboxypeptidase is determined to a large extent, by the choice of buffer used (Wolff et al., 1962; Erdos et al., 1964). It was also stated that salivary kallikrein would have a pH optimum between 7.8 and 6 (Lewis, 1963b), but the activity of other kallikrein preparations with synthetic or kininogen substrates decreases below neutrality (Wehster and Pierce, 1961; Habermann and Blennemann, 1964b; Trautschold and Rudel, 1963). The metabolism of kallidin is somewhat different from that of bradykiriin since the Lys1-Arg2bond of this peptide is susceptible to the effects of an aminopeptidase in blood serum (Webster and Pierce, 1963; Erdos et al., 1963b). According to Webster and Pierce (1963), kallidin can be rapidly converted to bradykinin before being destroyed by the carboxypeptidase in blood. Erdos et al. (1963b and unpublished results, 1962) found some biologically active bradykinin in their experiments when the
48
ERVIN G. ERDOS
LysI-Arg2 bond of kallidin was hydrolyzed by plasma, but they also observed the simultaneous release of arginine. This was taken as an indication that most of the kallidin was inactivated in blood. I n vitro, both bradykinin and kallidin are rapidly hydrolyzed in blood plasma (Erdos and Sloane, 1962; Erdos et al., 1963b) but met-lys-bradykinin seeins to be more stable (Elliott et al., 1963; Elliot and Lewis, 1965). Table IV shows some of the in aitro inhibitors of the enzymic metabolism of bradykinin in blood. Human and animal blood sera are different in this respect; for example, EDTA inhibits human plasma enzyme more than the variety found in the blood of various laboratory animals (Erdos et al., 1963b) but 1,lO-phenanthroline was a good inhibitor in either human or animal sera. The differences in inhibition by the various chelating agents probably are caused by structural, steric dissimilarities of the compounds. The inactivator of the peptides (kininase) also occurs in tissues (Werle et al., 1937), including the pancreas, and in body fluids (Werle and Grunz, 1939). The activity in the homogenized pancreas was attributed to a combined action of chymotrypsim, aniinopeptidase, and carboxypeptidase (Werle et al., 1950). Other tissue extracts, especially the kidney (Frey et al., 1950; Hamberg and Rocha e Silva, 1954; Halvorsen et al., 1960; Fasciolo, 1964; Carvalho and Diniz, 1964), also destroy kallidin or bradykinin. The presence of kininases was also observed in the perfused guinea pig lung (Brocklehurst and Lahiri, 1963), in leucocytes (Schwab, 1962), in brain (Hooper, 1963; Krivoy and Kroeger, 1964), in urine (Frey et al., 1950; Werle and Erdos, 1954; Horton, 1959b; Gadduni and Guth, 1960; Erdos et al., 1964), in lymph (Schachter, 1960; Edery and Lewis, 1963; Erdos et al., 1964), and in ascites (Werle and Grunz, 1939). Enzymes which destroy bradykinin are also present in a number of different microorganisms. Some of these enzymes are inhibited by EDTA (Aniundsen and Rugstad, 1965) (Section IV,C,2). A carboxypeptidase which cleaves basic C-terminal amino acids is present in urine, in lymph (Erdos et al., 1964), and in brain (Krivoy and Kroeger, 1964). Chlorpromazine inhibits the brain enzyme, possibly by chelating a metal cofactor. There is a catheptic carboxypeptidase in beef spleen which is activated by cysteine. This enzyme removes Args of bradykinin at pH 5 (Greenbaum and Yamafuji, 1965) (Table VI). Kidney contains several enzymes capable of inactivating bradykinin (Erdos and Yang, 1965, 1966) (see Section IV,C,2 and Table VI). One of them is a prolidase; the other is a carboxypeptidase. The third enzyme in kidney cortex hydrolyzes the Pro7-Phes bond in bradykinin; thus it is an endopeptidase. All three enzymes work at a neutral pH, and are inhibited by chelating agents. Interestingly, the Pro7-Phe*bond is also hydrolyzed by a collagenase preparation from C. hystolyticum.
49
HYPOTENSIVE PEPTIDES
TABLE IV INHIBITORS OF THE in Vitro HYDROLYSIS OF BRADYKININ IN BLOOD ~
Inhibitor Heavy metals
EDTA
1,lO-Phenant,hroline
Cysteine
Source of enzyme Human plasma fraction IV-1, human erythrocytes Human plasma fraction 1V-1, sera or plasma of various animals
Human plasma fraction IV-1, sera of various animals, human erythrocytes Serum or plasma of mail arid various animals
Chlorpromazine 2,2'-Dipyridyl
Bovine serum Human erythrocytes Human plasma
BAL
Rat plasma
8-Hydroxyquinoline-5sulfonic acid, 8-Hy droxyquinoline
Rat plasma and human serum
~~
Reference Erdos (1962, 1863a); Erdos and Sloane (1962); Erdos et al. (1963b) Arnistrong et al. (1955); Erdos (1962, 1963a); Erdos and Sloane (1862); Erdos et al. (1963b) Ferreira and Rocha e Silva (1962) ; Aarsen aiid Iiemp (1962) Erdiis (1962); Erdos and Sloane (1962); Erdiis et al. (196313) Werle and Grunz (1939); Van Arrnan (1955); Erdos (1962) ;Erdos and Sloane (1962) ; Aarsen and Kemp (1962) Krivoy and Kroeger (1964) Erdoa et al. (l963b) Amundsen and Nustad (1964) Perreira and Rocha e Silva (1962) Iperreira and Rocha e Silva (1962); Erdos and Wohler (1863a,h)
Arginine and related compounds Benzoylarginiiie, Human plasma frartioti E-amino-n-caprorir IV-1 or serum acid, a-amino-nvaleric acid, lysine heptoic acid, pimelic acid Urgocyton Human plasma and erythrocytes Citrate Bovine plasma
Erdijs (1962); Erdijs and Sloane (1962); Margolis and Bishop (1962); Erdos and Wholer (1963b); E. Bishop and Margolis (1963) Aniundsen et al. (1964) Klett (1962)
An enzyme which inactivates kinins was found in saliva, where it may originate froin the squainous epithelial cells of oral niucous nienibraiie (Aniundsen aiid Nustad, 1964). The inhibition pattern of the epithelial enzyme suggests similarities with the enzynie in human erythrocytes
50
ERVIN G. ERDOS
(Erdos e2 al., 1963b). The epithelial cells contain very little kininogenase activity (Amundsen and Nustad, 1964). Various types of cells and various cell fractions inactivate bradykinin at a different rate. For example, cells from the gastric mucosa are much less active than cells from the mucosa of the small intestine (Amundsen and Nustad, 1965). Among the subcellular fractions of the homogenized rat liver, the soluble fraction had the strongest kininase activity. Lysosomes froin rat liver were only slightly active (Amundsen and Nustad, 1965). In the rat kidney most of the activity was localized in the microsomal fraction (Erdos and Yang, 1966). Bradykinin has a very short half-life in the circulation. Based on the circulatory effects of the peptide (Saameli and Eskes, 1962) the calculated half-life in women is shorter than 0.5 minute; it is even less in dogs (Nicolaides et al., 1965). Kallidin has only a very slightly longer half-life in blood than bradykinin (McCarthy et aZ., 1965). Infusion of tritium-labeled bradykinin to rats a t a fairly high dose level (4.6 bg/minute) leads to accumulation of the peptide and its metabolites in kidney and liver (Table V) and to excretion in urine (Bumpus et at., 1964). Infusion of bradykinin into the artery of human forearm did not increase TABLE V TRITIATED BRADYKININ DISTRIBUTION I N NORMAL RATS~,~ Thirty minutes after end of infusion
End of infusion Organ
Electrophoretic Radioactivity mobility (cm)
Heart Lung Kidney
8,100 14,200 117,400
Adrenals Liver Spleen Brain Uterus Salivary glands Skeletal muscle Blood Urine
4,200 33,000 16,700 8,800 9,000 11,200 3,400 14,400 192,000
Bradykinin 0
9.5, 17.0 12.0, 17.0 18.5 21.5
14.5, 16.5 10.5, 1 6 . 5
Radioactivity 10,200 12,000 108,000 4,100 26,300 9,700 2,000 7,200 11,800 8,600 7,400 1,440,000
16.0-1 7 . 0
Values are averages of results obtained from eight rats. Bumpus et al. (1964).
Electrophoretic mobility (cm) 5.0 11.5 8.7, 13.5
9.0,20.0 14.0, 1 6 . 5 21 .o 16.0-17 .O
HYPOTENSIVE PEPTIDES
51
the peptide level in venous blood for any considerable period. This suggests a very rapid disappearance of the peptide from the circulation. The capillaries might play a prominent role both in the removal and in the subsequent inactivation of bradykinin (Sicuteri et al., 1963c; Sicuteri, 1964; Allwood and Lewis, 1964). The destruction of bradykinin and kallidin by human blood was studied in a variety of conditions. The results in pregnancy are somewhat controversial. Armstrong et a2. (1960) and Effkemann and Werle (1941) did not observe any increase in blood enzyme level while Centaro et al. (1963) and Werle in a later paper (1960) noticed that pregnant blood serum or plasma destroys the kinins quicker than does normal blood. The level of the enzyme in newborn was lower than in the mother. Blood samples of some of the asthmatic persons investigated inactivated less bradykinin than normal individuals (Sicuteri et al., 1962a). Erdos et al. (1965) studied the hydrolysis of hippuryl-L-lysine by the carboxypeptidase in the blood serum of over 400 individuals, The enzyme activity was above normal in pregnant women, and below normal in the newborn. Among hospitalized patients those with cirrhosis of the liver had lower values, while some neoplastic patients had higher values than healthy individuals. 2. Proteolytic E n z y m e s
Kinins are inactivated by some purified proteolytic enzymes (Table
VI). The activity of crude kallidin and bradykinin was destroyed by chymotrypsin (Werle et al., 1950). Snake venonis (Deutsch and Diniz) 1955), commercial kallikrein (Werle and Berek, 1950), and trypsin (Rocha e Silva et al., 1949))three of the kininogenases, can inactivate the peptides. The activity in trypsin was attributed to a chymotrypsin contamination (Werle, 1953), and in crude kallikrein to the presence of carboxypeptidase B (Erdos et al., 1962). The factor which inactivates bradykinin and the one which liberates it can be distinguished by heating snake venom (Hamberg and Rocha e Silva, 1957) (Section 11,F). Proteolytic enzymes were also used in degradation studies with purified or synthetic peptides. Chymotrypsin (Elliott et al., 1960b; Boissonnas et al., 1960a,b) was shown to hydrolyze the Phe8-Arg9bond of bradykinin quickly and the Phe6-Scf bond slowly. A carboxypeptidase preparat,ion released Argg first and then Phes from bradykinin (Boissonnas et al., 1960a; Zuber and Jaques, 1960). The first activity was due to carboxypeptidase B, the second to carboxypeptidase A. Leucine aminopeptidase did not attack the N-terminal Arg1-Pro2bond of bradykinin (Boissonnas ef al., 1960a). In biological studies bradykiiiin was also resistant to leucine aniinopeptidase (Erdos, 1962; Erdos et al., 1963b)) but it was inactivated by partially purified prolidase (iniidopeptidase). The latter enzyme destroyed the
TABLE V1 ENZYMIC HYDROLYSIS OF KININS ~
~~~~
~
1
Met-lys-bradykinin 5 6 7 8 9
3
2
4
1011
H-Met-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-OH 1
2
3
1
2
Enzyme
5 6 7 Kallidin 3 4 5 6 Bradykinin 4
7
8
9 Substrate"
T
T
Tb T T
Catheptic carbox-eptidase (spleen) Enzyme in kidney cortex Aminopeptidase (purified and plasma)
T
T t
t
F'rolidase (purified and erythrocytes) Snake venom (Crotalus adamanteus) (Agkistrodm halys blomhofii)
t =
met-lys-bradykinin.
I, 111 I
T
Carboxypeptidase B (pancreas CarboxypeptidaseN (plasma) and carboxypeptidase (kidney)
bradykinin; I1 = kallidin; I11
T
Tb
Carboxypeptidase A
=
10
I11 I1
Chymotrypsin
I
9
T t
Trypsin
0
8
I I, I1
I I I11 I1 I
T
* After removal of
I11 I
Reference Schroder (19%) Webster and Pierce (1963); Erdos et al. (1963b) Nicolaides et al. (1963~) Elliott et al. (1960a,b) Boissonnas et al. (1960a,b) Elliott and Lewis (1965) Zuber and Jaques (1960) Boissonnas et al. (1960a) Erdos (1962); Erdos et al. (1963b) Erdos (1962); Erdos and Sloane (1962) Erdos et al. (1963b) Erdos and Yang (1965, 1966) Greenbaum and Yamafuji (1965) Erdos and Yang (1965, 1966) Schroder (1964a) Webster and Pierce (1963) Erdos et al. (196313) Erdos and Yang (1965); Erdos el al. (1963b) Habermann and Blennemann (1964b) Suzuki et al. (196513)
C-terminal arginine.
HYPOTENSIVE PEPTIDES
53
activity of bradykinin by cleaving the first N-terniinal arginine (Erdos and Yang, 1966). Met-lys-bradykinin, however, is hydrolyzed by leucine aminopeptidase. The first two bonds of the peptides are broken by the purified enzyme (Schroder, 1964a). Snake venom (Agkistrodon halys blomhofii) contains a kininase, which is identical with proteinase B. It acts as an endopeptidase (Suzuki et al., 196511) (Table VI). Partially purified collagenase preparation froni L'. hzstolyticum also renders bradykiniii inactive by endopeptidase action. This bact crial eiizynic was inhibited by various chelatirig agents and heavy iiietals (Erdos, unpublished data, 1963). In biological studies Erdos (1962; Erdos et al., 1963a,l)) noted the very rapid inactivation of bradykinin arid kallidin by purified pancreatic carboxypeptidase B. The partially purified enzynie was 50 to 100 times faster in this respcct than crystallized chyniotrypsiri. This estimate is probably quite conservative and should be corrected upward when the two enzymes are compared oil the basis of the same degree of purity. The high affinity of pancreatic carboxypeptidase B to bradykiniii was also confirmed by Krivoy and Kroeger (1964) and by Nicolaides et al. (1965). The results of the in vitro studies led to the use of carboxypeptidase B in vivo to block the hypotensive effects of bradykinin, kallidin, and releasing enzymes such as kallikrein. Intravenous injection of purified paiicreatic carboxypept idasc B (Erdos el ul., 1963a,b) protected guinea pigs, rabbits, and cats against those peptides which have a basic C-terniinal amino acid by rapidly inactivating them in blood. Subcutaneous infiltration of the skin with carboxypeptidase B also decreased the effect of intradernial injection of bradykinin in the guinea pig. The blocking of the effect of the peptide and the eiizyiiie level in blood are in good correlation (Erdos et al., 1963a) (Fig. 9). Pretreatment with carboxypeptidase B can also prevent the hypotensive action of trypsin. Repeated injection of carboxypeptidase B to cats can block the effect of kinins for hours (Erdos and Yang, 1965,1966). The effect of iiiet-lys-bradykiriin was blocked by higher doses of injected carboxypeptidase B as well (Erdos, unpublished results, 1965). Injected carboxypeptidase B disappears froin the circulation rapidly. The half-life in cats is about 30 niinutcs; in rabbits it is even less (Erdos el al., 1963a). The active enzyme is taken up by the livcr and kidney and is excreted in the urine (Erdos arid Yang, 1965, 1966). Part of an infused carboxypeptidase B preparation was recovered from the lyiiiph of the dog hind leg (Webster et al., 1965). Trypsin can convert kallidin to bradykinin (Webster and Pierce, 1963; Nicolaides et al., 1963c; Erdos et al., 196313). Kallidin, however, is a rather poor substrate for trypsin, and not niore than 5-13y0 of it was converted to bradykinin (Webster and Pierce, 1963; Haberiiiann and Blenneniann, 1964b). Coriiniercial trypsin preparations can also destroy kallidin during
54
ERVIN G . ERDOS
I
I
I
2
I
I
3
la
2
I
Ib
I
lc
I
Id
.
I
2
lmin
FIG. 9. Blocking the action of bradykinin by intravenous injection of carboxypeptidase B. Return of the effect of bradykinin and decrease in circulating carboxypeptidase B level in blood in a 2.5-kg female cat. Doses of intravenous injections per kg body weight: (1) 18 pg bradykinin; (2) 0.56 pg histamine; (3) 3.8 mg (269 U) carboxypeptidase B. Injections (la), (lb), (lc), and (Id) were given 5, 50, 80, 110 minutes, respectively, after the administration of the enzyme. Vertical bars show enzyme activit,y in plasma. (Erdos et al., 1963a).
incubation (Werle) 1953; Erdos et al., 19631s; Webster and Gilmore, 1965). Met-lys-bradykinin can be converted easier to bradykinin by trypsin or snake venom (Elliott et al., 1963; Haberinann and Blennemann, 1964b; Schroder, 1964a), but the release of bradykinin from kininogen is much quicker than that from the undecapeptide (Habermann and Blennemann, 1964b). Oral feeding of proteolytic enzymes was claimed to antagonize partially the effects of bradykinin 011 capillary permeability, blood pressure, and smooth muscle (Innerfield et al., 1963).
3. I n V i v o Inhibitors Werle was the first to use cystcine in vivo to preserve the effect of kallidin in the dog ('Werle and Grunz, 1939). When carboxypeptidasc N was characterized in human plasma fraction IV-1 (Erdos, 1962, 1963a cf. Collier, 1963a; Erdos and Sloane, 1962) in vitro inhibitors of the enzyme were used extensively. The in vitro inhibition studies led to the application of these agents to guinea pigs in vivo (Erdos and Wohler, 1963a,b) and to other animals. Nine compounds pro-
55
HYPOTENSIVE PEPTIDES
TABLE VII POTENTIATION OF THE CARDIOVASCULAR EFFECTS OF BRADYKININ I N THE GUINEA PIC BY SOMEINHIBITORS OF THE ENZYMIC METABOLISM OF THE PEPTIDE"
No. of experiments
Dose of inhibitor (mg/kg)
Dose of bradykinin (.ug/kg)
2-Mercaptoethanol
15
6-Mercaptoethylamine
11
3-Mercaptopropionic acid
13
a-Thiogly eerol
13
2,3-Dimercaptopropanol (BAL)
15
Diethyldithiocarbamic acid
13
L-Penicillamine
13
67 (45-83) 90 (50-165) 41 (25-74) 242 (108429) 16 (t5-3 1) 71 (25-1 00) 130 (37-200) 126 (50-165) 175 (162-1 98) 88 (66-1 11)
1.o (0.03-2.0) 0.8 (0.4-1.2) 1.1 (0.5-1.8) 0.6 (0.3-1.7) 0.9 (0.3-1.8) 1.2 (0,3-2.0) 1.2 (0.4-2.2) 0.5 (0.3-0.6) 0.9 (0.5-1.2) 1.2 (1.1-1.2)
Inhibitor
8-Hydroxy-5-quinoline sulfonic acid Ca-EDTA
8
Gluthathione
4
a
9
Drop in mean blood pressure (yo) Before
After
10 (0-24) 10 (0-20) 13 (0-25) 11 (0-26) 13 (0-28) 13
42 (20-55) 16 (7-37) 44 (33-57) 32 (20-52) 48 (31-58) 38 (22-58) 23 (12-41) 31 (23-42) 27 (18-33) 24 (20-30)
(0-20) 11
(6-23) 5 (0-13) 8 (7-17) 10 (0-19)
From Erdos and Wohler (1963b).
longed the hypotension caused by bradykinin in guinea pigs (Table VII; Fig. 10). Some were tested and found equally active with kallidin. They are mostly chelating agents; thiols were especially effective (Ferreira and Rocha e Silva, 1962). Since these compounds effectively prevent the in oitro enzymic hydrolysis of the peptides, it was assumed that their in vivo action is also based on enzyme inhibition. I n the guinea pig mercaptoethariol and niercaptopropionic acid were very well toleratfed, but some other inhibitors had side effects. After the administration of the inhibitor, the drop in systemic blood pressure caused by the peptides was increased and greatly prolonged (Fig. 10) (Erdos and Wohler, 1963a,b). The action of inhibitors is different in the various experimental animals. Mercaptoethanol, for example, is very active in the rat (Erdos and Yang, 1965), BAL (2,3-dimercaptopropanol)(Ferreira et al., 1962; Corrado, 1963), thioglycolic acid (Werle et al., 1964), diethyldithio-
56
ERVlN G . ERDOS
2
I
3
I
2
30 sec
FIG.10. Potentiation of the effect of hradykirrin by mercaptopropionic acid in a 0.4-kg male guinea pig. Time between first mid lit& injection: 53 minutes. Dose per kg weight: (1) 0.8 pg histamine; ( 2 ) 1 pg lmdykiiiin; cd) 57 mg mercaptopropionk acid (Erdos and Wohler, 196313.)
carbamic acid, and 8-hydroxy-5-quinoline sulfonic avid (Erdos, unpublished results, 1964) prolong the hypotensive cf€cct, of kinins in the dog. lritraperitoneal injection of BAL increases thc effect of bradykinin in the rabbit as well (Erdos, unpublished results, 1963). The inhibition of the destruction of kinins is especially impressive when the peptides are injected in the splanchiiic circulation. A given effect of bradykinin requires a 2 to 5 tinies higher dose injected into a mesrnlerir vein than into tjhe femoral vein. This possibly is caused by the incrcased rate of destruction during passage through the liver. The effect of bradykinin after injection in a meseiiteric vein was greatly increased in dogs which were pretreated with BAL (Erdos and Yang, 1966). Inhibition studies, especially in dogs, also indicate that the enzymic metabolism is but one of the means the hody has at its disposal to inactivate circulating kinins. The peptides are protxhly rcmoved from the blood very rapidly.
r).
PATHOLOGY
Kiniris probably participate in various pathological processes. Although their presence has been shown in a variety of conditions, it has not been claimed yet that the appearance of the peptides “is good for you,” as suggested for histamine recently. But there arc some similarities between histamine and kinins. According to I,. R. Dragstedt (cited in Goodman for lhc participation of histamine and Gihnan, 1955, p. 644), the evickmc~c~ in certain physiological and pathological watt ions varies from unfounded assumption and illogical inference to subst ailtially concrete and undeniable proof. The same can be said about kinins. The difficulties between showing the presence of the peptides and proving its decisive role are very considerable. For example, in shock or in t)urns a number of active agents can be released. Some of them change the permeability of the capillaries which
HYPOTENSIVE PEPTIDES
57
may be followed by the extravascular appearance of plasma proteins including components of the systeni which liberates kinins. The release of peptides in turn may aggravate existing conditions. The usual experimental techniques do not allow distinction among the nona-, deca-, and undecapeptide congeners of the kinins. The participation of kiriins in pathological processes in blood and elsewhere may happen in one or more of the following ways: increase in kallikrein or kallikrein activator content ; decrease in kallikrein inhibitor level ; diiiiiriished kininogen level; increased appearance of free peptide; decreased inactivation of kinins, etc. These subjects have been briefly discussed in other parts of this review (see Sectioiis II,A,D, and E, 111, and IV,C). Table VIII summarizes some of the available information on the role of kinins in pathological processes. This question has been studied extensively in pancreatitis (Section I1,A) in conjunction with the clinical use of the pancreatic inhibitor TrasyIol. Another phenomenon in which the importance of kinins has frequently been nieritioried is localized tissue injuries such as inflaniniation or burns. Since the discovery of the dissociation of the kallikrein-inhibitor complex in acid niilieu, the role of acid pH in activating kallikrein in the body has been under consideration (Kraut et al., 1928; Frey, 1931). Another consequence of the increase in hydrogen ion concentration might be the decreased inactivation of kinins below neutrality (Section IV,C). These kinins in turn may be responsible in part for the syniptoms of inflammation by increasing capillary permeability, causing pain, edema, etc. The appearance of various toxic substances in burns has been known for a long time (C. L. Fox, 1963). In the lymph of the dog hind leg the amount of kininogenase (Edery and Lewis, 1963) or, more likely, kininogen (Jacobsen and Waaler, 1965) increases greatly after burns. Scalded skin releases bradykiniri among other agents (Rocha e Silva and Rosenthal, 1961). I n the rat paw heated to 45°C bradykinin appears before histamine is released, because histamine is liberated only a t higher temperatures (Rocha r Silva and Antonio, 1960). An additional instance in which the appearance of kinins niight be of iniportance is shock. The significance of the impairment of splanchnic circulation under experiment a1 conditions such as endotoxin shock in dogs has been suggested. The subsequent damage t o the intestinal wall has also been shown (Lillehei et al., 1964). Since kallikrein occurs in this tissue, the release of the enzyme in the local or in the general circulation might be a contributory factor (Kobold and Thal, 1963). Other coniponents of the kinin system are also involved, as indicated by the immediate increase in kiniri level in blood after the injection of endotoxin and by the decrease in kininogen (Meyer arid Werle, 1964). Pretreatment with kallikrein inhibitor
Ol
THEROLE OF Condition Anaphylactic shock
Species
Finding
Assay system
Dog
Appearance of kinin in blood
Rat
Animals sensitized with Bacillus pertussis vaccine are more sensitive to i.v. injection of bradykinin Kinin was found in blood, kininogenase Rat uterus in perfused lung and skin. Carboxypeptidase in blood below normal Chemical
Guinea pig
Angioneurotic edema (hereditary)
Man
Arthritis
Man
Asthma
Man
Blood transfusion
Man
Burns
Dog Rabbit Rat Man
00
TABLE VIII KININSIN PATHOLOGICAL PROCESSES
Guinea pig ileum
Level of inhibitor of kallikrein (also of Bio- and chemical C'-1-esterase and permeability facassay tor) is below and permeability factor is above normal in blood Synovia from knee joint of arthritic Rat uterus patients releases kinin Inhalation of bradykinin aerosol precipitates attack in asthmatic patients During transfusion the presence of a kinin and/or kallikrein can contribute to side effects Kininogen or kininogenase appears in lymph Thermal injury releases kinin from the skin Kinin appears in urine
Guinea pig ileum Guinea pig ileum Rat uterus Various Various
Reference Beraldo (1950); Back et (196313) Dawson and West (1965)
d.
Brocklehurst and Lahiri (1962); Erdijs et al. (1964) Jonasson and Becker (1965) (see also shock) Landerman et al. (1962); Donaldson and %sen (1964); Miles and Wilhelm (1960); Becker and Kagen (1964) Armstrong et al. (1957); Melchiorri (1963) H. Herxheimer and Stressemann (1961); Lecomte etal. (1962); Girard and Moret (1963) Mackay et al. (1962); Eisen and Keele (1965) Edery and Lewis (1963) Jacobsen and Waaler (1965) Rocha e Silva and Rosenthal (1961) Goodwin et al. (1963)
M
0
A Z
0 m
tl
8'
Carcinoid syndrome
Man
Cirrhosis of the liver
Man
Hageman trait
Man
Hemorrhagic shock
Rabbit Guinea pig Man
Hypertension (nephritis, Addison’s disease, damage to tubules in kidney) Ileus Infection by pathogenic organisms Inflammation
Irradiation
Rat
Iiallikrein was found in hepatic metasR a t blood pressure, Oates et al. (1964) tases of carcinoid tumor, kinin in rat uterus hepatic vein blood during flushes Sicuteri el al. (1962a) Bradykininogen level and carboxyBio- and chemical Dink and Carvalho (1963) peptidase level in blood below normal assay Erdos et al. (1965) Lacking a component which leads to the Rat uterus Margolis (1963) in vitro activation of kallikrein in blood Indirect evidence that bradykinin might Corticopial circula- Breda et al. (1962) (see also be involved tion shock) Decreased kallikrein excretion in urine. Dog blood pressure Frey et d. (1950); Werle and Appearance of kallikreinogen in Korsten (1938); Werle and urine Vogel (1958, 1960) Dobovicnik and Forell (1961)
Increase in kallikrein content of intestinal wall Laboratory Bradykininogen level in blood drops. Kinin appears in urine, plasma and animals tissues Laboratory Bradykinin in various animals can cause such symptoms of inflammation as animals pain, edema, hyperemia, etc. Rat Intrapleural administration of turpentine leads to appearance of kinin in inflammatory exudate Less kallikrein (1) is excreted in urine of Dog irradiated dogs Dog
Various
Various
m
20
il
9
Werle el ul. (1963)
v
Goodwin and Richards (1960) Tella and Maegraith (1962) Richards (1965) Spector (1964) (Sections IV,B,4 and 5)
2
Bioassay
Spector and Willoughby (196213)
Indirect UV spectrum
Fink (1954)
U M
u1
TABLE VIII (Colztinued) Condition Migraine
Species Man
Finding
S.C.fluid from the tender regions of the
scalp contains neurokinin, and releasing enzyme. Injection of blister fluid (bradykinin) contributes t o headache. Bradykinii causes headache in migraine patients Neoplastic conditions Man Carboxypeptidase level in serum above normal Pancreatitis Man, dog, Although the presence of free trypsin in rat pancreas is debatable, presumably kdlikrein is activated which in turn releases kinins. Decrease of bradykininogen in blood Pulmonary edema Rabbit Appearance of kinin in pulmonary edema induced by epinephrine Shock Dog, rabbit In various forms of shock kininogen level is below normal. I n endotoxin shock vasoactive material (probably kinins) is released. Trasylol increases surviva1 rate Shwartaman reaction Rabbit Soybean trypsin inhibitor inhibits, bradykinin potentiates reaction; Trasylol inhibits
Assay system
Reference
R a t uterus
Ostfeld et al. (1957) Chapman et al. (1961) Sicuteri et al. (196313, 196413)
Chemical
Erdos et al. (1965) (see also carcinoid) Forell and Dobovicnik (1964); Trautschold et d. (1964); Katz et al. (1964); Ryan et al. (1964) (Section II,A,P)
Various
Guinea pig ileum
D i Mattei (1962)
Various hioarnays
Lecomt,e (1961); Dinia and Carvalho (1963); Meyer and Werle (1964); Kobold et al. (1964) ;Lambert et d.(1964); Massion and Erdos (1965) Chryssanthou and Antopol (1961); Antopol and Chryssanthou (1963); Halpern (1964)
Rabbit skin
E 5
0 M
E0: u1
SOMEPOSSIBLE PATHWAYS OF
Injury
Inflammation
THE
TABLE IX LIBEEATION OF KINININ PATHOLOGICAL CONDITIONS"
Toxic metabolites
T:ziF1
Venoms
Heating
Irradiation
md
a 0 c3
Activation and liberation of proteases and kininogenases
Secretory cells-
\
i i
Interstitial space -Blood
Activation of kallikrein and release of kinins
i
Shock
0
According to Werle (1964a).
E z4 M
vessels
/
a M a
2 U M
m
62
ERVIN G. ERDOS
Trasylol increases the rate of survival of these dogs (Meyer and Werle, 1964; Massion and Erdos, 1965). Table IX, compiled according to Werle (1964a), suggests some possible pathways which may lead to the release of kallikrein. The liberated enzymes then would activate the kinin system intravascularly, in the tissues, and in the interstitial space. The end result could be pancreatitis, vasodilatation, edema, hemorrhage, and shock. Kinins would cause or contribute to these symptoms. The availability of inhibitors of the enzymic inactivation of kinins might make it possible to reinvestigate some of the problems in those cases where the lack of evidence for the presence of the peptides was attributed to rapid inactivation.
E. OTHERSOURCES OF KININS I n addition to bradykinin and kallidin, other kinin-like peptides have been found in nature. Some of these, such as neurokinin, have been discussed elsewhere (Section IV,B,5). The existence of kinins in venoms, in frog skin, and in the urine illustrates the few instances in which these types of peptides were shown to exist in a preformed, active condition. 1. Urine Werle and Erdos (1953, 1954)) while investigating the excretion of oxytocin in human urine, found a polypeptide which, against their expectations, was not identical with the pituitary hormone. The material lowered the blood pressure and contracted smooth muscle preparations of various laboratory animals. The substance was antidiuret,ic in the rat. Some subtle differences such as stability in alkali and action on rat colon seemed to distinguish this material from kallidin. The peptide was easily destroyed by blood plasma, hemolyzed erythrocytes, plant proteases, etc. Rat urine did not contain the peptide. The material in the human urine was named substance Z. Somewhat later, the suggestion was made that the substance might be identical with bradykinin (Gomes, 1955). The distribution of the partially purified peptide in countercurrent extraction was different from bradykinin. The substance was also relatively more active on rabbit blood pressure than bradykinin (Gomes, 1957, 1959). Walaszek purified the substance further on cellulose and aluminum oxide columns. Column chromatography yielded two fractions, named Z1 and Zz (Walaszek, 1957; Huggins and Walaszek, 1960). The separation of two components in the urinary peptide was confirmed by Jensen (1959). Gaddum and Horton (1959) attributed the two Feaks of activities to overloading of the chromatography column. This opinion is not shared by others and by this reviewer, who consistently received two activity peaks when separating the material on a CM-cellulose column (Erdos, unpublished data, 1962). Gaddum and
HYPOTENSIVE PEPTIDES
63
Horton ascribed all the biological effects of their preparations to bradykinin. They renamed the substance urinary kinin. Urinary kinin in their experiments was also relatively more active on rabbit blood pressure than on the guinea pig ileum. Taking the relative activities of synthetic bradykiriin and kallidin on rabbit blood pressure in consideration, we may assume with hindsight that the discrepancy between the hypotensive and the niusculotropic effects was caused by the kallidin content of this substance Z. It has been indicated recently that most of the urinary peptides consist of bradykinin and of a smaller amount of kallidin (Jensen et al., 1963). Horton (1959b, 1960) measured the excretion of urinary kinin in man. He found that in ternis of pure bradykinin about 10-30 pg was excreted in 24 hours. With little variation approximately the same rate of excretion was niaintained in some clinical subjects afiicted with various diseases (Abe et al., 1964; Jensen et al., 1965). A kinin-like material appeared in the urine of burned patients (Goodwin et al., 1963) and in the urine of laboratory animals after infecting them with some pathogenic organisms (Goodwin and Richards, 1960; Richards, 1965). Most of the effect of the urinary peptides was abolished by incubating them with carboxypeptidase B (Erdos et al., 1963b). Infusion of kallikrein did not increase the excretion of the peptide in man (Yoshinaga et al., 1964). Infusion of bradykinin in the renal artery of dog was not followed by urinary excretion of the material; thus, the peptide was probably reabsorbed from the tubules or destroyed there. The range of excretion is equivalent to 3.8-28 ng bradykinin per minute in man. The suggestion was made that the urinary kinin may originate from the renal tubular cells (Yoshinaga et al., 1964). In contrast to these observations, after the infusion of labeled bradykinin to rats, a material which was probably identical with the active peptide appeared in the urine (Bumpus et al., 1964) (Section IV,C). Beraldo (1952) found a substance in dog urine which was thermolabile and not destroyed by blood. The substance contracted guinea pig ileum and lowered the blood pressure of the cat. Subsequent (Beraldo, 1955) investigations showed that contrary to previous findings this material was destroyed by blood serum and could withstand heating. Proteolytic enzymes also inactivated this material. The active agent was named substance U. Substance U is formed by mixing urine with blood plasma. Beraldo et al. (1956) attributed most of the substance U-forming activity in urine t o kallikrein. 2. Ccloslrum Saliva or urinary kallikrein releases from bovine colostrum a smooth muscle-stimulating peptide, colostrokinin. The properties of the colostrokinin liberated by kallikrein resemble bradykinin (plasma kinin) more
64
ERVIN G. ERDOS
than the colostrokinin released by saliva (Guth, 1959). Serum also liberates a substance from colostruni (Werle, 1960). This material contracts smooth muscles, and is vasoactive. Human colostrum is a substrate of various kallikreins. I n addition, trypsin and chymotrypsin can release a substance from colostrum (Werle and Trautschold, 1960). 3. Venom Wasp venom contains a biologically active peptide which was named first wasp venom kinin or, simply, kinin (Jaques and Schachter, 1954; Schachter and Thain, 1954). Although this kinin resembles bradykinin, it behaves differently in Chromatography and it is inactivated by trypsiri (Mathias and Schachter, 1958). Studies with the crude kinin indicated that it might be even more potent than bradykinin (Schachter, 1964). Hornet venoni contains another type of kinin (Bhoola et al., 1961). Recent investigations showed that wasp venoni kinin niay consist of several fractions. One of them released Glyl-kallidin upon tryptic digestion (Pisano et al., 1965). This would be another occasion when a peptide was synthesized first (Schroder and Hempel, 1964) and found in biological material later. 4. Frog S k i n
Frog skin (Rana temporaria) is a rich source of bradykinin. It contains 35-280 pg of the peptide per gram of tissue. The structure of amphibian bradykinin is identical with the mammalian kinin. Highest concentrations were found in the rough glandular skin (Anastasi et al., 1965). Tadpoles had very little of the peptide. Crude skin extracts of various Japanese frogs, niostly Rana, also contain a bradykinin-like peptide (Erspamer et al. , 1964a). V. Eledoisin
A. STRUCTURE AND METABOLISM Erspamer in 1949 found a vasoactive substance in the posterior salivary gland of Eledone moschata and Eledone Aldrovandi. The crude extract of glands of the two molluscan species lowered the blood pressure of laboratory animals and contracted various smooth muscle preparations. The peptide was named moschatiii at first, but later was called eledoisin (Erspamer, 1952). The structure of the peptide was established after extracting it from more than 10,000 pairs of glands with methanol (Erspamer and Anastasi, 1962; Anastasi and Erspamer, 1963). The purification procedures included chromatography on alkaline aluniina coluiiin followed by precipitating eledoisin froin the solution as an albumin complex. Subsequently, the material was passed through Aniberlite CG-50 Hf column. Pure peptide was obtained in countercurrent distribution.
65
HYPOTENSIVE PEPTIDES
The peptide chain contained 11 aniino acids. It does not have a free N-terminal NH, group or a free C-terniinal carboxyl group. The first amino acid is pyroglutaiiiic acid, whose structure is shown in the acconipanying diagram : H,C-CH, I
1
O+cc,N/E\COOH H
Pyroglutamic acid
Met hioninaniide f o r m the C-terminal end of eledoisin. The structure was confirmed by synthesis (Sandrin a i d Boissoniias, 1962). The lack of free N-terminal aniino group or C-terminal carboxyl group renders the peptide resistant to aniinopeptidase or carboxypeptidase. Chymotrypsin, however, inactivates t,he peptide conipletely (Anastasi and Erspanier, 1962). Relatively low concentrations of trypsin inactivated eledoisin only partially (Anastasi arid Erspamer, 1962) by releasing a C-terminal heptapeptide (Erspanier and Anastasi, 1962; Anastasi and Erspanier, 1963). This peptide is less active than eledoisin in some tests (Bernardi et al., 1964a). The bonds in the peptide chain broken by these two enzymes are shown in Fig. 11. The peptide is relatively stable in blood (Sicuteri el al., 1963d; Eledoisin Pyr-Pro-Ser-Lys-Asp(0H) - Ala-Phe-Ile-Glg-Leu-Met-NH, Chymotrypsin
It
t
Trypsin P hysalae min
Pyr-Ala-Asp(OH)-Pro-Asp(NH,)- Lys-Phe-Tyr-Gly-Leu-Met-NH, Chymotrypsin Trypsin
t
t
FIG.11. Hydrolysis of eledoisin and physalaemin by tqypsin and chymotrypsin.
Stiirnier and Berde, 1963b), hut soine tissue extracts from octopoda (Anastasi and Erspamer, 1962) destroy the biological activity. Synthetic eledoisin is very rapidly inactivated by homogenized guinea pig kidney or a inicrosomal fraction of swine kidney cortex (Erdos, unpublished data, 1965). The enzyme in the guinea pig kidney is inhibited by various chelating agents. T h e skin of a South Aliierican frog, Physalaemus fuscumacuulatus, con-
66
ERVIN G . ERDOS
tains a peptide named physalaemin (Erspamer et al., 1962). The structure of physalaemin is closely related to eledoisin (Fig. ll),but six amino acids are different in the undecapeptide chains (Anastasi et al., 1964). The structure of this naturally occurring peptide has been also confirmed by synthesis (Bernardi et al., 1964b). Like eledoisin, physalaernin loses its activity very slowly when incubated in blood and it is resistant to amino- and carboxypeptidases. Liver and kidney homogenates, however, inactivate the peptide (Erspamer et al., 196413). Trypsin and chymotrypsin cleave various bonds in physalaemin (Fig. 11). Trypsin unexpectedly also splits a Tyr-Gly bond in the peptide. This was not attributed to a chymotrypsin contamination in trypsin, but to a rather unspecific hydrolytic activity (Anastasi et al., 1964). Eledoisin-like peptides are among the most potent hypotensive agents known; nevertheless, only a part of the eledoisin structure which is related to the C-terminal end is specific for the biological activity. This was shown by various groups which synthetized a large number of analogs of eledoisin. Some of the shorter derivatives of eledoisin turned out to be even more potent than the parent compound. Thus, many related peptides have the same or even higher activity than eledoisin. Since the shorter analogs have a lower molecular weight, the difference in activities is somewhat less striking when compared on the basis of molar concentrations. Among the various fractions of eledoisin the C-terminal pentapeptide still retains 1-3% of activity (Bernardi et al., 1964a). This activity increases with the length of the C-terminal residue. The octapeptide was as potent as eledoisin in several tests (Bernardi et al., 1964a). Schroder and Lubke (1964) found the C-terminal heptapeptide to be fully active on rabbit blood pressure. The nona- and decapeptide derivatives are even more effective than eledoisin (Bernardi et al., 1964a; Lubke et al., 1965). Lengthening the peptide chain of eledoisin a t the N-terminal end (Sandrin and Boissonnas, 1964) had no significant influence on activity. Substituting asparagine for aspartic acid (Sturmer et al., 1964; Sandrin and Boissonnas, 1964; Bernardi et al., 1964a; Lubke et al., 1965) also enhances the potency of the peptide. Replacing Asp5 with Gly5 increases the activity of eledoisin by about 50% (Lubke and Schroder, 1965). The Glu5-eledoisinis as active as the naturally occurring Asp6-eledoisin (Lubke et aZ., 1964). L-Alae could be substituted with n-Alas with relatively little loss, but replacing any of the last five C-terminal L-amino acid with a D-analog virtually abolished the activity of eledoisin (Schroder et al., 1965). The biological effect is not influenced by adding or removing acidic or basic groups, or, in other words, by changing the number of electrostatic charges in the peptide. The structure of the six C-terminal amino acids fragment is the one of real importance for activity (Sandrin and Boissonnas, 1964). Physalaemin, however, differs
HYPOTENSIVE PEPTIDES
67
froin eledoisin in two of the six C-terminal aniino acids, but the peptide is 3 to 4 times inore hypotensive than eledoisin (Erspanier et al., 196410). I n contrast to the changes which can be introduced a t the N-terminal end without a decrease in the biological potency of the peptides, shortening or lengthening the C-terminal end or changing methioninamide to niethionine rendered eledoisin inactive (Camerino et nl., 1963; Sandrin and Boissonnas, 1964). Substituting the C-terminal Met-NH2 with Eti-NHz, however, increases the biological activity (Bernardi el al., 1964a). The structural changes which alter the hypotensive activity do not necessarily influence the action on the isolated smooth muscles in a parallel manner (Schroder and Liibke, 1964). In some derivatives the hypotensive effect increases while the niusculotropic potency drops and vice versa (Sturmer et al., 1964). B. PHARMACOLOGY 1. Smooth Muscles
Eledoisin acts similarly to bradykinin in a number of assay systems, but it is easy to distinguish between the two types of peptides. Although both peptides contract the isolated uterus (Table 11),eledoisin is 200 times more potent than bradykinin on the rabbit uterus, but has only 0.2% of the activity of bradykinin on the rat uterus (Sturmer and Berde, 196310) (Table 11).Eledoisin contracts the isolated rat duodenum and hen rectal caecum while bradykinin relaxes them (Stunner and Berde, 196313). Eledoisin is 100 times more potent than bradykinin on the rabbit colon (Erspamer and Erspamer, 1962). Physalaernin is generally less active than eledoisin on the smooth rnuscles of isolated organs (Erspamer et al., 196413). In contrast to bradykinin, eledoisin (0.01-1.0 pg/kg i.v.) stiinulates the rabbit uterus in situ (Stiirmer and Berde, 1963b; Fregnan and Glasser, 1964). Eledoisin in similar experiments was ineffective on the rat, cat, and guinea pig uterus (Fregnan and Glasser, 1964). Eledoisin constricts the bronchiolar niuscles of the guinea pig. This effect, is not antagonized by aspirin (Erspanier and Erspamer, 1962; Stiiriner and Berde, 196310; Gjuris and Westermann, 1963, 1965). On the isolated guinea pig lung eledoisin is 3 to 5 times a stronger bronchoconstrictor than bradykinin. Both peptides were about equally potent in constricting the pulinonary artery of guinea pig, but bradykinin was more active on the pulmonary vein preparation (Moog and Fischer, 1964). Eledoisin was a much more effective vasoconstrictor than bradykinin or kallidin in the perfused rabbit lung. The peptide was strongly tachyphylactic in this preparation (Hague et al., 1964).
68
ERVIN G. ERDOS
2. Permeability
Intradermal injection of eledoisin (>1ng) in man causes pain, local edema, and erythema (de Caro, 1963). Eledoisin and physalaemin increase the capillary permeability of various animals (Sturnier and Berde, 1963b; de Caro, 1963; Erspamer et al., 1964b) (Table 11).Like bradykinin, eledoisin also causes swelling of the isolated rat liver mitochondria (Berndt et al., 1965). Arterial infusion of eledoisin increases the uptake of Ps2-labeled thio-Tepa (triethylenethiophosphoramide) in the dog skin (Berg et al., 1965). 3. Cardiovascular E f e c t s
Interest in research with eledoisin was stimulated by the unusually high potency of the peptide in lowering the blood pressure of man and animal. Eledoisin and its analogs are among the strongest hypotensive agents known, but in contrast to bradykiniri only a relatively small part of the peptide chain is specific for this activity (Section V,A). Although both peptides are hypotensive, there is not much structural resemblance between bradykinin and eledoisin. There are qualitative and quantitative differences in the actions of the peptides on the blood pressure. a. Laboratory Animals. Subcutaneous administration of eledoisin (10100 pg) causes a drop in systemic blood pressure of the anesthetized dog which lasts for hours (Erspamer and Glaesser, 1963), although the peptide disappears from the circulation in 10-15 minutes (Erspamer and Anastasi, 1962). Only 1 of 3 dogs injected survived when injected with 150-300 pg/kg eledoisin. Intravenously adniinistered eledoisin (0.1 pg/kg) abolished the hypertensive effect of 10 pg/kg norepinephrine, and 0.2 pg/kg blocked the effect of 5 pg/kg of angiotensin (Erspanier and Glaesser, 1963). Intravenous injection of eledoisin to anesthetized dogs lowers the systemic blood pressure. Eledoisin is niore potent, in this test than bradykinin. Eledoisin was estimated to be 50 to 100 times more active on the circulation of dog than bradykinin (Sturnier and Berde, 1963b; Berganiaschi and Glasser, 1963; Erspainer and Erspamer, 1962; Erspanier and Glaesser, 1963). On a weight basis eledoisin is 300 times more hypotensive than histamine (Erspamer and Glaesser, 1963). Nitroglycerin was 1/1,000 to 1/10,000 as active as eledoisin on coiiimon carotid blood flow (Berganiaschi and Glasser, 1964) (see Fig. 12). The threshold dose of eledoisin is about 0.4-2.0 ng/kg. Intravenous injection of this amount of the peptide causes a short-lasting drop in blood pressure. Large doses decrease the diastolic pressure more than the systolic. The heart rate, the myocardial contractility, and cardiac output increase. These phenomena are caused probably by reflex sympathetic stimulation
HYPOTENGIVE PEPTIDES
69
FIG. 12. Effects of intraarterial injections of eledoisin (0.00002 pg/kg) (anterior descending branch of the left coronary artery). Coronary blood flow increases without change in systemir blood pressure, myocardial contractility, or heart rate. (Bergamaschi and Glasser, 1963.) Figure reproduced by permission of the American Heart Association, Inc.
as well as direct effect on the adrenal medulla. Eledoisin enhances the coronary blood flow. The threshold dose after intracoronary injection to dog was 0.02 ng/kg (Bergamaschi and Glasser, 1963; Nakano, 1964b) (Fig. 12). The systemic blood pressure decreases, the pressure in the pulmonary artery (Broghamnier, 1963), the central veiious pressure, and the left atrial pressure (Nakano, 1965a) rise. The period of increased cardiac output and pulmonary pressure was followed by a period of slightly decreased cardiac output and pulmonary pressure (Nakano, 196413). A remarkable feature of the cardiovascular effects of eledoisin is the strong peripheral vasodilatation. According to Bergamaschi and Glasser (1964), arterial injection of approximately 10-lb niole/kg eledoisin causes a noticeable vasodilation in the fenioral artery. The vascular bed supplied by the brachial artery is somewhat less sensitive than the area of the femoral artery (Nakano, 1964a). Bradykinin is 5 to 10 times less active in increasing the femoral blood flow when snialler doses are compared, but becomes nearly as active as eledoisin at doses higher than 0.1 ng/kg i.a. (Bergamaschi and Glasser, 1964). The area supplied by the superior mesenteric artery in dog is also very sensitive to elcdoisin (Chou et al., 1965). Here, larger intraarterial doses of eledoisin (0.5 pg/kg), however, cause a triphasic response in perfusion pressure which resembles the effect of endotoxin. The pressure dropped first,
70
ERVIN G . ERDOS
then rose rapidly, then dropped again (Chou et al., 1965). Eledoisin also increased the blood flow through the skin, head, and muscle (Bergamaschi and Glasser, 1964). Experiments in the anesthetized intact dog yielded results which were somewhat different from those obtained with other methods. Maxwell (1964) used the Fick principle (measurement of gas exchange and blood gas concentration) in his studies. He observed that infusion of large doses of eledoisin (2-3 pglkg) lowered cardiac output, systemic and pulmonary arterial pressure, “index of cardiac efficiency,” and coronary blood flow. No changes in peripheral resistance were seen. The effects here were deemed comparable to those caused by injection of endotoxin. Bradykinin in the same system behaved differently in that it increased cardiac output and coronary blood flow (Maxwell et al., 1962). I n the conscious dog the effect of the intravenous injection of eledoisin on the circulation a t the 0.4 pg/kg dose level resembled those of bradykinin and kallidin. Eledoisin initially increased cardiac output, accelerated the heart, and lowered systemic blood pressure and peripheral resistance. The depressor phase was followed by an elevation in pressure and resistance. Large doses of eledoisin (4 pg/kg) reduced cardiac output and lowered systemic pressure and peripheral resistance for a prolonged period. The lowering of cardiac output was attributed to vasodilation which reduced the blood return to the heart. The threshold dose of intravenous eledoisin in the unanesthetized dog is below 0.04 pglkg. No tachyphylaxis was observed in these studies (Olmsted and Page, 1962). In the dog, cross-circulstion preparation eledoisin did not induce cardiovascular actions of central origin (Kato and Buckley, 1965). The differences in the potency of bradykinin and eledoisin are not so striking in other laboratory animals as in the dog, especially when the animal has been pretreated with one of the inhibitors of the enzymic metabolism of bradykinin (Erdos, unpublished data, 1964). Some of the ratios of hypotensive effects of the two peptides are shown in Table 11. Eledoisin lowers the systemic arterial blood pressure of the cat (Sturmer and Berde, 1963b). As in other animals pretreatment with atropine did not block the drop in blood pressure. When the intravenous injection of eledoisin was repeated at short intervals, the sensitivity to the peptide decreased and tachyphylaxis was observed. Eledoisin (20 pg) did not affect the rise in blood pressure caused by 10 pg of norepinephrine in this animal (Erspamer and Glaesser, 1963). Eledoisin also lowers the blood pressure of rat, guinea pig, and rabbit (Table 11). I n the chicken, eledoisin raises blood pressure after a short initial drop (Sturmer and Berde, 1963b). Since sympatholytic drugs and reserpine abolish this rise in pressure, it may be due to the release of catecholamines (Erspamer and Glaesser, 1963; Nakano, 1964a). The rise in
HYPOTENSIVE PEPTIDES
71
blood pressure which occasionally follows the injection of bradykinin in chickens is much smaller than that caused by eledoisin (Konzett and Sturmer, 196011; Nakano, 1964a). In hypotensive rats with a systemic blood pressure of 30-50 mm Hg, eledoisin is hypertensive, because it releases catecholamines from the adrenals (Parratt, 1964a). b. Man. The very strong hypotensive effect of eledoisin in dogs suggested clinical trials and possible therapeutic applications in man. Eledoisin induced arterial hypotension and hypertension of the spinal fluid in sensitive individuals a t the 1.5-3 ng/kg i.v. dose levels (Sicuteri et al., 196213; Sicuteri et al., 1963d). Higher doses of the peptide (e.g., 200-300 ng/kg) caused hot flushes of the face, throbbing headache, increased rate of respiration, intestinal hyperperistalsis, and a prolonged fall in blood pressure (Sicuteri et al., 1963d). In addition, intense erythema, hoarseness, and edema of the eyelids were observed. Infusion of more than 2 pg/minute of eledoisin was not tolerated well by healthy, young male volunteers (Kontos et al., 1964b). Infusion of smaller doses of the peptide (below 0.6 pg/minute) increased the blood flow in the hands and arnis and the cardiac output. The changes brought about by the infusion of 0.6 pg/niinute eledoisin were markedly above those caused by 25 pg/minute bradykinin (Kontos et al., 1964b). Eledoisin increases the rate of respiration even in doses which do not influence blood pressure (Sicuteri et al., 1963d). Broghaninier (1963) after intravenous infusion noticed a significant increase in the muscle blood flow in healthy persons arid in persons afflicted with peripheral vascular diseases. Gersnieyer (1964) also tested eledoisin in patients with impaired peripheral blood flow. Infusion of eledoisin (0.0084. I pg/kg/minute) increased cardiac output and lowered the peripheral resistance. The increase in the blood flow in the muscle and the skin became noticeable a t dose levels which do not influence systemic blood pressure. The condition of a limited number of patients suffering from peripheral vascular diseases (Sicuteri et al., 1962b; Gersnieyer et al., 1965) has been observed to improve after treating them with eledoisin. Some other investigators had less favorable results to report or contradicted the findings of others. In these experiments (Gerola et al., 1964; Ciampolini et al., 1964) infusion of 0.8-1.5 pg of eledoisin/minute decreased the systemic pressure, but also the cardiac output, while the total peripheral resistance varied and central venous pressure increased. No drop in blood pressure was observed by Mertz (1964) after the administration of small doses of eledoisin (0.002-0.033 pg/kg) to man.
4. Kidney Eledoisin in moderate doses (10-20 ng/kg/minute i.v. or 1-5 ng/kg/ minute i.a.) is diuretic in the dog (Heidenreich et al., 1963). In man, eledoisin did not influence the electrolyte excretion in the urine. The peptide
72
ERVIN G. ERDOS
increased vascular resistance in the kidney and decreased the urine excretion (Mertz, 1964). Intravenous injection of eledoisin did not change the blood flow in the renal artery of the dog (Bergamaschi and Glasser, 1964). 5. Miscellaneous Effects Intravenous injection of eledoisin to man (Sicuteri et al., 1964s) and dog (Holemans, 1965) increases fibrinolytic activity in blood. Although both hypertensive and hypotensive agents enhanced fibrinolysis in the dog, 5 pg/kg eledoisin was the most active substance tested, surpassing, among others, histamine, bradykinin, or vasopressin. I n unanesthetized animals eledoisin induced salivation and general depression. In addition, in dogs subcutaneous injection of 10-100 pg/kg of eledoisin increases the gastrointest,inal motility (Erspamer and Erspamer, 1962). VI. Conclusions
After completing this article the reviewer estimated references cited to be over six hundred. But even six hundred references do not make this review a complete survey of the literature. Many more publications discussed some aspects of the role of kinins and kininogenases in various processes. The progress niade toward the understanding of the structure, action, and fate of kinins and eledoisin is quite impressive, especially if we consider the number of vasoactive substances which have appeared under various names in the literature since the turn of the century but never have been characterized. Eledoisin has not yet been found in the mammalian body, but this should not exclude possible future developments in this direction. This peptide is t,he strongest hypotensive agent known in man and in some animals. It is puzzling how unspecific its structure is. Studies on kallikrein were prompted by a simple question-Why does injection of urine drop the blood pressure of experimental animals? Now, forty years after the initial discovery, most of the components of the kallikrein-kinin system are available in synthetic, pure, or purified state, but the questions to be answered are more numerous and more complex. For the chemically oriented investigator the amino acid sequence of kininogen, the mechanism of molecular biosynthesis of this protein in the liver (and perhaps elsewhere), the characterization of the ornitho-kallikrein-kinin system, and the participation of catheptic enzymes in the activation of kallikrein are still challenging problems. For the pharmacologist the status of kinins somewhat resembles that of acetylcholine or histamine. Bradykinin may never become a therapeutically important agent. Nevertheless, if kinins play a significantJ role in some
HYPOTENSIVE PEPTIDES
73
physiological or pathological conditions, agents which block their effects or inhibit their enzymic metabolism would be of prime importance. It remains the task of the clinical investigator to evaluate thoroughly the clinical usefulness of the kallikrein inhibitor, Trasylol, and to localize the site of its action. NOTE ADDEDIN PROOF New information was reported a t the International Symposium on Hypotensive Peptides, October 25-29, 1965, Florence, Italy; E. G. Erdos, N. Back, and F. Sicuteri (eds.), “Hypotensive Peptides.” Springer-Verlag, New York. (In press.) A. Anastasi, V. Erspamer and M. Bernardi described the structure and synthesis of phyllokinin of amphibian skin. Most derivatives of peptides remain active when structural changes are introduced a t the N-terminal end, but become inactive when the C-terminal end i s modified. In contrast, phyllokinin from amphibian skin has the structure of bradykinin and two additional amino acids (isoleucine and tyrosine-o-sulfate) a t the C-terminal end, but it is more hypotensive than bradykinin. A possible interrelationship of substance P with eledoisin is suggested by the presence of ten out of eleven amino acids of eledoisin in substance P. E. Werle indicated that the amino acid composition of a kinin which is released in the plasma of birds (ornitho-kinin) is different from mammalian kallidin. Rat and horse urinary kallikrein are inhibited by benzamidine HC1 (C. D. Diniz). J. Spragg and K. F. Austen prepared H3-acetyl bradykinin. The labeled peptide was bound to specific antibradykinin antibody. Replacements of labeled bradykinin in the complex with unlabeled peptide offers a sensitive method for measuring bradykinin. Pepsin can break down bradykininogen to smaller but still active fragments (E. Habermann). One of these derivatives of kininogen has the sequence of Met-LysBradykinyl-Ser-Val-Glu(NHz), as confirmed by synthesis by Schroder. Various kininogenases can release bradykinin or kinin I1 from this substrate. W. Vogt described the existence of two separate, parallel kininogen-kinin systems in blood plasma of man and some animals. J. V. Pierce discovered two kininogens in human plasma. One of them held a kinin in C-terminal position. According to T. Suzuki the bradykinin moiety is located between two cysteine molecules that form a disulfide linkage in bovine kininogen. H. Moriya found in parallel bioassays that the ratios of activities of bradykinin and colostrokinin can vary in different tests. A catheptic enzyme which can release a kinin from kininogen was extracted from leucocytes and from spleen by L. M. Greenbaum. The importance of the kallikrein-kinin system in various pathological conditions has been emphasized repeatedly. T. Shimamoto correlates the venoconstriction caused by bradykinin with the subsequent development of arteriosclerosis. He obtained favorable results by treating patients with pyridinolcarbamate. This compound prevents venoconstriction induced by bradykinin. According to F. Sicuteri in myocardial infarction released serotonin may sensitize to bradykinin, and thus contribute to pain. In subarachnoid hemorrhage the release of a kinin upon dilution of the blood in cerebrospinal fluid may also explain the accompanying intense pain. Plasma fractions given in infusion can contain a large amount of active kallikrein that can account for side effects. I n gouty arthritis, urate crystals may adsorb Factor XI1 and thus trigger the release of bradykinin and aggrevate symptoms (V. Eisen and C. A. Keele). P. Di Mattei found bradykinin in human pulmonary carcinoma. I. Trautschold and E. Werle attributed the beneficial effects of the administration of the proteolytic inhibitor Trasylol in pancreatitis to the inhibition of serum kallikrein and/or to the inhibition of the activation of serum kallikrein. They also proposed a new unit for kallikrein activity. One unit of
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kallikrein is that amount of enzyme which hydrolyzes 1 pmole of BAEe in a minute. In bioassay 1 milliunit of kallikrein releases a kinin equivalent in activity to 1.06 pg of synthetic bradykinin or 1.2 pg of kallidin. They also suggested the replacement of the biological kallikrein inhibitor unit (KIU). One international inhibitor unit is the amount of inhibitor that inhibits the action of one international trypsin unit in chemical assay. M. A. Webster compiled the report of an International Committee on Nomenclature. Accordingly kininogenases are enzymes which liberate a kinin from an inactive protein substrate. Kinins are hypotensive peptides which resemble bradykinin in structure and pharmacological activity. The continued use of the terms prekallikrein (kallikreinogen), kallikrein and kininogen was also suggested. REFERENCES Aarsen, P. N., and Kemp, A. (1962). Brit. J. Pharmacol. 19, 442. Aarsen, P. N., and Kemp, A. (1963). Nature 198, 687. Abe, K., Yoshinaga, K., Miwa, I., Aida, M., Maebashi, M., and Watanabe, N. (1964). Tohoku J . Exptl. Med. 82, 270. Abelous, J. E., and Bardier, E. (1909). Compt. Rend. Sac. Biol. 66, 511. Afonso, S., Rowe, G. G., Castillo, C. A., Lowe, W. C., and Crumpton, C. W. (1962). Federation Proc. 21, 94. Allgower, M. (1962). I n “Shock Pathogenesis and Therapy” (K. D. Bock, ed.), p. 240. Academic Press, New York. Allwood, M. J., and Lewis, G. P. (1964). J . Physiol. (London) 170, 571. Amundsen, E., Nustad, K., and Waaler, B. (1963). Brit. J . Pharmacol. 21, 500. Amundsen, E., and Nustad, K. (1964). Brit. J . Pharmacol. 23, 440. Amundsen, E., and Nustad, K. (1965). J. Physiol. (London) 179, 479. Amundsen, E., and Rugstad, H. E. (1965). Brit. J . Pharmacol. 26, 67. Amundsen, E., Waaler, B., Dedichen, J., Laland, P., Laland, S., and Thorsdalen, N. (1964). Nature 203, 1245. Anastasi, A,, and Erspamer, V. (1962). Brit. J . Pharmacol. 19, 326. Anastasi, A., and Erspamer, V. (1963). Arch. Biochem. Biophys. 101, 56. Anastasi, A., Erspamer, V., and Cei, J. M. (1964). Arch. Biochem. Biophys. 108, 341. Anastasi, A., Erspamer, V., and Bertaccini, G. (1965). Camp. Biochem. Physiol. 14, 43. Anderer, F. A. (1965). 2. Naturforsch. 2Ob, 462 and 499. Anderer, F. A., and Hornle, S. (1965). 2. Nalurforsch. 20b, 457. Andrade, S. O., and Rocha e Silva, M. (1956). Biochem. J . 64, 701. Antonio, A., and Rocha e Silva, M. (1962). Circulation Res. 11, 910. Antopol, W., and Chryssanthou, C. (1963). Ann. N.Y. Acad. Sci. 104, 346. Armstrong, D., and Stewart, J. W. (1960). Nature 188, 1193. Armstrong, D., and Stewart, J. W. (1962). Nature 194, 689. Armstrong, D., Dry, R. M. L., Keele, C. A., and Markham, J. W. (1953). J . Physiol. (London) 120, 326. Armstrong, D., Keele, C. A., Jepson, J. B., and Stewart, J. W. (1954). Nature 174, 791. Armstrong, D., Jepson, J. B., Keele, C. A., and Stewart, J. W. (1955). J . Physiol. (Londm) 129, 8OP. Armstrong, D., Jepson, J. B., Keele, C. A., and Stewart, J. W. (1957). J . Physiol. (London) 136, 350. Armstrong, D., Keele, C. A., and Stewart, J. W. (1960). J . Physiol. (London) 160, 2OP. Armstrong, D., Mills, G. L., and Sicuteri, F. (1965). Biochem. Pharmacol. 14, 1388. Asang, E. (1960). Arch. Klin. Chir. 298, 645.
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Uricosuric Drugs, with Special Reference to Probenecid and Sulfinpyrazone* ALEXANDER B. GUTMAN Department of Medicine, The Mozinl Sinai Hospital, New York, New York
I. Uricosuric Activity Defined . . . . . . . . . . . 11. Physiological Basis for Use of Uricosuric Drugs . . . . . . 111. Nature of Uricosuric Response in Normal and Gouty Man; SpeciesDependency . . . . . . . . . . . . . . IV. The Search for a Suitable Uricosuric Agent . . . . . . . A. Early Attempts to “Solubiliee” Urate Deposits . . . . . B. Salicylate . . . . . . . . . . . . . . C. The 2-Phenylcinchoninic Acids: Cinchophen and Analogs . . . D. Zoxaeolamine . . . . . . . . . . . . . V. The Alkylsulfonamidobenzoic and N-Alkylsulfamylbeneoic Acids . . A. The Alkylsulfonamidobenzoic Acids: Carinamide . . . . . B. The N-Alkylsulfamylbenzoic Acids: Probenecid . . . . . C. Structure-Activity Relationships of Probenecid Analogs . . . . VI. The Pyraeolidinediones . . . . . . . . . . . A. Phenylbutaeone . . . . . . . . . . . . B. Phenylbutazone Metabolites . . . . . . . . . . C. 1,2-Diphenyl~-(phenylthioethyl)-3,5-pyraeolidinediorie . . . . D. Sulhpyraeone . . . . . . . . . . . . . E. Structure-Activity Relationships in the Pyraeolidinediones . . . VII. Incidental Compounds Possessing Uricosuric Properties: Phenolsulfonphthalein, Mersalyl, Iodopyracet, Corticotropin and Adrenocortical Steroids, Coumarins and Indandiones, Chlorprothixene, Acetohexamide, Ethyl-pchlorophenoxyisobutyrate. The Paradoxical Action of Beneothiadiazines References . . . . . . . . .
91 92 96 99 99
100 103 105 107 108 110 116 117 118 120 122 124 128
132 134
I. Uricosuric Activity Defined
Uricosuric drugs, as here defined, increase urinary uric acid excretion by inhibiting renal tubular reabsorption of uric acid, and thereby reduce the plasma urate concentration and enhance the renal clearance of uric acid. The compounds possessing authentic uricosuric properties are of diverse structure, with no apparent coninion coniponent, but virtually all are organic acids; a striking and inexplicable exception is zoxazolamine, a weak base yet one of the most potent of the uricosuric drugs. The uricosuria produced by these various agents really represents a
* The author’s studies herein referred to were supported, in part, by Public Health Service Grant AM-00162 from the National Instit,ute of Arthritis and Metabolic Diseases. 91
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net renal response, since the acidic uricosuric drugs are all secreted by the same “organic acid system” of renal tubular transport, in common with uric acid, and in the process may also competitively inhibit, to greater or lesser degree, the tubular secretion of uric acid. When there is a dual action on tubular reabsorption and secretion of uric acid, the net effect, whether it is to augment or to diminish urinary elimination of uric acid, varies with the nature of the drug, the dosage employed, and the species treated. Many of the compounds with which we are here concerned because they are predominantly uricosuric in man have little effect in other animals, or cause distinct retention of uric acid. Even in man there is a wide spectrum of drug effects on the renal regulation of uric acid excretion. The most potent uricosuric agents, sulfinpyrazone and zoxazolamine, elicit no demonstrable uric acid retention in any dosage; many, like salicylate, exhibit a more or less pronounced “paradoxical” action over the dosage range in common use; and some compounds, like pyrazinoic acid, only suppress uric acid excretion, and do not produce uricosuria in any dosage. Of the uricosuric agents, probenecid and sulfinpyrazone will be considered in greatest detail since they have proved to be the most satisfactory yet brought forward, and are widely employed for therapy, principally in primary gout. A number of other compounds possess authentic uricosuric properties and will be described. Excluded by our definition, and not considered here, are compounds that increase urinary excretion of uric acid by augmenting uric acid production, such as preformed purines and amino acid precursors of uric acid taken in the diet, cytolytic agents that accelerate the degradation of nucleic acids, and the 2-substituted thiadiazoles that stimulate de novo purine biosynthesis. If the rate of uric acid formation is sufficiently enhanced, plasma urate levels tend to rise and urate spills over into the urine, presumably in part because a larger proportion of the urate filtered a t the glomerulus escapes tubular reabsorption, in part because of augmented tubular secretion of urate; Cur and C,,/GFR usually increase somewhat. Also excluded are such antimetabolites as allopurinol [4-hydroxypyrazolo (3,4-d)pyriniidine] and DON (6-diazo-5-oxo-~-norleucine) which inhibit uric acid biosynthesis and so lower the uric acid in both plasma and urine. Colchicine, the time-honored specific for acute gouty arthritis, likewise is extraneous to this review since it has no uricosuric effect or other discernible action on uric acid metabolism. II. Physiological Basis for Use of Uricosuric Drugs
Uric acid (2,6,8-trioxypurine), the final intact purine formed by biological oxidation of the purine ring, is the chief end product of purine metabolism in man. It owes its weakly acidic properties to ionization a t
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N-9 in the physiological p H range (Bergmann and Dikstein, 1955). With a pK, of 5.75 (Bergniann and Dikstein, 1955), uric acid is virtually completely dissociated at the pH of the plasma and circulates as the monovalent ion, whereas a t the lower pH of distinctly acid urine it is largely undissociated and is excreted in the urine for the most part as the free acid (reviewed by Seegmiller et al., 1963). The monosodium salt is soluble in water a t 37°C to a very limited extent, about 120 mg/100 ml, the free acid only to approximately 6 nig/lOO ml ; however, supersaturation occurs in the urine and other biological fluids which can thus hold higher concentrations in more or less stable solution (Peters and Van Slyke, 1932). Man normally has a conspicuously large body urate pool, averaging 1.2 gni, and a high plasma urate concentration, averaging about 5.0 mg% in adult males, as compared to most other mammals ; in the dog, for example, the plasma urate level is usually 0.2-0.5 mg%. This disparity is the result of a genetic mutation in the early evolutionary development of the anthropoids, which led to the loss of uricase from human tissues and hence of the main metabolic pathway for degradation of uric acid. The efficient reabsorption of uric acid by the renal tubules, which in most mammals operates to recycle uric acid to the liver for conversion there to more soluble allantoin, continued nevertheless, contributing to maintenance of the relative hyperuriceniia of normal man, and the even more pronounced hyperuriceniia of gouty man. Persistence of effective tubular reabsorption of the filtered urate does, however, serve a useful purpose in reducing the urinary uric acid, and thus the hazard of uric acid urolithiasis, by facilitating diversion of a substantial proportion of the uric acid into the bowel. Of the mean approximately 750 nig of uric acid formed daily by normal man on a diet low in purines and limited in proteins, a mean of about 420 f 80 mg daily is excreted in the urine (Gutman and Yu,1957a), the remainder very largely by way of the gut, where it is promptly degraded by the uricase, allantoinase, allantoicase, and urease of the intestinal bacteria (S@rensen, 1960). There is a further substantial increase in the body urate pool and plasma urate in primary gout, because of a superimposed inborn error of metabolism as yet not clearly defined; and also in a variety of other disorders, particularly of heniopoiesis such as polycytheniia Vera and the inyeloproliferative diseases, characterized by increased turnover of an expanded nucleic acid pool (for further discussion, see Wyngaarden, 1960; Seegmiller et al., 1963; Gutman and Yu,1965). So sparing is the solubility of uric acid and its sodium salt that any appreciable increase in the urate concentration of the circulating fluids, for whatever reason, favors deposition of the solid phase, chiefly as the monosodium urate monohydrate (Howell et al., 1963) a t certain tissue sites of predilection. Deposits form most readily on the articular
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ALEXANDER B. GUTMAN
cartilages and in the pinna (perhaps because of the presence in cartilage of acid polysaccharides), in the synovial membranes and fluid of the joints, in the bursae, tendons, and skin, and in the kidneys. In the joints, such microcrystalline precipitates play an as yet not altogether clearly defined role in eliciting the typical attacks of acute gouty arthritis (reviewed by Seegmiller and Howell, 1962; McCarty, 1964). In some cases, such tophaceous deposits increase to great size and cause the marked disabilities and deformities of advanced tophaceous gout and chronic gouty arthritis. If uric acid calculi form in the collecting passages of the urinary tract, they are composed very largely of the free acid (Howell et al., 1963) as long as the urine is distinctly acid, as it almost always is; and the propensity to uric acid urolithiasis is greater when the urine pH is unduly acid, the amount of uric acid eliminated in the urine is increased, or the urine volume is contracted. Deposition of uric acid crystals in the kidney contributes to the conglomerate of pathological changes collectively designated the gouty kidney. To combat this trend to precipitation of solid-phase urate in the tissues when hyperuriceniia is grossly excessive, it is necessary to lower the plasma urate concentration a t least to the upper limits of normal, or somewhat above, whereupon the deposition of solid-phase urate ceases. Indeed, if the plasma urate concentration can be made to fall low enough, the flow of urate may be reversed in adequately vascularized tophi, the urate deposits brought back slowly into solution, and caused gradually to decrease in size and even to disappear. There are two ways to reduce the plasma urate concentration: by slowing the rate of uric acid production, by accelerating the rate of uric acid elimination, or both. It was not feasible to suppress uric acid formation appreciably, except by dietary restriction of the purine and protein intake, and then usually inadequately, until the advent in 1963 of the xanthine oxidase inhibitor, allopurinol (Rundles et al., 1963, 1964; Klinenberg et al., 1963; Yu and Gutman, 1964). Acceleration of renal elimination of uric acid, by inhibiting tubular reabsorption of urate through the use of uricosuric drugs, has been possible for some time, although therapeutically not satisfactorily until the introduction of probenecid in 1950, and sulfinpyrazone in 1957. That the increased urinary excretion of uric acid produced by uricosuric drugs in man is in fact brought about by inhibition of tubular reabsorption of urate has been demonstrated by clearance studies showing a characteristic sharp rise in Cur and C,,/GFR, which in men norinally average 8.7 f 2.5 nil/min and 7.6 f 2.4%, respectively (Gutinan and Yu, 1957a). The physiological implications of the increase in uric acid clearance follow, since it is known that the renal mechanisms for regulation of uric acid
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excretion in nian involve a complex three-phase process of filtration of the plasma urate a t the glomerulus, reabsorption of the filtered urate by the tubules, and tubular secretion of urate (Gutman and Yii, 1961). Uricosuric drugs (at least probenecid and sulfinpyrazone) do not cause any significant increase in the filtered urate load, since there is no evidence that they produce metabolic overproduction of uric acid, they have no appreciable influence on the glonierular filtration rate in ordinary dosage, and they do not alter passage of uric acid across the glomerular membrane (uric acid is not bound to plasiiia proteins). They must therefore modify tubular transport of uric acid, either by reducing tubular reabsorption or accelerating tubular secretion. So far as can be determined, when uricosuric drugs have any detectable effect on tubular secretion of uric acid, it is not to increase but to diminish it. This leaves inhibition of tubular reabsorption of uric acid to be considered as the principal mode of action of uricosuric drugs. Even niinor inhibition of tubular reabsorption of urate by uricosuric drugs would appreciably enhance thc urinary excretion of uric acid in man, since the quantity of urate filtered a t the glomerulus and subsequently reabsorbed by the tubules is so much greater than that appearing in the urine. Thus the filtered urate load averages approximately 6.5 f 1.4 mg/niin in normal men, whereas a mean of only 0.49 f 0.16 nig/niin is excreted in the urine, under the conditions of clearance measurements and when the diet is low in purines and restricted in proteins; in primary gout the disparity is even greater, 10.1 f 2.8 mg/min and 0.66 f 0.24 ing/niin, respectively (Gutnian and Yii, 1957a). The amount of urate actually reabsorbed exceeds these differences between the filtered urate load and U,,V, because urate is concomitantly added to the tubular fluid by tubular secretion (Gutnian et al., 1959), hence the calculated difference represents only the net of bidirectional tubular transport of urate. It now seems likely that, under ordinary circumstances, virtually the entire filtered urate load is reabsorbed by the tubules, possibly also sonie that is secreted by the tubules. Consistent with this view is the large tubular capacity for reabsorption of uric acid in nian, estiniated by Berliner et al. (1950) to average approximately 15 nig/min/1.73 ni2, a figure well in excess of any filtered urate load likely to be presented to the tubules in normal or gouty subjects. Even this figure underestimates the true reabsorptive Tin for uric acid in man, since it does not take siiriultaneous tubular secretion of uric acid into account. Other than the large but liinited capacity of the tubules for reabsorption of urate, nothing is known of the nature of the reabsorptive process or how uricosuric agents inhibit it. Tubular reabsorption of urate in niaii is presumed to be acconiplished very largely in the proximal convolution and
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by active transport, in view of its limited capacity and the apparently competitive nature of the action of uricosuric drugs, but as Weiner and Mudge (1964) point out, there is also the possibility of diffusion mediated by a specific membrane carrier (Wilbrandt and Rosenberg, 1961). Nonionic back-diffusion plays a minor role, since there is little increase in urinary excretion of uric acid following a change from distinctly acid to distinctly alkaline urine pH. In respect to tubular secretion, the mechanism and kinetics of transport also are still obscure, but competitive inhibition by a number of organic acids secreted by an “organic acid system” (H. W. Smith et al., 1938; Weiner and Mudge, 1964), including various uricosuric drugs, suggests an active process. A proximal site of tubular secretion of urate can be demonstrated in the Dalmatian coach hound (Kessler et al., 1959a; Yu et al., 1960a, 1961) and rabbit (Beechwood et al., 1964), and is presumed to occur there in other species even when it cannot be demonstrated because of preponderant tubular reabsorption (Yu et al., 1960a). More distal tubular secretion of urate apparently also occurs (Yu et al., 1960a; Davis el al., 1965). Ill. Nature of Uricosuric Response in Normal and Gouty Man; Species-Dependency
When a potent uricosuric drug is given in adequate dosage to normal human subjects there is a prompt increase in renal excretion of uric acid, accompanied by a fall in plasma urate, but the urinary uric acid excess disappears after the first day or two, despite continued administration of the drug. This does not signify acquired refractoriness, since the reduction in plasma urate persists and the urate clearance remains above normal. The normal body pool of urate is so limited, however, that it is depleted after a day or two of substantially increased drainage through the kidneys. Thereafter, a steady state of balance between production and elimination is reestablished, and the rate of urinary excretion of uric acid is determined by the normal rate of uric acid production. The uricosuric response in patients with primary or secondary gout is more sustained, because the body pool of urate is expanded and the rate of uric acid formation is augmented. Thus in one case of advanced tophaceous gout, in which daily 24-hour urine collections were made and the urinary uric acid excretion determined over a year of regular uricosuric drug therapy, it was estimated that the excess uric acid eliminated during that period totaled approximately 100 gm (Gutman and Yu, 1955). The excess of uric acid disposed of under these circumstances derives from two sources: in part from solid-phase urate deposits redissolved by in vivo dialysis against a circulating fluid of sufficiently low urate concentration, in part from the urate produced in excess by the underlying metabolic error-urate which
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would ordinarily be reabsorbed by the renal tubules and recycled, but is not when reabsorption is inhibited by uricosuric drugs. There is now abundant documentation, from many sources, that it is entirely feasible by regular use of a suitable uricosuric agent to decrease the plasma urate concentration sufficiently, and long enough, to prevent the formation of t,ophaceous deposits in primary and secondary gout, apparently indefinitely, and to mobilize extensive tophi already present. To accomplish this, however, it is essential that the uricosuric drug be administered daily in effective dosage, hence that it be well tolerated, so inhibition of tubular reabsorption of urate can be maintained. When such drugs are discontinued, the patient, soon returns to positive uric acid balance and the plasma urate concentration resumes its initial level. It is also increasingly claimed that continued administration of uricosuric drugs prevents recurrence of attacks of acute gouty arthritis. How this is accomplished is not clear. Unlike colchicine, a generally effective prophylactic when given daily in low (suppressive) dosage, without altering the plasma urate concentration, probenecid and sulfinpyrazone are conipletely devoid of any ameliorative action in acute gouty attacks, despite marked lowering of the plasma urate level. In fact, when these uricosuric drugs are first administered in an at tack-free interval, precipitation of acute gouty arthrit,is is not unusual. Nevertheless, it is commonly inferred that reducing the plasma urate concentration to normal limits by the use of uricosuric drugs, and maintaining it there indefinitely, ultimately obviates acute gouty attacks. It is quite conceivable that dissolution of tophaceous deposits in and around the joints removes an incitant and may thus contribute to fewer seizures. The whole question deserves more careful examination than it has yet received. In some reports, for example, prophylactic colchicine has been given concurrently with uricosuric drugs, yet the ensuing prophylaxis has been ascribed solely or in large part to the uricosuric drugs. The experience with colchicine prophylaxis (Yu and Gutman, 1961) emphasizes the need, in a disease as capricious as gout, for sufficiently long-continued studies in a sufficiently large and well-controlled series of cases before any firm judgment in the matter can be made. Even when potent, regularly administered, and well-tolerated, the effectiveness of uricosuric drugs in gouty inan is subject to certain limitations. Probenecid and sulfinpyrazone have no analgesic or anti-inflammatory powers in uricosuric dosages, hence do not relieve the joint stiffness and pains of chronic gouty arthritis until the responsible tophaceous deposits have been slowly mobilized; and, as already mentioned, they are impotent in the treatment of acute gouty arthritis. In gouty subjects with seriously impaired renal function the diseased kidneys often are not amenable to the action of uricosuric drugs, and such patients therefore are usually
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found to be more or less refractory. On this account, and because of the extensive foreign-body connective tissue reaction to urate crystals, it is probably not possible to repair renal damage resulting from tophaceous deposits in the kidney, even when tophi in and around the joints disappear and joint functions are fully restored. It may be a forlorn hope even to prevent deposits of urate in the kidney parenchyma by the use of uricosuric drugs, since in untreated patients such deposits usually are found in the renal medulla, not in the proximal convolutions of the renal cortex where tubular reabsorption of urate presumably occurs in man, and the presumptive site of action of uricosuric agents. Localization in the renal medulla suggests that the chief seat of uric acid precipitation in such cases is the collecting tubule, where the concentration and acidity of the fluid of the lumen approach that of the final urine. I n fact, uricosuric agents tend to aggravate the formation of uric acid microliths in and around the distal sections of the nephron because, by inhibiting tubular reabsorption of urate, they add materially to the excretory burden of the urinary passages. An increase in the incidence of uric acid calculus in patients taking uricosuric drugs is well documented, but this hazard can be minimized by maintaining copious urine volumes and, if the urine is persistently very acid, by adding the further precaution of alkalinizing agents. When stones are already present in the urinary tract, or threaten to form, uricosuric drugs should not be employed except for the most compelling reasons; in these circumstances the use of allopurinol is preferable. For those who contend that the basic anomaly of primary gout is a n inborn metabolic error resulting in overproduction of uric acid (evidence summarized by Gutman and Yii, 1965), it may also be considered a deficiency of uricosuric drugs that their use in gouty subjects does not strike at the presumptive primary cause, but rather that they operate indirectly, by inducing what is in effect a compensatory abnormality of the kidneys. In this respect allopurinol comes closer to ideal corrective therapy but, by inhibiting xanthine oxidase, allopurinol suppresses the ultimate and penultimate steps in uric acid synthesis, whereas the metabolic error in primary gout is believed to reside in the first, committed reaction of de novo purine synthesis, in which the amide moiety of glutamine interacts with 5-phosphoribosylpyrophosphate to form 5-phosphoribosylamine. For those who contend that a t least a substantial minority of cases of primary gout have as their basic anomaly a defect in renal tubular transfer of uric acid (evidence reviewed by Seegmiller et al., 1963), this objection would not apply in the cases in question. I n species other than man, especially gouty man, the uricosuric drugs evoke a much less pronounced increase in Curand C,,/GFR, indeed may be entirely ineffective or even act in a reverse manner. I n the mongrel dog, for
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example, only a comparatively minor uricosuric response can be elicited with probenecid (Beyer, 1954; Yu et al., 1960a), sulfinpyrazone (Yu et al., 1963), or salicylate (Friedinan and Byers, 1948; Yu et al., 1963), and a “paradoxical” action of salicylate 011 uric acid excretion, so strikingly exhibited in man, cannot be denionstrated (Yu et al., 1963). In the Dalmatian coach hound, which is deficient in the tubular reabsorptive mechanism for urate, the same drugs are apt to decrease urinary elimination of uric acid when they have any effect at all (Friednian and Byers, 1948; Beyer, 1954; Yu et al., 1960a). In the rabbit, probenccid quite regularly diininishes renal excretion of uric acid (Poulsen, 1955; Beechwood et al., 1964). In the chicken, the elimination of uric acid is reduced by probenecid, sulfinpyrazone, and zoxazolaniine, and salicylate has little or no effect (Nechay and Nechay, 1959; Berger et al., 1960). The reasons for such marked species differences are not altogether clear but they seein to reflect, in the main, the inhibitory action of these drugs on bidirectional tubular transfer of uric acid, a dual effect on both reabsorption and secretion which is evident in some of them, salicylate, for example, even in man. In species in which the tubular apparatus for reahsorption of urate is defective (as in the Dalmatian coach hound) or poorly developed (as in the chicken), the predominant effect of drugs uricosuric in man is inhibition of tubular secretion of urate, whereas in man, whose tubular reabsorptive capacity for urate is large, the net effect of inhibition of both reabsorption and secretion of uric acid by the tubules ordinarily is uricosuric. However, this explanation does not account for all the discrepancies rioted in different species; full clarification niust await elucidat,ion of the still obscure nature of the mechanisms of bidirectional tubular transfer of uric acid, and how they are affected by the drugs in question. IV. The Search for a Suitable Uricosuric Agent
A. EARLY ATTEMPTS TO “SOLUBILIZE” URATEDEPOSITS The history of the long quest for means to coinbat the hyperuriceinia and tophaceous deposits of gout was recently reviewed (Gutman, 1963a). The earliest efforts were aimed a t “solubilizing” urate accumulations, particularly by conversion to the inore readily soluble lithium urate, through the internal and external applicatioii of natural and prepared lithiated waters. This approach was popularized by Garrod (1876), who reported beneficial results in several cases. However, although widely administered for a great many years, no credible allegations of success were recorded, and a t long last recognition of the serious toxic effects of lithium (reviewed by Schou, 1957) put an end to these endeavors. A prolonged effort to use for this same purpose the organic bases piperazine, piperidine,
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and other ethyleneamines, urate solvents in vitro, likewise proved unrewarding.
B. SALICYLATE Shortly after the discovery in 1876 of the striking antirheumatic properties of salicylate, it was found to ameliorate the inflammatory manifestations of gouty arthritis, and also to increase urinary uric acid excretion, with lowering of the plasma urate (SBe, 1877; Byasson, 1877; Campbell, 1878; reviewed by Gross and Greenberg, 1948, and Dixon et al., 1963). In fact it was claimed that after daily administration of salicylate for a few weeks or months, tophaceous deposits diminished in size and even almost vanished (SBe, 1877; Campbell, 1878). However, these enthusiasms began to wane when it was found that, despite its anti-inflammatory and analgesic qualilies, salicylate proved to be inferior to the specific, colchicine, for control of acute attacks of gouty arthritis. Moreover, to sustain its uricosuric effects required daily doses of 5 gm or more, which resulted in salicylate intoxication not long tolerated by most patients. To minimize toxicity, salicylate was given in interrupted dosage, on only 3 successive days of the week (Jennings, 1937), a regimen that was calculated to make a perceptible impact on the development of tophi, but in practice failed to do so. As subsequently disclosed, there is a “compensatory” retention of uric acid for a day or two when uricosuric doses of salicylate are discontinued, with little or no net loss of uric acid from the body. On this account, to deplete the body stores of urate in gout it is necessary to maintain large daily doses of salicylate regularly, for protracted periods if tolerated sufficiently long. This was finally partially accomplished by Stetten and associates (Benedict et al., 1950) who gave 2 . 6 4 . 0 gm aspirin daily for 3 months to a patient with advanced tophaceous gout and recorded a marked decline in the uric acid pool without, however, any demonstrable change in the tophaceous deposits. Shortly thereafter came documentation that administration of salicylate in 5-gm daily dosage, if sustained uninterruptedly for a t least 6 or 9 months, can prevent formation of fresh tophaceous deposits in gout, and can even cause long-established tophi to disappear (Yu and Gutman, 1951; Marson, 1952, 1953). This success with salicylate afforded the first documented and reproducible demonstration that it is entirely feasible to prevent the deposition of urate in gout, indeed even to niobilize tophi long present, by daily administration of a potent uricosuric agent for the protracted periods required. The encouraging results with salicylate thus stimulated a continuing search that led ultimately to probenecid and sulfinpyrazone. The use of salicylate as a uricosuric agent was soon generally abandoned, however, because it was found to necessitate a therapeutic tour de force
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impracticable for all but the most hardy. Salicylate in sufficient and prolonged dosage is well known to disaffect several basic metabolic processes, uncoupling oxidative phosphorylation, inhibiting transaminases and dehydrogenases, producing a severe metabolic acidosis following transient respiratory alkalosis (reviewed by M. J. H. Smith, 1963). The list of side reactions it may elicit is a formidable one; in addition to the traditional and inevitable symptoms of acute salicylism, there may be gastric bleeding, anemia, insidious mental aberration, hepatic and renal damage, desquaniation of epithelial surfaces, etc. In smaller, analgesic dosage, salicylate is still occasionally employed to combat the stiffness and aching pains of chronic gouty arthritis, since the uricosuric agents presently available are devoid of any analgesic or anti-inflammatory properties. Even here the use of salicylates is severely limited by the circumstance that it counteracts the uricosuric action of probcnccid and sulfinpyrazone, hence should not be used in conjunction with them. This objection does not, however, apply to allopurinol. Extensive studies have been made of the renal clearance of salicylate, taking advantage of t,he ready availability of methods for measuring salicylate in plasma and urine (Brodie et al., 1944;Schachter and Manis, 1958), and these studies have contributed much to our understanding of the renal mechanisms for handling organic acids generally, including other acidic conipounds with uricosuric properties and uric acid itself. Salicylate is filtered at the glomerulus (approximately 1/3 of the plasma salicylatc, over a range of 2-35 mg%, is not bound to albumin in man) and is both secreted and reabsorbed by the tubules (Gutman et al., 1955). When the urine is acid there is net tubular reabsorption since, by virtue of its pK, of 3.0, salicylate is very largely undissociated in distinctly acid urine and is rapidly reabsorbed by nonionic diffusion (Milne et al., 1958). If the urine is made alkaline, there is net tubular secretion since the salicylate in the tubular lumen, derived by active tubular secretion (Weiner et al., 1959) as well as glomerular filtration, is largely “trapped” in ionized form which does not readily back-diffuse. The clearance of salicylate is thus greatly enhanced when the urine is alkaline, and SO-SO% or more of the augmented total salicylate excreted is unconjugated; this is, in fact, the only circumstance in which salicylate is appreciably eliminated in the urine unchanged. Ordinarily, i.e., in acid urine, most of the free salicylate of the tubular fluid having been reabsorbed, what remains is largely conjugated forms which are filtered a t the glomerulus and also actively secreted by the tubules (Schachter and Manis, 1958)but are not as freely diffusible as free salicylate, hence are not so rapidly reabsorbed. Salicyluric acid, a glycine conjugate, constitutes 5565% of the total salicylate in acid urine, salicyl phenolic and acyl glucuronides constitute 20-30%, the remainder is free
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salicylate; also present in small amounts are salicyl oxidation products, gentisic acid (2,5-dihydroxybenzoic acid), 2,3-dihydroxybenzoic acid, and 2,3,5-trihydroxybenzoic acid (Milne, 1963, see Fig. 2). Salicylate has a striking iLparadoxicaI1'effect on renal excretion of uric acid in man (Fauvel, 1907; Yu and Gutman, 1955, 1959b). In oral doses of 1-2 gm/day, salicylate causes more or less retention of uric acid, in contrast to the markedly uricosuric action of daily doses of 4-5 gm or more; intermediate doses have little or no net effect. This phenomenon is illustrated in Table I, which summarizes the results of slow, sustained infusion of TABLE I EFFECTOF SLOW,SUSTAINEDINFUSION OF SODIUMSALICYLATE ON URIC ACID EXCRETION IN MAN' Salicylate
0 0.23 0.69 1.32 1.78 2.89 4.23
0 1.4 2.8 5.7 9.0 12.4 17.7
Urate
0 0.04 0.24 0.41 0.59 0.90 1.73
9.1 9.3 9.3 9.2 8.9 8.9 8.8
0.88 0.52 0.32 0.37 0.62 0.90 1.65
9.7 5.6 3.4 4.0 6.9 10.1 18.8
7.5 5.2 3.1 4.1 6.8 9.6 17.7
Salicylate infusion stopped
uv
uv
Time (hours)
P (mg%)
P (mg%)
(mg/min)
C (ml/min)
Cur/GFR
(mg/min)
0-4 4-8 8-12 12-16 16-20
20.2 18.2 14.8 12.0 8.0
1.05 0.98 0.49 0.41 0.33
8.6 8.0 7.8 7.9 8.4
1.23 1.32 0.54 0.40 0.42
14.3 16.5 6.9 5.1 5.0
16.8 14.6 7.4 4.2 5.1
(%)
From Yu and Gutman (1959b).
sodium salicylate on the urinary uric acid excretion in a gouty subject. The first response, while salicylate levels in the plasma and urine were still low, was a reduction in Curand CJGFR. As the infusion was continued, and plasma and urinary salicylate levels rose, Curand C,,/GFR returned to preniedication figures, then distinctly exceeded them when the salicylate load reached uricosuric proportions. Upon discontinuing the infusion, the same effects were noted in reverse order, and could be correlated more
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closely with the rate of renal excretion of unconjugated salicylate than with the plasma salicylate concentration-explaining the “compensatory” retention of uric acid previously mentioned as occurring with intermittent medication with salicylate. This “paradoxical,” dosage-dependent action of salicylate in nian, which incidentally is not demonstrable in the dog (Yu et al., 1963), has been explained (Yu and Gutnian, 1955, 1959b) by assuming that small doses of salicylate inhibit tubular secretion of urate and larger doses also inhibit tubular reabsorption of urate. If tubular reabsorption of urate is sufficiently suppressed, the net response is markedly uricosuric because so much more urale ordinarily is reabsorbed than is excreted, but with intermediate doses the two effects more or less cancel out to give little or no net change in urinary excretion of uric acid. The “paradoxical” action of salicylate relates to the rate of renal excretion of unconjugated salicylate, not to the plasma salicylate concentration, and therefore is chiefly due to an effect upon renal tubular transport. This is especially evident from the marked potentiation of salicylate uricosuria that ensues when the urine is alkalinixed and urinary excretion of unconjugated salicylate is greatly enhanced, at the expense of the plasma salicylate. Whether salicylate (or any other uricosuric agent) inhibits tubular reabsorption of urate in nian by conipetition for energy sources, limited receptor sites or specific carrier, etc., is not known; in fact, as already mentioned, no information is available as to the very nature of tubular reabsorption of urate except that it appears to be active and is presumably located in the proxinial convolution. Inhibition of active tubular secretion of urate [and of a number of other acidic compounds (Weiner and Mudge, 1964)] by active tubular secretion of salicylate presumably results from competition for transport by the “organic acid system.”
c. THE2-PHENYLCINCHONINIC ACIDS: CINCHOPHEN AND
ANALOGS
Cinchophen (2-phenylcinchoninic acid) (Fig. 1), first synthesized in 1887 hy Doebner and Giesecke, was reported in 1908 by Nicolaier and COOH
FIG.1. Cinchophen (2-phenylcinchoninic acid).
Dohrn to increase the urinary excret)ion of uric acid in normal man. In 1911, Weiritraud applied the drug to the management of gout, noting a
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more sustained uricosuric response in gouty subjects and also marked analgesic and anti-inflammatory properties useful in ameliorating gouty arthritis. I n 1913, Folin and Lyman reported that the increased renal elimination of uric acid was accompanied by a distinct reduction in blood urate levels. Thereafter, cinchophen was extensively employed in the treatment of gouty and other forms of arthritis. Its efficacy in mitigating the symptoms of acute and chronic gouty arthritis was fully substantiated (reviewed by Hueper, 1948), as was its effectiveness in prevention of recurrence of acute attacks (Bartels, 1943), and also its uricosuric potency, although Weintraud’s initial claim of mobilization of tophaceous deposits in a patient treated regularly for several weeks was not confirmed by subsequent experience (G. Graham, 1920; Hueper, 1948). However, despite its usefulness in the management of gout, it gradually became apparent that cinchophen was hepatotoxic, in low incidence but with a high rate of mortality in affected persons (reviewed by Hueper, 1948). This finally necessitated virtual abandonment of the drug. Realization of the limitations of cinchophen usage because of hepatotoxicity led to an extensive program of preparation and testing of a large number of cinchophen derivatives and analogs (listed by Hueper, 1948), in the hope of finding a congener possessing the desirable but not the toxic properties of the parent compound. Certain structure-activity relationships pertinent to the present review emerged from the enterprise. It was found (reviewed by Hueper, 1948) that the uricosuric properties of this series of compounds depended upon the presence of a substitution in the quinoline nucleus, most effective when in the 4-position as a carboxyl group, together with introduction of one or another ring structure in the %position of quinoline-4-carboxylic acid. A second substitution in the quinoline nucleus decreased the uricosuric potency, hydroxylation of the benzene ring abolished it. From these various compounds, the ethyl ester of 6-methyl-2-phenylcinchoninic acid (neocinchophen) was selected for extended clinical trial, but it proved to be quite as toxic in man as cinchophen, and less potent as a uricosuric agent (reviewed by Goodman and Gilman, 1955). Subsequently, 3-hydroxy-2-phenylcinchoninicacid (HPC) was introduced as a substitute. It was found to be as effective as the parent conipound in analgesic and antirheuniatic properties (Jager, 1952; Ross, 1954) and also to be uricosuric in man (Ross, 1954), although not in the dog (Beyer et al., 1951b); but it caused troublesome skin eruptions and diarrhea in such high incidence that it had to be withdrawn. No acceptable alternative cinchophen analog has been forthcoming. Information about the physiological disposition of cinchophen in man is limited to the study of Axelrod and Chenkin (1954), who developed a spectrophotometric method for its estimation in biological materials. The drug is rapidly and almost completely absorbed from the gastrointestinal
URICOSURIC DRUGS : PROBENECID, SULFINPYRAZONE
105
tract, peak plasma levels being reached in man in 1.5-3 hours; only 1% of the oral dose is found in the feces, only 2% of the dose is recoverable as cinchophen in the urine. At plasma cinchophen concentrations in the range of 10 nig/liter, about 95% of the drug is firmly bound to plasma proteins, and the distribution of the drug is largely confined to the extracellular fluid. The plasma half-life in man is brief, about 4 hours; taken together with the negligible elimination of cinchophen as such, this signifies rapid and almost complete nietabolisni of the drug. The products of its metabolic conversions have not, however, been clearly identified. The physiological disposition of 3-hydroxy-2-phenylcinchoninicacid (Marshall and Dearborn, 1950) is very similar to that of cinchophen except that the drug is morr slowly metabolized, hence persists longer as such in the plasma. Beyer et al. (1951b) made detailed studies of the renal clearance of 3-hydroxy-2-phenylcinchoninicacid. Like cinchophen, there is negligible excretion of unchanged drug in the urine (Marshall and Dearborn, 1950). Filtration a t the glomerulus is likewise limited by more than 90% binding to plasma proteins. HPC inhibits tubular secretion of phenolsulfonphthalein, p-aminohippurate (PAH) and penicillin (Beyer et al., 1951b); the last, however, apparently more by depression of GFR than by competition for tubular secretion. Information on these latter points is lacking for cinchophen, but in view of the collective evidence, and the low pK, of cinchophen and its analogs, it may be assumed that they are filtered a t the glomerulus to the limited extent that they are not bound to plasma proteins, actively secreted by the tubular “organic acid system,” and in acid urine readily reabsorbed in the tubules by nonionic back-diffusion. Coombs el al. (1940) cited a clearance study indicating that cinchophen, in 3-gm oral dosage, increases urate clearance 2- to %fold. Of interest is the (‘compensatory’’ retention of uric acid noted by Nicolaier and Dohrn (1908) and Weintraud (1911) for a day or two after discontinuance of cinchophen administration; this is reminiscent of the “paradoxical” effect of salicylate on urinary uric acid excretion, already referred to, and may well reflect a similar mode of action on the tubular secretory and reabsorptive mechanisms for uric acid.
D. ZOXAZOLAMINE Zoxazolamine (2-amino-5-chlorobenzoxazole) (Fig. 2) was introduced in 1956 as a muscle relaxant for the relief of skeletal muscle spasm, a n action effected centrally by depressing transmission through subcortical,
clm iJ
0’ ‘NH,
FIQ.2. Zoxazolamine (2-amino-5-chlorobenzoxaaole).
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ALEXANDER B. GUTMAN
brain stem, and spinal polysynaptic pathways. The drug is fairly rapidly absorbed from the gut, in man giving peak plasma levels of 3-12 mg/liter 1 to 3 hours after oral administration of a 1.0-gm dose (Burns et al., 1958a). Plasma and tissue drug levels are negligible within 6 hours, despite insignificant urinary excretion of the unaltered compound. Virtually complete biotransformation is implied, the chief metabolite being 6-hydroxyzoxazolamine, which is recovered in the urine as the glucuronide, and also, in small amounts, 2-hydroxychlorobenzoxazole (chlorzoxazone) (Conney et al., 1960). The latter derivative retains the muscle relaxant properties of the parent compound, whereas hydroxylation in the benzene ring forfeits this activity. Neither metabolite is uricosuric. I n 1958 Reed et al. reported that zoxazolamine possesses potent uricosuric properties, an observation all the more unexpected since the compound is a weak base, unlike all other known uricosuric drugs, which are acidic. After administration of a single oral dose of 0.25 gm, or even less, increased excretion of uric acid could be demonstrated in the first 2-hour aliquot of urine, the response being sufficient to augment the 24-hour output about 500/0, with an associated decline in plasma urate concentration (Burns et al., 1958s). Renal clearance studies after similar oral dosage showed a n approximately threefold rise in C,,/GFR ratios after 20-30 minutes, soon increasing to a peak approximately fivefold or more than the premedication C,,/GFR ratios (Burns et al., 1958a). Indeed, in terms of the minimum dose required to elicit a pronounced increase in urate clearance, zoxazolamine was found to exceed probenecid and at least to equal sulfinpyrazone in potency (Burns et al., 1958a; Reed et al., 1961; Rivera, 1961). This effect on the kidney was noted to be specific for uric acid, not extending t o electrolyte and other components of the urine, and to be blocked by pyrazinamide and small doses of salicylate but not by probenecid or sulfinpyrazone (Burns et al., 1958a). Alkalinization of the urine had no significant effect on zoxazolamine uricosuria. Also consistent with its expected properties as a base presumably not secreted by the tubular “organic acid system,” zoxazolamine caused no detectable change in the renal excretion of phenolsulfonphthalein or penicillin (Reed et al., 1961). Extensive clinical trials substantiated the efficacy of zoxazolamine as a uricosuric agent, markedly lowering the plasma urate concentration in gouty subjects sometimes even when refractory to probenecid, preventing the development of new tophi, and strikingly mobilizing established tophi (Seegmiller and Grayzel, 1960; de SBze et al., 1961;Kuhlback and Harjanne, 1961; Reed, 1962; Yii and Gutman, unpublished experiments, 1960). However, disturbing reports of hepatotoxicity (reviewed by Carr and Knauer, 1961) and nephrotoxicity (Streitz, 1959) began to appear, particularly
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107
following the larger doses administered when the drug was used as a muscle relaxant. The incidence of severe hepatotoxicity increased to proportions sufficient to warrant proscription, and in 1961 the drug was withdrawn. V. The Alkylsulfonamidobenzoic and N-Alkylsulfamylbenroic Acids
The first drug to achieve general acceptance as a uricosuric agent was probenecid, although it was originally introduced for a quite different purpose, t o diminish urinary excretion of penicillin and thus to maintain higher than otherwise attainable blood penicillin levels, a n especially important therapeutic objective when the penicillins were still costly and in short supply. The approach to development of a pharmacological agent designed to inhibit the rapid renal elimination of penicillins was a then novel one, involving application of the physiological principles of renal tubular transport of organic acids (Beyer, 1954). These principles apply also t o the uricosuric effects of the drugs in question, and, as is now generally appreciated, have broad implications in the planned development of many new pharmacological agents. The basis for this approach lies in the observations of H. W. Smith and his colleagues (1938) that a variety of organic acids are not only filtered a t the glomerulus, insofar as they are not bound to plasma proteins, but are also secreted by the tubules; that tubular secretion of these organic acids is effected by a common tubular transport mechanism which is of limited capacity (secretory Tm) ; that there is quantitative competition among t,hese organic acids for this transport mechanism, a n excess of one inhibiting tubular secretion of the others; and that the various organic acids differ in their affinity for the common tubular secretory mechanism, hence in their power to limit the transport of the others. The properties of this (I organic acid system” have since been further elaborated (Taggart, 1950; Weiner and Mudge, 1964) but its nature and modus operandi remain obscure. Rammelkamp and Bradley (1943) first suggested the possibility of slowing the renal elimination of penicillins by simultaneous administration of a substance that would compete with penicillins for tubular secretion, and demonstrated that iodopyracet could accomplish this. PAH was subsequently found t o have a similar effect (Beyer el al., 1944). Neither compound, however, proved to be feasible for clinical application, but the results were deemed by Beyer and his associates to be sufficiently promising t o launch a program of drug synthesis, which yielded carinamide (an alkylsulfonamidobenzoic acid) and later the more potent probenecid, a n N-alkylsulfamylbenzoic acid. The development of these compounds was directed entirely t o their use in selectively enhancing the blood levels of penicillins and other anti-
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ALEXANDER B. GUTMAN
bacterial agents. Interest in this clinical application diminished, however, as the penicillins became more readily available and other antibiotics unaffected by these drugs were introduced, and turned in an unanticipated direction, that of their initially unrecognized concomitant uricosuric properties. This clinical application was foreshadowed by the observations of Wolfson et al. (1948), who, with the same general principle of competition for tubular secretion in mind, employed carinamide t o test their hypothesis that uric acid is eliminated by the kidneys in man solely by tubular secretion. Contrary to expectation, and to its effect on the excretion of penicillins, carinamide was found to increase urinary excretion of uric acid in normal man. This result subsequently was confirmed in normal man and extended to use of the drug in gouty subjects (Gutman and Yu, 1950; Gutman, 1950), for which purpose it was later replaced by the more effective probenecid. The details follow. A. THEALKYLSULFONAMIDOBENZOIC ACIDS: CARINAMIDE Carinamide (4’-carboxyphenylmethanesulfonanilide) (Fig. 3), originally designated caronamide, was initially synthesized by Sprague and Ziegler Q H , S o z N H e
COOH
FIG.3. Carinamide (4'-carboxyphenylmethanesulfonanilide) .
in 1946 and, as already indicated, introduced as an agent for enhancing blood penicillin levels by blocking the rapid loss of penicillins via the kidneys (Beyer et al., 1947a,b). Shortly thereafter, the physiological disposition of the drug could be elucidated by methods made available for estimating carinamide concentrations in biological materials (Brodie et al., 1947; Ziegler and Sprague, 1948). Despite its very sparing solubility in water, carinamide was found to be rapidly and virtually completely absorbed after oral administration, and to have a volume of distribution in excess of that of mannitol (Beyer et al., 1948). About 60% of the plasma carinamide is bound to plasma proteins in the dog (Earle and Brodie, 1947). Carinamide is rapidly eliminated by the kidneys, about 60% of the dose being recoverable in the urine unaltered (some 35% in the first 24 hours), and a large part of the remainder as unidentified metabolites, probably including glucuronide conjugates because the urine acquires reducing properties (Earle and Brodie, 1947; Boger e l al., 1948). After correction ratios in the dog averaged for binding to plasma proteins, C,,,i,,,id,/GFR 1.30 (Earle and Brodie, 1947), implying tubular secret,ion of the drug; thus, as pointed out by Earle and Brodie (1947), accounting for its capacity
URICOSURIC DRUGS: PROBENECID, SULFINPYRAZONE
109
to inhibit tubular secretion of penicillins (Beyer et al., 1947a,b; Shaw et al., 1947), PAH (Beyer et al., 1947b), PSP (phenolsulfonphthalein) (Beyer et al., 1947b; Boger and Crosson, 1949), p-aminosalicylic acid (Home arid Wilson, 1949), pantothenic acid (Roholt and Schmidt, 1951), and the like. Caririamide was found not to influence GFR, the tubular reabsorption of glucose or amino acids, or the conservation of essential electrolytes (Beyer et al., 1947b; other properties reviewed by Beyer, 1954). Carinamide has other inhibitory effects, among them inhibition of the conjugation of p-aminobenzoic acid with glycine (Beyer et al., 1950), by blocking the preliminary activation of the benzoate as benzoyl CoA (Schachter and Taggart, 1953). More relevant to the present context, however, is its capacity to inhibit tubular reabsorption of uric acid in man. This uricosuric property, as already noted, was discovered by Wolfson et al. (1948), who reported a 50-12070 increase in urinary acid excretion, accompanied by a slight fall in plasma urate and doubling of the urate clearance, in normal adults given 4.6 gm carinamide in a single oral dose. Wolfson et al. (1948) attrihuted this response to decreased binding of the plasma urate to plasma proteins, with a consequent increase in the filtered and excreted uric acid, but it is now well established that uric acid is not significantly bound to plasma proteins (evidence reviewed by Gutman and Yu, 1961), and that the uricosuric response to carinamide must indeed be due t o inhibition of tubular reabsorption of urate. In 13 gouty subjects given carinamide, the increase in 24-hour urinary uric acid excretion was found t o average about 40% with 8-gm divided daily dosage and 60% with 12-gm divided daily dosage, accompanied by a decline in serum urate of 20 and 28%, respectively (Gutman, 1950; Gutman, 1963a). For a few years after its introduction carinamide enjoyed some clinical usage in the treatment of a variety of infections, especially those due t o penicillin-sensitive organisms (reviewed by Boger and Crosson, 1950), notably bacterial endocarditis (Boger et al., 1947; Eisman et al., 1949; Stuart-Harris et al., 1949), gonococcal urethritis (Merren et al., 1948), and meningitis. Its use in gout seems to have been limited almost entirely to our own experience, so far as can be ascertained from the literature. However, despite seemingly general satisfaction with the results (Boger and Crosson, 1950), it soon became evident that in clinical usage carinamide left much to be desired. For one thing, because of its abbreviated biological half-life of only a few hours, due to rapid renal elimination and biotransformation, it was necessary to administer orally 8-12 gm in divided daily dosage in order to maintain effective blood carinamide levels of 15-40 mg% (Boger et al., 1948). Administration of the drug could usually be maintained for weeks or months but it was an ordeal to swallow 4 to 8 large (0.5-gm) tablets every 3 or 4 hours, and nausea and vomiting commonly ensued
110
ALEXANDER B. GUTMAN
(Boger and Crosson, 1950; Gutman, 1950). Moreover, despite a low order of toxicity (Beyer et al., 194713; Boger and Crosson, 1950), the large dosages required for protracted periods induced a small but significant incidence of drug fever and rash (Boger and Crosson, 1950; Gutman, 1950). When the limitations of carinamide became evident, Beyer and his associates began to search for a drug with similar properties but biologically more effective in lower dosage. Using as a guide the capacity to inhibit both the tubular secretion of certain organic acids and their conjugation with glycine, it was found that, in general, the N-alkylsulfamylbenzoic acids were more potent in these respects than the corresponding alkylsulfonamidobenzoic acids related to carinamide, and that the dialkylsulfamylbenzoic acids surpassed their monoalkyl congeners (Beyer et al., 1951a). These observations led to the preparation and testing of several dialkyl derivatives, including the di-n-propyl analog.
ACIDS:PROBENECID B. THEN-ALKYLSULFAMYLBENZOIC Probenecid, p-(di-n-propylsulfamy1)-benzoic acid (Fig. 4), was initially synthesized by Miller, Ziegler, and Sprague as one of a series of N-alkylCH,CH,CH, H O , + =)NSO (, C H,C H,C H,
FIG.4. Probenecid (p-[di-n-propylsulfamyl] benzoic acid).
sulfamylbenzoic acids prepared, as just mentioned, in the course of a planned program designed to evolve a compound more therapeutically effective than carinamide in maintaining high blood penicillin levels, by inhibiting the renal tubular secretion and hence the rapid urinary loss of the antibiotic. One of the desiderata was a drug of more prolonged biological half-life than carinamide. To study the physiological disposition of the most promising of the new compounds, probenecid, spectrophotometric and colorimetric methods for its estimation in biological materials were soon devised (Tillson et al., 1950, 1954). This methodology, although of limited sensitivity and specificity because of large blanks and other difficulties, sufficed to reveal that probenecid is well absorbed following oral administration, that it is excreted extremely slowly “if a t all,” and that hydrolyzable metabolites gradually appear in the urine (Tillson et al., 1950). More precise studies of the physiological disposition of probenecid in man had to await the development of a more refined spectrophotometric method of sufficient sensitivity and specificity (Dayton et al., 1963). That probenecid, although difficultly soluble in water, is rapidly and virtually completely absorbed from the gastrointestinal tract is evident
URICOSURIC DRUGS: PROBENECID, SULFINPYRAZONE
111
from its prompt appearance in the plasma, within an hour of ingestion, and the general correspondence of blood drug levels after oral and intravenous administration (Boger et al., 1950; Dayton et al., 1963). A single 2.0-gm dose given orally to man yields peak plasma probenecid levels of 15-20 mg%, ordinarily in about 4 hours (Boger et al., 1950; Dayton et al., 1963). Plasma drug concentrations then fall off rapidly, some 7oy0 of the drug, on the average, disappearing from the circulation in man in 24 hours (Dayton et al., 1963), but detectable levels often persist for as long as 48 hours (Beyer et al., 1951a; Dayton et al., 1963). The half-life of the plasma disappearance curves is dose-dependent within the range of 0.5-2.0-gm dosages in man, the customary limits being at 6-12 hours (Dayton et al., 1963). At 2-10 mg% plasma probenecid levels, 89-94y0 of the drug is bound t o plasma proteins, chiefly albumin, in man (Dayton et al., 1963), some 75y0 in the dog (Boger and Pitts, 1950a; Beyer et al., 1951a; Tillson et al., 1954). In its distribution in the tissues, therefore, the bulk of the drug is confined to the extracellular fluid compartment, of the body water (Dayton et al., 1963). The fairly rapid turnover of probenecid is not accounted for by renal elimination of the unaltered compound, of which less than 5% can be recovered in the urine, both in the dog (Beyer et al., 1951a; Tillson et al., 1954; Beyer, 1954; Weiner et al., 1960) and in man (Dayton et al., 1963). Consequently, the major portion of the drug must be conjugated or degraded. In the initial studies, Tillson et al., (1950, 1954) noted the appearance of hydrolyzable metabolites in the urine of the dog, including a nonfermentable reducing substance suspected t o be a glucuronide conjugate. Schachter (1957) confirmed this finding, recovering from the urine of a human subject the major portion of a probenecid dose as the acyl glucuronide (see also Dayton et al., 1965). The nature of the ultimate products of the metabolic degradation of probenecid is still obscure. As anticipated from its pK, of 3.4 (Shore et al., 1957), the rate of renal elimination of probenecid is urine pH-dependent. When the urine is distinctly acid, excret>ed/filteredratios of the drug are very low, indicating net tubular reabsorption; when the urine is made alkaline, excretion of the drug increases substantially, especially at high rates of urine flow, and excreted/filtered ratios unequivocally exceed 1.0, indicating net tubular secretion (Weiner et al., 1960; Braun and Schniewind, 1962; Dayton et al., 1963). It may therefore be concluded that probenecid is both filtered a t the glomerulus (to the extent that it is not bound t o plasma proteins) and secreted by the tubules, the latter by t,he “organic acid system” resident in the proximal convolution (Weiner et al., 1960; Dayton el al., 1963; Weiner and Mudge, 1964). Tubular reihbsorption from the luminal fluid may be considered to occur by nonionic diffusion, in more distal segments of the
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ALEXANDER B. GUTMAN
nephron, a t a rate determined by probenecid’s pK,, its Kp, the pH of the urine, the rate of urine flow, and other factors less well defined (Weiner et al., 1960; Gutman et al., 1960; Weiner and Mudge, 1964). The minimal excretion of the compound in normally acid urine in both dog and man thus is the result, not of failure of tubular secretion, as initially supposed, but of virtually complete back-diff usion of both filtered and secreted probenecid (Weiner et al., 1960; Dayton et al., 1963). Probenecid causes no significant alteration in glomerular filtration rate (Beyer et al., 1951a; Sirota et al., 1952) or renal blood flow-its markedly suppressant action on C p A H and PSP excretion does not signify any deleterious effect on renal hemodynamics but simply reflects inhibition of the tubular secretion of these compounds. Except for a modest salt and water diuresis in some persons (Sirota et al., 1952), notably those with congestive heart failure (Bronsky et al., 1955), the excretion of electrolytes is not affected by the drug (Beyer et al., 1951a; Sirota et al., 1952; Spurr et al., 1954);however, the hyperphosphatemia of hypoparathyroidism seems nevertheless to be corrected (Pascale et al., 1954; Kolb and Rukes, 1954; Dubin et al., 1956). Renal conservation of glucose (Beyer et al., 1951a), amino acids (Beyer et al., 1951a; Bearn and Kunkel, 1954), and other useful metabolites is not disturbed. The excretion of purines other than uric acid, such as xanthine and hypoxanthine, is not enhanced (Gjgrup and Poulsen, 1955), and in fact is reduced when initially high (Goldfinger et al., 1965). The elimination of a variety of organic bases secreted by the distinct tubular “organic base system” is not in any way affected (reviewed by Weiner and Mudge, 1964). Probenecid is known to inhibit several enzymic processes, notably the conjugation of a variety of benzoic acid derivatives with glycine (Beyer et al., 1950), by what was thought at first to be suppression of a “conjugase,” with formation of a probenecid complex refractory and obstructive to the tubular secretory mechanism for organic acids (Beyer et al., 1950). It was subsequently shown, however, that glycine conjugation is in fact a three-phase process, involving intermediary formation of an acyl CoA through the agency of glycine N-acylases (Schachter and Taggart, 1953; reviewed by Taggart, 1958, and Williams, 1959). At present there is no clear evidence that conjugation plays a role in the inhibitory effects of probenecid on tubular secretion of organic acids, and since it is itself freely secreted, competitive inhibition for transport by the common “organic acid systJem” seems t o be an entirely adequate explanation (Weiner et al., 1960). Probenecid is still occasionally employed as a therapeutic adjunct t o inhibit the tubular secretion, hence diminish the urinary elimination and enhance the blood levels of the penicillins (Boger et al., 1950; Beyer et al.,
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113
1951a,b; Waldo and Tyson, 1951; Burnell and Kirby, 1951; Nichols et al., 1957; Henderson et al., 1962) and of p-aminosalicylic acid (Boger and Pitts, 1950a,b; Janssen, 1951; Breitenbucher et al., 1952; Huang et al., 1960). Given in divided doses of 1.0-2.0 gm/day, probenecid produces a two- to tenfold increase in plasma penicillin levels (Boger et al., 1950, 1951a) and a 15-50% increase in plasma p-aminosalicylic acid levels (Boger et al., 1950). For aid in the intensive treatment of refractory cases particularly of bacterial endocarditis and tuberculosis, the use of probenecid therefore has a place despite the current ready availability of both antibiotics. Probenecid does not affect the elimination or blood levels of sulfonamides, streptomycin, chloramphenicol, or the tetracyclines (Boger et al., 1951a). Tubular secretion of a number of other organic acids is diminished by probenecid, among them p-aminohippuric acid (Beyer et al., 1951a), phenolsulfonphthalein (Beyer, 1950; Boger et al., 1951a; Peck and Beyer, 1954; Blondheim, 1955; Benedek, 1961; Newcombe and Cohen, 1963), and salicylic acid and its acyl and phenolic glucuronides (Gutman et al., 1955; Schachter and Manis, 1958; Yu and Gutman, 195913; Weiner et al., 1959). Additional compounds of biological interest similarly affected by probenecid (reviewed by Weiner and Mudge, 1964) are pantothenic acid (Boger et al., 1953; Markkanen et al., 1963), androsterone (Gardner et al., 1951; Bongiovanni and Eberlein, 1957), corticotropin (Bonar and Perkins, 1962), diiodotyrosine (Huang, 1961), phlorizin and its conjugated glucuronide (Braun et al., 1957), and acetasolamide (Weiner et al., 1959). Secretion of uric acid by the tubular “organic acid system” also is competitively inhibited t o greater or lesser extent by probenecid, as can best be demonstrated in species in which the tubular reabsorptive mechanism for uric acid is defective or rather poorly developed, as, for example, in the Dalmatian coach hound (Beyer et al., 1951a; Beyer, 1954; Kessler et al., 1959a; Yu et al., 1960a), rabbit (Poulsen, 1955; Beechwood et aZ., 1964) and chicken (Berger et al., 1960). In man, however, so preponderant is the uricosuric action of probenecid that a “paradoxical” effect, brought about by inhibition of tubular secretion of uric acid in low drug dosage, can be shown only equivocally, if at all (Yu and Gutman, 1955). Recognition of the uricosuric properties of probenecid derived from the prior experience with the uricosuric effects of the related carinamide in normal man (Wolfson et al., 1948) and in gouty subjects (Gutman arid Yu, 1950; Gutman, 1950). When, late in 1949, it was rumored that a n improved carinamide had been developed, a compound that was much more effective in sustaining augmented blood penicillin levels, it seemed appropriate to determine whether the uricosuric activity of the new drug was likewise enhanced. That this was indeed the case was soon apparent. Initial reports of the experience in gouty subjects (Gutman, 1950, 1951a,b; Gut-
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ALEXANDER B. GUTMAN
man and Yu,1951; Talbott et al., 1951; Bishop et al., 1951) revealed that the uricosuric response t o 2.0 gm daily dosage of probenecid was about the same a s to 12-gm daily dosage of carinamide-a mean increase of some 60% in urinary uric acid excretion, accompanied by a mean fall of approximately 45y0 in serum uric acid over the first week. I n 1.0-gm daily dosage of probenecid the corresponding figures approximated 50 and 33%, respectively; in 0.5-gm daily dosage they approximated 45 and 25%, respectively (Gutman and Yu,1951). Renal clearance studies (Sirota et al., 1952) showed that a single oral 2.0-gm dose of probenecid elicited a uricosuric response within 40 minutes (Table 11),which soon reached a n approximately fourTABLE I1 EFFECTOF PROBENECID (SINGLE ORALDOSE,25 I N MANO Time (min)
Pur bg%)
0 20 40 60 80 100 120
6.7 6.8
-
6.6 6.2
MG/KG)
ON
URATECLEARANCE
cur
Cw/GFR
(ml/min)
(%I
5.0 5.4 14.5 24.3 25.8 29.9 27.3
5.0 5.2 13.5 22.3 22.7 25.8 25.5
From Sirota et al. (1952).
fold maximum, Cur rising from a mean of 8.0 ml/min to a mean peak of 32.7 ml/min, C,,/GFR from 0.07 to 0.33. These data indicated that probenecid was the most potent uricosuric agent, per unit weight, that had been brought forward up to that time. Confirmatory reports by many investigators were soon forthcoming (Pascale et al., 1952; Gutman et al., 1954; Kuzell et al., 1955; Mason, 1956; Ogryzlo and Harrison, 1957; Robinson, 1957, 1965; Bartels and Matossian, 1959; Seegmiller and Grayzel, 1960; G. R. Thompson et al., 1962; de S h e et al., 1963), and the chief use of probenecid has since been in the management of gout. This does not apply to termination of acute attacks of gouty arthritis, on which the drug has no effect whatsoever, despite prompt lowering of the serum urate level; indeed, for reasons still obscure, initiation of probenecid therapy in the quiescent stages of the disease sometimes is accompanied by a flare-up of acute gouty arthritis. The principal indication , for probenecid in gout is to prevent the formation of tophaceous deposits and t o mobilize any already present, by lowering the serum urate sufficiently (to about 7.0 mg% or less) to reverse the hyperuricemic trend to
URICOSURIC DRUGS: PROBENECID, SULFINPYRAZONE
115
net transfer of urate from the circulating fluids to susceptible tissues. This can be accomplished in enough cases to improve greatly the outlook for most of those afflicted, in whom the deformities and disabilities of chronic gouty arthritis can be obviated or ameliorated. Optimal dosage of the drug varies, and must be judged by the uricosuric response in the individual patient; in one large series, 1.0 gm/day proved to be sufficient in 50% of cases, 1.5-2.0 gm/day in 25%, 2.5-3.0 gm/day in 15%, and 0.5 gm/day in 10% (Gutman and Yu, 1957b). However, diminution in size of the tophi occurs very slowly at best, and only partially or not perceptibly in some cases, even with the highest probenecid dosage tolerated, notably when renal damage is present (Gutman and Yu, 1955, 1957b; Bartels, 1957; Bartels and Matossian, 1959; Smyth et al., 1960; G. R. Thompson et al., 1962; de S&zeand Ryckewaert, 1960; Talbott, 1964). I n such refractory cases allopurinol may be employed advantageously together with probenecid (Yu and Gutman, 1964), or probenecid can be combined with sulfinpyrazone for an additive uricosuric effect (Yu and Gutman, 1959a; Seegmiller and Grayzel, 1960). Probenecid is often used to normalize the serum urate when it is inordinately high in gout, even when tophi are not yet in evidence, or when hyperuricemia develops in nongouty subjects taking such drugs as the benzothiadiazines (Healey et al., 1959; Warshaw, 1960; Duarte et al., 1961; Smilo el al., 1962; Bryant et al., 1962). Some physicians employ probenecid prophylactically in this way in gouty subjects, for the express purpose of minimizing recurrence of acute gouty arthritis by preventing accumulation of presumptively inciting microcrystals of monosodium urate monohydrate; acute gouty attacks often do seem to bec,ome less frequent as the deposits in chronic tophaceous gout diminish (Bartels and Matossian, 1959; Bartels, 1960; de S&zeand Ryckewaert 1960). The usual practice, however, is t o depend on the more reliable colchicine prophylaxis, with or without concomitant uricosuric therapy, for the prevention of recurrence of acute gouty arthritis (Talbott and Coombs, 1938; Gutman and Yu, 1952; Robinson, 1957; Talbott, 1959; Yu and Gutman, 1961; Talbott, 1984). A curious incompatibility of probenecid (and other uricosuric drugs) is in its relation to salicylate, which suppresses the uricosuric effect of probenecid (Gutman and Yu, 1951; Pascale et al., 1952, 1955; Seegmiller and Grayzel, 1960) but not its capacity to inhibit tubular secretion of penicillin (Boger et al., 1955). A wholly satisfactory explanation for this interference has not yet been forthcoming. It may be assumed that the uricosuric activity of probenecid is counterbalanced in some measure by simultaneous inhibition of tubular secretion of urate by salicylate in ordinary dosage (Gutman and Yu, 1958), but in quantitative terms this mecha- ' nism would not seem to sufice (Yu and Gutman, 1959b). T o judge by the
116
ALEXANDER B. GUTMAN
results of more detailed investigation of the similar abolition of sulfinpyrazone uricosuria by salicylate (Yu et al., 1963), the interaction of probenecid and salicylate is probably complex, involving competition not only at the renal level but also at other sites. No such suppression of uricosuria is noted when probenecid is administered together with sulfinpyrazone; indeed the effects are additive or substitutive (Yu and Gutman, 1959a; Seegmiller and Grayzel, 1960). It would appear that probenecid inhibits tubular secretion of sulfinpyrazone, like that of other organic acids, thereby prolonging its uricosuric action (Dayton et al., 1965). The clinical application of probenecid, particularly in gout which requires sustained daily administration over a span of many years, depends of course also upon its low order of toxicity. This was early established in acute and chronic animal toxicity studies, which indicated an LDbOof 1.6 gm/kg in rats, death occurring ultimately by central nervous system effects (McKinney et al., 1951). Convulsions and coma also characterized a unique case of attempted suicide by ingestion of 47.5 gm probenecid (Rizzuto et al., 1965). I n ordinary therapeutic dosage, however, the drug is usually well tolerated for indefinite years. In one series of 169 gouty patients receiving protracted probenecid therapy the most frequent side reactions were gastrointestinal complaints (go/,), drug hypersensitivity with fever and rash (5%), flank pain or passage of stone or gravel associated with uricosuria (9yo), and precipitation of acute gouty attacks in some 10% of cases (Gutman and Yu, 1957b). Most reports cite comparable figures (Austrian and Boger, 1956; Talbott, 1964), although de S8ze et al. (1963), in a series of 156 cases, record a higher incidence of gastrointestinal complaints (18%, necessitating discontinuance of tthe drug in 12%) and of precipitation of acute attacks (20Q/,).A few investigators ascribe even more frequent reactions to probenecid (Marson, 1954; Kuzell et al., 1955). Apart from isolated case reports of hepatic necrosis, in a patient who continued to receive full doses despite clear indications of drug hypersensitivity (Reynolds et al., 1957), nephrotic syndrome (Ferris et al., 1961) and the precipitation of calculi and subsequent renal damage that may ensue from uncontrolled uricosuria unless proper precautions are taken, the use of probenecid seems not to have been associated with irreparable toxicity.
C. STRUCTURE-ACTIVITY RELATIONSHIPS OF PROBENECID ANALOGS I n a study of a series of N-dialkylsulfamylbenzoates, Beyer (1954) found that as the length of the N-alkyl substitutions increased, the renal clearance of the compounds decreased. This association of structure with rate of urinary excretion was interpreted by Beyer to signify that the length of N-alkyl substitution was inversely related to rate of tubular
URICOSURIC DRUGS : PROBENECID, SULFINPYRAZONE
117
secretion, and directly related to rate of tubular reabsorption. Weiner et al. (1960), however, consider the structural relationship to be limited to tubular reabsorption. They showed that the chloroform/water partition coefficient of these probenecid analogs increased, as anticipated, as the length of the N-alkyl substitutions increased, and pointed out that the enhanced lipid solubility imparted by N-alkyl substitutions of greater length would readily account for their more complete back-diffusion in acid urine. The pK, of all these compounds was the same, 3.3-3.4, so the differences in tubular reabsorption could not be ascribed to differences in the degree of ionization at the same (acid) urine pH (Table 111). TABLE: 111
N-DIALKYLSULFAMYLBENZOATES: RELATIOXSHIP O F LIPII)SOLUBILITY (K,) CLEARANCE IN
THE
TO
RENAL
Doci'~b~c
R1
/ ' \ N S O ? ~ C O O H R2
H H CHI CH3
H CH, CHI
C2Hs
C:Ha
C2Hs C3H7
C3H7
C3H7
CnHs
3.1 2.6 2.3 1.5 0.7 0.1 <0.01
< O . 002 0.12 6.0 13 75 ca. 250 >2,000
,
3.3 3.3 3.3 3.3 3.3 3.3 3.4
From Weiner et al. (1960). Partition coefficients were determined in a chloroform-N HCI system. c Listed in ascending order of K,. a
The diethyl analog of probenecid possesses modest uricosuric properties, approaching probenecid in potency, and has been employed in the management of gout (Kersley et d., 1958; Horn and Thompson, 1960; M. Thompson and Horn, 1961). It offers no apparent advantage over probenecid. VI. The Pyrazolidinediones
The introduction of sulfinpyrazone as a therapeutically useful uricosuric drug (Burns et al., 1957) was an outcome of exploration of a large series of pyrazolidinediones, more than 80 in all. The investigation necessitated the development of sensitive and specific methods for the estimation in biological materials of each of a number of pyrazolidinediones, by extraction in appropriate solvents, purification by countercurrent distribution, and spectrophotometric measurement. The availability of these methods made
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ALEXANDER B. QUTMAN
possible physiological distribution studies in man, by which the course of the investigation was guided. The results, taken as a whole, endorse the usefulness of physiological distribution studies in uncovering new pharmacological agents and in establishing structure-activity relationships of predictive value (Burns et al., 1960; Gutman et al., 1960; Bloom and Laubach, 1962; Perel et al., 1964). The starting point of the venture, by Brodie, Burns and their associates, was phenylbutazone, the initial objective to find an analog that would retain the striking antirheumatic powers but would not elicit the toxic side reactions of the parent compound, or of those of the anti-inflammatory steroids. Later, interest shifted to the accompanying uricosuric action of the analogs tested, in part because acute gouty arthritis had been selected for convenient assay of antirheumatic potency since it offers a sharper pharmacological end point than the customary patient with rheumatoid arthritis.
A. PHENYLBUTAZONE Phenylbutazone (1,2-diphenyl-4-n-butyl-3,5-pyrazolidinedione) (Fig. 5) was first synthesized by Stenel et al. (1950). The voluminous literature on
Metabolite II FIG.5. Phenylbutarone, metabolite I, and metabolite 11.
URICOSURIC DRUGS : PROBENECID, SULFINPYRAZONE
119
this compound has been reviewed by von Rechenberg (1961)) who gives a detailed account of the strange history, antirheumatic triumphs, classic animal toxicology, and clinical pharmacology of the drug. For the purposes of this review, three properties of phenylbutazone need be noted, and their variable transmission t o key analogs traced: its remarkable capacity to ameliorate a number of arthritic disorders (reviewed by von Rechenberg, 1961), a marked tendency t o cause sodium and water retention (Chenkin et al., 1953), and modest uricosuric activity. Phenylbutazone is rapidly and quite completely absorbed from the gastrointestinal tract, peak plasma concentrations being reached within 2 hours of ingestion of the drug (Burns et al., 1953). At plasma phenylbutazone levels of 5-15 mg%, approximately 98% is bound to plasma proteins in man (about 92% in the dog), and the drug accordingly is largely distributed within the extracellular fluid compartment. Urinary elimination of phenylbutazone as such is negligible in man (Burns et al., 1953; Gutman et al., 1960), signifying virtually complete metabolic transformation. This, however, occurs at a slow rate in man, since the biological half-life is about 72 hours, as compared to about 6 hours in the dog (Burns et al., 1953). Analysis of the mechanisms of renal excretion of phenylbutazone is unsatisfactory in man because of the extremely small quantities appearing in the urine, even less than calculated to be filtered at the glomerulus, but in the dog, net tubular reabsorption could readily be demonstrated in acid urine, net tubular secretion in alkaline urine (Gutman et al., 1960). It may be inferred that the plasma phenylbutazone is filtered a t the glomerulus to the very limited extent that it is not bound to plasma proteins; that it is actively secreted by the tubules, apparently in the proxima1 section of the nephron as localized by stop-flow studies in the dog (Gutman et al., 1960); and that it is freely reabsorbed by nonionic diffusion in the more distal nephron, a site likewise localized by stop-flow studies in the dog. Phenylbutazone seems to compete fairly successfully for transport by the tubular secretory “organic acid system,” as indicated by its depressant action on tubular secretion of p-aminohippurate (Yu et al., 1953; King, 1953) and phenolsulfonphthalein (Brodie et al., 1954). In respect to the uricosuric properties of phenylbutazone, they were noted initially when the drug was found to be efficacious in terminating attacks of acute gouty arthritis (Kuzell et al., 1952). However, it was not clear at first whether phenylbutazone was truly uricosuric, since it would sometimes reduce the plasma urate concentration without increasing urinary elimination of uric acid, indeed might even cause renal retention of uric acid (reviewed by von Rechenberg, 1961). These discrepancies were probably due t o variations in drug dosage. Renal clearance studies subsequently demonstrated moderate but definite uricosuria, i.e., increased
120
ALEXANDER B. GUTMAN
Cur and C,,/GFR, when the drug dosage was sufficient to give plasma phenylbutazone levels of 10 mg% or more (Yu et al., 1953; Huffman et al., 1955; Ogryzlo and Harrison, L957), whereas at plasma phenylbutazone levels of 2 4 mg% a transitory decline in C,,/GFR occurred (Yu and Gutman, 1955; Yu et al., 1956); a t plasma phenylbutazone levels of 1-2 mg% there was no direct effect on urate clearance, but sufficient retention of salt and water took place to lower the plasma urate concentration by hemodilution (Yu et al., 1956). The data thus indicate that phenylbutazone has modest but distinct uricosuric powers; however, it apparently exercises a dose-dependent “paradoxical” effect on renal excretion of uric acid like that of salicylate, although less marked (Yu and Gutman, 1955). Presumably, there is much the same dual action on tubular secretion and tubular reabsorption of uric acid as previously discussed. As is well known, the clinical usefulness of phenylbutazone is limited by a high incidence of a variety of toxic side effects, some fatal (reviewed by von Rechenberg, 1961; Am. Med. ASSOC., 1963-1964). In gout, it is still employed for its striking antirheumatic powers in the short-term treatment of acute attacks, terminating them as effectively as colchicine, and without its troublesome sequelae. Phenylbutazone has no place, however, in longterm uricosuric therapy in gout, in view of the availability of the more potent and less hazardous uricosuric drugs, probenecid and sulfinpyrazone.
B. PHENYLBUTAZONE METABOLITES As already mentioned, study of the physiological disposition of phenylbutazone indicated that the drug was slowly but virtually completely metabolized in man. A search for metabolites in the urine yielded two compounds, designated phenylbutazone metabolites I and I1 (Burns et al., 1955) (Fig. 5). Metabolite I was characterized as a derivative in which one of the benzene rings of phenylbutazone was hydroxylated in the para position, and was found to be identical with authentic l-(p-hydroxypheny1)2-phenyl-4-n-butyl-3,5-pyrazolidinedionesynthesized by Pfister and Hafliger (1957). In metabolite 11,a hydroxyl group had been introduced in the 3-position of the butyl side chain (Burns et al., 1955), as was demonstrated by identity with the L-isomer of 1,2-diphenyl-li-(a-hydroxy-n-buty1)-3,5pyrazolidinedione prepared synthetically by Denss et al. (1957). 1. Metabolite I (Oxyphenbutazone)
The physiological disposition of oxyphenbutazone in man closely resembles that of phenylbutazone. Like the parent compound, it is rapidly and almost completely absorbed from the gastrointestinal tract; at plasma levels of 5-15 mg% approximately 9801, is bound t o plasma proteins; despite the hydroxylation of a phenyl ring, it has a similarly prolonged biological
URICOSURIC DRUGS
PROBENECID, SULFINPYRAZONE
121
half-life in man, averaging about 3 days; and there is negligible urinary elimination of the intact compound (Burns et al., 1955; Brodie et al., 1956; Yu et al., 1958b). Slow but virtually complete biotransformation is implied, one conjugation product being the glucuronide, which accounts for 1-5% of the dose in the first 24-hour urine sample (Perel et al., 1964). I n the dog, in contrast, the biological half-life of oxyphenbutazone is only 0.5 hour, due to rapid urinary elimination of the drug and its glucuronide (Perel et al., 1964). Oxyphenbutazone also retains the pharmacological properties of phenylbutazone. It is a potent antirheumatic agent, causes marked retention of salt and water, and is moderately uricosuric (Yii et al., 195813). In fact, it is likely that oxyphenbutazone contributes appreciably to the effects attributed t o phenylbutazone, since there is substantial accumulation of this major metabolite of phenylbutazone in the blood after administration of phenylbutazone (Burns et al., 1955). Renal clearance and 24-hour urinary excretion studies of the effects of oxyphenbutazone on climination of uric acid, water, and electrolytes revealed no significant quantitative differences from phenylbutazone (Yu et al., 1958b). As in the case of the parent compound, slow, sustained infusion experiments showed significant decreases in the renal clearance of uric acid, sodium, chloride, and water initially, at low plasma concentrations of oxyphenbutazone; when the plasma drug levels rose to exceed 10 mg%, salt and water retention continued but the clearance of uric acid increased t o exceed the initial C,,/GFR-again a “paradoxical” effect on urinary uric acid excretion (Yii et al., 1958b). Although specific data are lacking, it seems wholly likely that the mechanisms involved in the renal regulation of oxyphenbutazone excretion are much the same as described for phenylbutazoIie. Possessing similarly striking anti-inflammatory and analgesic properties (Brodie et al., 1956; Yu et al., 1058b; Vaughan and Howell, 1958; Vaughan et al., 1959; Cardoe, 1959; Hart and Burley, 1959; Graham, 1960; Domenjoz, 1960; Leng-LBvy and David-ChaussB, 1963), oxyphenbutazone offers a therapeutic alternative for phenylbutazone, and indeed has now replaced it t o a considerable extent in the treatment of a variety of arthritides and other disorders. How much of this preference rests on psychological and how much on pharmacological grounds is difficult to say. Animal toxicity studies (Domenjoz, 1960) indicate that oxyphenbutazone is less ulcerogenic than phenylbutazone, and this seems to be borne out by clinical experience (Hart and Burley, 1959; Graham, 1960). However, like the parent compound, oxyphenbutazone presents a threat of bone marrow damage (Smyth, 1960; Armstrong and Scherbel, 1961; Kennedy, 1962) and other toxicity, particularly when administered on a continuing schedule. I n gout its use is
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ALEXANDER B. GUTMAN
restricted to short-term therapy, the abolition of attacks of acute gouty arthritis, which it accomplished within 48 hours in about 85% of 200 attacks, without appreciable incidence of significant toxicity (Gutman, 1965). I n fact, since use of oxyphenbutazone is not followed by the unpleasant gastrointestinal reactions of colchicine, it is the drug of choice in the termination of acute gouty arthritis in many circumstances. 2. Metabolite I1 In contrast to metabolite I, the properties of metabolite I1 were found to differ markedly from those of phenylbutazone (Burns et al., 1955; Brodie et al., 1956; Yu et al., 195813). It proved to be much more insoluble, and so poorly absorbed from the gastrointestinal tract after oral administration that it had to be given intravenously for studies of physiological disposition. Although also largely bound to plasma proteins in man (about 93% at plasma levels of 5-15 mg%), the biological half-life was much shorter than that of phenylbutazone, averaging about 10 hours; this was due to more rapid metabolic transformation, not to appreciable urinary excretion of unchanged drug. Metabolite 11, given intravenously, did not exhibit the pronounced antirheumatic properties of phenylbutazone and oxyphenbutazone, nor did it cause appreciable retention of salt and water. On the other hand, metabolite I1 had acquired enhanced uricosuric properties, causing a three- to fivefold increase in C,,/GFR a t plasma drug levels of 5-9 mg%, a distinctly greater effect than produced by the parent compound or metabolite I (Yu et al., 195813). This latter finding suggested the possibility that other structural alterations in the side chain of phenylbutazone might yield compounds of even greater uricosuric potency, and such congeners accordingly were investigated. Among those tested in renal clearance studies in man (Yu et al., 1955) were the isopropyl and phenylthiopropyl analogs, which caused little and transient uricosuria, and the phenylthioethyl and isopropylthioethyl analogs, which proved to be markedly uricosuric. Later it became clear that the enhanced uricosuric activity of metabolite I1 was more directly related t o the increased acidity of the molecule imparted by hydroxylation in the butyl side chain, and that decreasing the pK, by appropriate substitution anywhere in the phenylbutazone molecule would have a similar effect.
c. ~,~-~IPHENYL-~-(PHENYLTHIOETHYL)-~,~-PYRAZOLIDINEDIONE This compound, in which a phenylthioethyl group replaces the butyl side chain of phenylbutazone, was synthesized by Pfister and Hafliger (1961) and designated G-25671 in the pyrazolidinedione series (Fig. 6). For
URICOSURIC DRUGS: PR.OBENECID, SULFINPYRAZONE
G-25671
123
Sulfinpyrazone
p -Hydroxysulfinpyrazone FIG.6. Metabolic conversion of G-25671 to sulfinpyraaone, and sulfinpyraaone to p-hydroxysulfinpyraaone.
reasons already set forth, interest in G-25671 was early directed toward examination of its uricosuric activity, which was discovered to be at least as great as that of probenecid (Brodie et al., 1954; Yu et al., 1956, 1958b; Ogryzlo and Harrison, 1957). It also possesses some degree of antirheumatic activity, much less than phenylbutazone or oxyphenbutazone (Wilhelmi and Currie, 1954; Brodie et al., 1954; Yu et al., 1956; Domenjoz, 1960), and insufficient t o abolish acute gouty attacks consistently (Yu et al., 1956), but enough t o exert an early and distinctly beneficial effect on the stiffness and aching pains of chronic gouty arthritis (Yu et al., 1956; Ogryzlo and Harrison, 1957). Retention of salt and water was found to be negligible (Brodie et al., 1954; Yii et al., 1956). Side reactions encountered with protracted usage were neither unduly frequent nor severe (Yu et al., 1956; Ogryzlo and Harrison, 1957). These several qualities confer upon G-25671 a rather unique usefulness in the management of chronic gouty arthritis, since probenecid and s u l h -
124
ALEXANDER B. GUTMAN
pyrazone are devoid of antirheumatic effect in uricosuric dosage, and salicylate and cinchophen, while combining both antirheumatic and uricosuric properties, are too toxic for protracted use a t uricosuric dosage levels. It is t o be regretted that G-25671 has not been made available for more extensive clinical trial. G-25671 is readily and virtually completely absorbed from the gastrointestinal tract, and is then rapidly metabolized, with a biological half-life of about 3 hours in man (Brodie et al., 1954). Unaltered drug is excreted by the kidneys in minimal amounts, by mechanisms not studied in detail but which may be surmised to be much the same as described for other pyrazolidinediones. Secretion by the tubular “organic acid system” is implied by the capacity to inhibit tubular secretion of phenolsulfonphthalein (Brodie et al., 1954) and p-aminohippurate (Yu et al., 1956). A uricosuric effect is exerted at plasma G-25671 levels as low as 1 mg% (Yu et al., 1956). This makes a marked “paradoxical” action on uric acid excretion unlikely, but there is some suggestion of such a n effect in renal clearance studies with slow, sust,ained infusion of the drug, and in the distinct urate retention noted upon cessation of medication (Yu et al., 1956). The unexpectedly rapid biotransformation of G-25671 in man prompted a search for its metabolites in the urine. This yielded a sulfoxide derivative (Burns et al., 1957) which was shown to possess exceedingly potent uricosuric properties and was subsequently designated sulfinpyrazone (Fig. 6). Metabolic conversion to a derivative in which one of the benzene rings is hydroxylated in the para position, as in the case of phenylbutazone, does not occur t o a detectable extent (Dayton et al., 1961). D. SULFINPYRAZONE
As just indicated, sulfinpyrazone, (l12-diphenyl-4-(2’-phenylsulfinethyl)-3,5-pyrazolidinedione) (Fig. 6), was first discovered as the result of a systematic search in the urine for metabolites of l12-diphenyl-4(phenylthioethyl)-3,5-pyrazolidinedione1of which it proved t o be the sulfoxide derivative (Burns et al., 1957). Isolation of the sulfoxide was accomplished (Burns et al., 1957) by extraction of the acidified urine with ethylene dichloride, removal of the major portion of the urine “blank” of the organic phase with citrate (0.1 M)-phosphate (0.2 M) buffer, pH 4.8, and extraction of the sulfoxide from the organic phase into 2.5 N NaOH; quantification was by spectrophotometry at maximum absorption, 260 mp. The product was shown to be identical with authentic l12-diphenyl-4(2‘-phenylsulfinethyl)-3,5-pyrazolidinedionesynthesized by Pfister and Hafliger (1961). By virtue of the presence of a sulfoxide group, the compound occurs as two optically active isomers, and both D- and L-enantiomorphs have been prepared (Pfister and Hafliger, 1961). The sulfoxide
UIZICOSURIC DRUGS: PROBENECID, SULFINPYRAZONE
125
isolated from urine proved to be a mixture of isomers, predominantly the D-form (Dayton et al., 1961). Absorption of sulfinpyrazone from the gastrointestinal tract is rapid and virtually complete; plasma sulfinpyrazone levels are about the same 2 hours after oral and intravenous administration of a single 600-mg dose of the drug (Burns et al., 1957). This oral dosage yields peak plasma sulfinpyrazone levels of 6-10 mg%, usually within 1 hour. Then the plasma drug levels rapidly decline, giving a short biological half-life averaging 3 hours in man, and in some instances as brief as 1hour (Burns et al., 1957; Dayton et al., 1961). Like the parent compound phenylbutazone arid many of its other derivatives, approximately 98% of the plasma sulfinpyrazone a t concentrations of 10 mg% is bound to plasma proteins in man, about 92% in the dog (Gutman et al., 1960; Dayton et al., 1961). In its distribution, therefore, sulfinpyraeone is very largely confined to the extracellular compartment of the body water. The rapid turnover of sulfinpyrazone is accounted for in appreciable part by renal elimination of the unaltered drug, 2&45y0 of a 600-mg oral dose being recovered in the urine in 24 hours, most of it in the first 6 hours (Burns et al., 1057; Gutmari et al., 1960). Of the drug not excreted as such, the largest proportion appears in the urine as p-hydroxysulfinpyrazone (Fig. 6), but not in detectable amounts as the sulfonc (Dayton et al., 196l), even though a variety of other sulfoxides are known to be oxidized to sulfones (Brodie et al., 1958). Administration of racemic sulfinpyrazone yields optically inactive drug in the urine, indicating that both isomers are handled in the same may; this is further indicated by recovery of the optically active isomer as such, without racemization, after intravenous administration (Dayton et al., 1961). Since sulfinpyrazorie circulating in the plasma is almost wholly bound to plasma proteins in man a t ordinary therapeutic blood levels, very little can be filtered at the glomerulus, hence renal elimination must occur by tubular secretion, presumably by way of the “organic acid system.” This inference was substantiated in renal clearance studies by the demonstration of very high excreted/filtered ratios of the drug (Gutman el al., 1960), notably in man, with indications that the secretory T m is approximated a t the usual therapeutic blood levels of the drug. Stop-flow studies in the dog showed a proximal site of secretion. Tubular reabsorption, by backdiffusion, is very limited even in acid urine, in which sulfinpyrazone is largely dissociated (pK, 2.8), and allialinieation of the urine does not enhance the urinary elimination of the drug significantly (Gutman et al., 1960). That there is nevertheless some diminution of nonionic diffusion in the distal portion of the nephron when the urine is alkalinized could be shown ill stop-flow studies in the dog (Gutman et al., 1960).
126
ALEXANDER B. GUTMAN
Sulfinpyrazone has little or no effect on renal hemodynamics. The glomerular filtration rate is usually unaltered and, while CPAHis markedly reduced, this presumably reflects decreased renal extraction of PAH, the result of competition for secretion by the tubular “organic acid system,” and does not signify any decline in renal blood flow (Burns et al., 1957). Phenolsulfonphthalein excretion is similarly suppressed (Domenjoz, 1960). The drug in effective uricosuric dosage elicits no significant change in urinary excretion of sodium in man (Burns et al., 1957) but apparently increases sodium, chloride and potassium excretion in the dog (De Koster et al., 1963). Renal conservation of other electrolytes or metabolites seems not to be disturbed. Sulfinpyrazone is devoid of analgesic or antipyretic properties, and is ineffective in relieving inflammatory manifestations in the human arthritides, although reported to be anti-inflammatory in animal preparations (Domenjoz, 1960). It has uniformly been found to have no discernible beneficial effect in acute gouty arthritis, despite sharp lowering of the serum urate level, and the pain and stiffness of chronic gouty arthritis respond only after the long interval required for mobilizing tophaceous deposits. The sole therapeutic use, then, of sulfinpyrazone is as a uricosuric agent, by virtue of its striking capacity to suppress tubular reabsorption of urate in man (Burns et al., 1957). The potency of sulfinpyrazone as a uricosuric drug was first clearly brought out in clearance studies which showed t,hat intravenous infusion of 420-450 mg (about 5 mg/kg body weight) rapidly produced a mean sevenfold peak increase in C,,/GFR (Table IV), as compared to a fouror fivefold increase effected by probenecid in about 25 mg/kg dosage, and that as little as 35 mg sulfinpyrazone given intravenously sufficed to elicit detectable uricosuria, as ccmpared to a minimum requirement of some 100 TABLE IV EFFECTOF SULFINPYRAZONE O N URATECLEARANCE IN MAN“ Time (min) 0 044 44-64 68-85 85-111 111-129 129-144 144-164 0
From Burns et a2. (1957).
8.4 Infusion of 420 mg 7.4 6.9 6.3 5.9 5.6 5.5
10.2 6.2 (4.6 mg/kg) sulfinpyrazone 69.5 51.1 71.2 54.8 72.6 46.6 72.6 46.6 63.0 44.4 63.0 44.4
URICOSURIC DRUGS:
PROBENECID,
SULFINPYRAZONE
127
nig for probenecid (Burns et al., 1957). Administered orally in 400 or 800 mg daily dosage to gouty subjects (in divided doses because of the short half-life of the drug), the 24-hour urinary output in the ensuing days about doubled, and the serum urate fell precipitously to almost half the initial level (Burns et al., 1957). A similar response of the urinary uric acid excretion and serum urate level was found by Ogryzlo and Harrison (1957), who estimated that, per unit weight, sulfinpyrazone was about 6 times more potent than probenecid. The efficacy of sulfinpyrazone as a uricosuric agent, which is in part ascribable to the p-hydroxy metabolite rapidly formed and also possessed of potent uricosuric properties, has since been consistently confirmed by many investigators (Kersley et al., 1958; Yu et al., 195813; Ruiz Moreno, 1959; Council on Drugs, 1960; de SBze and Ryckewaert, 1960; Kersley and Gibbs, 1960; G6mez Carpio et al., 1961; G. R. Thompson et al., 1962; Mellinghoff and Gross, 1962; Leng-LBvy et al., 1962; Emmerson, 1963; Kuzell et al., 1964; Robinson, 1965). I ts capacity, on protracted administration, to prevent tophaceous deposits and slowly to mobilize those already formed (Yu et al., 195813) has also been generally confirmed (for testimonials, see Lega Intern. Reumat., 1961; G6mez Carpio et al., 1961; G. R. Thompson et al., 1962; Emmerson, 1963; Kuzell et al., 1964; Robinson, 1965). I n the management of primary gout, sulfinpyrazone is employed in the same manner as already described for probenecid but, because of its greater potency, it is given in lower dosage. In the usual prescription, 400 mgjday in divided doses, sulfinpyrazone elicits a mean increase of 65% in the 24-hour urinary uric acid excretion and a mean fall of 30% in serum urate levels (Yu et al., 1958b), a response of the same order of magnitude as ordinarily obtained with 1.5-2.0 gm/day probenecid; in some cases 200 mg sulfinpyrazone/day suffices, in others 600-800 mg/day is required. As a n alternative to probenecid when uricosuric therapy is indicated, sulfinpyrazone is particularly useful in patients who do not tolerate or do not respond satisfactorily to probenecid, and is often a more effective substitute (Yu et al., 195813; de SBze et al., 1963; Kuzell et al., 1964). However, when renal damage is sufficiently advanced, significant uricosuria may not be obtainable even with sulfinpyrazone. In some such cases a better response may follow the combined use of sulfinpyrazone and probenecid; as previously noted, this combination exerts an additive effect (Seegmiller and Grayzel, 1960), apparently because probenecid inhibits the tubular excretion of sulfinpyrazone, and thus prolongs its uricosuric action (Dayton et al., 1965). I n refractory cases allopurinol is efficacious in lowering the Serum urate level, and sulfinpyrazone may be given concomitantly (Yu and Gutman, 1934). Salicylate cannot be administered concomitantly with sulfinpyrazone,
128
ALEXANDER B. GUTMAN
to compensate for its lack of analgesic effect, because the uricosuric action of sulfinpyrazone, like that of probenecid previously discussed, is thereby abolished in man (Ogryzlo and Harrison, 1957; Kersley et al., 1958; Seegmiller and Grayzel, 1960; Yu et al., 1963; Emmerson, 1963) but not in the dog (Yu et al., 1963). Two sites of interaction of salicylate and sulfinpyrazone have been demonstrated (Yu et al., 1963). There is competition for binding sites on plasma proteins, salicylate displacing sulfinpyrazone, in the concentrations employed therapeutically, in both man and dog. Sulfinpyrazone competes successfully with salicylate for tubular secretion in man, but salicylate, in some as yet obscure manner, apparently blocks the inhibitory effect of sulfinpyrazone on tubular reabsorption of uric acid. The toxicity of sulfinpyrazone is of low order, comparable in kind and frequency with that of probenecid. The median lethal oral dose in acute toxicity studies in the mouse and rat was found by Domenjoz (1960) to be 300-375 mg/kg, and the ulcerogenic tendency in the rat was, like that of oxyphenbutazone, substantially less than that of phenylbutazone. In man, the incidence of gastrointestinal complaints is l0-15% (Yu et al., 1958b; de SBze and Ryckewaert, 1960; Emmerson, 1963; Kuzell et al., 1964). Drug rash and fever occur rarely. As with probenecid and other potent uricosuric agents, and to similar degree, there is the danger of provoking uric acid stone formation with renal colic, unless the urinary volume and pH are maintained at appropriate levels. There is also a like propensity to precipitate acute gouty attacks in the early stages of therapy, particularly if initiated with large doses. Sulfinpyrazone has, however, a somewhat greater tendency to depress hemopoiesis (Yu et al., 1958b; Glick, 1961; Persellin and Schmid, 1961); therefore, although this effect has thus far been reversible when the drug was discontinued, blood cell counts should be obtained a t intervals, as a precaut,ionary measure, when sulfinpyrazone is continuously administered.
E. STRUCTURE-ACTIVITY RELATIONSHIPS IN
PYRAZOLIDINEDIONES Burns, Brodie, Dayton, and their associates have investigated the structure-activity relationships of the pyrazolidinediones by analysis of the effects of substitutions in the benzene rings and/or butyl side chain of the parent compound, phenylbutazone, on its potent antirheumatic powers, its marked tendency to cause salt and water retention, and its modest uricosuric properties. Table V lists some of the analogs examined in greater detail. Various aspects of the results have been summarized by Burns et al. (1960), Gutman et al. (1960), Bloom and Laubach (1962), and Perel et al. (1964) These studies were initiated with the observation that the distinct structural alterations characterizing the two major metabolites of phenylTHE
URICOSURIC DRUGS : PROBENECID, SULFINPYRAZONE
129
butazone in man (Fig. 5) had brought about quite different effects. Metabolite I, formed by hydroxylation in the para position of a benzene ring, retained the pharmacological at tributes of the parent compound virtually without change, whereas metabolite 11, in which carbon-3 of the side chain had been hydroxylated instead, had acquired much more pronounced uricosuric activity (Burns et al., 1955; Brodie et al., 1956; Yii et al., 1958a). It was first inferred that the uricosuric activity of the pyrazolidinediones depends solely on the composition of the side chain, but this was not borne out when analogs representing a variety of changes in the side chain were tested. Finally it was realized that structural alterations, whether in the side chain or benzene rings, which appreciably increase the acidity of the compound, as, for example, by affecting electron transfer (Bloom arid Laubach, 1962), also enhance uricosuric activity (Burns et al., 1958b). This inverse relationship is illustrated in Table V, where it is apparent that) analogs with pZ<, < 4.0 are markedly uricosuric, whereas those with pK, > 4.0 are distinctly less so. The association of uricosuric potency with acidity in this series is all the more striking because it is the only correlation thus far apparent. There is 110 readily discernible relationship to lipid solubility or to biological half-life of the various compounds-although both factors presumably play some role-and the binding to plasma proteins in man is about the same for all the analogs. How the pk’, of the compounds in question, i.e., the degree of their dissociation in the body fluids, affects the tubular system for reabsorption of uric acid, and perhaps also that for tubular secretion (Gutman et al., 1960), is still wholly obscure. There is also a general relationship of the pK, to the enhanced rate of renal excretion of the more acidic pyrazolidinediones (Table V), but this clearly reflects their lesser rionionic back-diffusion a t the pH of the urine. This latter phenomenon is common to a variety of foreign organic acids, those of lower pK, being eliminated more expeditiously, those of higher pK, more slowly; the latter are more freely reabsorbed by lionionic back-diff usion, and usually are then converted to conjugates and metabolites of suitably lower pK,. [This “increased acidity” hypothesis of detoxication of organic acids is discussed by Williams (1959), Gutman et al. (1960), and Weiner and Mudge (1964).] I n regard t o the antirheumatic and sodium-retaining properties of phenylbutazone, these are but little affected by substitution of a chloro, methyl, or nitro group in the para position of the benzene ring. However, if substitutions are made in the meta position of the benzene rings or in the butyl side chain the resulting compounds are found to have lost much or all of their antirheumatic and salt-retaining activity (Burns et al., 1960). Of course, the structure-activity relationships of the pyrazolidinediones are markedly affected by many other determinants. Some, like metabolite
c
0
URICOSURIC POTENCY, RATE OF
TABLE V 0 URINARY EXCRETION, A N D BIOLOGICAL HALF-LIFE,IN MAN, OF SOMEPYRAZOLIDINEDIONES~*~
I
I
0 4
c=o
\ /
H-C-Ra
M
T1/2 Designation G-34208 G-35716 G-13838 G-15235 Oxyphenbutazone G-27463 G-26924 Phenylbutazone G-32170 '2-29665 G-25592 G-25903 G-33378
RI
Rz
OH OH H CI4 OH OH H H F OH H H OH
H H H CH3 H OH H H F H H H H
It3
PK=
KP
(W
8.0
1 12 72 24 72 36 36 72
0.4
3.4 0.6 -
2.2 1.o 0.01 2.4 0.4
40
4
3 17
%-hour urinary excretion (% dose) <2 <3 <1
-
<2 <1 <1 <1
>50 <2
sxz u uricosuric potency" >1200 >1000 800-1Ooo 1000
-
80(r1000 <800
-
800-1000 <400
E W
G-15140 Metabolite I1 G-25671 G-34764 G-28234 G-32642 Sulfinpyrazone G-31442 G-32567 G-29701 G-30249
c1 H H C&SOz NO2 OH H H C&SOz
OH OH
c1 H H H H H H H C&SO, H H
(CJWCH3 (CH2)zCHOHCHs (CHd zSC& (CH2)sCHa (CHzhCHa (CH2)zSOCd% (CHz)zSOCsHs (CHI)& O Z C ~ H ~ (CHz)*CIt CO(CH2)zC& COCHzC6Hs
4.0 4.0 3.9 3.4 3.2 3.1 2.8 2.7 2.6 2.3 2.0
83 0.6 1.6 1.o 0.01 0.5
-
0.6 0.4 -
20 12 3 24 20 1 3 3 1 8 3
<1
8 <3 <2 <2 58 43 35 40 40
-
1000 150-300 150-300 150-300 30-100 100-1 50 30-70 100-150 30-70
a Data compiled from Burns et al. (1960); Gutman el al. (1960); Seegmiller et al. (1960); Perel et al. (1964); P. G. Dayton (oral communication, 1965). * All substitutions in the benzene rings of phenylbutazone are in the para position, as indicated, except '2-27463 which is meta substituted. Determinations of pK, were made by the method of Flexser et al. (1935); K, is the partition coefficient for the system peanut oil-Sorensen buffer, pH 7.4, determined by the method of Mark et al. (1958), as modified by Perel et al. (1964). 6 Uricosuric potency is defined as the approximate dosage required to elicit 100% increase in uric acid clearance.
C
!2
n
?i Q
U C
8 W M
z
2
-t3 v,
3
z r
N
0
2
n
132
ALEXANDER B. GUTMAN
I1 and G-28551 (which differs from phenylbutazone in having a keto group in the side chain), are so insoluble and poorly absorbed from the gastrointestinal tract that, although the pK, is low, they elicit comparatively little uricosuria after oral administration. The biological half-life, which varies greatly in man (Table V) as a reflection of significant differences in the rate of biotransformation and renal excretion, has an obvious influence on the pharmacological activities of the compounds, as do the properties of their respective metabolites. The rates and routes of metabolic conversions play an especially important role in the striking differences in biological half-life and response to the pyrazolidinediones in species other than man (Burns et al., 1960; Perel et al., 1964). Nevertheless, the structure-activity relationships described are useful in prediction of the properties of newly prepared pyrazolidinediones. This was illustrated in the synthesis of such analogs as the p-nitro congener of phenylbutazone, which displays not only the marked antirheumatic and sodium-retaining activities of the parent compound but, being more acidic, also possesses potent uricosuric properties (Yu et al., 1959; Burns et al., 1960). VII. Incidental Compounds Possessing Uricosuric Properties: Phenolsulfonpthalein, Mersalyl, lodopyracet, Corticotropin and Adrenocortical Steroids, Coumarins and Indandiones, Chlorprothixene, Acetohexamide, Ethyl-p-chlorophenoxyisobutyrate. The Paradoxical Action of Benzothiadiazines
There is a miscellany of compounds possessing incidental uricosuric properties, some of sufficient interest to merit mention a t this point. These include phenolsulfonphthalein (Talbott, 1943), mersalyl (Coombs et al., 1940), iodopyracet (Talbott, 1943; Bonsnes et al., 1944), corticotropin (Forsham et al., 1948), certain coumarins and indandiones (Sougin-Mibashin and Horwitz, 1955), and some benzothiadiazines. In respect to corticotropin, apart from its effectiveness as an antiinflammatory agent in terminating attacks of acute gouty arthritis, it also increases urinary elimination of uric acid if given in sufficient dosage (Forsham et al., 1948), with an accompanying fall in plasma urate concentration (Gutman and Yu, 1950). This would suggest that whatever increase in uric acid formation corticotropin may induce, whether by enhanced degradation of cellular riucleic acids or otherwise, its principal effect on uric acid metabolism is a t the renal level, to augment urinary excretion of uric acid presumably by inhibiting tubular reabsorption. The renal function studies of Ingbar et al. (1950) in man and Friedman and Byers (1950) in the rat support this view. Of the anticoagulant drugs, ethyl biscoumacetate [3,3’-carboxymethyl-
URICOSURIC DRUGS: PROBENECID, SULFINPYRAZONE
133
ene-bis-(4-hydroxycoumarin) ethyl ester] also possesses pronounced uricosuric properties, of the same order of magnitude as probenecid (SouginMibashan arid Honvitz, 1955; G. R. Thompson et al., 1959). Dicoumarol [3,3’-rnethylene-bis-(4-hydroxycoumarin)], too, is uricosuric but less so (Hansen and Holten, 1958; Dreyfuss and Czaczkes, 1958; Christensen, 1964) ; acenocoumariri [3-(a-p-nitrophenyl-p-acetyl ethyl)-4-hydroxycoumarin] has no uricosuric activity (G. R. Thompson et al., 1959). Phenylindandione (2-phenyl-indandione-1,3)is distinctly uricosuric whereas inisindiorie [2-(p-methoxyphenyl)indandione-l,3]is not (G. R. Thompson et al., 1959). The references cited give the results of supporting renal clearance studies. Chlorprothixene, the trans-isomer of 2-chloro-9-(3-dimethylaminopropy1idene)-thioxanthene,has been found in the usual tranquilizer dosages t o produce moderate uricosuria (Healey et al., 1965). The drug increased C,, t o about 20 ml/min and appreciably reduced the serum urate levels in gouty subjects. It is notevorthy that chlorprothixene, like zoxazolamine, is a weak base. The sulfonylurea, acetohexamide, (l-[p-acetylbenzenesulfonyl]-3-cyclohexylurea), has a dual hypoglycemic and uricosuric effect, the former exerted promptly, the uricosuric action not until 3-4 hours after administration (Yu and Gutman, 1965). In doses of 1.0 gm daily, C,,/GFR increased about 70%, the 24-hour urinary uric acid excretion rose a mean of 33% and the serum urate fell about 30Oj,. This association of activities, which is not shared by tolbutamide, has been found useful in the management of the occasional patient who has both diabetes and gout. Ethyl p-chlorophenoxyisobutyrate, which lowers serum triglycerides and cholesterol, has been found also to have uricosuric properties in hyperuricemic subjects, but with little or no accompanying reduction in serum urate levels (Oliver, 1962; Hellman et al., 1963; Trevaks and Lovell, 1965). The uricosuric effect is abolished by concomitant administration of salicylate (Trevaks and Lovell, 1965). The benzothiadiazine, phthalimidine, and quiriaeoline diuretic agents also deserve notice here in connection with their effects on urinary uric acid excretion (reviewed by Fuchs et al., 1960; Beyer and Baer, 1961). Best known in this regard is the frequent development of hyperuricemia associated with the administration of chlorothiazide (Laragh et al., 1958; Oren et al., 1958; Monroe et al., 1959; Demartini et al., 1962) or hydrochlorothiazide (Healey et al., 1959), and encountered also with flumethiazide, benzydroflumethiazide, trichlormethiazide, and cyclothiazide (Swartz et al., 1963), and also quinethazone (Bryant et al., 1962). It seems quite likely that this drug-induced hyperuricemia, which can be counteracted by giving probenecid or sulfinpyrazone concurrently (Healey et al.,
134
ALEXANDER B. GUTMAN
1959; Smilo et al., 1962; Bryant et al., 1962; Sperber et al., 1965), is due chiefly to inhibition of tubular secretion of urate (Borhani, 1960;Demartini et al., 1962; Bryant et al., 1962; but see also Ayvazian and Ayvazian, 1961; Smilo el al., 1962).The benzothiadiazines, phthalimidines, and quinazolines presumably are secreted by the common tubular “organic acid system.” Tubular secretion has been demonstrated for chlorothiazide (Beyer, 1958; Kessler et al., 1959b; Baer et al., 1959), occurring in the proximal tubule in the dog (Kessler et al., 1959b); moreover, chlorothiazide has been shown to inhibit tubular secretion of p-aminohippurate competitively (Essig, 1961). The action of potassium salts in correcting thiazide hyperuricemia does not appear to be by affecting renal excretion of uric acid, but by some other, as yet undetermined, mechanism (Zweifler and Thompson, 1965). However, if chlorothiazide is injected intravenously in substantial dosage, thus presumably effecting higher plasma and urinary drug concentrations, the prevailing response in respect to uric acid is distinctly uricosuric (Demartini et al., 1962; Bryant et al., 1962; Duarte and Bland, 1965). The same may apply to hydrochlorothiazide (Healey et al., 1959), although more equivocally (Duarte et al., 1961; Bryant et al., 1962). Ethacrynic acid (methylenebutyryl phenoxyacetic acid) is another diuretic agent that may cause renal retention of uric acid, with hyperuricemia, when given orally (Cannon et aZ., 1963), but is uricosuric when administered intravenously (Demartini, 1965). REFERENCES Am. Med. Assoc. (1963). “New and Nonofficial Drugs” (Council on Drugs), Lippincott, Philadelphia, Pennsylvania. Armstrong, F. B., and Scherbel, A. L. (1961). J. Am. Med. Assoc. 176, 614. Austrian, C. R., and Boger, W. P. (1956). Arch. Internal Med. 98, 505. Axelrod, J., and Chenkin, T. (1954). Proc. SOC.Exptl. Biol.Med. 86, 401. Ayvazian, J. H., and Ayvazian, L. F. (1961). J. Clin. Invest. 40, 1961. Baer, J. E., Leidy, H. L., Brooks, A. V., and Beyer, K. H. (1959). J. Phurmacol. Exptl. Therap. 126, 295. Bartels, E. C. (1943). Ann. Internal Med. 18, 21. Bartels, E. C. (1957). Metab. Clin. Exptl. 6, 297. Bartels, E. C. (1960). Med. Clin.N. Am. 44,447. Bartels, E. C., and Matossian, G. S. (1959). Arthritis Rheumat. 2, 193. Beam, A. G., and Kunkel, H. G. (1954). J. Clin. Invest. SS, 400. Beechwoocl, E. C., Berndt, W. O., and Mudge, G. H. (1964). Am. J. Physiol. 207, 1265. Benedek, T. G. (1961). Am. J. Med. Sci. 242, 448. Benedict, J. D., Foraham, P. H., Roche, M., Soloway, S., and Stetten, D., Jr. (1950). J . Clin. Invest. 29, 1104. Berger, L., Yii, T. F., and Gutman, A. B. (1960). Am. J. Physiol. 198, 575. Bergmann, F., and Dikstein, S. (1955). J . Am. Chem. SOC.77, 691. Berliner, R. W., Hilton, J. G., Yu, T. F., and Kennedy, T. J., Jr. (1950). J. Clin. Invest. 29, 396. Beyer, K. H. (1950). Pharmacol. Rev. 2 , 227.
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Synthetic Anti-Inflammatory Drugs: Concepts of Their Mode of Action R. DOMENJOZ Institirte of Pharniacolog?j, Rheinische Friedrich- Wilhelms liniversitat, Bonn, Germany
I. Int,roduction . . . . . . . . . . . . . . 11. Former Int,erpretations of the Effects of Antipyret.ic/Non-Narcotic Analgesic Drugs . . . . . . . . . . . . . . A. The Ant,iseptic Effect as t,he Hypothetical Cause of the Antipyretic Action . . . . . . . . . . . . . . . B. Central Nervous Site of Action . . . . . . . . . 111. The Pituitary-Adrenal Axis aiid Drug-lnduced Inhihitinn of Inflammatioii A. General Considerations . . . . . . . . . . . B. Clinical Findings in Man . . . . . . . . . . C. Experimental Findings in the Animal . . . . . . . . D. Discussion . . . . . . . . . . . . . . IV. The Inflammatory Focus as Site of Action of Anti-Inflammatory Drugs . A. Outline of the Inflammatory Process . . . . . . . . B. Trauma arid Lesion . . . . . . . . . . . . C. Tissue Alterathis Associated with Inflammation . . . . . D. The Inflammatory Reaction . . . . . . . . . . E. Peripheral Actions of Antiphlogistics . . . . . . . . V. Summary . . . . . . . . . . . . . . . References . . . . . . . . . . . . . .
143 144 144 148 156 156 157 160 166 171 172 173 175 181 186 204 204
I. Introduction
The synthetic anti-inflammatory drugs, i.e., the antiphlogistics, as they are still called today, belong to distinct chemical groups. From the therapeutic point of view the most important ones are (1) salicylates, (2) pyrazoles and pyrazolidines, and (3) quinoline derivatives. This enumeration might be supplemented by indole derivatives of the indomethacine type, the anti-inflammatory properties of which have rerently been discovered. One further group represented mainly by acetanilide and phenacetin is no longer used in anti-inflammatory therapy, although phenacetin as well as acetyl-p-aminophenol display excellent anti-inflammatory activities. The compounds belonging to this group are characterized by toxic effects owing t o the appearance of methemoglohinizing metabolites (acetanilide : M. Gross, 1946; phenacetin: P. K. Smith, 1957; Sarre et al., 1958; acetyl-paminophenol: Baader et al., 1981; Kiese and Menzel, 1962). A compound belonging t o one of the above-mentioned chemical groups of course, does 143
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not necessarily possess anti-inflammatory properties. There are many pyrazole derivatives without any antiphlogistic activity. The systematic research carried out in the last twenty years has demonstrated the presence of anti-inflammatory effects in other series of drugs, such as the sulfonamides, antibiotics, monoamine oxidase inhibitors, cytostatics, flavonoids, proteolytic enzymes, etc. ;nevertheless, these substances cannot be regarded as anti-inflammatory agents. In the current textbooks of pharmacology, synthetic drugs used in the treatment of inflammatory states are discussed in the chapters dealing with antipyretics and analgesics. This is due partly to the historical development in this field, partly to the characteristic properties of these drugs. From a purely schematic point of view, the anti-inflammatory drugs show three principal qualities of action. Under certain conditions, they act as antipyretics, analgesics, and antiphlogistics. These three principal properties are apparently not correlated quantitatively, as is evident from comparative experimental studies : in some drugs the antipyretic effect is predominant, while others are excellent anti-inflammatory agents without particular antipyretic or analgesic properties. This seems to justify the conclusion that the above-mentioned actions are fundamentally not correlated and are based on different mechanisms. This opinion ascribes the surprising coincidence of these three properties to mere chance, a n interpretation which is rather disappointing. The favorable therapeutic actions of some antipyretic-analgesic agents in inflammatory conditions and, in particular, in rheumatic fever were well known in the last century. The specific anti-inflammatory component of these agents, however, has been recognized and defined on the basis of appropriate pharmacological methods only in the last twenty years. Before that the favorable action in inflammatory conditions was ascribed to the antipyretic and analgesic properties. Recent studies seem to justify the interpretation that drug-induced inhibition of inflammation is due mainly to a local action of the agent within the inflammatory focus. II. Former Interpretations of the Effects of AntipyretidNon-Narcotic Analgesic Drugs
A. THEANTISEPTICEFFECT AS THE HYPOTHETICAL CAUSE OF THE ANTIPYRETIC ACTION The main indication of anti-inflammatory drugs is concerned with acute and chronic rheumatism, the most important therapeutic effect consisting in the inhibition of the inflammatory manifestations : diminution and disappearance of swelling, resorption of exudates, improvement of
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active and passive mobility, disappearance of pain, and, in case of fever, normalization of body temperature. The pharmacological interpretation of this therapeutic action has, in the course of time, been modified corresponding to the progress of medical knowledge and the development of pharmacological research. This fact is particularly evident from the changing ideas about the mode of action of antipyretic-analgesic drugs such as the salicylate derivatives. I n the therapeutic use of salicylates, two historical periods can be distinguished, aside from the use of salicylates in homeopathic doses, the therapeutic application of salicylate-containing drugs dates from ancient times up t o the 1870’s. The second period, characterized by systematic scientific research on their mode of action, began in 1873 with the discovery of a chemical synthesis of salicylic acid practicable on an industrial scale. In 1858 Kolbe (see 1860) succeeded in defining the chemical structure of salicylic acid as o-hydroxybenzoic acid. In December, 1859, in joint work with one of his assistants (Kolbe and Lautemann, 1860), he was able to synthesize salicylic acid from the sodium salt of phenol. This method, however, was not suited for the production of salicylic acid in larger quantities. Only in 1873 was Kolbe (see 1874) able to develop a method practicable on an industrial scale, for which he was granted a patent in 1877. Kolbe himself tried to find possibilities of application for the new drug. The suggestion for therapeutic use of salicylic acid was also his. The name of salicylic acid was given by Piria (1839a,b), one of J. B. Dumas’ assistants, who in 1838 degradated salicin, the active ingredient of willow bark, to a substance t o which he gave the name “acide salicylique.” Kolbe (1874) reported that the physical and chemical properties of salicylic acid were thoroughly studied and well known, but not its physiological properties, save for the observation of Bertagnini (1856) that high doses of salicylic acid (6 gm in 2 days) produce tinnitus and impaired hearing and that in the organism the substance is partly transformed to salicyluric acid. His words seem to indicate that Kolbe did not interpret the already well-known therapeutic action of willow bark as a n effect of salicylic acid. As a matter of fact, the curative action of willow bark in “agues” had been described a long time before by Stone (1763), while the chemical and therapeutic aspects of salicin had been investigated by Fontana (1825), Buchner (1828), Leroux (1830), and by Gay-Lussac and Magendie (1830). In personal investigations, Kolbe (1874) demonstrated that salicylic acid has antiseptic properties comparable to those of quinine and carbolic acid. He was able to show that it inhibits certain enzymic reactions, such as yeast fermentation, degradation of amygdalin to benzaldehyde and sugar,
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liberation of ally1 mustard oil from the sinigrin of ground mustard, etc. Moreover Kolbe (1874) rioted that fresh meat could be preserved by salicylic acid. He annotated his observation with the following: “thus the use of salicylic acid promises to become a means that will allow a piece of meat to be packed in barrels in South America a t low costs and to arrive here tastier, better preserved, and cheaper than after transformation into meat-extract a t Fray-Bentos by help of Liebig’s method which is in use now.” He added: (‘Theremarkable properties of salicylic acid, which inhibit the growth of fungi and render enzymes inactive and harmless, allow me to kelieve that it may find application in the therapy of certain diseases. It is certainly worth while to investigate what effects smaller or larger doses of salicylic acid may have on the course of the disease if they are administered t o the patient per os, intramuscularly, or rectally at the first signs of cholera.” Kolbe had made sure that his drug was innocuous by taking it himself in daily doses up to 1.5 gm. In March, 1875, he was able to persuade his colleague, the surgeon Thiersch of Leipzig, to try salicylic acid as a n antiseptic for the local treatment of wounds in the place of carbolic acid as recommended by J. Lister in 1867. Thiersch (1875) applied salicylic acid experimentally t o 160 cases of infected injuries and wounds. He used salicylic acid for irrigation and cleaning of wounds as well as for impregnation of dressing materials with excellent results. In his publication he maintained that salicylic acid has the valuable antiseptic properties of carbolic acid without the unpleasant side effects. Learning of the laboratory investigations of Kolbe and of the clinical experiments of Thiersch, (‘and since salicylic acid is distinguished by excellent antiseptic properties,” C. E. Buss, a physician in St. Gallen, “had the idea that this substance might also act as a n antipyretic.” The priority for the internal use of salicylic acid must, without doubt, be ascribed to Buss (1875a,b,c, 1876, 1878). I n scientific literature this merit is erroneously attributed to Stricker (1876), whose publication on the same subject appeared in January, 1876. In his paper, however, Stricker (1876) mentions the results that Buss obtained with salicylic acid in rheumatic polyarthritis; therefore the issue touching the priority is clear. Buss reconfirmed his results in numerous clinical cases and very soon observed the specific and favorable influence of salicylic acid on rheumatic fever; his first clinical paper on the internal use of salicylic acid was published in 1875. The communications of Kolbe (1874) and Thiersch (1875) concerned a problem of actual interest. In the same issue of the Centralblatt der Medizinischen Wissenschaften which contained the first publication of Buss, a paper by Furbringer (1875) of the Heidelberg Medical Clinic appeared, describing the antipyretic action of salicylic acid in experiments
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on animals. Shortly afterward Buss’s observations were confirmed by Riess (1875, 1876), Stricker (1876), and Riegel (1876) from three Berlin clinics. At the time of Kolbe, Thiersch, and Buss, particular importance was attached l o the antipyretic effect, owing to Liebermeister’s (1875) theory of t h c origin and pathologiral meaning of fever. The antipyretic effect of salicylic acid was attributed to its antiseptic properties. This hypothesis corresponded to the contemporary interpretation of the antipyretic action of quinine. Binz (1 867) had published experimental findings with quinine under the title: “Zur Wirkung antiseptischer Stoffe auf Infusorien von Pflanzenjauche.” He reported that quinine has a characteristic lethal effect on unicellular organisms. These findings suggested to him that thc favorable action of yuiriiiic in malaria might bc due to an analogous phenomenon. Moreover, he concluded that the cause of malaria, as yet unknown, might be found in an infection by a unicellular etiological agent (Fuhner, 1930; Bachem, 1932). With this ingenious hypothesis Binz was in advance of his time. Only in 1880 did the discovery of the Plasmodium malariae by A. Laveran establish the truth of Binz’s theory. Buss’s hypothesis concerning the antipyretic effects of salicylic acid was completely in line with these views. He was so firmly convinced that antipyresis results from antiseptic action that he was amazed by the fact that alcohol, in spite of its good antiseptic qualities, had no effect at all on hyperthermia. For several decades the interpretation that antipyretic action was due to antiseptic properties, as Binz (1867) formulated first for quinine and then extended to salicylic acid (Binz, 1877), was considered valid. It is worth mentioning that Ellinger (1%3), in his contribution to HeffterHeubner’s “Handbuch der experimentellen Pharmaltologie” does not say anything about a specific anti-inflammatory property of salicylic acid. Regarding the mechanism of action of this compound, Ellinger states: ii. . . in rheumatic arthritis, whose etiology is as yet unknown, the universally acknowledged therapeutic success justifies the conception of a causal action due to an internal disinfection, occurring after intake of both salicylic acid and sodium salicylate.” Similarly, Auer (1922) reports that metamizol (Novalgine) possesses “. . . a strong analgesic effect and, to a certain extent, an anti-inflammatory action inasmuch as . . . in the organism it probably Causes the intermediary appearance of formalin, by mealis of which it exercises an antiseptic and germicidal action against pathogenic organisms. . . .” Likewise Schottmuller (1927, 1929), who introduced the treatment of rheumatic polyarthritis with aminophenazone, was still under the influence of the old theories. He summarized his views concerning the
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mechanism of action of aminophenazone as follows: “. . . there is no doubt about the specific anttimicrobialaction of the above-mentioned drugs (i.e., salicylate and aminophenazone),” In his opinion aminophenazone, too, was to be regarded as a drug active against the etiological factor of rheumatic arthritis. I n scientific literature, however, early doubts about the validity of this theory on “antipyresis through antisepsis” had appeared a considerable time before. In regard to phenazone, introduced into therapy by Filehne (1884), Engel (1886, cited in Rohde, 1923, p. 1107) was able to demonstrate that concentrations that would be necessary to kill pathogenic microorganisms were difficult to obtain in the body. On the basis of in vitro experiments he calculated that, e.g., an inhibition of Friedlander’s pneumococcus in vivo would require a therapeutic dose of about 30 gm. A similar limitation exists in regard to the antiseptic action of salicylates. Furthermore, it should be considered that their antibacterial action, when occurring at characteristically low concentrations, is caused by a specific inhibition of the synthesis of pantothenic acid (Ivanovics, 1942, 1943a,b), and therefore applies only to microorganisms dependent on pantothenic acid. Decisive objections to the conception of a causal significance of the antiseptic effect in the therapeutic and particularly in the anti-inflammatory action of the antipyretics/analgesics arose from the observation that these drugs are active also in noninfectious inflammations, and that they exercise their interesting action principally in rheumatic affections not due to active infectious processes. Furthermore, several excellent antiphlogistics, entirely devoid of significant antiseptic activity, are known today. Thus, it was never possible to demonstrate any antibacterial activity for aminophenazone. In the case of phenylbutazone, there are only the results of Gaudin (1956, 1957), according to which it inhibits the growth of Saccharomyces cerevisiae at concentrations of 1 :5000. Undoubtedly, this effect must be interpreted as an unspecific antiseptic activity devoid of practical importance. B. CENTRAL NERVOUS SITE OF ACTION The conceptions of Binz and Buss concerning the mechanism of druginduced . antipyresis, especially by sodium salicylste, due to antiseptic properties, were soon modified by results obtained in the course of investigations on the physiology of thermoregulation. Bergmann (1845) maintained that body temperature is controlled by regulatory mechanisms. His hypothesis, founded entirely on speculation, took into consideration two possibilities : In hyperthermia caused by an increase of basal metabolism, heat dissipation is effected by a direct peripheral vasodilatation,
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especially of skin vessels. Second, he considered the existence of a thermosensitive cerebral structure effecting, by reflex action, a n increased loss of heat from the body surface. The existence of the central regulatory mechanism was demonstrated by Tscheschichin (1866), Naunyn and Quincke (1869), Heidenhain (1870), and others, who were able to induce fever by artificial lesions of certain cerebral zones. Aronson and Sachs (1885) reported particularly convincing results obtained with hyperthermia induced by the so-called “heat puncture.” This experimental procedure was later almost universally used in pharmacology as a means of investigating the temperature-lowering mechanism of salicylic acid, phenazone, and quinine. Thus, in his classic experiments Gottlieb (1890) was able to demonstrate that salicylic acid as well as phenazone, but not quinine, abolish “heat-puncture fever.” Since he was able to obtain similar results with morphine, he concluded from these experiments that salicylic acid and phenazone may act as specific narcotics of the thermoregulatory center. I n regard t o quinine he supposed that hypothermia was not due to a n “interaction with central nervous regulation,” but resulted from a decrease in heat production by direct action on tissue metabolism. Among other things he mentioned that phenazone ‘(induced a definite state of analgesia and somnolence.” He referred to LBpine’s (1886) observations on the favorable influence of phenazone on the shooting pains of tabetics. L6pine had emphasized the concomitance of the antipyretic and analgesic effects of acetanilide and phenacetin and postulated a common origin for these different kinds of action. Particularly interesting in this connection is the statement of Gottlieb (1890) that the striking concomitance of antipyretic and analgesic actions of these drugs cannot be considered as accidental. By his investigations Gottlieb (1890) furnished an experimental demonstration of a central site of action for the synthetic antipyretics/analgesics. His findings were confirmed by Bondi and Katz (1911), Hashimoto (1915a,b), Barbour and Wing (1913), and others. These results, as well as the experimental investigations on the significance of nervous factors for the inflammatory reaction, led to the conception that the central action of the antipyretics/analgesics might be responsible for their favorable therapeutic action in inflammatory diseases. In this regard, various possibilities were discussed, in which, at the beginning, special attention was attached to the analgesic component. 1. Analgesia as the Hypothetical Cause of Anti-InJlammatory Activity
This point of view was maintained in its purest form by Spiess (1906) on the basis of his experiences concerning the influence of anesthesia and analgesia on the course of inflammatory conditions. He was surprised at the observation that local inflammatory reactions could be controlled by
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application of local anesthetics such as orthoform, Anaesthesin (ethyl-paminobenzoate), and procaine. His idea was that inflammation occurs within a reflex mechanism, the centripetal impulses being conducted through the sensory .fibers, the centrifugal ones in the vasomotor fibers being responsible for the dilatation of the small blood vessels. His explanation of these observations was formulated as follows: “An inflammation will not become manifest if one succeeds in interrupting the reflexes that start from the phlogistic focus and whose impulses are conducted by the centripetal sensory nerves.” He stated more precisely : “anesthesia must inhibit only the sensory fibers without interfering with the normal activity of the sympathetic nerves.” To support his point of view he referred to the earlier investigations of Samuel (1890b), who in his extensive experimental work on inflammation had observed that a strong thermal stimulus delivered t o a rabbit’s ear did not induce inflammation if the nerves had been severed previously. I n addition, Spiess observed that certain inflammatory reactions can be prevented by morphine. The decisive factor in this case was, in his view, the elimination of painful sensations: “If one succeeds in removing pain, inflammation will be prevented.” He actually ascribed such a mechanism of action to salicylic acid and phenazone, and stated literally: “The antipyretics widely used in inflammatory diseases may owe their activity principally t o their analgesic properties; maybe they are mainly anesthetics.” I n accordance with Spiess, Gaisboeck (1917) interpreted a n interesting clinical case: I n a patient suffering from strong articular pains caused by a subacute febrile arthritis, a sensory-motor hemiparesis appeared suddenly as a consequence of apoplexy localized in the medulla and pons. The apoplexy eliminated the articular pains. At the same time, the inflammatory manifestations disappeared; afterward they reappeared, though only within the limits of the hypo- and hyperesthetic zones. They did not reappear at all in the monolateral anesthetic areas. This behavior was interpreted by Gaisboeck as due to a lesion of the sensory pathways. The evident inhibition of inflammation observed in this case was considered by him a s a proof of the existence of central nervous regulation of inflammation. I n his opinion the inhibition of inflammation by anti-inflammatory drugs was also due to an action on the central regulatory mechanism. The opinion held by Spiess induced a whole series of investigations on the part played by the central nervous system in the inflammatory reaction. An experimental contribution to this problem was furnished by Bruce (1910), who showed that an elimination of pain sensation alone was not as important as Spiess had supposed. Bruce (1910) was able to demonstrate, in a n extensive series of animal experiments, that the early phases of inflammation are not influenced by an acute section of the spinal cord
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or of the dorsal roots or of a peripheral sensory nerve. Bruce noted a n inhibition of the inflammatory reaction in the following cases: ( a ) when the peripheral nerve had degenerated after its section; (6) when the function of sensory nerve terminations was inhibited by local anesthesia. From these findings he concluded that the initial vasodilatation in the early phases of inflammation is probably induced by an axonic reflex. At first the hypotheses of Bruce (1‘310) were confirmed by Breslauer (1919, 1920a,b). He observed in clinical cases that the inflammatory reaction is indeed inhibited only after degeneration of a severed peripheral nerve, and that inflammation may be suppressed by infiltration anesthesia, but not by nerve block. Subsequent investigations performed by Groll (1923) with phlogogcnic stimuli applied within paralytic, hyperemic vascular zones in the frog showed, however, that even during complete elimination and paralysis of the vasomotor nerves, inflammation occurs in the same way as during normal function of the vasomotor nerves. Groll therefore concluded that the nervous elements of the vasomotor apparatus have no essential significance for the induction of inflammation. Similar inferences were drawn by Ratschow (1933) from experiments performed on denervated vascular zones (cervical sympathetic). He considered the posttraumatic stasis as a consequence of the liberation of vasoactive substances from the damaged tissues. It is interesting to note that a similar hypothesis had been formulated as early as 1923 by Ebbecke. 2. Antipyresis and Inhibition of Inflammation
It has been known for a long timc that application of cold brings about a subjective and objective improvement in cases of inflammation. As early as 1892 Samuel had performed animal experiments in order to substantiate this empiric knowledge. In his investigations he used the croton oil dermatitis of rabbit’s ear. By cooling the ear hc was able to delay the appearance of inflammation, even in the contralateral ear. Further, he obtained “antiphlogosis a t a distance,” as he termed it, by cooling a single limb or the whole body of his animals. A similar mechanism of action was discussed by Fuerst (1925) for the inhibition of inflammation by phenylquinolincarbonic acid. In experiments with mustard-oil inflammation performed on rabbits, inhibition of inflammation occurred only when the body temperature simultaneously decreased for several degrees. The anti-inflammatory effect was indeed present, to a n appreciable extent only when skin temperature fell to 36°C. If, on the contrary, the antipyretic effect was prevented by artificial heating, the anti-inflammatory action of phenylquinolincarbonic acid was abolished. Fuerst suggested that lowering of the temperature results in a remarkable diminution of skin responsiveness leading to an inhibition of
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the inflammatory reactions. In fact, in these experiments doses of 400 and 500 mg/kg of phenylquinolincarbonic acid were required to obtain consistent anti-inflammatory effects. Fuerst (1925), of course, was quite aware that tthe mode of action of these rather toxic doses might not apply to the situation of human patients. The conception of Fuerst concerning an intimate relation between lowering of body temperature and inhibition of inflammation did not meet with general assent. Starkenstein (1920) demonstrated in experiments with papaverine that decrease of body temperature is not necessarily connected with a simultaneous inhibition of inflammation. In this regard it should be noted that the temperature-lowering effects obtained by Fuerst with phenylquinolincarbonic acid were considerably stronger than those resulting from the doses of papaverine administered by Starkenstein. Moreover, today numerous compounds of different chemical groups, which display intense and long-lasting hypothermal effects but do not exert any influence on inflammatory reactions are at our disposal. This fact, of course, does not clarify the relation between antipyretic and anti-inflammatory mechanisms unless the mechanism causing the hypothermia is considered. In this regard an antipyretic effect by loss of heat conditioned by a direct peripheral vasodilatation has, of course, to be distinguished from the antipyresis resulting from a primary action on the thermoregulatory centers. At any rate, papaverine, chosen by Starkenstein, was not at all suited for clarification of this problem. 3. Alterations o j Blood and Tissues Resulting from Respiratory Changes, and Inhibition of InJEammation First observations on the inflammation-inhibiting and delaying effects of narcotics were published by Shimura (1924). Peng (1930), who examined the activity of ethyl urethane on the mustard-oil dermatitis in rabbits, stated that an appreciable inhibition of inflammation was obtained only when the respiratory activity was simultaneously strongly diminished. Such an effect, incidentally, was noted with doses that were not sufficient to induce a significant decrease of body temperature. In addition to these investigations, Guggenheim (1930) was able to demonstrate that hypnotics with different chemical structures, such as diethylallylacetamide (Novonal), induced similar effects. On the other hand, the respiratory stimulant pentylenetetrazol (Cardiazol), at a dose of 0.1 gm/kg by mouth, enhanced inflammation in 66% of the animals, and in 83% blocked the inhibition of inflammation produced by urethane if administered simultaneously with the pentylenetetrazol. Artificial respiration also blocked the anti-inflammatory effect of urethane. Guggenheim observed the interesting detail that urethane induces a delay of anti-inflammatory action that lasts several days longer than the respiratory inhibition.
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Winkler (1930), another author belonging to the same research team, was able to demonstrate that bromides, without inducing variations of body temperature, also caused an inhibition of respiration and inflammation, which can be antagonized by pentylenetetrazol. In addition, Frohlich (1930) showed that urethane- or bromide-induced inhibition of respiration causes a remarkable alkalization of the blood, measurable by increase of pH and alkaline reserve values. These findings were interpreted by Lipachitz (1930) in the sense that the shift of blood reaction toward alkalinity caused by inhibition of respiration “demonstrates a passage of alkaline valences from the tissues to the blood” and that the resulting “relative acidification” of the tissues might explain the decrease in reactivity against the inflammatory stimulus. For many reasons, however, the interpretation of Lipschitz seems unsatisfactory today . 4. Central Nervous System and Control of In$ammation
After checking the validity of Lipschitz’s conceptions, Hofmann (1940) came to different conclusions. The influence of different narcotics on respiratory functions seemed to this author less important than the suppression of sensory stimuli. The inhibition of inflammation induced by different narcotics was abolished by pentylenetrazol proportionate to its antagonistic action against narcotics. In the case of sodium barbital, the anti-inflammatory effect increased “abruptly only when complete analgesia appeared in addition to the diminution of lung-ventilation.’’ Hofmann therefore ascribed the anti-inflammatory effect of narcotics to their central depressant action, believing in the existence of a “neurogenic regulation of inflammation.” The effects of narcotics on respiration and tissue metabolism were regarded by Hofmann as of secondary importance. The existence of central nervous mechanisms involved in the drug-induced inhibition of inflammation has recently been reconsidered by Tanaka and Mishima (1957), who indicated interesting perspectives in this field. These authors studied the anti-inflammatory effect of morphine on four different types of experimental inflammations in the rabbit. Consistent inhibition was obtained in all cases (histamine and mustard-oil conjunctivitis, mustard oil and cantharidin dermatitis) with a dose of 5 mg/kg i.v. If morphine was administered intracisternally, the same effect was obtained with doses as small as 0.5 mg/kg. With chlorpromazine, the proportion of equivalent doses amounted to 1 :20 (Mishima and Aoi, 1956). From these findings the authors deduced the existence of a central nervous site of action for the anti-inflammatory effect of morphine. Concerning the efferent pathways, it was possible to demonstrate that local anesthetics and atropine had no effect, but that inhibition of inflammation was reduced or even abolished by adrenergic blocking drugs or by
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extirpation of the sympathetic ganglia. Tanaka and Mishima (1957) therefore hold that the efferent, pathways concerned with the anti-inflammatory effect of morphine are sympathetic fibers. Participation of the adrenal medulla by liberation of adrenaline could not be excluded (Theobald and Domenjoz, 1956), since sympathetic blockade did not abolish inhibition of inflammation regularly and totally. Riechert and Barghorn (1954) have recently recalled the hypothesis of a central regulation of inflammation. From experiments performed with tribromoethyl alcohol (Avertin) on egg-white edema they conclude that peripheral edema formation depends on a central factor. After administration of tribromoethyl alcohol the reactive hyperemia developed normally, while the edematous phase was retarded until the anesthesia decreased. Subsequent investigations of Riechert and Eschbach (1955) concerned reserpine, which prevents the appearance of, but is inactive against an existing, edema; these results, too, were explained by the existence of a central control, an interpretation that will probably have to be modified when confronted with the present state of knowledge. The above-mentioned findings concerning a participation of the central nervous system in drug-induced inhibition of inflammation encouraged many investigations on the existence of a central control of inflammation. According t o the present point of view, as formulated by Heilmeyer and Kahler (1962), the nervous system exercises a modifying action on inflammation. Its function, however, is not essential, because inflammatory vascular reactions can also be obtained in denervated tissue (Lange et al., 1930). I n highly differentiated organisms, of course, the central nervous system must be ascribed a certain control function in regard to the inflammatory reaction. Certain observations gained in medical practice seem to prove the participation of the nervous system in the regulation of inflammation. Among these may be listed the localized tissue reactions that may be induced by conditioned reflexes (Metallnikow, 1932; Koslowski, 1942), the influence of the psyche on the clinical course of some inflammatory diseases, and, finally, the suppressive effect obtained in certain inflammatory conditions by hypnosis (Chapman et al., 1963). An experimental detail of notable interest is also represented in regard to this problem by the results obtained with antidromic excitation conducted through afferent sensory fibers. By means of experimental stimulation of the peripheral segments of severed sensory nerves it was possible to obtain topographically limited tissue lesions coexistent with increased tissue sensitivity to external stimuli resulting in enhanced disposition t o inflammation. Reilly et al. (1935a,b) produced intestinal lesions by stimulation of the peripheral portion of the transected splanchnic nerve. Similar effects were obtained by Tine1 et al. (1937) after stimulation of the dorsal roots through which the afferent
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fibers of the splanchnic nerve pass. These results recall earlier experiments of Forster (1927), who observed in man that stimulation of the peripheral portions of a severed posterior root caused painful sensations. These experiments are interesting insofar as the ant idromic stimulation applied may be compared to the effect of exogenous noxious stimuli on a limited zone of tissue, as Chapman et al. (19G3) and others were able to show: Immersion of the lower extremities in water at 43°C caused an extensive cutaneous vasodilatation. During the active phase of this t hermoregulat ory reaction mediated by the diencephalon, vasodilatation occurred also in some nonimmersed areas. In these remote zones, reacting with hyperemia, the pain threshold was lowered, and stimuli tolerated under normal conditions caused tissue damage, which proved the existence of a n increased disposition to inflammation. Bilisoly et al. (1954) also observed a lowering of the pain threshold in zones in which a flare reaction was induced by means of noxious stimulation. These findings suggest that tissue damage leads to an increased seiiaitivity of lhe pain receptors, probably by means of humoral mechanisms. In this connection, investigations of Habgood (1950) should be mentioned. He used flaps of frog skin with intact innervation. Stimulation of an isolated cutaneous nerve produced potentials of excitation in distinct sensory fibers originating from the same area. The results obtained by Chapman et al. (1963), Bilisoly et al. (1954), and Habgood (1950) are of special importance in regard to the “methods of measurement of analgesic activity on inflamed tissue,” described by Randall and Selitto (1957), and Labelle and Tislow (1950). These procedures allow us to distinguish the “analgesic” effects of certain antiphlogistics from those of morphine, its derivatives, and its synthetic substitutes. The existence of a central nervous site of action of the antiphlogistics is suggested mainly in connection with narcotics and analgesics whose principal effectsare limited to nervous substrates. The results of Tarmka and Mishima (1957) with intracisterrial application of morphine have been discussed. I n this connection we also have to recall the investigations concerning the anti-inflammatory action of morphine, ketobemidon, etc. on egg-white, formalin, and dextran edemas, performed by F. Gross (1950a,b), Meier et al. (1951), Domenjoz (1953, 1954, 1955), Domenjoz et al. (19551.)), Theobald (1954, 1955), and Frank (1955). These anti-inflammatory effects, while still present after hypophysectomy (Domenjoz et al., 1955b), were notably reduced by adrenalectomy (Meier et al., 1951; Domenjoz, 1954, 1955; Theobald and Domenjoz, 1956). The investigations of Domenjoz (1953, 1954, 1955) and Theobald (1954, 1955) demonstrated a n intense inhibition of formalin edema by ketobemidon, which exceeded the effect of high doses of ACTH (adrenocortivotropic hormone) or cortisone. Since
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these results were obtained in animals under aprobarbital (Numal) anesthesia, it is obvious that the observed inhibition of inflammation could not be ascribed to the analgesic properties of ketobemidon. Ill. The Pituitary-Adrenal Axis and Drug-Induced Inhibition of Inflammation
A. GENERALCONSIDERATIONS
As a consequence of Selye’s investigations on the physiological effects of stress, and as a result of the research work leading up to the discovery of the corticosteroids, numerous efforts have been made to clarify the question of whether certain drugs-in particular, antiphlogistics-are able to activate the pituitary-adrenal system, thus effecting a liberation of cortical hormones. The literature dealing with this subject is so extensive that it cannot be discussed in detail. As early as 1948 Forman et al. (1948) pointed out that the toxic effects of high doses of salicylates might be interpreted as symptoms of an alarm reaction. In connection with their investigations on the inhibition of hyaluronidase by aromatic compounds, Calesnick and Beutner (1949) considered the possibility that the therapeutic effect of salicylates might result from a stimulation of the adrenals, leading to a liberation of a hormone active against rheumatic manifestations. One of the first systematic investigations on this problem was that of Kelemen et al. (1950). These authors were able t o demonstrate in rat,s that high doses of sodium salicylate (500-600 mg/kg, s.c.) cause the following effects: (1) decrease of the total number of eosinophils by 70 to 90% within 4 hours; (2) appearance of the histological signs of an alarm reaction in thymus, spleen, and adrenals; (3) adrenal depletion of ascorbic acid and lipoids; and (4) inhibition of formalin and hyaluronidase edemas (bull testicular extract) and inhibition of the local reaction to histamine. Lower doses (200-300 mg/kg, s.c.) were ineffective. After adrenalectomy the administration of salicylates no longer produced eosinopenia, and the effects described in point (4) were lacking. In man, too, an eosinopenia of about 50% was observed after single administrations of 6 gm, corresponding to a salicylemia of 30-35 mg%. Higher doses caused stronger effects, whereas single doses of 4-5 gm were ineffective. Kelemen et al. (1950) interpreted the effects observed as an unspecific defense reaction depending primarily on the function of the adrenals. They claimed that high doses of salicylates are able to mobilize cortisone in effective amounts. The publication of Kelemen et al. (1950) treated in a critical manner the different aspects of the problem, and stressed the important observa-
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tion that adrenal activation is to be taken into consideration only for high doses of salicylate (see also Kelemen et al., 1952; Kelemen, 1956, 1957, 1960). This extremely interesting hypothesis was further confirmed by numerous other authors, and was also extended to other drugs with antiinflammatory action.
For salicylates see: Champy and Demay (1951) Blanchard et aZ. (1950a,b) Hetsel and Hine (1951) Cochran et aZ. (1950) Pasqualini (1950), Pasqualini et al. (1951) Roskam et aZ. (1951a) Pfeiffer et al. (1950a) Van Cauwenherge (19511, Van Cauwenberge and Heusghern (1951a,b), Bets and Robinson (1950, 1951) Van Cauwenberge (1951a,b). Bertolani et al. (1951)
For aminopyrine see: Blanchard et a2. (1950a) Pfeiffer et al. (1950b)
Kuzell and Schaffarzick (1951)
For phenazone see: Pfeiffer and Hasegawa (1951)
For butupyrin (Irgapyrin) see: Fellinger et al. (1951)
For phenylbutazone see: Basai and Giangrasso (1953)
Agolini et al. (1952)
For phenylquinolincarbonic acid and derivatives see: Meier et al. (1951)
Blanchard et al. (1950a)
For some thiosemicarbazone derivatives see. Schuler and Meier (1951).
B. CLINICALFINDINGS i~ MAN Various arguments have been suggested to demonstrate the hormonal mechanism of action of the salicylates. From the beginning, some clinicians were impressed by the striking analogy between the clinical indications and effects of cortisone and ACTH and those of the salicylates and other antiphlogistics (Heteel and Hine, 1951; Bywaters and Rutstein, 1953). This analogy may also be gathered from the report of the “Joint Committee of the Medical Research Council and Nuffield Foundation on Clinical Trials of Cortisone, ACTH and Other Therapeutic Measures in Chronic Rheumatic Diseases” (see Joint Committee, 1954). This thoroughly critical investigation considered all objective and subjective criteria concerning the evolution
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of the disease and the side effects of medical treatment, and led to the conclusion that “there appears to have been surprisingly little to choose between cortisone and aspirin.” The analogy between cortisone and the salicylates extends partly to the side effects of these drugs. One of the first publications on this subject is that of Cochran et al. (1950). They observed in a 12-year-old girl under treatment with acetylsalicylic acid at a dose of 5 gm daily the appearance of symptoms similar to Cushing’s syndrome, which occurs very regularly in a more or less manifest form during treatment with cortisone. This observation, based on a single clinical case, has been extensively cited in literature, and thereby has gained in power of psychological persuasion but not in statistical significance. The symptoms of adrenocortical hyperfunction (moon face, weight gain, hypertension, acne, tendency toward infections, etc.) observed during treatment with salicylates by Roskam (1954), Roskam and Van Cauwenberge (1953, 1954a,b,c), and others, are not very impressive from a statistical point of view; even if they do appear, they are of little importance in comparison with the common and relatively frequent side effects of salicylates. Besides these interesting parallels between the actions of cortisone and ACTH and the salicylates some opposite effects of these drugs have been described. Hailman (1952) presented a systematic survey on this subject. He stressed that cortisone increases (M. J . H. Smith, 1952a), while salicylate decreases (Lutwak-Mann, 1942), liver glycogen, and that fundamental differences exist in the therapeutic effect of both drugs when applied in cases of anaphylactic shock. Ingle (1950), too, described antagonistic effects after examining the elimination of glucose and the glycemia in diabetic dogs. M. J. H. Smith (1952a, 1953) was able to demonstrate that the disturbance of carbohydrate metabolism arising during cortisone therapy can be normalized by simultaneous administration of salicylate. In clinical cases treated with salicylates, the following reactions were accepted as suitable criteria for a participation of the pituitary-adrenal system: (1) decrease of the number of eosinophils according to Thorn’s test; (2) increase of the plasma concentration and elimination of steroids; and (3) uricosuric effect. The eosinopenia following the administration of high doses of salicylates described in man by Kelemen et al. (1950) was confirmed by Roskam et al. (1951a,b,c) (see also Roskam and Van Cauwenberge, 1952a,b, 1953, 1954a,b,c; and Roskam, 1956) in normal subjects, and by Delaville et al. (1954) in patients. These findings, however, could not be confirmed by Meade and Smith (1951), Coste et al. (1953a), Marson (1953), Thrift and Traut (1958), and others. According to Heilmeyer et al. (1953), this may partly be explained by the fact that Thorn’s test (Thorn et al., 1948; Thorn
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159
and Forsham, 1949) for technical and general reasons, is of limited value as a means of investigating the function of the adrenal cortex (Lutzenkirchen, 1951; Hitzelberger et al., 1952). Indeed, administration of adrenalin may cause eosinopenia even in adrenalectoniized subjects; such results were obtained in animals by de Fossey and Deltour (1950), Ruppel and Hitzelberger (1951). These findings suggest that eosinopenia does not prove a participation of the adrenal cortex; the absence of eosinopenia, however, may furnish a n argument against such participation. Using the method of Heard and Venning (1946), a n increase of urine elimination of reducing corticosteroids was observed by Van Cauwenberge arid Heusghem (1951a) during treatment with acetylsalicylic acid. The elimination of 17-ketosteroids, however, determined by the method of Cahen and Salter (1944), did not shorn any correlation with the treatment (see also Van Cauwenberge and Heusghem, 1952; Roskam and Van Cauwenberge, 1952a,b). Employing a modification of the technique described by Nelson and Samuels (1952), Roskani el al. (1955) were able to demonstrate an increase of blood and urine concentration of 17-hydroxycorticosteroids after daily administration of 8 gm of different salicylates for 15 days or more. BBnard et al. (1956) also found a significant increase in plasma 17-hydroxycorticosteroids after a 3 weeks’ adniiriistration of sodium salicylate a t a dose of 4 gm daily, 1 hese findings could not be confirmed by a series of other authors, although they obtained (with relatively low doses of ACTH) significant effects on the urinary concentratioii of 17-hydroxycorticosteroids (Coste et al., 1953a). After prolonged treatment with acetylsalicylic acid and after high single doses corresponding to plasma levels of 20 mg% or more of salicylic acid, Bayliss and Steinbeck (1954) did not find any increase in the plasma level of 17-hydroxycorticosteroids. Thrift and Traut (1958) also did not observe an increase in elimination of 17-ketosteroids after daily doses of 8-10 gm, equivalent to a plasma level between 12.5 and 41.5 mg%. Kelemen et al. (1952), using Cope’s (1951) biological method in adrenalectomized mice, found no increase of cortisonelike substances in the urine of normal persons receiving 7-9 gm daily of sodium salicylate for between 1 and 5 days. The uricosuric effect wa:, proposed by Rosltam et al. (1951~)as a criterion for t,he pituitary-adrenal mediation of salicylate action. His idea was based on the data furnished by Thorn et al. (1947, 1948) about a uricosuric action of cortisone and AC’TH. Blanchard et al. (1950a) also assumed a pituitary-adrenal mediation of the uricosuric effect of phenylquinolincarbonic acid, salicylate, etc. Marson (1953) observed a satisfactory uricosuric action after administration of salicylate, but not a n eosinopenia. In his experiments, t he influence of salicylates on the uric acid/creatinine quotient was even more iiiteiise than that of ACTH. After administration of
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ACTH, eosinopenia and the uricosuric effect were regularly and simultaneously present. In one case of Simmonds’ disease, in which a diminished response was to be expected, salicylate revealed itself as even more active than ACTH. On the basis of these findings, Marson rejected the hypothesis that the uricosuric effect of salicyIates is mediated by the pituitary-adrenal system. These data collected in clinical experiments permit no clear conclusion for or against the hypothetic hormonopoietic action of salicylate therapy. There is no doubt that the affirmative findings presented by the group of Roskam, Van Cauwenberge, and collaborators deserve much attention. Nevertheless, it is impressive that Kelemen et al. (;L952) formulate their viewpoint as follows: “it is not possible to explain the therapeutic action of salicylates only on the basis of a cortisone-like effect; even with high doses of salicylates the quantity of cortisone-like substances the organism disposes of does not attain the level sufficient to induce remission of rheumatoid arthritis.” Even if one admits the activation of the pituitary-adrenal system by high doses of salicylates in the sense of an unspecific stress reaction, the antiexudative and anti-inflammatory effects of small and medium doses of salicylates, clearly demonstrated in clinical cases, still remain unexplained. C. EXPERIMENTAL FINDINGS IN
ANIMAL The experimental investigation of the hormonal mechanism of action were mainly based (1) on the histological and analytical changes in the adrenals, such as arise in stress reactions or under the influence of toxic doses of pharmacodynamic agents; and (2) on the anti-inflammatory action of salicylates apparent in experimentally induced exudative and inflammatory reactions. The phenomena described by Kelemen et al. (1950) have been mentioned above: ascorbic acid and lipoid depletion of the adrenals, inhibition of the local reaction to formalin, histamine, and hyaluronidase, particularly bull testicular extract, as well as the absence of these “anti-inflammatory effects” after adrenalectomy. THE
1. Depletion Reactions of the Adrenals The decrease of ascorbic acid in the adrenals (determined by the method of Roe and Kuether, 1943) after administration of salicylate was also reported by Blanchard et al. (1950a), evidently independent of Kelemen et al. (1950),and probably even before these authors (“publication received Dec. 7, 1949”). In the same series of experiments, these authors studied a group of quinolinic acid derivatives in addition to aminophenazone. In the discussion of the results, Blanchard et al. (1950a) refer to Svirbley’s
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161
(1939) and Frommel et al.’s (1945) earlier observatioiis concerning a decrease of adrenal ascorbic acid caused by aminophenazone, acetanilide, and related antipyretics. In the case of 3-hydroxy-2-phenylquinolincarbonic acid and of 3-hydroxy-2-phenylquinolin-4,8-dicarbonic acid, it was demonstrated that hypophysectomy inhibits the depletion of ascorbic acid. Blanchard et al. (1950a) deduce from their findings that the anterior hypophysis mediates the adrenotropic effect of the quinolinic acid derivatives, and suppose that the same mechanism of action can be ascribed to salicylic acid and aminophenaaone. The adrenal depletion of ascorbic acid and cholesterol caused by salicylates was described and confirmed by numerous authors. In regard to the intensity of ascorbic acid depletion, it was possible to demonstrate an evident relation to the dose administered or to the blood level of salicylates (Hetzel and Hine, 1951; Van Cauwenberge, 1951; Van Cauwenberge and Heusghem, 1951a; Ungar et al., 1952; Van Cauwenberge and Beta, 1952); a similar correlation is said to exist in the case of cholesterol depletion (Van Cauwenberge, 1951; Van Cauwenberge and Beta, 1952). The depletion of both cholesterol (Robinson, 1950, 1951) and ascorbic acid (Pasqualini et al., 1951) can be demonstrated just 30 minutes after the administration of salicylates. In the case of ascorbic acid it reaches its maximum after 2-6 hours (Pasqualini et al., 1951). Hypophysectomy abolishes these depletion effects (Hetzel and Hine, 1951; Betz and Van Cauwenberge, 1951a; Cronheim et al., 1952). The eosinopenia occurring in normal animals also disappears after hypophysectomy, as well as the histological changes observed in the lymphatic system after treatment with salicylates (Van Cauwenberge, 1951; Beta and Van Cauwenberge, 1951b). According to some authors, salicylates induce, even after hypophysectomy, a depletion of adrenal ascorbic acid. In the majority of cases, however, the decrease of ascorbic acid is only a slight one, according to Coste et al. (1953a,b), Domenjoa (1954, 1955), Domenjoa et aZ. (1955a). Weidmann (1955), on the other hand, obtained a high degree of depletion. These results were ascribed both by Coste el al. (1953a,b) and Weidmann (1955) t o a direct adrenotropic effect of salicylates. There are various observations concerning the influence of other drugs on salicylate-induced depletion. Pretreatment with cortisone decreased the liberation of ascorbic acid (Hetzel and Hine, 1951; Cronheim and Hyder, 1954). As was demonstrated by Van Cauwenberge (1952), the simultaneous administration of dibenamine does not influence the depletion of ascorbic acid or of cholesterol, in contradistinction to what occurs in adrenalineinduced depletion. p-Aminobenzoic acid has no effect on ascorbic acid
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depletion (Cronheim et al., 1953; Forbes et al., 1954). While the simultaneous administration of glucuronic acid was ineffective, itl was possible tto intensify the effect of salicylates by glycine (Cronheim, et al., 1953). It is an interesting observation that the depletion effects are influenced by anesthesia. According to Van Cauwenberge and Beta (1952), allobarbital narcosis prevents the depletion of ascorbic acid and cholesterol as well as the eosinopenia, whereas the salicylate-induced nuclear pyknoses of lymphatic tissues are only diminished in number. Kelemen et al. (1952) observed an absence of eosinopenia after prolonged anesthesia of rats with hexobarbital. Braun (1954, 1955) obtained, with equal doses of sodium salicylate administered to rats under aprobarbital anesthesia, a depletion of ascorbic acid of 36.5% as compared to 62.9% observed in nonanesthetized animals. According to Cronheim and Hyder (1954), in deep thiopental anesthesia ascorbic acid depletion is blocked, while under “incomplete anesthesia” no influence on the ascorbic acid reaction was observed. Cronheim and Hyder (1954) as well as Van Cauwenberge and Beta (1952) concluded from their results that the depletion reaction is controlled by a hypothalamic site of action. This hypothesis seems to be confirmed by the findings of George and Way (1957), who were able to block totally the adrenal ascorbic acid depletion induced by acetylsalicylic acid, by means of a lesion produced in the “median eminence” of the hypothalamus. These results correspond to the experiments and hypot,heses of Hume and Wittenstein (1950) and other authors concerning the importance of this area for the “adrenocortical response to stress.” In the discussions of the adrenal depletion of ascorbic acid and cholesterol induced by pharmacodynamic agents, it is frequently overlooked that changes in ascorbic acid levels are not confined to the adrenals (Domenjoz, 1955). An investigation of the ascorbic acid and cholesterol content of heart, liver, lung, kidneys, and adrenals after administration of different anti-inflammatory agents, formalin, carbon tetrachloride, and diphtheria toxin was performed by Morsdorf (1955) and Stenger et al. (1955). I t was noted that cholesterol depletion occurs selectively only in the adrenals without involving other organs. The situation was entirely different in regard t o ascorbic acid. Although in this case there is a striking adrenal depletion, there are also significant variations in other organs. Thus increases of ascorbic acid concentration in liver and kidneys are to be observed after administration of aminophenazone, and in a lesser degree after phenylbutazone. The results obtained after administration of sodium salicylate, acetylsalicylic acid, and p-aminosalicylic acid show an entirely different pattern, which corresponds to that induced by ACTH. The biological significance of these findings is obscure, but at least these results suggest
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that there are interesting qualitative differences in the pattern of action of the various anti-inflammatory agents. The results obtained by Allison (1955) are related to this question, who demonstrated, in animals under pentobarbit a1 anesthesia and subjected to electrical stimulation of the cervical sympathetic trunk distal of the superior cervical ganglion, that the increase of the ascorbic acid plasma level can be considered as a “most reliable index of stress.” I t is interesting that in these experiments anesthesia did not abolish the stress reaction mediated by the hypophysis. 2. Inhibition of Experimental Inflammation
The literature concerning the anti-inflammatory properties of salicylates as gathered from animal experiments is so extensive that it can be discussed only in part. From a survey of the most important publications on this subject published by Domenjoz et al. (1955a,c) it is obvious that sodium salicylate is more or less active on various types of experimental inflammations and in different species of animals. The following edemas showed distinct reaction to salicylates: Rat egg-white edema Domenjoa and Wilhelmi (1951) Ungar et al. (1952) Wilhelmi (1952) dextrari edema Dornenjoz (1953, 1954, 1955) Frank (1955) serotonin reaction Kelemen (1956) Theobald and Domenjoa (1958) Bode (1958) Van Cauwenberge et al. (1958) Mijrsdorf and Bode (1959)
hyaluronidase edema Kelemen el al. (1950) Domenjoz (1955) Dewes (1955) chloroform erythema Van Cauwenberge et al. (1954) carrageenin edema Benitz and Hall (1963) Wint,er et al. (1963)
Mouse yeast edema Weis (1963) formalin edema Northover and Subramanian (1961a) Weis (1963) Guinea pig Thurfyl erythema Haining (1963) “antiguinea-pig” serum reaction Ungar et al. (1959)
serotonin edema Northover and Subramanian (1961a) Weis (1963)
I
hist,amine reaction IJngar et al. (1959) bradykinin reaction Ungar et al. (1959)
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Rabbit chloroform erythema Van Cauwenberge and Lecomte (1952) Man Thurfyl erythema Truelove and Duthie (1959) Adams and Cobb (1963)
Other experimental inflammations responded in a less specific manner to salicylates : Rat formalin edema Kelemen et al. (1950) F. Gross (1950b) Kelemen et al. (1952) Wilhelmi (1952) Domenjos (1953, 1954, 1955) Theobald (1954, 1955) Domenjos et al. (1955a) incorporation of 5 3 6 (sponge implantation) D u Boistesselin and Porcile (1963) Mouse croton oil reaction Wilhelmi (1950, 1952) Guinea pig W-erythema Wilhelmi (1950, 1952) Adams and Cobb (1958) Haberland (1959) Rabbit mustard-oil dermatitis F. Gross (1950b) Wilhelmi (1952) Man UV-erythema Seidel and Knobloch (1957) Winder el al. (1958)
Several details of procedure, especially the dosage of the inflammatory agent as well as the mode of administration of the anti-inflammatory drug, proved t o be determining factors in the intensity of inhibition, Opitz and Schutz (1960) found orally administered doses of sodium salicylate (2 X 300 mg/kg) to be completely inactive on rat formalin and dextran edema, whereas the same doses produced a significant inhibition if injected subcutaneously. However, higher doses of sodium salicylate as well as of acetylsalicylic acid administered orally are, as Kornfeld (1964) demonstrated, able to cause a good inhibition of formalin edema. Experimental inflammations on adrenalectomized animals were used
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to verify the hypothesis of the pituitary-adrenal mechanism of action of salicylates. After extirpation of the adrenals, salicylates became almost inactive in the following tests, among others: Rat hyaluronidase edema (sodium salicylate) Kelemen et a2. (1950) Kelemen et aE. (1952) hyaluronidase wheal, dye accumulation (acetylsalicylic acid) Mathies (1958) histamine reaction (sodium salicylate) Kelemen et al. (1950) Kelemen (1956)
formdin edema (acetylsalicylic acid) Bacchus and Bacchus (1953) formalin edema (sodium salicylate) Domenjoz (1955) (aprobarbital anesthesia) chloroform erythema (sodium salicylate) Van Cauwenberge et al. (1954) Van Cauwenberge and Lecomte (1954) cotton pellet granuloma (sodium salicylate) Van Cauwenberge and Lecomte (1957)
Under different experimental conditions, however, the decrease in activity after adrenalectomy was only partial. Thus Kelemen et al. (1950) observed that the action of sodium salicylate on formalin edema considerably diminished after adrenalectomy, but did not disappear. Concerning serotonin edema, Kelemen (1957) reported a decrease only of salicylate activity after adrenalectomy. In investigations by Northover and Subramanian (1961a) adrenalectomy only diminished the salicylate inhibition of dye accumulation in the histamine wheal of mice. Van Cauwenberge et al. (1954) and Van Cauwenberge and Lecomte (1954) observed an inhibitory effect of sodium salicylate on the cutaneous reaction to chloroform in rats even after adrenalectomy. Furthermore, Ungar et al. (1952) obtained an inhibition of egg-white edema after adrenalectomy of equal intensity as observed in normal animals. In experiments on formalin edema performed by Domenjoz (1960) acetylsalicylic acid administered subcutaneously at a dose of 350 mg/kg was only slightly less active in adrenalectomized rats (inhibition of 29%) than in normal animals (inhibition of 35%). In the case of sodium salicylate this effect seemed to be dose related: a dose of 250 mg/kg, s.c., showed only a limited but approximately equal activity in both adrenalectomized and normal animals (23% in the former and 26% in the latter). Higher doses (350 mg/kg, s.c.), however, caused an inhibition of 35% in normal animals, but proved almost inactive after adrenalectomy (inhibition of 12%). Anesthesia modifies the anti-inflammatory properties of, as it influences the deplet,ion reaction induced by, sodium salicylate. With subcutaneous doses of 500 mg/kg Domenjoz (1954, 1955), Domenjoz et al. (1957) obtained only a slight but not significant inhibition of formalin edema in rats under aprobarbital anesthesia. Dextran edema, however, was satisfactorily inhibited under the same experimental conditions. This insufficient
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inhibition of formalin edema was ascribed by Roskam (1956) to a blockade by anesthesia of the suprapituitary site of action of salicylates. A systematic investigation by Domenjoz et al. (1957) showed that aprobarbital in the case of dextran edema only decreases, but in the case of formalin edema in fact inhibits, the anti-inflammatory effects of sodium salicylate. It is interesting tto note that the anti-inflammatory action of phenylbutazone under the same experimental conditions was not at all modified. Inhibition of edema
Phenylbutaeone, 200 mg/kg S.C. Sodium salicylate, 500 mg/kg S.C.
Type of edema
(Normal animals)
(Anesthetized animals)
Formalin
60%
64%
Formalin Dextran
39 % 71%
55 %
4%
The inhibition of dextran edema obtained with salicylates is presumably based not on an act,ivation of the adrenal cortex, but on their antihistaminic properties (Van Cauwenberge and Lecomte, 1954; Domenjoz, 1954, 1955). The fact that this effect is also diminished by aprobarbital anesthesia cannot be considered as a proof of a pituitary-adrenal mediation. D. DISCUSSION Since the effects of salicylates are partly similar to those observed after administration of ACTH, it seemed logical to ascribe their therapeutic action to an activation of the pituitary-adrenal system. These conclusions, explaining the therapeutic effect of salicylates in the sense of an endogenous cortisone therapy, were formulated by Champy and Demay (1951) on the basis of histological investigations. A critical review of the abovementioned findings concerning the depletion effects and the anti-inflammatory properties demonstrable in the animal shows, however, that several experimental results are in conflict with that conception. In a systematic investigation of the depletion effects it was noted that the decrease of the adrenal ascorbic acid is regularly observed not only after administration of salicylates, but also, with equal intensity, after administration of the therapeutically inactive compounds m-hydroxybenzoate and p-hydroxybenzoate (M. J. H. Smith, 195213). The investigations of Lowenthal and Jaques (1953) with twenty different derivatives of benzoic acid, namely, with its hydroxy, methoxy, dihydroxy, and methoxyhydroxy derivatives, demonstrated that the changes occurring in the level of the adrenal ascorbic acid had no relation to the therapeutic properties of these
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agents. Similar results are reflected by the data of Cronheiin et al. (1952). The results obtained by Feeney et al. (1955) with other hoinologs of salicylic acid, including gentisic and y-resorcylic acids, confirm the same fact. The studies of Braun (1954, 1955) on ascorbic acid depletion, and of Schulz (1953) and Schule and Stenger (1955) on cholesterol depletion, using a comparatively large number of anti-inflammatory agents, showed that no correlation exists between the therapeutic efficiency and the depletion phenomena. In this connection it must be remembered that Stenger et al. (1955) obtained a 34y0 depletion of ascorbic acid after a subcutaneous injection of fornialin in the sanie concentration and dose as was used to induce formalin inflammation. Furthermore, no correlation was noted between edema inhibition and depletion effect (Donienjoz, 1954, 1955) in rats anesthetized with aprobarbital, in which both formalinedema inhibition and adrenal ascorbic acid and cholesterol depletion were investigated siinultaneously. In these experiments sodium salicylate produced only a nonsignificant, 10% inhibition of edema, although it induced an ascorbic acid depletion of 63% and a cholesterol depletion of 35a/,. From these and many other results it must be deduced that the so-called depletion effects are to be interpreted as symptoms of a nonspecific reaction, whose intensity has no relation to the anti-inflammatory properties of the drugs examined. I1 was furtherniore demonstrated that intense depletion can be obtained even with substances entirely devoid of anti-inflaniiiiatory properties. For this reason the depletion reaction cannot be regarded as a criterion for the functional activation of the adrenal cortex and its incretory activity. It seems unjustified to evaluate ACTH-containing preparations on the basis of their depletive activity, as introduced by Sayers d al. (1948). As is denionstrated by the above-mentioned results in hypophysectomized animals, the depletive effect is certainly mediated by the pituitary gland. It is, therefore, clear that a site of inipact of the salicylates is located in the pituitary region or in the hypothalamic suprapituitary structures. This fact deserves attention because an accuiriulation of C14-labeled salicylate in the hypophysis was demonstrated by Pallot et al. (1956a,b), Pallot, and Eberhardt (1956)) and Eberhardt (1963). This site of action, however, does not seeni to play any part in the anti-inflamri~atoryniechanisni of salicylates. The depletion reaction is to be interpreted as a consequence of stress induced by chemical means, and it niust be taken into consideration in pharniacodynaiiiics and especially in the toxicological appreciation of salicylates. This conclusion that the depletion reactions are unspecific in character may also be drawn from investigations on hypophysectoniized animals anesthetized with aprobarbital, in which the depletion effect and the dextran-edema inhibition were siniultaneously examined (Domenjoz et al.,
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1955a). Administration of sodium salicylate in a single dose of 500 mg/kg resulted in only a slight depletion of ascorbic acid (17%) without any significant variation of the cholesterol level, while at the same time it induced a 40% inhibition of dextran edema. The fact that the anti-inflammatory properties are entirely preserved after hypophysectomy was also confirmed in nonanesthetized animals (Domenjoz, 1960). Like some other anti-inflammatory agents, sodium salicylate and acetylsalicylic acid proved to be more active on formalin edema in hypophysectomized than in intact animals. These results are in accordance with the findings of Ungar et al. (1952) that egg-white hyperergia is equally well inhibited by sodium salicylate in hypophysectomized and normal guinea pigs. The inhibition of hyaluronidase by acetylsalicylic acid, evaluated by dye spreading, is not affectcd by hypophysectomy either (Mathies, 1958). The above-mentioned results obtained with hypophysectomized animals prove that the anti-inflammatory properties as well as the anti-hyaluronidase effect of salicylates are obviously not mediated by the pituitary gland. The range of anti-inflammatory action of salicylates as it can be demonstrated in animal experiments shows important differences if compared to that of cortisone and ACTH. Van Cauwenberge and Lecomte (1954) observed that the skin reaction obtained in the rabbit by application of chloroform can be inhibited by salicylates, ACTH, and promethazine, but not by cortisone. In the rat the reaction to chloroform was attenuated by salicylates and by promethazine, while adrenocortical extract, cortisone, and ACTH accelerated its appearance. The action of histamine on permeability in the rat was not influenced by ACTH, cortisone, and adrenocortical extract, while salicylates and promethazine induced an appreciable inhibition. In acute experiments on rabbits, cortisone, adrenocortical extract, and proinethazine proved devoid of antihyaluronidase action, while salicylate and ACTH caused an evident delay of spreading. Donienjoz (1955) observed that sodium salicylate caused a strong inhibition (53y0) of dextran edema in aprobarbital-anesthetized rats; the same dose of acetylsalicylic acid produced a weaker inhibitory effect (38y0).It is interesting to note that under the same experimental conditions cortisone and ACTH exhibited no significant action a t all. In nonanesthetized rats the action of sodium salicylate on dextran edema was still more intense, with an inhibition of about 70% (Domenjoz et al., 1957); this observation was confirmed by investigations of Theobald (see Domenjoz, 1960). Lecomte et al. (1959) also reported that cortisone and hydrocortisone are completely ineffective against dextran edema. Although it has to be considered that the adrenal cortex of the rat produces corticosterone instead of cortisone, it is obvious that salicylates and ACTH exhibit evident qualitative differences. S.C.
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From the reported results it must be deduced that salicylates are certainly able to inhibit an entire series of local inflammatory reactions, but that this inhibition is not due to an adrenocortical activation but rather to other mechanisms of action (Van Cauwenberge and Lecomte, 1954; Domenjoz, 1954, 1955). The conflicting results obtained in inflanimation experiments on adrenalectomized animals have been reported above. The complete loss of the anti-inflammatory properties after adreiialectoniy described by numerous authors is inconsistent with other results suggesting a weakening of this action only (Domenjoz, 1960), as well as observations about an action of practically equal intensity on both normal and adrenalectomized animals (Ungar et al., 1952). These conflicting results may be partly ascribed to technical differences of methods and dosages. The fact that adrenalectomy conditions a decrease in the resistance to toxic agents is generally acknowledged. The high doses coniinonly used in inflammation experiments, reaching 500 mg/kg, s.c., and more, are alniost within the toxic ranges. It is, of course, not to be expected that under these conditions the reaction to a local insult exhibits the normal pattern. Domenjoz (1960) reported that in adrenalectoniized animals a dose of 250 nig/kg of sodiuni salicylate, s.c., induced a stronger anti-inflammatory effect than a higher dose of 350 mg/kg. This decrease in activity may be ascribed to the toxic effect of the higher dosage: 14 of the 31 adrenalectomized rats died after administration of 350 nig/kg, while normal animals tolerated this dose without any particular symptoms. It is to be noted that adrenalectoniy can be used with the intention to prove participation of the adrenal cortex only with certain restrictions. I n the appreciation of results obtained on adrenalectomized animals it must be taken into account that in these animals the adrenal cortex has been removed together with the medulla; the latter plays a particularly important part in the regulation of exudation through the capillary walls. M. J. H. Smith (1955) demonstrated in rats that intraperitoneal administration of sodium salicylate regularly resulted in a hyperglycemia lasting some hours, which seems to be caused by a stimulation of the adrenal medulla, that is, by a liberation of adrenaline. In this regard it is especially important that in rats evident inhibition of serotonin as well as dextran and formalin edemas is obtained with adrenaline and noradrenaline (Schmidt, 1963). According to Geschickter et al. (1960) egg-white edema is also inhibited by adrenaline. In mice, too, formalin edema can be inhibited by administration of noradrenaline, isoprenaline, etc., as was demonstrated by Northover and Subramanian (1962). Kelemen et al. (1952) had already established that in the experiments in which salicylates and dibenarnine were administered together, most of
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the animals died. They concluded that the damage caused by the administered salicylate can only be overcome if the animals dispose of an intact adrenaline-noradrenaline system. The specific part played by the adrenal medulla in the inhibition of inflammation was clearly demonstrated by Tanos et al. (1953). They showed that after adrenaI demedullation salicylates lose their anti-inflammatory action. In order to explain this observation, Kelemen (1960) suggested a “permissive” function of the medulla, as Ingle had proposed as early as 1952. At any rate results obtained after adrenalectomy do not allow conclusions about a participation of the adrenal cortex in the anti-inflammatory effect of the salicylates. This negative conclusion is consistent with the results of Mathies and Jankowski (1957) concerning the inhibition of hyaluronidase by adrenaline and salicylates. Inhibition of spreading by salicylates and pyrazole derivatives is ascribed to an adrenal liberation of catecholamines, because this effect is abolished by administration of sympatholytic and ganglion blocking agents (Mathies el al., 1959; Mathies and Krott, 1960). Inhibition of hyaluronidase by chloroquine seems to be caused by a different mechanism (Mathies and Will, 1962). Briefly, it may be said that the organism responds with a stress reaction when challenged by high doses of salicylates. This stress reaction explains the ascorbic acid and cholesterol depletion observed after administration of salicylates. According to numerous and carefully performed investigations, the depletion phenomenon represents an unspecific component of action not related to the anti-inflammatory action of salicylates. Extirpation of the pituitary gland abolishes the depletive effects but not the antiinflammatory action. The effect of adrenalectomy is more complex. In certain experiments, although not regularly, extirpation of the adrenal gland weakens, and sometimes even abolishes the anti-inflammatory action. This situation is consistent with the following three alternatives : 1. The anti-inflammatory action of salicylates is independent of the function of the pituitary-adrenal system, the effects observed after adrenalectomy being explained as unspecific. 2. Salicylates act as anti-inflammatory agents by means of a direct corticotropic effect. 3. The depletive effects and the anti-inflammatory action are mediated by distinct “pituitary,” adrenotropic factors; after hypophysectoniy release of the depleting factor stopped, but the secretion of anti-inflammatory steroids is still mediated by factors liberated from suprapituitary structures. While the two last-mentioned explanations seem not very likely, they still cannot be excluded with certainty. Nevertheless it is obvious that
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salicylates inhibit a series of local inflammatory reactions that are not influenced by cortisone and ACTH. The action of salicylates, at least in these cases, cannot depend on a mechanism mediated by the pituitaryadrenal system. The existence of a L‘hormonopoietic”effect of salicylates, which could furnish a satisfactory explanation of the anti-inflammatory properties of the salicylates, has as yet not been sufficiently proved. It can be gathered from the results now available that the hypothesis of a pituitary-adrenal mechanism of action of the anti-inflammatory drugs cannot be applied generally. This statement is supported by the findings obtained with phenylbutazone and oxyphenbutazone. The anti-inflammatory action of these compounds in the rat is not weakened by anesthesia (Domenjoz et al., 1957); it is also little (Domenjoz, 1955; Theobald and Domenjoz, 1956) or not a t all modified (Domenjoz, 1960) by adrenalectomy. The anti-inflammatory action of phenylbutazone and oxyphenbutazone, therefore, seems, at least in the rat, not to depend on a n activation of the pituitary-adrenal system. One must, however, concede that this supposition is based on experiments in which the adrenocortical function was examined by indirect methods only. In recent investigations, however, Bernauer and Schmidt (1963) demonstrated that phenylbutazone administered to rats in doses of 25-200 mg/kg i.m. induces a dose-parallel increase of corticosterone depletion. This effect must be taken into account in respect to the general action of this drug in man; it is certainly of interest in regard to the characteristic side effects on fluid balance. The question of its significance as a n accessory component of the anti-inflammatory action of therapeutic doses is left open to discussion. IV. The Inflammatory Focus as Site of Action of Anti-Inflammatory Drugs
As it became possible to define the anti-inflammatory components of the antipyretic-analgesic drugs by more adequate techniques, the hypothesis of a peripheral action of these agents within the inflammatory focus has gained more and more importance. As early as 1880 the French physician and physiologist Vulpiari (1880, 1881a)b) had ascribed to sodium salicylate a specific mode of action on articular tissues: “La substance organisde et vivante de ces dldrnents est inodifide par les salicylates . . . ces dldments, par l’incorporation de ce sel, deviennent rdfractaires B 1)irritation particulikre qui tend B y provoquer le rhumatisme articulaire aigu.” Some early observations concerning a peripheral site of action of the anti-inflammatory drugs were published by Januschke (1913). He was able to inhibit the chemosis induced by mustard oil in rabbits with phenazone, sodium salicylate, and quinine. This led him to the question: “if the action of salicylates in rheumatic diseases should not also be ascribed to an
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elective inhibition or anesthesia of the peripheral inflammatory apparatus.” Experiments performed by Starkenstein and Wiechowski (1913) and by Starkenstein (1920) with phenylquinolincarbonic acid invited similar considerations. Winternitz-Koranyi (1930), one of the assistants of G. von Bergmann in Berlin, discussed a specific anti-inflammatory action of aminophenazone and quinine. She demonstrated on the cantharidin blister that these two agents influence the inflammatory process both qualitatively and quantitatively, an effect which could not be explained by their analgesic and antipyretic properties. Some years later, important investigations concerning the specific mode of action of aminophenazone were performed by Eppinger et al. (1934). These authors suggested that aminophenazone could be applied with success in the treatment of serous inflammations because it inhibits exudation, probably by decreasing the permeability of the capillary wall. The importance of these findings is to be seen in the fact that they suggest the existence of a peripheral site of action of the anti-inflammatory drugs. A. OUTLINEOF
THE
INFLAMMATORY PROCESS
With the progress of knowledge about the physiology and pathogenesis of inflammation, research on drug-induced anti-inflammatory mechanisms proceeded more and more in a systematic and precise manner. Ample information about the pathophysiology of the inflammatory reaction can be gathered from the reports of Ehrich (1956) and of Heilmeyer and Kahler (1962). According to a scheme elaborated by Ehrich (1956) and adopted here in a modified form, the following factors and phases are to be differentiated in the course of inflammation: I. Cause of inflammation, causative agent. 11. Direct effects of the causative agent = primary alteration of tissues: injury of tissue, denaturation of tissue substrates, disturbance of local balance. 111. Indirect effects of the causative agent = secondary alterations of tissues, inducing inflammation. Physicochemical characteristics: primary acidosis disturbances of electrolyte balance. Chemical characteristics: enzyme activation, initiation of catabolic processes (glycolysis, proteolysis, depolymerization and breakdown of mucopolysaccharides, lipolysis, degradation of nucleic acids). Consequences: appearance of biologically active breakdown products, i.e. local formation of mediators inducing inflammation.
1
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IV. Local reactions of the vascular system and connective tissues, general reactions of the organism = clinical inflammation. 1. Initial phase local: (a) Disturbances of circulatory dynamics: hyperemia, stasis, inflammatory thrombosis, increase in capillary permeability: formation of exudate and edema. Increase in osmotic and mechanical tissue pressure. (b) Swelling and basophilia of fibrocytes and endothelial cells, indicating increased cellular activity; hydration (soaking of water, “Verquellung”), increased affinity for dye. (c) Secondary “inflammatory” acidosis Increase of local metabolism, especially of synthetic activity: formation of tissue substrates e.g. of ground substance (accumulation of energy-supplying nucleotides, increased synthesis of mucopolysaccharides), beginning formation of new connective tissue. 2. Late phase belonging in part to the phase of resorption and healing. Infiltration of the inflamed area by immigration of granulocytes, monocytes, histiocytes (macrophages) from the blood; the latter possibly originating locally from undifferentiated mesenchymal cells. Proliferation of connective tissue in the sense of an intensification of the processes in 1 c. Neo-formation of vessels. 3. General reactions of the organism (“Fernwirkungen”) changes in vegetative regulation alteration of the cellular and chemical composition of blood activation of bone marrow and lymphatic system activation of the pituitary-adrenal system fever changes in circulatory regulation (blood pressure, blood distribution, etc.) 4. Recovery (cannot be clearly distinguished from the late phase). Resorption: Fixation (plasma proteins). Phagocytosis, segregation, intracellular digestion, deposition or accumulation (granulocytes, macrophages, histiocytes). Topical angiotaxis (endothelial cells). Repair and restoration regeneration of differentiated tissues, cicatrization, substitution of specific structures by connective tissue.
B. TRAUMA AND LESION The cause of inflammation is the action of a noxa which upsets the physiological equilibrium in a limited zone of tissue. The noxa may be of physical or chemical nature, represented by living organisms (fungi, bacteria, virus), or caused by an antigen-antibody reaction.
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The primary alteration caused by the inflammatory agent modifies the milieu intkrieur in a limited area of tissue conditioning a denaturation of the tissue substrates, thus Starting inflammation as a chain reaction. Under experimental conditions, the influence of the noxa can be controlled in regard to intensity and duration, so that the injury is terminated or eliminated before its effect becomes evident. This demonstrates the important fact that the inflammatory reaction is the direct consequence of the tissue damage and is only indirectly related to the etiological agent. This means that inflammation is mediated by vectors produced by the metabolism of damaged tissues, or by tissue disintegration, respectively (Zweifach, 1953; Menkin, 1950, 1953, 1959; Ehrich, 1956). A very early hypothesis concerning the participation of endogenous vectors in the inflammatory reaction was proposed by Ebbecke (1923). He supposed that in the “stimulated epithelium” an agent is produced which, diffusing through the tissues, dilates the capillaries and smaller arterioles. The assumption of a humoral regulation of the inflammatory reaction through mediators formed in the damaged tissue corresponds to practical experiences insofar as the clinical symptoms-regardless of the cause-are almost alike. Actually, the clinical status permits reliable conclusions concerning the causative agent only in a small proportion of cases. Precise examination, however, might detect-especially in the case of experimentally induced inflammations-certain qualitative differences : The different inflammatory agents act by distinct mechanisms (Meier et al., 1955; Meier, 1959). The differences in reaction to different kinds of noxae can be recognized in some cases even when applied simultaneously t o the same animal (Domenjoz et al., 1955~).With respect t o certain defined noxae, the nuances of the inflammatory reaction are due to selective damage done to distinct tissue components conditioning various types of “secondary tissue alterations.’’ Furthermore, the differences in the inflammatory reaction depend on the dosage of the phlogogenic stimulus (Rocha e Silva and Rosenthal, 1961; Rocha e Silva, 1962) and, of course, on differences in local reactivity. The importance of quantitative factors for the induction of “secondary tissue alterations” and of the subsequent inflammatory reaction is widely known. The functional alterations resulting from slight stimulation, as in the case of hyperemia, are still within the limits of physiological regulations and cannot as yet be considered as inflammatory reactions, even though they are induced by humoral mediators formed ad hoc. Inflammation is consequent to tissue damage, e.g., to a noxious stimulus strong enough to initiate the whole of the chain reaction. It is not the participation of humoral mediators that characterizes inflammation, but their quantity, quality, and possibly also the site of their formation. The quantitative and qualitative variants of the inflammatory reaction, depending on their
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specific biological mechanisms, can explain the differences of response to particular anti-inflammatory agents, a fact well known from animal experiments as well as clinical experiences.
C. TISSUE ALTERATIONS ASSOCIATED WITH INFLAMMATION 1. Physicochemical Characteristics
The primary lesion conditions the “secondary alteration of tissue, inducing inflammation.” This secondary alteration is apparent in the form of chemical and physicocheniical changes as soon as a few seconds after the injury. It is characterized by catabolic processes, which, from a teleological point of view, may be interpreted as destined to remove the denaturated substrates. Among the physicocheniical alterations inducing the inflammatory reaction, acidosis was one of the earliest to attract attention (Ewald, 1873). Disturbance of the ionic balance is evidently coexistent with an alteration of the electrolyte equilibrium. Among the phlogogenic factors, acidosis is undoubtedly of primary importance. Its characteristics have been studied extensively by Frunder (1953), whose results have been confirmed by Eckstein et al. (1960) and others. Frunder (1953) demonstrated that tissue injury, after an interval of only a few seconds, induces a local acidosis corresponding to a pH of 6.8-6.0. This reaction is not influenced by variations of glycemia, although its intensity decreased when the tissue level of glycogen was lowered. The origin of primary acidosis is ascribed by Frunder (1953) to glycogenolysis. The special importance of primary acidosis in regard to the induction of inflammation is t o be seen in the following consequences: activation of enzymes, hydration of the ground substance (Schade, 1927, 1935), facilitation of its depolymerization, induction of hyperemia and increase of capillary permeability (Fleisch, 1921), emigration of leucocytes, phagocytosis, etc. (for references see Ehrich, 1956). According to Frunder (1953), the primary acidosis is followed b y a secondary inflammatory acidosis, corresponding in degree to the intensity of inflamniation and to the level of glycemia, a fact which may explain the increased disposition to inflammation observed in diabetics. According to the same author (Frunder, 1953), the secondary acidosis, which is a coinponent of the inflammatory reaction and not a n inducing agent, is to be ascribed to a n increase of anaerobic glycolysis. 2. Chemical Aspects
An outstanding chemical peculiarity of the “secondary tissue alteration” consists in the intensification of local metabolism connected with catabolic processes. I n the beginning this metabolic activity is apparent as a n en-
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hancement of respiration, i.e., an increase of oxygen consumption and of COz elimination (Gessler, 1921), and furthermore as aerobic glycolysis (Druckrey, 1936; Brock et al., 1938), while in a later state it manifests itself as anaerobic glycolysis (Ehrich, 1956). Injured tissues differ from normal ones by their increased metabolism leading to a n exhaustion of the local resources (Schade, 1935). In this connection recent findings of Kalbhen (1963b) are to be mentioned. By column chromatography (ionic exchange) of tissue homogenates of inflamed rat paws (2 hours after injection of dextran) the author noted an appreciable diminution of phosphates connected with glycolysis as shown in the following tabulation. Decrease (%) Uridine diphosphoglucose Glucose phosphates Dihydroxyacetone and a-glycerophosphate 2,3-Phosphoglyceric acid
75 24 60 16
As Rubel (1936) and Menkin (1938) demonstrated, in inflamed tissues glycolysis and proteolysis are closely related : proteolysis increases or decreases proportionally to the intensity of glycolysis. According to Rubel (1936) accumulation of lactate, the final metabolite of glycolysis, enhances proteolysis. Causative relations between proteolysis and inflammation were discussed as a hypothesis by Mirsky and Freiss (1944) : “With extensive tissue damage some proteolytic enzyme is released or activated which in turn may be responsible not only for changes a t the site of the injury, but also for production of catabolic factors. . . .” The special significance of proteolysis for the induction of inflammation was stressed in a very affirmative manner by Ungar (1952, 1953, 1963) and Menkin (1953). The latter author (V. Menkin, 1942, cited in Heilmeyer and Kahler, 1962, p. 9) actually classified proteolysis among the cardinaI symptoms of inflammation. Among the catabolic processes occurring in inflamed tissues, protein breakdown has been up to now the most extensively studied. It is assumed that, following “denaturation,” degradation to albumoses, peptones, and peptides is effected by means of proteases, peptides in turn being degraded to amino acids by peptidases. Accumulation of these degradation products in the inflamed area had already been described by Schade (1923) and has been repeatedly confirmed since. Menkin (1938) found in the pleural exudate of dogs, produced by injection of turpentine oil, an increased local concentration of amino acids of about 6.6% (serum concentration = 5.0 mg%) in the first 6 days, and a level of 10.7 mgyo between days 7 and 10, with a serum concentration of 5.5 mg%.
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It was demonstratcd by nieans of chroniatographic investigations of skin slices (Lindner, 1957) and of inflamed tissues (Kalbhen, 1963a) that intense protein degradation takes place in the early stage of inflammation, parallel t o the degree of swelling. Kalbhen (1963a) worked on rat paws with formalin, serotonin, and dextran edemas. I n normal tissues he found a total concentration of free aniino acids of 1.0-1.4 nig/gni, and of 0.4 mg/ml of serum. In the case of dextran reaction the increase in total amino acids in paw tissues reached 29.3a/, as quickly as 10 minutes after the injection of the phlogogenic agent, the maximuin concentration (increase of 38.5a/0) being attained after 60 minutes. Fornialin edema, developing more slowly than dextran-induced swelling, did not lead to a notable alteration of the tissue level of free amino acids within 10 minutes. After 90 minutes the total increase amounted to l8.60/,; within 120 minutes after the injection of fornialin the increase was still 12.3y0 as compared to normal paws. In experiments with serotonin edema a rise of 33.7y0 was observed within 20 minutes, the maximum (38.40/,) being attained within 90 niinutes; the increase still amounted to 24.7% 120 minutes after the injection. The serum figures were not modified in the course of these experimental inflammations. From mathematical estimations it may be concluded that the observed increase in aniino acids is due to a local proteolysis and not to a n importation froin the blood. Froni the different responses to pharmacological agents observed in the so-called primary and delayed reactions to tissue injury and from nuiiierous other experiniental results one can deduce that the inflanimatory reaction is controlled and niodulated by a series of humoral factors. Besides the classic mediators histamine and serotonin in which release from stores as well as formation by proteolysis has to be taken into account, the peptides with a chain length of 8 to 14 aniino acids particularly appear to play an important part (Spector, 1951). This question was investigated by Menkin (1953, 1960), who isolated from inflaniniatory exudates a whole series of polypeptide-like substances, which he defined by biological criteria : leucotaxine, exudin, leucopenin, necrosin, pyrexin, leucocytosis factor, etc. According to Menkin (1960) all these factors have to be considered as inflammation-inducing agents. Unfortunately, it was not possible to establish their chemical identity and to define their function as biological vectors. Consequently, the question as to which of the “pyrexines” is responsible for the hyFerpyrexia observed in certain inflaniinatory conditions is still undecided. As early as 1890, Saniuel (see 1890a) denionstrated that inflammatory exudates contain pyrogenic substances; he induced hyperpyrexia in normal animals by iiijecting inflaniniatory exudate. Atkins (1960) also came to the conclusion that “fever is caused by pyrogenic materials released from injured cells in the host.” According to King and Wood (1958) the pyrogens active in inflammation are liberated from polymorphonuclear leucocytes.
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The origin, chemical constitution, and biological action of the “polypeptides which affect smooth muscle and blood vessels” has been thoroughly studied in the last few years by several groups of scientists. Their investigations concerning agents biologically related to the inflammatory reaction, led to remarkable results, e.g., to the chemical identification of bradykinin and kallidin. Detailed reports on these investigations have been published by Lewis (1960), as well as by Schachter (1959,1963) in the Symposiumon Polypeptides and the New York Academy of Sciences volume on structure and function of biologically active polypeptides, respectively. Important results concerning the part played by kinins in the mechanism of functional hyperemia were obtained by Hilton and Lewis (1955), who experimented on the salivary glands of the cat. As a result of stimulation of the chorda tympani, the glandular cells liberate a proteolytic enzyme (salivary kallikrein) present in saliva. As early as 1936 Ungar and Parrot identified this enzyme as kallikrein. They ascribed to it a mediator function in regard to the local vasodilatation consequent to the stimulation of the lingual nerve. As Hilton and Lewis (1955) demonstrated, this protease is not only liberated into the saliva eliminated through the secretory duct of the gland, but it also penetrates into the interstitial space. There it reacts with a kinin precursor belonging to the plasma globulins. The proteolysis of this globulin leads to the appearance of the biologically active degradation product bradykinin, which, a t the site of its formation, induces a functional vasodilatation and hyperemia. Similar processes must also be assumed as responsible for the induction of functional hyperemia in other exocrine glands, as well as for several forms of reactive hyperemia and for certain reactive mechanisms after tissue injury. The functional hyperemia of skeletal muscle seems to depend on other mechanisms (Trautschold and Rudel, 1963). According to Lewis (1960), tissue injury leads to a disturbance in the function of the cell membrane. As a consequence, precursors of the kinin-forming enzyme penetrate from the blood into the interstitial space, where they are transformed by an activator furnished by the damaged cells. The enzyme, thus formed, particularly the kinins produced, induce, as biological mediators, the local changes in circulation and capillary peremeability characteristic of inflammation. Hilton and Lewis (1957) (see also Elliot et al., 1960) held that bradykinin has all the characteristics of a “mediator of inflammation.” Important features of its action are vasodilatation, increase of permeability (Holdstock et al., 1957), pain-producing effect (Armstrong et al., 1957; Keele, 1960), chemotaxis, and stimulation of leucocyte migration (Lewis, 1962). Similar characteristics may be ascribed to substance P (Bisset and Lewis, 1962) as well as t o the polypeptide eledoisin isolated from the octopus (Erspamer and Anastasi, 1962).
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From the results of Fox and Hilton (1958) (see also Fox et al., 1960) it may be concluded that bradykinin is liberated from human skin as a consequence of thermic stimulation. After thermal injury Rocha e Silva and Rosenthal (1961) could identify histamine, serotonin, and bradykinin in the diffusatesfrom traumatized rat skin. When the temperature of heating was exactly dosed at 44”45”C, there was no liberation of histamine and serotonin from rat skin; the “thermic edema” appearing under these conditions can be ascribed to the liberation of bradykinin (Rocha e Silva, 1962, 1963). Investigations of Edery and Lewis (1962a) demonstrated that in the process of kinin formation very complex synergistic correlations have to be considered. In experiments with lymph draining from areas of tissues subjected to dosed thermal stimulation and other trauma they were able to demonstrate a notable increase in the kinin-forming enzyme concentration. In spite of a high lymph histaminase level, liberation of histamine could still be detected. Arterial injection of histamine caused an increased flow of lymph as well as an increase of kinin-forming enzyme. These effects could be inhibited by mepyramin. According to Edery and Lewis (1962a), the histamine liberated after tissue injury is the cause of the increase observed in enzyme activity. Possibly this effect is due to the influence of histamine on permeability, which facilitates the reaction of activator and precursor (Lewis, 1960). After intravenous administration of histamine Copley and Tsuluca (1962) observed in the guinea pig a bradykinin-induced increase of local capillary permeability. At present it is not possible to give a precise account of all the factors that are involved in the activation of enzyme precursors after tissue injury. Acidosis occurring in the area of damaged tissue may be sufficient to activate the kallikrein circulating in the blood. Edery and Lewis (1962b) could demonstrate that kininase activity responsible for inactivation of bradykinin-like polypeptides is inhibited even by a slight acidification. It follows that local acidosis of the inflamed area appears to provide favorable conditions for the formation and accumulation of bradykininlike polypeptides and possibly for the production of an “acid-formed kinin” more resistant against inactivation (Lewis, 1963). The increased breakdown of tissue substrates in the inflamed area comprises also the mucopolysaccharides. The connective tissue reaction a t this point is characterized by the terms h’ntleimung and Entmischung of the ground substance (Schallock and Lindner, 1957). From a physicochemical point of view, this reaction is characterized by an enzymic depolymerization of the macromolecular ground substance (Meier, 1959). This depolymerization is rendered possible and even enhanced by the alteration of pH in the inflammatory focus. How the cleavage of mucopolysaccharides from their mucoproteins, their depolymerization, and breakdown influence the local processes is as yet not known in detail.
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Certainly, these degradation processes have important consequences for enzyme activity and for buffer capacity within the area of inflamed tissues. Other local processes are probably influenced, too, e.g., water binding, osmotic properbies, and diffusibility, as well as leucocyte phagocytosis and blood coagulation. Lindner (1957) isolated from inflamed cutaneous tissue a series of mucopolysaccharide degradation products. Theoretically, the degradation of mucopolysaccharides also may condition the appearance of “inflammation-inducing” or, at least, of biologically active metabolites. In regard to the alteration of connective tissue, depolymerization and degradation of hyaluronic acid and chondroitin-sulfate by hyaluronidase have been studied more thoroughly. When hyaluronidase is simultaneously introduced into the connective tissue with a chemical irritant (paraphenylenediamine, allylisothiocyanate), the resulting tissue irritation is significantly intensified, and the inflammatory reaction spreads over a larger area than it would without hyaluronidase (Glassmann et al., 1953). Westphal et al. (1953) emphasized the particular significance of hyaluronidase for Fernwirkungen (actions at a distance) of the inflammatory focus. An activation of hyaluronidase in the paraphenylene dermatitis of guinea pigs was observed by Mayer (1950). In experiments performed by Zweifach (1953) on the mesentery of rats the first consequence of a local chemical or mechanical stimulation was the conversion of connective tissue from a gellike to a sol-like state; the same phenomenon was produced by microinjection of proteolytic enzyme, of purified preparations of hyaluronidase, snake venom, and several bacterial toxins (Shiga, Clostridium). In connection with the secondary, inflammation-inducing tissue alterations, Ehrich (1956) took the increased breakdown of fat and nucleic acids into consideration. In this regard the more recent experiments of Harvengt (1961, 1963) demonstrate an intense lipid mobilization as a consequence of burns, which results in an increased level of serum lipids. Schade (1935) had already reported an increased concentration of fatty acids in inflamed tissue. By measuring the adenosine-deaminase activity in inflamed lymph nodes Wagner and Ehrich (1950) demonstrated an increased degradation of nucleic acids. The importance of these processes for the induction of the inflammatory reaction has as yet not been evaluated. Among the breakdown products of nucleic acids, ATP (adenosine triphosphate) and adenosine phosphoric acid-owing to their well-known vasoact,ive properties-may very well be relevant in the inflammatory reaction of blood vessels. The strong vasodilatatory property of ATP was already reported by Drury and Szent-Gyorgyi (1929). For this reason, ATP has been considered by Fleisch and Domenjoz (1940) and other authors as the mediator of reactive as well as functional hyperemia. Wedd and Drury (1934) as well as Cul-
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lumbine (1947), cited in Ehrich, 1956, p. 78) noted a high increase in adenosine blood level consequent to burns. According to Best and Taylor (1950), adenylic acid and adenosine have chemotactic effects on leucocytes; these properties, however, could not be confirmed in vitro (Meier, 1959). According to Ludany (1955), nucleic acids, even in high dilutions, stimulate phagocytosis. Green and Stoner (1950) claimed that adenosine is responsible for multiple reactions consequent to tissue damage. The significance of these pharmacological properties is certainly limited in comparison with the energetic function of nucleotides in the metabolism of inflamed tissue. Kalbhen (1963b) found in rat-paw homogenates (2 hours after subcutaneous injection of dextran) a significant rise of nucleotide level as shown in the following tabulation. Average increase (%) Adenosine triphosphate (ATP) Adenosine diphosphate (ADP) Adenosine monophosphate (AMP)
41 60 55
The fractions containing cytidine nionophosphate/diphosphopyridine nucleotide showed an increase of about 400%, corresponding to a n estimated increase of 200-300% of diphosphopyridine nucleotide (DPN). These findings demonstrate that even in t8heearly stages of inflammation there is a remarkable intensification of energy-supplying processes.
D. THE INFLAMMATORY REACTION 1. Vascular System: Hyperemia, Stasis, Edema
The reaction of the vascular system and of the connective tissue corresponds to the clinical state of inflammation. This reaction is principally characterized by constructive and regenerative processes. The modifications of circulatory dynamics and of vascular permeability, which mark the beginning of the inflammatory reaction, are mediated by vasoactive metabolites and breakdown products of the tissue substrates. Present knowledge confirms this explanation concerning the initiation of hyperemia and stasis. I n the case of exudation and edema formation, however, more complex processes than the permeability enhancing action of polypeptides and other protein nietabolites have to be taken into consideration. As Schade (1923, 1935) showed, an alteration of the osmotic equilibrium in the sense of an increase of osmotic as well as of mechanical pressure can be demonstrated very early in the inflamed area. As we believe today, these alterations are connected with the enzymic depolymerization of the macromolecular mucopolysaccharides of the ground substance, which results in
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a higher capacity for hydration. The formation of edema must, therefore, be ascribed not only to an increase of vascular permeability, but also to the mechanisms conditioning the hydration of ground substance. According to Meyer (1947, 1954), Meyer and Rapport (1951), nonsulfated mucopolysaccharides, especially hyaluronic acid, are of primary significance for the degree of hydration of the ground substance. The reversibly dePO ymerizable ground substance represents, in this regard, a n important water-binding system between blood and tissues (Grauniann, 1964).
2. Metabolism during the Reaction Phase From a morphological point of view, the reaction phase is characterized by swelling and basophilia of fibrocytes and endothelial cells, by increased hydration, higher affinity for dye, and by augmentation of ground substance. I n studies by Ernst (1926) on turpentine oil inflammation in rats, these modifications were histologically demonstrable within a time between 30 minutes and 2 hours after the injury. I n regard to the time of their appearance, these symptoms seem to belong to the reactive phase-in contrast to Ehrich’s (1956) hypothesis-and not to the “secondary” inflaniniation-inducing tissue alterations. This view is confirmed by the present interpretation of these phenomena in the sense of increased cellular activity : basophilia resuking from increased synthesis of ribonucleic acids, metachromotropism indicative of proliferative activity (Graumann, 1964), i.e., formation of connective tissue. This conception is particularly in accordance with the results obtained in classic experiments by Gersh and Catchpole (1949), in which augmentation of ground substance was observed only several hours after tissue injury: “Ground substance becomes replaced perhaps through the secretory activity of fibroblasts” (Gersh and Catchpole, 1949). I n the course of in vitro investigations with connective tissue of different age, Layton (1950a) established that embryonic tissues are distinguished by particularly high levels of S36incorporation (see also Takahashi, 1928). He obtained analogous results (Layton, 1950b) with granulation tissue and concluded that “sulfate is required for tissue formation , probably being bound in the newly forming connective tissue substance , . and hence is necessary for wound healing.” Layton (1951) emphasized that the increase of ground substance in arthritis and in other so-called collagen diseases is closely related to the clinical symptoms and the pathological pattern of the disease. Bunting (1950) demonstrated by histochemical methods an increase of mucopolysaccharide content in the inflamed area. Consden et al. (1953) observed an increased level of hexosamine in human rheumatic nodules. Numerous experimental investigations have established the increase
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in the concentration of hexosamine and niucopolysaccharides in granulation tissue (surveys are to be found in Delaunay, 1962; Delaunay and Bazin, 1960a,b, 1963; Graumann, 1964). By subcutaneous injections of crystallized trypsin in rabbits, Schlamowitz and Degraff (1950) produced tissue alterations niorphologically resembling rheumatic nodules that were characterized by an increased hexosamine content of the granulation tissue (Schlamowitz et al. 1950a). Similar results were obtained after injection of streptokinase (Schlamowitz et al., 1950b), but not after heat-inactivated trypsin, casein, crystallized bovine serum, crystallized lysozyme, crystallized chymotrypsin (Schlamowitz et al., 1950a), or hyaluronidase (Schlamowitz et al., 1950~) .These investigations were performed with the intention of supplementing earlier studies of Mirsky (1945). By subcutaneous injection of crystallized trypsin (1 ml, 5%), Mirsky (1945) had obtained local alterations resembling rheumatic nodules in 700/, of rheumatic patients while nonrheumatic subjects did not respond in this characteristic way. The systematic investigations by Boas and Foley (1954) showed, after subcutaneous injection of different phlogistic agents (formalin, ammonia water, ethyl alcohol, turpentine oil, and tannic acid) as well as after mechanical injury, an increase of tissue hexosamine that was limited to the traumatized area. This result was ascribed by these authors to be caused by an increased synthetic activity of the injured connective tissue. According to Romani (1954), the fibroblasts of inflammatory granulomas produce acid mucopolysaccharides, the accuniulation of which is said to be typical of several kinds of inflammation. Dunphy and Udupa (1955) and Dunphy (1956) found, in healing wounds of rats, an increase in the hexosamine ccntent of granulation tissue from 450 nigyo (normal) to 956 nig% of dry weight ; the metachromasia apparent in the histological picture was interpreted as a sign of tissue neoformation. According to these authors, wound healing is characterized by an initial productive phase (day 1to 5) followed by a collagenous phase, during which the tissue concentration of mucopolysaccharides decreases toward normal values. I n guinea pig experiments with carrageenin edema, the newly formed connective tissue showed maximum values of sulfur incorporation and hexosamine level on days 5 and 6, thus indicating an early and intense mucopolysaccharide synthesis (Slack, 1957). Evaluated on the basis of weight, the formation of granuloma reached its maximum within 7 to 9 days; complete resorption of granulation tissue followed within 4 to 6 weeks (Jackson, 1957). Total collagen attained its highest concentration after about 21 days. Chronologically, a t the beginning the formation of insoluble collagen was predominant, later on neutral salt-soluble collagen appeared, and finally, acid-soluble collagen was present in increasing quantities (Jackson, 1957). Houck and Jacob (1958) noted a n increased hexosamine content in rat tissues reacting to
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croton oil. Bollet et a2. (1958, 1959) found that in the polyvinyl granuloma the formation of acid niucopolysaccharides reached its highest level toward the end of the second week; glucosamine and galactosamine were identified as metabolites of the niucopolysaccharides. With increasing age of the granuloma, the concentration of chondroitin-sulfate B was gradually augmented. In experiments performed by Hershberger et al. (1959), the incorporation of sulfur in the cotton-pellet granuloma was distinctly more intense than in normal connective tissue; this result was explained by inflammation-induced proliferation of fibroblasts. Similarly, increased incorporation of labeled sulfur was observed by Du Boistesselin (1960) and Du Boistesselin and Porcile (1963) in granulation tissue after implantation of gelatin sponges; by Junge-Hulsing and Hauss (1960) and by Wirz et al. (1962) in the granuloma pouch after injection of diluted croton oil. At present it is generally established that in the inflamed area there is an increased production of mucopolysaccharides resulting from the synthetic activity of granulation tissue and especially of the fibroblasts (for references see Delaunay, 1962; Delaunay and Baain, 1960a,b, 1963; Graumann, 1964). The question is still undecided if the increased serum level of hexosaniine and polysaccharides, observed in certain inflammatory diseases, metastasiaing tumors, etc., may be ascribed to the passage of these substances from the inflammatory area into the blood. It has been suggested that serum mucopolysaccharides may reflect connective tissue activity (Graumann, 1964). On the other hand, it has been pointed out that serum polysaccharides are chemically different from those of the ground substance (Catchpole, 1950). Papers on the so-called Eiweisszucker and Aminozuclcer (hexosamine of the serum) are to be found in the literature since the end of the 1920’s. According to Nilsson (1937) and Blix et al. (1941), plasma glucosamine is notably increased in pneumonia. West and Clarke (1938) found an increase of glucosamine in numerous cases of acute rheumatic arthritis, rheumatic fever, different infectious processes (tuberculosis, bacterial endocarditis, gonococcal arthritis), infectious mononucleosis, lymphogranuloma, sterile infarction, etc. Kerby (1958) reported an increase of acid mucopolysaccharides and particularly of hexuronic acid in patients with rheumatic arthritis, pulmonary and urinary infections, and post-traumatic inflammations. Rosenberg and Schloss (1949) observed increased serum hexosamine values in rheumatic and other inflmmatory conditions as well as in multiple myeloma. Similar findings were reported by Ganip (1955) in cases of active rheumatic diseases in which the serum glucosamine level increased parallel to the activity of the inflammatory process. Stidworthy et al. (1957) aIso found a close correlation between the clinical activity of rheumatic arthritis and the increase of serum glycoproteins.
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I n animal experiments, Schlaniowitz et al. (1950b,c) deinonstrated a n increased plasma level of hexosainine after subcutaneous injection of crystallized trypsin, streptokinase, lysozyme, as well as after injection of casein and albumin. Boas and Peternian (1953) found increased values of plasma hexosaniine after turpentine oil abscess and experimental fracture of the tibia. Shetlar et al. (1958) noted a characteristic increase of serum glycoprotein concentration and of proteins during the active phase of experimental arthritis in the pig (infection by Erysipelothrix rhusiopathiae). Boas et al. (1952) supposed that the regulation of the serum hexosamine level is mediated by a pituitary hormonc. Increased urinary elimination of hexosamine in inflammatory conditions was reported by Di Ferrante et al. (1957) in lupus erythematosus, and by Di Ferrante (1957), and Loewi (1959) in rheumatic arthritis. As revealed by systematic biochemical and histochemical investigations, inflammatory granulation tissue is characterized not only by increased synthesis and turnover of inucopolysaccharides, but also by a n increased production of collagen, protein, ribonucleic acids (Daniel-Moussard, 1957), and deoxyribonucleic acids (Forscher and Cecil, 1957; Bollet et al., 1958). Surveys of the extensive literature on this subject are given by Delaunay (1962) and Delaunay and Bazin (1960a,b, 1963). The results generally point out a fundamental aspect of the inflammatory reaction, namely, the appearance of productive processes subsequent to the catabolic processes that are characteristic of the secondary alterations inducing the inflammatory response. Evidently, in the course of inflammation the initial catabolic processes conditioning tissue degradation are reversed and substituted by anabolic processes resulting in tissue neoformation. These latter processes are indicative of recovery and healing. The production of mucopolysaccharides by the granulation tissue reveals the capacity of the newly formed connective tissue to syrithetize the constituents of ground substance and intercellular substance with a sufficiency superior to normal. From a biochemical point of view the qualitative aspects of this activity need further elucidation. Inflammatory granulation tissue is evidently characterized by a certain degree of dedifferentiation. As in embryonic tissues, more hyaluronic acid is formed, a t least in the initial stage of granuloma formation. Synthesis of chondroitin-sulfuric acid becomes evident only at a later stage (Delaunay, 1962; Delaunay and Bazin, 1960a,b, 1963). According to Hershberger et al. (1959) this phenomenon results from a “shift of the cellular population” and fibrocytes become predominant. It is certain that the alterations of the circulatory system and of the vascular permeability represent only part of the inflammatory reaction. The formation of vasoactive polypeptides and their function as mediators of the vascular reaction are only responsible for certain defined
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components of the reactive process. It is of fundamental significance that, in the course of the reactive phase and as a result of tissue injury, cellular and tissue metabolism is modified in the sense of increased synthetic activity. It would be interesting to know how these productive processes are initiated, and if the synthetic efEciency of granulation tissue is induced by defined mediators. According to Meier (1959), this function cannot be ascribed to the classic inflammation-inducing agents. Therefore it may be supposed that growth of granulation tissue and its biochemical activity are controlled by specific mediators liberated by the catabolic processes of tissue disintegration. As can be demonstrated in cultures of fibroblasts, there are numerous bioactive substances with more or less characteristic growth-stimulating properties. There are certain factors contained in leucocytes, in cartilage, and in specific organs, such as liver, kidney, and pancreas, which might have a biological function of this sort (for references see Meier, 1959).
E. PERIPHERAL ACTIONSOF ANTIPHLOGISTICS The infiammatory response is based on a controlled interplay of numerous functions and performances of the cells and tissues involved. The individual mechanisms of this response may be modified quantitatively, occasionally even qualitatively. An essential aspect of this reaction is its apparent tendency to reestablish the status quo, i.e., the equilibrium of normal tissue. The aim of anti-inflammatory therapy is to favor this process of recovery by influencing partial functions as well as regulatory mechanisms. The various physiopathological processes concerned with the inflammatory response offer many points of impact to the anti-inflammatory drugs, so that there is some justification in ascribing the therapeutic activity of these agents to a peripheral action within the inflammatory focus. 1. Concentration at the Site of Action
In this regard it is interesting that in the inflamed area as well as in the inflammatory exudate, phenylbutazone, but not salicylate, can be detected in higher concentrations than in normal tissues (Wilhelmi and Pulver, 1955; Wilhelmi et al., 1959). Sedlmayr et al. (1956) and others emphasized that in regard to the particular chemical and physicochemical properties of phenylbutazone (Pulver et al., 1956) changes in pH might determine its tissue distribution. These authors observed in yeast cells and in cells of the Ehrlich carcinoma that acid reaction facilitates the passage of phenylbutazone across the cellular membrane, thus conditioning an accumulation of the undissociated ketonic form within the cells. Similar considerations are to be found in Wallenfels and Sund (1959). They suppose
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that phenylbutazone is transported with the proteins, bound in complex form to the zinc atonis of serum albumin. As could be demonstrated in model experiments with phenylbutazone-zinc complexes, a t a pH similar to that of blood phenylbutazone is to be found almost entirely in bound form (Wallenfels and Sund, 1957). With a shift toward acid pH values, corresponding to those of inflammatory acidosis, the complex is dissociated, and free phenylbutazone appears mainly in neutral, undissociated form (ketonic). 2. Influence on Vascular Response I n regard to the peripheral action of anti-inflammatory drugs in the inflamed area two possibilities require consideration : ( a ) Action on circulatory dynamics and vascular perineability within the inflamed area. (b) Action on metabolism of inflamed tissues, on growth and synthetic capacity of granulation tissue. Among the agents that, in regard to their vascular actions, may be considered as mediators of inflammation, the metabolites deriving from protein degradation seem to be most attractive. They may be classified as follows: (1) The vasoactive amines, histamine, and serotonin, which are either released from stores or produced in the course of inflammatory proteolysis; (2) the so-called kinins, i.e. polypeptides such as bradykinin, kallidin, etc., possibly also the basic polypeptide with leucotactic and permeabilityenhancing properties, recently isolated by Frimmer and Hegner (1963) ; (3) kinin-forming enzymes, such as kallikrein and other tissue proteases, as well as the so-called permeability factors or permeability globulins described by Miles and Wilhelm (1955, 1960), Wilhelni (1962). The lastmentioned agents may also be considered as proteolytic enzymes. Their biological activity seems to be due to as yet unknown products of protein degradation (Spector and Willoughby, 1963; Miles, 1963). According to Schachter (1963) these permeability factors might be identical with kallikrein. Indeed, Davies and Lowe (1963) demonstrated the presence of kallikrein in the 7-globulin permeability factor. Recently, however, Miles (1963) reported that the permeability factor may very well be prepared in a kallikrein-free form. The biological function of these mediators may be modified by drugs as follows : ( a ) by antagonisms between antiphlogistics and biogenic amines and polypeptides, or by interference of drugs with the release of preformed mediators; or (b) by inhibition of enzymes involved in tissue degradation, respectively in the formation of mediators. a. Antagonisms between Mediators and Anti-Injammatory Drugs. It has been demonstrated by chemicoanalytical methods that the biogenic amines histamine [Schachter and Talesnick (1952) ; Halpern and Briot
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(1952); F. Hahn and Wellmann (1952)l and, in the rat also, serotonin [Bhattacharya and Lewis (1956); Parratt and West (1957); Halpern et al. (1959) ; Morsdorf (1959, 1961c)l are liberated consequent to tissue injury. Their biological function in the inflammatory response requires still further investigation (see also Morsdorf, 1960, 1961a,b). Both aniines seem to be important for the initiation of the vascular reaction and for edema formation, while the polypeptides and the permeability factors seem to control the later phases of inflammation (Meier, 1959; Wilhelm, 1962). Nevertheless, if one considers the rapid inactivation of the polypeptides, this conception wants further proof; the same objection applies to the permeability factors (Wilhelm and Mason, 1960). Possibly histamine is involved in the “immediate response” (Halpern, 1953; Rocha e Silva, 1953; Ungar, 1953). This conception is supported by the fact that pretreatment with antihistaminics or histamine-depletion induced by compound 48/80 inhibits the increase of capillary permeability in turpentine oil pleurisy of guinea pigs without suppressing subsequent exudate formation (Spector and Willoughby, 1959). Numerous results concerning the influence of antihistaminics, histamine and serotonin liberators, protease inhibitors, etc., on the immediate and delayed responses obtained after thermal injury in rats, guinea pigs, and rabbits are reported by Wilhelm and Mason (1960). For a detailed discussion of the literature on the function of histamine and serotonin in inflammation, see Meier (1959) ;the mediator function of serotonin is discussed by Morsdorf (1961~). The significance of histamine and serotonin for the mediation of inflammation is limited by the fact that these amines are not involved in the delayed response and have no influence on growth and metabolism of granulation tissue. Moreover, the biological function of serotonin seems to be species-dependent ; in man its participation in inflammatory diseases could not be demonstrated. Nevertheless, there are observations of Van Cauwenberge et al. (1962) and others suggesting that serotonin may be involved in the pathogenesis of rheumatic inflammation in man. I n connection with the mediator functions of histamine and serotonin, not only the specific effects of these amines but their reciprocal interactions (Sparrow and Wilhelm, 1957; Sciuteri et al., 1959) as well as their interferences with other inflammation-inducing agents have to be considered. The influence of histamine on the activity of the kinin-forming enzyme has been mentioned above (Edery and Lewis, 1962a; Copley and Tsuluca, 1962). Data concerning the antihistaminic properties of antiphlogistics have been provided by a series of authors. Danielopolu et al. (1946) seem to have been the first to report that not onIy the antihistaminics but also aniinophenazone are able to inhibit several biological effects of histamine.
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Swyer (1948) was able to prevent capillary damage due to intradermal injection of histamine by administration of salicylates. Jaques and Domenjoz (1950) and Domenjoz (1952, 1953, 1960) described an antihistaminic effect of aniinophenazone and phenylbutazone on perfused rabbit’s ears ; a similar effect was reported by Donienjos (1960) for sodium salicylate, too. The forniation of a histamine wheal in the guinea pig was not inhibited by arniiiophenazone and phenylbutazone (200 mg/kg s.c.) (Wilhelmi and Domenjox, 1951). In investigations with dye spreading, Wilhelmi (1952) found, in the histamine wheal of the guinea pig, a 50% inhibition after subcutaneous administration of sodium salicylate (100 mg/kg), while aminophenazone and phenylbutazone, given a t the same dose, were inactive. I n experiments on rats, Van Cauwenberge el al. (1959b) also observed a moderate but distinct inhibition by salicylate. In ihe histamine asthma of guinea pigs Zicha et al. (1961) found that even on the third day after single intraperitoneal adniinistration of 10 nig/kg of sodium salicylate the shock response was still delayed from 27.5 (normal) t o 47.5 seconds. Similar effects, though even more pronounced, were recorded for aminophenazone and phenylbutazone, the action of the former being demonstrable for 11 days. Coniparative investigations, performed with the same method, but with intraniuscular administration (30 mg/kg), revealed, especially in regard to the duration of action, a clear superiority of oxyphenbutazone to phenylbutaxone. The effect of oxyphenbutazone was detectable for as many as 32 days (Zicha and Bregulla, 1962). Domenjoz (1952, 1953) described a protective action of phenylbutazone in guinea pigs in the so-called histamine-detoxication experinient : After pretreatnient with 100 mg/kg of phenylbutazone the animals tolerated histamine a t doses 15-20 times higher than the lethal dose. An inhibition of histamine release in sensitized aninials by salicylate was reported by Trethewie (1951), Ungar and Damgaard (1955), Haining (1956), Mongar and Schild (1957), Trethewie and Morris (1959), and others. I n the investigations of Haining (1956) a similar but more intense effect was noted for 3-hydroxy-2-phenylquinolincarbonic acid. Trethewie (1957) observed that phenylbutaxone (2.5-5 ing/lOO ml of perfusion solution) also inhibits the liberation of histamine and of “slow reacting substances” from lungs of sensitized guinea pigs, perfused with a solution containing the antigen. A survey of antiallergic effects was given by Austen (1963) for salicylates, by von Rcchenberg (1961) for phenylbutazone. Kelemen (1957) seems to have been the first to describe serotoninantagonistic properties of salicylates. He succeeded in inhibiting the serotonin edema of the rat paw by sodium salicylate administered at a dose of 690 mg/kg, S.C. This effect was also obtained in adrenalectomized
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animals. Theobald and Donienjoz (1958) found distinct inhibitions of serotonin edema in the rat after subcutaneous administration of phenazone (200mg/kg) , aminophenazone (200mg/kg), phenylbutazone (200mg/kg), cinchophen solubile (200 mg/kg), acetylsalicylic acid (500mg/kg), sodium salicylate (500 mg/kg), and quinine sulfate (50 mg/kg). In these experiments, aminophenazone (72% inhibition) and acetylsalicylic acid (67% inhibition) proved the most effective. Bode (1958)and Morsdorf and Bode (1959),who studied dye spreading in the area of a subcutaneous injection of serotonin, noted good inhibition by aminophenazone (200 mg/kg, s.c.). Phenazone (200 mg/kg, s.c.) and acetylsalicylic acid (500 mg/kg, s.c.) were appreciably less active; sodium salicylate (500mg/kg, s.c.) was totally inactive. This discrepancy with the results of Theobald and Domenjoz (1958) seems to correspond to the observation of Parratt and West (1958)concerning the dissimilar influences of phenothiazine derivatives on inhibition of bluing and edema formation. In exreriments of Van Cauwenberge et ul. (1958,1959a)increase of vascular permeability due to serotonin was not modified by phenylbutamone. Eckhardt et al. (1958)described an inhibition of serotonin edema by aminophenazone. Hillebrecht (1959) also found phenylbutazone and Valmorin [2-(~-chloroethyl)-2,3-dihydro-4-oxo-(benzo-l,3-oxazine)]active against serotonin edema. Buch (1959)described an inhibition of serotonin edema by proformiphen (y-phenylpropylcarbamate). In man, Glover et al. (1957)blocked the constrictor response of forearm and hand vessels to intraarterial infusions of serotonin by means of intraarterial administration of sodium salicylate. This effect became evident only when a general blood level of 10-20 mg% of salicylate was reached. This phenomenon was evidently caused by a secondary effect since local arterial salicylate concentrations of 3 M O mg% were inactive. Under various experimental conditions it was possible to demonstrate antagonistic effects of anti-inflammatory drugs against the potentially phlogogenic polypeptides. Collier et al. (1959,1960)and Collier and Shorley (1960)reported that bradykinin induces bronchoconstriction in the guinea pig and that this effect can be inhibited even by small doses of acetylsalicylic acid, phenylbutazone, and aminophenazone. A participation of histamine and serotonin in this antagonism could be excluded with certainty. The characteristics of this surprising antagonism have since been studied in detail. Bronchoconstriction induced by injection of antigen in sensitized guinea pigs was inhibited by mepyramine, but not by the abovementioned antiphlogistics. Listed in the order of their average active dose (EDbo)after intravenous administration, the following scale was obtained : acetylsalicylic acid as calcium or sodium salt (2 mg/kg), phenylbutazone sodium (4 mg/kg) ,aminophenazone and phenazone (8 mg/kg) , paracetamol
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(16 nig/kg), cincophen (32 mg/kg), sodium salicylate (64 mg/kg) (Collier, 1962, 1963a; Collier and Shorley, 1960). This gradational orJer recalls the degree of activity of the anti-intlanimatory drugs found in the UV (ultraviolet) erythema test (Winder et al., 1958) and corresponds to the clinical efficiency in rheumatisni (Collier, 1962, 1963a; Collier and Shorley, 1960). Similar effects in regard to bradykinin and kallidin could be demonstrated for inefenamic acid [N-(2,3-xylyl) anthranilic acid] and flufenamic acid [N-cr,a,a-trifluoro-mtoly1)anthranilic acid]. Both compounds displayed a n intensity of action corresponding to that of acetylsalicylic acid. The hypotension produced by bradykinin was not influenced by any of these drugs (Collier and Shorley, 1963). The inhibition of bradykinin bronchoconstriction may also be obtained on the isolated guinea pig lung (Bhoola et al., 1962). In the rabbit lung in situ, on the other hand, on which bradykinin also induces bronchoconstriction, it was not possible to denionstrate a n antagonism (Bhoola et al., 1962), neither with acetylsalicylic acid nor with phenylbutazone (Collier, 1963a,b). The strictly specific significance of this antagonism is revealed by the fact that other bradykinin effects are not inhibited in a corresponding manner. The effect of intradernial injections on blued guinea pigs is rather resistant to antiphlogistics: Acetylsalicylic acid (150 and 200 mg/kg) influences neither the reaction to bradykinin nor to histamine, while mepyramine abolishes the effect of histamine, but not of bradykinin. Phenylbutazone (100-200 nig/kg) and aminophenazone (75 and 150 mg/kg) slightly inhibit, but do not suppress, the reactions to bradykinin and histamine (Collier and Shorley, 1960). On the isolated guinea pig ileum in zitro, phenylbutazone and aminophenazone at equal concentrations inhibit both the bradykinin and the histamine reactions. Siniilar results were obtained on the rat duodenum (Collier and Shorley, 1960). Pain or nociceptive responses in man, dog, and guinea pig, induced by intradernial injection of bradykinin, could not be modified by acetylsalicylic acid, aminophenazone, and phenylbutazone, but morphine and codeine proved effective (Collier and Lee, 1963). Recently Collier (196313) was able to demonstrate that the comparative potencies of antiphlogistics found in the bradykinin bronchospasm correspond closely to those recorded in guinea pig bronchoconstriction induced by slow reacting substances. The antagonisms between antiphlogistics and bradykinin as well as other kinins and slow-reaching substances on the bronchial muscles are certainly extremely interesting. But unfortunately it has not yet been possible to discover any concrete correlation with the inflammatory response. This antagonism, of course, is conducive to speculation about the biological
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significance of polypeptides in asthmatic attacks, all the more so as there are clinical experiments that confirm a beneficial effect of acetylsalicylic acid, phenazone, aminophenazone, and phenylbutazone on bronchial asthma (Collier, 1963a,b). Lecomte and Troquet (1960) performed investigations on the antagonism between phenylbutazone and bradykinin. In animals anesthetized with Nembutal these authors showed that natural bradykinin (trypsh 7-globulins of bovine serum) a t doses of 0.5 to 2 mg/kg induces hypotension, an effect that is abolished after pretreatment with 100 mg/kg of phenylbutazone. Dye spreading a t the area of an intradermal injection of bradykinin was notably retarded by 75 mg/kg of phenylbutazone. The vasoconstriction of the pulmonary vessels induced in vivo with bradykinin was inhibited by phenylbutazone a t a perfusion concentration of 50 p g / m l . Furthermore, Lecomte (1960) reported that phenylbutazone (75 mg/kg i.p.) inhibits hypotension and the alteration of capillary permeability resulting in sensitized rabbits from injection of antigen. In perfusion experiments with isolated lungs of sensitized rabbits, phenylbutazone added previously to the perfusion liquid suppressed the constriction of pulmonary vessels induced by antigen as well as by bradykinin. Further antagonisms between bradykinin and kallidin and anti-inflammatory drugs were demonstrated by Gjuris and Westermann (1963). The increase of the respiratory minute volume induced in rabbits and guinea pigs by small doses ( 1 4 y/kg, i.v.) of bradykinin and kallidin reaching values two or three times above normal, as well as the apnea induced by higher doses of bradykinin (20-60 y/kg, i.v.), can be suppressed by acetylsalicylic acid (2-40 mg/kg, i.v.). The stimulating action of small doses of kinins was ascribed to an effect on the afferent vagal pathways, the inhibition of respiration by high doses to a central site of action. As Gjuris et al. (1964) demonstrated, bradykinin and kallidin produced tachypnea characterized by tachyphylaxis. The tachypnea was suppressed by bivagotomy, but not by bronchiolytic agents (isoproterenol, adrenaline, papaverine). In contrast to the similar respiratory effects of veratrine, the kinin tachypnea was antagonized in a characteristic way by the antipyretics/analgesics. Gjuris el a,?. (1964) established the order of potency given in the following tabulation :
o-Acetylsalicylic acid Phenylbutazone Aminophenazone Oxyphenbutazone Cinchophen Sodium salicylate
E D L(mg/kg) ~
Index of activity
1.25 2.75 5.0 25.0 25.0 20.0
100 77 32 8.6 6.8 5.5
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It is interesting to note that the effect of phenylbutazone appeared sooner (approximately 5 minutes after i.v. injection) than that of acetylsalicylic acid (about 10-15 minutes after i.v. injection). The above mentioned results concerning the antagonisms between anti-inflammatory drugs and bradykinin/ltallidin do not allow conclusions to be drawn about the physiopathological significance of these kinins in regard to the iriflaniiiiatory response or about the peripheral site of action of the antiphlogistics. b. Inhibition o j Kinin Formation. Ungar ef al. (1952) reported that the antipyretics/analgesics cause a characteristic inhibition of fibrinolysin. This enzyme, however, does not seem to play an important part in the inflammatory response (for references see Heilmeyer and Kahler, 1962) ; a t least it does not influence capillary permeability (Bhoola et al., 1960; Schachter, 1962) and apparently has no relation to kinin formation by kallikrein (Schachter, 1962). It is interesting, however, that fibrinolysin abolishes the actions of bradykinin, histamine, and serotonin on capillary pernieability (Copley and Tsuluca, 1962). For plasminogen, on the other hand, the latter demonstrated a permeability enhancing effect. Miles and Wilhelm (1955) were able to inhibit the effects of the permeability factor, which has also enzymic properties, by means of a subcutaneous injection of salicylate. Obviously the permeability factor, or factors (Mill et al., 1958; Wilhelni, 1962), as we11 as kallikrein, can be inactivated in vitro and in vivo by sodium salicylate (Spector and Willoughby, 1959). This phenomenon, however, seems to be unspecific, since the effects of histamine and serotonin are inhibited siniultaneously. The action of locally applied sodiuni salicylate seems to be more selective. Local salicylate niainly inhibits kallikrein and the globulin permeability factor ; it influences the serotonin effects only partially, and those of histamine not a t all (Spector and Willoughhy, 1963). Inhibition by salicylate of hypotension induced by kallikrein in dogs was recorded by Guth (1960). Northover and Subranianiari (1961b) described inhibition of kinin formation by antipyretics/analgesics (kallidinogen from guinea pig and ox serum; kallikrein from guinea pig seruni and human saliva). From these experiments resulted the following gradation of potencies listed in decreasing order : phenylbutazone, acetylsalicylic acid, sodiuni salicylate. I n vitro inhibition occurred a t low concentrations which, on the isolated guinea pig ileum, were ineffective against bradykinin, substance P, histamine, serotonin, and acetylcholine. The hypoterisive action of human salivary kallikrein in dogs under chloralose anesthesia was significantly reduced by phenylbutazone (76 mg/kg) and also by acetylsalicylic acid and sodium salicylate administered a t doses about twice as high as those of phenylbutazone (Northover and Subramanian, 1961b). By these doses the hypotensive effects of brady-
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kinin, kallidin, histamine, serotonin, and acetylcholine were influenced only very slightly or not at all. The increase of capillary permeability induced by intradermal injection of bradykinin in the rabbit, was scarcely reduced by the above mentioned anti-inflammatory drugs. On the other hand, accumulation of dye in the salivary kallikrein wheal was inhibited by phenylbutazone and, to a slightly lesser extent, by acetylsalicylic acid and sodium salicylate. Northover and Subramanian (1961b) concluded from their results that the anti-inflammatory effect of the antiphlogistic drugs they tested is partly due to a specific inhibition of kinin formation. Systeniatic investigations on kinin formation from pancreatic, salivary, and serum kallikrein on the esterolytic effect (toluene-p-sulfonyl-L-arginine methyl ester) of pancreatic and salivary kallikrein, as well as the possible inhibition by sodium salicylate and acetylsalicylic acid, have recently been performed by Hebborn and Shaw (1963). They observed, contrary to Northover and Subramanian (1961b), no inhibition of kinin formation by salicylates. Salivary kallikrein was inhibited to about 50% by sodium salicylate only a t concentrations as high as 50 mM. At concentrations up to 5 m M neither sodium salicylate nor acetylsalicylic acid influenced the esterolytic properties. According to the results of Hebborn and Shaw (1963), acetylsalicylic acid and sodium salicylate apparently do not inhibit the in vitro kinin formation conditioned by the different kallikreins. The experimental data available at present seem to exclude an interpretation of the mode of action of anti-inflammatory drugs by inhibition of kinin formation. 3. Analgesic Component of Action
The discussion of the peripheral mechanisms of action of anti-inflammatory drugs, in the sense of an antagonism with mediators of the polypeptide type particularly of an interference with their formation, may be extended to the analgesic properties of these drugs. Most of these drugs have well-known central points of impact, which can explain the suppression of inflammatory pain. Their selective action in pain of rheumatic and inflammatory origin is, however, surprising. The painful sensations in inflammatory conditions are probably, to a large extent, caused by the enzymic liberation of pain-producing substances within the inflamed area. An explanation of the analgesic properties of anti-inflammatory drugs must, therefore, take into account their central analgesic components as well as their possible interferences with formation and effects of pain-producing substances liberated within the inflammatory focus. In the therapeutic application of antiphlogistics, inhibition of inflammation and analgesia are so intimately connected that a separate evaluation of these two components is almost impossible. Thus, it is not surprising
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that specific analgesic qualities have been ascribed even to cortisone and ACTH, which are known to act mainly within the peripheral tissues (Jacob and Szerb, 1951, 1952; Plag, 1953; Eichholtz and Alexander, 1961). These findings, however, could not be confirmed by other authors (Winter and Flatacker, 1951; Lee and Pfeiffer, 1951). With a modification of RBgnier’s (1923) method (Eichholtz and Slizys, 1947), Eichholtz and Alexander (1961) were able to demonst,rate an analgesic action of cortisone. The investigations of Jacob and Szerb (1951, 1952) were performed on mice with a modification of the hot plate method described by Woolfe and MacDonald (1944) in which the reaction time was used as a criterion for thermalgesia. The experiment showed an evident analgesic action both for morphine and for aniinophenazone (100 ing/kg, i.p.), furthermore, a statistically significant thermoanalgesia induced by cortisone (0.05-2.5 mg/aninial) as well as by ACTH (Corticotrophin Organon, especially ACTH-Arniour, 1 mg of standard La IA per animal, given 5-6 times a t intervals of 1 hour). According to Jacob and Szerb (1952), this effect should also be taken into account in clinical conditions. It may indeed explain the subjective improvement that in most cases is noted so soon after the injection that it cannot reasonably be ascribed to a n inhibition of inflammation. To explain the niechanism of this analgesic effect, the authors considered, in addition to the specific pain-relieving action, the possibility of a peripheral inhibition of the biochemical processes responsible for the stimulation of pain receptors. According to Lim (1963a,b), visceral pain, induced in animals by intraarterial injection of bradykinin, can be inhibited even by low doses of acetylsalicylic acid. This effect has certainly to be ascribed to a peripheral site of action, as may be concluded from the particular experiniental procedure: pain reactions were recorded in a dog whose spleen was kept in normal nervous connections, but was perfused by a donor dog. Bradylcinin was injected into the lienal artery. Pain was manifested by vocalization and by modification of the action potentials in the splenic nerves. 4. Action on Tissue Cultures A consideration of the biochemical effects of anti-inflammatory drugs should be preceded by a discussion of their action on edema formation and granuloma growth. The inhibition of these coniponents of inflammatory response is a well-proven fact. In regard to the specific peripheral effects on granulation tissue and other mesenchymal substrates and on growth and regeneration processes in general, the demonstration of these properties in experiments in vitro is of primary importance. Material from investigations of this type will be discussed in connection with the effects on the
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metabolism and on the synthetic activity of granulation tissue. At this point we shall only discuss the effects of anti-inflammatory drugs on the growth of tissue cultures. The influence on tissue cultures as well as the cytotoxicity, as observed in vitro, have occasionally been considered in regard to the side effects of the antiphlogistics, especially in connection with tissue damage resulting from local injection. There can be no doubt, however, that the effects noted in tissue cultures on fibroblasts are indicative of a component of action which may partially be responsible for the therapeutic efficiency. There are surprisingly few reports (Saito, 1935; Schuniacher, 1953) about the influence of salicylates on cellular proliferation and tissue growth. In investigations performed by Haberland (1959, 1960), no characteristic result was obtained with salicylic acid at a concentration of 1:4000 (25 mg%) on chick fibroblasts, rat and human fibrocytes, and rat bone marrow tissue. Investigations on tissue cultures with phenylbutazone were performed by Heilmeyer et al. (1953). These authors had observed favorable therapeutic effects in several cases of leukemia and lymphogranulomatosis; consequently, they tried to obtain precise information about a possible “cytostatic” effect in fibroblast cultures. Significant decrease of tissue growth as well as alterations of fibroblasts were recorded even at high dilutions (3-30 mgyo). These effects were partially observed with dilutions which are equal to, or even lower than, blood concentration of phenylbutazone during therapeutic application. Bucher (1955) was correct in expressing certain reserves in regard to the significance of the results obtained in cell cultures for man. He reported decrease of growth intensity as well as visible niorphological alterations of nuclei and nucleoli a t a dilution as low as 1: 100.000 (1 mg%). Haberland (1959, 1960) observed growth inhibition of chick fibroblast cultures by phenylbutazone a t concentrations of 25 nig%. Albrecht et al. (1960) found inhibition of mitosis as well as significant alterations of fibroblast cultures only at concentrations of 75-150 mg% of phenylbutazone. Lower concentrations (5-10 mg%) did not influence mitosis, but induced slight cellular alterations. Schellenberg (196l), experimenting with mouse and chick fibroblasts, inastocytoma P-815, and HeLa cells, reported a notable inhibition of cellular proliferation as well as morphological alterations with concentrations of 10 mg% and more of phenylbutazone. With chloroquine diphosphate, Haberland (1959, 1960) demonstrated an evident inhibition of growth on chick fibroblasts a t dilutions as low as 1:40.000 (2.5 mgY0), while in human and rat fibrocytes and in rat bone marrow the same effect was obtained with only 25 mg%.
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5. Metabolic Effects I n the discussion of the peripheral action of anti-inflammatory drugs within the inflamed area, the effects on tissue metabolism deserve special attention. In recent years numerous investigations on these properties have been performed. Valuable inforniation on the mechanisms of action of anti-inflammatory drugs was expected particularly from research work on their interference with enzymic functions. For reviews on this topic for salicylates: see M. J. H. Sniith (1963) and for phenylbutazone: Stenger (1958) and von Rechenberg (1961). The anti-inflammatory drugs, e.g., the salicylates and phenylbutazone, are able to influence distinct steps of reactions in the intermediate metabolism of carbohydrates, especially in the tricarboxylic acid cycle and the so-called respiratory chain. In addition, they act on various transaniination processes; furthermore, they interfere with phosphate and purine metabolism, with the inactivation of corticosteroids, and, especially in the case of phenylbutazone, with the biotransforniation and elimination of numerous drugs, such as acetylsalicylic acid, aniinophenazone, novocaine, p-aniinosalicylic acid, etc. (Pulver, 1954; see also von Rechenberg, 1961). a. Interference with the Biotransformation of Corticosleroids. An influence on the biotransformation of anti-inflammatory corticosteroids in the sense of an inhibition of degradation was suggested by Hiller (1955, 1956). In previous investigations with liver homogenates, Hiller and Strauss (1954) had obtained an inhibition of fructose turnover and of DPN-H2 oxidation by aminophenazone and phenylbutazone, which could be abolished by addition of carboxylase. In a complementary investigation, aniinophenazone and butapyrine a t doses corresponding to therapeutic blood levels caused ti concentration-related inhibition of DPN-H2 oxidation and of oxygen consumption of liver homogenates. These results suggested the conclusion that the anti-inflammatory action might be due to an interference with energy-yielding processes, and possibly to an inhibition of the DPNdependent biotransformation of cortisone. Kersten and Staudinger (1956, 1957) demonstrated that in corticosteroid inactivation the hydrogenation of ring A effected by an enzyme isolated from the rat liver (Tonikins, 1957), functioning with T P NH as hydrogen donator was inhibited by phenylbutazone and, a t higher concentrations, also by aniinophenazone and acetylsalicylic acid. I n addition, Korus et al. (1956), who studied cortisone inactivation by rat-liver homogenates, showed that phenylbutazone not only inhibits the transformation of the a,@-unsaturated ketonic group of ring A, but also the degradation of the a-ketolic side chain of ring D. Dirscherl and Lutzniann (1959) found
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that aminophenazone interferes with the reduction of ring A only, but not with the transformation of the ketolic side chain. The above mentioned studies revealed that phenylbutazone inhibits or retards biotransformation of two components of the cortisone molecule, which are fundamental for its antirheumatic activity. These results imply that the retardation, particularly the inhibition, of cortisone biotransformation must be considered as a possible component of the therapeutic action of phenylbutazone. This possibility has to be taken into account not only for the biological function of endogenous corticosteroids, but also for the efficiency of drug associations applied in rheumatic diseases. Interference with the biotransformation of steroids, however, seems to be only an accessory mechanism of action, as is evident from animal experiments in which phenylbutazone displays an intense anti-inflammatory action after hypophysectomy as well as after adrenalectomy (Domenjoz, 1953, 1955, 1960). b. Injluence on Metabolism of Inflamed Tissues. Anti-inflammatory drugs interfere with the intermediate metabolism of carbohydrates mainly by more or less specific inhibitions of certain enzymic reactions. Several examples of activation, however, have also been described. Concerning salicylates, Brody (1955, 1956a,b) and Falcone (1959) described an activation of adenosinetriphosphatase, attributed by Charnock and Opit (1962) to a facilitation by salicylates of ATP penetration through the mitochondrial membrane. With phenylbutazone concentrations lower than, and up to, 10 mg%, Exer (1956) and Pulver et al. (1956) observed, in some experiments, an increased oxidation of isocitric, succinic, malic, and fumaric acids. In investigations of Denis and Means (1916) and Cochran (1952) on man, and of J. Reid (1952, cited in Sproull, 1954, pp. 262 and 264) on rabbits, it was demonstrated that administration of salicylates caused a notable increase of oxygen consumption. For this reason Cochran (1952) characterized salicylate as a powerful metabolic stimulant. In complementary experiments on mouse-liver slices, Sproull (1954) was able to confirm M), corresponding to that low concentrations of salicylate (3.5 X therapeutic blood levels, enhance respiration particularly oxygen consumpM ) he obtained tion; with hig,her concentrations (3 X up to 7.5 X inhibition. He concluded from these experiments that the salicylate-induced stimulation of metabolism results from peripheral mechanisms, and may well be compared to the action of 2,4-dinitrophenol. These as well as earlier results concerning the influence of salicylates on the respiration of tissue slices (Lutwak-Mann, 1942; LBvy, 1946; Fish1951) were confirmed by Brody (1955, 1956a,b). After treatment gold et d., with salicylate, and similarly, after 2,4-dinitrophenol, he observed stimula-
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tion of respiration of brain slices, as well as inhibition of the phosphorylation activity of cerebral mitochondria during pyruvate oxidation. Brody interpreted these results in the sense of an uncoupling of oxidative phosphorylation. I n his opinion, afterwards confirnied by numerous authors (see M. J. H. Smith, 1963), the metabolic effects of salicylate are responsible for certain clinical symptoms observed in salicylate intoxication. M. J. H. Smith and Jeffrey (1956) were the’first to suggest a causal relationship between the anti-inflammatory effects of salicylate and the uncoupling of oxidative phosphorylation. On the basis of his results concerning the inhibition of phosphate incorporation in surviving corneal tissue, Kohler (1955) had already considered that phenylbutazone might cause uncoupling of phosphorylation in the respiratory chain, and that this effect might explain its anti-inflammatory action, too. By systematic investigation of various salicylate and phenylbutazone derivatives it was demonstrated that clinically active substances in fact induced uncoupling of oxidative phosphorylation. Adams and Cobb (1958), therefore, put forward the hypothesis that uncoupling of phosphorylation may be considered as the fundamental mechanism of anti-inflammatory action. I n more recent studies on the metabolism of cartilage tissue, Whitehouse and Bostrom (1962), Whitehouse and Haslani (1962), and Whitehouse (1964) confirnied that both salicylates and phenylbutazone, as well as oxyphenbutazone, and cinchophen are uncoupling agents. Incidentally, pyrazole derivatives cannot be delined as “metabolic stimulants” in the same sense as this term has been applied to the salicylates. The basic perturbation characteristic of uncoupling of oxidative phosphorylation consists in a diminished biosynthesis of ATP. The result is an inhibition of substrate phosphorylation, which may occur without a modification of the intensity of respiration, and even in the case of decreased oxygen consumption. I n this connection it should be mentioned, however, that 2,4-dinitrophenol and other uncoupling agents, such as Dicuniarol and other vitamin-K antagonists (Martius and Hess, 1951, 1952), as well as thyroxine (Martius, 1955), have no anti-inflammatory action a t all in both animal and man. These facts, no doubt, conflict with the hypothesis formulated by Adams and Cobb (1958), a t least limiting its general application (Marks and Smith, 1960; M. J. H. Smith, 1963). The effects of the anti-inflammatory drugs on intermediate metabolism of carbohydrates extend to a series of enzymic reactions, depending, among other things, on di- (Hiller and Strauss, 1954; Wallenfels and Sund, 1957, 1959) and triphosphopyridine nucleotide, as well as on cytochrome C (Pennial, 1958), acting as cofactors. The consequences of these metabolic effects and particularly of the uncoupling of oxidative phosphorylation consists in a n inhibition of energy supply and exchange. Perturbation of
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carbohydrate utilization results in a limitation of synthesis of ATP, which, in regard to tissue metabolism, must be considered the most important energy source. This ATY deficit is probably even increased by the activation of adenosinetriphosphatase observed with salicylates (Brody, 1956a,b). It may be supposed that this particular metabolic situation results in an inhibition of numerous anabolic processes involved in tissue growth. At least, the specific inhibition by anti-inflammatory drugs of a series of ATP-dependent reactions may be explained in this sense: Inhibition of phosphate incorporation in the surviving cornea by phenylbutazone (Kohler, 1955); inhibition of sulfur incorporation into niucopolysaccharides of cartilage and cornea by salicylate in vitro (Bostrom and Mansson, 1955; Whitehouse and Bostriim, 1961), by salicylate, phenylbutazone, oxyphenbutazone, cinchophen, and flufenamic acid in vivo (Bostrom et al., 1964);decrease of glutathione blood level by phenylbutazone (Gros, 1956); inhibition of glutamine biosynthesis in the guinea-pig brain by salicylate (Messer, 1958); inhibition of choline acetylation in the guinea pig by salicylate (Kuriaki and Marumo, 1959); inhibition of glucose incorporation into uridine diphosphoglucose (yeast) by salicylate (Moses and Smith, 1960); inhibition of acetate incorporation into various tissues by salicylate (M. J. H. Smith and Moses, 1960); and inhibition of incorporation of inorganic phosphate into cartilage by clinically active salicylates and phenylbutazone (Whitehouse, 1964). c. Effects on Biosynthesis of Mucopolysaccharides. The above mentioned effects of the clinically active salicylates, of phenylbutazone, and cinchophen on sulfur and phosphate incorporation into cartilage may be correlated with an important action component of the anti-inflammatory drugs. The implications of intensified mucopolysaccharide synthesis by granulation tissue for the inflammatory response have already been discussed. As was demonstrated by Sobel et al. (1953) and other authors, acetylsalicylic acid (intake of 0.3% salicylic acid in the diet for 95 days of rats) and cortisone cause an evident decrease of the hexosamine content in skin. The mechanisms responsible for the inhibition of mucopolysaccharide synthesis were investigated in detail by the research teams of Bollet and of Bostrom. Bollet et al. (1959) demonstrated that granulation tissue obtained by implantation of polyvinyl sponges contains all enzymic systenis necessary for the biosynthesis of uridine diphosphoglucuronic acid (for synthesis scheme, see Dorfman, 1955; Gibian, 1959; Bostrom, 1960). The first steps in the biosynthesis of glucuronic acid (formation of glucosamine-6-phosphate from glucose, particularly fructose-6-phosphate and glutamine), effected by connective tissue homogenates, could be inhibited by auric chloride. This effect was obtained with concentrations corresponding to the blood level of patients during gold therapy (Bollet and Shuster, 1960).
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With gold sodium thiomalate, too, it was possible to demonstrate a similar inhibition of glucosamine-6-phosphate biosynthesis by connective tissue of rats an vivo, a n effect which could not be produced in liver hornogenates (Bollet and Shuster, 1960). Furthermore, Bollet (1961) was able to show that the synthesis of glucosamine-6-phosphate by granulation tissue in vitro could be inhibited by sodium salicylate as well as by phenylbutazone, oxyphenbutazone, and sulfinpyrazone. In this regard it is interesting that transamination of a-ketoglutaric acid with alanine to glutamic acid is also competitively inhibited by phenylbutazone (Pulver et al., 1956). It was denionstrated by Bryant and Smith (1962) that salicylate, too, is able to inhibit important transamination processes and hence glutamine synthesis. These effects deserve consideration because glutanline, which furnishes the amino groups for the biosynthesis of glucosamine and galactosamine, may be a limiting factor in mucopolysaccharide synthesis (Whitehouse, 1963). Obviously anti-inflammatory drugs have further characteristic points of impact within mucopolysaccharide synthesis. Sulfur incorporation into acid mucopolysaccharides of the chondroitin type is of particular importance for the biogenesis of chondroitin-sulfuric acid, which is a n important constituent of ground and intercellular substance. It is still undecided at what stage of the synthesis this sulfation takes place. Chondroitin is possibly not the obligatory substrate for sulfur fixation, as mono- as well as oligosaccharides can also function as sulfur acceptors. Sulfur incorporation is, however, accelerated with increasing chain length (Suzuki and Strominger, 1960a,b). Furthermore, it has been denionstrated on cartilage slices in vitro that with a simultaneous intensified sulfur incorporation degradation of chondroitin-sulfuric acid is increased (Coelho and Chrisman, 1960). These results indicate that sulfur incorporation can be used as a criterion for de novo mucopolysaccharide synthesis with certain restrictions only (Muir, 1961). Suzuki and Stroniinger (1960a,b) observed that fully sulfated polysaccharides, such as chondroitin-sulfate A and C, are able to fix and to exchange sulfur or both. The sulfur-exchange phenomenon, which may be studied in vitro on cartilage slices with routine methods, appears to be mediated by an enzynie: it shows all the characteristics of a n enzymic reaction and, moreover, may be specifically inhibited by agents able to block SH groups, carbonyl groups, and metals. At any rate, it may be taken for granted that S36offered in the form of inorganic sulfate, both in vivo and in vitro, is absorbed and distributed in a characteristic pattern. The highest concentration is to be found in those areas of connective tissue in which metabolism and growth are increased. Two interesting applications have resulted from the knowledge of this fact. Increased incorporation of 535 into neoplastic tissues may facilitate diagnosis in cases of chon-
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drosarcoma (Gottschalk and Allen, 1952), and, under certain circumstances, may permit radiotherapy (Gottschalk et al., 1959). In experimental research on inflammation, the intense fixation of P5by inflamed tissue and by granulation tissue in vitro and in vivo (Layton, 1950a,b; Kodicek and Loewi, 1955; Drenkhahn and Meissner, 1956; Slack, 1957) can be used for the demonstration of certain action components of anti-inflammatory drugs. Thus, an evaluation of the activity of anti-inflammatory agents by means of S35incorporation in granulation tissue was performed in vitro: by Layton (1951) for cortisone and in vivo: by Verne and Du Boistesselin (1958) for azulene sulfonate, salicylate, and phenylbutazone; by Hershberger et al. (1959) for cortisone, Nilevar, and phenylbutazone; by Du Boistesselin (1960) for phenylbutazone, oxyphenbutazone, salicylate, and cortisone; by Wirz et al. (1962) for phenylbutazone and prednisone; and by Du Boistesselin and Porcile (1963) for phenylbutazone and oxyphenbutazone. The papers quoted confirm Layton’s previously expressed viewpoints (1951) : The anabolic processes of tissue neoformation, such as those occurring in granulation tissue, are characterized by an increased need for sulfur; the rate of sulfur incorporation may be used as a criterion for the intensity of the inflammatory reaction; the fixation of sulfur may be characteristically inhibited by antiinflammatory drugs. On the other hand, it is still an open question whether sulfur-binding capacity can become a limiting factor in wound healing, as supposed by Layton (1950b). Obviously, in granulation tissue, acid inucopolysaccharides may be formed which must be considered as definitely “undersulfated,” without evident inhibition of the reactive processes, as Grossfeld et al. (1957) and others pointed out (see also Whitehouse and Bostrom, 1961). In this connection it should be noted that mixtures of polysaccharides obtained from connective tissues are different when the animals had been treated by hormones or suffered from deficiencies. Ascorbic acid deficiency depresses biosynthesis of chondroitin-sulfate but not that of hyaluronic acid (Muir, 1961). I n investigations on the granuloma pouch after administration of phenylbutazone and prednisone Wirz et al. (1962) found evident diminution of granulation tissue, particularly retardation of granuloma growth, as well as a significant reduction of S35incorporation. The weight content of mucopolysaccharides, expressed in grams of granulation tissue, was not modified. The relation between acid and total rnucopolysaccharides showed no characteristic modifications, whereas the percentage of sulfated as compared to acid mucopolysaccharides was, after treatment with phenylbutazone, prednisone, or a combination of both, higher than in the control animals. Phenylbutazone, which conditions evident inhibition of S35incorporation in granulation tissue, exhibited no characteristic action on normal xiphoid cartilage, in contrast to prednisone, which was active
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on both tissues. Similar results were reported by Hershberger et al. (1959) for normal cutaneous tissue. The results obtained by Bostrom et al. (1964) suggest that the differences observed between sulfur incorporation in normal and granulation tissues may result from differences in dosage and modes of administration. The effect of anti-inflammatory drugs on S35incorporation into normal tissue was investigated by Layton (1951) on embryonal tissue with cortisone; by Bostroni and Odeblad (1953) and Bostrom and Mansson (1955) on cartilage slices with cortisone and salicylate; by Schiller and Dorfman (1957) on skin with cortisone and hydrocortisone; by Munich (1961) on cornea with phenylbutazone; by Lash and Whitehouse (1961) on cartilage cultures with cortisone and derivatives; by Whitehouse and Bostroin (1961) on cartilage and cornea with salicylate and cortisone; by Whitehouse and Bostroni (1962) on cartilage, cornea with salicylate, phenylbutazone, cortisone, hydroxychloroquine, and chloroquine ; by Whitehouse (1964) on cartilage slices and liver mitochondria with eighty salicylate analogs; and by Bostroni et a2. (1964) on rib cartilage in vivo (rat) with salicylates, phenylbutazone, oxyphenbutazone, and chloroquine. The most important results of these investigations may be summarized as follows : (1) Inhibition of mucopolysaccharide metabolism, as nieasurable in vitro by means of the incorporation rate of P5, P32,C14-labeled glucose and C14-labeled acetate (Whitehouse, 1964; Bostroni et al., 1964), seems to correspond to a characteristic action component of synthetic anti-inflammatory agents. Its intensity seenis to reflect their therapeutic efficacy. Among the steroids, however, not only cortisone and hydrocortisone are active, but also progesterone, deoxycortone, and Compound S, which, although active when applied topically, do not exhibit anti-inflammatory properties. In in vitro experiments, prednisone proved inactive. (2) Inhibition of SF incorporation in vitro by all anti-inflammatory drugs except choroquine and hydroxychloroquine proved reversible. In the case of hydrocortisone and cortisone, inhibition appeared after a certain delay. I n vivo chloroquine was ineffective. In the case of salicylate, in vitro, the active concentration for the incorporation of S35,and C14-glucoseand acetate amounted to about 2 mM. In experiments in vivo, sodium salicylate at doses of 100 nig/kg was significantly active (Bostroni et al., 1964). (3) Inhibition of S35incorporation seems not to he due to the “competition” for “active sulfate,” as supposed by Greiling and Schuler (1961) and Greiling and Dorner (1962). As Whitehouse and Bostroin (1962) and Whitehouse (1963) demonstrated, the interference with mucopolysaccharide metabolism is to be ascribed to an inhibition of ATP-synthesis, “either by inhibiting cellular respiration or by uncoupling oxidative phosphorylation” (Bostrom et al., 1964). Since salicylate inhibits P32 incorporation
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a t concentrations ineffective in regard to oxidation of (l-C14) octanoate, (C14) glucose, (l-CI4) acetate, (2-C14) pyruvate, and (l-C14) lactate, the primary action seems to be due to an uncoupling of oxidative phosphorylation (Whitehouse, 1963). These investigations on mucopolysaccharide metabolism, which we owe largely to the research teams of Bostrom and of Whitehouse, have considerably enriched our knowledge about the action of anti-inflammatory drugs. From these results, it may be gathered that all clinically active antiinflammatory drugs may interfere with mucopolysaccharide metabolism in normal, noninflamed tissue, too. The distinct mechanisms demonstrated in vitro and in vivo seem to condition the action of these drugs on the inflammatory granuloma. Therefore, it seems sensible to consider the examination of mucopolysaccharide metabolism in vitro as a valuable screening technique for new anti-inflammatory agents (Bostrom et al., 1964). Incidentally, the interference with the biosynthesis of mucopolysaccharides is significant not only for the therapeutic efficiency of antiinflammatory drugs, but also for the appreciation of their side effects. V. Summary
As is evident from clinical experience, it is possible to obtain considerable improvement and even complete recovery in inflammatory conditions by a drug-induced inhibition of the inflammatory response. Present interpretations of this therapeutic action on the basis of precise pharmacodynamic mechanisms are still unsatisfactory. Numerous and convincing data seem to indicate that synthetic anti-inflammatory agents act, to a large extent, by means of peripheral points of impact within the inflamed area. Although many details are known about the partial mechanisms of inflammatory response as well as about the pharmacodynamics of antiinflammatory drugs, we are still in search of an adequate explanation for their specific mode of action. REFERENCES Adams, S. S., and Cobb, R. (1958). Nature 181, 773. Adams, S. S., and Cobb, R. (1963). I n “Salicylat,es” (A. St. J. Dixon et al., eds.), p. 127. Churchill, London. Agolini, G., Bertelli, A., and Cavicchini, G. (1952). Boll. SOC.Ital. Biol. Sper. 28, 1761. Albrecht, M.,Eschenbach, J., and Kretschmer, V. (1960). Arzneimittel-Forsch. 10, 606. Allison, J. E. (1955). Proc. Soc. Exptl. Biol. Med. 90, 277. Armstrong, D., Jepson, J. B., Keele, C. A., and Stewart, J. W. (1957). J. Physiol. (London) 136, 350. Aronson, E., and Sachs, J. (1885). Arch. Ges. Physiol. 37, 232. Atkins, E. (1960). Physiol. Rev. 40, 580.
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Biochemistry of Drug Oxidation and Reduction by Enzymes in Hepatic Endoplasmic Reticulum JAMES R.
GILLETTE
Laboratory of Chemical Pharmacology, National Heart Institute, National Institutes of Health, Bethesda, Maryland
I. Introduction . . . . . . . . . . . . . . 11. Oxidation of Foreign Compounds by Enzymes in Hepatic Endoplasmic
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Reticulum . . . . . . . . . . . . . . A. Localization of the Enzyme Systems . . . . . . . . B. Oxidative Reactions . . . . . . . . . . . . C. Endogenous Substrates of NADPH-Dependent Oxidat,ive Enzymes . 111. Reduction of Foreign Compounds by Enzymes in Hepatic Endoplasmic Reticulum . . . . . . . . . . . . . . A. Azo Reduction . . . . . . . . . . . . . B. Kit,ro Reduction . . . . . . . . . . . . IV. Mechanisms of Oxidation and Reduction by Enzymes in Hepatic Endoplasmic Reticulum . . . . . . . . . . . . . A. Mixed Oxygeiiase Mechanism . . . . . . . . . B. Microsomal P-450 . . . . . . . . . . . . C. P-450 Reductases . . . . . . . . . . . . D. Mechanisms of “Active Oxygen” Transfer . . . . . . . . . . . E. Mechanisms of Hydroxylation by Model Systems . F. Inhibitors of the Oxidative and Reductive Enzyme Systems in Hepatic Endoplasmic Reticulum . . . . . . . . . . . G. How Many Oxidative Enzyme Systems are Present in Liver Microsomes? V. Factors Which Limit Drug Metabolism in Living Animals . . . . . . . . . . . . . . . . . . References
220 220 22 1 228 23 1 231 232 234 234 236 240 242 243 244 253 254 255
I. Introduction
In studying the action of drugs in the body, it is important not only t o determine the mechanisms through which drugs elicit their pharniacological effect, but, also to study the factors which control their concentration at active sites. The conccntration of some drugs are controlled mainly by their relative rates of absorption and excretion. Most drugs, however, are converted to other substances before they are excreted into air, bile, and urine, and thus their rates of metabolism may be a major factor in limiting their action. Indeed, the pharmacological effects of highly bound drugs, such as chlorpromazine, phenylbutazone, and thiopental, would last for virtually a lifetime if they were not converted to inactive metabolites which were readily excreted. 219
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The metabolism of drugs in the body does not always result in detoxification. Some drugs, like chlorpromazine, codeine, and ephedrine, are N-demethylated to form metabolites that have pharmacological properties similar t o those of the parent compounds. Other drugs, like parathion, tremorine, and prontosil, produce their action only through the formation of pharmacologically active metabolites. Moreover, the toxicity of foreign conipounds may be caused not by the compounds themselves but by their metabolites. Thus, the carcinogenic properties of certain aniines are now thought to be mediated by the formation of N-hydroxylamine derivatives and other active metabolites. Studies on drug metabolism have thus led to a better understanding of drug action as well as the mechanisms of drug inactivation. During the past several years, numerous studies have shown that most drugs are oxidized or reduced by enzymes in the endoplasmic reticulum of tissues. Though these enzymes are localized mainly in liver, they are also present in minor amounts in other tissues, such as kidney, lung, and the gastrointestinal tract. The oxidative enzymes require oxygen and reduced nicotinamide adenine dinucleotide phosphate (NADPH). For the metabolism of many substrates, however, reduced nicotinamide adenine dinucleotide (NADH) may replace NADPH, though the rate of metabolism is invariably slower. This review describes the oxidative and reductive reactions catalyzed by enzymes in endoplasmic reticulum, some of the properties of the enzymes, and the current views of their mechanism of action. II. Oxidation of Foreign Compounds by Enzymes in Hepatic Endoplasmic Reticulum
A. LOCALIZATION OF
THE
ENZYME SYSTEMS
The hepatic endoplasmic reticulum is a network of submicroscopic tubules, which extends into almost all regions of the cytoplasm and comprises two major components : A rough-surfaced form consisting of lipoid tubules studded with ribosomes and a smooth-surfaced form devoid of ribosomes (Palade and Siekevitz, 1956). On homogenization, the reticulum disintegrates to form small vesicles which may be isolated as “rough”- and “smooth”-surfaced microsomes by centrifugation of discontinuous gradient systems (Fouts, 1961; Dallner, 1963). Though ribosomes serve an important role in synthesis of protein, they do not contain the relatively nonspecific enzyme systems which catalyze the oxidation of drugs. Treatment of liver microsomes with ribonuclease, which destroys protein synthetic mechanisms in ribosomes, does not alter the activity of the drug-metabolizing enzymes; in contrast, treatment with deoxycholate, which solubilizes the lipoidal membranes, destroys the drug
DRUG OXIDATION AND REDUCTION BY ENZYMES
22 1
enzymes (Gillette et aZ., 1957). Moreover, smooth-surfaced microsomes metabolize drugs more rapidly than do the rough-surfaced microsomes (Fouts, 1961; Remmer and Merker, 1963, 1965a,b). Whether the nonspecific enzymes are localized in the lumen of the reticulum or in the lipoid membranes has not been completely resolved, but it seems likely that the enzymes reside in the membranes. Treatment of niicrosonies with sonic oscillations or with hypotonic solutions, which presumably rupture the microsomal vesicles, fails to solubilize the oxidative and reductive enzymes (J. R. Gillette, unpublished results, 1955); in contrast, these treatments solubilize microsonial albumin, which presumably exists in the lumen (Peters, 1962). Moreover, certain polar drugs which are presumably not able to penetrate the lipid membrane are oxidized by liver microsonial enzymes.
B. OXIDATIVEREACTIONS 1. Deamination
Axelrod (1955) found that amphetamine and a number of its analogs are converted to ketones and ammonia by an enzyme system in liver microson~esof rabbit, but not of dog or rat. The enzyme is apparently stereospecific, for the 1-isomers are deaniinated more rapidly than are d-isomers. The enzyme differs from monoamine oxidase not only in its requirement for NADPH, but also in its ability to deaminate substrates that are not metabolized by monoamine oxidase. 2. O-Dealkylation Axelrod (1955) found that aIkyl groups of alkylaryl ethers are removed to form aldehydes and phenols. For example, codeine is converted to morphine and formaldehyde, and p-ethoxyacetanilide is oxidized to p-hydroxyacetanilide and acetaldehyde. The finding that alkyl derivatives of o-nitrophenol (Netter, 1959, 1960) and p-nitrophenol (McMahon et al., 1963) are similarly dealkylated is of particular interest, because the phenolic products have an intense color in slightly basic solutions (pH 8.0). Thus, the rate of dealkylation of these derivatives may be measured spectrophotometrically . I n addition to lipid soluble compounds, the enzyme system also O-dealkylates certain polar substances, such as metanephrine (Axelrod, 1965). There is evidence that a number of enzymes in liver microsomes of various mammals may catalyze this reaction. Axelrod (1956) found that rabbit and guinea pig niicrosomes metabolize p-ethoxyacetanilide equally well, but that rabbit microsomes convert codeine to morphine ten times faster than do guinea pig microsomes. Moreover, SKF-525A blocks the
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O-dealkylation of p-ethoxyacetanilide by rat liver microsomes (J. J. Burns, personal communication, 1962), but not that by guinea pig microsomes (Axelrod, 1956).
3. Hydroxylation of Alkyl hydrocarbons The alkyl side chain of barbiturates is oxidized to primary and secondary alcohols (J. R. Cooper and Brodie, 1955, 1957; s. Toki et al., 1962). Moreover, the side chain of p-nitrotoluene (Gillett,e, 1959) and presumably of other arylalkane compounds (Williams, 1959) are similarly converted to alcohol derivatives. The primary alcohols, formed by the hydroxylation of side chains, are oxidized by dehydrogenases present in the soluble fraction of liver. For example, p-nitrobenzyl alcohol is oxidized to p-nitrobenzoic acid by alcohol dehydrogenase and aldehyde dehydrogenase (Gillette, 1959). However, 3-hydroxyhexobarbital, formed from hexobarbital by the microsomal enzyme, is oxidized to 3-ketohexobarbital by another dehydrogenase which requires NADP (K. Toki and Tsukamoto, 1964). 4. Aromatic Hydroxylation This reaction introduces a hydroxyl group in a n aromatic ring. Naphthalene and other polycyclic hydrocarbons can thus be converted to a number of different phenolic derivatives. Arylamines, such as acetanilide, are hydroxylated mainly to ortho and para derivatives (Mitoma et al., 1956; Booth and Boyland, 1957). Iniipramine and chlorproniazine are hydroxylated in positions para to the nitrogen in the ring systems. Hydroxylation is not restricted l o lipid-soluble compounds, however, for certain phenylazonaphtholsulfonates are converted to hydroxyphenylazonaphtholsulfonates in living rats (Barrett et al., 1965) and hydroxyphenylethylamines are hydroxylated to catecholamines by rat liver microsomes (Axelrod, 1963). Hydroxylation of acetanilide in the ortho and para positions appear to be catalyzed by different enzymes. Rabbit liver niicrosoines metabolize the compound niainly to p-hydroxyacetanilide, whereas cat liver niicrosomes form mainly o-hydroxyacetanilide (Posner et al., 1961a). I n addition, pretreatment of rats with 3,4-benzpyrene enhances ortho hydroxylation, but does not affect para hydroxylation of biphenyl (Creavin et al., 1964). Moreover, ortho hydroxylation of aniline by rabbit liver microsomes is much more sensitive to the impairing effects of semicarbazide and cupric chloride than is para hydroxylation (Bauer and Kiese, 1964). 5. Epoxidation
I n this reaction oxygen is added across a double bond. For example, chlorinated hydrocarbons, such as aldrin, isodrin, and heptachlor, are
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converted to epoxides (Wong and Terriere, 1965), which are relatively stable in animals (Cueto and Hayes, 1962; Ludwig et al., 1964). Epoxides are presumably intermediates in the format ion of dihydrodiols from aromatic hydrocarbons, but these epoxides are hydrolyzed so rapidly that they have not been detected in liver preparations (Booth and Boyland, 1957). The dihydrodiols may he precursors of catechol analogs, for Ayengar et al. (1959) found an enzynie in liver which catalyzes the dehydrogenation of dihydrodiols. It also seems probablc that epoxide derivatives are precursors of preniercapturic acids (Booth et ul., 1960). It is possible that niany, but not necessarily all, aromatic hydroxylations are mediated through the forniation of epoxides, and that the final products formed in the system depend on the relative rates of rearrangement and hydrolysis. 6. N-Dealkylation
In this reaction alkyl groups are removed from secondary and tertiary anlines to forni aldehydes and primary aniines (La Du et al., 1955; Mueller and Miller, 1953). The enzynie system acts on niany lipid-soluble foreign substances including aniinopyrine, methylaniline, diniethylaminoazobenzene, but does not remove the methyl groups of sarcosine and other N-methylamino acids (Gaudette and Brodie, 1959). These findings led Gaudette and Brodie to suggest that only lipid-soluble compounds were metabolized by liver niicrosonies. In accord with this view, McMahon (1961) studied the metabolism of a homologous series of alkyl bufynamines and found a correlation between lipid solubility and the rate of metabolism of the conipounds. Nevertheless, Maze1 and Henderson (1965) found that a number of polar conipounds including 1-methylguanosine and puroniycin aminoriucleoside are deniethylated by rat liver microsonies. It is possible that the polar substances are metabolized by different enzymes than the lipid-soluble conipounds, for H. A. Sasanie and J. R. Gillette (unpublished results, 1965) found that carbon monoxide blocks the N-deniethylation of aminopyrine inore effectively than that of N-diniet hyladenosine. The finding that diinethylnitrosaniine niethylates RNA (ribonucleic acid) in a number of tissues in addition to liver (Lee et ul., 1964) suggests that N-denicthylases are also present in other tissues. This conipound is deniethylated first to forni niononiethylnitrosaniine, which decomposes spontaneously to form azoniethane, a potent alkylating agent. Since it is unlikely that appreciable anlourits of niononiethylnitrosaniine and azomethane diffuse out of cells in which they are formed, the determination of niethylated HNA and protein niay be used as an indirect measure of N-demethylase activity (Magee, 1963, 1965; Miller and Miller, 1965). Recent evidence suggests that rat liver inicrosonies metabolize tertiary
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and secondary amines by different enzymes. For example, rat liver niicrosonies rapidly convert imipraniine to desmethylimipramine, but only slowly convert desmethylimipramine to the primary amine (Dingell et al., 1964). Similarly, rat liver microsomes convert chlorpromazine and amitriptyline more rapidly to secondary ainines than their secondary amine derivatives are converted to primary amines (McMahon, 1964). Moreover, McMahon (1964) found that pretreatment of rats with phenobarbital enhances the demethylation of certain tertiary amines by liver niicrosomes to a greater extent than it enhances the metabolism of their secondary amine analogs. Recent evidence also suggests that N-methylbarbiturates may be demethylated by other demethylases in liver microsomes. Rat liver microsoines usually demethylate these compounds slowly. Pret,reatment of rats with phenobarbital enhances the N-deniethylation of certain barbiturates about 20- to 50-fold, whereas it enhances the metabolism of certain hydantoins only about 3- to 4-fold (Smith et al., 1963). It is possible, however, that many of the variations in the relative rates of demethylation of various secondary and tertiary amines may be caused by product inhibition. Certain primary amine analogs of tertiary amines, such as iniipramine (J. V. Dingell and Gillette, unpublished results, 1961) and Lilly 18947 (McMahon and Mills, 1961; McMahon, 1962), are potent inhibitors of N-demethylation reactions; thus, demethylation of tertiary amines would presumably proceed to a greater extent in incubation mixtures than demethylation of secondary amines. Moreover, it is also possible that the rate-limiting step in the N-dealkylation reactions may be either the dissociation of the complex of the enzyme and the hydroxymethyl intermediate or the deconiposition of the intermediate to the amine and the aldehyde. Thus, the reaction may be inhibited by the hydroxymethyl intermediate rather than the amine. 7. Formation of Alkylol Derivatives Though alkylol derivatives are presumably intermediates in N-dealkylation reactions, certain alkylols are stable and, hence, do not readily form aldehydes. Hodgson and Casida (1961) found that a microsomal enzyme metabolizes a series of N-dimethyl carbaniates to hydroxyniethyl derivatives, which decompose in strongly acidic solutions to form formaldehyde (see Dorough and Casida, 1964). The enzyme system, however, does not act on N-monomethyl carbamates. O’Brien (1957) found that schradan (octamethyl pyrophosphoramide) is converted to a potent cholinesterase inhibitor by liver microsonies. It was once thought that the active metabolite was schradan N-oxide (Hartley, 1951), but is now believed to be hydroxymethyl schradan (Heath et al., 1955).
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The conversion of nicotine to cotinine may be viewed as a type of alkylol formation since 2-hydroxynicotine is presumably the intermediate in this reaction. The 2-hydroxynicotine is converted to cotinine by a liver microsomal enzyme, presumably a dehydrogenase or a n aldehyde oxidase (Hucker et al., 1960). Treniorine is another drug metabolized along this pathway. Though devoid of pharmacological activity itself, tremorine is converted to oxotremorine (Cho et aZ., 196l), a metabolite which evokes central as well as peripheral cholinergic effects (George et al., 1962).
8. N-Oxide Formation I n this reaction, oxygen reacts with the nitrogen atom in tertiary amines to forin N-oxides. For example, liver microsomes convert trimethylamine t o trimethylamine N-oxide (Baker and Chaykin, 1962), dimethylaniline to dimethylaniline N-oxide (Ziegler and Pettit, 1964) and tremorine t o tremorine N-oxide (Cho et al., 1964). In addition, N-oxide derivatives have been isolated from urine of patients receiving chlorpromazine (Fishman et al., 1962) and imipramine (Fishman and Goldenberg, 1962). Fish et aZ. (1956) proposed that N-oxides may be intermediates in the N-dealkylation reactions. This seemed unlikely to us, however, because we found that rabbit liver microsomes dealkylate the N-oxide of dimethylaniline much more slowly than they do dimethylaniline (see Brodie et al., 1958; Gillette, 1962, 1963a). Interest in this proposed mechanism was renewed by the recent findings of Pettit and Ziegler (1963) that liver microsomes of rat and beef catalyze the conversion of dimethylaniline N-oxide to nionoinethylaniline and formaldehyde. Since the apparent Michaelis constant of the enzyme catalyzing this reaction is very high (0.139 M ) , however, it is likely that the rate of rearrangement is insignificant a t the concentrations of dimethylaniline N-oxide found in incubation mixtures containing dimethylaniline and microsomes (see Ziegler and Pettit, 1964). In addition, McMahon and Sullivan (1964) showed that the N-demethylation of Z-propoxyphene by rat liver niicrosonies proceeds more than ten times faster than the demethylation of its N-oxide derivative. Moreover, on incubation of microsomes with Z-propoxyphene-N-meth~1-C'~and large amounts of unlabeled l-propoxyphene-N-oxide, only insignificant amounts of radioactivity were trapped in the N-oxide pool. These findings thus suggest that N-oxidation and N-demethylation are catalyzed by different enzymes.
9. N-Oxidation of Primary, Secondary Amines, and Their Derivatives This reaction attaches a hydroxyl group to nitrogen atoms of amines and amides to form derivatives of hydroxylamine. For example, microsomes
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form hydroxylamine derivatives of 2-acetylaminofluorene (Irving, 1962), certain other carcinogenic amines (Uehleke, 1963; Booth and Boyland, 1964), and various aniline derivatives (Kiese and Uehleke, 1961a,b; Kampffnieyer and Kiese, 1963, 1964a; Booth and Boyland, 1964). The microsomal enzymes that catalyze the N-hydroxylation of aniline derivatives differ from the niicrosomal enzymes that catalyze N-dealkylation and p-hydroxylation in a number of ways. Using aniline, N-methylaniline, N-ethylaniline, and N-butylaniline as substrates, Kiese and co-workers (see Kiese, 1965) have shown that p-chloromercuribenzoate and N-ethylnialeimide stimulate N-hydroxylation by rabbit liver microsomes, but inhibit N-dealkylation and p-hydroxylation. These workers also found that the affinity of oxygen for the N-dealkylation and p-hydroxylation enzymes is an order of magnitude greater than that for the N-hydroxylation system (Kampffnieyer and Kiese, 1964b). Moreover, carbon monoxide blocks the N-dealkylation and p-hydroxylation of N-ethylaniline, but does not inhibit its N-hydroxylation by rabbit liver inicrosomes (Kampffmeyer and Kiese, 1965). Although hydroxylamine derivatives are apparently not intermediates in p-hydroxylation reactions, they may be intermediates in o-hydroxylation. Miller and Miller (1960) found that N-hydroxyacetylaminofluorene could be transformed to its o-hydroxyl derivative in rats. Moreover, Booth and Boyland (1964) have shown that the hydroxylamine derivatives of a number of aromatic amines are converted to o-hydroxyaromatic amines by an enzyme in the soluble fraction of liver. The enzyme apparently required NAD, NADH, or NADPH, but the function of the coenzymes in the transformation reaction is obscure. The formation of hydroxylaniine derivatives is especially important in pharmacology, for these derivatives may account for the toxicities caused by aromatic arnines. For example, Kiese and co-workers found that the N-hydroxylaniine derivatives of aniline and its analogs mediate the oxidation of hemoglobin to methemoglobin (see Kiese, 1965). Moreover, the Millers and a number of other workers have correlative evidence suggesting that 2-acetylaniinofluorene, 4-acetylaminobiphenyl, 2-acetylaminophenanthrene, 4-acetylaminostilbene, and 2-naphthylamine evoke their carcinogenic activity through the formation of N-hydroxylamine derivatives (see Miller and Miller, 1965). 10. S-Demethylation Alkyl thio ethers, such as 6-methylmercaptopurine, are S-demethylated to form thiol derivatives and formaldehyde in incubation systems containing the 9000 X g supernatant fraction of liver and a NADPH generating system (Maze1 et al., 1963). Strangely, formaldehyde is not formed when
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6-niethylniercaptoprine is incubated with rat, liver ~nicrosoniesand NADPH (P. Mazel, personal communication, 1965; H. A. Sasaiiie and J. R. Gillette, unpublished results, 1965)) suggesting that a component in the soluble fraction of liver plays an important, but, as yet. unknown, role in S-demethylation reactions. 11. S-Oxidation
Aryl t,hio ethers, such as chlorprornazine arid 4,4’-diaminodiphenyl sulfide, are oxidized to the corresponding sulfoxide derivatives (Gillette and Kanini, 1960). Liver niicrosonies, however, convert chlorproniazine sulfoxide to other products, which are presumably hydroxylated and N-deniethylated derivatives. Thus, in vitro studies based solely on the disappearance of chlorpromazine should be interpreted with care, because they may reflect N-dealkylation and hydroxylation reactions in addition to sulfoxidation. 12. Phosphothionate Oxidation Parathion, Guthion, and a nuniber of other phosphorothionates are metabolized to potent cholinesterase inhibitors by converting phosphothionyl groups to phosphates (Davison, 1955; O’Brien, 1959; Murphy and Du Rois, 1957, 1958). Although most of the niicrosonial oxidative systenis are not found in fish and certain aniphibia (Brodie and Maickel, 1962), the enzymes catalyzing this reaction are present in a wide variety of species, representing a nuniber of phyla. Indeed, liver slices of hrook trout form as niuch paraoxon from parathion as do those of guinea pig and mouse (Potter and O’Brien, 1964; O’Brien, 1965). 13. Conversion of Thiobarbiturates 20 Oxybarbiturates
I n this reaction, a sulfur atom is replaced by an oxygen atom (Raventos, 1954; Taylor et al., 1952; Winters e2 al., 1955). For example, thiobarbital is converted to barbital in nia~i(Bush ef al., 1961). Spector and Shidenian (1959) reported that an enzyme in rat liver niicrosonies catalyzes the conversion of thiopental to pentobarbital. Since thiopental is readily oxidized to pentobarbital by peroxides, however, it is possible that pentobarbital isolated by Spector and Shideniari was not formed enzyiiiically but arose during the process of isolation (Bush et al., 1961). 14. Dehalogenation
Chlorine is removed from met hoxyfluorane, arid halot hane by enzyme systems in the 9000 x g supernatant fraction of rat liver which appear to require NADPH (Van Dyke and Chenoweth, 1965). In the presence of an
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NADPH generating system, microsomes dechlorinated the substrates several tinies faster than did the soluble fraction, thus suggesting that the enzyme is localized mainly in the endoplasmic reticulum. Glutathione did not enhance the activity of the soluble fraction; thus these compounds are apparently not dehalogenated as a result of the formation of glutathione derivatives by glutathiokinase. Renson (1964) reported that rat liver microsomes catalyze the conversion of p-fluoroaniline to p-aniinophenol and inorganic fluoride salts. This reaction is thus reminiscent of the action of phenylalanine hydroxylase on fluorophenylaniline (Kaufman, 1961, 1962) and of proline hydroxylase on trans-4-fluoroproline (see Renson et al., 1965).
15. Dealkylation of Metalloallcanes Liver microsomes catalyze the conversion of tetraethyl lead to triethyl lead (Cremer, 1959). Presumably, tetramethyl lead and tetraethyl tin are similarly dealkyled by liver microsomes. This reaction is especially significant, because the triethyl and trimethyl metabolites are more neurotoxic than their parent compounds.
C. ENDOGENOUS SUBSTRATES OF NADPH-DEPENDENT OXIDATIVEENZYMES Mammalian liver contains a host of NADPH-dependent enzymes which catalyze the oxidation of endogenous substances, some of these are discussed below. 1. Formation of Unsaturated Fatty Acids
The CoA derivatives of long-chain, saturated fatty acids are converted to unsaturated fatty acids by enzymes that require NADPH and oxygen. For example, stearyl-CoA is converted to oleyl-CoA. Though the reaction was originally detected in yeast (Bloomfield and Bloch, 1960), it is also present in inicrosomes of rat liver (Holloway et al., 1963; Bernhard et al., 1959; Marsh and James, 1962). Unlike many of the NADPH-dependent oxidative systems, however, the yeast enzyme is not blocked by carbon monoxide (W. Comirier and R. W. Estabrook, personal communication, 1965). 2. w-Oxidation
The 10,000 X g supernatant fraction of liver contains NADPHdependent enzymes which catalyze the oxidation of long-chain fatty acids, including stearate, oleate, palmitate, myristate, and laurate, to hydroxy acids and dicarboxylic acids (Preiss and Bloch, 1964). For example, stearic acid is converted to 17- and 18-hydroxystearate and octadecane-l,l& dioate; oleic acid is converted mainly to 9-octadecene-1,18-dioate;and
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palmitate is oxidized mainly to hexadecane-l,l6-dioate. There appears to be a n inverse relationship between the formation of double bonds and w-oxidation. Fresh enzyme preparations convert fatty acids mainly to unsaturated fatty acids, whereas preparations which have been frozen and thawed oxidize fatty acids mainly by w-oxidation. In similar experiments, Wakabayashi and Shimazono (1963), showed that aniides of decanoic, sorbic, and octatrienoic acids are converted by NADPH-dependent enzymes to w-hydroxy acid aniides. 3. Formation of Estrogens The aromatization of androgens to estrogens is catalyzed by an enzyme system in placenta that requires both NADPH and oxygen (Ryan, 1959). The pathway leading to estrogen forniation goes through the 19-hydroxymethyl and 19-aldehyde analogs of the androgens (Longchampt et al., 1960; Morato et al., 1961). However, the removal of the C-19 and arornatization appear to occur simultaneously, since analogs of 19-nortestosterone are not readily converted to estrogens. It would be of interest to determine whether these reactions also occur in liver microsomes. 4. Synthesis of Cholesterol A number of NADPH-dependent oxidative enzymes in liver microsomes are involved in the synthesis of cholesterol. Tchen and Bloch (1957a,b) showed that squalene is converted to lanosterol by squalene-oxidocylase I. Moreover, the 600 X g supernatant fraction of liver, comprising mitochondria, niicrosonies, and soluble fraction, catalyzes the conversion of lanosterol to cholesterol in the presence of NADPH and oxygen (Olson et al., 1957). During the reaction, C-30, C-31, and C-32 of lanosterol are converted to carbon dioxide by enzymes in niicrosonies. Presumably, the methyl groups are converted to alcohols by NADPH-dependent hydroxylases first arid then transformed by dehydrogenases to carboxylic acids, which are cleaved to form carbon dioxide. Though aldehydic or acidic intermediates have not yet been isolated, suprort for this view was obtained by showing that under anaerobic conditions microsomes convert 4-hydroxymethylene-A’cholesten-3-one-2-H3 to carbon dioxide and A7-cholenten-3@-ol-H3(Pudles and Bloch, 1960). The product, A7-cholesten-3@-ol,may be a n important intermediate in cholesterol synthesis, for it is converted first to As,’cholesten-/3-ol and then to cholesterol by enzymes present in the soluble fraction (Denipsey et al., 1964). 5. Conversion of Chdesterd lo Cholic Acid In this reaction, hydroxyl groups are introduced into the C-7a and c - 1 2 ~positions, ~ the 3P-OH is inverted to 3a-OH, the A5 double bond is reduced and the side chain is oxidized and cleaved to form a C-24 carboxyl
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group and a fragment which may be propionic acid (see Danielsson, 1963; Mitropoulos and Myant, 1965). The enzymes catalyzing these reactions are present mainly in liver mitochondria and apparently require both NAD and NADPH. The 7a-hydroxylation, however, may be catalyzed by a NADPH-dependent enzyme in liver microsomes. There is a sex difference in rats and mice in the conversion of cholesterol to bile acids. But unlike the sex difference in the oxidation of drugs by rat liver microsonies, the mitochondria of female rats and mice oxidize cholesterol faster than do those of males (Kritchevsky et al., 1963). In addition to cholic acid, mammals form a number of minor bile acids, some of which contain a hydroxyl group in the 6a- or 6p-positions (see Danielsson, 1963). 6. Metabolism of Estrogens, Androgens, and Glucocorticoids Liver microsomes contain a number of NADPH-dependent enzymes which catalyze the hydroxylation of a wide variety of steroids. For example , estradiol is hydroxylated to form 6p-hydroxyestradiol (Mueller and Rumney, 1957; Breuer et al., 1962), 6a-hydroxyestradiol (Breuer et al., 1962), 16a-hydroxyestradiol (Pangels and Breuer, 1962), and 2-hydroxyestradiol (King, 1961); in the presence of methionine, liver homogenates also form 2-niethoxyestradiol, presumably by the action of O-methyltransferase (King, 1961). Liver iiiicrosomal enzymes also convert testosterone to about twenty different metabolites, including 7a-, 6p-, and 2p-hydroxytestosterone, and metabolize A4-androstene-3,17-dioneto a t least nine metabolites, including 7a-, 16a-, 6p-, and 11p-hydroxy A4-androstene-3,17-dione (Conney and Klutch, 1963). Recently, Kuntzman and Jacobson (1965) reported that liver preparations convert progesterone to 6p- and 16a-hydroxyprogesterone and other metabolites. There are a number of similarities between the enzymes which hydroxylate steroids and those which catalyze the aliphatic hydroxylation of barbiturates. Both types are more active in male rats than in females; both are inhibited by p-diethylaminoethyl diphenylpropylacetate (SKF-525A) ; and both are enhanced by pretreatment of rats with phenobarbital and chlorcyclizine, but not by pretreatment with 3-methylcholanthrene (Kuntzman et al., 1964; Conney et al., 1965a). These findings thus suggest that the same microsornal enzynies niay metabolize steroids as well as barbiturates and certain other foreign compounds. Whether the niicrosomal enzymes convert steroids to physiologically active substances remains to be elucidated, but the wide diversity of products formed by liver microsomes suggests that the reactions are mainly catabolic. Steroid hormones may also be inactivated by reductases and other enzynies in liver. The importance of the hydroxylating enzymes
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23 1
in regulating the levels of the physiologically important steroids thus depends on the relative activities of the hydroxylases and these reductases. In female rats, progesterone, corticosterone, cortisone, and hydrocortisone are metabolized niairily by reduction reactions; thus, pretreatment of female rats with phenobarbital does not alter the rate of metabolism of these steroids by liver niicrosomes, though it modifies the pattern of the various metabolites formed (Conney el al., 1965a; Kuntznian and Jacobson, 1965). On the other hand, in guinea pigs, progesterone is apparently metabolized niairily by hydroxylation reactions; thus, pretreatment of guinea pigs with inducers, like phenobarbital, enhances the metabolism of progesterone by liver preparations (Kuntzman and Jacobson, 1965). These findings suggest that clinical tests of adrenocortical function, based solely on the urinary excrct ion of 17-hydroxycorticosteroids, may lead to erroneous intcrpretations. Pretreatment of aninials or patients with inducers, such as phenobarbital, may enhance the hydroxylation of the metabolites of adrenocortical steroids, such as cortisol, and thus lead to a decrease in nonpolar steroids in urine. Since the urinary test measures mainly nonpolar metabolites, the results of the assay would imply that the pretreatment impaired adrenocortical function (Rledsce et al., 1964; Conney t t al., 1965b). Ill. Reduction of Foreign Compounds by Enzymes in Hepatic Endoplasmic Reticulum
A. Azo REDUCTION A wide variety of azo dyes are reductively cleaved in the body to form primary aroniatic amines. The best known example of this pathway of drug metabolism is the classic discovery of Tr6fon61 et al. (1935) that prontosil is reduced to form sulfanilamide. Other well-known examples are the reductive cleavage of azobenzene (Elson and Warren, 1944) and dimethylaniiiioazobenzene (Stevenson et al., 1942). However, it would be a mistake to assume that all azo dyes arc reduced by mammalian azo reductase. Tartrazine, an azopyrazolone dye currently used in coloring foodstuffs, is reductively cleaved after oral adniinistration (Daniel, 1962), but is excreted essentially unchanged after iritraperitoneal injection (Jones, 1964). Other azopyrazolone dyes, such as l-(p-sulfophcnyl)-3-carboxy-4phenylazo-5-pyrazolone and l-phenyl-3-carboxy4-(p-sulfophenylazo)-5pyrazolone, arc reductively cleaved after oral adniinistration, but not after intraperitoneal injection. Similarly, Rarrett et al. (1965) found that 2-phenylazo-1-naphthol-4-sulfonic acid and 2-phenylazo-1-naphthol-5sulfonic acid were hydroxylated, but not reduced, following intraperitoneal adniinistration to rats. Thus, azopyrazolone dyes and certain phenylazo-
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naphthol dyes are apparently reduced by bacterial azo reductases, but not by mammalian enzymes. Liver contains NADPH-dependent enzymes which form primary amines from a variety of azo compounds, including dimethylaminoazobenzene (Mueller and Miller, 1950), prontosil, and azobenzene (Fouts et al., 1957). The enzymes, however, do not reduce appreciable amounts of 3-methyl-4methylaminoazobenzene (Mueller and Miller, 1953) or azopyrazolone dyes (Sirayavirojana, 1963). Though Fouts et al. (1957) reported that azo reductase was localized mainly in the soluble fraction, it is now known that the enzyme is mainly in the endoplasmic reticulum (J. J. Kamm, unpublished results, 1957). Relatively minor amounts of azo reductase are also present in a variety of other tissues including kidney and lung (Fouts et al., 1957). Treatment of liver microsomes with steapsin solubilizes an azo reductase (Kamm, 1964). On purifying the solubilized azo reductase about 40-fold, Hernandez et al. (1965) found that only those preparations which were high in NADPH-cytochrome c reductase could catalyze the reduction of neoprontosil. Moreover, Kamm (1964) found that highly purified preparations of porcine liver NADPH-cytochrome c reductase, obtained from Kaniin (see Williams and Kamin, 1962), could also reduce azo compounds. These findings thus strongly indicate that microsomal NADPH-cytochrome c reductase catalyzes the reduction of azo compounds in addition to the reduction of FAD (flavin adenine nucleotide) (Kamm and Gillette, 1963a), neotetrazolium compounds, 2,6-dichlorophenolindophenol,and other electron acceptors (Williams and Kamin, 1962). However, the following evidence indicates that azo compounds are reduced through additional pathways: (1) The ratio of the activities of azo reductase and cytochrome c reductase decreased after microsomes were treated with steapsin (Hernandez et al., 1965). (2) Pretreatment of rats with methylcholanthrene enhances azo reductase considerably more than it does NADPH-cytochrome c reductase (von der Decken and Hultin, 1960; R. Kato, unpublished results, 1963). (3) Though carbon monoxide does not inhibit NADPH-cytochrome c reductase, it partially blocks the reduction of Neoprontosil (S. Wright and J. R. Gillette, unpublished results, 1964). Thus, there are apparently two pathways of azo reduction in liver microsomes: one through NADPH-cytochronie c reductase and the other through P-450 (see Section IV,B).
B. NITROREDUCTION Under anaerobic conditions, nitro compounds such as chloramphenicol, p-nitrobenzoic acid, and nitrobenzene are reduced to primary amines by an enzyme system which can use either NADH or NADPH as its hydrogen
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donor (Fouts and Brodie, 1957). The enzyme is localized mainly in microsomes of liver, though it is also found in relatively minor amounts in kidney and lung. Nitroso and hydroxylamine derivatives are presumably intermediat,esin the reduction of nitro compounds, since liver microsomes reduce nitrosobenzene and phenylhydroxylamine to aniline more rapidly than they do nitrobenzene (Kamm, 1964). Thus, phenylhydroxylamino derivatives, which presumably cause methemoglobinemia and other toxicities, may be formed not only by N-hydroxylation but also by nitro reduction. Unlike most reductases, nitro reductase is active only under anaerobic conditions; indeed, it is virtually inactive in air. Part of this oxygen sensitivity, however, may be only apparent ; the phenylhydroxylamine intermediate is rapidly autoxidized in air to a nitrosobenzene derivative, which in the presence of NADPH is nonenzymically reduced back to phenylhydroxylamine. This cycle of oxidation and reduction thus leads to a depletion of NADPH, which in turn causes a cessation of nitro reduction (Kamm and Gillette, 196313). At first glance, this cycle of oxidation and reduction of phenylhydroxylamine derivatives could also occur in red blood cells and thus might be important in the formation of methemoglobin, but Kiese (1965) believes that in red blood cells the reduction of nitrosobenzene is mediated mainly by an NADPH-dependent reductase rather than by the nonenzymic react ion. Treatment of liver microsomes with pancreatic lipase destroys most of the activity of nitro reductase, but solubilizes an enzyme that in the presence of large amounts of FAD M ) reduces p-nitrobenzoate to p-aminobenzoate (Kamm and Gillette, 1963a,b). The enzyme catalyzing this reaction is probably NADPH-cytochrome c reductase, for purified preparations of this liver enzyme reduced FAD to FADHZ, and FADHz is known to reduce p-nitrobenzoate nonenzyniically. This mechanism thus explains the finding of Fouts and Brodie (1957) that large amounts of flavins stimulate the reduction of nitro compounds by liver microsomes. In the absence of added FAD, the reduction of nitro compounds by intact microsomes is not mediated through this mechanism, but is apparently mediated by another microsomal electron transport system. Carbon monoxide, which does not inhibit FAD reduction by NADPH-cytochrome c reductase, almost completely blocks the NADPH-dependent nitro reductase in mouse liver (Gillette and Sasame, 1965) and inhibits the NADPH-dependent enzyme in rabbit about 50% (Kamm, 1964). Moreover, carbon monoxide also markedly inhibits the NADH-dependent enzyme in mouse liver. Thus, both the NADPH- and the NADH-dependent systems are apparently mediated through P-450 (see Section IV,B).
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IV. Mechanisms of Oxidation and Reduction by Enzymes in Hepatic Endoplasmic Reticulum
A. MIXEDOXYGENASE MECHANISM Many oxidative enzymes in various mammalian tissues are known to require reducing agents, such as pyridine nucleotides and ascorbic acid. In general, these enzymes catalyze hydroxylation reactions. Phenylalanine is converted to tyrosine by an NADPH-dependent enzyme in the soluble fraction of liver (Kaufman, 1957). Tyrosine is converted to dopa by an adrenal enzyme which requires tetrahydropteridine derivatives (Nagatsu et al., 1964). Dopamine is converted to norepinephrine by an adrenal enzyme which requires ascorbic acid and fumarate (Levin et al., 1960). Tryptophan is oxidized to kynurenine by tryptophan pyrrolase, which is apparently activated by hydrogen peroxide (Tanaka and Knox, 1959). Methanol is converted to formaldehyde by catalase and a peroxide-generating system, such as xanthine oxidase (Tephley et al., 1961), or microsoma1 NADPH-oxidase (Gillette et al., 1957). Cholesterol is converted to pregnenolone by NADPH-dependent 20a- and 22-hydroxylases (Constantopoulos and Tchen, 196l), present in adrenals, testes, ovaries, and placenta, but not in liver (Shimizu et al., 1961). Progesterone is transformed in adrenals to a variety of corticoid hormones by the action of NADPH-dependent 17a-, 21-, 11&, and 18-hydroxylases (see Hayano, 1962). The finding that these enzymes as well as the drug-oxidizing enzymes in hepatic endoplasmic reticulum require both a reducing agent and oxygen suggests that they all may be classified as mixed oxygenases (Mason, 1957). In accord with this view, atmospheric oxygen and not the oxygen of water is incorporated in the conversion of squalene to lanosterol (Tchen and Bloch, 1957b), 1l-deoxycorticosterone to corticosterone (Hayano et al., 1955a,b), acetanilide to p-hydroxyacetanilide (Posner et al., 1961b), and trimethylamine to trimethylamine oxide (Baker and Chaykin, 1962). Unfortunately, it has not been possible to demonstrate incorporation of atmospheric oxygen into many of the other drug substrates. Oxygen in formaldehyde and other aldehydes rapidly exchanges with aqueous oxygen ; thus, 0l8studies of N-deamination, N-dealkylation, O-dealkylation, and S-demethylation would be fruitless. With the use of 0l8,however, Renson et al. (1965) disproved the possibility that O-demethylation of p-methoxyacetanilide proceeded by replacement of the methoxy group by molecular oxygen. It is also of interest that most of the reactions catalyzed by NADPHdependent oxidative enzymes in hepatic microsomes may be visualized as hydroxylation reactions (Table I).
DRUG OXIDATION AND REDUCTION BY ENZYMES
235
TABLE I HYDROXYLATION MECHANISMS IN DRUGMETABOLISM
-
Aromatic hydroxylation
[OH1
CH3CO-NH-CaH6 Aliphatic hydroxylation
CH3-CO-NH-CsH4-OH
[OH1
R-CH3 --t R-CHz-OH N-Dealkylation R-NH-CH3 O-Dealkylation R-0-CH, Deamination
PII1
[OH1
R-CH(NHS)--CHs Sulfoxidat ion R-&R’ N-Oxidation (CH8)aN
[R-NH-CHzOH]
+
LOHI
[OH1
[R--O-CHzOH]
ROH
--t
[OH1
-
ROH
+ CHZO
-
[R-C(0H) (NH+CH3]
[R-SOH-R’]+
+R-SO-R’
-
[(CHs)3NOH]+
(CHa)3NO
+ CH2O
R-CO-CHa
+ NH3
$. H+
+ Ht
According to the mixed-oxygenase mechanism, NADPH reduces a component in niicrosomes that reacts with oxygen to form a n ‘(active oxygen” intermediate. The “active oxygen” is then transferred to the drug substrate.
+ + + + + + A + HzO NADPH + Oz + drug = NADP+ + HzO + oxidized drug
1. NADPH A H+ -+ AH2 NADP+ 2. AH2 O2 + “active oxygen” 3. “Active oxygen” drug --* oxidized drug
The mechanism proposed above implies that equivalent amounts of NADPH, oxygen, and substrate are utilized in the reaction. Evidence for these stoichiometric relationships has been obtained for the hydroxylation of phenylalanine by liver preparations (Kaufman, 1957) and the hydroxylation of 17-hydroxyprogesterone by 2 1-hydroxylase of adrenal microsomes (D. Y. Cooper et al., 1963). But definitive evidence supporting the mixed-oxygenase mechanisni for hepatic microsomal systems has been difficult to obtain, because liver microsonies contain enzymes which rapidly oxidize NADPH (Gillette et al., 1957) and utilize oxygen (Hochstein and Ernster, 1963) even in the absence of drug substrate. Some investigators have reported that the addition of drug substrate stimulates the rate of NADPH oxidation by an amount which is equivalent to the amount of drug metabolized. For example, Baker and Chaykin (1962) showed that trimethylamine stimulated NADPH oxidation by an amount equivalent to the amount of trimethylamine oxide formed. Similarly, T rims and Spirtes (1965) reported that hexobarbital enhanced NADPH oxidation
236
JAMES R. GILLETTE
by an amount equivalent to the amount of hexobarbital metabolized. In our laboratory, however, we have found that the degree of stimulation of NADPH oxidation may not necessarily be correlated with drug metabolism. Sometimes the addition of a drug caused no stiniulation of NADPH (Gillette et al., 1957); other times the addition of the drug increased NADPH oxidation to a much greater extent than can be accounted for by metabolism of the drug (Gillette, unpublished results, 1962, 1963, 1965). A part of this variability may be related to peroxidation of lipids in liver microsomes. Recently, Hochstein and Ernster (1963) showed that NADPH induced the formation of malonaldehyde, which presumably arises from peroxidation of unsaturated fatty acids in phospholipids (Bernheim et al., 1948; May et al., 1965). Moreover, the addition of ironpyrophosphate chelates markedly enhanced the formation of malonaldehyde, the oxygen uptake, and the oxidation of NADPH (Hochstein et al., 1964; Orrenius et al., 1964), but the addition of codeine and aminopyrine decreased malonaldehyde formation (Orrenius et al., 1964). These findings may account for the observation that preincubation of microsomes with NADPH in the absence of drug substrate causes an impairment of the microsomal enzyme systems which does not occur when substrates, such as aniline, are present (Gillette and H. A. Sasame, unpublished results, 1964). Thus, variations in lipid peroxidation, caused perhaps by differences in the concentration of microsonial nonheme iron, would complicate studies on the stoichiometry of the drug oxidation reactions. Gillette et al. (1957) showed that hydrogen peroxide is formed during the oxidation of NADPH by microsomes in the absence of drugs. Although a t first it, seenied possible that this hydrogen peroxide niight be used by a number of nonspecific peroxidases to oxidize drugs, there are a number of facts which make it difficult to accept this view. For example, cyanide, which inhibits most heme peroxidases, does not appreciably affect the oxidation of drugs (J. R. Cooper and Brodie, 1955; Gillet8te et al., 1957; Gillette and Kamm, 1960). Moreover, a hydrogen peroxide-generating system cannot replace the requirement of NADPH in the various oxidative pathways (Gillette et al., 1957). It therefore became apparent several years ago that ‘(active oxygen” was not hydrogen peroxide, but probably was an intermediate leading to the formation of peroxide (Gillette and Brodie, 1961; Gillette, 1962, 1963a). Hydrogen peroxide, however, might be an intermediate in peroxidation of rnicrosonial lipid.
B. MICROSOMAL P-450 Liver microsonies contain a pigment, first detected by G. R. Williams (see D. Y. Cooper et al., 1965a), and later by Klingenberg (1958), Garfinkel (1958) and Omura and Sat0 (1962, 1964a,b), that is now thought to be
DRUG OXIDATION AND REDUCTION BY ENZYMES
237
important in the nietabolisni of steroids and drugs. This pigment is also present in adrenal microsonies and niitochondria (Estabrook et al., 1963; D. Y. Cooper et al., 1965a; Oniura et al., 1965a) and in microsomes of kidney and intestinal mucosa (Sato et al., 1965). Unlike cytochrome bb, the pigment is reduced about six times more rapidly by NADPH than by NADH (Omura and Sato, 1964a; Cooper et al., 1965a; H. Sasanie and J. R. Gillette, unpublished results, 1965). The pigment is unusual in that its reduced form has virtually thc same spectrum as its oxidized form. Moreover, its oxidized form does not form a complex with cyanide, whereas its reduced form readily combines with carbon monoxide to forin a coniplex having an absorption niaximurii at 450 nip and a niinimuni at about 405 mp. Indeed, its presence in niicrosonies may be detected only in a n atmosphere of carbon monoxide. For this reason, it has been named P450. Under anaerobic conditions, the reduced form of 1’450 avidly binds carbon inonoxide ; Oinura et al. (1965a) found the dissociation constant to be about 2 X 10-7 M . Nevertheless, under aerobic conditions the concentration of carbon monoxide must be several orders of magnitude greater than 10-7 M before the absorption band a t 450 nip appears (Omura and Sato, 19f34a). Thus, in the absence of carbon monoxide, but in the presence of air and NADPH, P 4 5 0 must exist mainly in the oxidized form or as a complex with oxygen. Reduced P-450 (CO)
NADPH
co
reduced P-450
T
+ P-450 reductase
or
reduced P-450 ( 0 2 )
?I
oxidized P-450
In adrenal niicrosoines, carbon monoxide does not block endogenous NADPH oxidation, suggesting that the P 4 5 0 ( 0 2 ) coniplex is stable (Estabrook et al., 1963). In contrast, H. A. Sasanie in our laboratory (unpublished results, 1964) found that high concentrations of carbon monoxide block NADPH oxidase of mouse liver niicrosonies as much as 80%; this suggests that liver microsonies contain endogenous substrates or that considerable amounts of P 4 5 0 may exist in the oxidized form and that the rate-limiting step may be the reduction of P450. In accord with the latter view, Sasanie showed that in an atmosphere containing oxygen (15y0) and carbon monoxide (3Oy0), the absorbance of 450 nip was greater when NADPH was used as a reducing agent than when NADH was used, whereas in an atmosphere of carbon monoxide (lOOOj,), the absorbance was the same whether NADPH or NADH was used as the reducing agent (Table 11). The finding of Ryan and Engel (1957) that carbon monoxide blocked the C-2 1 hydroxylation of 17-hydroxyprogesterone by adrenocortical niicrosonies suggested to Estabrook et al. (1963) that P 4 5 0 might be a
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JAMES R. QILLETTE
TABLE I1 P-450-CO COMPLEX IN MOUSELIVERMICROSOMES AFTER REDUCTION WITH NADH AND NADPH Atmosphere
30% C0-15% 02 0.D: 450490 mb NADH NADPH 4
0.170 0.320
100%
co
0.D: 450-490 mp 0.780 0,780
O.D. = optical density.
component of the 21-hydroxylase system. Accordingly, they showed that the inhibitory effects of carbon monoxide on this system could be reversed more effectively by monochromatic light having a wavelength of 450 mp than by light of other wavelengths. In subsequent studies, Estabrook and co-workers have solubilized and purified components of the 1lp-hydroxylase system in adrenal mitochondria, and have shown that P-450 is also the terminal oxidase of this enzyme system (Omura et al., 1965a,b). Presumably, the reduced P 4 5 0 functions in these enzyme systems by reacting with oxygen to form “activated oxygen” which in turn is transferred to the steroid substrate. There is now considerable evidence favoring the view that P 4 5 0 is also a component of the electron transport system in liver microsomes which forms “active oxygen.” Treatment of liver microsomes with deoxycholate, steapsin, or the venom of Trimeresums jlavoviridis, which inactivated NADPH oxidase and the drug-metabolizing enzymes (Gillette el al., 1957; Gillette, 1963a; Posner et al., 1961a), changes the maximum of the carbon monoxide complex from 450 mp to 420 m p (Omura and Sato, 1964a,b). Moreover, cutscum (isooctylphenoxypolyethoxyethanol),which clarified liver microsomes, decreases both the activity of the drug-metabolizing enzymes and the absorption peak at 450 mp about 30% (J. J. Kamm, H. A. Sasame and J. R. Gillette, unpublished results, 1963, 1964). Carbon monoxide inhibits the metabolism of drugs by microsomes of various species. For example, it inhibits the microsomal enzymes that catalyze the N-demethylation of 3-methyl4-monomethylazobenzene (Conney et al., 1957), aminopyrine (Orrenius et al., 1964), and monomethyl-4-aminoantipyrine1 the O-demethylation of codeine, and the hydroxylation of acetanilide (D. Y . Cooper et al., 1965b). It also inhibits the enzymes in rabbit liver which catalyze the deamination of amphetamine (H. A. Sasame and J. R. Gillette, unpublished results, 1965), the hydroxylation of aniline and N-ethylaniline, and the N-dealkylation of N-ethylaniline, but not the enzyme which catalyzes N-hydroxylation of aniline and
239
DRUG OXIDATION AND REDUCTION BY ENZYMES
N-ethylaniline (Kampffmeyer and Kiese, 1965; Kiese, 1965). In our laboratory, H. Sasame and I have found that carbon monoxide inhibits microsonial enzynies in mouse liver which catalyze N-demethylation, O-demethylation, S-dernethylation, aromatic hydroxylation, and aliphatic hydroxylation reactions, but not the sulfoxidation of diaminodiphenyl sulfide (Table 111).We also found that carbon monoxide blocked the reducTABLE 111 INHIBITORY EFFECTSOF CARBON MONOXIDEON METABOLISM OF DRUGS BY MOUSELIVER MICROSOMES ~
_
_
_
_
Atmosphere
8 u bstrate
Reaction
GO
02
(%)
(%I
CO/O2 2 2 2 2 2
~~
p-Nitroanisole p-Acetanisidine Aminopyrine N-Methylaniline p-Nitrotoluene Aniline 3,4-Benepyrene Hexobarbi t a1 Chlorpromaeine Chlorpromaeine Diaminodiphenyl sulfide 6-Methylmercaptopurine 6-Methylmercaptopurine riboside 0-Ethyl 0-(4-nitrophenyl) phenylphosphonothionate Amphetaminea 5
Inhibition
(%)
~~
77
O-Demethylation O-Demethylation N-Demethylation N-Demethylation Aliphatic hydroxylation Aromatic hydroxylation Aromatic hydroxylation Aliphatic Oxidation N-Dealkylation Sulfoxidation Sulfoxidation S-Demethylation S-Demethylation
30 30 30 30 30 30 30 50 50 50 50 50 50
15 15 15 15 15 15 15 10 10 10 10 10 10
2 2 5 5 5 5 5 5
Desulfuration
50
10
5
28
Deamination
50
10
5
91
43 54 45 54 50 69 47 63 61 0
49 51
Rabbit liver microsomes.
tion of p-nitrobenzoate and, to a lesser extent, the reduction of neoprontosil (S. Wright and J. R. Gillette, unpublished results, 1964). Moreover, D. Y . Cooper et al. (1965b) showed that light of 450 nip reverses the inhibitory effects of carbon monoxide on the iiietabolisiii of codeine, nionomethyl-4aminoantipyrine, and acetanilide. These findings thus indicate that P-450 plays a major role in the oxidation of many drugs, and in the reduction of azo and nitro compounds, but apparently not in N-oxidation and certain S-oxidation reactions. Since NADH slowly reduces P-450, it seemed possible that P-450 might mediate a t least a part of the NADH-dependent drug-oxidizing systems in liver microsonies. H. Sasanie in our laboratory found that carbon monoxide
240
JAMES R. GILLETTE
blocked NADH-dependent aniline hydroxylation almost as much as it did the NADPH-dependent reaction, though in the absence of carbon monoxide the reaction was considerably slower in the NADH system than in the NADPH system (Table IV). Similarly, carbon monoxide blocked the reducTABLE IV
EFFECTS OF CARBON MONOXIDEON ANILINEHYDROXYLATION BY
MOUSELIVERMICROSOMES
p-Arninophenol formed (mfimole/mg microsomal protein/lO min) Inhibition Cofact or
30% C0-15% Oa
NADH NADPH
3.73 7.14
15% 0%
7.14 19.8
(%I 48 64
tion of p-nitrobenzoate by NADH almost as effectively as it blocked nitro reduction by NADPH, though again, in the absence of carbon monoxide, the reduction was slower in the NADH system than in the NADPH system. These findings further suggest that the rate-limiting step in the NADH system may be the reduction of P-450.
C. PA50 REDUCTASES The possibility that NADPH-cytochrome c reductase might be a component of the microsomal oxidizing systems was first suggested by the finding that cytochrome c and other electron acceptors inhibited drug metabolism by liver microsomes. In the current view, NADPH-cytochrome c reductase acts by reducing P-450 either directly or indirectly through an unidentified carrier. Evidence for this view rests on the following indirect evidence : (1) Microsomal NADPH-cytochrome c reductase in liver endoplasmic reticulum has no known function, because cytochrome c is not present in these organelles. (2) Pretreatment of rats with phenobarbital increases the activity of NADPH-cytochrome c reductase in addition to enhancing the drug-metabolizing enzymes in microsomes (Remmer and Merker, 1963, 1965a; Orrenius and Ernster, 1964; Kamm, 1964). (3) On treatment of liver microsomes with various concentrations of steapsin at O'C, the per cent of NADPH-cytochrome c reductase retained in the microsomes was approximately the same as the per cent of the P-450 reductase and aniline hydroxylase retained (Sato et al., 1965). (4) NADP, but not NAD, inhibits the NADPH-dependent oxidation of chlorpromazine, monomet hyl-4-aminoantipyrine , naphthalene, and p-nitroanisole (J. Booth, J. J. Kamm, J. R. Gillette, unpublished results, 1961; see
DRUG OXIDATION AND REDUCTION BY ENZYMES
241
Gillette, 1962, 1963a), suggesting that, like NADPH-cytochrome c reductase, NADPH-P-450 reductase is very specific. Acceptance of the view that P-450 is reduced by NADPH-cytochrome c reductase has been hampered by an apparent lack of correlation between the kinetic constants of NADPH-cytochrome c reductase and those of the drug oxidation systems. For example, Ernster and Orrenius (1965) reported that the apparent K , (NADPH) for the metabolism of aminopyrine by rat liver microsomes was about 2.5 x lop5M , whereas Phillips and Langdon (1962) found that the K , (NADPH) of purified NADPH-cytochrome c reductase was about 1 X M . Moreover, Ernster and Orrenius (1965) reported that the K I (NADP) for the demethylation of aminopyrine was about 2.8 X lo-’ M , whereas Phillips and Langdon (1962) found that the Kr (NADP) of NADPH-cytochrome c reductase was about 1 x 10-6 M . Discrepancies such as these, however, may be caused by inadequate NADPH-generating systems or by enzymes in liver microsomes that destroy NADPH (see Section IV,F). Accordingly, Gillette and Sasame (1966) found that the apparent K , (NADPH) for the oxidation reactions in rat liver microsomes varied with the enzyme concentration in the assay system and the time of incubation. When systems containing 1.0 mg of microsomal protein from rat liver were incubated for 10 minutes, the apparent K , (NADPH) for the hydroxylation of 3,4-benzpyrene was about 6X M . At lower microsomal concentrations (0.1 mg/ml), however, the apparent K , (NADPH) for the reaction was about 2.6 X lo-’ M and that for cytochrome c reduction was about 3.5 X lo-’ M . The high apparent K , (NADPH) value for the hydroxylation reaction obtained at high microsomal concentrations is presumably caused by NADPH pyrophosphatase, which lowers the effective concentration of NADPH. The NADPH-cytochrome c reductases of liver microsomes and adrenocortical mitochondria are apparently different enzymes. Omura et al. (1965a,b) have isolated three different components from adrenocortical mitochondria : one is a flavoprotein, which the authors have designated Y-2 ; another is an iron-containing protein, designated nonhenie iron protein (NHI), which has spectral characteristics similar to ferredoxin; and the third is P-450. In the presence of NADPH, the flavoprotein (Y-2) reduces dichlorophenolindophenol, but not cytochrome c nor P-450. Addition of NHI to the flavoprotein forms a system, however, which catalyzes the reduction of dichlorophenolindophenol, cytochrome c and P450. These authors also showed that in the presence of NADPH, cortexone was hydroxylated in the 1ID-position when all three components were present, but not when either P-450 or NHI was omitted. The NADPH-cytochrome c reductase of liver microsomes has been solubilized by various workers (Horecker, 1950; Williams and Kamin.
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JAMES R. GILLETTE
1962; Kamm and Gillette, 1963a; Nishibayashi el al., 1963; Phillips and Langdon, 1962; Hernandez et al., 1965). Purification of this enzyme system revealed that NADPH-cytochrome c reductase activity was associated with a single protein, which can also reduce ferric ions, ferricyanide, tetrazolium compounds, menadione, FAD, and azo compounds. The purified enzyme, however, does not catalyze the reduction of the P-420 form of P450 isolated from liver niicrosomes (Omura and Sato, 1964a,b; Sato et al., 1965) or the P-450 isolated from adrenocortical mitochondria (Omura et al., 1965a). If NADPH-cytochrome c reductase is a component of P-450 reductase, it must catalyze the reduction of several molecules of P450, because Mason et al. (1965) have shown that the concentration of flavins in smooth-surfaced microsomes of rabbit liver is less than a tenth of the concentration of P450. Since the NADPH-dependent oxidative systems in microsomes are not blocked by NAD, it seems likely that NADH reduces P450 by another reductase. Probably, the NADH-dependent P-450 reductase is a combination of NADH-cytochronie bs reductase and cytochrome bg, but definitive evidence for this view is lacking. D. MECHANISMS OF “ACTIVEOXYGEN” TRANSFER
A number of workers have pointed out that P-450 differs from other cytochroines in a number of ways. For example, in its native form, PA50 has no a- or 0-bands, though P-420, its denatured form, has absorption maxima at 559, 530, and 427 mp in the reduced state and 414 mp in the oxidized form (Omura and Sato, 1964b). Since the native form of P-450 in adrenal microsomes is converted to P-420 by sulfhydryl reagents (D. Y. Cooper et al., 1965b), these spectral differences are apparently associated with -SH groups or components that complex with --SH groups. Moreover, the addition of carbon monoxide to P-450 causes the disappearance of an absorption band having a maximum at a wavelength (405 mp) which is shorter than the wavelength of the band that appears at 450 m p ; whereas the action of carbon monoxide on other hemoproteins, such as cytochrome oxidase, horseradish peroxidase, and hemoglobin, causes disappearance of bands at wavelengths that are longer than the absorption bands which are formed (Omura et al., 1965a). Mason et al. (1965) have suggested that these unusual properties of P450 may be caused by interaction of the heme-CO complex with other components, such as flavin, heme, nonheme iron, copper, or manganese. Though these authors favor the view that the other component is copper, the evidence thus far obtained is not conclusive. Since there are about two molecules of P-450 heme per atom of copper in smooth-surfaced microsomes of rabbit h e r , these authors have proposed that the functional unit of
DRUG OXIDATION AND REDUCTION BY ENZYMES
243
P-450 comprises two molecules of P-420 and one niolecule of the copperprotein complex. It seems just as likely, however, that the functional unit consists of P-420 and a rionheine iron protein or a complex between two molecules of P-420. Whatever the composition of the functional unit may be, the concept that it consists of two reducible groups is attractive, for such a relationship could account for the formation of “active oxygen” without implicating the formation of hydroxyl and hydroxoniuin free radicals.
E. MECHANISMS OF HYDROXYL.4TION BY MODELSYSTEMS Studies with model systems have been undertaken as an aid in elucidation of the mechanisms of the microsonial enzyme systems. Fenton’s reagent, which consists of hydrogen peroxide and ferrous iron, produces free radicals that can hydroxylate aromatic compounds (Stein and Weiss, 1950). Udenfriend et al. (1954) added verserie and ascorbic acid to the hydrogen peroxide-iron system, and found that this modified Fenton’s reagent hydroxylated aromatic compounds a t a greatly increased rate. The system differs from Fenton’s reagent in that oxygen may also serve as the oxidizing agent, although the rate of hydroxylation is relatively slow. At least three oxidative reactions that are catalyzed by the liver inicrosoines occur in this system : hydroxylation of aromatic compounds, O-dealkylation of aroniatic ethers (Udenfriend el al., 1954; Brodie et al., 1954), and N-deniethylation of 4-N-dirnethylaminoazobenzene (Hanaki and Ishidate, 1962). The N-demethylation reaction may proceed through formation of N-oxides (Terayania, 1963). Barbiturates, alkylamines, and primary amines are also oxidized, but the expected end products are unstable in the reaction mixture (Brodie el al., 1954). It was first thought that the system hydroxylated aromatic compounds only in electronegative positions of the aromatic ring and that the reaction occurred by a n ionic mechanism. Thus, Udenfriend et al. (1954) postulated HOf as the “active oxygen” and Mason and Onoprienko (1956) proposed FeO(OH),. The finding that hydroxyl groups are also introduced into other positions, however, suggested to Breslow and Lukens (1960) that the ‘Lactiveoxygen” was probably a free radical rather than a positive ion. In accord with this view, Staudinger et al. (1965) found that the hydroxylation of acetanilide by the Udenfriend system was blocked by radical trapping agents, such as sulfite. Staudinger et aE. (1965) presented evidence indicating that the Udenfriend system (Os/Fe+++/ascorbate/EDTA) hydroxylated acetanilide by the action of both hydroxyl free radicals (HO.) and hydroxoniuin free radical (HO,.). They found that Fenton’s reagent, which produces mainly HO. radicals, hydroxylated acetanilide equally well in the o- and p-positions but very little in the m-position (o-, 45.501,;m-, 2.5y0; p - , 52a/0) whereas a
244
JAMES R. GILLETTE
system consisting of Ti+++ and 02,which forms only HOT free radicals, hydroxylated acetanilide predominantly in the 0- and m-positions (0-,40% ; m-,4oyO; p-, 20y0).The relative amount of the m-hydroxyacetanilide formed in the Udenfriend system is between these extremes; thus, both HO. and HOz. niust participate in the Udenfriend system. These findings are thus in accord with the prediction of Diner (1964) that the relative amounts of the various products produced by hydroxoniuni free radicals would differ from the amounts formed by hydroxyl free radicals. Staudinger and co-workers (see Staudinger et al., 1965; Staudinger and Ullrich, 1964) have also shown that various coenzymes, enzymes, and cytochromes can replace components of the Udenfriend system. For example, hydroxylation of acetanilide occurred in the following systems : (1) 02, Fesft, EDTA, NADH or NADPH, and FAD, or F M N (flavin mononucleotide) (2) 02, Fe+++, EDTA, NADH, and cytochrome bg ascorbic acid, and cytochrome bs. (4) 0 2 , NADH, cytoreductase. (3) 02, chrome b, reductase, and cytochrome b,. However, the relative amounts of the products formed in systems 1 and 2 resembled the pattern formed in the Udenfriend system, whereas the relative amounts of the products formed in systems 3 and 4 resembled the pattern produced by HOz. generating systems. It seems unlikely, however, that these free radical model systems represent the major hydroxylase mechanisms of drug metabolism in intact liver microsomes. Rat liver microsomes hydroxylate acetanilide almost exclusively in the p-position; thus, the pattern of metabolites does not parallel either of the patterns produced by free radicals. Moreover, Nilsson et al. (1964) found that the luminescence produced by luminol in the presence of NADPH and liver microsomes was not blocked by carbon monoxide; hence, free radicals, produced by liver microsomes, are probably associated with the radical form of NADPH-cytochrome c reductase (see Masters et al., 1965; Kamin et al., 1965) rather than the P-450-oxygen complex. These observations, however, do not preclude the possibility that very short-lived radicals or atomic oxygen arising from the P-450-oxygen complex are transferred t o enzyme-bound substrates. Indeed, quantum-mechanical calculations indicate that the substrates as well as oxygen must be activated to account for the various metabolites formed by the liver microsomal enzyme systems (Diner, 1964).
F. INHIBITORS OF THE OXIDATIVE AND REDUCTIVE ENZYME SYSTEMS IN HEPATIC ENDOPLASMIC RETICULUM The metabolism of drugs by liver microsomes may be inhibited in a number of different ways. Hepatic homogenates contain a host of enzymes
DRUG OXIDATION AND REDUCTION BY ENZYMES
245
which limit the concentration of reduced coenzymes or alter components of the drug-metabolizing systems. Some substances added to incubation mixtures may alter the steady-state concentration of the “active oxygen” complex by serving as electron acceptors or by inactivating either P-450 or PA50 reductase. Other substances may interfere with the formation of enzyme-drug complexes. Many of these mechanisms of inhibition are discussed below. 1. Enzyme Systems That Limit the Concentration of Reduced NADPH and NADH
Undialyzed preparations of the 9000 X g supernatant fraction of liver from well-fed animals contain a number of enzyme systems which maintain NADP in its reduced form; indeed, the activity of the drug enzyme systems in these preparations is seldom enhanced by the addition of isocitrate (Clouet, 1964) or glucose-6-phosphate (Roth and Burkovsky, 1961). Dialyzed preparations or preparations from starved or morphine-treated animals, however, must be supplemented with isocitrate or glucose-6phosphate to achieve maximal activity of the oxidative drug-metabolizing enzyme systems in liver niicrosonies. When glucose-6-phosphate is used as the substrate of NADPH-generating systems, it should be added in considerable excess, because it is rapidly hydrolyzed by the glucose-6phosphatase present in liver microsomes. Other enzymes in hepatic microsonies block the oxidative enzyme systems by destroying NADP and NADPH. NADase hydrolyzes NADP (Zatman et al., 1953) but not NADPH (Hofmann and Rapoport, 1955) to form nicotinamide and 2’-phosphoadenosine-5‘-diphosphoribose (PADPribose). When NADPH-generating systems are used to maintain NADPH in incubation mixtures, it is necessary to add nicotinamide, a compound which blocks the action of NADase (Mann and Quastel, 1941). However, nicotinaniide cannot be added with impunity, for in high concentrations (10-2 M ) it inhibits the oxidative drug enzyme systems in niicrosonies (J. Booth and J. R. Gillette, unpublished results, 1961). Moreover, when preformed NADPH is used in the absence of a generating system, it may be desirable to omit nicotinamide to prevent the accumulation of NADP, since NADP is known to inhibit drug metabolism (see Gillette, 1962; 1963a; Ernster and Orrenius, 1965). This is not certain, however, for the possibility that PADP-ribose and 2’-AMP (adenosine monophosphate) may also inhibit drug metabolism (see Phillips and Langdon, 1962) has not been investigated. The liver of various species contains pyrophosphatases that cleave NAD and NADP to nicotinamide mononucleotide (NMN) and NADH and NADPH to reduced nicotinamide mononucleotide (NMNH) (Jacobson
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and Kaplan, 1957a,b; Gillette et al., 1963). Phosphatases in liver then remove phosphate from NMN and NMNH to form the respective pyridine ribosides (Jacobson and Kaplan, 1957b; Gillette et al., 1963). Since the extinction coefficients of NMNH and reduced pyridine riboside (PRH) at 340 mp are identical to that of NADPH, the absorbency of incubation mixtures at this wavelength may reflect the presence of NMNH and PRH as well as NADPH. Thus, NADPH in these preparations is assayed by measuring the decrease in absorbency caused by the reduction of oxidized glutathioiie by glutathione reductase. The activity and the distribution of the pyrophosphatases that catalyze the hydrolysis of pyridine dinucleotides varies widely from one species to another. Jacobson and Kaplan (1957a,b) found that the NAD and NADH pyrophosphatases are about six times more active in rat liver homogenates than in rabbit liver homogenates. Moreover, the pyrophosphatases are localized mainly in the microsomal and nuclear fractions of liver homogenates of rats, mice, and hamsters, but in the soluble and nuclear fractions of rabbit liver homogenates. Similarly, Gillette et al. (1963) found that NADP and NADPH are hydrolyzed much more rapidly by rat liver homogenates than by rabbit liver homogenates, and showed that the NADP and NADPH pyrophosphatases of rat liver are localized mainly in nuclei and microsomes. Though destruction of NADPH by the pyrophosphatases may be important in incubation systems containing preformed NADPH and NADH, it may not necessarily be important when NADPH-generating systems or the soluble fraction of liver are used. Undialyzed preparations of the 9000 X g supernat,ant fraction of rat liver contain AMP, ADP, and ATP, which partially inhibit the pyrophosphatases. Moreover, the K , of the M which may NADPH pyrophosphatases of rat liver is about 3.5 X be as much as one or two orders of magnitude higher than the K , (NADPH) of the oxidative microsomal drug enzyme systems, such as aniline hydroxylase. Thus, the pyrophosphatases are rather ineffective at the relatively low concentrations of NADPH required by these drug enzyme systems. Nevertheless, they are sufficiently active to play havoc with the determination of the K , (NADPH) of the oxidative drug enzyme systems (see Section IV,C). Similarly, they may partially account for the inability of low concentrations of NADPH to reduce P-450 fully. 2. Inhibition of Drug Metabolism by Stimulation of NADPH Oxidation
Since the ateady-state level of NADPH in incubation mixtures depends not only on its formation by NADPH-generating systems, but also on its rate of oxidation by NADPH-oxidases, the addition of any substance which enhances its oxidation would be expected to decrease the metabolism
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of drugs. Accordingly, R. Kato in our laboratory (unpublished results, 1963) found that rat liver mitochondria, which rapidly oxidize NADPH and NADH via cytochronie oxidase, inhibited the metabolism of drugs by rat liver microsomes, and that the inhibition could be reversed by the addition of cyanide. Similarly, Krisch and Staudinger (1961) showed that cyanide, which inhibitled NADH oxidation by rat liver microsomes, enhanced the hydroxylation of acetanilide by the NADH-dependent system in rat liver microsomes. The addition of catalytic amounts of cytochrome c to microsomal preparations inhibits the metabolism of drugs (Gillette et al., 1957; J. R. Cooper and Brodie, 1955, 1957; Gillette and Kamm, 1960; Hucker et al., 1960; Krisch and Staudinger, 1961). Cyanide reverses this inhibition presumably by inhibiting the cytochronie c oxidase in mitochondria, which invariably contaminate microsomal preparatJions (Gillette et al., 1957). These reactions are shown as follows: 2 NADPH
microsomal NADPH + 4 ferricytochrome c cytochrome c reductase t
4 ferrocytochrome c
4 Ferrocytochrome c
+ O2 + 4 H+
-
+ 2 NADP+ + 2 H+
cytochrome oxidaee
4 ferricytochrome c
+ 2 HzO
Other electron acceptors which are reduced by microsomal NADPHcytochrome c reductase and hence inhibit the oxidation of drugs are methylene blue, 2,6-dichlorophenol-indophenol1menadione (Gillette et al., 1957), riboflavin, FMN, FAD (Kanim and Gillette, 1962), tetrazolium compounds (Williams and Kamin, 1962; Harper and Calcutt, 1961). However, ferric-pyrophosphate complexes, which are slowly reduced by NADPHcytochronie c reduct,ase, do not appreciably inhibit drug metabolism (Orrenius et al., 1964). The electron acceptors that inhibit drug metabolism may act not only by decreasing the level of NADPH in the incubation mixture, but also by decreasing the rate of reduction of P450. Accordingly, the addition of large amounts of glucose-6-phosphate and glucose-6-phosphate dehydrogenase does not completely reverse the inhibitory effects of cytochrome c, methylene blue, or tetrazolium compounds (J. R. Gillette and H. A. Sasame, unpublished results, 1964).
3. Inhibitors of P-460 Reductase
2,6-Dichloro-6-phenoxyethylamine(DPEA) apparently blocks microsoma1 drug enzymes by inhibiting the reduction of P-450 by NADPH and NADH. Since the inhibitor does not inhibit either NADH-cytochrome c
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reductase or NADPH-cytochrome c reductase, but blocks the reduction of P-450 by dithionite, it apparently acts by combining with P-450 and not with the reductase (H. A. Sasame and J. R. Gillette, unpublished results, 1965). Accordingly, addition of DPEA to microsomes in the absence of NADPH causes the formation of a peak at 430 mp, though it does not alter the spectrum of P-450-carbon monoxide complex, formed on the addition of NADPH. 4. Inactivators of P-450 It has long been known that NADPH oxidase and the drug-metabolizing enzymes in liver microsomes are inactivated by metallic salts of mercury, copper, and silver, by treatment with detergents, such as digitoxin, potassium cholate, and sodium lauryl sulfate (Gillette et al., 1957), or by treatment with enzymes, such as trypsin, pancreatic lipase (Gillette, unpublished results, 1955; Posner et al., 1961a), and the venom of Trimeresums JEavoviridis (see Gillette, 1962, 1963a; Sat0 et al., 1965). It now seems likely that many of these treatments destroy microsomal enzymes, at least in part, by inactivating P-450. Omura and Sato (1962, 1964a,b) showed that treatment of microsomes with deoxycholate, steapsin, or venom of Trimeresurus JEavoviridis changes P-450 to a form which in its reduced form combines with carbon monoxide to form a complex having an absorption maximum at 420 mp (see Section IV,B). Moreover, Narasimhulu et al. (1964) reported that addition of sulfhydryl reagents to adrenocortical microsomes also converts P450 to this form (P-420). Further, Mason et al. (1965) found that addition of bathocuproine sulfonate, which preferentially reacts with cuprous ions, enhances the conversion of P-450 to P-420. Preincubation of liver microsomes with NADPH in air also inactivates the drug-metabolizing enzymes. Recently, H. A. Sasame in our laboratory (unpublished results, 1964) observed that the decrease in the activity of the oxidative enzymes in mouse liver microsomes paralleled a decrease in P-450. In this system, however, there was no apparent formation of P-420. Addition of substrates, such as aniline or hexobarbital, prevents the impairing effects of air (H. A. Sasame and J. R. Gillette, unpublished results, 1964), possibly by preventing lipid peroxidation (Orrenius et al., 1964). Like air, JB-516 (p-phenylisopropylhydrazine) apparently inhibits the drug-metabolizing enzymes in mouse liver microsomes by destroying P-450 without the formation of P-420. Preincubation of SKF-525A (p-diethylaminoethyl diphenylpropylacetate) with mouse liver microsomes in the presence of air and a NADPHgenerating system irreversibly inactivates the oxidative drug-metabolizing enzymes and nitroreductase by altering P-450 in still another way (J. R. Gillette and H. A. Sasame, 1964, 1965). During the preincubation with
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SKF-525A, P-450 was converted to a substance which had a n absorption maximum a t 450 mp even in the absence of carbon monoxide. Since the relative amount of P-450 converted to this form paralleled the per cent of inhibition of nitro reductase, it appears that this form of P-450 is inactive. This form of P-450 was also obtained by preincubation of mouse liver microsomes in air with a NADPH-generating system and P-monoethylaminoethyl diphenylpropylacetate (the monoethyl analog of SKF-525A), 2,4-dichloro-6-phenylphenoxyethyl diethylamine (Lilly 18947), 2,4-dichloro-6-phenylphenoxyethyldimethylamine, but not with 2,4-dichloro-6phenylphenoxyethylamirie, the primary amirie analog of Lilly 18947 (see Section IV,E,3) (H. A. Sasame arid J. R. Gillette, unpublished results, 1964). The similarity of the spectrum produced in the presence of these inhibitors with that caused by carbon monoxide suggests that the inhibitors may cause the formation of carbon monoxide, but this has not been adequately investigated. 5. Competitive Inhibitors of Drug Metabolism The N-deiiiethylation of Ai-niethylbutynaniine by rat liver niicrosonies is competitively inhibited by a number of conipounds including SKF-525A, deniet hylmeperidine, Lilly 18947, and DI'EA, the primary aniine analog of Lilly 18947 (McMahon arid Mills, 1961; McMahon, 1962). Similarly, Rubin et al. (1964) have found that the N-deiiiethylation of ethylmorphine by rat liver niicrosonies is inhibited by hexobarbital, chlorpromazine, zoxazolaniine, phenylbutazone, and acetanilide and that ethylmorphine conipetitively inhibits the metabolisni of chlorproniazine. Moreover, these workers reported that the inhibitory constant of hexobarbital in the deniethylation of ethylmorphine was virtually the same as the K , of hexobarbital, and that the inhibitory constant of ethylniorphine in the metabolism of chlorpromazine is virtually the same as the K , of ethylmorphine. These findings lend support for the view that hexobarbital, chlorproniazine, and ethylmorphinc are metabolized by the same enzyme, but, as the authors point out, the data should not be regarded as conclusive. Competitive inhibition could occur even when there are several enzyme systems, if the systenis have a conin~oninterniediate, such as P-450 or P-450 reductase. In accord with the latter view, the Michaelis constants of the substrates of the oxidative enzyme systems do not always correspond to their inhibitory constants in the metabolism of other substrates. Rubin et al. (1964) found, for example, that the K , for chlorpromazine was three times higher than its K I in the metabolism of ethylniorphine. In similar experiments with rabbit liver niicrosonies, aniinopyrine was found to competitively inhibit the O-dealkylation of p-nitroanisole, yet there was no correlation between the Kr and the K , of aminopyrine in these reactions
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(J. Booth and J. R. Gillette, unpublished results, 1961). Moreover, despite the fact that the N-demethylation of ethylmorphine by rat liver microsomes is competitively blocked by zoxazolamine and acetanilide, there is considerable evidence that morphine and acetanilide are metabolized by different enzymes (Kato and Gillette, 1965a,b) (see Section IV,F). There is a species difference in the effects of inhibitors. For example, SKF-525A blocks the demethylation of aminopyrine competitively when rat liver microsomes are used in incubation mixtures (R. Kato, H. A. Sasame and J. R. Gillette, unpublished results, 1964) but noncompetitively when rabbit or mouse liver microsomes are used (La Du et al., 1954; La Du, 1962; R. Kato, H. A. Sasame and J. R. Gillette, unpublished results, 1963). It would therefore be a mistake to assume that substances which competitively inhibit oxidative enzyme system in one species also competitively inhibit these enzyme systems in others.
6. The h’flect of Binding in Studying the Kinetics of Drug Metabolism Binding of a drug to intracellular components in incubation mixtures lowers its free concentration and thus decreases its rate of metabolism. For example, about 60% of the imipramine added to 10% liver homogenates is bound to nuclei and mitochondria, and about 3ooj, is bound to microsomes (Dingell et al., 1961; Gillette, 1962, 1963a, 1965). The binding of imipraniine to mitochondria and nuclei partially explains why this drug is metabolized more slowly by whole homogenates than by 9000 X g supernatant fractions of liver. Binding of a drug to niicrosonies may also cause a n apparent decrease in the activity of an enzyme system as the concentration of the enzyme is increased. For example, the rate of metabolism of irnipramine is proportional to the enzyme concentration only a t very low concentrations of niicrosonies. Since imipraniine is bound very tightly to microsomes, however, it occurred to us that the apparent inhibit,ion obtained with high enzyme concentrations may partially be caused by a decrease in the free concentration of the drug. Accordingly, H. A. Sasanie in our laboratory (unpublished results, 1962) showed that the apparent Michaelis constant increased as the concentration of the enzyme is increased. Thus, the kinetics of the inhibition resembled those of an endogenous competitive inhibitor. When the free concentration of the drug was measured, however, it was found that the Michaelis constants obtained at high and low enzyme concentrations were the same. Thus, most of the inhibitory effect of high enzyme concentration on the metabolism of imipramine may be attributed to a decrease in the free concentration of the substrate (see Gillette, 1963a, 1965). A number of other substrates commonly used in studying the drug-
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metabolizing enzymes are also highly bound to microsomes. Among these are chlorproniazine and other phenothiazines, atabrine, thiopental, chloroquine, SKF-525A, and Lilly 18947 (see Gillet,te, 1965). Thus, unless corrections are made for binding, the values for the Michaelis constants determined for the metabolism of these substrates are wrong, particularly when large amounts of microsomes are used in the incubation mixtures. Reversible binding of substances to microsomes may also complicate the study of inhibitors of the drug-metabolizing enzymes. When the ratio of unbound inhibitor to bound inhibitor remains constant as the concentration of the inhibitor is increased, the Dixon plot of the data (l/u vs I) may be linear, but the apparent Kr will vary with the concentration of the microsomes (see Gillette, 1965). On the other hand, when the ratio of unbound inhibitor to bound inhibitor increases as the inhibitor concentration is increased, the Dixon plot will curve upward, which differs from the usual types of inhibition (see M. Dixon and Webb, 1958; Webb, 1963). Furthermore, the inhibitor may be so tight,ly bound to microsomes that it is not readily removed by dialysis techniques and thus suggest irreversible inhibition; yet, kinetic experiments may still provide data suggesting that the inhibitor acts competitively. For example, SKF-525A and DPEA are very tightly bound to rat liver niicrosomes and are not easily removed by prolonged dialysis; yet, these substances competitively inhibit the N-demethylation of N-methylbutynamine by rat liver microsomes in vitro (McMahon, 1962).
7. General Comments Numerous compounds are now known to inhibit the metabolism of drugs i n vitro. Indeed, Kato et al. (1964) studied the inhibitory effects of over 40 different compounds on the oxidative microsomal systems of rat liver. Little is known of the mechanism of actions of niost of these inhibitors. As indicated above, however, the inhibitors may act in a variety of ways: some compounds may interfere with NADPH-generating systems; some may interfere with reduction of P4 5 0 ; some may block the transfer of active oxygen from P-450 to the drug substrate. Studies of inhibition are complicated by the fact that an inhibitor may block drug metabolism through several mechanisms. Sulfhydryl inhibitors, such as p-chloroniercuribenzoate, inactivate glucose-6-phosphate dehydrogenase, NADPH-cytochrome c reductase, and P450. SKF-525A and Lilly 18947 presumably combine with the active site of an N-dealkylase in rat liver microsonies, since these substances competitively inhibit the N-demethylation of N-methylbutynaniine and aminopyrine ; but SKF525A also is converted to a substance which inactivates P450, and Lilly
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18947 is converted to a compound, presumably DPEA, which slows the reduction of P-450 (H. A. Sasame and J. R. Gillette, unpublished results, 1964). Complications such as these may account for apparent discrepancies in the literature. Desmethylimipramine inhibits the conversion of tremorine to oxotremorine in rats, but not in mice (Sjoqvist and Gillette, 1964). SKF-525A inhibits the O-dealkylation of phenacetin by liver microsomes of rat (J. J. Burns, personal communication, 1962), but not by those of rabbit (Axelrod, 1956). Moreover, SKF-525A noncompetitively inhibits the metabolism of aminopyrine by liver microsomes of rabbit (La Du et al., 1954; La Du, 1962; Gillette, 1965) and mice (H. A. Sasame and J. R. Gillette, unpublished results, 1963), but competitively inhibits the metabolism of this substrate by those of rat (H. A. Sasame and J. R. Gillette, unpublished results, 1964). In contrast, the administration of SKF-525A to rats causes irreversible inhibition of the drug-metabolizing enzymes (Rogers and Fouts, 1964; H. A. Sasame and J. R. Gillette, unpublished results, 1963). Reports have appeared indicating that a number of conipounds are potent inhibitors of the microsomal enzyme systems in vitro, but evoke little inhibitory action in vivo; for example, propyldiphenylpropylacetate (SKF-525A acid), diphenylpropylethanol, diphenylpropylethylamine (La Du et al., 1954; La Du, 1962), triparanol (Gillette and Davenport, 1961) inhibit microsomal enzymes of rabbit liver more effectively than does SKF-525A in vitro, but do not appreciably alter hexobarbital sleeping time in mice. From these observations, it is obvious that mechanisms of inhibition which occur in one species may not necessarily occur in other species and that those which occur in vitro may not necessarily occur in living animals. Another complication in studying inhibition of the drug-metabolizing enzymes is the fact that drugs are often converted to products which are in turn metabolized. If an inhibitor blocks both the formation and the destruction of the product, the amount of the intermediate that accumulates in incubation mixtures may not be significantly changed, thus leading to the conclusion that the inhibitor was ineffective. For example, SKF-525A does not appreciably change the amount of aniline accumulating in microsoma1 enzyme assay systems containing methylaniline, even though it inhibits N-demethylation of methylaniline and the hydroxylation of aniline (H. A. Sasame, unpublished results, 1963). Moreover, in rats SKF-525A and desmethylimipramine block both the formation of oxotremorine from tremorine and its metabolism to inactive metabolites (H. Schumacher and F. Sjoqvist, unpublished results, 1964); hence, the onset of effects of tremorine is delayed by these inhibitors but once the-actionappears, it is markedly prolonged (Sjoqvist and Gillette, 1964, 1965). Furthermore, in
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mice SKF-525A inhibits both the conversion of parathion to paroxon and the hydrolysis of paroxon to inactive metabolites; thus, SKF-525A actually potentiates rather than blocks the action of parathion (O’Brien, 1961,1962). G. How MANYOXIDATIVE ENZYME SYSTEMS ARE PRESENT IN LIVERMICROSOMES? The wide variety of drugs metabolized by liver niicrosomes suggests that the oxidative enzymes possess an extraordinary degree of nonspecificity. In recent years, however, considerable evidence has accumulated for the view that a number of oxidative enzyme systenis are present in liver niicrosonies. Sonie of the evidence is based on species differences in the metabolism of drugs. For example, aniline is hydroxylated niainly in the para position by liver rnicrosonies of rabbits and rats, but in the ortho position by those of cats (Posner et al., 1961a). Amphetamine is mainly deaminated in rabbits, but is hydroxylated in dogs and rats (Axelrod, 1954). Iniipramine is mainly deniethylated by rat liver inicrosomes and presumably hunian liver niicrosonies, but is hydroxylated by those of rabbits (Dingell et al., 1964). These species differences thus indicate that there are various amounts of a number of different enzymes in liver niicrosonies of different species, or that there are species differences in the substrate specificity and properties of the liver niicrosomal enzymes or both. There is considerable evidence that liver niicrosomes contain a number of different enzymes. For example, there is marked sex difference in rats in the inetabolisni of a number of substrates, such as hexobarbital, pentobarbital, and aniinopyrine, but no sex difference in the metabolism of aniline and zoxazolamine (Kato and Gillette, 1965a,b). Moreover, starvation of male rats decreases the metabolism of aniinopyrine and hexobarbital, but does not alter the metabolism of zoxazolaniine (Conney and Garren, 1961; Kato and Gillette, 1965a) or aniline (Kato and Gillette, 1965a). Similarly, the ability of rat liver inicrosonies to metabolize aminopyrine and hexobarbital is impaired by adrenalectomy or the administration of epinephrine, morphine, thyroxine, or alloxan, but none of these treatments impairs their ability to metabolize aniline and zoxazolaniine (Kato and Gillette, 196513). In fact, the administration of alloxan enhances the metabolism of aniline (R. L. Dixon el al., 1963; Kato and Gillette, 1965b). The presence of a variety of drug oxidative enzymes in liver niicrosonies has hampered studies on the metabolism of the drug-nietabolizing enzymes. Several groups of workers have attempted to correlate the ability of drugs to increase the levels of NADPH-cytochronie c reductase and P-450 in niicrosonies with their ability to enhance the inetabolisni of various drugs (Remmer and Merker, 1965a; Orrenius and Ernster, 1964; Orrenius et al., 1965; Clouet, 1965), but it is obvious that such correlations will seldom
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be exact. Indeed, it would be difficult to explain the ability of phenobarbital to increase the N-demethylation of metharbital fiftyfold (Smith et al., 1963) solely by its ability to increase either NADPH-cytochrome c reductase and P450. Moreover, the finding that pretreatment of rats with methylcholanthrene doubles the P-450 in liver microsomes does not explain why methylcholanthrene enhances the hydroxylation of 3,4-benzpyrene. several fold, but does not alter the metabolism of hexobarbital, aminopyrine, and steroids (Conney et al., 1957; 1965a, Gillette, 1963a,b; R. Kato and J. R. Gillette, unpublished results, 1964). The effects of starvation of male rats on the levels of microsorrial P 4 5 0 and NADPH-cytochrome c reductase cannot account for all the changes in drug metabolism, for starvation decreases the rate of metabolism of hexobarbital and aminopyrine, but enhances the rate of hydroxylation of aniline by liver microsonies (Kato and Gillette, 1965a). Such discrepancies as these can be explained, however, by the concept that either liver microsomes contain a number of enzymes, each of which has P-450 as its prosthetic group, or by the concept that a common P-450 serves a number of drug enzymes. At the present time, however, there is no unequivocal evidence that distinguishes between these two concepts. V. Factors Which limit Drug Metabolism in living Animals
Since a t therapeutic doses, the concentration of drugs in animals is seldoni high enough to saturate the drug-metabolizing enzymes, the drugs are usually metabolized at rates proportional to their plasma levels. Thus, the rate of drug nietabolisni is lowered by factors which decrease the effective concentration of the drugs a t the active site of the enzymes. The effective concentration of drugs may be altered in the following ways: 1. The drug may be reversibly bound to plasma proteins; for example, plasma proteins bind about 98% of the phenylbutazone present in blood (Brodie and Hogben, 1957; Anton, 1960; Gillette, 1965). 2. The drug may be dissolved in fat; for example, lipid-soluble compounds, like thiopental, become localized almost entirely in adipose tissues (Brodie and Hogben, 1957; Richards and Taylor, 1956). 3. The drug may be localized in tissues; for example, atabrine is localized in liver, and imipramine is localized in lung, brain, and spleen (Brodie and Hogben, 1957; Herrmann and Pulver, 1960; Dingell et al., 1964; Gillette, 1965). 4. Insoluble drugs in the gastrointestinal tract may dissolve so slowly that their rate of metabolism is limited by their rate of absorption; thus, the half-life of zoxazolamine is markedly longer when the drug is given orally than when given intravenously (Burns et al., 1958). The route of administration of drugs can markedly alter their pharmaco-
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logical effects. For example, iritraperitoneal administrattion of parathion causes a greater blockade of brain cholinesterase than does intramuscular administration ; in cont'rast, this enzyme is blocked more effectively when paraoxon, the active metabolite of parathion, is given intramuscularly than when it is administered intraperit'oneally (West*ermann,1962; Holtz and Westerniann, 1959). Similarly, oxotreniorine is more effective when injected intravenously than when given intraperitoneally (Sjoqvist and Gillette, 1965). The reason for these differences becomes obvious when the anatomy of the circulatory system is considered. Drugs administered intraperitoneally are absorbed directly into portal blood, which then flows through the liver before it reaches the systemic circulation; accordingly, if a drug is metabolized in liver so rapidly that it is virtually cleared from the blood passing t'hrough this organ, little of t,he drug enters the systemic circulation. In contrast, drugs inject'ed intramuscularly, subcutaneously, or intravenously enter the systemic circulation directly; thus, the concentrations of t'he drugs are virtually the same in systemic and portal blood. It is obvious from t,hese considerations that the metabolism of a drug in vivo represents a coniplex interplay among a variety of factors, including the activity of the drug-metabolizing systems, the degree of binding of the drug to tissues, and the rate of excretion of the drug. REFERENCES Anton, A. H. (1960). J. Pharmacol. Exptl. Therap. 129, 282. Axelrod, J. (1954). J. Pharmacol. Exptl. Therap. 110, 315. Axelrod, J. (1955). J. Biol. Chem. 214, 753. Axelrod, J. (1956). Biochem. J . 63, 634. Axelrod, J. (1963). Science 140, 499. Axelrod, J. (1965). Proc. 2nd Intern. Pharmacol. Meeting, Pragur, 1963, Vol. 4, p. 309. Pergamon Press, Oxford. Ayengar, P. K., Hayaishi, O., Najajima, M., and Tomida, I. (1959). Biochim. Biophys. Acta33, 11. Baker, J. R.., and Chaykin, S. (1962). J. Biol. Chem. 237, 1309. Barrett, J. F., Pitt, P. A., Ryan, A. J., and Wright, S. E. (1965). Biochem. Pharmacol. 14, 873. Bauer, S.,and Kiese, M. (1964). Arch. Exptl. Pathol. Pharniakol. 241, 317. Bernhard, K., von Bulow-Koster, J., and Wagner, H. (1959). Helv. Chim. Acta 42, 152. Bernheim, F., Bernheim, M. L., and Wilbur, K. M. (1948). J. Biol. Cheni. 174, 257. Bledsoe, T., Island, D. P., Ney, R . L., and Liddle, G. W. (1964). J. Clin. Endocrinol. 24, 1303. Bloomfield, D. K., and Bloch, K. (1960). J. Biol. Chem. 236, 337. Booth, J., and Boyland, E. (1957). Biochem. J . 66, 73. Booth, J., and Boyland, E. (1964). Biochem. J. 91, 362. Booth, J., Boyland, E., Sato, T., alrd Simms, P. (1960). Biochem. J . 77, 182. Breslow, R., and Lukens, L. N. (1960). J. Biol. Chem. 236, 292. Breuer, H., Knuppen, R., and Pangels, G. (1962). Biochim. Biophus. Acta 66, 1. Brodie, B. B., and Hogben, C. A. bl. (1957). J. Pharm. Pharmacol. 9, 345.
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Experimental and Clinical Chemoteratogenesis RAYMONII L. CAHEN Pharmacology Department, Pjizer-Clin-Research Center, Arnboise (Indre et Loire), France
I. Introduction . . . . . . . , . . . . . . 11. General Survey , . . . . . . . A. Historical Data . . . . . . . . . . B. Bibliographical Survey . . . . . . . . . . . . . 111. General Principles . . . . . . . . A. Laws of Teratogenesis . . . . . . . . . . . B. Determining Factors of Terat!ogenesis . . . _ . . . IV. Experimental Conditions . . . . . . . . . . . A. Species and Strains . . . . . . . , . . . B. Number of Animal Species . . . . . . . . . . C. Number of ilnimals . . . . . . . . . D. Doses Used . . . . . . . . . . . . E. Routes of Administration . . . . . . F. Timing of Administration . . . G . Experimental Precautions . . . . . V. Experimental Techniques . . . . . . . . . A. In Vivo Studies . . . . . . . . . . . . B. In Vitro Studies . . . . . . . . . VI. Teratogenic Drugs . . . . , . . . . . . . A. Drugs Teratogenic in Man . . . . . . . . . . B. Drugs Whose Teratogenic Effect in Man Has Not Been Confirmed . C. Drugs Which Pass through the Placental Barrier but Are Not Terato. . . . . . . . . . . . . . genic VII. Nature and Mechanism of Action of Teratogenic Drugs . . . . A. Nature of the Observed Malformations . . . . . . . B. Selectivity of the Effects . . . . . . . . . . C. Relationship between Teratogenic Action and Chemical Structure or Pharmacological Action. . . . . . . . . . . D. The Mechanism of Teratogenic Action , . . . . . . VIII. Conclusions . . . . . . . . . . . . . . Glossary . . . . . . . . . . . . . . References’ . . . . . . . . . . . . . . ,
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I. Introduction
During recent years the field of modern therareutics has made stupendous progress, marked by the development of new drugs which possess tremendous therapeutic activity, as well as complex pharmacological action. Despite this development and the systematic pharmacological screening entailed (Cahen, 1957), it has not been possible to avoid an 1
The survey of literature pertaining t o this review wm completed in March, 1965.
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appalling biological reaction, which has brought the pharmacologist face to face with problems of a teratological nature. Because of the difficulty of extrapolating data from animals to man, the clinician must be doubly vigilant, not only to recognize important clinical signs, but also to detect unanticipat,ed manifestations and to determine their etiology. In 1961 a drug which has been proved to be innocuous both pharinacologically and clinically was found to be extremely dangerous to the fetus when taken by a pregnant woman. This observation urgently refocused attention on the teratogenic capabilities of drugs. Both clinical practice and pharmacology have long known that certain drugs, although not lethal, may produce malformations to the embryo. This monster-producing reaction has been called the teratogenic effect of drugs. The history of the concept of teratogenesis will be discussed in Section 11. The laws and conditions of teratogenesis are well known a t the present time, and will be mentioned in Section 111. Nevertheless, the detection of teratogenesis is extremely difficult and will be elaborated in Section IV. The highly complex nature of t,his problem is revealed by the multiplicity of techniques, as described in Section V, and also by the frequently contradictory results, discussed in Section VI. Neither the relationship between the chemical structure and pharmacological action of a teratogenic substance nor the mechanism of teratogenesis is as yet clearly understood. Consequently, research of the teratogenic potentiality of drugs has by no means attained a final solution to the problem. II. General Survey
A. HISTORICAL DATA Fetal abnormalities have been known since antiquity. Achondroplastic dwarfs are depicted on Egyptian frescoes. Conditions of hydrocephalus were attributed to witchcraft. For centuries, monsters were considered to be possessed by demons; they were expelled from society and often burned alive with their families and possessions. The hypothesis that malformations may be inherited or may develop as the result of environmental factors was first advanced by Ambroise Par6 in 1678. It was not until the nineteenth century, however, that Geoffroy Saint-Hilaire (1832) succeeded in producing experimental fetal abnormalities (anencephaly and spina bifida) by puncturing duck embryos through the shell. The same technique, in a more accurate and highly developed form, was employed by Dareste (1891) on the chick embryo, but fetal malformations in mammals were not produced experimentally until 1907 : von Hippel and Pagenstecher obtained fetuses with microphthalmia
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and cataracts after irradiating pregnant rabbits. X-ray teratagenic effects were discovered in man (Aschenheirn, 1920) and systematically studied in mice (Rugh, 1952, 1958; Russell, 1950). Using X-rays as the traumatic agent, Etienne Wolff (1933a,b) carried out a series of important experimental investigations on chick embryos. By selectively irradiating minute areas of the embryo he produced highly localized malformations. The role of maternal malnutrition as a teratogenic factor was reported by Hale (1935) in the pig exposed to avitaminosis A during pregnancy. This effect, confirmed in laboratory animals by Warkany and Nelson (1947), Wilson and Barch (1949), Wilson et al. (1953), and by A. Giroud and Martinet (1959), was later extended to other avitaniinoses: folic acid (Giroud, 1952; Giroud and Lefebvres, 1951; Sansone and Zunin, 1954), paritothenic acid (Giroud et al, 1951, 1954))Vitamin E (Gallison and OrentKeiles, 1951) and riboflavin (Kalter, 1959a,b; Warkany and Deuschle, 1955; Warkany and Schraffenberger, 1944), and also to hypervitarninosis A (Kalter, 1960a; Cohlan, 1953; A. Giroud and Martinet, 1955a,b; Yukioka et d.1959; Kalter and Warkany, 1961). Wolff and Ginglinger (1935) succeeded in producing intersexuality in the chick embryo by means of estrone, thereby demonstrating the elective action of a chemical substance on the normal development of certain anlagen. The endocrinologists Courrier and Jost (1942) were responsible for our knowledge of the teratogenic role of the adrenal, pancreatic, and sex hormones. Studies of the biological aspect of fetal malformations extended into virology with the research of Gregg (1941). This author established the relationship between certain birth defects and a maternal rubella infection. At that point it had been recognized only that either vitamin or hormonal imbalance might provoke repercussions. In 1952, with the rise of an effective chemotherapy of malignant disease, a new category of teratogenic agents, namely, the antiinetabolites such as aminopterin, appeared (Thiersch, 1960; Murphy and Karnofsky, 1956; Tuchmann-Duplessis and Mercier-Parot, 1958a). Actually, a common and atoxic drug, thalidornide, often taken during the early days of pregnancy, exposed the problem of the teratogenicity of drugs as a most crucial one (Lenz, 1961, 1962a,b; McBride, 1962). Never before had the association been made between the use of drugs innocuous to the mother and the occurrence of malformations in her child. The thalidomide drama was followed by an eruption of fear of most drugs, old and new, from which many patients, as well as many physicians, have not yet recovered. A posteriori, many drugs employed for years have been suspected, more or less justifiably, of being teratogenic. Attempts have been
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made, more or less successfully, to reproduce embryopathy by animal experimentation. The research of the teratogenic effect poses an especially complex problem to the pharmacologist since such an action varies according to the period of administration of the compound and to the animal species, and even to the animal strain used. The odds of obtaining in animals results applicable to man are extremely slight. Therefore it is through the investigation of families of abnormal children (Barnes, 1964) that statisticians and clinicians are striving to uncover the potential teratogenicity of drugs.
B. BIBLIOGRAPHICAL SURVEY Numerous publications have been devoted to the problem of the teratogenic effect, and its central points have been discussed in a series of general reviews. In 1950, Ancel wrote a book on chemoteratogenesis in the chick embryo. In 1959, Kalter and Warkany compiled a noteworthy study of the congenital malformations induced in mammals by metabolic disorders. In 1960, Baker described a phenomenon whose nature differs from that of the teratogenic effect: the toxic effect of drugs on the fetus. In 1962, A. Giroud and Tuchmann-Duplessis published a complete review of the experimental and clinical research of the causes of congenital malformations. In 1964, Fave compiled a list of all drugs so far identified as having provoked embryopathies in mammals. The same year the author published his own study, “Evaluation of the teratogenic effect of drugs” (see Cahen, 1964b), in which the techniques used and the results obtained by teratologists were described and critically evaluated. Recently (1965) an excellent review by Karnofsky appeared. Valuable contributions were also made at such gatherings as the Ciba symposium (see Ciba, 1960) and the first (London, see Fishbein, 1962) and second (see Proc. 2nd Intern. Med. Congr., 1964) international conferences on congenital malformations, the report (Chicago, see Commission on Drug Safety, 1963) on prenatal effects of drugs, and the meeting (see Proc. European SOC.Drug Toxicity, 1963) on congenital embryopathies. Research is steadily progressing, but despite the publication of many studies during 1964, a final solution is yet to be achieved. Ill. General Principles
A. LAWSOF TERATOGENESIS After Geoffroy Saint-Hilaire (1832) produced anencephaly and spina bifida in ducks by puncturing the embryo through its shell, and without any decisive evidence, he formulated certain general laws of teratogenesis :
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in particular, that the initial fact of teratological evolution is arrest of development. Wolff (1937) confirmed this law by using X-rays, which were known to arrest development, as a teratogenic agent. The same author established three other laws: 1. Any embryo may be transformed into a monster of predetermined type, even without possessing a hereditary basis for certain malformations. 2. Teratogenic factors act on undifferentiated anlagen between the time of determination and the time of differentiation. 3. The different tissues composing the region of injury vary in their sensitivity to the teratogenic agent.
B. DETERMINING FACTORS OF TERATOGENESIS The school of A. Giroud et al. (1954) in France and those of Kalter (1954), Nelson et al. (1956) and Wilson and Warkany (1949) in the United States have demonstrated that, regardless of what agents are employed, three factors are essential before teratogenesis can take place. These factors are the period of teratogenic sensitivity, the teratogen dosage, and the nature of the animal’s genotype. 1. Period of Teratogenic Sensitivity
During a certain period of its development, the embryo is sensitive to teratogenic agents, although it is resistant a t other periods (Giroud, 1960; Wilson, 1964a). Teratologists have established that the sensitive period of organogenesis is of very limited duration. During the first period (first to seventh day in the rat, and first to tenth day in man) the ovum becomes segmented and is transformed into blastocytes. At that period the agent may cause death but not nialformation of the embryo. This was observed in hypervitaminosis A in rats (A. Giroud and Martinet, 195513) and after administration of thalidomide to monkeys (Lucey and Behrman, 1963). The second period, the gastrula stage (seventh to fourteenth day in the rat, and fourteenth to sixtieth day in man), during which the various organs develop, is the sensitive period of teratogenesis. It is then that hypervitaminosis A (Giroud and Martinet, 1955b, 1956b), folk acid antagonists (Nelson et al., 1956), thalidomide (Lenz and Knapp, 1962a,b; Hay, 1964), virus H (Ferni, 1964b), and virus HVJ (Ohba, 1957) exert their teratogenic effects. During the third period, the growth process, teratogenic agents are no longer active, except in the genitourinary system and, in some cases, in the central nervous system (Legrand et al., 1961).
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2. Teratogen Dosage
Since 1955, A. Giroud has insisted on the importance of what he calls the (‘subtlety of the teratogenic factor.” Embryopathy may be induced by a physical, chemical, or viral agent in such a small quantity that toxic phenomena do not occur in the mother. In larger doses, these teratogenic factors kill the embryo. This is also true of the rubella virus, which may produce fetal malformations, even if asymptomatic in the mother. P. and A. Giroud (see P. Giroud et al., 1954a, 1963) observed experimentally that a rickettsiosis pursuing an unapparent course in a pregnant female rat could lead to fetal malformations and abortions. “The clearest demonstration of the subtlety of the teratogenesis agent is given by the study of deficiency diseases” (A. Giroud, 196310). In the course of deficiency of vitamin B1 (Warkany and Nelson, 1947), riboflavin (Giroud et al., 1950, 196l), and pantothenic acid (A. Giroud et al., 1955), the vitamin reserves in the maternal liver need only be reduced 10% to produce embryonic malformations. Another typical example is that of actinomycin D (Tuchmann-Duplessis and Mercier-Parot, 1958b), which is teratogenic even in doses one-tenth as large as those used therapeutically. Delatour et al. (1965) administered thalidomide to dogs in a dosage of 30 mg/kg and observed malformations of the skeleton, face, and nervous system, while Weidman et al. (1963), using doses 3 and 6 times larger, obtained only mild malformations, but also a high neonatal mortality and a high incidence of sterility. Recent experiments (Delahunt and Lassen, 1964) have shown that thalidomide is teratogenic in the monkey if even very small doses are used (10 mg/kg per 0s). 3. Nature of the Genotype
The nature of the embryo, i.e., its genetic constitution, also influences the reaction to a teratogenic agent. Consequently, the teratogenic effect does not necessarily occur in every species of animal. In some cases, the sensitivity to teratogenic agents is the same regardless of animal species. Folic acid deficiency leads to fetal malformations in the rat (A. Giroud, 1952), as well as in the mouse, the cat (TuchmannDuplessis and Lefebvres-Boisselot, 1957), and man (Thiersch, 1952). Hypervitaminosis A is equally teratogenic in the rat, the mouse, the rabbit, and the guinea pig (A. Giroud and Martinet, 1959). Conversely, in many cases, the teratogenic effect is specific to certain animal species. Cortisone is teratogenic in the mouse and rabbit, but not in the rat (Lohmeyer, 1961). Insulin is teratogenic in the mouse (Smithberg et al., 1956) and rabbit (Brinsmade et al., 1956), but not in the rat. Thalidomide, which causes
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fetal einbryopathies in the mouse and rabbit (Lichtenstein et al., 1951), is generally not tcratogenic in the rat (A. Giroud et al., 1962a,b). Moreover, in the same species, a drug may be more teratogenic to one inbred st rain than another. Many such examples can be cited : the incidence of cortisone-induced cleft palate in mice differs according to the strain, as follows: A/JAX (lOOy0), DBA (92%), C 57 BL (19yo), CBA (12%) (Fraser and Fainstat, 1951; Kalter, 1954, 1965). Sensitivity to trypan blue also varies from one strain of rats to another (Gunberg, 1958; TuchmannDuplessis and Mercier-Parot , 1959d). The incidence of diaphragmatic hernia in rats with vitamin A deficiency varies from 1 to 20%, depending on the strain (Anderson, 1941). The appearance of thyroxin-induced cataracts varies considerably, according to the strain in rats (A. Giroud and De Rothschild, 1951, A. Giroud and Martinet, 1954b,c). Furthermore, certain strains of rabbits, e.g., Silver Gray, are refractory to thalidomide, while others, e.g., New Zealand, are sensitive (Taussig, 1962a; Seller, 1962). Thalidoniide is teratogenic only in certain strains of rats, e.g., SpragueDawley (King, 1962) and Wistar SM (Bignami el al., 1962). Thalidomide is teratogenic in mice of Swiss A strain (DiPaolo, 1963), but inactive in strain C 57 BL (Trasler and Fraser, 1963). Finally Kalter (1959a) pointed out that in cortisone-treated mice maternal weight is of great significance in determining the number of abnormal offspring. IV. Experimental Conditions
Investigations undertaken to secure the experimental conditions necessary for screening the teratogenic effect of drugs have engendered a new branch of pharmacology that has grown tremendously. Working empirically, investigators had to invent and develop new methods (Fraser, 1959). Different scientific societies have met for the purpose of comparing opposing results and coordinating the research. Nevertheless, 4 years after the occurrence of the accidents responsible for this frantic research, many points remain obscure and on many occasions experimentation has to be conducted by a process of trial and error. The search for the special techniques necessary for elucidating the teratogenic effect of a new drug poses many difficult problems; we shall list them briefly, but cannot hope to solve them.
A. SPECIES AND STRAINS Pharmacological literature describes the teratogenic effect of drugs in different species of birds and mammals. Terat,ologists use chickens, rats, mice, and hamsters frequently, guinea pigs rarely, and cats, dogs, and monkeys exceptionally. It is impossible at present, however, to decide which
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species is best suited for experimentation. From the theoretical point of view, the choice of the animal species is based on similarities with the human species : placentation, reproduction pattern, metabolism, and enzyme systems of the embryo. 1. The Chick
From the practical point of view, the chick embryo appears to be the most convenient test object; it is available in large numbers most of the year; it is inexpensive; but above all, it is sensitive to thalidomide (Boylen et al., 1963; Kemper, 1962a,b; Somers, 1963a,b; Landauer, 1953, 1954; Clavert, 1963; Karnofsky and Lacon, 1964). Opinions vary concerning the merit of chick embryos in teratogenic trials. Some teratologists reject this species on theoretical grounds, because its embryo, lacking a placenta, cannot be compared with the mammalian embryo. It was for this reason that the French Experts Committee (see Ministry of Health, 1962) advised against its use. Moreover, from the practical point of view, Somers (1963a,b) and Delatour (1964) consider that it may give false positive results inasmuch as, according to Williamson et al. (1963), injection of any insoluble compound may produce malformations of the embryo. In the opinion of other authors, the chick embryo is, on the contrary, an excellent material : “Irreplaceable, provided you know how to interpret it” (J. Clavert, personal communication, 1964). It is true that some malformations which can be induced also by saline, e.g., celosomia and microphthalmia, are not specific. On the other hand, only a true teratogenic substance can induce specific malformations. For instance, cyclophosphamide (or endoxan) gives rise to abnormalities in the chick embryo (Gerlinger et al., 1962) (see Fig. l), but it has no action on the mouse. Greenberg and Tanaka (1964), however, have recently described the teratogenic effect of this antimitotic agent in a pregnant woman suffering from Hodgkin’s disease. Cortisone moreover, causes specific malformations in the chick: unibilical hernia and hare lip (Clavert et al., 1961). McLaughlin et al. (1963) have also demonstrated that certain pesticides induce chick malformations, in contrast to solvents and saline which are ineffective. Two still more convincing examples are nitrogen mustard (Salzgeber, 1962) and thalidomide (Salzgeber and Salaun, 1963a,b) which produced malformations of the chick’s limb, i.e., the same phocomelia found in man. The Food Drug and Administration (F.D.A.) recognizes this method of investigation as an auxiliary one for the screening of teratogens. If experimentation is conducted carefully, compared with controls, and analyzed critically, the utilization of the chick embryo may prove of value in ascertaining, in the event of negative results, that drugs are not teratogenic.
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F I ~ 1.. Cyclophosphamide. A . Chick. Right, control; left, 11-day embryo treated with cyclophosphamide: hypotrophy, niicromelia, hypognathia. (After Gerlinger at al., 1'362; courtesy of Conipt. Rend.) B. Rabbit. Right, control; left and center, 20-day embryos treated with cyclophosphamide: hypotrophy, mirromelia, hypognathia. (After Gerlinger and Clavert, 1964. (Courtesy of Compt. Rend.)
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On the other hand, all positive results must be confirmed on a species of placentalia before they can be applied to man. 2. Mammals Mammals possessing a placenta are theoretically closer to man than is the chick; thus, they are theoretically preferable for the detection of teratogens. In actual fact, considerable variations exist in the placental exchange of the fetuses and in the enzymic systems of different animals. a. Rodents. Rodent fetuses exhibit differences of individual sensitivity to teratogenic agents such as thalidomide, cortisone (Kalter and Warkany, 1959; Woollam and Millen, 1957), and hydrocortisone (Jost, 1956). The rat fetus is susceptible to vitamin A and alkylating agents but not to thalidomide. The resistance of the rat fetus to the teratogenic effect of thalidomide (Christie, 1962; A. Giroud et al., 1962d; Pliess, 1962; Seller, 1962; Somers, 1962a,b) is probably due to its drug metabolic behavior, which differs from that of man (B. B. Brodie, personal communication, 1965). Therefore teratologists generally prefer to use rabbits and mice. Highly selective results have recently been obtained by Tuchmann-Duplessis and Mercier-Parot (1964c,d) in experiments with the purine antimetabolite, azathioprine (Imuran). They noted 57% severe malformations in the rabbit, and even more in the rat or mouse. The significance of the frequency of spontaneous malformations in the rabbit (Greene and Saxton, 1939; Staemmler, 1962; Tuchmann-Duplessis and Mercier-Parot, 1964d; Yeary, 1964; Staemniler et al., 1964) and in some strains of mice (Bignami el al., 1963) cannot be overemphasized. This occurrence stresses the necessity of adequate controls from the same litter. Furthermore, as we have seen in the preceding section, there are many differences of sensitivity among strains and this makes an already complex problem more difficult. b. Carnivores. The cat is rarely used because of the difficulty in obtaining vaginal smears. As far as we know, only Somers has used this animal in experiments with thalidomide (1963a,b). The dog, because of placental similarities to man, is preferred to the cat, but its use has so far been restricted for financial reasons. Weidman et al. (1963) reported abnormalities in puppies (tail vertebrae, sternebrae, cleft palate) when thalidomide (100 mg/kg) had been administered orally to the bitch from day 1through day 21 of pregnancy. Delatour et al. (1965) have recently observed more definite malformations in the dog (celosomia, exencephaly, hypoplasia) produced by thalidomide (30 mg/kg from the eighth until the twentieth day) when laparotomy was performed at mid-gestation. Such experiments should be confirmed by an adequate number of controls sacrificed before term; moreover, they should be applied to other drugs, both teratogenic and nonteratogenic to man.
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c. Primates. So far, very few investigations have been conducted on the monkey. Lucey and Behrman (1963) obtained only disappointing results on rhesus monkeys, with thalidomide; a dose of 50-200 mg/kg, given for a period of 35 and 45 days, did not produce malformations but merely prevented gestation. Under such conditions thalidomide probably prevented implantation since a high dose was given at too early a stage of embryonic development. Delahunt and Lassen (1964), however, obtained typical skeletal malformations in the rhesus monkey by oral administration of 10 mg/kg thalidoiiiide during the sensitive period. Another species, the marmoset, was suggested by Benirschke (1963) for a long-range screening program. Such experiments may be prohibitively costly but may yield dividends. d . Armadillo. The armadillo, whose placenta resembles that of the primates, has heen recommended by Benirschke (1963). This animal is reasonably priced in the United States. Its gestation is well understood but it is difficult to obtain in Europe. It has recently been studied by MarinPadilla and Benirschke (1963). Thalidoniide given orally a t a dosage of 100 mg/kg for 15 days soinetinies produced phocomelia, sometimes ahortion. More recently Marin-Padilla (1964) observed degeneration of the eriibryoblastic cells of the blastocyte. Undoubtedly the screening of teratogenic drugs in this animal species would be of interest, were it universally procurable. In brief, there is no rule for selecting the teratogenosensitive species; consequently, various species are used more or less empirically, e. Man. Many drugs which are teratogenic in animals appear safe in man (Wilson, 1964a). Conversely, thalidomide, which is teratogenic in humans, is ineffective in certain animals species. As Fraser (1963) has emphasized, final proof of whether a drug is likely to be teratogenic in man should be sought in man. The Chicago coriference on congcriilal malforinations (see Proc. Rrid Intern. Conf., 1964) placed on its agenda the problem of clinical testing of teratogens and discussed the possibility of carrying out such tests with the minimum of risk. The best method was discussed a t the Lausanne conference (Proc. European SOC.Drug Toxiczty, 1964). Some participants contemplated a clinical study of potential teratogens in countries where abortion is legally permitted. The moral and legal implications of such investigations cannot be overlooked.
B. NUMBEROF ANIMALSPECIES Because of differences in the sensitivity to teratogens the fetuses of different animal species must be used for screening new drugs. As Fraser (1964a) rightly points out, although little is known about comparative teratology there is enough to show that the teratogenic effect in one species
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cannot be extrapolated to another. We do not know the mechanism of teratogenesis, and every day we discover considerable differences of drug metabolism in various animal species. It is possible that a drug which apFears not to be teratogenic in one species may, nevertheless, be teratogenic in another. It is mandatory that experiments not be limited to a single species, but for practical reasons not more than two or three species can be used. The French Ministry of Health previously required two and recently three animal species. Moreover, it must always be kept in mind that, regardless of the number of animal species used in the experiments, the result may be applied to man only with utmost caution.
C. NUMBER OF ANIMALS The study of teratogenesis may yield variable results within a single species or within a pure strain. Even in the case of classic teratogens the response of the fetus is inconstant, and the percentage of malformations is not always very high. Finally, with most laboratory species, the possibility of spontaneous malformations must he taken into consideration (Tuchmann-Duplessis and Mercier-Parot, 1964d). Therefore, it is essential to use an adequate number of animals and analyze statistically the results obtained in experimental and control groups. The Experts Committee of the French Ministry of Health has specified that at least 50 animals and 3 different dosages must be used; t,he time required for such an investigation is apparent. D. DOSESUSED 1. Importance of Using Small Doses
One fact is well established. In order to uncover a teratogenic effect, small doses must be used. The subtlety of the teratogenic factor, which has already been emphasized earlier, is of paramount importance. Large doses may induce abortion or resorption of the fetus. In practice, therefore, it is possible to attempt to find the abortive dosage and then to reduce it to determine whether or not a teratogenic effect has been obtained. Unfortunately, the problem of species’ sensitivity plays an important role in this issue. Moreover, many drugs which are abortive in large doses are not necessarily teratogenic a t a therapeutic dosage level. It is essential, therefore, to look for a teratogenic effect in the animal in a dosage corresponding to that administered clinically. Theoretically the proper course is to determine the median active dose, i.e., the dose which, if given to pregnant females, produces malformations in the embryo. Frequently,
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however, the teratogenic action of a drug has no relationship to the pharmacological action for which it is clinically used. I n such a way, thalidomide is teratogenic in doses that have no apparent sedative action, either in the rabbit (Fraser, 1964b) or in the dog (Weidman et al., 1963). Furthermore, the toxicity of a drug to the mother and its effect on the morphology of the embryo are absolutely independent. Atrican (a-thenoylaniino-2-nitro-5-thiazole) produces no detectable malformation in the embryos of the mouse, rat, or rabbit, even at a dose level toxic to the mother (LDGB) (Tuchniann-Duplessis and Mercier-Parot, 1958b). 2. Dose-Efect Relationship In order to obtain quantitative information, more than one dose must be used and the relationship between dose and teratogenic effect must be noted. In some cases, the difference between the abortive and nonabortive dose is very small. Once the abortive dosage has been found, it must be reduced progressively until the teratogenic dose has been determined. Extrapolation of results from animal to man presents many difficulties. Cohlan (1962), after calculating the relationship between the teratogenic dose in the rat and the teratogenic dose in man, as had been found for thalidomide by Somers (1962a), and for aspirin by Warkany and Takacs (1959), concludes that thalidomide is only one-twelfth as teratogenic as aspirin. Of course this conclusion assumes that all drugs are metabolized a t the same rate in rats as in man, and that variations between species are related to differences in activity. The fallacy of this assumption became apparent when Brodie and Erdos (1962) demonstrated that the clear-cut species difference in response to lipid-soluble drugs is the result of differences in the rate of metabolic breakdown; hence the teratological effect in the rat cannot correlate with that in man.
E. ROUTESOF ADMINISTRATION Theoretically, the route of administration to be followed for testing teratogenic substances must be the one that is used clinically. The effect of a drug may vary depending on the route of administration. Fraser (1964a) recalls that the teratogenic effect often varies with the route of introduction. Excessive doses of vitamin A when administered orally may produce malformations, whereas none are produced after intraperitoneal or subcutaneous injection. On the other hand, Woollam and Millen (1960) obtained highly significant fetal nialformations following injections of a water-miscible preparation of vitamin A. With thalidomide, the teratogenic effect is seen after gastric but not after intraperitoneal administration.
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F. TIMINGOF ADMINISTRATION As noted above, teratogenic substances are effective only during the critical embryonic period, i.e., the period of organogenesis. During the first stage of segmentation and during blastocyte formation, teratogens can only kill the embryo, as is the case of thalidomide (Lucey and Behrman, 1963; Lutwak-Mann and Hay, 1962; Adams et al., 1961). During the critical period, the type of malformation varies according to the time when the treatment is given, as demonstrated with vitamin A, by A. Giroud (1963a,b) and Hicks (1954). Central embryopathies appear sooner than skeletal or visceral ones. I t therefore appears that the drug must be adniinistered throughout the entire critical period. On the other hand, it seem that the prolonged administration of thalidomide throughout the period of gestation is less teratogenic than if its administration is limited to a few days of the critical period. This fact explains the discrepancy between the negative and positive results by FBlisati (1962) and King (1962) using the same strain of rats (Sprague-Dawley). Similarly, (King et al. 1965), noted a greater teratogenic effect of chlorcyclizine when given from days 1 to 15 of pregnancy. If this observation applies to all t,eratogens, it raises a serious objection to the test recommended by the F.D.A.
G . EXPERIMENTAL PRECAUTIONS The investigation of the teratogenic effect of a drug calls for an experimental technique free from artifact. Many factors other than drugs are capable of disturbing the development of the embryo. Consequently, these must be eliminated in all experiments. They include physical factors such as hypo- and hyperthermia, vitamin imbalance (Warkany, 1944, 1954, 1957, 1960), malnutrition, virus infection, and modifications of the environment. 1. Physical Factors
The thermal effect is for practical purposes the only one to be considered. Since the work of Courrier and Marois (1953)) the influence of hyperthermia on the fetal development of the rat has been recognized. Conversely, hyperthermia (MacFarlane et al., 1957) increases the risk of fetal resorption. In the chick, the work of Ancel (1950) revealed numerous malformations-celosoma, brachygnathia, and anophthalmia-produced by hypo- and hyperthermia. 2. Vitamin Imbalance
The teratogenic action of hypovitaminosis A has been known since the classic observations of Hale (1935) on the pig. It has been confirmed in
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various laboratory animals (J. G. Wilson and Warkany, 1948; Millen et al., 1953; A. Giroud et al., 1961). Deficiencies of vitamin Bl2 (A. Giroud and Boisselot, 1947a,b; Overholser et al., 1954; Newberne and O’Dell, 1958; O’Dell et al., 1954), of pantothenic acid (A. Giroud, 1952), of folic acid (A. Giroud et al., 1951), and of riboflavin (Kalter and Warkany, 1957) induce fetal malformations. Moreover, hypervitaminosis A (Cohlan, 1953; Giroud, 1964; A. Giroud and Martinet, 1954a; Deuschle el al., 1959; Kalter, 1960a) may cause embryopathies.
3. Nutritional Dejciencies Fast,ing (Runner and Miller, 1956), deficiencies in minerals, e.g., manganese (Landauer, 1940), and in such amino acids as tryptophan (Pike, 1951) are teratogenic. 4. Injections
The fetus is sensitive to virus infections which may break out in the animal house, such as those caused by herpes virus in the rabbit (Biegeleisen and Scot,t, 1958) and by HVJ virus in the mouse (Ohba, 1958). The fetus of the rat is very sensitive to certain parasites, such as toxoplasma (P. Giroud et al., 195413) and rickettsia (P. Giroud et al., 1954a,b). 5. Modification of the Environment Seasonal variations (Kalter, 1959c), modifications of food supply, stress induced in the rat by immobilization (Hartel and Hartel, 1960; Goldman and Yakovac, 1964), and scent of a strange animal (Parkes and Bruce, 1961) may strengthen the teratogenic effect or even inhibit implantation. In humans, however, maternal emotional stress is not related to the occurrence of cleft lip or cleft palate or to the lack of a prenatal nutritional supplement (Fraser and Warburton, 1964). 6. Clinical Observations All these factors play an important role in huniaii pathology, and may even produce true embryopathies (Ingalls, 1960) such as anophthalmia, associated with d a m i n A (Sarma, 1959), or folic acid deficiency (Thiersch, 1952), or hypervitaminoses A (Marie el al., 1955). Embryopathies due to viruses are more serious. The teratogenic effect of rubella virus (microphthalmia and cataract dealness, persistence of the patent foramen ovale) has been known since the observations of Gregg (1941a) and Gregg et al. (1945);it appears in a t least 50% of women exposed during the first 2 nionths of pregnancy (Lamy and Serot, 1956; Campbell, 1961; Kaye and Reaney, 1962; Lock et al., 1961). The viruses of chickenpox, mumps (Kaye et al., 1953), and influenza (Do11 et al., 1960; Saxen et al.,
278
RAYMOND L. CAHEN
1960) also have been suspected of producing anencephaly and encephalocele. In a survey of 1735 cases of German measles, 2775 of influenza, 300 of. polio, 159 of measles, 615 of mumps. 345 of chicken pox, German measles emerges as the only viral disease with an established effect (Biancone, 1964). Inquiries conducted in various countries concerning drug-induced malformations have not only demonstrated their existence, but have also established virus infections as responsible and suggested the existence of malformations of alimentary origin. These investigations must be collated and their results confirmed by statistical analysis. Clinical studies of this type have begun in many countries (Justin-Besancon et al., 1964) (particularly in France) on about 35,000 cases, under the aegis of the National Institute of Health and Medical Research and of the Statistical Research Unit (Professor Schwartz;). In the United States, an inquiry of the same type is taking place. In Denmark (Winberg, 1963) and Belgium (Derom, 1963) similar investigations are being carried out. Many years must pass before conclusions can be drawn from such a vast study. In the United States in 1960, of 110,000 deaths of infants under 1 year of age, only 700 were due to infectious diseases, but 15,000 were due to congenital malformations. To sum up, the differential diagnosis of drug-induced teratogenesis must proceed by the elimination of other embryopathic factors. To be significant, animal experiments must be accompanied by parallel observations on controls kept in the laboratory under the same conditions. In clinical practice, observations must be verified by a systematic and complete etiological investigation. V. Experimental Techniques
The production of experimental malformations in the fetus by administration of a teratogen to the mother is theoretically extremely simple. In practice, however, the investigator must work accurately in definite and standardized conditions during mating, verification of gestation, and investigation of teratogenesis. As in all biological tests, the choice of the standard is of prime importance. Unfortunately, at the present stage of our knowledge, the standard is still undetermined. The experimental techniques used by teratologists are varied; some are applied in vivo, others, in vitro. These techniques will be examined in the following sections.
A. In Vivo STUDIES The numerous methods used can be divided into three types, depending on whether the teratogen is administered directly to the embryo, or indirectly to the mother during the period of gestation.
EXPERIMENTAL AND CLINICAL CHEMOTERATOGENESIS
279
1. Administration of Teratogens to the Embryo
Among the embryos of the highter vertebrates, bird embryos are the niost accessible for experimental purposes. The duck's egg used by Dareste (1891) has now been replaced by the hen's egg, niost frequently the White Leghorii (Ancel, 1950; Wolff and Ginglinger, 1935). a. ELperimental Procedure. Two procedures have been carried out successively: injection through a hole made in the shell, and deposition of the teratogen on the exposed embryo. i. Injection through a hole in the shell (Ancel, 1950; Salzgeber, 1957, 1963; Delatour, 1964). The egg is kept for 24 hours a t laboratory teniperature and is perforated by means of a dental drill. The hole made in the shell does not damage the shell nieriibrane; it is situated a t a point equidistant froiii the edge of the air chamber and the center of the egg. The drug to be studied is injected by nieans of a microsyringe into the albumen, pointing the needle obliquely, and taking all aseptic precautions. I t is advisable to inject a volume of only 0.1 cc in order to reduce the premature mortality rate and the nuinber of celosoniia. At the end of the operation the hole used for the injection is sealed with niolten paraffin wax. The eggs are then placed in the artificial incubator on racks so that the axis of the egg is a t a 30" angle to thc horizontal and the air chamber is upperillost. The eggs are turned and transilluminated daily. Sterile eggs or those containing dead embryos are opened for exaniinat,ion of their contents. Under these conditions Keniper (1962a) and Boylen et al. (1964) observed malformations caused by thalidomide. McLaughlin et al. (1963) studied the effect of various pesticides. At present the method is recognized by the F.D.A. for auxiliary screening of teratogenic substances (Ellenhorn, 1964, and personal communication, 1965). ii. Administration to the exposed embryo. In this method, originated by Wolff and Ancel (1933), the teratogen is deposited on the ernbryo exposed around the forty-eighth hour of incubation. This niethotl has been used by Salzgeber (1957) with various aiitiniitotic poisons, by Salzgeber and Salaun (1963a,b) with nitrogen mustard and thalidoniide, and recently by Caujolle et al. (1965) with various sulfoxides. 2. Adniinistrakon o j Teratoyens to the Pregnant Peniale The techniques used are, in principle, the same in all mammals; they differ only in detail, depending on whether the rat, mouse, rabbit, dog or monkey is used. I n every case problenis arise and these must be examined in succession : the mating procedure, the verification of fertilization and of gestation, the administration of the drug, the verification of teratogenesis and the choice of a standard.
280
RAYMOND L. CAHEN
a. Mating Procedure. First, all external conditions must be cleared: the physiological state of the animals, their aptitude for fertilization and reproduction, a vitamin-balanced diet, the selection and verification of the homogeneity of the strain used. Second, only animals with a regular estrous cycle should be chosen. Third, the exact period of the sexual cycle must be determined. In the rat and mouse daily vaginal smears are taken in the period immediately following the estrous cycle to insure that the animals will be mated at the most favorable time of the cycle, i.e., the proestrus. In t,he rabbit, on the other hand, it is unnecessary to deterniine the cycle for, in theory, ovulation is automatically brought about by copulation. The most reliable method for mice and rats is to place a female in the cage of the male for the night. A more convenient and effective method is the harem method: 4 or 5 females are placed in a cage with 2 males and left together for several days until verification of copulation has been obtained. The bitch will copulate only during the 3 days following the end of estrus, at the time when the blood-stained vulvar discharge is replaced by a viscid, mucous discharge (Delatour et al., 1965). b. Verification of Fertilixation. The verification of fertilization is important; determination of its exact date is absolutely essential. The verification of fertilization is easily determined in the mouse by the appearance of a vaginal plug. If several animals have copulated in the same cage, this investigation should be carried out daily. In the case of the rabbit, all that is generally required is observation of copulation; if successful, it takes place very shortly after the female has been placed in the cage with the male. The discovery of spermatozoa in the vagina of the rabbit niust be made very quickly, because of their fragility in this qecies. In order to insure fertilization, it is advisable to repeat copulation several times. It should be noted that the signs of copulation in the rat and especially in the rabbit, are not necessarily followed by fertilization of the animal; rather pseudocopulation, the frequency of which is increased if the atmosphere is suddenly chilled, may occur (Klein, 1961; Aron et al., 1963). In the bitch, in theory, copulation may be regarded as tantamount to fertilization, provided that sexual union has been duly witnessed (Delatour el al., 1965). c. Verification of Gestation. The verification of gestation must be carried out well before term. In the mouse and rat, microscopic examination of vaginal smears enables the essential condition of gestation to be established, namely, the presence of a permanent diestrus, since the normal estrous cycle is resumed in unfertilized females. In the rat, physiological hemorrhage on the fourteenth day is an unequivocal proof of gestation. In all animals, the most reliable criterion of gestation is exploratory
EXPERIMENTAL AND CLINICAL CHEMOTERATOGENESIS
28 1
laparotoniy, which niust be performed a t mid-term, i.e. on the ninth to tenth day in the rat and the eleventh to twelfth day in the mouse. This operation causes neither ill effects, nor resorption of the embryo, nor fetal abnormalities (Bertrand, 1960). The body weight curve of the animals confirms the evidence of gestation, for weight, increase in the rat and mouse is observed chiefly during the last quarter of pregnancy. In the rabbit, the verification of gestation is more difficult; the weight curve does not give very precise results. Verification by palpation of the abdomen, when the embryos can be felt as hard masses, 15-25 days after fertilization, is a delicate test. In the case of the bitch, there is no test for pregnancy that gives constant and reliable results at an early stage (Delatour et al., 1965). The diagnosis cannot be niade by abdominal palpation before the thirtieth day, which is relatively late. d . Administration of Potentially Teratogenic Drugs. It is extreniely important to administer the test substance during the critical period of organogenesis of the embryo i.e., froni the seventh until the fourteenth day. The administration niust be regular, not missing a single day. According to the route of clinical administration, the drug is given either orally or parenterally. If given orally, the drug should be administered by stomach tube and not mixed with the food. e. Verification of Teratogenesis. It is very useful to perform a Caesarian section on at least half of the aninials shortly before natural delivery would have occurred. In this way, the fetuses can be counted, the number of resorptions determined, and the errors resulting froni the frequent occurrence of cannibalism can be avoided. For this purpose, rats and mice are sacrificed on the twentieth day and rabbits on the twenty-ninth day. Depending on the strain, the operation is performed on the bitch between the thirtieth and thirty-fifth day (Delatour et al., 1965). Caesarian section under ether anesthesia is preferable to sacrifice of the mother, for living fetuses can be obtained. The necessity of avoiding the caesarian section before these dates cannot be overemphasized : younger fetuses have abnormal body form and open eyes, signs which could be falsely interpreted as malformations. The presence of nialforniations is determined by examination of the fetus with a magnifying glass. The limbs, tail, axial skeleton, face, palate, eyes, and head are systeniatically inspected; the presence of edema and of heinatonla is sought. This examination niust always be completed by dissection for the detection of visceral abnormalities, and by a niicroscopic examination of the organs fixed in Bouin’s fluid. The skeleton is examined after digestion of the tissucs with alcoholic potassium hydroxide and alizarin (method of Crary, 1962). i. Resorption of fetuses. Whether ~nalforniationsare present or not, it is
282
RAYMOND L. CAHEN
important to look for signs of fetal resorption, which frequently occurs if the drug has been given at a subtoxic dosage level. The uterus is examined with a magnifying glass for traces of implantation of the placenta, and the points of resorption are counted; Kopf and Salewski’s (see 1964) Kopf et ~ l . staining , method facilitates niacroscopic localization of the implantation sites. Resarptians are rarely total, and often occur in the control. It is important, therefore, to determine the percentage of resorptions and to compare it with the corresponding control figures. ii. Observaticns e n the ycung aster natural delivery. One group of animals is allowed to proceed to natural delivery. Observations are maintained on the young until they attain sexual maturity. In this way, certain visual, auditory, or central nervous systems disturbances may be detected. Under such conditions, dental and craniomandibular malformations have been observed in 21-day-old rats and mice (Paget and Thorpe, 1964) after administration of a sulfonamide (sulfadimethoxypyrimidine) to the mother. After prolonged administration of a monoamineoxidase inhibitor (nialamide) to a female rat, Tuchmann-Duplessis and Mercier-Parot (1963b) discovered that the young females of the first filial generation would not receive the male but practiced pseudocopulation with each other. iii. Investigation of the abortive efect. If the limit of toxicity is exceeded, a teratogenic drug may become abortive. Abortion takes place before or near term. If, after sacrificing the animals, no sign of fetal implantation is found although gestation was certainly present, it may be concluded that abortion or fetal resorption occurred. The abortive effect is different from the teratogenic effect, but the occurrence of an abortion does not mean that the fetus was necessarily normal. In such cases the experiment must be repeated using a smaller dose. f . Interpretation of Results. All positive results, whether teratogenic or abortive in nature, are relatively easy to interpret. On the other hand, if the experiment reveals that a new drug shows neither teratogenic nor abortive effect, the evaluation of its teratogenic potentialities for the human fetus is difficult. In such cases, the French Ministry of Health (Circular 2282) stipulates that the exFerirrierits must be repeated on a t least 50 litters of rats, as many mice, and 20 litters of rabbits, using a t least three different dosages. g. Chcice of Standard. The use of a standard of reference is necessitated by the variability of all biological tests. This obligation is capital since the teratogenic effect of a drug varies, according to the species and the strain. The choice of a standard of reference must be determined by the reliability, the uniformity, and, if possible, the multiplicity of the teratogenic effects. The choice of thalidomide is questionable inasmuch as its teratogenic effect does not induce the typical skeletal malformations described in man.
EXPERIMENTAL AND CLINICAL CHEMOTERATOGENESIS
283
Trypan blue induces more consistent and general embryopathies (Gillman et al., 1948;Hamburgh, 1952; Christie, 1961; Wilson, 1954; Cahen et al., 1964; Carpent, 1958) in a wide range of animal species: mouse, rabbit (Ferm, 1964a; Gunberg, 1956), rat, and hamster. The intensity of the effect, however, varies with the strain of mouse (Waddington and Carter, 1953) or rat (Tuchmann-Duplessis and Mercier-Parot 1959d). The teratogenic effect in the rat is illustrated in Fig. 2. However, the use of trypan blue as a standard preseiits a serious difficulty: its teratogenic effect varies with the origin of the sample and with its state of purity (Tuchniann-Duplessis and Mercier-Parot, 1959c; Jelen, 1964; Kelly, 1964) (see Table I). Hypervitaniinosis A (A. Giroud and Martinet, 1954b, 1956c, 1959; Cohlan, 1953) apparently has a more general teratogenic effect since anomalies are produced in the central nervous system, the eye, the face and the mouth (Kalter, 1960a) the palate, the skeleton and the viscera. Its effect has been described in three species of laboratory animals: the rat, the mouse and the rabbit (Kalter and Warkany, 1959). Figure 3, for which we are indebted to Professor A. Giroud, illustrates the teratogenic effect in the rat. The principal results observed are given in Table 11. In the light of our present knowledge, vitamin A in large doses appears of interest because of the constancy of its action, although its teratogenic effects have only seldom been described in man. However, a drug that is teratogenic in animals as well as in man, such as an antitumor agent, would theoretically be a preferable standard. In our ignorance of the intricacies of teratogenesis, the choice of a standard of reference is difficult. The use of a provisional standard is essential, however, in order to train the pharmacologist in teratogenic testing and to assess the sensitivity of strains or litters available in the laboratory (Cahen, 1964a,b). 3. Administration of Teratogens before and during Pregnaiicy
The Food and Drug Administration recommends the following “rat litter test” (Ellenhorn, 1964). The procedure consists of a long-term administration of the drug to the parent rats for 2 months before mating and throughout the gestation and lactation pcriods. This procedure is continued through three generations of rats. A sample of the young rats is sacrificed at the weaning stage; the number of offspring, the duration of survival, and the growth rate are determined. According to M. J. Ellenhorn (personal communication, 1965), this niethod is preferable. Thalidomide, if given to rats under these conditions, reduces the number of offspring as a result of embryo resorption (Kopf et al., 1964). It would seem that this procedure is practical and easy to carry out; it
284
RAYMOND L. CAHEN
FIG.2. Effect of trypan blue on the rat. A. Right, exencephaly in a fetus receiving trypan blue; left, control. B. Section through the brain of a rat fetus receiving trypan blue. A defect of the cranium is seen in the region of the vertex. The meningeal spaces are in contact with the epidermis, which is abnormally thin and devoid of appendage (alopecia circumscripta). This anomaly corresponds to a minor degree of failure of closure of the cranium, while the exencephaly shown in A represents a major degree (After Cahen et al., 1964. Courtesy of Med. Ezptl.). C. Skeleton of a rat fetus treated with potash and stained with alizarin (Crary’s method, 1962). Torsion of the axial skeleton can be seen.
EXPERIMENTAL AND CLINICAL CHEMOTERATOGENESIS
285
has the advantage of eliminating tedious cytological examinations for the verification of pregnancy, which are obligatory in the preceding method. On the other hand, this procedure has the drawback of studying the sterilizing effect rather than the teratogenic effect. Moreover, there is the risk that the chronic administration of a drug may stimulate its own nietabolism and increase the synthesis rate of catabolic enzyines, so that, when finally given during the critical period of organogenesis, it is catabolized too rapidly to exert its usual teratogenic effect. Brodie and Erdos (1962) and Conney and Burns (1962) have cited numerous examples of drugs whose metabolism is accelerated by repeated administration, which increases the amount of specific enzymes in the liver microsonies. We have demons strated in detail (Cahen, 196410) the risks of obtaining false negative result-
E.3
m
TABLE I
Q,
STANDARDS OF REFERENCE FOR TRYPAN BLUE
Species Hamster Rat
Strain
Source of sample
Days of Malforadminis- mations tration (yo)
Golden ? Stock colony Gnlbler
7-10
Wistar Long Evans Long Evans
7-9 7-8 3-7
? ?
?
19
Fetuses resorbed Nature of malformations Hydrocephalus, exencephaly Hydrocephalus, spina bifida, regression of tail, clubfoot Anophthalmia, hydrocephalus Anophthalmia, heart Spina bifida, Arnold Chiari syndrome Spina bifida, myeloschisis, myelomeningocele Anencephaly, microphthalmia, reduction of tail
(%)
Author
Year
Ferm Gillman et al.
1957 1951
J. G. Wilson Fox et al.
1955
Gunberg
1958 1958
Warkany
1958
Wistar
National Aniline ?
Wistar
R. A. L.
7-9
6
Wistar
National Aniline
7-9
4
Spina bifida, microphthalmia, reduction of tail
30
Wistar
Blue Wilson
7-9
28
Spina bifida, microphthalmia, reduction of tail
33
Wistar
Coleman Bell
7-9
38
Spina bfida, microphthalmia, reduction of tail
Tuchmann, Duplessis 1959d and MercierParot Tuchmann-Duplessis 1959d and Mercier-
Stock colony Coleman Bell
7-9
I1
Anencephaly, microphthalmia, torsion of tail and axial skeleton
3
Tuchmann-Duplesiss 1959d and MercierParot
21
25
Tuchmann-Duplessis 1959d and MercierParot Tuchmann-Duplessis 1959d and Mercier-
Parot
Parot
r4 1: U
6m
Mouse
Rabbit
Wistar
Coleman Bell
7-9
37
Black
Coleman Bell
7-9
80
August
Coleman Bell
7-9
10
PUC
Coleman Bell
7-9
33
Long Evans
Coleman Bell
7-9
54
Wistar
Ethereal extract
0
Wistar
Alcoholic extract
0
Wistar
Kuhlman R. A. L.
7-9
? ? ?
? ? ?
8
? Pelger
?
8 5-13 19
?
16
8 8
22
Anencephaly, microphthalmia, torsion of tail and axial skeleton Anencephaly, microphthalmia, torsion of tail and axial skeleton Anencephaly, microphthalmia, torsion of tail and axial skeleton Anencephaly, microphthalmia, torsion of tail and axial skeleton Anencephaly, microphthalmia, torsion of tail and axial skeleton Anencephaly, torsion of tail and axial skeleton None Microphthalmia, clubfoot, defective closure of cranium Exencephalia, spina bifida Exencephalia, spina bifida Exencephalia, spina bifidn Exencephalia, anophthalmia Umbilical hernia, exophthalmia Encephalocele, hydrocephalus
29
0
0
20
14
30
0
30
Tuchmann-Duplessis and MercierParot Tuchmann-Duplessis and MercierParot Tuchmann-Duplessis and MercierParot Tuchmann-Duplessis and MercierParot Tuchmann-Duplesuis and MercierParot Tuchmann-Duplessis and MercierParot Tuchmann-Duplessis and Mercier Parot Cahen Cahen et al. Gillman et al. Hamburgh Waddington and Carter Barber el al. Harm Ferm
1959d 1959d
1959d
1959d 1959d
1959d
1959d
1964b 1964 1948 1952 1953 1959 1954 1956
288
RAYMOND L. CAHEN
FIG.3. Hypovitaminosis A in the rat. A. 21-day embryo presenting anencephaly and anophthalmia. Shortening of the facial prominence and protrusion of the tongue can be seen. B. Frontal section at the level of the eyes of an embryo presenting bilateral microphthalmia. Note the presence of a cleft palate. C. External view of a bilateral ureterohydronephrosis. D. Frontal section showing two very dilated calices. (After A. Giroud and-Martinet, 1955a,b).
inherent in the method advocated by the F.D.A. This opinion has been affirmed by Burns (1965).
B. In Vitro STUDIES The methods described above are all indirect methods involving the injection of teratogenic substances either into the embryo (chick) or the mother (mammals). Wolff and Haffen (1951) were the first to propose direct administration of the teratogen a t the level of tissues or organs excised and cultivated in vitro. Their method, known as “organotypical culture,’’ enables a certain organ to be explanted and a t a certain stage of its development, to be placed in direct contact with the teratogen. In this way it is
TABLE I1 STANDARDS OF REFERENCE Fon VITAMINA
Species Mouse
Rat
Rabbit I
Guinea pig
Single dose (units)
Route
50,000 50,000
Per Per
0s
35,000
Per
08
75,000 15,000 15,000 60,000
Per as I.P. S.C. S.C.
60,000
S.C.
11-13
60,000 60,000 60,000 60,000
S.C. S.C. S.C. S.C.
14-16 18-20 14-16 10-12
200,000 50,000
S.C. S.C.
8-10 8-10
500,000 125,000 150,000
Per 0s Per 0s Per 0s
5-10 5-10 10-13
50,000
Per 0s
1&13
0s
Days of administration &13 8-10 11-13 2-16
10 10 10 8-10
Malformations
Malformations (yo)
Kalter, 1959d A. Giroud, 1960 A. Giroud, 1960 Cohlan, 1953
Anencephaly, cleft palate Exencephaly Cleft palate Exencephaly, cleft palate, anophthalmia, cataract Spina bifida Anencephaly, cleft palate. cataract, anophthalmia Anencephaly, cleft palate, cataract Cleft palate, cataract Cleft palate, cataract Cleft palate Anencephaly, cleft palate, cataract Anomalies of ears and teeth Anencephaly, hydronephrosis, cardiac anomaly
-
Face Anencephaly, anophthalmia, syndactyly Maxillary fissure
References
4
Cohlan, 1953 Gehauer, 1954 Woollam and Millen, 1957 A. Giroud and Mart,inet, 1955a,b
92
A. Giroud and Martinet, 1956a,h,c
49 9 100
A. Giroud and Martinet, 1956a,b,c A. Giroud and Martinet, 1956a,h,c Filippi and Mela, 1957 A. Giroud and Martinet, 1956a,c
A. Giroud et al., 1958 Roux, 1961 A. Giroud and Martinet, 195!) A. Giroud and Martinet, 1959 A. Giroud and Martinet; 1959 A. Giroud and Martinet, 1959
d
E
E % ca
290
RAYMOND L. CAHEN
possible to localize the teratogen’s action by finding out whether its application in vitro to a particular organ can induce specific lesions. Other less valid methods have recently been suggested. They consist essentially of studying toxicity in monocellular organisms or single cells (flagellates, leucocytes). A more specific method involves the blastocytes of the preimplantation embryo (Lutwak-Mann and Hay, 1962). 1. Organotypical Cultures In this method the chosen organ or tissue is taken from 9-day-old chick embryos and kept alive in a natural medium of agar dissolved in Gey’s fluid, Tyrode’s solution, and chick embryo extract. This tissue culture has proved a fruitful research tool in studying the direct action of teratogenic substances on cells (fibroblasts, nerve tissue, bone marrow). It was employed by Salzgeber (1957) to differentiate between the actions of a mitotic poison (colchicine), nuclear poisons (urethane, narcotine), and cytoplasmic poisons (nicotine, quinine). Ruch and Rumpler (1964) demonstrated the specific action of dexamethasone, a synthetic steroid, on the growth of cardiac fibroblasts. De Meyer and Isaac-Mathy (1958) disclosed the anomalies produced by a hypoglycemic sulfonaniide (carbutamide) on the anlagen of the eye in 12-day-old chick. 2. B. Miller (1963) recently used a monolayer culture of mouse enibryonic tissues. With this method he studied the effect of thalidoniide and of one of its metabolites, N-3-hydroxyphthalimidoglutariniide,which causes total inhibition of ribonucleic acid (RNA) synthesis and inhibits protein synthesis by 80%. The result with thalidoniide is questionable, however, because the product was dissolved in dioxane, which per se inhibits cell cultures. 2. Nonspecific Cultures ’ For the sake of completeness we may mention the techniques of leucocyte (Roath et al., 1962, 1963), flagellate (Frank et al., 1962, 1963a,b), and Ehrlich ascites tumor cell (DiPaolo, 1964) cultures as tests of teratogenicity. Roath et al. (1962) observed inhibitory effects and morphological anomalies of leucocytes treated by thalidomide, but the nature of the solvent is not described in their paper. It seems difficult to extrapolate the result obtained with this method to human embryopathy.
3. Blastocytes of the Rabbit Lutwak-Mann and Hay (1962) described a method completely different from the preceding methods-the study of the effect of teratogenic agents transmitted from the mother rabbit to the rabbit embryo a t the blastocyte stage. The method consists essentially of histological examination of fixed
EXPERIMENTAL AND CLINICAL CHEMOTERATOGENESIS
29 1
and stained hlastocytes taken from the uterus. Thalidoniide and 6-mercaptopurine, injected into a female rabbit, cause degeneration of the preiniplantatiori enibryo, mainly affecting the zone of the embryonic disc. The method is rapid, simple, and sensitive. The teratogenic specificity allowed by the procedure remains to be determined. So far as we know, no one else has yet used this attractive technique. However, it seems that this niethod is concerned essentially with the effect produced on the ovum before organogenesis, which is a probleni different from that of teratogenesis. VI. Teratogenic Drugs
The most widely varying drugs given during pregnancy have been suspected, rightly or wrongly, of producing embryopathies. Certain drugs which pass through the placental barrier are toxic to the fetus, but do not give rise to fetal malformations. An important list of substances teratogenic in animals was given by Ancel (1950) in his book. More recently, objective and critical lists of teratogens have been published by various authors (Kalter and Warkany, 1959; A. Giroud and Tuchniann-Duplessis, 1962; Chassagne and Lechat, 1962; Tuchmann-Duplessis, 1963; Tuchniann-DupIessis and MercierI’aroi , 1963a, 1964a; Fave, 1964; Cahen, 1964b; Karnofsky, 1965). I n the following sections we shall review the principal drugs that have been described as capable of giving rise to malformations of the fetus. This list is not exhaustive and it does not specify all the technical details described previously (Cahen, 196413). It is believed necessary to classify drugs into three groups: drugs teratogenic in man, drugs whose teratogenic effect in man has not been confirmed, and drugs which pass through the placental barrier but which are not teratogenic. IN MAN A. DRUGSTERATOGENIC
1. Antitumor Agents
The antitumor agent,s were the first to be ident,ified as teratogenic. In their well-documented reviews, Sokal and Lessmann (1960), Chassagne and Georges-Janet (1962), Tuchmann-Duplessis and Mercier-Parot, (1964a), Karnofsky (1965) describe various embryopathies caused by niany antitumor agents possessing different mechanisms of action : alkylating agents, antiniit,otic agents (spindle poisons), and antimetabolites. The teratogenic or abortive effects of the principal alkylating and antimitotic agents are summarized in Table 111; the antinietabolites, which are described last, are listed in Table IV. a. Alkylating Agents. Alkylating agents (or radiomimetic substances),
TABLE 111 ALEYLATING AND ANTIMITOTIC AGENTS ~
Species
Dose (mg/kg)
Days of administration
Abortion Malformations
(%I
Author
Year
Alkylating Agents Hat Mouse
1 2 37
13-14 10-12 10-15
Rat Chick
0.3-0.5 0.6-16
11-16 2-3
Chick
0.1
Rabbit
2-50
Before placing in incubator 10-13
Rat Mouse Rabbit Rat Rat
0.3 1.2-2.7 1.5 0.55 0.1-0.4
11-12 7-9 12 7-9
Rat
10
12
Rat
2 4
6-8
10-20
12-13
Mouse
Nitrogen mustard Deformed limbs, syndactyly, exencephaly Atrophy of limbs, hydrocephalus Polydactyly, oligodactply, cleft palate Syndactyly, exencephalocele Phocomelia, hypotrophy
1956 1963a,b
Gerlinger et al.
1963
Gerlinger and Clavert
1964
Thiersch Jurand Didcoketal. Murphy Tuchmann-Duplessis and Mercier-Parot
1957a 1959 1956 1958 1960a
10
Murphy
1962
50
Tuchmann-Duplessis and Mercier-Parot Didcok et al.
1964a
50
Cy clophosphamide Brachymelia, hypotrophy, cardiac lesions, absence of eyelids Hypognathia, hypotrophy, cleft palate, complete dieappearance of germ cells Triethylenemelamine None Hypotrophy, meningocele Hypotrophy Anencephaly Hypotrophy, microphthalmia Chloram bucil Reduction in number of ribs, syndactyly, torsion of tail Craniorachischisis, multiple malformations Cleft palate, umbilical hernia
1948 1954 1955
Haskin Danforth and Center Thalhammer and HellerSziillosy Murphy and Karnofsky Sslzgeber and Salaun
100 75 75 50 50
30
1956
Rat Rat
10 34
14-16 12
Busulfan Sterile progeny Atrophy of limbs
Bollag Murphy
1953 1960
Sinclair Tuchmann-Duplessis and Mercier-Parot Nishimura and Kuginuki Nishimura and Nakai Blattner et al.
1950 1958a
100
Didcok et al.
1956
100 10 100 100
Didcok et al. Didcok et al. Wiesner et. al. Tuchmann-Duplessis and Mercier-Parot
1956 1956 1958 195813
Tuchmann-Duplessis Mercier-Paro t Tuchmann-Duplessis Mercier-Paro t Tuchmann-Duplessis Mercier-Parot Tuchmann-Duplessis Mer cier-Parot Tuchmann-Duplessis Mercier-Parot Tuchmann-Duplessis Mercier-Parot
and
195913
and
1959b
and
195%
and
1960a
and
1960a
and
1960a
0 50 Antimitotic Agents
15 2S50
7-8
Rat Mouse
1.5000
9-12
Rat
-
2
Urethane Meningocele Clubfoot
70
Cleft palate, syndactyly None Colchicine None Desacet ylmethylcolchicine None None None None
75
Chick
10/egs
Mouse
1
Rabbit Rabbit Mouse Rat
2-8 0.05 0.15
Rat
0.010
1-9
Rat
0.015
5-6
Rat
0.015
10-14
Rabbit
0.030
6-8
None
33
Rabbit
0.150
6-10
Exencephaly
45
Rabbit
0.200
10
Spina bifida
45
1
2-1 1 13-16 13-16 6-12 7-9
Actinomycin D Exencephaly, anopht,halmia, cleft palate None Spina bifida, microphthalmia
20 50
0
1958 1958 1960
TABLE I V ANTIMETABOLITES
Species
Dose (mg/kg)
Resorption of fetuses
Days of administration
Malformations
(%I
Author
Year
50 100 100 5 100
Murphy and Karnofsky Murphy and Karnofsky Thiersch Thiersch Friedman
1956 1956 1957a 1957s 1957
Glutamine Antagonists Rat Rat Rat Rat Dog
2.5 2.5 10 2.5 5
1&13 6-9 7-12 15-16 19-21
Azaserine Cleft palate, syndactyly, fusion of ribs None None Hydrocephalus, syndactyly, cleft palate None
7-15 7-12 15-16
510 5-10
Rat
50-200 4-75
Rat
5-10
4-5 7-8 12-13 2-6 5-9
U
c
None None Hypotrophy, cleft palate, anencephaly
100 100 5
Murphy and Karnofsky Thiersch Thierach
1956 1957b 1957b
Thiersch Thiersch Thiersch Zunin and Borbone Zunin and Borbone
1954 1954 1954 1955 1955
Purine Antagonists Rat Rat Rat
2,
d
D.O.N. 0.5-1 0.5-5 0.1-0.5
~
5
F
Adenine Antagonists
Rat Rat h t
!a
c
Mercaptopurine Hemangiomas 12 Hemangiomas 80 Torsion of axial skeleton 12 Hydrocephalus, hypotrophy, microphthalmia Hydrocephalus, microphthalmia, anophthalmia
X
Rat Rat Rabbit
12.5 0.10-12 150-200
7-1 1 7-9 11-13
Peribuccal hemorrhage None
100
None
Rat Rat Rat
100 100 5-20
4-7 11-12 8-10
Chloropurine None Hydrocephalus, ventral hernia None
Rat
5-20
9-12
Circular constriction of body
2.5-15
6-14
Rat
25
100 10 100 .50
Imuran None
30
Rat
10
None
100
Mouse
20
None
20
Rabbit
5-15
6-14
Polydactyly, syndactyly, amelia, phocomelia
0
Zunin and Borbone Tuchmann-Duplessis and Mercier-Parot Didcok et d.
1955 1958a
Thiersch Thiersch Tuchmann-Duplessis and Mercier-Parot Tuchmann-Duplessis and Mercier-Parot
1957a 1957a 195913
Tuchmann-Duplessis and Mercier-Parot Tuchmann-Duplessis and Mercier-Parot Tuchmann-Duplessis and Mercier-Parot Tuchmann-Duplessis and Mercier-Parot
1964c
1956
1959b
1964c 1964d 1964d
Folic A d Antagonists Aminopterin
Rat Rat Rat
Rat
0.2 0.2 0.2
9-1 0 11-13 1-7 11 15
100 Cleft palate None Anencephaly None
100 0
Murphy Murphy Thiersch Thiersch Thiersch
1960 1960 1960 1960 1960
296
RAYMOND L. CAHEN
like irradiation, cause cellular lesions manifested by the delay or inhibition of development. i. Nitrogen mustard. This substance causes malformations principally of the linib in rodents, and occasionally anomalies of the central nervous system; it is also abortive (Murphy and Karnofsky, 1956; Murphy, 1963). However, it is not teratogenic in humans (Chassagne and Georges-Janet, 1962). ii. Endoxan (cyclophosphamide). Another nitrogen mustard, endoxan, produces malformations of the limbs associated with hypotrophy in the chick embryo and the rabbit fetus (Gerlinger et al., 1962). This antitumor agent, which a t the present time is used extensively in the treatment of cancer because of its low toxicity, leads to the total disappearance of the germ cells in apparently normal embryos (Gerlinger and Clavert, 1964). A case of fetal malformation (hand and foot defects) was described in a patient treated with endoxan (Greenberg and Tanaka, 1964). iii. Triethylenemelamine. Triethylenemelaniine (TEM) is primarily abortive (Thiersch, 1960) but it rarely produces malformation in the surviving fetuses (in part,icular, hypotrophy) if administered late in small doses (Tuchmann-Duplessis and Mercier-Parot, 1960; Kageyama, 1961). When used to treat a woman with Hodgkin’s disease, T E M did not prove to be embryopathic (Chassagne and Georges-Janet, 1962). iv. Chlorambucil. The teratogenic effect of chlorambucil is much more powerful if administered late in the embryonic development; it produces polymorphic, especially skeletal malformations (Didcok et al., 1956; Murphy, 1962; Tuchmann-Duplessis and Mercier-Parot, 1964a) and absence of the kidiiey (Monie, 1961; Shotton and Monie, 1963). u. Busulfan. Not only is busulfan teratogenic (Murphy, 1960), but it also inhibits the male germinal line of t,he adult and leads to sterility of the progeny (Bollag, 1953). A clinical observation of Diamond et al. (1960) describes the discovery of marked hypoplasia of the ovaries and thyroid in a hypotrophic infant. b. Antimitotic Agents. i. LTrethane. Among the antimitotic agents, urethane is teratogenic in animals (Sinclair, 1950) but not in women (Chassagne and Georges-Janet, 1962). In fact, urethane has little clinical application, but it must he remembered that it is frequently used as a solvent for drugs. ii. Cclchicine. Colchicine (Ferm, 1958) and its derivatives (deacetylmethylcolchicine) (Thiersch, 1958a,h) are primarily abortive, because in addition to their cytotoxic action, they are also oxytoxic. The rare clinical observations have not revealed a single fetal malformation. iii. Aclinomycin. Actinomycin D is teratogenic in the rat, causing polymorphic malformations in doses smaller than those employed in
EXPERIMENTAL AND CLINICAL CHEMOTERATOGENESIS
297
clinical practice. Moreover, depending on the stage of enibryonic development at which the drug is injected, either resorption or anomalies may be observed, the nature of the malforinatioiis depending on the precise date of administration (Tuchmann-Duplessis arid Mercier-Parot, 1959b, 1960). The teratogenic effect in the rat is illustrated in Fig. 4, which we reproduce through the courtesy of Professor Tuchmann-Duplessis.
FIQ.4. Effect of actinomycin D on the rat. A. Litter of rats with severe malformations of the nervous system, the face, and the viscera, resulting in some cases in veritable monsters. Treatment often causes a slowing of development. The difference in size of two fetuses from the same litter will be rioted; on the left, an anomaly of the tail, and on the right, celoaoma. (After Tuchmann-Duplessis and Mercier-Parot, l958b. Courtesy of Compt. Rend.)
Rufocroniomycine is teratogenic in chickens (Maraud el al., 1963). c. Antimetabolites. Whether they are antagonists of glutanline, adenine, purines, or folic acid, all the antiriietabolites are terat,ogenic. The results of observations on animals are summarized in Table IV. i. Glutamine antagonists. In t,his group azaserine (0-diazoacetyl-Lserine) has proved to be teratogenic if' administered at the critical stage of embryogenesis. ii. Adenine antagonists. One of this group, namely, D.O.N. [diazo-6oxo-5(norleucine)], is primarily toxic to the embryo. However, a sniall dose administered between the eighth and the ninth days of gestation produces malformations of the face and brain (Thiersch, 1957b).
298
RAYMOND L. CAHEN
iii. Purine antagonists. 6-Mercaptopurine, when given on the seventh and eighth days of gestation, produces resorption. At other days it gives rise to hemangiomas or to torsion of the axial skeleton (Thiersch, 1954, 1962) and decrease in placental weight (Bragonier et al., 1964). Zunin and Borbone (1955), on the other hand, observed hydrocephalus and anophthalmia. The teratogenic action is considerably reduced in man. Depending on the time of itjs administration, 6-chloropurine may be either abortive or teratogenic; in the latter case, the malformations are typical and consist of a circular constriction of the body located a t the origin of the forelimbs (Tuchmann-Duplessis and Mercier-Parot, 1959a). Similar malformations are noted in the human fetus and are related to the presence of amniotic bands. Another purine antagonist, azathioprine (Imuran) [6-(l-methyl-4nitro-5-imidazolyl)-thiopurine]is interesting because it is the only compound capable of producing arnelia- or phocomelia-type limb malformations in the rabbit (Tuchmann-Duplessis and Mercier-Parot, 1964c,d) (see Fig. 5). It is not teratogenic in the rat or mouse. iv. Folic acid antagonists. One of these compounds, aminopterin, produces either resorption or malformations (Thiersch, 1952) depending on whether it is administered before or after implantation. Its use for the induction of therapeutic abortion (Bourne et al., 1957; Thiersch, 1960) led to the developmcnt of anencephaly. Observations of clubfoot (Meltzer, 1956), hypotrophy, and cranial dystosis (Warkany et al., 1959, Jorgensen, 1964) have confirmed the teratogenic effect of aminopterin in women. Another folic acid antagonist, X-methylfolic acid, is a powerful teratogen in the rat (Nelson et al., 1952). The nature of the ensuing malformations (nervous, ocular, visceral) depends on the period of administration (Nelson, 1960). It mainly produces abnormalities of the central nervous system in mice and of the skeleton in cats (Tuchmann-Duplessis and LefebvresBoisselot, 1957). v. Nicotinamide antagonists. Nicotinamide antagonists are toxic to the adult and possess little teratogenic activity. The compound 6-aminonicotinamide is teratogenic in the rat (Murphy, 1960) and especially in the mouse (Ingalls et al., 1963) in which it produces cleft palate. Similar malformations have been produced in the rat by thiadiazole and triazene (Murphy, 1960). 2. Hormones Many hormones may exert a teratogenic action on the fetus, whether they be endogenous maternal hormones produced in abnormal quantity, exogenous hormones taken by the mother, or specific hormones induced by drug administration.
I EXPERIMENTAL
AND CLINICAL CHEMOTERATOGENESIS
299
FIG.5 . Effect of Imuraii (aeathioprine) iii the rabbit. Fetus with severe malformations of the face (hare lip) and limbs, which are reduced to two stumps for the forelimbs; the hind limbs are shortened and syndartyl. (After Tuchmaiin-Duplessis and MercierParot, 1964c,d. Courtesy of C”ompt. Rend.)
Moreover, horniones niay act directly either on the endocrine gland itself, or on the receptors of the fetus. The principal results are summarized in Table V. a. Direct Action on a n Endocrine Gland of the Fetus. Certain drugs, when they reach the fetus, prevent an endocrine gland froin exerting its normal physiological function. Radioactive iodine administered to a pregnant mouse (Speent et al., 1951) or injection of the antithyroid drug, thiouracil, into a pregnant rat (Weiss and Noback, 1949; Jost, 1953a) produces severe anonlalies in the embryo as a result of the radiothyroidectorny and deficient production of hormone.
w
8 TABLE V HORMONES
Species
Rabbit Rat Castrated rabbit Mouse Mouse
Strain
Dose (mg/kg)
Days of administration
? ?
70 5-20
11-30 14-20
?
Webster
O.OO06 5-7.5
12-28 13-14
0.14.2
11-16
Resorption of fetuses Malformations
(%I
1 7-Ethin yl Testosterone Masculinization of fetus Masculinization of fetus
Estradwl Spina bifida, anencephaly Arrested development of eyelids Cleft palates
Author
Year
Courrier and Jost Marois
1942 1960
Courrier and Jost Raynaud
1939 1943
Nishihara
1958
A. Giroud et al. A. Giroud and Martinet
1951 1954b
Fraser et al. Fraser e t d . Fraser et al. Grumbach et al.
1954 1954 1954 1959
Thyroxin Rat
Mouse Mouse Mouse Mouse
0.250.50
1-20
Cataracts Cataracts (20%)
Commentry Norvegicus
0.25
9-20
Cataracts (2.2%) Cataracts (1.7%)
C57/BL/6 DBA/1 A/JAX
2.5 2.5 2.5 5
11-15 11-15 11-15
? Wistar
?
0-6
Cortisone Cleft palates (20%) Cleft palates (80%) Cleft palates (100%) None
20 20 20
100
F4.
B
2,
U F
Bm E
Rat Rat Rat Castrated rabbit Castrat,ed rabbit Mouse Rat Castrated rabbit
Mom Mouse Rat Rat
? ? ? ? ?
25 0.5 20 20 25 25-40
10-23 16-19 6-16 9-20 10-23 14-18
None None Hypotrophy None Hypotrophy Cleft palates (50%)
Long Evans ?
Various
14-21 11-14
Hydrocortisone Cleft palates Hypotrophy Cleft palates, spina bifida, exencephaly ACTH
A
5 5
13-16 11-15
Cleft palates Cleft palates
15-18
Adrenalin Hemorrhage, necrosis of limbs
? ? ?
10 u
5-16
Hypophyseal Somatotropic Hormone Pseudogigantism, prolongation of gestation
100 100 -
80
70
Courrier Evans and Clingen Mercier-Parot Kalter Courrier Fainstat
1951 1953 1957 1960b 1951 1954
Kalter Gunberg Clavert el al.
1962 1957 1961
Fraser et al. Ingalls and Curley
1954 1957
m
t iz
E 5 $ +z tc
d F
3
5
d
Jost
1953a
K z
M Tuchmann-Duplessis and MercierParot
1955
3 1
F
z
m
302
RAYMOND L. CAHEN
A clinical case of congenital cretinism has been reported following administration of radioactive iodine to the mother (K. P. Russel et al., 1957; Valenci and Nahum, 1958). The ingestion of propylthiouracil is goit,rogenic in the fetus (Aaron, 1955). b. Progesterone Dejiciency. Administration of an insufficient dose of progesterone to a castrated rabbit (Courrier and Jost, 1939) permits gestation but leads to the development of malformations in the offspring (celosomia, spina bifida, exencephaly). In the castrated rat it leads to compression and deformity of the fetus (Kroc et al., 1959). c. Action on the Receptors of the Fetus. Androgens. Besides the immediate morphological effects (masculinization of the fetus) of testosterone (Raynaud, 1942, 1955; R. R. Greene et al., 1940), long-term effects have also been reported, such as permanent estrus in the rat when reaching adult life (Turner, 1939) or modifications of the sexual instinct in the guinea pig (Phoenix et al., 1959). Many clinical reports have been published attributing masculinization of the female fetus to androgens (Zander and Muller, 1953; Hofmann et al., 1955; Hayles and Nolan, 1958; Lelong et al., 1959; Grumbach et al., 1959). Estrogens. The effect depends on the species. In the mouse (Raynaud, 1942) and rat (R. R. Greene et al., 1938; Grcene and Burrill, 1939), estradiol feminizes the male and masculinizes the female; in the rabbit it produces abortion. In man it has no abortive effect (0.W. Smith and G. Van Smith, 1949). Injection of diethylstilbestrol into the pregnant woman masculinizes the fetus (Bongiovanni et al., 1959). d . Synthetic Progesterones. In 1942, Courrier and Jost observed masculinization of fetuses following administration of 17-ethinyl testosterone to the pregnant rabbit. Such a teratogenic effect due to the crossing of the placental barrier (Courrier, 1924) has been consistently confirmed (Courrier and Gros, 1932; Courrier and Kehl, 1937; Courrier, 1961; Courrier, 1963). Since then, the use of artificial progesterones by pregnant women has not been advised. Nevertheless, this product marketed under various names) has induced many cases of pseudohermaphroditism; results have been summarized in 1958 by Wilkins et al. and in 1960 by Wilkins. Because of these observations, the search for other synthetic progesterones has continued and their virilizing action on the fetus of the rat (Jost, 1950a, 1955, 1961a, 1963; Jost and Moreau, 1963; Mey, 1963; Marois, 1960; Johnstone and Franklin, 1964) and dog (Curlis and Grant, 1964) has been studied. Allylestrenol (l7-a-allyl-l7~-hydroxyestrene) is less virilizing than normet handrolone (17-a-me t hyl- 19-nortestosterone) or even than norethindrone (17-a-ethinyl-19-nortesterone) (Grumbach et al., 1959; Wilkins, 1960). Among the progesterone derivatives, one has an antimasculinizing effect
EXPERIMENTAL AND CLINICAL CHEMOTERATOGENESIS
303
on the fetus: -6-chloroAs-l,2-cu-methyIene-l7-cu-hydroxyprogesterone acetate (Junkniann and Neumann, 1964). The great practical interest of these artificial progesterone is related to their efficiency following oral route of administration and to their inhibition of ovulation. However, the irregular taking of these anovulatory tablets may produce unexpected pregnancy. Nearly 600 cases of pseudohermaphroditisni have now been reported in young girls following administration of these progestins (hypertrophy of the clitoris, fusion of the labia minora) (Wilkins and Jones, 1958;Reilly, 1958;Hayles and Nolan, 1958;Grumbach el al., 1959;J. W.Jones, 1957; Wilkins, 1959;Grumbach and Ducharne, 1960;Bongiovanni and McPadden, 1960;Grepinet, 1964). It is hoped that clinical trials will confirm the absence of virilizing effect of synthetic derivatives. e. Unforeseen Teratogenic Action of Certain Hormones. There are many such examples : cortisone and the corticoids, epinephrine and vasopressin, thyroid and hypophyseal hormones, estrone. Incidentally, cholestrol which is hornionally inactive is teratogenic in the rat (Buresh and Urban, 1964; Peer et al., 1958;Baxter and Fraser, 1950;Fraser, 1959). i. Cortisone. Baxtrr and Fraser (1950),Fraser aiid Fainstat (1951), Courrier and Colonge, (1951),Donini and Leroy (1955),Doig and Coltman (1956),Clark (1956),Curtis el al. (1961),Isaacson and Chaudry (1963), Miller (1962),and Walker and Fraser (1956,1957) have observed that cortisone in the mouse and rabbit produces cleft palates, cardiac anomalies, and hypotrophy. The rat and monkey, however, are resistant to this action (Courrier et al., 1951;P.E.Smith, 1955,Mercier-Parot, 1957).Among the other corticoids, ACTH (Jost, 1951 ; Heiberg et al., 1959), hydrocortisone (Gunberg, 1957), and soludecadron (Clavert et al., 1961;Buck et al., 1962) produce various fetal abnormalities (spina bifida, exencephaly). The teratogenic effect of cortisone has been confirmed clinically (De Costa and Abelman, 1952;Harris and Ross, 1956;Wells, 1953;Bickel and Secretan, 1955;Fraser, 1962a). ii. Epinephrine and vasopressin. These two horinones, when injected into a rat fetus, cause hemorrhages followed by necrosis of the limbs or the tail (Jost, 1953a,b;Thompson and Olian, 1961). iii. Thyroid hormones. An incidence of 20% cataracts of the offspring was observed after giving thyroxin to the rat (A. Giroud et al., 1951;A. Giroud and Martinet, 195413). Clinically, hyperthyroidism primarily causes disturbances of fertility or of gestation. iu. Hypophyseal hormones. The injection of a hypophyseal somatotropic preparation into a pregnant rat (Watts, 1935; Hultquist and Engfeldt,
304
RAYMOND L. CAHEN
1949; Jost], 1950a) causes gigantism because of the increased production of soniatotropic hormones acting on the protein metabolism of the mother or fetus. Injection of somatotropic hormone into the pregnant rat from the third to the sixteenth day (Tuchniann-Duplessis and Mercier-Parot, 1955) prolongs gestation: the delivery takes place 2 to 4 days after the normal dates. Furthermore, Steiniger (1940) observed that administration of crude anterior pituitary hormone increases the frequency of cleft palate in strains of niice presenting this anomaly spontaneously. v. Estrone. I n the mouse, injection of estrone into the mother produces anomalies of the eyelids in the embryos (Raynaud, 1943). Estradiol benzoate produces cleft palate (Nishihara, 1958). After injection of large doses of folliculin into women during the first 2 months of pregnancy, three cases of fetal nialformations were descrited (Uhlig, 1959). 3. Thalidomide (a-N-Phthalimidoglutarimide)
It is unnecessary to recall that one of the most innocuous drugs (Somers, 1960b; Robertson, 1962) has caused the severest deformities of the human embryo. It)is unnecessary to recall how Leriz (1961, 1962a,b) and McBride (1961) were led to incriminate thalidomide in the etiology of limb nialformations of the fetus. Only then did pharmacologists begin to produce fetal malformations experimentally by giving thalidomide to laboratory animals. We shall describe the various human enibryophathies and malformations in the offspring of various laboratory animals. a. H u m a n Embryopathies. As McBride (1961) has remarked, the malformations produced by thalidomide essentially affect the organs derived from the mesenchyme. They are most common in the upper limbs, which are reduced to the state of stumps ending in malformed hands, with the number of digits reduced to four. X-rays reveal shortening of the hunioral segment and, not rarely, extreme hypotrophy or absence of the distal segment (Lenz, 1964; Pfeiffer, 1963). The lower limbs may show abnormalities such as shortening of different segments and reduction in the number of toes (Weicker and Hungerland, 1962; Ward, 1962; Bergamaschi, 1963; Yamamoto, 1944). All the joints are oriented haphazardly, and the skeleton resembles that of a seal; hence the nanie phocomelia first given to it by Geoffroy Saint-Hilaire (1832). Besides these malformations, syndactyly and triphalangia of the thumb have been reported (Taussig, 1962b; Lenz, 1964). Other embryopathies are nearly always associated : absence or dysplasia of the ear (Knapp et al., 1962; Lenz, 1964; Pfeiffer, 1963; Kleinasser and Schlothane, 1964), facial henmngioiiia (Pfeiffer, 1963; Knapp, 1963), and
EXPERIMENTAL AND CLINICAL CHEMOTERATOGENESIS
305
more rarely, cleft palate (Farizon and Mashernard, 1962; Kajii and Goto, 1963), and abnornialities in dentition (Dalderup, 1962). Monsters developing after administration of thalidoniide to pregnant women are illustrated in Figs. 6 and 7, for which we are indebted to Professor Lenz and Dr. Pfeiffer. Internal inalforniations are also frequent ; atresia of the esophagus or duodenum (Lenz, 1961), aplasia of the gall bladder (Kajii, 1962), anorectal anomalies (Inidahl et al., 1963), tetralogy of Fallot)and renal agnesis
FIG.6. Effect, of t.halidomide in man. A. Triphalangia of the thumbs, which are aligned with the other digits. B. Roentgenogram. C. Phocomelia and facial paralysis. D. Absence of radius. (After Lenz, 1964. Courtesy of Intern. Med. Congr., LW.)
306
RAYMOND L. CAHEN
FIG.7. Effect of thalidomide in man. A. Aplasia of radius and hemangioma of the center of the face. B. Phocomelia tetramela. C. Phocomelia brachialis and ameba, convergent strabismus. D. Mandibular hypoplasia. Note absence of the auricle and facial paralysis. (After Pfeiffer, 1963. Courtesy of Ada Med. Belg.)
(Lens, 1961) may be present. Above all, the sensory organs are affected: microphthalmia (Kajii, 1962),absence of the semicircular canals (Rosental, 1963). Only the central nervous system is not adversely affected.
EXPERIMENTAL AND CLINICAL CHEMOTERATOGENERIS
307
The conditions determining the appearance of malformations were accurately established in most cases. The critical period of thalidomide administration was from the fourth to the seventh week of pregnancy, i.e., the critical period of embryonic development. Small doses are no less dangerous than large doses (50-100/mg) (Rosental, 1963). Fortunately only 20y0 of the women exposed to thalidomide have been found teratogenosensitive. b. Malformations Prcduced in Laboratory Animals. Numerous experiments have been carried out in order to produce a posteriori enibryopathies similar to those occurring in man. But, first of all, it was necessary to find the reliable laboratory animal with which to study the teratogenic effect of thalidomide (Cahen, 1964b). As is shown in Table VI, which summarizes our results, the action of thalidomide varies considerably from one species to another and from one strain to another; it also depends on the time of administration of the drug and on experimental techniques used. I n the chick most authors (Kemper, 1963) have observed neural nialformations; only Salzgeber (see Salzgeber and Salaun 1963a,b) have systenlatically obtained phocomelias. These nionsters are illustrated in Fig. 8, for which we are indebted to the kindness of Professor E. Wolff. The effects on the chick embryo have not been consistent. Feeding hens with thalidomide increased the number of nonfertile eggs but not the incidence of gross malformations (Shorb et al., 1963). I n the rat, fetal resorption has been observed, but fetal nialformations are exceptional. Only certain strains appear to be sensitive to the teratogenic effect of thalidomide (Bignami et al., 1962; King, 1962). Minor skeletal malformations (scrambled vertebrae) have been reported (Klein-Obbink and Dalderup, 1964; McColl et al., 1963). The results in the mouse are contradictory. According to many authors, thalidomide is not teratogenic. A. Giroud et al. (1962a,b), however, have observed malformations in this species : cleft palate, hare lip, cataract, and, rarely, malformations of the limbs (see Fig. 9). Amelia has been reported only by Murad and Alvarenga (1964), who describe two cases. However, these small numbers are not significant, especially in the absence of controls for comparison. The rabbit fetus is sensitive to the teratogenic action of thalidomide (A, Giroud et al., 1962a,b,c,d) presenting neural malformations (anencephaly and torsion of the limbs) (see Fig. 9). The skeletal nialformations observed clinically (amelia or phocomelia) have not been found. Heart and kidney anomalies have been recently noted (Roux et al., 1965). There is also a difference in the sensitivity of the strain (Seller, 1962). I n the dog large doses produce prenatal death (Weidman et al., 1963). In markedly different conditions, however, e.g., by administration of small
TABLE VI TEWLLDOMIDE Order or class Aves
Species Chick Chick Chick Chick Chick
Strain White Leghorn Darby Hamn White Leghorn White Leghorn
Chick Chick Chick
Rodents
Resorption of fetuses
Dose (mg/kg)
Days of administration
1 4
5
Brachygnathia
Cameron
1962
0.510
1
Exencephaly
Kemper
1962a
Anophthalmia, microphthalmia
Ehmann
1963
None Cyclopia, micromelia
Somers Boylen et al.
1963a,b 1963
Anencephaly, oligodactyly None Micromelia, phocomelii
Yang et nl. 1963 Yangetal. 1963 Salzgeber and 1963a,b Salaiin
0.2-1
2-5
25&500 2
3 4
2.5 2.5 0.3
2 9 11
Hamster Rabbit Rabbit Rabbit Rabbit
2000-8000 New Zealand 50 Silver Grey 50 New Zealand 150 25-500
Rabbit Rabbit
Himalaya
0.5 50
1-12 1-12 8-1 6 6-14 8-14 1-7
Malformations
Mammals None Encephalocele, torsion of limbs None Shortening, torsion of limbs Anencephaly, torsion of dislocation of limbs Clubfoot Hydrocephalus, deformity of limbs
(%I
38 14 50 50
Author
Somers Seller Seller Somers A. Giroud et al. Spencer F6lisati
Year
1963a,b 1962 1962 1962a,b 1962a,b,c,d 1962 1962
Rabbit Rabbit
Hybrid New Zealand
500 150
8-15 8-16
Renal agenesis, pes z'arus Clubfoot, phocomelia
-
Rabbit
Common
250
8-12
20
Rat Rat Rat
Royal Wistar August,
50 200 500
6-9 9-1 1 6-14
Anencephaly, malposition of limbs None Malformation of limbs and tail None
Rat
Long Evans
250
1-14
None
30
Rat
SpragueDawley Spra gu eDawley Wistar SpragueDawley
Rat Rat Rat Rat Rat Rat
Wistar Wistar
Rat,
Wistar
Mouse
NAz
Mouse Mouse
DK Black A/Hc
Mouse
Swiss
15
60
4
Ingalls et al. Dekkerand Mehrizi Lechat et al.
1964a,b 1964 1964
m
Christie Bigriami el nl. A. Giroud el al. A. Giroud el al. King
1964 1962 1962a,b
! [
1962c,d
3
King
1962
1962
10-50
3
20-50
7-14
Hamartoma, abnormal orientation of limbs Absence of tail
50 50
1-15 1-21
None None
30 40
Seller FBlisati
1962 1962
Y
-
None Malformations of spine None
40 50 33
-
1962 1963a.b 1964a,b 1964 1964
d
Anomalies of cervical vertebrae and fontanels
Pliess Somers Cahen Cahen et d. Klein-Obbink and Dalderup Nishimura et al. Seller A. Giroud et al. A. Giroud et al.
250 800 50-250 1-2000
8-12 1-15
6-2 1
d
0-17
Kone
50 50-75
1-15 1-14
None Cleft palate, hare lip
64
50-75
1-14
Craniorachischisis. cataracts
50
100
2U F
2K 31
i2
sm
3
5
0 D
1962
m
1962 1962a
$
1962b
3
w
z
w +
TABLE VI (Continued) Order or class
Species
Mouse Mouse Mouse Mouse Mouse Mowe Mouse Mouse
Strain
Malformations
1-21 6-8 1-21 s21 5-21 1-24 1-14
20-200
&21
None
400-4000
0-21
Reduction in size of litters
?
Mouse
Monkey
Days of administration
Strong A 50 A 31-61 Schofield 4000 C57-Black 5 5 Strong A Swiss 62 CNRS 50 XLII/Gif ? 100-500
Mouse
Mouse Carnivores Cat Dog Dog Dog Dog Primates Monkey
Dose (mg/kg)
Swiss
None Hydrocephalus, clubfoot None None None Absence of tail, clubfoot Celosoma Amelia, micromelia
Mongrel Mongrel Mongrel Mongrel Macaque
250 0.5 100 200 30 60 50
1-15 la30 1-21 1-25 8-20 10-25 0-33
Macaque
10
3242
0
Resorption of fetuses
None Anomaly of ear, pes vurus Anomaly of caudal vertebrae Prenatal death Exeneephaly, hare lip None Sterility (absence of implantation) A d a , phocomelia, facial hemangioma
(%I 66 73 5 46 0 50 0
0 25 40 0 100 100
Author
Year
Woollam DiPaolo Somers Hagen Hagen Cahen Cahen et al.
1962a,b 1963 1963a,b 1963 1963 1964b 1964
Muradand Alvarenga Mauss and Stumpe Maussand Stumpe Lechat e t a l . Somers Weidman et al. Weidman et al. Delatour et al. Delatour et al. Lucey and Behrman Delahunt and Lassen
1964
E 3
1963
d
.= %
F 1963 1964 1963a,b 1963 1963 1965 1965 1963 1964
*m
FIG.8. Effect of thalidomide on tlLe chick emhryo. A. Chick embryo of 11 days treated with thalidomide (deposited in powder form); gross destruction of left leg resembling phocomelia. B. Embryo of 11 days treated with thalidomide dissolved in distilled water (hot water to procure solution) ; right leg is very underdeveloped and is reduced to 2 digits. (Mter Salzgeher arid Salaun, 1963b. Courtesy of Compt. Rend.)
312
RAYMOND L. CAHEN
FIG.9. Effect of thalidomide on the mouse and rabbit. A. Mouse with hare lip. B. Rabbit with anencephaly. The meninges can be seen in the posterior part. (After A. Giroud el ul., 1962a. Courtesy of Compt. Rend.)
FIG.LO. Effect of thalidomide in the monkey. Amelia on the left side. The right upper limb is reduced to a fleshy bud terminating in a digit composed of two phalanges. (Aft,er Delahunt and Lassen, 1964. Courtesy of Science.)
EXPERIMENTAL AND CLINICAL CHEMOTERATOGENESIS
313
doses and by performing laparotomy at niid-term, Delatour et al. (1965) have recently observed nialforniations : more comnionly facial and neural, rarely skeletal. I n the nionkey, large doses administered early in pregnancy only prevent implantation of the fetus (Lucey and Behrman, 1963). Sniall doses, on the contrary, given from the thirty-second until the forty-second day of gestat,ion may produce phocomelia, anielia, and even facial hemangiomas. (Delahunt and Lassen, 1964) see Fig. 10. In the armadillo thalidoniide affects the blastocyste (Marin-Padilla and Benirschke, 1963). In brief, thalidoniide is sonietinies teratogenic in certain species or strains of animals, but attempts to reproduce phoconielia in rodents have received a partial setback. 4. Hyperglycemic and Hypoglycenaic Agents Besides vitamin imbalances, disturbances of carbohydrate metabolism involving hot h hyperglycomia and hypoglycemia (Table VII) may be teratogenic. a. Hgperglycemic Agents. The teratogenic role of diabet,es is established beyond doubt (Joslin et al., 1940; Lawrence and Oakley, 1942; A. Giroud and Tuchniann-Duplessis, 1962; Tuchmann-Duplessis, 1963), but the percentage of enibryopathies in diabetic women varies considerably, froin 1 to 10% depending on the clinician reporting them (Cardell, 1953; Hagbard, 1961; J o s h et nl., 1940). In a series covering 853 infants of diabetic mothers and 1212 infants of normal niothers, Pedersen et ul. (1964) observed 13 times more malformations among the infants of the diabetic mothers. Ross and Spector (1952), Koskenoja (1961), and Barashnev (1964) produced alloxan diabetes experimentally in mice and observed an increase in the frequency of ocular anomalies. Kreshover e f ul. (19rj3) also observed certain malformations in the rat. Tuchmann-Duplessis and Mercier-Parot (1962) producing hyperglyceniia in the rat by injections of glucagon (300 pg) from the seventh to the ninth day, observed glaucomas, and after injecting higher doses (400500 pg) noted microphthalmias. De Meyer (1961) observed fetal resorption and various malformations by administration of galactose,2-deoxyglucose,and fluoroacetate. b. Hypoglycemic Agents. Insulin (see Table VII) is teratogenic, but its effects vary from animal to animal. In the mouse, insulin (Smithberg et al., 1956) produces exophthalmia, unibilical hernia, and fusion of the ribs. However, as Kalter and Warkany (1959) and A. Giroud and TuchmannDuplessis (1962) point out, the significance of these results is difficult to assess, for similar anomalies have been obtained in mice of the same strain purely by starvation.
TABLE VII HYPOGLYCEMIC AGENTS
Species
Strain
Resorption of fetuses
Dose Days of (mg/kg) administration
Malformations
(%)
Carbutamide Anophthalmia, hydrocephalus
Mouse Rat
800
10-12
Anophthalmia, anencephaly, spina bifida
200
11-12
None
250
11-12
Dimethylbiguanide Anophthalmia, anencephaly
50
Author
Year
De Meyer and Isaac 1958 Mathy Tuchmann-Duplessis and 195% Mercier-Par0t
Chlorpropamide Rat
Wistar
Rat
Rat Rat
Slbino
Mouse Rabbit Various Rabbit Various Rabbit Various
71 U 8U 0.14 U 20U 20U 21 U
2-21 6-9 8 6 el1 6-13
Chick Mouse
800
1-12
20
Tuchmam-Duplessis and 1961c Mercier-Parot
Insulin Mild skeletal anomalies None
1951 Lichtenstein et 01. Tuchmann-Duplessis and 195% Mercier-Parot Smithberg et al. 1956
Exencephaly, fusion of ribs, umbilical hernia Microcephaly, ectopia of spine None Microcephaly, agnathia, spina bifida Malformations of limbs and mouth Tolbutamide Spina bifida, anophthalmia
Tuchmann-Duplessis and 1959d Mercier-Parot
Chomette Chomette Brinsmade Landauer 40
1955 1955 1957 1953
Tuchmam-Duplessis and 195% Mercier-Parot
EXPERIMENTAL AND CLINICAL CHEMOTERATOGENESIS
315
In the rat, insulin produces few malformations. With slow-acting insulin, Lichtenstein et al. (1951) observed retardation of fetal development and skeletal anomalies. On the other hand, Tuchmann-Duplessis and Mercier-Parot (1958a) okserved no malformations in rats whose mothers had been treated during the critical period from the sixth to the ninth day of gestation. I n the rabbit, following administration of insulin between the sixth and the thirteenth days, Choniette (1955) and Brinsmade (1957) observed embryopathies : microphthalmia, spina bifida, and, in particular, absence of the vitreous body and agnathia. The hypoglycemic sulfonamides and the biguanides may be much more highly teratogenic, but this depends on the species. TuchmannDuplessis and Mercier-Parot studied carbutamide (BZ 55), tolbutamide, and chlorpropamide in the rat (1958a, 1959a), the mouse, and the rabbit (1963~) ; they observed that the t,eratogenic effect varies considerably depending on the hypoglycemic agent used. The most dangerous is carbutamide, which produces 23Q/, of anomalies in the rat, whereas chlorpropamide is not teratogenic. The malformations mainly concern the eye (microphthalmia, anophthalmia), with anomalies of the lens and retina and atrophy of the optic nerve (Tuchniann-Duplessis and Mercier-Parot, 1958a). I n the mouse (De Meyer and Isaac-Mathy, 1958; De Meyer, 1963) the embryopathies affect the eye (anophthalmia, cataract). The nervous system, the face, and the kidneys may also be attacked (De Meyer, 1961). In the rabbit, the malformations are polymorphic : celosoma, torsion of the limbs, shortening of the maxilla, cleft palate, umbilical hernia (Tuchmann-Duplessis and Mercier-Parot , 1963a). Among the biguanides, diniethylbiguanide is by far the most teratogenic (Tuchmann-Duplessis and Mercier-Parot, 1961~). In the mouse and rat, the malformations affect mainly the eye (microphthalmia, anophthalniia) and rarely the central nervous system (anencephaly, spina bifida). In man, many cases of teratogenesis produced by hypoglycemia agents have been reported. Y. Larsson and Sterky (1960) described the case of a woman treated with tolbutamide, who gave birth to a n infant with multiple and neural cardiac malformations. These results have been disputed by Ghanem (1961). G. D. Campbell (1963) incriminated chlorpropamide in the etiology of human malformations. There are differences of opinion concerning the action of insulin in women. Embryopathies have been reported by Wickes (1954) and Sobel (1960), but none were observed by Cardell (1953), or, more recently, by Sterne (1963), who conducted a long clinical investigation. It seems that insulin is the least teratogenic of the known hypoglycemic agents.
316
RAYMOND L. CAHEN
B. DRUGSWHOSETERATOGENIC EFFECTIN MANHAS NOT BEEN CONFIRMED Many drugs have been suspected of having a teratogenic potentiality in laboratory animals or man, but their effect in man has not been proved. In most cases a teratogenic effect is certainly absent, but, in others, prudence demands that research be intensified in order that every guarantee may be given to the obstetrician. 1. Teratogenic Effect in Laboratory Animals Attention has been drawn to the teratogenic effect of various drugs as a result of trials, some more systematic than others. a. Mineral Salts. Salts of barium (Fabre et al., 1958), strontium (Baba and Araki, 1959), mercury (Murakami et at., 1955), and lead (Ridgway and Karnofsky, 1952; Murakami et al., 1955), sodium fluoride (Fleming and Greenfield, 1954; Knouff et al., 1935-1936), and selenium salts (Westfall et al., 1938) are teratogenic in the chick. An organic mercury salt (the plenylacetate) is teratogenic in the mouse when administered per vaginam (Eastman and Scott, 1944; Murakami et al., 1955). b. Alkaloids. I t was first shown by Ancel (1950) that several alkaloids used in therapeutics are teratogenic. Only a small number of these substances has been tested in animals: caffeine in the mouse (Nishimura and Nakai, 1960), ergotamine in the rat (Shelesnyak and Davies, 1955; Sommer and Buchanan, 1955), nicotine (Vara and Kinnunen, 1951 ; Landauer, 1960; Nishimura and Nakai, 1958), quinine in the rat (West, 1938; Winckel, 1948; Neuweiler and Richter, 1964), veratrum alkaloids in lambs (Binns et al., 1964; Keeler and Binns, 1964), and reserpine and deserpidine in the rat (Budnick, 1955; Tuchmann-Duplessis and Mercier-Parot, 1956, 1961a; G. B. West, 1964; Werboff and Kesner, 1963) and the rabbit (Kehl et al., 1956a,b; Arnaud, 1963). c. Neuroleptics. Chlorpromazine is abortive (Chamhon, 1955; Arnaud, 1963; Courrier and Marois, 1953; Murphee et al., 1962). G. B. West (1964) and Roux (1959) observed rare malformations in the rat affecting the nervous system and palate. In mice of strain C57 BLlo treated throughout gestation, Ordy et al. (1963) observed a decrease in fertility, a reduction in the number of young in the litter, but no malformations. On the other hand, another phenothiazine derivative, prochlorpromazine, produced numerous nmlformations in the rat (Roux, 1959) : cleft palate, craniorachischisis. Levopromazine and promazine likewise produced fetal malformations in the rat (Murphy, 1962; ROUX,1959) : ectopic kidneys. These results are inconstant, however. Despite all these observations in animals, the phenothiazines and, in
EXPERIMENTAL AND CLINICAL CHEMOTERATOGENESIS
317
particular, chlorproniazine have never, to our knowledge, induced human embryopathies. Aniong the other neuroleptics, suspicion has also fallen on glutethimide (a-phenyl-a-ethylglutarimide). Tuchniann-Duplessis and Mercier-Parot (1963c,f) studied the action of increasing doses in three species-rat, rabbit, and mouse-and found that it is abortive but never teratogenic. Another glutariniide derivative, aturbane[a-phenyl-a-(0'-diethylaniinoethyl)-glutarimide] is embryotoxic but not teratogenic (Tuchniann-Duplessis and Mercier-Parot, 1964a). A systematic investigation conducted by Favre-Tissot et al. (1964) in pregnant women led to the conclusion that not one of the classic neuroleptics, whether associated with sedatives or not, gives rise to a teratogenic effect. d. Sedatives. Pentobarbital exhibits antimitotic activity in the niouse (Setala et al., 1963). According to McColl et al. (1963) it produces skeletal malformations in the rat. No teratogenic action has ever been described clinically. Meprobaniate has been the cause of impairing intelligence in the rat (Werboff and Kesner, 1963). This has been denied by Berger (1963). e. Monoamine Oxidase Inhibitors. Tuchniann-Duplessis and MercierParot (1961b) observed that doses corresponding to those used clinically produced resorption of the fetus but no malformations. However, prolonged adniinistration, for 12 to 15 months to rats of both sexes, reduces fertility and produces a disturbance of sexual behavior of the Fz generation. The feniales refuse the niale and practice a forni of pseudocopulation with each other (Tuchniann-Duplessis and MercierParot, 1963d). f. Hypocholesteremic Agents. Triparanol produces multiple malforniations in the rat, notably of the central nervous system and the face (Roux and Dupuis, 1961). g. Analgesics and Antiphlogistics. A teratogeiiic effect has been described in the rat, but only in enornious doses which cannot be conipared with clinical therapeutic doses; sodium salicylate (Jackson, 1948; Warkany and Takacs, 1959; Gulienetti et al., 1962; S. Larsson et al., 1963) and phenylbutazone (Triebold et al., 1957) were used. Recent tests have denionstrated no teratogenic effect of acetylsalicylic acid at the inaxinial tolerated dose (250 nig/kg) in both the rabbit (Earley and Hayden, 1964) and the rat (West, 1964). h. Chemotherapeutic Agents. i. Bacteriostatic suljonamides. Sulfadiazine (Bass et al., 1949, 1951) is toxic to the fetus but is not teratogenic; the same is true of sulfanierazine (Bass et al., 1949, 1951). K. G. Green (1963) drew attention to the possible teratogenic effect of Biniez (a combination of sulfadimidine and sulfamoprine) in the pregnant woman. Paget and Thorpe (1964) have recently observed that sulfanioprine (sulfadiniethoxypyrimi-
3 18
RAYMOND L. CAHEN
dine) alone, when administered to pregnant rats and mice, produces malformations of the teeth and skull in the young. No malformations are observed in the case of the rabbit. With the exception of Gantrisin (sulfisoxazole), reported to be teratogenic by Ziamim and Findland (1957), no clinical embryopathy has yet been described following the administration of the sulfonamides which are still used on a wide scale. ii. Antibiotics. Penicillin (Filippi and Mela, 1957; Carter, 1963) and tetracycline (Mela and Filippi, 1957) have been accused of producing skeletal malformations (Carter and Wilson, 1962) (micromelia, syndactyly). The small number of trials and the lack of the experimental conditions make these conclusions dubious (Walford, 1963). Hurley and TuchmannDuplessis (1963) observed no malformations in the rat even with doses 100 times larger than doses used clinically. Atrican (a-thenoylamino-2-nitro-5thiazole), a trichomonacide, is not teratogenic in the rat, the mouse, or the rabbit, even in a dosage toxic to the pregnant female (Tuchmann-Duplessis and Mercier-Parot, 1964d). In mice streptomycin does not induce fetal malformations in the central nervous system (Ericson-Stradnvik and Gyllensten, 1963; Rubjn et al., 1951). i. Antihistamines. Various antihistamines, namely Benadryl, pyrolazote (Shelesnyak and Davies, 1955), and cyclizine (Tuchmann-Duplessis and Mercier-Parot, 1964e) are teratogenic in the rat, the mouse (anophthalmia), and the rabbit (spina bifida, encephalocele) ;negative findings were reported in the mouse by Goldstein and Hazel (1955). The responsibility of meclizine and chlorcyclizine in producing brachygnathia, cleft palate (Kendrick and Weaver, 1963), and micromelia has been recently confirmed in the rat (King et al., 1965). However, neither McBride (1963) nor Mellin (1963) found any human embryopathy in significant clinical investigations. j . Antidepressants. I n the rabbit, imipramine has been claimed to be teratogenic by Robson and Sullivan (1963), but noriteratogenic by Larsen (1963). Clinically, scrutiny of all the cases published (13,107) in 1963 confirmed the teratogenic innocuousness of imipramine. This illustrates the difficulty of extrapolating to man the results obtained in animals. In the rabbit and mouse, imipramine is rapidly metabolized into numerous degradation products, but, as Brodie and Erdos (1962) have shown, it is not converted into demethylimipramine, the active antidepressant metabolite, in man. As Fraser (1963) has remarked, the crucial demonstration of the teratogenic effect of a drug must be sought in man. 2. Unjustifiable Suspicion of Teratogenesis in M a n Several medical publications have reported human embryopathies caused by meclizine, phenmetrazine, and dominal.
EXPERIMENTAL AND CLINICAL CHEMOTERATOGENESIS
319
a. Meclizine. According to G. I. Watson (1962), James (1963), King (1963) and G. David et al. (1963), this antiemetic, which is frequently used a t the beginning of pregnancy, has been responsible for fetal malformations. On the other hand, Carter and Wilson (1962), Lask (1962), Lenz (1962b), Leck (1962), Weicker et al. (1962), McBride (1963), BieringSorenson (1963), Diggory (1964), Moraiidi and Marchesoni (1963), and, in a study of 3200 pregnancies, Mellin (1963) and Mellin and Katzensteiri (1964) observed no fetal malformations. A more recent study by Smithells and Chinn (1964) and Smithells et al. (1962) comprising 219 cases, has confirmed the absence of its teratogeriic effect in women. Furthermore, Bovet-Nitti et al. (1963) have shown experimentally that meclizine is not teratogenic in the rat. Burns (1965), who used smaller doses (25 mg/kg/day) and gave the drug to three generations of rats as in the F.D.A., test, observed no congenital malformations. b. Phenmetrazine. The first report of the teratogenic action of this drug was given by Powell and Johnstone (1962), who described two cases of fetal malformations : diaphragmatic hernia appeared in two brothers, not twins, whose mother had been treated with phenmetrazine during her pregnancies. Two other cases of malformations subsequent to the same treatment have been reported, one by Lenz (1962b), who observed the same fetal malformation, and the other by Moss (1962), who described a deformity of both lower limbs and of the hand. On the other hand, Notter and Delande (1962) found no anomalies in the infants of mothers who had taken phenmetrazine during pregnancy. This problem has not been discussed in any more recent clinical publications. I n the course of an experimental investigation of the teratogenic effect of another anorexic agent, 2-diethylarninopropiophenonel given a t the critical teratogenic period to rats and mice, no embryopathy was observed (Cahen et al., 1964). These results have been confirmed by clinical experience. c. Dominal. The psycholeptic drug, dominal, [N-(3-dimethylaminopropy1)-thiophenylpyridylaniine hydrochloride] has recently been reported by Tuchmann-Duplessis and Mercier-Parot (1964a) as responsible for multiple malformations in an infant whose mother had taken the drug since the beginning of pregnancy. To our knowledge, no other case has been reported. WHICH PASSTHROUGH THE PLACENTAL BARRIERBUT ARE C. DRUGS NOT TERATOGENIC Although many analgesics, such as morphine (Snyder, 1949) and meperidine (Way et al., 1949; J. Barnes, 1947) and central depressants
320
RAYMOND
L.
CAHEN
such as scopolamine (Snyder, 1949) chloral hydrate (Bernstine et al., 1954), and barbiturates (Dille, 1934), anticonvulsants (Janz and Fuchs, 1964) and antibiotics pass through the placental barrier, they do not produce congenital malformations. The reader is referred to Baker’s (1960) excellent review of this subject. All that is presently known is that the placenta behaves toward most drugs as an inert barrier with lipoid properties, permitting liposoluble substances to pass freely through the placental barrier. Nevertheless, it is important to note that, without causing malformations, anticoagulants administered to the mother may give rise to severe hemorrhages in the fetus (Merger et al., 1961). This is true of anticoagulants of the dicoumarin (Mahairas and Weingold, 1963) and phenylindanedione (Kraus et al., 1948; R. Joseph et al., 1960) series. In two exhaustive reviews, Bocquet (1961, 1964) has recently examined the risk of hemorrhaging to the infant as a result of anticoagulant therapy during pregnancy. Furthermore, vitamin K and nienadione have been responsible for a case of hyperbilirubineniia in a newborn infant (Hill et al., 1961). I t is best to avoid the use of dicouniarin derivatives during pregnancy and to replace them with heparin, which does not pass through the placental barrier. VII. Nature and Mechanism of Action of Teratogenic Drugs
The preceding study has demonstrated the diversity of the nature of the teratogenic drugs. The pharmacologist’s problem is to draw general conclusions concerning the nature and specifity of the teratogenic action, its relationship to chemical structure or to pharmacological action, and, above all, the niechanism of teratogenesis. Only if the cause of embryopathies is known can they be prevented.
A. NATUREOF
THE
OBSERVED MALFORMATIONS
The malformations produced by a teratogenic agent are polymorphic (A. Giroud and Martinet, 1960). All three parts of the gastrula may, in fact, be affected. In most cases the central nervous system is involved, with the formation of anencephaly by hypervitaminosis A (Giroud and Martinet, 1957; Baba and Araki, 1959) and of spina bifida by trypan blue (Gillman et al., 1948; Tuchmann-Duplessis and Mercier-Parot, 1959d; Cahen et al., 1964; Cahen, 196413). However, neither thalidomide nor iinuran act a t this level. The face is less often involved; cleft palate is produced by cortisone (Fraser and Fainstat, 1951; Jost, 1956; Kalter, 1954), hydrocortisone (Gunberg, 1957), estradiol (Nishihara, 1958), hypervitaminosis A (Cohlan, 1953; A. Giroud and Martinet, 1955b), and by a deficiency of folic acid (A. Giroud, 1952) or of riboflavin (Walker and Crain, 1961). Hydrocephalus
EXPERIMENTAL AND CLINICAL CHEMOTERATOGENESIS
32 I
is induced in niice by galactoflavin, an antimetabolite of riboflavin (Kalter, 1963). Malforniat ions of the eye are coinnion : anophthalniia, induced by vitaniin A deficiency (Hale, 1935); niicropthalniia by the same deficiency (Zunin and Borbone, 1955) and also by trypan blue (Tuchmann-Duplessis and Mercier-Parot, 1959c; Cahen et al., 1964); cataract caused by thyroxin (A. Giroud et al., 1951) or by hypervitaminosis A (Giroud and Martinet, 195510). They niay also be observed in association with the synthetic hypoglycemic agent, tolbutaiiiide (De Meyer and Isaac-Mathy, 1958; Tuchmann-Duplessis and Mercier-Parot , 1958a). Malforniations of the ear are unconiiiion: absence or dysplasia of ear by hypervitaminosis A (Kuzukawa, 1960) and thalidomide (Lenz, 1962a,b). Various anomalies of the skeleton are found (Millen, 1962): club-foot by trypan blue (Gillman et al., 1051) or thalidomide (Pfeiffer, 1963); absence of tail and axial torsion by trypan Hue (Tuchniann-Duplessis and Mercier-Parot, 1 9 5 9 ~Cahen ; e2 al., 1964; Cahen, 1964b); syndactyly by azazerine (Thiersch, 1957b); polydactyly by nitrogen mustard (Murphy and Karnofsky, 1956). The typical phoconielia of thalidoniide in the huinan fetus (Lens, 1962a,b; McBride, 1962; Pfeiffer and Kosenow, 1962) has not been produced in laboratory aniniak, with the exception of the chick (Salzgeber and Salaun, 1963a,b). I t is of interest to note that a recent study by Tuchmann-Duplessis and Mercier-Parot (1964c,d) has shown that another drug, Imuran (azathioprine) is capable of producing linib anomalies in the fetus (polydactyly, syndactyly, amelia, and rarely, phoconielia). Teratogens niay also give rise to visceral nialformations : umbilical hernia by insulin (Smithberg el al., 1956); atresia of the anus by insulin (Brinsniade et al., 1956). The cardiovascular system is also often affected by actinoniycin D (Tuchniann-Duplessis and Mercier-Parot , 1959b,d), hypervitaminosis A (Roux and Dupuis, 196l), and, in particular, vitamin A deficiency (J. G. Wilson and Warkany, 1949), folic acid deficiency (Monie et al., 1954), and chloranibucil (Monie et al., 1954). The urogenital system is frequently involved : hydronephrosis by hypervitaminosis A (A. Giroud et al., 1958; Roux and Dupuis, 196l), deficiency of vitaniin A (Wilson and Warkany, 1948) or of fulic acid (Monie el al., 1954); horseshoe kidney by carbutamide (De Meyer and Isaac-Mathy, 1958) and by thalidomide (Roux et al., 1965). Even the peripheral liiiib vessels may be affected by epinephrine and hypophyseal preparations (Jost, 1950b), and by pantothenic acid deficiency (A. Giroud et al., 1955). Whatever chemical agent is used, a teratological complex that affects most tissues of the embryo may be induced. Plurifocal malformations niay
322
RAYMOND L. CAHEN
involve many systems. As Warkany (1963) has demonstrated, a drug may produce a complete sFectrum of congenital malformations. B. SELECTIVITY OF
THE
EFFECTS
Most drugs, if administered at the same critical period of organogenesis, cause the same embryopathies. Consequently, hypovitaminosis A produces the same fetal malformations as hypervitaminosis A (Millen and Woollam, 1963). There are exceptions to this rule: some drugs produce specific malformations in a definite organ: cortisone (palate), vitamin A (central nervous system, palate, eye), thalidomide and imuran (long bones of the skeleton), steroids (eye, face). It is unnecessary to specify that these comparisons are valid only if the drugs have been given a t the same stage of embryonic development. C. RELATIONSHIP BETWEEN TERATOGENIC ACTION AND CHEMICAL OR PHARMACOLOGICAL ACTION STRUCTURE The various drugs which produce human embryopathies differ so greatly in their chemical structure (alkaloids, nitrogen mustard, purine bases, steroids, terpenes, sulfonamides, dioxo-2,6-piperidine derivatives) that their teratogenic action cannot be correlated with a particular chemical moiety or a specific function. 1. Relationship between Chemical Structure and Teratogenic E$ect
Even when restricted to a particular chemical agent, such as that of thalidomide, the systematic studies of Faigle et al. (1962). Williams (1963), Bignami et al. (1963), Fabro et al. (1963, 1964a,b,c), and Lechat et al. (1964) have not yielded significant results. a. Glutarimide Derivatives. Thalidomide is a-phthalimidoglutarimide, or phthalimidi-3-dioxo-2, 6-piperidine. It was logical, therefore, to compare the teratogenic action of various known glutarimide derivatives. Neither glutethimide (a-phenyl-a-ethylglutarimide) nor aturbane [phenyl-a-(8’-diethylaminoethy1)-glutarimide] which have been used therapeutically for several years, is teratogenic (Keberle et al., 1962; TuchmannDuplessis and Mercier-Parot, 1963c, 19648,; McColl et al., 1963). Similarly the 3-substituted amino-3-glutarimide, derived from thalidomide by hydrolysis (Faigle et al., 1962) is not teratogenic (Fabro et al., 1964a,b,c). Therefore, with the exception of thalidomide, no other 3-substituted glutarimide has been found to be teratogenic. b. Phthalimide Derivatives. Thalidomide may also be considered as an N-substituted derivative of phthalimide. Accordingly, the teratogenic activity of the alkyl- and amido substitution derivatives of N-phthalimide has been investigated.
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None of the N-alkylpthaliiiiides is teratogenic, neither the methyl (Lechat et al., 1964) nor the isopropyl, n-butyl, isobutyl, n-aniyl, or isoamyl substitution products (Bignami et al., 1962, 1963). None of the N-aiiiidophthalimides that have been studied is teratogenic ; neither pht halimido-3-glutariniide nor pht halimido-4-glutarimide is teratagenic in the rat, although the latter is teratogenic in the chick (Misiti et al., 1963). Among the other thalidomide honiologs, neither phthalimidoaspartamide, resulting from substitution of the glutariniide moiety by aspartamide, nor the anhydride of N-phthalimidoglutaric acid, an oxygenated homolog of thalidomide, is teratogenic (Fabro et al., 1964a,b,c; Fabro, 1964). Neither hexahydrothalidomide (Wuest, 1964) nor succidoglutarimide, which have lost their benzene ring, is teratogenic. In short, the structure responsible for the teratogenic effect in the thalidomide series is not yet known. It may be conjectured that it is phthalimide substituted at the nitrogen atom by a hexagonal grouping of unknown nature. 2. Relationship between Pharwtacologzcal Action and Teratogenic Efect
The teratogenic effect and the pharmacological action are completely independent. The most striking examples of this are the hypoglycemic sulfonamides, of which only one (carbutamide) is teratogenic. The enibryopathic action is not linked with the hypoglycemic property. Neither is the teratogenic action linked with antitumor properties. Chlorambucil and 6-niercaptopurine exert, a more marked emhryopathic action, while triethylenemelai.riine and aniinopterin are more embryotoxic, bisulfan and cyclophosphamide are more sterilizing than teratogenic. Only one neuroleptic from the group of phenothiazine derivatives, prochlorpromazine, is teratogenic. As we have seen above, of all the known hypnotics only one is teratogenic. Among the anticoagulants, only the dicouniarin derivatives give rise to fetal hemorrhages. We do not know the cause of this specificity in the enibryopathic action of drugs. Nevertheless, two working hypotheses may be proposed : (a) selective catabolic degradation into teratogenic metabolites, and ( b ) the presence of particular physical properties (polarity, molecular weight, liposolubility) determining ability to cross the placental barrier. These hypotheses should be explored.
D. THEMECHANISM OF TERATOGENIC ACTION As may be gathered, the mechanism of teratogenic action has been the subject of many investigations. Only with its elucidation will it be possibIe to foretell with a reliable degree of assurance the teratogenic potentiality of
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a drug. Investigations of this type have been carried out chiefly on thalidomide, with special emphasis on its metabolism. A recent paper by Burns (1965) emphasizes the importance of data relating to the metabolic transformation of a drug in the evaluation of its teratogenic potentialities. We shall begin our study of this problem by describing the numerous attempts that have been made to identify the metabolite responsible for the teratogenic effect of thalidomide. (Recently Narrod et al. (1965) found that norchlorcyclizine is the metabolite responsible for the teratogenic effect of nieclizine in rats.) An opposing hypothesis, however, is that embryonic malformations may be produced by conipetit)ive inhibition of cellular components. The essence of this concept is that an antimetabolit,e, if substituted for an essential component, may interrupt a chain of biochemical reactions, and thereby disturb the morphogenesis of the embryo. Even the transient action of an antimetabolite is sufficient because of the very low reserves of metabolite in the embryo, especially when the action takes place a t the crucial periods of fetal development (A. Giroud and Tuchmann-Duplessis, 1962). We shall summarize the investigations of competitive inhibition of the essential constituents of the cells (glutamic acid, folic acid, pantothenic acid, which were observed in the course of fetal enibryopathies. I n the last section we shall briefly review the other hypotheses elaborated to explain the mechanism of action of teratogenic factors. 1. T h e Metabolism of Thalidomide
A knowledge of thalidomide metabolism is essential, for the specific effects of this drug may be due to a metabolite. Thalidomide is a rapidly acting tranquilizer possessing no anticonvulsant properties (Kuhn and Van Maanen, 1961), no secondary action, and practically no toxicity in animals (Somers, 1960a) and man; nevertheless, it is teratogenic in therapeutic doses (Lenz, 1961, 1962a,b; McBride, 1961). A few micrograms of circulating thalidomide (0.9 pglml) is sufficient to produce malformations in the human fetus (Beckmann and Kampf, 1961). It may therefore be suggested that the teratogenic effect of thalidomide is due to its transformation into a metabolite teratogenic to the embryo. a. Hydrolysis Products. Using C'*-labeled thalidomide, Bassi1 et al. (1962) demonstrated that the most important metabolites are produced by hydrolysis of the peptide bonds present in the thalidomide molecule. Thalidomide (V) (N-phthalimidoglutarimide)is hydrolyzed spontaneously at a physiological pH to yield 12 hydrolysis products (Williams, 1963; Schumacher et al., 1964). A list of these metabolites isolated by Williams and Parke (1964) is given in Table VIII. The first metabolite to be isolated was 4-phthaliniidoglutarimic acid or phthaloylisoglutamine (X) (R. L. Smith et al., 1962), an essential metab-
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TABLE VIII HYDROLYSIS PRODUCTS OF THALIDOMIDE V X XI XI1 XI11 XIV XV XVI XVII XVIII XIX XX XXI
a-Phthalimidoglutarimide or thalidomide 4-Phthalimidoglutaramic acid or phthaloylisoglutamine 2-Phthalimidoglutaramic acid or phthaloylglutamine a-(o-Carboxybenxamido) glutarimide 2-Phthalimidoglutaric acid or phthaloylglutamic acid 4-(o-Carboxybensamido) glutaramic acid 2-(o-Carboxybensaniido) glutaramic acid 2-(o-Carboxybensamido) glutaric acid Phthalic acid a-Aminoglutarimide 4-Aminoglutaramic acid or ivoglutamine 2-Aminoglutaramic acid or glutamine 2-Aminoglut,aric acid or glutamic acid
olite of thalidomide in nian, the rat and the rabbit. The other products isolated are probably nothing more than the products of spontaneous hydrolysis (Williams, 1963). The various processes of metabolic degradation of t halidoniide are summarized in Fig. 11 (Williams and Parke, 1964). When thalidomide is given to the rat and the rabbit, its metabolites may be isolated from the urine, the blood, and the brain 1 hour after administration. The teratogenic effect of these inetabolites has been studied by Fabro et al. (1964a,b,c) in female rabbits of two particularly sensitive strains: New Zealand and Chinchilla. None of these metabolites proved to be teratogenic, whereas thalidomide had been teratogenic in both strains chosen. As Fabro et al. (1964a,b,c) note, all of these metabolites are polar, and possibly do not penetrate as far as the fetus. The recent researches of Keberle et al. (1965) have resulted in the isolation of other metabolites by means of incubation techniques. These include three monocarboxylic acids, two dicarboxylic acids, and one tricarboxylic acid. Only one of these, phthalylglutamic acid (VI), is embryotoxic. b. Hydroxylation Products. R. L. Smith et al. (1962) have also observed hydroxylation products of thalidomide, i.e., derivatives of 3-hydroxyphthalic acid, and suggest that thalidoniide undergoes hydroxylation in vivo. These products have been found in the urine of the rabbit (Schumacher el al., 1964). The teratogenicity of these hydroxylated derivatives on the hen’s egg has been cIaimed by Boylen et aZ. (1963), but this experimental material has been questioned by Willianis and Parke (1964). c. Nonmetabolized Thalidomide. i. Pregnant rat. Labeled thalidoniide administered to pregnant rats by gastric tube is absorbed very rapidly and
I
c
//
I
C Q H CQH C Q H
(XIX)
(XXI)
(XX)
FIG.11. The pathways of hydrolysis of thalidomide. (After Williams and Parke, 1964. Courtesy of Ann. Rev. Phannacol.)
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almost completely (Beckmann, 1962; McKenzie and McGrath, 1962). The niaxiniurn concentration is attained in the blood in 15 minutes, in the brain in 1hour. Thalidomide is also excreted in the urine, from which it can be recovered intact (R. L. Smith et al., 1962). The studies of Faigle et al. (1962) in the pregnant rat showed that 80% of the labeled thalidomide administered is excreted equally in the urine and feces within 24 hours. In the dog, 28% radioactivity is found in the urine and 64% in the feces. In this species, too, 97% radioactivity in the feces is due to intact thalidomide. ii. Fetus. Thalidomide passes through the placental barrier. Closely similar levels of radioactivity in the fetus and the mother after administration of C14-labeled thalidoniide to thc latter were found by E’aigle et al. (1962) in the rat, by Beckniann (1963) in the rat aiid mouse, and by Koransky aiid Ullberg (1964) in the mouse. Determination by Fabro et al. (see 1964a,b,c) of the radioactivity in blastocytes taken the tenth day of gestation from the uterus of a pregnant rabbit, which had received 6 nig/kg of labeled thalidomide per os (see 1964a,b,c), reveaIed that 70% of the thalidomide was intact. Similar results were obtained by Keberle et al. (1965) with blastocytes taken between the sixth and eighth day from a pregnant rabbit receiving 100 mg/kg of labeled thalidoniide. d . Differences of Metabolism in Varying Species. Different species of animals axe known to react differently to certain classic drugs. The reason for this phenomenon remained obscure until it was demonstrated that it could he attributed to differences of drug metabolism in aniinals of different species, or even of different strains. These differences are most likely related to genetic factors (A. Giroud, 1963b). The variations in the teratogenic effects of thalidoniide among animals of different species are probably due to differences in its metabolic degradation (Cahen, 1964b; Brent, 1964; Burns, 1965). Before significant conclusions can be drawn, the metabolism of thalidoniide must be investigated in different species, especially teratogenosensitive ones. The comparison of thalidoniide metabolites in sensitive species (man and rabbit) and in resistant species (rat) has already been the object of several studies. R. L. Smith et al. (1962) isolated from the urine of man, rabbit, and rat the same two essential metabolites, N-phthalylisoglutamine arid a derivative fluorescent ultraviolet light, probably derived from hydroxy-3-phthalic acid. More recently, from the urine of man and the rat, Beckmann (1963) isolated eight metabolites of identical nature but in different proportion. The most abundant metabolite in man is N-phthalylisoglutaniine; in the rat, N-phthalyoylglutarimide. Research along these lines is worth pursuing. No decisive conclusions can yet be drawn froin these long and difficult
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studies; whether thalidomide or one of its metabolites is the teratogenic factor cannot yet be ascertained. Study of the mechanism of the teratogenic action is more the concern of enzymology and biochemistry than of analytical chemistry. Recently, advances have been made by using techniques based on quantitative determinations in the blastocyte and fetus. One vital fact seems to have been forgotten by some researchers: No valid conclusions can be drawn from a study unless it is carried out on a teratogenosensitive species and at a teratogenosensitive stage. e. Direct or Indirect Action of Thalidomide on the Fetus. One last point remains to be considered: Once thalidomide has crossed the placental barrier, are its teratogenic effects produced by direct action on the embryo or by a metabolite formed in the tissues of the embryo. 2. Thalidomide metabclites in the embryo. As stated above, Fabro et al. (1964a,b,c) found 70% intact thalidoniide in the blastocyte of the rabbit a t the tenth day of gestation. In addition, the same authors isolated five of its hydrolysis products, i.e., a(a-carboxybenzaniido)glutarimide, 2-phthaliinido-glutarimic acid, 4-phthalimido-glutariniic acid, 2-phthalimidoglutarimic acid, and phthalic acid. From these results it appears that during the sensitive period of teratogenesis the blastocyte accumulates thalidomide metabolites. However, this study does not explain the passage of thalidomide into the blastocyte nor does it state specifically whether the metabolites described are teratogenic. ii. Penetration of a thalidomide metabclite into the embryo. A more elaborate and significant investigation has just been undertaken by Keberle et al. (1965) using the rabbit blastocyte, but continuing observations until the fetal stage (twelfth day). These authors have shown that nionocarboxylic acids, i.e., catabolites of thalidomide do penetrate into the fetal membranes and consequently may induce fetal malformations. The polarit,y of these metabolites is such that they are liberated slowly from the fetal membranes and they tend to accumulate to a greater degree than thalidomide itself. “In this way, all the metabolites are smuggled into the embryo, whereas they would not be able to penetrate were they administered as such” (Keberle et al., 1965). These observations provide an excellent approach to the study of the mechanism of the teratogenic effect of thalidomide. However, the essential question remains unanswered : Is the teratogenic factor thalidoniide itself or one of its metabolites? iii. Metabolism of thalidomide in the fetus. No study of this problem has been completed. The observations of Jondorf et al. (1959), Fouts and Adamson (1959), and Brodie and Erdos (1962) have shown, however, that the microsomal enzymes of the liver are completely absent from the fetus of the guinea pig and rabbit. Hence, the fetus is not equipped for metabolizing drugs.
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2. T h e Competitive Inhibition of Glutaniic Acid Assuming that thalidomide is hydrolyzed in vivo into glutamic acid and homologs of glutaniine, it is tempting to suppose that the degradation products of t halidoniide behave as competitive antagonists of these amino acids, which are found normally in the body (Bassi1 et al., 1962). This hypothesis has also been considered by Faigle et al. (1962), who attempted to denionstrate that thalidomide’s teratogenic effect is due to its interference with the glutaniic acid or glutamine metabolism. These findings may be paralleled with those on antinietabolites such as azaseririe arid D.O.N. (Murphy, 1960; Thiersch, 1957b). The teratogenic effect also results from inhibition of the synthesis of glutaniine, a n essential metabolite of the cell. In other words, the teratogeiiic action of thalidoniide was attributed to the intervention of one of its metabolites behaving as a glutaiiiic acid antagonist. This hypothesis is based on the fact that certain thalidoniide nietabolites (Nos. XIII, XIV, and XVI in Table VIII) inhibit certain enzymes (glutaniinase, glutamate synthetase, and dehydrogenase) actively involved in glutamic acid nietabolisni (Fabro et al., 1964a,b,c). Boylcn et al., (1963) observed that treatment with N-glutaniine protects the chick embryo against the teratogenic effect of hydroxylated thalidoiiiide derivatives. Narrod and King (1963) studied the effect of thalidoniide on the pyridine nucleotides of the liver [nicotirianiideadeiiiiie denucleotide (NAD) arid nicotinamideaderiirie nucleotide phosphate (NADP)] arid observed no inhibition of glutaniine. On the other hand, Misiti et al. (1963) observed a teratogenic effect 011 chick and rat embryos with A/‘-phthaloyl derivatives of aspartic acid, as well as with the same derivatives of glutaniic acid.
3. Competative Inhibition of Folic Acid Certain thalidoinide metabolites resemble folic (pteroylglutamic) acid in their cheiiiical configuration. Accordingly, Keinper (1962a), who studied the effects of thalidoniide on the sexual development of the chicken, suggested that its teratogenic effect might be explained by the competitive inhibition of folk acid. This hypothesis is likewise supported by recent studies of the excretion of forniinoglutamic acid (Nystroni, 1963), but no other observations have confirmed these findings. On the contrary, neither Evered and Randall (1963), who used an enzyniic method in vitro, nor FQlisati (1962), who determined the teratogenic activity t n vivo, observed any antifolic effect of t halidoniide. In order to explain the teratogenic effect in aniiiials arid inan of the aiititunioral folk acid antagsnists, arniiiopterin and X-inethylfolic acid, it
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has been suggested that a folic acid deficiency may disturb prenatal development. I n fact, it seems that the folic acid requirements of the embryo are much greater than those of the adult (Nelson, 1960). 4. Pantothenic Acid Deficiency The teratogenic effect of thalidomide has also been attributed to panthothenic acid deficiency, but attempts by Tuchmann-Duplessis and Mercier-Parot (1964a) to neutralize this action by administration of the vitamin proved ineffective. Recently, Toivanen (1964), measuring the pantothenic acid content of the liver, noted a decreased content of this vitamin in the chick embryo and rat fetus after administration of thalidomide to the mother. Similar experiments on another animal species would be more significant. 5. Miscellanecus Hypctheses For the sake of completeness we shall now cite numerous suggestions which have been put forward in an attempt to explain the teratogenic effect of certain drugs. a. Alteration of Protein Metabolism. Changes in the metabolism of maternal proteins have been implicated in the teratogenic effect of trypan blue (Gillman et al., 1948, 1951) but not of Congo red (Beaudoin, 1964). The serum a- and P-globulins are raised (Langman and Van Drunen, 1959) and the albumin fraction is lowered (Brown et al., 1963). On the other hand, in the case of cortisone, Molteni and Loevy (1963) observed no correlation between the changes in the protein metabolism and the appearance of cleft palate. Finally, it has been suggested that the teratogenic effect of alkylating antitumor agents is the result of denaturation of nucleoproteins and precipitation of deoxyribonucleic acid, similar to that produced by the action of irradiation (Tuchniann-Duplessis and Mercier-Parot, 1964a). b. Changes in L i p i d Metabolism. It has not been possible to demonstrate that diabetic embryopathies and fetal malforniations caused by hyper : and hypoglycemic agents are the direct result of the disturbance of carbohydrate metabolism. However, in connection with their observation of the teratogenic effect of triton (W.R. 1339) (see Fig. 12), a drug producing changes in the lipoproteins comparable to those found in diabetic states, Tuchmann-Duplessis and Mercier-Parot (1964~)suggest that a disturbance of lipid metabolism may be implicated in the enibryopathology of diabetic states. c. Selective Destructive or Antimitotic Action. Thalidomide may act on fetal cell division (Hunter, 1962). Cultures of human leucocytes are inhibited by thalidomide, but not by phenobarbital or glutethimide (Roath
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FIG.12. Ttratogenic effect of a disturbance of lipid metabolism (Triton. W.R. 1339). A. Anencephaly. B. Celesomia. (After Tuchmann-Duplessis and Mercier-Parot, 1964c. Courtesy of Compt. rend.)
et ul., 1963). Certain antimitotic antitumor agents (colchicine) inhibit cell division by blocking it at the stage of metaphase; these are the spindle depolarizers (Lits, 1934). d. Action on the Stability of Oxygenation of the Intracellular Hemoglobin. Metcalf (1962) observed that trypan blue and Evans blue have a niarked and immediate action on the stability of oxygenation of the intracellular henloglobin in the rat. He considers that the effect of these teratogenic agents is due to interference with the protein component of the flavoprotein enzymes. e. Chromosomal Anomalies. Ingalls et al. (1963) found that the incidence of cleft palate aniong mouse fetuses following administration of another metabolite, 6-aniinonicotanamide, is correlated with the incidence of chromosomal anomalies : polyploidy and fragmentation of the chromosomes. The same phenomenon has been reported in a chick eiiibryo after adniinistration of thalidomide (Villa and Eridani, 1963). It may be questioned, however, whether this anomaly may not be the result of stress rather than of a genetic anomaly. f. Maternal Stress. Stress due to restraint potentiates the teratogenic effect of vitamin A (Hartel and Hartel, 1960). Recent experiments by Goldnian and Yakovac (1963) suggest that immobilization of the pregnant
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rat, producing the characteristic changes of stress, potentiates the teratogenic effect of salicylate. The audiogenic stress, however, does not induce cleft palate or other congenital malformations in mice (Warkany and Kalter, 1961). g. Changes in Spermatogenesis or in Seminal Line. Lutwak-Mann (1964) administered thalidomide to male rabbits and in all cases observed a reduction in fertility and an increase in the prenatal mortality rate. It must be emphasized, however, that no embryopathies result,ed. Dams et al. (1963, 1964) tested the fertility of a malformed cock receiving hydrocortisone sirice the embryonic period; after crossing it with genotypically healthy hens, these workers observed numerous malformations in the descendants. Hence, the germinal line is not entirely shielded from the toxic action of a teratogen. Whether this property is specific to the teratogens or if it may also be observed with nonteratogenic poisons remains to be studied. Moreover, an objective confirmation of the results should be undertaken. 6. Production of Tissue Antibodies
Closely linked to chernoteratogenesis, a new field of imniunoteratogenesis has opened up. Gluecksohn-Waelsch (1957), W. L. Miller (1958), Fowler arid Clarke (1960), Brent el al. (1961), G. David et a,?. (1963), and Mercier-Parot et al. (1963) have observed fetal abnornialities by injecting a pregnant rat with a heterologous antikidney serum obtained from injection of a rabbit with a hoinogenate of rat kidney. Aiioinalies chiefly involved the central nervous system and the eyes and extended to the production of actual monsters. In the mouse the rat kidney antiserum is inactive whereas t,he mouse kidney antiserum is teratogenic. In these cases teratogenesis results from the formation of specific tissue antibodies. Blizzard et al. (1960) discovered antibodies to thyroglobulin in the serum of mothers whose infants exhibited athyrotic cretinism. Ectodermal anomalies were produced by W. L. Miller (1958) in the rabbit and by Langxnan (1959) and Maisel and Langman (1961) in the chick by means of an antilenticular serum. Perhaps a transfer of maternal antibodies is the nierhanisni responsible for fetal nialforniations.
7. Interaction between the Various Teratogenic Factors It had been demonstrated experimentally that certain associations of teratogenic factors may be additive in their effects; this occurs with starvation and trypan blue (Runner and Dagg, 1960) or with irradiation (Runner and Dagg, 1960). The siniultaneous administration of various teratogens sometimes may produce potentiation of their effect, e.g., starvation and
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cortisone (W. L. Miller, 1958), insulin and nicotinaniide (Smithberg, 1961), constraint, stress and vitamin A (Hartel and Hartel, 1960),hypervitaminosis A and trypan blue (J. G. Wilson, 1964b), 5-fluouracil and trypan blue (Wilson, 1964b), cyclophosphamide and 5-fluouracil (Wilson, 1964b), thalidoniide and riboflavin or trypan blue (Bertrand et al., 1964), thyroxine and antimitotic agents (Faucouneau et al., 1964). Human teratogenesis may be considered as resulting from the conibination of exogenous, genetic, or metabolic factors and the agents supposed to be solely responsible. Many unknown factors are undoubtedly still unsuspected. VIII. Conclusions
Despite the nunierous investigations carried out during the last quarter of a century, the problem of the experimental investigation of the teratogenic potentialities of drugs is far from being resolved. From the practical point of view, it may be concluded from an analysis of the clinical observations that the danger of drug-induced fetal malformation is, for the time being, not as great as had been feared. Besides thalidomide, there are today two classes of drugs which should he banned from obstetric practice: antitumor agents and, above all, steroids. It is obvious, however, that systematic experiniental investigations and intensive trials must be carried out before a new drug can be authorized for prescription to the pregnant woman. From the theoretical point of view, the general impression acquired from a reading of the innumerable publications on this topic is the total lack of cohesion. There is no pharmacological or chemical correlation among the various teratogenic drugs. For the same teratogenic drug there is no correlation of effect among different species or different strains of the sanie species, even when they are tested with the same experimental procedure. There is often no correlation between the teratogenic effect observed in laboratory animals and in humans. This may perhaps account for the futility of the efforts of all who are attempting to produce experimental fetal malformations, to study the metabolism of teratogenic drugs and their mechanism of action, and especially to understand what happened in the case of thalidoniide. The aspirations for progress are realistic providing that research is designed and organized on the basis of normal standards, using determined animal species sensitive to drug-induced teratogenesis. Only after standardization of experimental methods can collaboration among biochemists, embryologists, geneticists, pharmacologists, and clinicians be effective. Only in such conditions would drug metabolism studies ascertain the parameters responsible for the teratogenic potentiality of this drug.
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In assessing the present state of our knowledge of the mechanism of drug-induced teratogenesis, it has to be admitted that, unfortunately, very few of the tempting hypotheses which have been put forward have led to the creation of constructive working models; not one has produced an intellectually satisfactory solution. Not only must research continue along the same lines, but also, in so difficult a problem, attempts must be made to explore other directions. Kalter et aZ. (1942), who first considered the theoretical problems of drug-induced teratogenesis, developed the concept that congenital malformations result from interference with metabolic processes during pregnancy. This still remains a valid working hypothesis in 1965, but in actuality it has not yet been demonstrated. A new idea has appeared on the horizon: congenital malformations result from niultiple causes whose effects were cumulative. The problem a t issue today is the following: what are the metabolic systems of the mother or fetus t>hatare manifestly disturbed? GLOSSARY Agnathia: Absence of the jaw Anencephaly: Absence of the brain Anophthalmia: Absence of the eyes Bmchydactyly: Abnormal shortening of digits Craniwachischisis: Fissure of the skull and vertebral column Cranioschisis: Fissure of the skull Ectrodactyly: Absence of digits Exencephaly: Absence of closure of the cranium (but with closure of the brain) Gastroschisis: Defect of the abdominal muscles Hydronephrosis: Distension of renal calyces and pelvis by urine Hydroureter: Distension of the ureter by urine Lordosis: Exaggeration of the normal lumbar curvature
Meningocele: Hernial protrusion of the meninges Micromelia: Shortening of the limbs Microphthalmia: Reduction in the size of the eye Phocomelia: Atrophy of the intermediate segments of the limbs, resembling the limbs of a seal Omphalocele: Umbilical hernia Rachischisis: Fissure of the vertebral column Spina bifida: Opening of the vertebral column with protrusion of the meninges or of the spinal cord Syndactyly: Fusion or webbing of the digits Teratogenesis: The production of monsters
REFERENCES Aaron, H. H. (1955). J. A m . Med. Assoc. 169, 848. Adams, C. E., Hay, M. F., and Lutwak-Mann, C. (1961). J.Ernbryol. Exptl. Morphol. 9, 468. Ancel, P. (1950). “Chemoteratogenesis in Vertebrates.’] Paris. Anderson, D. H. (1941). A m . J. Diseases Children 62, 888. Arnaud, G . (1963). Compt. Rend. SOC.Biol. 167, 1585. Aron, C., Asch, G., and Asch, L. (1963). Gyneco2. Obstet. (Paris) 62, 53.
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Author Index Numbers in italics indicate the pages on which the complete references are listed.
A Aaron, H. H., 302, 304 Aaronson, S., 335 Aarsen, P. N. 47, 49, 74 Abe, K., 63, 74, 90 Abelman, M. A., 303, 337 Abelous, J. E., 4, 74 Adamovics, A., 330, 336 Adams, C. E., 276, 334 Adams, S. S.,164, 199, SO4 Adamson, R. H., 328, 338 Afeman, C., 287, 335 Afonso, S., 36, 37, 74, 86 Agolini, G., 157, 204 Agostino, D., 13, 76 Aida, M., 63, 74 Akama, G., 265, 349 Albrecht, M., 196, 204 Alexander, K., 195, 208 Allen, H. C., 202, 209 Allen, R. T. J., 34, 81 Allgower, M., 13, 74 Allisoni, J. E., 163, 204 Allwood, M. J., 51, 74 Alms, U., 189, 217 Alpert, L. K., 202, 209 Alvarenga, R. J., 307, 310, 344 Amarger, J., 316, 342 Amatuzio, D. S., 113, 135 Ames, R. P., 134, 130 Amundsen, E., 20, 46, 48, 49, 50, 74 Anastasi, A., 22, 64, 65, 66, 67, 68, 74, 78, 178, 208 Ancel, P., 276, 279, 291, 316, 334, 349 Anderer, F. A., 12, 74 Anderson, D. H., 269, 334 Anderson, D. V., 184, 185, 205 Anderson, M. A., 296, 337 Andrade, S. O., 22, 74 Anselmi, B., 21, 38, 51, 71, 87 Anton, A. H., 254, 255 Antonio, A,, 36, 57, 74, 86 Antopol, W., 13, 60, 74, 76
Aoi, H., 153, 212 Appel, W., 11, 12, 84, 89 Araki, E., 316, 320, 336 Arias, F., 19, 81 Armstrong, D., 15, 16, 20, 21, 33, 34, 42, 46, 49, 51, 58, 74, 88, 178, 204 Armstrong, F. B., 121, 134 Arnaud, G., 316, 334 Aron, C., 280, 334 Aronson, E., 149, 204 Artz, C. P., 13, 80 Asang, E., 13, 74 Asch, G., 280, 334 Asch, L., 280, 334 Ascheim, E., 32, 75 Aschenheim, E., 265, 335 Asling, C. W., 298, 344 Atkins, E., 177, 204 $ubertin, J., 127, 139 Audibert, A,, 316, 342 Aucr, A,, 147, 205 Austen, K. F., 189, 206 Austrian, C. R., 116, 134 Averich, E., 332, 336 Axelrod, J., 104, 134, 221, 222, 226, 243, 252, 253, 256, 266, 269, 261 Axelrod, L. R., 229, 250 Ayengar, P. K., 255 Ayvazian, J. H., 134, 134 Ayvazian, L. F., 134, 134
B Bander, H., 143, 205 Baba, T., 316, 320, 335 Bacchus, A,, 165, 206 Bacchus, H., 165, 205 Bachem, C., 147, 205 Bachmann, K. D., 319, 348 Back, N., 15, 30, 58, 7 5 Baer, J. E., 133, 134, 134, 135 Baggenstoss, A. H., 13, 87 Baird, C. D. C., 267, 344
351
352
AUTHOR INDEX
Baker, H., 290, 338 Baker, J. B. E., 266, 320, 336 Baker, J. R., 225, 234, 235, 266 Balart, L., 13, 83 Banting, B. M., 332, 336 Barac, G., 39, 76 Barashnev, Yu, I., 313, 336 Barber, A. N., 287, 336 Barbour, H. G., 149, 206 Barch, J., 265, 349 Barchow, D., 13, 82 Bardier, E., 4, 74 Barer, G. R., 39, 76 Barghorn, H., 154, 213 Barnafi, L., 19, 77 Barnes, A. C., 266, 320, 336, 340 Barnes, J., 319, 336 Barone, R., 332, 337 Barraclough, M. A,, 39, 76 Barrett, J. F., 222, 231, 266 Barrot, J., 320, 344 Bartels, E. C., 104, 114, 115, 134 Bartholomew, L. G., 13, 87 Bass, A. D., 317, 336 Bassil, G. T., 324, 329, 336 Bataille, J., 158, 2W Battaglia, G. B., 21, 76 Bauer, E., 4, 6, 10, 11, 32, 57, 82 Bauer, S., 222, 266 Baumann, W., 118, 140 Barter, H., 303, 336 Bayliss, R. I. S., 159, 206 Bayne, G. M., 113, 115, 136 Basin, S., 183, 184, 185, 207 Bazzi, Cl., 157, 206 Beach, V. L., 302, 342 Bear, D. M., 313, 342 Bearis, W., 277, 340 Bearn, A. G., 112, 134 Beatty, J. O., 112, 113, 136 Beaudoin, A. R., 330, 336 Beaudry, P. H., 298, 348 Beck, E., 12, 13, 76, 86 Beck, I. T., 7, 13, 76 Becker, E. L., 11, 16, 17, 58, 76, 82, 83 Beckfield, W . J., 180, 209 Beckmann, R., 324, 327, 936 Beechwood, E. C., 96, 99, 113, 134 Been, M., 164, 191, 217
Behrman, R. E., 267, 273, 276, 310, 313, 3@ Beilenson, S., 39, 76 Beisel, W. R., 115, 134, 140 Belle, M. S., 133, 139 Belmar, J., 37, 41, 77 BBnard, H., 159, 206 Benedek, T. G., 113, 134 Benedict, J. D., 100, 13.4 Benirschke, K., 273, 313, 336, 943 Benits, K.-F., 163, 206 Berakha, G. J., 13, 87 Beraldo, W. T., 7, 14, 18, 26, 51, 58, 63, 76, 81, 86, 86
Berck, U., 4, 19, 51, 89 Berde, B., 22, 27, 29, 37, 39, 65, 67, 68, 70, 76, 76, 87, 88 Berg, D. C., 68, 76 Bergmaschi, M., 27, 29, 36, 37, 68, 69, 70, 72, 76 Bergamaschi, P., 304, 336 Berger, F. M., 317, 336 Berger, L., 95, 96, 99, 106, 110, 111, 112, 113, 115, 118, 119, 125, 129, 131, 133, 134, 134, 136, 138, 142, 254, 266 Berghoff, A., 12, 76 Bergmann, C., 148, 206 Bergmann, F., 93, 134 Berkinshaw-Smith, E. M . I., 32, 76 Berliner, R. W., 95, 134 Bernardi, L., 65, 66, 67, 76 Bernauer, W., 171, 206 Berndt, G., 68, 76 Berndt, W. O., 96, 99, 113, 134 Bernhard, K., 228, 266, 322, 342 Bernhard, W. G., 303, 346 Bernheim, F., 236, 266 Bernheim, M. L., 236, $66 Bernstein, E., 108, 136 Bernstein, K., 200, 203, 204, 206 Bernstine, J. B., 320, 336 Berry, P. A., 40, 41, 43, 44, 76 Berrod, F., 538 Bertaccini, G., 22, 64, 66, 67, 68, 74, 78 Bertagnini, C., 145, 206 Bertelli, A. 157, 204 Bertha, I., 32, 83 Bertrand, M., 281, 333, 336 Best, C. H., 181, 206 Bets, H., 157, 158, 161, 162, 206, 214, 216
AUTHOR INDEX
Beutner, R., 156, 206 Beyer, K. H., 99, 104, 105, 107, 108, 109, 110, 111, 112, 113, 116, 133, 134, 134,
353
Boger, W. P., 108, 109, 110, 111, 112, 113, 115, 116, 134, 135, 137, 140 nogiovanni, A. M., 113, 135 Bohr, D. F., 38, 86 135, 139, 141 Bhargava, N., 12, 82 Boime, A., 309, 310, 322, 343 Bhattacharya, B. K., 188, 205 Boisselot, J., 277, 339 Bhoola, K. D., 15, 30, 32, 33, 40, 42, 43, Boissonnas, R. A., 22, 23, 51, 52, 65, 66, 64, 75, 191, 193, 205 67, 76, 85, 86, 88 Bollag, W., 293, 296, 335 Biancone, S., 278, 335 Bickel, G., 303, 335 Bollet, A. J., 184, 185, 200, 201, 205 Bonaccorsi, A,, 33, 76 Bickerton, R. K., 31, 76 Biegeleisen, J. Z., Jr., 277, 335 Bonar, J. A,, 113, 135 Biering-Sorensen, K., 319, 335 Bonati, B., 205 Bignami, G., 269, 272, 307, 309, 319, 322, Bondi, S., 149, 205 Bongiovanni, A. M., 302, 303, 335 323, 329, 335, 344 Bonsnes, R. W., 132, 135 Bilisoly, F. N., 155, 205 Booth, J., 222, 223, 226, 255 Binia, A,, 48, S1 Binns, W., 316, 335, 342 Borbone, C., 294, 295, 298, 321, 349 Binz, C . , 147, 205 Bordeaut, L. F., 286, 338 Borhani, N. O., 135 Biro, L., 43, 79 Bischoff, F., 4, 76 Borniche, P., 302, 343 Bosisio, G., 65, 66, 67, 75 Bishop, C., 114, 135, l4l Bosscr, C., 13, 83 Bishop, E. A., 16, 26, 49, 75, S4 Bostrom, H., 199, 200, 202, 203, 204, 205, Bishop, J. M., 37, 38, 75 217, 317, 343 Bisset, G. W., 30, 75, 178, 205 Blanchard, K. C . , 157, 159, 160, 161, 205 Bowel, M., 158, 159, 161, 206 Bourne, F. M., 298, 335 Bland, J. H., 134, 157 Bovet, D., 231, 261, 269, 272, 307, 309, Blattner, R. J., 270, 293, 335, 349 319, 322, 323, 329, 335,344 Blau, R., 128, 138 Bovct-Nitti, F., 269, 272, 307, 309, 319, Bledsoc, T., 231, 255 322, 323, 329, 335, 344 Blennemann, G., 6, 14, 17, 29, 47, 52, 53, Bowen, A,, 330, 336 54, 80 Boyland, E., 222, 223, 226, 255 Bliss, J. Q., 16, 87 Boylen, J. B., 270, 279, 308, 325, 329, 335 Blix, G., 184, 205 Bradley, S. E., 107, 139 Blix, S., 12, 75 Bragonier, J. R., 298, 335 Blizzard, R. M., 332, 335 Brand, L., 131, 159 Bloch, J. H., 7, 57, 83 Brandi, C. M. W., 8, 22, 85 Block, K., 228, 229, 234, 255, 260, 261 Braun, C., 30, 34, 35, 43, 80, 83 Blondheim, S. H., 113, 135 Braun, H., 162, 167, 206 Bloom, B. M., 118, 128, 129, 135 Braun, W., 111, 113, 135 Bloomfield, D. K., 228, 255 Breda, G., 59, 76 Blumenthal, A., 180, 209 Bregulla, B., 189, 217 Breitcnbucher, R. B., 113, 135 Board, J. A., 162, 208 Brent, R. L., 327, 332, 336 Boas, N. F., 183, 185, 205 Breslauer, F., 151, 206 Bochey, J. M., 116, 139 Breslow, R., 243, 255 Bocquet, L., 320, 335 Brest, A. N., 115, 133, 134, 137, 1.40 Bodanszky, M., 24, 31, 75, 76, 82 Breuer, H., 230, 255, $60 Bode, H. H., 163, 190, 205, 212
354
AUTHOR INDEX
Brinsmade, A. B., 268, 314, 315, 321, 336 Briot, M., 187, 209 Brock, N., 176, 806 Brocklehurst, W. E., 48, 58, 76 Brodie, B. B., 101, 108, Ill, , 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 131, 132, 136, 136, 137, 139, 140, 141, 221, 222, 223, 225, 227, 232, 233, 234, 235, 236, 238, 243, 247, 248, 250, 252, 254, 266, 266', 267, 268, 261, 275, 285, 318, 328, 336, 341 Brody, T. M., 198, 200, 206 Broghammer, H., 38, 69, 71, 76 Bronsky, D., 112, 115, 136, 137, 139 Brooks, A. V., 134, 134 Broussole, P., 317, 338 Brown, A. V., 184, 200, 206 Brown, B., 114, 116, 119, 138 Brown, D. V., 330, 336 Brown, J. W., 109, 140 Brown, R. R., 238, 254, 266 Bruce, A. N., 150, 151, 206 Bruce, H. M., 277, 346 Bryan, W. H., 303, 346 Bryant, C., 201, 206 Bryant, J. M., 115, 133, 134, 136 Buchanan, A. R., 316,346 Bucher, O., 196, 106 Buchner, A., 145, 206 Buck, P., 270, 301, 303, 336' Buckley, J. P., 31, 70, 76, 82 Budnick, I. S., 316, 336' Buch, O., 190, 206 Buchner, F., 268, 321, 336 Buhrer, G., 184, 202, 217 Bumpus, F. M., 18, 31, 50, 63, 76, 81 Bundy, R. E., 54, 81 Bunot, O., 309, 310, 322, 343 Bunting, H., 182, 206 Burch, G. E., 34, 36, 76 Burckhardt, D., 33, 76 Buresh, J. J., 303, 336 Burkovsky, J., 245, 260 Burley, D., 121, 138 Burnell, J . M., 113, 136 Burnett, C. H., 132, 138 Burns, J. J., 96, 106, 112, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 131, 132, 136, 136, 138, 139,
140, 141, 142, 254, 266, 285, 288, 319, 324,327, 336 Burr, V., 164, 191, 217 Burrill, M. W., 302, 340 Burrows, B. A., 132, 138 Bush, M. T., 227, 266 Buss, C. E., 146, 206 Butler, T. C., 224, 254, 260 Byasson, H., 100, 136 Byers, S. O., 99, 132, 137 Byham, B. B., 116, 139 Bywaters, E. G. L., 157, 206
C Cahen, R. L., 159, 206, 263, 266, 283, 284, 285, 287, 291, 307, 309, 310, 319, 320, 321, 327, 336, 346 Cain, J. C., 13, W Calcutt, G., 247, 267 Calewick, B., 156, 206 Calle, J. D., 15, 32, 33, 42, 64, 76, 193, 206
Callison, E. C., 336 Calvert, M., 279, 336 Camerino, B., 67, 76 Cameron, J. M., 308, 336' Campbell, G. D., 315, 336 Campbell, H., 100, 136 Campbell, M., 277, 336 Campbell, P. C., Jr., 113, 138 Campbell, W. L., 27, 83 Campomanes, C. I., 131, 133 Canlorbe, P., 302, 343 Cannon, P. J., 134, 136 Cano, G., 36, 80 Capek, R., 31, 45, 76 Cardell, B. S., 313, 315, 336 Cardoe, N., 121, 136 Carleton, J., 113, 138 Carlier, J., 37, 76 Carlo, P.-E., 167, 208 Carpent, G., 283, 336 Carpi, A., 36, 76 Carr, H. J., Jr., 106, 136 Carr, J., 26, 76 Carretero, O., 39, 76 Carretero, 0. A., 48, 81 Carter, M. P., 318, 319, 336' Carter, T. C., 283, 287, 348
AUTHOR INDEX
Carvalho, I. F., 20, 21, 26, 48, 59, 60, 76, 77 Carver, M. J., 298, 335 Casida, J. E., 224, 256, 257 Castenholz, A., 71, 80 Castillo, C. A,, 36, 37, 74, 86 Catchpole, H. R., 182, 184, ZOG, 208 Caujolle, D., 279, 336 Caujolle, F., 279, 336 Cavicchini, G., 157, 204 Cecil, H. C., 185, 208 Cei, J. M., 66, 67, 68, 74, 75 Cendron, J., 302, 343 Centaro, A,, 20, 51, 75, 76, 85 Center, E., 292, 337 Cerletti, A,, 27, 75 Cesano, L., 30, 38, 79 Chain, E., 31, 77 Chambers, J., 227, 256 Chambon, Y., 316, 336 Champlin, B., 116, 138 Champy, C., 157, 166, 206 Chandler, R. W., 332, 335 Chapman, L. F.,34, 38, 60, 76, 85, 154, 155, 206 Charnock, J. S., 198, 206 Chasis, H., 96, 107, 140 Chassagne, P., 291, 296, 336 Chaudhry, A. P., 303, 341 Chaykin, S., 225, 234, 235, 255 Chen, W., 110, 111, 112, 118, 119, 121, 125, 128, 129, 131, 132, 136, 138, 139 Chenkin, T., 104, 119, 120, 121, 123, 124, 134, 135, 136, 141 Chenoweth, M. B., 227, 261 Chen-Yu-Sung, 319, 348 Chiandussi, L., 30, 38, 79 Chiesara, E., 251, 258 Chillemi, F., 65, 66, 67, 75 Chinn, E. R., 319, 346 Cho, A. K., 225, 256 Chomette, G., 314, 315, 336 Chou, C. C., 36, 69, 70, 76 Chrisman, 0. D., 201, 206 Christensen, F., 133, 136 Christensen, J. F., 31, 76 Christie, G. A., 272, 283, 309, 336 Chryssanthou, C., 13, 60, 74, 76 Ciampolini, E., 71, 7 6 Clark, C. T., 243, 261
355
Clark, K. H., 303, 336 Clark, W. R., 88 Clarke, S. H., 184, 217 Clarke, W. M., 332, 338 Clavert, J., 270, 271, 292, 296, 301, 303, 336, 339 Clemente, P., 12, 84 Cliffton, E. E., 13, 76 Clifton, E. E., 13, 86 Clingen, G., 301, 337 Clough, 0. W., 313, 342 Clouct, D. H., 245, 253, 266 Cobb, R., 164, 199, 204 Coburn, A. F., 101, 136 Cochran, J. B., 157, 158, 198, 206 Coelho, R. R., 201, 206 Coffman, J. D., 38, 76, 82 Cohcn, A. S., 113, 139 Cohlan, S. Q., 265, 275, 277, 283, 289, 320, 336 Cohn, A. E., 154, 211 Cohn, C., 108, 109, 113, 141 Collier, H. 0. J., 22, 26, 27, 30, 33, 40, 41, 42, 43, 44, 54, 75, 76, 77, 85, 190, 191, 192, 2006, 206 Colonge, A,, 303, 336, 337 Colopy, J. E., 15, 86 Colowick, S. P., 245, 261 Colquhoun, J., 109, 140 Coltman, 0. McK., 303, 337 Combridge, B. S., 58, 83 Compagnon, A,, 154, 213 Concioli, M., 36, 44, 59, 77 Conney, A. H., 106, 136, 230, 231, 238, 253, 254, $56, 268, 285, 336 Consden, R., 182, 206 Consolazio, W. V., 105, 132, 136 Constantopoulos, G., 234, 256 Contzen, C., 9, 77 Cook, E. R., 117, 127, 128, 138 Coombs, F. S., 105, 115, 132, 136, 140 Cooper, D. Y., 235, 236, 237, 238, 239, 241, 242, 248, 256, 259, 260 Cooper, J. R., 222, 236, 247, 256 Cope, C. L., 159, 206 Copley, A. L., 32, 33, 42, 43, 44, 76, 77, 179, 188, 193, 206 Cormia, E., 34, 77 Corrado, A. P., 31, 36, 37, 40, 55, 76, 77, 79, 86
356
AUTHOR INDEX
Coste, F., 158, 159, 161, 206 Couland, H., 297, 343 Courrier, R., 265, 276, 300, 301, 302, 316, $36, 337 Cradic, H., 13, 83 Craft, M. K., 24, 25, 27, 29, 37, 52, 53, 86 Craig, S. G., 106, 139 Craighead, C. C., 13, 83 Crain, B., Jr., 320, 348 Crary, D. D., 281, 284, 337 Craver, B. N., 24, 66 Crawford, M. A,, 101, 139 Creavin, P. J., 222, 266 Cremer, J., 228, 266 Creutzfeldt, W., 12, 13, Y7 Crigler, J. R., Jr., 113, 137 Crivetz, D., 188, 207 Cronheim, G., 161, 162, 167, 207 Cros, S., 279, 336 Crosson, J. W., 109, 110, 136, 140 Crotatto, H., 19, 37, 41, Y7 Crumpton, C. W., 36, 37, 74, 86 Cruz-Horn, A., 159, 206 Cuenod, C. L., 161, 208 Cueto, C., 223, 266 Culp, H. W., 221, 269 Curley, F. J., 298, 301, 309, 331, 341 Currie, J. P., 123, 141 Curtis, E. M., 302, 303, 337 Czaczkes, J. W., 133, 137
Davenport, L., 252, 267 David, A., 337 David, G., 319, 332, 337, 344 David-Chausse, F., 124, 127, 139 Davies, A. M., 316, 318, 346 Davies, G. E., 9, 77, 187, 207 Davis, B. B., 96, 136 Davis, N. C., 317, $40 Davison, A. N., 227, 266 Dawson, W., 58, 7Y Dayton, P. G., 96, 99, 103, 110, 111, 112, 116, 118, 119, 121, 124, 125, 127, 128, 129, 131, 132, 136, 13S, 139, 140, 141,
142
Dearborn, E. H., 105, 139, 153, 159, 160, 161, 206 De Azevedo, D. F., 38, 77 de Caro, G., 65, 66, 67, 68, 76, 76, 77 De Carvalho, I. F., S4 de Castiglione, R., 65, 66, 67, 76 Decker, J. L., 115, 133, 134, 138 Decortis, A., 158, 214 De Costa, E. J., 303, 337 Dedichen, J., 20, 49, 74 de Fossey, M., 159, 207 de Freitas, F. M., 38, 77 Degraff, A. C., 183, 185, 214 De Graft, A. C., 134, 140 De Jonge, G. A,, 337 Dekker, A., 309, 337 De Koster, J. P., 126, 136 D Delahunt, C. S., 268, 273, 310, 312, 313, Dagg, C. P., 332, 346 337 Dalderup, L. M., 307, 309, 337, 342 Delande, M. S., 319, 344 Dallner, G., 220, 236, 238, 247, 248, 266, Delatour, P., 268, 270, 272, 279, 280, 281, 310, 313, 333, 536, 337 260 Damgaard, E., 161, 163, 165, 168, 169, Delaunay, A,, 183, 184, 185, 207 189, 193, 216 Delaville, M., 158, 207 Dammin, G. J., 116, 139 Delbsrre, F., 158, 161, 206 Dams, R., 268, 272, 280, 281, 310, 313, Del Bianco, P. L., 34, 42, 87 332, 337 Deleau, D., 309, 310, 322, 343 Dana, E., 132, 136 Delmas, A., 265, 267, 339 Danforth, C. H., 292, 337 Deltour, G., 159, 207 d’Anglejan, G., 114, 116, 127, 136 Demartini, F. E., 133, 134, 136, 138 Daniel, J. W., 231, 266 Demay, M., 157, 166, 206 Daniel-Moussard, H., 185, 207 De Meyer, R., 290, 313, 314, 315, 321, Danielopolu, D., 188, $07 337 Danielsson, H., 230, 266 Dempsey, M. E., 229, 266 Dareste, C., 264, 279, 337 Denis, W., 198, 207 Daumer, J., 10, 88 Denss, R., 120, 136
357
AUTHOR INDEX
De Pasquale, N. P., 34, 36, 76 Derom, R., 278,337 De Rothschild, B., 265, 269, 277, 300, 303, 321, 339 Desaulles, P., 155, 157, 174, 212 de Siize, S.,106, 114, 115, 116, 127, 128, 13G
Dcuschle, F. M., 265, 277, 337, 348 Deutsch, E., 12, 77 Deutsch, H. F., 18, 51, 7 7 , 81 De Wald, H. A,, 22, 23, 24, 27, 29, 37, 52, 53, 77, 85 Dewes, R., 163, 207 Diamond, I., 296, 337 Dickerson, G. D., 34, 35, 43, 83 Didcok, K., 292, 293, 295, 337 Di Ferrante, N., 185, 207 D i George, A. M., 302, 335 Diggory, P. L. C., 319, 337 Dikstein, S., 93, IS,$ Dill, L., 132, 135 Dille, J. M., 320, 337 Di Mattei, P., 41, 60, ?7 Diner, S., 244, 256 Dingell, J. V., 224, 250, 253, 254, 25G Dingwall, A,, 131, 137 Diniz, C. R., 18, 20, 21, 26, 48, 51, 59, 60, YG, 77, 84 Di Paolo, J. A,, 269, 290, 310, 337 Di Paolo, P. D., 341 Dirscherl, W., 197, 207, 211 Dixon, A. St. J., 100, 1% Dixon, M., 46, 77, 251, 256 Dixon, R. L., 253, 25G Dobovicnik, W., 7, 12, 59, 60,Y7, Y9 Dobozy, E., 41, S7 Dobriner, K., 231, 260 Doebner, O., 103, 137 Doering, W. von E., 234, 257 Dorner, G., 203, 209 Dohrn, M., 103, 105, 139 Doig, R. K., 303, 337 Doll, R., 277, 337 Domenjoz, R., 121, 126, 128, 137, 154, 155, 161, 162, 163, 164, 165, 166, 167, 168, 169, 171, 174, 180, 189, 190, 198 207, 210, 215, 217 Domm, L. V., 303, 337 Donaldson, V. H., 16, 58, YY Dorfman, A,, 200, 203, 207, 614
Dorfman, R. I., 229, 234, 257, 258, 659, 260
Dorough, H. W., 224, 256 Dougherty, J. W., 34, 7Y Douglas, W. W., 42, 85 Doxie, J., 158, 196, 210 Drabkin, D. L., 13, 81, 84 Dragstedt, C. A., 14, 88 Drapiewski, V. A., 332, 336 Dreifus, L. S., 115, 134, 137 Drenkhahn, F. O., 202, 207 Dresse, A,, 41, 83 Dreyfuss, F., 133, 137 Drnckrey, H., 176, 206, 207 Drury, A. N., 180, 207, 216 Dry, R. M. L., 33, 74 Duarte, C. G., 115, 134, 137 Dublin, A., 112, 114, 115, 136, 137, 139 Du Bois, K. P., 227, 2558 Du Boistesselin, R., 164, 184, 202, 20Y, 21 G Du Buit, H., 154, 213 Ducharme, J. R., 300, 302, 303, 340 Duckert, F., 12, 75 Duff, I. F., 114, 115, 127, 141 Dumont, L., 317, 338 Duncan, G. G., 109, 140 Duncan, G. M., 162, 208 Duncan, W. A. M., 250, 256 Dunphy, J. E., 183, 207 Dupuis, R., 268, 277, 307, 317, 321, 339, 340, 345 Duthie, E . S., 31, Y7 Duthie, J. J. R., 164, 216
E Eanes, E. D., 93, 94, 138 Earle, D. P., Jr., 108, 137 Earley, P. A,, 317, 33Y Eastman, N. J., 316, 33Y Ebbecke, U., 151, 174, 207 Ehrrhardt, H., 167, 207, 213 Eberlein, W. R., 113, 135 Eckhardt, E. F., 190, 207 Eckstein, M., 175, 20Y Edery, H., 32, 42, 45, 47, 48, 57, 58, 77, 179, 188, 207, 208 Edwards, L. F., 316, 342 Effkemann, G., 19, 51, 77
358
AUTHOR INDEX
Ehmann, B., 308, 337 Ehrenstein, M., 229, 268 Ehrich, W. E., 154, 156, 172, 173, 176, 180, 181, 187, 208, 221, 216 Ehringer, H., 38, 7 8 Eichenberger, E., 180, 217 Eichholtz, F., 195, 208 Eisen, V., 14, 15, 16, 58, 78 Eisenstein, S., 13, 76 Eisman, S. H., 109, 136, 137 Elder, J. M., 16, 78, 84, 193, 212 Elion, G. B., 94, 140 Ellenhorn, M. J., 279, 283, 337 Ellinger, A., 147, 208 Elliot, A. H., 4, 36, 41, 76, 78 Elliott, D. F., 6, 10, 22, 23, 24, 29, 31, 34, 35, 48, 51, 54, 78, 178, 208 Elliott, R. B., 37, 70, 84 Ellsworth, H., 319, 348 Elson, L. A,, 231, 266 Elves, M. W., 290, 330, 346 Emek, J. F., 35, 43, 78 Emmart, E. W., 9, 88 Emmerson, B. T., 127, 128, 137 Engel, L. L., 237, 2G0 Engfeldt, B., 303, 340 Engle, R. J., 34, 35, 43, 83 Eppinger, H., 172, 207 Erdos, E. G., 13, 20, 24, 25, 26, 27, 30, 33, 36, 39, 40, 42, 43, 44, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 58, 59, 60, 62, 63, 78, 84, 89, 275, 285, 318, 328, 336 Ericson-Strandvik, B., 317, 318, 337, 343 Ericsson, J. L. F., 253, 260 Eridani, S., 331, 348 Ernst, T., 182, 208 Ernster, L., 235, 236, 238, 240, 241, 244, 245, 247, 248, 253, 266, 267, 269, 260 Ernster, S., 253, 260 Erspamer, G. F., 29, 67, 68, 72, 78 Erspamer, V., 22, 29, 64, 65, 66, 67, 68, 70, 72, 74, 76, 78, 178, 208 Eschbach, H., 154, 213 Eschonbach, J., 196, 204 Eskes, T. K. A. B., 50, 86 Essig, A., 134, 137 Estabrook, R. W., 235, 236, 237, 238, 241, 242, 866, 260 Evans, H. J., 301, 337
Evans, H. M., 267, 298, 321, 344 Evered, D. F., 329, 337 Ewald, C. A., 176, 208 Exer, B., 184, 186, 198, 201, 202, 208, 213, 217
F Fabre, R., 316, 337, 338 Fabro, S., 322, 323, 325, 327, 328, 329, 338 Faigle, J. W., 321, 324, 325, 327, 328, 329, 336, 338, 342 Fainstat, T. D., 269, 300, 301, 303, 320, 338, 339 Fairhurst, A. S., 225, 266 Falcone, A. B., 198, 208 Falk, A., 113, 136 Faltitscheck, J., 172, 208 Fanciullacci, M., 34, 42, 51, 59, 65, 71, 72, 87 Faraco, E. Z., 38, 77 Farizon, F., 305, 338 Fasciolo, J. C . , 5, 21, 27, 39, 48, 76, 78, 81 Faucounau, N., 333, 338 Fauvel, P., 102, 137 Fave, A., 266, 291, 338 Favre-Tissot, M., 268, 272, 280, 281, 310, 313, 317, 337, 538 Feeney, G. C., 167, 208 Feichtmeir, T. V, 106, 139 Feinstein, R. N., 338 Feldberg, W., 7, 31, 37, 38, 41, 42, 63, 76, 78, 79 Feldman, M., 338 FBlisati, D., 276, 308, 309, 329, 338 Felix, J., 4, 36, 7 9 Fellinger, K., 157, 808 Ferguson, A. W., 338 Ferm, V. H., 267, 283, 286, 287, 296, 338 Frrras, de Oliveira, M. C., 18, 20, 81 Ferreira, S. H., 44, 45, 49, 55, 79 Ferris, T. F., 116, 137 Ferstl, A., 157, 208 Fertig, H., 115, 133, 134, 136 Feruglio F. S., 30, 38, 79 Fichman, M., 18, 81 Field, J., 198, 208 Field, J. B., 96, 136 Filehne, W., 148, 208 Filippi, B., 289, 318, 338, 3.43 Findland, M., 318, 349
359
AUTHOR INDEX
Fink, K. F., 59, 79 Finland, M., 113, 159 Fisbein, M., 266, 358 Fisch, S., 134, 140 Fischer, J., 37, 40, 67, 84 Fish, M. S., 225, 267 Fishgold, J. T.,198, 208 Fishman, J. A., 104, 105, 136 Fishman, J. K., 110, 111, 141 Fishrnan, V., 225, 267 Fitzhugh, O., 270, 279, 343 Flatackerfi L., 195, 217 Fleisch, A., 175, 180, 208 Fleming, H. S., 316, 358 Fletcher, L., Jr., 115, 133, 134, 136 FIexser, L. A., 131, 137 Flippin, H. F., 112, 113, 256 Florio, R., 332, 333, 336, 337 Flosdorf, K., 41, 89 Fock, D., 338 Forster, O.,155, 208 Foley, J. B., 183, 206 Fohn, O., 104, 137 Folk, J. E., 27, 45, 46, 79, 80, 90 Fontana, 145, 208 Forbes, J . C.,162, 208 Ford, R. V.,112, 140 Forell, M. M., 6, 7, 12, 13, 14, 36, 59, 60, 77, 79, 89 Forman, C., 156, 208 Forscher, B. K., 185, 208 Forsham, P. H., 100, 115, 132, 134, lS4, lSY, 140, 158, 159, 216 Fouts, J. R., 220, 221, 232, 233, 252, 253, 266, 267, 260, 328, 338 Fowler, I., 332, 338 Fox, C. I,.,Jr., 57, 79 Fox, M. H., 286, 338 Fox, R. H., 34, 38, 79, 179, 208 Franchi, G., 21, 34, 38, 42, 51, 59, 60, 65, 71, 87, 188, 214 Franchirnont, P., 188, 216 Frank, E., 155, 163, 208 Frank, L., 43, 79 Frank, L. S., 115, 140 Frank, O., 290, 338 Franklin, R. R., 302, S4l Frascr, F. C.,269, 273, 275, 277, 300, 301, 303, 318, 320, 3S6, 3S7, 338, 339, 340, 347, 3-48
Frazer, M. L., 317, 336 Freedlander, S. O., 13, 81 Freedman, S. O.,298, 3% Fregnan, G. B., 27, 67, 79 Freiss, E. D., 176, 212 Freudenthal, R. F., 134, 140 Freund, J., 32, 79 Frey, E. K., 2, 4, 5, 6, 7, 10, 11, 12, 13, 17, 19, 21, 22, 32, 35, 36, 38, 40, 48, 57, 59, 79, 82 Friedman, M., 99, 132, 187 Friedman, M. H., 294, 339 Friend, D. G., 339 Frimrner, M., 31, 32, 33, 68, 76, 79, 208 Frohlich, H., 153, 208 Frohlich, E. D., 36, 69, 70, 76 Frornageot, C., 19, 86 Frommel, E., 161, 208 Frunder, H., 175, 208 Fuchs, M., 133, 137, 140 Fuchs, U.,320, 341 Fuhner, H., 147, $08 Furbringer, P., 146, 2U8 Fuerst, K., 151, 152, 208 Furano, E., 64, 86 Furuyama, T., 63, 90
G Gaddurn, J. H., 48, 62, 79 Gage, C., 316, 842 Gaisboeck, F., 150, 2008 Galindez, H., 114, 115, 127, 141 Gallagher, C., 337 Galletti, R., 34, 79 Camp, A., 184, 208 Garattini, S., 33, 76 Garberi, J. C.,157, 161, 213 Gardner, L. I., 113, 137 Garfinkel, D., 236, 267 Garren, L., 253, 266 Garrod, A. B., 99, 1SY Gass, R. W., 110, 111, 141 Gass, S. R., 107, 109, 112, 113, 136 Gatlings, H. B., 277, 34s Gaudette, L. E., 223, 267, 268 Gaudin, G., 148, 208 Gaudin, O., 158, 2OY Gay-Lussac, L.-J., 145, 208 Gebauer, H., 289, 339 Geiger, J. F., 277, 337
360
AUTHOR INDEX
Geoffroy Saint-Hilaire, L., 264, 266, 304, 339 George, R., 162, 20208, 225, 267 Georges-Janet, L., 291, 296, 336 Gerall, A. A., 302, 346 Gerlinger, P., 270, 271, 292, 296, 339 Gerola, A., 71, 76, 79 Gersh, J., 182, 208 Gersmeyer, E. F., 36, 37, 71, 79, SO Geschickter, C. F., 169, 209 Gessler, H., 176, 209 Ghanem, M. H., 315, 339 Giangrasso, F., 157, 206 Gibbs, A. R., 127, 128, 138 Gibian, H., 66, 83, 200, 209 Gibson, Q. H., 244, 26S, 269 Giesbrecht, A. M., 34, 86 Giese, W., 36, 90 Giesecke, M., 103, 137 Gilbert, C., 283, 286, 287, 320, 321, 330, 339 Gillespie, L., 7, 41, 59, 85 Gillette, J . R., 221, 222, 224, 225, 227, 232, 233, 234, 235, 236, 238, 241, 242, 245, 246, 247, 248, 250, 251, 252, 253, 254, 255, $66, 257, 26268, 260 Gillette, S. R., 125, 136 Gillman, J., 283, 286, 287, 320, 321, 330, 339 Gillmas, T., 283, 286, 287, 320, 321, 330, 339 Gilman, A., 56, 104, SO, 137 Gilmore, J. P., 27, 39, 54, SS Gimble, A. I., 319, 34s Ginglinger, A., 265, 279, 349 Girard, J. P., 58, SO Girault, M., 316, 337, 338 Girgis, S., 143, 206 Giroud, A., 265, 266, 267, 268, 269, 272, 276, 283, 288, 289, 291, 300, 303, 307, 308, 309, 312, 312, 313, 320, 321, 324, 327, 339, 340 Giroud, P., 268, 277, 340 Gjerup, S., 112, 137 Gjuris, V., 30, 40, 43, 44, 67, 80, 192, 209 Gladner, J. A., 27, 45, 80, S6 Glaesser, A., 29, 65, 68, 70, 75, 78 Glasser, A. H., 27, 29, 36, 37, 67, 68, 69, 70, 72, 76, 79
Glassmann, J. M., 180, 209 Glatzel, H., 12, 76 Gleiss, J., 319, 34s Glick, E. N., 128, 137 Glickman, F. S., 43, 79 Globus, M., 307, 317, 322,343 Glover, R., 128, 138 Glover, W. E., 196, 209 Gluecksohn-Waelsch, S., 332, 340 Glynn, L. E., 182, 206 Goblet, J., 163, 165, 216 Godman, G., 202, 809 Goldel, L. F., 7, 88 Gotze, W., 4, 5, 20, 45, 47, 48, 89 Goffredo, O., 65, 66, 67, 76 Goldenberg, H., 225, 267 Goldfingen, S., 94, 112, 137, 138 Goldman, A., 119, 136 Goldman, A. S., 277, 331, 340 Goldring, W., 96, 107, 140 Goldsmith, R., 34, 38, 7Q, 179, 208 Goldstein, A., 318, 340 Goldstein, M. B., 340 Golenhofen, K., 10, SO Gomes, F. P., 39, 62, 80 G6mez Carpio, M., 127, 137 Gonick, H. C., 116, 139 Goodell, H., 34, 38, 60, 76, 86, 154, 155, 206, 206
Goodman, L. S., 56, 104, SO, 137 Goodspeed, A. H., 337 Goodwin, J. F., 184, 185, 200, 206 Goodwin, L. G., 58, 59, 63, SO Goodwin, S., 120, 121, 122, 129, 136 Goss, C. M., 286, 338 Goto, K., 34, 35, 43, S3 Goto, M., 305, 341 Gottlieb, R., 149, 209 Gottschalk, R. G., 202, 209 Govier, W. M., 190, 207 Goy, R. W., 302, 346 Grace, W. J., 116, 140 Graham, G., 103, 137 Graham, R. C., Jr., 32, 80 Graham, W., 121, 137 Grant, L. H., 133, 139 Grant, R. P., 302, 337 Grassmann, W., 24, 70 Graumann, W., 182, 183, 184, ,209
361
AUTHOR INDEX
Grayzel, A. I., 106, 114, 115, 116, 127, 128, 140 Grazi, S., 71, 76, 79 Greco, F., 30, 38, 79 Greeff, K., 37, 40, 43, 80 Green, H. N., 181, 209 Green, K.-G., 317, 340 Greenbaum, L. M., 19, 20, 27, 48, 52, 80 Greenberg, L. A,, 100, 137 Greenberg, L. H., 270, 296, 340 Greene, H . S. N., 272, 3$0 Greene, R. R., 302, 340 Greenfield, V. S., 316, 338 Gregg, N., 265, 277, 340 Greiling, H., 203, 209 Grepinet, J., 303, 340 Grieb, W., 246, 257 Groainger, K. H., 13, SO Groll, H., 151, 209 Gros, G., 302, 33G Gros, H., 199, 209 Gross, F., 155, 157, 164, 209, 212 Gross, M., 38, 8G, 100, 137, 143, 209 Gross, R. H., 127, 139 Grossfeld, H., 202, 209 Grossiord, A,, 154, 215 Grumbach, M. M., 300, 302, 303, 335, 340 Grunz, M., 4, 20, 35, 45, 46, 48, 49, 54, 89 Gual, C., 229, 258 Guest, G. M., 269, 314, 315, 343 Guggenheim, K., 152,209 Guimarais, J. A,, 38, 41, 78, 80 Gulienetti, R., 317, 340 Gunberg, D, L., 269, 283, 286, 301, 303, 320, 340 Gurd, R. S., 96, 113, 134, 138 Gut, M., 234, 2GO Guth, P. S., 11, 15, 21, 30, 36, 48, 58, 64, 75, 79, 80, 193, 209 Gutman, A. B., 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 106, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 131, 132, 133, 134, 156, 13Y, 138, 140, 141, 254, 256 Guttmann, S., 22, 23, 51, 52, 76, 85 Guzman, F., 30, 34, 35, 43, 80, 85 Gylfe, J., 113, 115, 135 Gyllensten, L., 318, 337
H Haberland, G. L., 164, 196, 209 Habermann, E., 6, 8, 9, 10, 11, 12, 14, 17, 18, 20, 23, 24, 26, 29, 47, 52, 53, 54, 80
Habgood, J. S., 155, 209 Hafliger, F., 120, 122, 124, 139 Hacndlc, H., 12, 13, 60, 88 Hiirtel, A,, 277, 331, 333, 340 Hartel, G., 277, 331, 333, 340 Haffrn, K., 288, 349 Hagbard, L., 313, 340 Hagcn, E. O., 310, 340 Hahn, F., 188, 209 Hailman, H . F., 158, 209 Haining, C. G., 163, 189, 209 Hakosalo, H., 277, 34G Hakosalo, J., 277, 346 Hale, F., 265, 276, 321, 340 Halkin, F., 189, 190, 215 Hall, L. M., 163, 206 Hall, V. E., 198, 208 Halliday, R. P., 31, 76 Halpcrn, B. N., 13, 60, 81, 187, 188, 209 Halvorscn, K., 5, 78 Hnlvorscn, K. A,, 27, 48, 81 Hamberg, U., 18, 22, 23, 27, 48, 51, 81 Hambuechen, R., 20, 45, 89 Hamburger, M., 113, 138 Hnmburgh, M., 283, 287, 340 Hammett, L. P., 131, 137 Hammond, A. R., 43, 77 Ilammond, W. S., 317, 355 Hanaki, A., 243, 267 Hancock, J. A., 342 Hancock, J. E. H., 234, 257 Hanscn, L., 184, 185, 202, 203, 210 Hnnsen, 0. E., 133, 138 Hnrjanne, A,, 106, 13s Harm, H., 287, 340 Harper, K. H., 247, 957 Harpman, J. A,, 34, 81 Harris, P., 37, 38, 75 Harris, S. W., 303, 340 Harrison, J., 114, 120, 123, 127, 128, 139 Harrison, M., 133, 138 Hart, F. D., 121, 138
362
AUTHOR INDEX
Hart, L. G., 253, 266 Hartenbach, W., 7, 10, 13, 79, 89 Hartley, G. S., 224, 267 Harvengt, C., 180, 209 Harvey, A. M., 157, 206 Harvy, R., 302, 346 Harwerth, H.-G., 158, 196, 210 Hasegawa, A., 157, 213 Hashimoto, M., 149, 809 Hashimoto, Y., 242, 248, 269 Haskin, D., 292, 340 Haslam, J. M., 199, 217 Haslett, W. L., 225, 266, 267 Hauge, A., 30, 37, 67, 81 Hauss, W. H., 184, 210 Hay, M. F., 267, 276, 290, 340, 334, 343 Hayaishi, O., 266 Hayano, M., 229, 234, 267, 269, 260 Hayashi, K., 309, 344 Hayden, J., 317, 337 Hayes, W. J., Jr., 223, 266 Hayles, A. B., 302, 303, 340 Hayman, H. B., 320, 336 Hazel, M. M., 318, 340 Healey, L. A., 115, 133, 134, 136, 138 Heard, R. D., 159, 209 Heath, D. F., 224, 267 Heath, R. G., 31, 81 Heaton, A., 225, 267 Hebborn, P., 11, 81, 194, 210 Hector, O., 234, 267 Heeg, E., 38, 81 Hegner, J., 208 Heiberg, K., 303, 340 Heicke, B., 30, 40, 43, 44, 80, 192, 209 Heiderhain, R., 149, 210 Heidenreich, O., 39, 71, 81 Heidrich, H. G., 24, 90 Heilmeyer, L., 154, 158, 172, 176, 193, 196, 110
Heinemann, H. O., 133, 138 Heinkel, K., 7, 87 Heller-Szollosy, E., 292, 3.46 Hellman, H., 133, 138 Helm, F., 272, 346 Hemery, N., 320, 341 Hemmens, E. S., 157,813 Hempel, R., 21, 24, 25, 29, 64, 66, 83, 87 Henderson, J. F., 223, 226, 869
Henderson, W. R., 113, 138 Henning, N., 7, 87 Henriques, 0. B., 18, 20, 44, 45, 81, 86 Hensel, H., 10, 41, 80, 86 Herken, H., 176, 206 Hermann, R. E., 13, 81 Hermanns, A., 305, 341 Hernandez, P. H., 232, 242, 267 Herrmann, I%.,186, 198, 201, 213, 217, 254, 267
Herschlein, IJ. J., 12, 13, W Hershberger, L. G., 184, 185, 202, 203, 210 Herxheimer, A., 32, 81 Herxheimer, H., 40, 58, 81 Herzog, P., 38, 78 Heseltine, M., 277, 340 Hem, B., 199, 211 Hetzel, B. S., 157, 161, 210 Hetzel, H., 19, 88 Heusghem, C., 157, 158, 161, 214, 216 Hierholzer, K., 96, 113, 134, 138 Hicks, S. P., 276, 340 Hill, A. B., 277, 337 Hill, R., 120, 138 Hill, R. M., 320, 340 Hillebrecht, J., 190, 210 Hiller, J., 197, 199, 210 Hills, A. G., 132, 137, 159, 216 Hilton, J. G., 95, 134 Hilton, S. M., 7, 34, 38, 41, 63, 76, 79, 81, 178, 179, 208, 210 Hine, D. C., 157, 161, 210 Hitchings, G. H., 94, 140 Hitzelberger, A., 159, 110 Hjelt, H., 277, 346 Hochberg, R., 54, 81 Hochrein, M., 35, 81 Hochstein, P., 235, 236, 267 Hochstrasser, K., 19, 20, 55, 89 Hodgson, E., 224, 267 Homle, S., 12, 74 Hoferichter, J., 13, 81, 84 Hoffman, W . S., 112, 114, 115, 139 Hoffmann, K., 322, 342 Hofman, H. L., 13, 81 Hofmann, E. C. G., 245, 267 Hofmann, F., 302, 340 Hofmann, H., 153, 210 Hofmann, K., 22, 81
363
AUTHOR INDEX
Ragan, A. G., 277,346 Hogben, C . A. M., 111, 140, 254, 266 Holdstock, D. J., 178, 210 Holemans, R., 15, 72, 81 Holgate, J. A., 26, 30, 40, 77, 190, 206 Hollenberg, M. J., 13, 88 Hollis, A. U.,13, 80 Holloway, P. W., 228, 267 Holman, G . H., 302, 348 Holten, C., 133, 138 Holta, P., 9, 18, 77, 81, 268 Holzer, H., 186, 214 Hooper, K. C . , 48, 81 Horecker, B. L., 241, 268 Horn, D. B., 117, 138, 141 Horne, H. H., 270, 279, 308, 325, 329, 336 Horne, N. W., 109, 138 Homing, E. C., 120, 121, 122, 129, 136, 225, 250, 252, 267, 268 Hornstein, S., 298, 348 Horsach, I., 13, 77 Horton, E. W., 14, 22, 23, 30, 31, 34, 35, 48, 51, 52, 62, 63, 78, 79, 81, 178, 208 Horwood-Barrett, S., 43, 77 Hosoda, T., 19, 20, 27, 80 Houck, J. C., 183, 210 Howard, J. E., 157, 206 Howell, D. S., 121, l 4 l Howell, R. R., 93, 94, 98, 138, 140 Horwitz, M., 132, 133, 140 Huang, K. C., 113, 138 Hucker, H. B., 225, 247, 268 Huddlestun, B., 108, 109, 113, 141 Hueper, W. C., 104, 138 Hurter, J., 5 , 89 Huffman, E. R., 115, 120, 138, 140 Huggins, C. G., 19, 62, 81 Hujanena, O., 317, 346 Hultin, T., 232, 261 Hultquist, G. T., 303, 340 Hume, D. M., 162, 210 Hummel, F. P., 161, 163, 165, 168, 169, 193, 216 Hungerland, H., 304, 348 Hunter, T. A. A., 330, 341 Hurley, L. E., 318, 341 Hutner, S. H., 290, 338 Hutzel, M., 7, 88 Hyder, N., 161, 162, 167, 207 Hyman, C., 36, 86
I Imdahl, H., 305, 341 Inaba, T., 265, 349 Inanovics, G., 148, 210 Indovina, D., 30, 38, 79 Ingalls, T. H., 277, 298, 301, 309, 331, 341 Ingbar, S. H., 132, 138 Ingenito, E. F., 298, 309, 331, 341 Ingle, D. J., 158, 170, 210 Inglesby, T. V., 116, 1.40 Innerfield, I., 14, 54, 81, 88 Inzunza, Bascunan, I., 127, 1 f l Irving, C. C., 226, 268 Isaac-Mathy, M., 290, 314, 315, 321, 337 Isaacson, R. J., 303, 341 Ishidate, M., 243, 267 Island, D. P., 231, 266 Israels, M. C . G., 290, 330, 346 Itoiz, J. E., 27, 78 Ivanyi, J., 156, 158, 160, 163, 164, 165, 210 Ivy, A. C., 302, 340 Iwanaga, S., 18, 20, 52, 53, 86, 88
J Jackson, A. V., 317, 341 Jackson, D., 292, 293, 295, 337 Jackson, D. S., 183, 210 Jacob, J., 195, 210 Jacob, R. A., 183, 210 Jacobs, J., 13, 81 Jacobsen, S., 57, 58, 81 Jacobson, K. B., 245, 246, 268 Jacobson, M., 230, 231, 254, 266, 268 Jankalii, E. O., 40, 81 Jager, B. V., 104, 138 James, A. T., 228, 269 James, J. R., 319, 341 James, L. F., 316, 336 Jankowski, A. J., 170, 211 Januschke, A., 171, El0 Janz, D., 320, 3.41 Jaquenoud, P.-A., 22, 51, 52, 76 Jaques, L. B., 166, 211 Jaques, R., 18, 22, 51, 52, 64, 81, 82, 90, 189, M O Jaramillo, J., 36, 80 Javett, S. L., 38, 76, 82 Jeffrey, S . W., 199, 200, 216 Jelen, P., 283, 341
364
AUTHOR INDEX
Kampffmeyer, H., 226, 239, B 8 Kaplan, N. O., 245, 246, 258, 261 Kappeler, H., 87 Kara, E., 133, 138 Karnofsky, D. A., 265, 266, 270, 291, 292, 294, 296, 316, 321, 342, 344, 346 Kass, E. H., 132, 138 Kato, H., 31, 70, 76, 82 Kato, R., 250, 251, 253, 254, 268 Kato, T., 316, 344 Kattus, A., 157, 205 Katz, H., 149, 205 Katz, W., 7, 60, 82 Katzenstein, M., 319, 343 Kaufman, S., 10, 87, 228, 234, 235, 258 Kaufmann-Boetsch, B., 9, 10, 11, 89 Kaunitz, H., 172, 208 Kay, C. F., 109, 136, 137 Kaye, B. M., 277, 342 Kaymakcalan, S., 27, 88 Kazmers, N., 13, 82 Keberle, H., 322, 323, 324, 325, 327, 328, 329, 335, 338, 342 Kedes, L. H., 96,136 Keele, C. A,, 15, 20, 21, 33, 34, 42, 46, 49, 51, 58, 74, 78, 178, $04, 210 Keeler, R. F., 316, 342 Kehl, R., 48, 51, 89, 302, 316, 337, 342 Keiderling, W., 180, 216 Kelemen, E., 156, 157, 158, 159, 160, 162, 163, 164, 165, 169, 170, 189, 210, 215 Keller, C. J., 35, 81 K Keller, P., 39, 71, 81 Kahler, H. J., 154, 172, 176, 193, 210 Kelly, J. W., 283, 342 Kagen, L., 16, 17, 58, 75 Kemnitz, A., 186, 214 Kagen, L. J., 11, 17, 82, 83 Kemp, A., 47, 49, 74 Kageyama, M., 296, 309, $41, 344 Kemp, R. L., 104, 105, 136 Kahn, M. F., 106, 136 Kemp, W. W., 109, 140 Kajii, T., 305, 306, 341 Kemper, F., 270, 279, 307, 308, 329, 342 Kalbhen, D. A., 176, 177, 181, 210 Kendrick, F. J., 318, 342 Kaliampetsos, G., 6, 59, 89 Kennedy, R. D., 121, 138 Kalter, H., 265, 266, 267, 269, 270, 277, Kennedy, T. J., Jr., 95, 134 283, 289, 291, 300, 301, 303, 313, 317, Kennell, J. H., 320, 340 320, 321, 332, 334, 339, 840, 341, 342, Keppler, A,, 4, 5, 20, 45, 47, 48, 89 Kerby, G. P., 184, 210 348 Kameyama, Y., 316, 344 Kersley, G. D., 117, 127, 128, 138 Kamin, H., 232, 241, 244, 247,268, 269,261 Kersten, H., 197, 210 Kamm, J. J., 227, 232, 233, 236, 240, 242, Kesner, R., 316, 317, 348 247, 257, 268 Kessler, G., 133, 138 Kessler, R. H., 96, 113, 134, 138 Kampf, H.H., 324, 335
Jenden, D. J., 225, 256, 257 Jennings, G. H., 100, 138 Jensen, K. B., 62, 63, 82 Jepson, J. B., 15, 20, 21, 33, 46, 49, 58, 74, 178, $04 J i m h e z Elqueda, L., 127, 137 Johnson, N. M., 225, 267 Johnson, R., 316, 346 Johnson, W. J., 270,279, 308, 325, 329,335 Johnstone, E. E., 302, 341 Johnstone, J. M., 319, 346 Jonasson, O., 58, 82 Jondort, W. R., 328, 341 Jones, C. R., 58, 63, SO Jones, H. W., 302, 303, 348 Jones, J. W., Jr., 303, 341 Jones, R., 231, 268 Jonszen, H., 113, 138 Jorgensen, G., 298, 341 Jori, A,, 33, 76 Joron, G. E., 298, 336 Joseph, A,, 341 Joseph, R., 320, 341 J o s h , E. P., 313, 341 Jost, A., 265, 267, 272, 299, 300, 301, 302, 303, 304, 320, 321, 336, 337,341, 343 Junge-Hulsing, G., 184, 210 Junkmann, K., 303, 341 Jurand, A,, 292, 341 Jurkiewicz, A., 19, 86 Justin-Besancon, L., 278, 341
AUTHOR INDEX
Ketterer, H., 12, 77 Khairallah, P. A., 31, 45, 50, 63, 76, 82 Kidd, D. J., 34, 38, 79, 179, 808 Kiel, H., 272, 346 Kiem, I. M., 121, 141 Kiese, M., 143, 204, 210, 222, 226, 239, 255, 268 King, C. T. G., 269, 276, 307, 309, 318, 319, 324, 329, 342, 344 King, J. St., 161, 162, 167, 207 King, M. E., 119, 138 King, M. K., 177, 211 King, R. J. B., 230, 258 Kinnunen, O., 316, 348 Kiran, B. K., 27, 88 Kirby, W. M. M., 113, I50 Kitchin, P. C., 316, 342 Klein, M., 280, 342 Klein-Obbink, H. J., 307, 309, 842 Kleinsasser, O., 304, 34% Klett, W., 20, 45, 47, 49, 80, 82 Klieger, E., 24, 87 Klinenberg, J. R., 94, 112, 137, 138 Klingenberg, M., 236, 258 Klupp, H., 37, 43, 82 Klutch, A,, 231, 266 Kluthe, R., 143, 814 Knapp, K., 267, 304, 342, 343 Knauer, Q. F., 106, 13G Kneebone, G. M., 37, 70, 84 Knesslova, V., 45, 76 Knighton, H. T., 342 Knobloch, H., 164, 214 Knouff, R. A., 316, 342 Knowles, R. C., 13, 81 Knox, W. E., 234, 260 Knuppen, R., 230, 255 Kobold, E. E., 7, 13, 36, 57, 60, 82, 88 Kobrin, S., 163, 216 Koch, W., 305, 341 Kodama, R., 115, 134, 137 Kodicek, E., 202, 211 Koebke, K., 48, 51, 89 Kohler, V., 199, 200, 211 Korbel, W., 5, 82 Korbel-Enkhardt, R., 12, 82 Koets, P., 116, 138 Kohn, J., 58, 63, 80 Kolb, F. O., 112, 138 Kolbe, H., 145, 146, 211
365
Kontos, H. A., 36, 38, 71, 82 Konaett, H., 22, 30, 36, 37, 38, 42, 43, 71, 76, 78, S2 Kook, Y., 39, 71, 81 Kopf, R., 282, 283, 342 Koransky, W., 327, 348 Kornfeld, H., 164, 211 Korsenow, K., 342 Korsten, H., 6, 29, 59, 89 Karte, F., 223, 268 KO~LIS, W., 197, 211 Kosenow, W., 321, 346 Koskenoja, M., 313, 342 Koslowslti, L., 13, 82, 154, 811 Kovics, B. A., 43, 44, 82 Kovacs, K., 156, 158, 160, 163, 164, 165, 210 Kovalcik, V., 36, 82 Kraus, A. P., 320, 342 Krauss, R., 158, 196, 210 Kraut, H., 4, 5, 6, 7, 9, 10, 11, 12, 17, 19, 21, 22, 35, 36, 40, 48, 57, 59, 79, 82 Krayer, O., 4, 35, 36, 40, 82 Krell, R., 32, 34, 84 Kreshover, S. J., 313, 342 Kretschrner, V., 196, 204 Kricgel, A,, 267, 343 Krisch, K., 247, 258 Kritchevsky, D., 230, 258 Krivoy, W., 38, 88 Krivoy, W. A,, 31, 48, 49, 53, 82 Kroc, R. I,., 302, 342 Kroeger, D. C., 38, 48, 49, 53, 82, 88 Kromer, H., 213 Kroneberg, G., 35, 36, 83 Kroft, H. M., 170, 211 Krych, G., 33, 7B Kuether, C. A., 160, 213 Kuginuki, M., 293, 344 Kuhlback, B., 106, 138 Kuhn, W. L., 43, 44, 53, 324, 342 Kung, H. L., 317, 347 Kunkel, H. G., 112, 134 Kuntzman, R., 230, 231, 254, 966, 268 Kupfer, S., 96, 99, 113, l4S Kuriaki, K., 200, 211 Kushner, D. S.,112, 156, 1.97 Kuzell, W. C., 114, 116, 119, 129, 138, 57, 211
366
AUTHOR INDEX
Kuzukawa, S., 321, 342
L Labarca, E., 37, 77 Labelle, A., 155, 211 Lacon, C. R., 270, 342 LaDu, B. N., 125, 136, 221, 223, 225, 234, 235, 236, 238, 247, 248, 250, 252, 266, 267, 268
Laftan, R. J., 24, 86 LaGrange, C., 13, 83 Lahiri, S. C., 48, 58, 76 Laki, K., 3, 27, 45, 80, 83, 86 Laland, P., 20, 49, 74 Laland, S., 20, 49, 74 Lambert, P. H., 60, 83 Lambert, P. P., 126, 136 Lamming, G. E., 276, 344 Lamy, M., 277, % 43' Landauer, W., 270, 277, 314, 316, 342 Lane, D. W . J., 224, 267 Langdon, R. G., 241, 242, 245, 260 Lange, F., 154, 211 Lande, S., 24, 31, 76, 76, 82, 83 Landerman, N . S., 27, 58, 83 Landrau, M., 124, 125, 136 Langman, J., 330, 332, 342, 343 Lanz, P., 24, 88 Lapane, R., 154, 213 Lapiere, C. M., 163, 188, 189, 190, 216 Lapras, M., 332, 337 Laragh, J. H., 133, 134, 136, 138 Larsen, V., 318, 342 Larsson, S., 317, 343 Lareson, Y., 315, 343 Lash, J. W., 203, 211 Lask, S., 319, 343 Lassarre, M., 127, 139 Lamen, L. J., 268, 273, 310, 312, 313, 337 Lasten, L., 93, 98, 140 Latanick, A,, 316, 346 Laubach, G. D., 118, 128, 129, 136 Lautemann, E., 145, 211 Lawrence, F. P., 225, 267 Lawrence, R. D., 313, 343 Layton, L. L., 182, 202, 203, 211 Lechat, P., 291, 309,310, 322, 343 Leck, I. M., 319, 343 Leclercq, R., 33, 43, 83 Lecomte, J., 21, 37, 40, 41, 43, 44, 58, 60,
83, 84, 86, 163, 164, 165, 166, 168, 169, 188, 189, 190, 192, 211, 216 Leddy, J. P., 17, 82 Lee, I. R., 43, 77, 191, 206 Lee, K. Y., 223, 268 Lee, P., 119, 136 Lee, R. E., 157, 195, 211, 213 Leevy, C. M., 290, 338 Lefebvres, J., 265, 268, 277, 300, 303, 321, 339
Lefebvree-Boisselot, J., 265, 267, 268, 277, 298, 339, $40 Legrand, J., 267, 343 Leidy, H. L., 134, 134 Lelong, M., 302, 343 Leme, J. G., 43, 44, 45, 86 Leng-LBvy, J., 121, 127, 139 Lenthall, J., 36, 86 Lens, W., 265, 267, 304, 305, 306, 319, 321, 324, 34.2, 343 Leocani, B., 21, 86 Leonhartsberger, F., 157, 208 LBpine, 149, 221 Lergier, W., 24, 88 Leroux, 145, 211 Leroy, P., 303, 337 Lessmann, E. M., 291, 346 Levenberg, B., 234, 268 Levin, E. Y., 234, 268 Levin, S., 238, 239, 266 Levine, M. I., 24, 25, 33, 40, 42, 43, 47, 51, 53, 54, 55, 59, 60, 78 Levine, R., 108, 109, 113, 141 Levitin, H., 116, 137 Levitt, M., 234, 269 Levy, B., 27, 83, 108, 136 Levy, G., 268, 339 U v y , J., 198, 211 Lewis, G. P., 6, 14, 22, 23, 24, 29, 30, 31, 34, 35, 37, 38, 42, 44, 47, 48, 51, 52, 54, 57, 58, 74, 76, 77, 78, 79, 81, 83, 178, 179, 188, 206, 207, 208, 210, 211 Lewis, R. E., 43, 44, 83 Leysath, G., 22, 23, 55, 89 Liacopoulos, P., 188, 209 Liacopoulos-Briot, M., 188, 209 Liang, H. M., 308, 349 Libretti, A., 71, 76, 79 Lichtenstein, H., 269, 314, 315, 343 Liddle, G. W., 231, 266
367
AUTHOR INDEX
Liebermeister, C., 147, 211 Lijinsky, W., 223, 268 Lillehei, R. C., 7, 57, 83 Lim, R. K. S., 30, 34, 35, 43, 80, 83, 195, 211 Lindberg, M. C., 229, 234, 267, 259 Lindbroos, B., 317, 346 Linder, J., 207 Lindner, H., 177, 180, 211 Lindner, J., 175, 179, 214 Ling, W. S. M., 109, 140 Linker, A., 202, 209 Lioy, F., 36, 37, 86 Lipschitz, W., 153, 211 Lisin, N., 33, 43, 83 Lits, F. J., 331, 343 Littmann, D., 133, 139 Liu, T. Y., 22, 81 Lloyd, S., 37, 83 Lock, F. R., 277, 349 Lockie, L. M., 114, 141 Loffler, F., 11, 89 Loevy, H., 330, 344 Loewi, G., 185, 202, 211 Lohmeyer, H., 268, 343 Lombardi, V., 72, 87 Longchampt, J. E., 229, 268 Longerbeam, J. K., 7, 57, 83 Lorbek, W., 13, 85 Lorenz, D., 282, 283, 342 Lorenzini, R., 206 Lotspeich, W. D., 113, 136 Louatalot, P., 325, 327, 328, 342 Loutifi, M., 161, 208 Lovell, R., 7, 60, 82 Lovell, R. R. H., 133, 141 Lowe, J. S., 9, 77, 187, 207 Lowe, W. C., 37, 74 Lowenthal, J., 166, 211 Lowman, E. W., 119, 136 Lucey, J. F., 267, 273, 276, 310, 313,343 LudBny, G., 32, 83, 181, 211 Ludwig, A. W., 185, 2U6 Ludwig, G., 223, 268 Lubke, K., 22, 66, 67, 83, 87 Liideritz, O., 180, 217 Lugo, J. E., 36, 37, 86 Lukens, L. N., 243, 266 Lund, F., 10, 83 Lund, P. G., 307, 346
Lunde, P. K. M., 30, 37, 67, 81 Lutwak-Mann, C., 158, 198, 211, 276, 290, 332, 334, 343 Lutz, H. R., 270, 349 Lutzenkirchen, A,, 159, 811 Lutzmann, L., 20'7 Lyman, H., 104, 137
M McBride, W. G., 265, 304, 318, 319, 321, 324, 343 McCarthy, D. A,, 23, 24, 27, 29, 50, 53, 83, 86
McCarty, D. J. Jr., 94, 139 McCay, P. B., 236, 269 McColl, J. D., 307, 317, 322, 343 McConnell, D. J., 11, 83 McCreadie, S. R., 296, 337 McCutcheon, A. D., 13,83 MacDonald, A. D., 195, 217 MacFarlane, W. V., 276, 343 McGrath, W. R., 43, 44, 83, 327, 343 McHardy, G., 13, 83 MacKay, M. E., 16, 17, 58, 83, 89 McKelway, W. P., 319, 348 McKenna, R. D., 7, 13, 76 McKenzie, R. D., 327, 343 McKinney, S. E., 116, 139 McLaughlin, J., Jr., 270, 279, 343 McMahon, R. E., 221, 223, 224, 225, 249, 251, 268, 269 McMennamin, M. A., 51, 78 McPadden, A. J., 303, 336 Machin, A., 277, 340 Maebashi, M., 63, 74 Maegraith, B. G., 59, 88 Magat, A., 333, 336 Magee, J. H., 36, 38, 8.9 Magee, P. N., 223, 268, .969 Magendie, F., 145, 208 Magid, G. J., 115, 133, 134, 138 Mahairas, G. H., 320, 343 Maickel, R. P., 227, 266, 328, 341 Maier, L., 6, 10, 11, 12, 14, 36, 89 Maisel, H., 332, 343 Majoros, M., 156, 157, 158, 159, 160, 162, 163, 164, 165, 169, 210 Maller, R. K., 325, 327, 328, 342 Mallet-Guy, P., 13, 83 Malnic, G., 39, 86
368
AUTHOR INDEX
Manat, W. G., 7, 57, S3 Manis, J. G., 101, 113, 140 Mankle, E. A., 114, 116, 119, 138 Mann, P. J. G., 245, 259 Mannering, G. J., 234, 249, 260, 261 Mansson, B., 200, 203, 205 Maraud, R., 297, 333, 338, 343 Marble, A., 313, 341 Marcelle, R., 58, S3 Marchesoni, M., 319, 344 Maren, T. H., 157, 159, 160, 161, 205 Margolis, J., 15, 16, 21, 26, 32, 49, 59, 76, 84 Mariani, L., 43, 44, 59, 84 Marie, J., 277, 343 Marin-Padilla, M. D., 273, 313, 343 Mark, L. C., 131, 139 Markham, J. W., 33, Y 4 Markkanen, P., 113, 139 Marks, V., 199, 211 Marliac, J. P., 270, 279, 343 Marmorstone, J., 200, 215 Marois, M., 276, 300, 302, 316, 337, 343 Marra, N., 34, 79 Marrazzi, A. S., 25, 31, S5 Marschner, I., 12, 77 Marsh, J. B., 228, 259 Marshall, E. K., 157, 159, 160, 161, 206 Marshall, E. K., Jr., 105, 139 Marshall, F. J., 221, 259 Marshall, R. J., 190, 209 Marson, F. G. W., 100, 116, 139, 158, 159, 211 Martin, B. K., 100, 136 Martinet, M., 265, 267, 268, 269, 277, 283, 288, 289, 300, 303, 320, 321, 339, 340 Martinez, A. R., 83 Martius, C., 199, $11 Marumo, H., 200, 211 Marx, R., 12, 84 Masbernard, A,, 305, 338 Mason, B., 188, 217 Mason, D, T., 7, 41, 59, 85 Mason, H. S., 234, 242, 243, 248, 259 Mason, R. M., 114, 139 Mamion, W. H., 13, 60, 62, 84 Massopust, L. C., 316, 345 Masters, B. S . S., 244, 258, 259 Mathias, A. P., 64, 84, 178, 210 Mathies, H., 165, 168, 170, 211, 218
Matossian, G. S., 114, 115, 134 Matteucci, W. V., 113, 135 Mattis, P. A,, 107, 135 Mauck, H. P., 38, 71, 82 Maurer, W., 13, 84 Mauss, H. J., 310, 343 Maxwell, G. M., 37, 70, 84 May, H. E., 236, 259 Maycock, W. d'A., 58, 85 Mayer, R. L., 180, 212 Mazel, P., 223, 226, 227, 232, 242, 266, 257, 259 Meade, B. W., 158, 212 Means, J. H., 198, 207 Mrhrizi, A., 309, 33Y Meier, R., 18, 41, 81, 88, 155, 157, 174, 179, 181, 186, 188, 212 Meissner, J., 202, 207 Mela, V., 289, 318, 338, 343 Melchiorri, P., 58, 84 Mellin, G. W., 318, 319, 343 Mellinghoff, C. H., 127, 139 Melmon, K., 7, 41, 59, 85 Melon, J., 40, 58, 83, 84 Meltzer, H. J., 298, 343 Melville, K. I., 43, 44, 82 Meng, K., 38, 81 Menkin, V., 31, 84, 174, 176, 177, 218 Menzel, H., 243, 210 Mercier-Parot, L., 265, 268, 269, 272, 274, 275, 282, 283, 286, 287, 291, 292, 293, 295, 296, 297, 298, 299, 301, 302, 304, 307, 308, 309, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 330, 331, 332, 337, 340, 343, 344, 347 Merger, R., 320, 344 Merker, H. J., 221, 240, 253, 260 Mrrren, D. D., 109, 139 Merrifield, R. B., 23, 84 Mertz, D. P., 39, 71, 72, 84 Mcsser, M., 200, 212 Metallnikow, S., 154, 212 Metcalf, W. K., 331, 344 Mey, R., 302, 344 Meyer, A,, 7, 21, 57, 60, 62, 84 Meyer, A. E., 320, 335 Meyer, K., 182, 202, 209, 812 Meyers, E., 277, 340 Michelacci, S., 59, 65, 71, 72, 87, 188, g14 Michoulier, J., 13, 83
369
AUTHOR INDEX
Migeon, C. J., 113, 137 Mikkelsen, W. M., 114, 115, 127, 133, 141 Miles, A. A., 16, 17, 31, 32, 58, 76, 79, 83, 84, 86, 89, 187, 193, 212 Miles, E. M., 31, 84 Mill, P. J., 16, 32, 79, 84, 193, 2lb Millen, J. W., 272, 275, 277, 289, 321, 322, 344, 349
Miller, A. K., 108, 109, 110, 111, 112, 113, 136
Miller, E. C., 223, 226, 238, 254, 266, 250 Miller, J., 94, 1S8 Miller, J. A., 223, 226, 232, 238, 254, 256, 259
Miller, J. R., 277, 303, 344, 345 Miller, P. O., 202, 209 Miller, W. L., 332, 333, 344 Miller, Z. B., 290, 344 Mills, G. L., 39, 74 Mills, J., 221, 224, 249, 259 Milne, M. D., 101, 102, 139 Mirsky, I. A., 176, 183, 212 Mishima, H., 153, 154, 155, 216 Misiti, D., 323, 329, 344 Mitchell, J. C., 32, 34, 84 Mitoma, C., 222, 234, 238, 248, 253, 259, 260
Mitropoulous, K. A., 230, 259 Miwa, I., 63, 74, 90 Miyoshi, T., 265, 349 Mizushima, Y., 18, 20, 86, 88 Moench, A., 153, 214 Morsdorf, K., 155, 161, 162, 163, 164, 165, 166, 167, 168, 171, 174, 188, 190, 207, 212, 216 Moffat, J. G., 60, 86 Moloshok, M. D., 300, 302, 303, 340 Molteni, A., 330, 344 Mongar, J. L., 189, 212 Monie, I. W., 296, 321, 344, 345 Monier, R., 19, 86 Monroe, B. B., 348 Monroe, B. L., 316, 344 Monroe, K. E., 133, 139 Montague, D., 38, 86 Montagne, J., 283, 284, 287, 309, 310, 319, 320, 321, 3% Moog, E., 37, 40, 43, 67, 80, 84 Moore, K. B., 113, 138
Morandi, E., 319, 344 Morato, T., 229, 269 Morchesoni, M., 344 Moreau, M. G., 302, 341 Moret, P., 58, 80 Morgan, R. S., 32, 76 Morgan, W. S., 116, 137 Moriya, H., 8, 9, 84, 88 Morley, J., 30, 32, 76 Morris, C. W., 189, 216' Morris, H. C., 14, 88 Morrison, E., 183, 185, $14 Moses, J. M., 32, 80 Moses, V., 200, 212, 216 Moss, P. D., 312, 344 Moyer, J. H., 112, 115, 133, 134, 137, 140 Mudge, G. H., 96, 99, 101, 103, 107, 111, 112, 113, 1117, 129,134,141 Mueller, G. C., 223, 230, 232, 269 Munich, W., 203, 212 Muir, H., 201, 202, 212 Muller, E., 317, 347 Muller, H. A., 302, 349 Multhaupt, G., 36, 89 Munson, A. E., 15, 30, 58, 76 Murad, J. E., 307, 310, 344 Murakami, U., 316, 344 Muratori, F., 30, 38, 79 Murphee, 0. D., 316, 344 Murphy, M. L., 265, 292, 293, 294, 295, 296, 298, 316, 321, 329, 344 Murphy, S. D., 227, 269 Murtaugh, P. A., 27, 45, 80 Muscholl, E., 42, 84 Mutchler, M. K., 270, 279, 343 Mutsers, A., 158, 159, 214 Myant, N. B., 230, 259
N Nagatsu, T., 234, 259 Nagcl, W., 7, 84 Najayima, M., 266 Nahum, A,, 302, 316, 348 Nakai, K., 293, 316, 344 Nakano, J., 37, 38, 69, 70, 71, 84 Narasimhulu, S., 236, 237, 238, 239, 248, 256, 259
Narrod, S. A., 276, 318, 324, 329, 342, 344 Nasjletti, A., 39, 76
370
AUTHOR INDEX
Naugler, W. E., 116, 138 Naunyn, B., 149, 212 Nechay, B. R., 99, 139 Nechay, L. J., 99, 139 Neher, G. M., 185, 214 Nehrbauer, E., 13, 84 Nelson, D. H., 159, 212 Nelson, M. M., 298, 321, 330, 344 Nelson, R. C., 265, 267, 268, 334, 342, 348 Nemir, P., Jr., 13, 81, 84 Netter, K. J., 221, 269 Neumann, F., 303, 341 Neurath, H., 10, 87 Neuweiler, W., 316, 344 Newberne, P. M., 277,344 Newcombe, D. S., 113, 139 Newman, B. E., 133, 137 Newman, E. V., 157, 206 Ney, R. L., 231, 266 Nicolaides, E. D., 22, 23, 24, 27, 29, 37, 50, 52, 53, 83, 86 Nicolaier, A,, 103, 105, 139 Nicholas, R. L., 113, 139 Nicolay, M., 71, 80 Nilsson, J., 184, 212 Nilsson, R., 244, 669 Nishibayashi, H., 237, 240, 242, 248, 269, 260
Nishihara, G., 300, 304, 320, 344 Nishimura, H., 293, 309, 316, 344 Nitti, F., 231, 261 Noback, C. R., 299, 3.48 Nolan, R. B., 302, 303, 340 Norcross, B. M., 114, l4l Nordenbrand, K., 236. 267 Norlind, L. M., 330, 336 Norris, R. F., 109, 137 North, J. C., 242, 248, 269 Northover, B. J., 11, 86, 163, 165, 169, 193, 194, 212, 213 Notter, A., 319, 344 Nowack, E., 304, 342 Nuss, G. W., 163,217 Nustad, K., 46, 50, 74 Nuaum, F. R., 4, 36, 41, 78 NystrGm, c.,329, 846
0 Oakley, W., 313, 343 Oates, J. A., 7, 41, 59, 86
O’Brien, R. D., 224, 227, 253, 269, 260 Odeblad, E., 203, 206 O’Dell, B. L., 277, 344, 346 Ogryzlo, M. A., 114, 120, 123, 127, 128, 139
Ohba, N., 267, 277, 346 Ohlmeyer, P., 213 O’Keefe, E., 24, 86 Okon, M. E., 6, 23, 24, 80 Olian, S., 303, 347 Oliveira, M. C. F., 44, 45, 86 Oliver, M. F., 133, 139 Olmsted, F., 29, 36, 37, 70, 86 Olson, J. A., 229, 269 O’Malley, W. E., 169, 209 Omura, T., 236, 237, 238, 240, 241, 242, 248, 269, 260 Ondetti, M. A., 24, 76, 76, 86 Onesti, G., 133, 140 Onoprienko, I., 243, 269 Opit, L. J., 198, 206 Opitz, K., 164, 213 Orahood, R. C., 13, 87 Ordy, J. M., 316, 346 Oren, B. G., 133, 139 Orent-Keiles, E., 336 Orrenius, S., 236, 238, 240, 241, 244, 245, 247, 248, 253, 266, 269, 260 Osbahr, A, J., 45, 86 Ostfeld, A. M., 34, 60, 86 Otto-Servais, 60, 83 Overholser, M. D., 277, 346 Overaier, c., 302, 340
P Page, I. H., 18, 29, 31, 36, 37, 45, 50, 63, 70, 76, 81, 82, 86 Pagenstecher, H. E., 264, 348 Paget, G. E., 282, 317, 346 Palade, G. E., 220, 260 Paladini, A. C., 27, 78 Paldino, R. L., 36, 86 Pallot, G., 167, 213 Pangels, G., 230, 266, 260 Papenberg, J., 10, 41, 80, 86 Papper, E. M., 131, 139 Paradiso, M., 76 Parent, B., 320, 344 Park, P. O., 224, 267
371
AUTHOR INDEX
Parke, D. V., 222, 266, 324, 325, 326, 348 Parkes, A. S., 277, 546 Parks, R. E., Jr., 234, 261 Parratt, J. R., 30, 37, 38, 71, 85, 188, 190, 215 Parrot, J. L., 38, 88, 154, 216 Pascale, L. R., 112, 114, 115, 137, 189 Pashkina, T. S., 16, 86 Pasqualini, C. D., 157, 161, 215 Pasqualini, R. Q., 157, 161, 215 Patch, E. A., 108, 109, 110, 136 Paton, B. C., 119, 120, 121, 122, 123, 124, 127, 128, 129, 156, 141 Patterson, J. L., Jr., 36, 38, 71, 82 Pavesi, L., 19, 81 Payne, R. W., 184, 185, 214, 216 Peck, G . C . , 303, 545 Peck, H. M., 113, 116, 159 Pecora, L. J., 105, 132, 136 Pedersen, J., 313, 346 Pedersen, L. M., 313, 545 Peer, L. A., 303, 546 Peluffo, R., 228, 267 Peng, D., 152, 215 Pennial, R., 199, 215 Pennycuik, P. R., 276, 545 Pequignot, H., 278, 341 Pereda, T., 37, 77 Perel, J. M., 111, 116, 117, 118, 121, 124, 125, 126, 127, 128, 131, 132, 156, 139 Periti, P., 21, 34, 51, 59, 60, 76, 76, 86, 87 Perkins, W. H., 113, 156 Perlow, S., 320, 342 Persellin, R. H., 128, 159 Pessonnier, J., 283, 284, 287, 309, 310, 319, 320, 321, 336 Peterman, A. F., 185, 205 Peters, J. P., 93, 139 Peters, L., 107, 156 Peters, T., Jr., 221, 260 Petit, J. M., 58, 85 Petit, M. D., 332, 536 Petit, P., 302, 545 Petras, H. S., 24, 87 Petroff, J. R., 4, 85 Pettit, F. H., 225, 260, 261 Pfeilfer, C . C., 157, 195, 211, 213 Pfeiffer, R. A., 304, 306, 319, 321, 342, 546, 348
Pfister, R., 120, 123, 124, 139 Phillips, A. H., 241, 242, 245, 260 Phoenix, C. H., 302, 346 Picarelh, z. P., 18, 20, 44, 45, 81, 86 Pierce, J. V., 6, 8, 9, 10, 11, 12, 14, 15, 18, 20, 22, 27, 37, 47, 52, 53, 84, 86, 88 Pieri, L., 25, 31, 86 Piguet, B., 159, 161, 206 Pike, R. L., 277, 546 Pinsky, M. F., 340 Pinter, E. J., 7, 13, 76 Piquet, J., 208 Piria, R., 145, 215 Pisano, J. J., 64, 86 Pitt, A. A,, 110, 111, 141 Pitt, P. A., 222, 231, 166 Pitts, F. W., 111, 112, 113, 156 Pitts, R. F., 134, 158 Plag, M., 195, 213 Pless, J., 23, 86 Pliess, G., 272, 309, 546 Poisner, A. M., 42, 86 Pollard, W., 307, 346 Popesco, M., 188, 207 Popper, H., 172, 208 Porcile, E., 164, 184, 202, 207 Posner, H. S., 222, 234, 238, 248, 253, 269, 260
Potter, D. E., 24, 27, 29, 83, 86 Potter, G. D., 30, 34, 35, 50, 53, 80 Potter, J. L., 227, 260 Poulantzas, J., 170, 212 Poulsen, H., 99, 112, 113, 137, 139 Powell, P. D., 319, 346 Powell, W. J., Jr., 43, 53, 88 Poyer, J. L., 236, 269 Prado, E. S., 8, 19, 22, 26, 86 Prado, J. L., 8, 19, 22, 86 Preiss, B., 228, 260 Preisser, F., 19, 89 Preston, D. W., 316, 342 Prost, H., 265, 267, 268, 321, 339 Prunty, F. T . G., 132, 137, 158, 159, ,916 Pudles, J., 229, 260 Pulver, R., 186, 197, 198, 201, 213, ,917, 254, 257
Q Quan, R. B. E., 115, 133, 134, 136
372
AUTHOR INDEX
Richter, R. H. H., 316, 344 Ridgway, L. P., 316, 346 Riechert, W., 154, 613 Riegel, F., 147, 213 Riess, L., 147, 213 R Riess, W., 322, 327, 329, 338 Ringelmann, E., 89 Race, D., 13, 83 Rammelkamp, C. H., 107, 139 Rinvik, S. F., 63, 86 Ramos, A. O., 31, 34, 37, 40, 60, 76, 86, 86, Risley, E. A., 163, 217 154, 155, 606 Rittel, W., 22, 87 Ramos, L., 34, 86 Ritterband, A., 117, 124, 125, 126, 127, 136 Rand, R., 114, 136 Rivalier, E., 154, 213 Randall, H. G., 329, 337 Rivera, J. V., 106, 139 Randall, L. O., 155, 213 Rizsuto, V. J., 116, l 4 O Randolph, V., 114, 138 Roath, S., 290, 330, 346 Ranney, R. E., 184, 185, 202, 203, 210 Robbins, W. C., 185, 207 Rapoport, S., 245, 267 Robel, K. P., 7, 84 Robert, J. M., 317, 336 Rapp, Y., 43, 79 Robertson, N., 293, 336 Rapport, M., 182, 616 Ratcliffe, H. E., 58, 83 Robertson, W. F., 304, 346 Ratnoff, 0. D., 15, 16, 32, 86, 88 Robinson, F. B., 157, 161, 613 Ratschow, H., 151, 213 Robinson, S., 307, 317, 322, 343 Raudonat, H. W., 9,77 Robinson, W. D., 114, 115, 127, 140, 141 Raventos, J., 227, 260 Robson, J. M., 292, 293, 295, 318, 337, 346 Raynaud, A., 300, 302, 304, 346 Rocha e Silva, M., 10, 14, 18, 19, 22, 26, Reaney, B. V., 277, 346 30, 31, 32, 36, 37, 39, 40, 41, 42, 43, Reeke, T., 7, 38, 86 44, 45, 46, 48, 49, 51, 55, 57, 58, '74, Reed, E. B., 106, 139 79, 81, 86, 86, 174, 179, 188, 613 R6gnier, J., 195, 613 Roche, M., 100, 134 Reichenthal, J., 120, 121, 122, 129, 136 Rochlin, D. B., 68, 76 Reid, J., 157, 158, 206 Rodgers, D. W., 30, 34, 35, 43, 80, 83 Reilly, J., 154, 613 Rodman, G. P., 96, 136 Reilly, W. A., 303, 346 Roe, J. H., 160, d l 3 Reit, E., 30, 42, 83 Roesky, N., 298, 336 Reits, H. C., 222, 269 Rogers, L. A., 252, 253, 266, 260 Relman, A. S., 132, 138 Rohde, E., 148, d l 3 Remmer, H., 221, 240, 253, 260 Roholt, K., 108, 140 Renfrew, A. G., 27, 30, 42, 43, 46, 47, 48, Rolle, J., 13, 86 49, 50, 51, 52, 53, 54, 63, 78 Romani, J. D., 183, 213 Renson, J., 163, 190, 616, 228, 234, 260 Root, H. F., 313, 34i Reynolds, E. S., 116, 139 Roeas, R., 38, 86 Rhoads, C. P., 231, d60 Rose, R. K., 119, 120, 121, 122, 129, 136 Rhode, H., 68, 76 Rosen, F. S., 16, 58, '77 Ribiere, M., 320, 841 Rosenberg, C., 184, 213 Rich, C., 185, 607 Rosenberg, M. L., 109, 139 Rich, M., 133, 139 Rosenbcrg, T., 96, 141 Richards, R. K., 227, 254, 260 Rosenbusch, G., 20, 80 Richards, W. A., 113, 139 Rosenfeld, G., 14, 18, 51, 86 Richards, W. H. G., 58, 59, 63, 80, 86 Rosenfeld, R. S., 133, 138 Richie, A. C., 7, 13, 76
Quastel, J. H., 245, 669 Quimby, E. H., 299, 346 Quincke, H., 149, 216
AUTHOR INDEX
373
Rosenthal, O., 235, 236, 237, 238, 239, 241, Ryan, J. W., 60, 86 242, 248, 256, 259, 260 Ryan, K. J., 229, 237, 260 Rosenthal, S . R., 57, 58, 86, 174, 179, 213 Ryckewaert, A., 106, 114, 115, 116, 127, Rosenthal, T., 306, 307, 345 128, 136 Rosiere, C. E., 164, 191, 217 Roskam, J., 157, 158, 166, 213, 214 5 Rosnati, V., 269, 272, 307, 309, 322, 323, 329, 335, 345 Saameli, K., 27, 50, 76, 86 Rosner, D. C., 277, 342 Saba, N., 234, 257 Ross, D. N., 104, 240 Sackers, E., 38, 79 Ross, H., 319, 348 Sachs, J., 149, 204 Ross, 0. A., 313, 345 Saito, K., 196, 214 Ross, I. P., 303, 340 Sakagishi, P., 242, 248, 259 Roth, C. B., 265, 349 Sakula, J., 277, 337 Roth, J. S., 245, 260 Salaun, J., 270, 279, 292, 307, 308,311, 321, Rothberg, S., 234, 238, 260 346 Roubacky, E. P., 169, 209 Salewski, E., 282, 283, $42 Roudonat, H. W., 18, 81 Salmon, J., 21, 60, 83, 86 Roux, C., 278, 289, 307, 316, 317, 321, 339, Salter, W. T., 159, 206 Salzgeber, B., 270, 279, 290, 292, 307, 308, 341, 346 Rowe, G. G., 36, 37, 74, 86 311, 321, 346 Rowley, D. A,, 36, 86 Samuels, L. T., 150, 151, 159, 177, 218, 224 Rubel, W. M., 176, 814 Sanchez, H. W., 268, 313, 314, 321, 346 Rubin, A,, 249, 200, 318, 345 Sanders, E., 238, 241, 260 Rubin, B., 24, 86 Sandrin, Ed., 22, 65, 66, 67, 76, 86, 88 Rubin, I. L., 133, 138 Sansone, G., 265, 346 Ruch, J. V., 270, 271, 290, 292, 296, 339, Santandrea, E., 36, 77 Santini, D., 21, 51, 87 34 Rubsaamen, H., 268, 321, 336 Sardi, G., 79 Rudel, G., 6, 11, 13, 26, 47, 88, 178, 216 Sarma, V., 277, 346 Ruhl, A,, 4, 35, 36, 40, 82 Sarre, H., 143, 214 Rugh, R., 265, 345 Sasahara, A. A., 133, 139 Rugstad, H. E., 74 Sasame, H. A., 233, 241, 246, 248, 267 Ruiz Moreno, A,, 127, 140 Sato, R., 236, 237, 238, 240, 241, 242, 248, Rukes, J. M., 112, 138 259, 260 Rumney, G., 230, 269 Sato, T., 18, 20, 52, 53, 86, 88, 223, 266 Rumpler, Y., 270, 290, 301, 303, 336, 546 Sautai, M., 283, 284, 287, 309, 310, 319, Rundles, R. W., 94, 240 320, 321, 336 Runner, M. N., 268, 277, 313, 314, 321, Sauvant, R., 277, 343 332, 345, 340 Sayers, G., 167, 214 Ruppel, W., 159, 210, 214 Sayers, M. A., 167, 214 Rush, B., Jr., 13, 86 Saxen, L., 277, 346 Russel, J., 270, 349 Saxton, J. A., Jr., 272, 340 Russel, K. P., 302, 345 Schachter, D., 101, 109, 111, 112, 113, 140 Russell, L. B., 265, 345 Russo, H. F., 104, 105, 108, 109, 110, 111, Schachter, M., 2, 5, 14, 15, 16, 17, 21, 26, 30, 31, 32, 33, 39, 40, 42, 45, 48, 64, 112, 113, 136 75, 77, 81, 82, 83, 84, 86, 178, 187, 190, Rutledge, M. L., 318, 345 191, 193, 205, 206, 210, 214 Rutstein, D. D., 157, 206 Schade, H., 175, 176, 180, 181, 21.4 Ryan, A. J., 222, 231, 265
374
AUTHOR INDEX
Schar, B., 155, 174, 212 Schatale, W., 167, 813 Schaffarzick, R. W., 114, 116, 119, 138, 157, 211 Schallet, R., 302, 343 Schallock, G., 179, 214 Scheiffarth, F., 189, 217 Schellenberg, H., 196, big Scherbel, A. L., 121, 194 Schild, H. O., 189, 2lb Schiller, S., 203, 21.4 Schimmel, N. H., 113, 156 Schirmer, E. W., 47, 90 Schlamowitz, S. T., 183, 185, bl4 Schlant, R. C., 116, 199 Schloss, B., 184, 213 Schlothane, R., 304, 348 Schmid, E., 32, 34, 47, 90, 189, 217 Schmid, F. R., 128, 139 Schmid, J., 157, 208 Schmid, K., 322, 324, 325, 327, 328, 329, 336, 338, 342
Schmidt, D., 170, 812 Schmidt, J., 169, 214 Schmidt, H., 12, 77 Schmidt, L., 171, 206 Schmidt, V., 109, 140 Schmiechen, R., 66, 83 Schmutaler, R., 12, 13, 76, 86 Schneider, C., 43, 77 Schneider, D., 86 Schneidman, K., 230, 231, 254, 266, 868 Schniewind, H., 111, 136 Schon, H., 7, 87 Schott, G., 189, 217 Schottmuller, H., 147, 214 Schotton, W., 12, 82 Schou, M., 99, 140 Schraffenberger, E., 265, 348 Schricker, K. T., 32, 34, 90 Schriefers, H., 197, 211 Schroder, E., 21, 22, 23, 24, 25, 29, 37, 52, 53, 54, 64, 66, 67, 83, 87 Schroepfer, G. J., Jr., 229, 266 Schubert, M., 183, 185, 214 Schuchardt, G. S., 104, 105, 108, 110, 111, 116, 136, 139, 141 Schuck, S., 80 Schiitz, E., 164, 219 Schuler, B., 157, 203, 209
Schulert, A., 119, 136 Schultz, F., 7, 10, 11, 12, 79, 82 Schuls, W., 167, 214 Schumacher, H., 196, 214, 322, 323, 324, 325, 327, 328, 329, 338, 346 Schutt, A. J., 13, 87 Schvartz, N., 115, 133, 134, 136 Schwab, J., 48, 87 Schwartz, M. S., 115, 133, 134, 136 Schwert, G. W., 10, 87 Schwyzer, R., 22, 87 Sciuteri, F., 188, 214 Scott, A. B., 316, 3% Scott, L. V., 277, 336 Scribner, B. H., 101, 139 Scrobot, L., 143, 204 Seager, A. D., 316, 3.44 Seaton, J. D., 229, 266 Sebaoun-Zuanan, M., 302, 3@ Sebening, H., 12, 13, 60,88 Secretan, P., 303, 336 Sedlmayr, G., 186, 214 Ske, G., 100, 140 See, G., 277, 3.43 Seegmiller, J. E., 93, 94, 98, 106, 112, 114, 115, 116, 127, 128, 131, 137, 138,140 Seeman, A., 159, 206 Segel, N., 37, 38, 76 Seidl, K., 164, 814 Seifter, J., 156, 180, 208, 209 Selitto, J. J., 155, 213 Seller, M. J., 269, 272, 307, 308, 309, 346 Seller, R. H., 133, 140 Seror, M. E., 277, 942 Setilia, K., 317, 346 Sezesny, B. R., 163, 216 Shanaman, J., 35, 43, 78 Shaner, G., 108, 109, 110, 136 Shapiro, W., 36, 38, 71, 82 Shaw, B., 11, 81, 194, 210 Shaw, C. C., 109, 140 Sheehan, J. T., 24, 76, 76 Shelemyak, M. C., 316, 318, 846 Shetlar, C. L., 184, 185, 214, 816 Shetlar, M. R., 184, 185, 214, 816 Shideman, F. E., 227, 260, 261 Shimazono, N., 229, 261 Shimizu, K., 234, 260 Shimura, K., 152, 216 Shorb, M. S., 307, 346
AUTHOR INDEX
375
Shore, P. A., 111, 140, 243, 266 Smithells, R. W., 319, 346 Shorley, P. G., 22, 26, 30, 33, 40, 43, 76, Smyth, C. J., 115, 120, 121, 138, 140 77, 86, 190, 191, 206, 206 Smyth, D. G., 6, 22, 23, 29, 48, 54, 78 Shotton, D., 296, 346 Snell, M. M., 111, 116, 118, 121, 127, 128, 131, 132, 136, 139 Shupe, J. L., 316, 336 Shuster, A., 200, 201, 206 Snoke, J. E., 10, 87 Sicam, L. E., 112, 118, 119, 124, 125, 128, Snyder, F. F., 319, 320, 346 Sobel, D. E., 315, 346 129, 131, 136, 138 Sicuteri, F., 21, 34, 38, 42, 51, 59, 60, 65, Sobel, H., 200, 216 71, 72, 74, 76, 86, 87 Sobotka, H., 290, 338 Sieber, P., 22, 87 Soling, H. D., 12, 77 SGrensen, L. B., 93, 140 Siekevitz, P. S., 220, 260 Silberman, H. R., 94, 140 Soffer, L. J., 185, 206 Silverstein, M., 60, 82 Sokal, J. E., 291, 346 Soloway, S., 100, 134 Simon, C., 118, 140 Simonsen, L., 293, 336 Soltesz, R., 157, 159, 160, 162, 164, 165, Simms, P., 223, 226 169, 170, 210, 216 Simpson, W. F., 184, 185, 206 Solymar, J., 7, 13, 76 Sinclair, J. G., 293, 296, 346 Somers, G. F., 270, 272, 275, 304, 308, 309, 310, 324, 346 Singer, K., 320, 342 Sommer, A. F., 316, 346 Sirayavirojana, A., 232, 260 Sirota, J. H., 101, 112, 113, 114, 119, 120, Sotaniemi, E., 113, 139 138, 140, 141 Sougin-Mibashin, R., 132, 133, 140 Sparrow, E. M., 188, 216 Sisson, J. H., 132, 138 Spector, E., 227, 260, 261 Sivo, R., 41, 87 Sjoqvist, F., 252, 255, 260 Spector, S., 313, 346 Spector, W. G., 11, 16, 17, 32, 33, 42, 59, Sjoerdsma, A., 7, 41, 59, 86 87, 90, 177, 187, 188, 193, 216 Sjostedt, J. E., 277, 346 Speent, H., 299, 346 Skinner, N. S., Jr., 43, 53, 88 Spence, I., 283, 286, 287, 320, 321, 330, 339 Slack, H. G. B., 183, 202, 216 Spencer, K. E. V., 308, 346 Slizys, S. M., 195, 208 Sloane, E. M., 27, 30, 42, 43, 46, 47, 48, Sperber, R. J., 134, 140 Spiess, G., 149, 216 49, 50, 51, 52, 53, 54, 58, 63, 78 Spirtes, M. A., 235, 861 Smaje, L. H., 30, 39, 75 Spitzbarth, H., 36, 37, 80 Smeby, R. R., 31, 50, 63, 76 Spitzer, S., 133, 140 Smilo, R. P., 115, 134, 140 Sprague, J. M., 108, 142 Smith, C., 307, 346 Springorum, P. W., 86 Smith, H. W., 96, 107, 1.40 Smith, M. J . H., 100, 101, 136, 140, 158, Sproull, D. H., 198, 816 166, 169, 197, 199, 200, 201, 206, 211, Spurr, C. L., 112, 140 Stabler, F., 319, 346 212, 216 Staemmler, M., 272, 346 Smith, J. A., 224, 254, 260 Stagg, R. B. L., 322, 323, 324, 325, 327, Smith, M. I., 316, 348 328, 329, 338, 346 Smith, 0. W., 302, 346 Stamm, H., 317, 347 Smith, P. E., 303, 346 Stanier, W. M., 182, 206 Smith, P. K., 143, 167, 208, 214 Staple, E., 230, 268 Smith, R. B. III., 13, 87 Smith, R. L., 322, 323, 324, 325, 327, 328, Starr, P., 302, 946 Starkenstein, E.. 152, 172, 216 329, 338, 346 Staub, A., 118, 140 Smithberg, M., 268, 313, 314, 321, 346
376
AUTHOR INDEX
Staudinger, H. J., 197, 210 Staudinger, Hj., 243, 244, 247, 268, 260 Steele, J. M., 100, 119, 121, 122, 123, 124, 129, 136, 136, 141 Steger, R., 15, 76 Steichele, D. F., 12, 13, 87 Stein, G., 243, 260 Stein, I. F., 277, 342 Steinbeck, A. W., 159, 206 Steinetz, B. G., 302, 342 Steiniger, F., 304, 346 Stempfel, K. S., 302, 348 Stenger, E. G.,155, 161, 162, 163, 164, 165, 166, 167, 168, 171, 197, 2U7, 216 Stenal, H., 118, l4O Sterky, G., 315, 343 Sterne, J., 315, 346 Stetten, D., Jr., 134 Stevenson, E. S., 231, 260 Stewart, J. M., 13, 16, 46, 49, 51, 58, 74, 87, 178, 204 Stewart, P. B., 16, 87 Stidworthy, G., 184, 216 Stoepel, K., 35, 36, 83 Stohlmann, E. E., 316, 348 Stoll, R., 297, 333, 338, 34.9 Stone, E., 145, 216 Stoner, H. B., 181, 209 Strauss, E., 197, 199, 210 Strean, L. P., 303, 346 Streitz, J. M.,106, 140 Stresemann, E., 30, 40, 44, 58, 81, 87 Stricker, F., 146, 147, 216 Strickland, S. C., 115, 136 Strominger, J. L., 216 Stuart-Harris, C. H., 109, 140 Sturmer, E., 22, 23, 29, 30, 36, 37, 39, 65, 66, 67, 68,70, 71, 76,82, 86, 87, 88 Stumpe, K., 310, 343 Subramanian, G., 11, 86, 163, 165, 169, 193, 194, 212, 213 Sullivan, F. M., 318, 346 Sullivan, H. R., 225, 269 Sulser, F., 224, 253, 254, ,266 Sund, H., 186, 187, 199, 216 Sutherland, B. S., 348 Suzuki, C., 63, 90 Suzuki, S., 201, 216 Suzuki, T., 18, 20, 52, 53, 86, 88 Svensson, H., 184, 206
Svirbley, J. L., 160, $16 Swartz, C., 133, 140 Sweeley, C. C.,225, 267 Swenson, C. B., 214 Swyer, G. I. M., 189, 216 SzakEL11, A., 39, 88 Saent-Gyorgyi, A., 180, 207 Szerb, J., 195, 210
T Tabern, D. L., 227, 260 Taggart, J. V., 107, 109, 112, 140 Takacs, E., 275, 317, 348 Takahashi, M., 182, 216 Talbott, J. H., 105, 114, 115, 116, 132, 136, 136,140,141 Talesnick, J., 187, 214 Tamura, Z., 64, 86 Tanaka, K., 153, 154, 155, 216 Tanaka, K. R.,270, 296, 340 Tanaka, T., 234, 260 Tanos, B., 159, 160, 162, 164, 165, 169, 170, 210, 216 Tauber, K., 7, 13, 89 Taussig, H. B., 269, 304, 346 Taylor, J. D.,227, 254, 260 Taylor, N. B., 181, 206 Tcher, T. T., 229, 234, 266, 261 Tedeschi, G., 186, 217 Tella, A., 59, 88 Tephley, T. R., 234, 249, $60, 261 Tepper, S. A., 230, 268 Terayama, H., 243, 261 Terriere, L. C., 223, 261 Texter, E. C., Jr., 36, 69, 70, 76 Thain, E. M., 2, 64,86 Thal, A. P., 7, 13, 36, 57, 60, 82, 88 Thalhammer, O., 292, 346 Theobald, W., 154, 155, 161, 163, 164, 165, 166, 167, 168, 171, 174, 190,207, 216 Thiersch, C,, 146, 216 Thiersch, J. B., 265, 268, 277, 292, 294, 295, 296, 297, 298, 321, 329, 346, 347 Thomas, B. G., 190, 207 Thompson, A. G., 60, 86 Thompson, G. R., 114, 115, 127, 133, 134, 141, 142 Thompson, M., 117, 138 Thompson, W. R., 303, 346
377
AUTHOR INDEX
Thorn, G. W., 132, 137, 158, 159, 215 Thorogood, E., 105, 132, 136 Thorpe, E., 282, 317, 345 Thorsdolen, N., 20, 49, 74 Thrift, C. B., 158, 159, 215 Thrift, E., 276, 343 Tillson, E. K., 107, 108, 109, 110, 111, 112, 113, 155, 141 Tinel, J., 154, 216 Tiselius, A,, 184, 205 Tislow, R., 155, 211 Toivanen, A,, 113, 139, 330, 347 Toivanen, P., 113, 139 Toki, K., 222, 261 Toki, S., 222, 261 Tollon, Y., 279, 336 Tomida, I., 255 Tomkins, G. M., 197, 216 Tonioke, H., 265, 349 Topis, D., 13, 83 Torosdag, S., 115, 133, 134, 136 Tovey, D. C., 117, 127, 128, 138 Trader, D. G., 269, 347 Traut, E. F., 158, 159, 215 Trautschold, I., 4, 8, 9, 11, 12, 13, 22, 23, 26, 47, 55, 60, 64, 88, 89 Trautschold, J., 178, 216 TrbfoniY, J., 231, 261 Trbfonsl, Mme. J., 231, 261 Trethewie, E. R., 13, 88, 189, 216 Trevaks, G., 133, 141 Trevor, Burnett, N., 319, 346 Triebold, H., 317, 347 Tripod, J., 41, 88 Trivus, R. H., 235, 261 Trockman, R. W., 229, 256 Troquet, J., 37, 41, 43, 44, 58, 83, 192, 211 Trousof, N., 106, 131, 136, 139, 223, 250, 252, 258 Truelove, L. E., 164, 616 Truhaut, R., 316, 337, 338 Tscheschichin, J., 149, 216 Tsukamoto, H., 222, 281 Tsuluca, V., 32, 33, 42, 43, 44, 76, 77, 179, 188, 193, 206 Tuchmann-Duplrssis, H., 265, 266, 268, 269, 272, 273, 275, 282, 283, 286, 287, 291, 292, 293, 295, 296, 297, 298, 299, 301, 304, 307, 308, 309, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322,
324, 330, 331, 332, 33Y, 339, 340, 341,
344, 347 Tiirker, K., 27, 88 Turba, F., 19, 88 Turner, C. D., 302, S47 Turner, W. A,, 9, 88 Tygstrup, I., 313, $46 Tyson, J. T., 113, 141
U Udenfriend, S.,64, 85, 101, 136, 222, 228, 234, 238, 243, 248, 253, 256, 259, 260, 261 Udupa, K. N., 183, 207 Uehleke, H., 226, 258, ,261 Uhde, G., 302, 340 Uhlig, H., 304, 347 Ullberg, S., 327, 342 Ullrich, V., 243, 244, 260 TJngar, G., 38, 88, 154, 161, 163, 165, 168, 169, 188, 189, 193, 616 Urakawa, N., 64, 78 Urban, T. J., 303, 336 ITrhahn, K., 5 , 89
V Vajda, G., 32, 85 Valenci, G., 302, 348 Van Arman, C . G., 20, 46, 49, 88 Van Cauwenbergc, H., 157, 158, 159, 161, 162, 163, 164, 165, 166, 168, 169, 188, 189, 190, 205, 211, 213, 214, 216 Vanderveiken, 126, 136 Van Drunen, H., 330, 343 Van Dyke, R. A., 227, 261 Van Maanen, J., 324, 342 Van Slyke, D. D., 93, 139 Van Smith, G., 302, 346 Vara, P., 316, 348 Vasaitisv, B., 307, 346 Vassanelli, P., 251, 258 Vaughan, P., 121, 141 Vecchiet, L., 34, 79 Vennerod, A. M., 63, 86 Venning, E. H., 159, 209 Veragut, U. P., 13, 88 Vereerstraeten, P., 126, 136 Verne, J., 202, 216 Verrett, M. J., 270, 279, 3@
378
AUTHOR INDEX
Verwey, W. F., 107, 108, 109, 110, 112, 113, 136 Vest, S. A., 109, 139 Vick, R. L., 38, 88 Vickery, D., 277, $40 Villa, L., 331, 348 Villani, R., 36, 44, 77 Virtama, P., 40, 81 Vivario, R., 157, 158, 159, 214 Vliers, M., 159, 168, 211, 214 Vogel, R., 6, 7, 59, 88, 89 Vogler, K., 24, 88 Vogt, M., 42, 84, 88 Vogt, W., 15, 20, 88 von Biilow-Koster, J., 228, 266 von der Decken, A., 232, 261 von Hippel, E., 264, 348 von Kerekjarto, B., 243, 244, 260 von Rechenberg, H. K., 119, 120,141,189, 197, 216 von Roden, P., 5, 6, 38, 89 von Schweinitz, H.-A., 175, 207 von Ziegesar, L., 6, 88 Vulpian, M. A,, 171, 216
W Waaler, B., 20, 49, 74 Waaler, B. A., 30, 37, 57, 58, 67, 81 Waaler, B. Z., 30, 88 Waddell, W. J., 224, 254, 260 Waddington, C. H., 283, 287, 338, 348 Wagner, B., 180, 216 Wagner, D. E., 68, 76 Wagner, H., 228, 266 Wakabayashi, K., 229, 261 Wakil, S. J., 228, 267 Wakim, K. G., 13, 87 Walaszek, E. F., 62, 88 Walaszek, E. J., 62, 81 Waldo, J. F., 113, 141 Walford, P. A., 318, 348 Walker, B. E., 300, 301, 303, 320, 339, 348 Walker, J. C., 303, 346 Wallach, D. P., 227, 261 Wallenfels, K., 186, 187, 199, 216 Wangensteen, S. L., 13, 87 Warburton, D., 277, 303, 337, 339 Ward, S. P., 304, 348 Warkany, J., 265, 266, 268, 269, 272, 275,
276, 277, 283, 286, 291, 298, 313, 314, 315, 317, 321, 322, 332, 334, 337, 343, 343, 348, 349 Warren, F. L., 231, 266 Warshaw, L. J., 115, 141 Waschkeit, G., 13, 82 Washington, J. A., 11, 101, 111, 112, 113, 117, 141 Watanabe, N., 63, 74 Watson, R. D., 157, 158, 206 Watson, G. I., 319, 337, 348 Watts, M. R., 303, 348 Waugh, M. H., 24, 86 Wax, J., 164, 191, 217 Way, E. L., 162, 208, 319, 348 Weaver, S. A., 276, 318, 342 Webb, E. C., 46, 72, 251, 266 Webb, J. L., 251, 261 Webster, M. E., 6, 8, 9, 10, 11, 12, 14, 15, 18, 20, 21, 22, 27, 37, 39, 43, 47, 52, 53, 54, 58, 83, 84, 86, 88 Wedd, A. M., 180, 216 Weicker, H., 304, 319, 348 Weidmann, H., 161, 216 Weidman, W. H., 268, 272, 275, 307, 310, 348
Weiner, I. M., 96, 101, 103, 107, 111, 112, 113, 117, 129, 141 Weingold, A. B., 320, 343 Weintraud, W., 103, 105, 141 Weis, J., 163, 216, 223, 268 Weiss, J., 243, 260 Weiss, R. M., 299, 348 Weissbach, H., 228, 234, 260 Weissbecker, L., 159, 210 Weissenbach, R., 158, 159, 161, 206 Wellmann, A., 188, $09 Wells, C. N., 303, 348 Wells, H. B., 277, 343 Wells, J. A., 14, 88 Werboff, J., 316, 317, 348 Werle, E., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 17, 19, 20, 21, 22, 23, 30, 32, 35, 36, 38, 39, 40, 41, 45, 46, 47, 48, 49, 51, 54, 55, 57, 59, 60,61, 62, 64, 77, 79, 82, 84, 86, 88, 89
Werner, S. C., 299, 346 Wernitsch, W., 38, 76 Wesson, R. L., 13, 80 West, C. D., 332, 336
379
AUTHOR INDEX
West, G . B., 58, 77, 188, 190,213, 316, 317, 348 West, L. A., 110, 111, 112, 136 West, R. A., 316, 348 West, R., 184, 216 Westermann, E. O., 30, 40, 43, 44, 67, 80, 192, 209, 255, 268, 261 Westermann, M. P., 47, 51, 59, 60,78 Westfall, B. B., 316, 348 Westphal, O., 180, 216 Wheaton, E. A., 133, 134, 136 Whelan, R. F., 190, 209 White, P., 313, 341 Whitehouse, M. W., 199, 200, 201, 203, 204, 206, 211, 217, 230, 268 Whitley, J. R., 277, 346 Whittle, B. A,, 34, 89 Whittaker, V. P., 113, 136 Wickes, I. G., 315, 348 Wiebelhaus, V. D., 104, 105, 109, 112, 136 Wiechowski, W., 172, 216 Wiesner, B. P., 293, 348 Wilbrandt, W., 96, 141 Wilbur, K. M . , 236, 266 Wilhelm, D. L., 16, 17, 26, 31, 32, 58, 76, 76, 78, 79, 83, 84, 89, 90, 187, 188, 193, 212, 216, 217 Wilhelmi, G., 123, 141, 163, 164, 186, 189, 207
Wilhoyte, K. M., 109, 112, 136 Wilk, L., 324, 344 Wilkins, L., 302, 303, 348 Will, E., 170, dl2 Willett, F. M., 106, 139 Williams, C. H., Jr., 232, 244, 247, 265, 269, 261 Williams, R. A. D., 324, 325, 327, 346 Williams, R. T., 112, 129, 1.41, 222, 241, 266, 261, 322, 323, 324, 325, 326, 327, 328, 329, 338, 346, 348 Williamson, A. P., 270, 293, 336, 349 Willig, F., 7, 84 Willis, J., 287, 336 Willis, P. W., 111, 133, l 4 l Willoughby, D. A., 11, 16, 17, 32, 33, 42, 59, 87, 90, 187, 188, 193, 216 Wilson, F. W., 318, 319, 336 Wilson, G. M., Jr., 120, 138 Wilson, J. G., 265, 267, 273, 277, 283, 286, 321, 333, 3.49
Wilson, K. H., 27, 83 Wilson, W. M., 109, 138 Winberg, J., 278, 349 Winckel, C. W. F., 316, 349 Winder, C. V., 164, 191, 217 Wing, E. S., 149, 206 Winkler, H., 153, 217 Winston, J., 318, 346 Winter, C. A., 163, 195, 217 Winternitz-Koranyi, M., 172, 217 Winters, W. D., 227, 261 Wirz, H., 184, 202, 217 Witte, S.,32, 34, 90 Wittenstein, C. J., 162, 210 Wohler, J. R., 24, 25, 27, 30, 33, 36, 40, 42, 43, 44, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 58, 59, 60, 63, 78 Wolfe, M., 293, 348 Wolff, E., 265, 267, 279, 288, 349 Wolff, E. C., 47, 90 Wolff, H. G., 34, 38, 60, 76, 86, 154, 156, 206, 206 Wolfson, W. Q., 108, 109, 113, 141 Wong, D. T., 223, 261 Wood, H. B., 250, 252, 268 Wood, P. H . N., 100, 136 Wood, W. B., 177, 211 Woodard, R., 107, 136 Woodbury, L. A., 167, 214 Woolfe, G., 195, 217 Woollam, D. H . M., 272, 275, 277, 289, 310, 321, 344, 349 Woolley, D. W., 87 Wright, G. P., 32, 76 Wright, H. V., 267, 344 Wright, L. D., 113, 136 Wright, S. E., 222, 231, 266 Wunsch, E., 24, 90 Wuest, H. M., 323, 349 Wyngaarden, J. B., 93, 94, 140, 141
Y Yadkins, J., 293, 348 Yajima, H., 22, 81 Yakovac, W. C., 277, 331, 340 Yamafuji, K., 19, 20, 27, 48, 52, 80 Yamamoto, H., 265, 304, 349 Yamano, T., 242, 248, 269 Yanaihara, N., 22, 81 Yang, H. T., 43, 48, 50, 52, 55, 56, 78
380
AUTHOR INDEX
Yang, T. J., 308, 349 Yang, T. S., 308, 349 Yeary, R. A., 272, 349 Yeoman, E. E., 109, 135 Yi, R. M. M., 32, 76 Yntema, C. L., 317, 336 Yoshinaga, K., 63, 74, 90 Young, H. H., 268, 272, 275, 307, 310, 348 Young, W. C., 302, 345 Yii, T. F., 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 106, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 131, 132, 133, 134, 134, 135, 136, 137, 138, 140, 141, 142
Yukioka, K., 265, 349
Z Zamelis, A., 13, 87 Zander, J., 302, 349 Zappasodi, P., 309, 341
Zatman, L. J., 245, 261 Zetler, G., 31, 90 Ziaim, M., 318, 349 Zicha, L., 189, 217 Ziegler, C., 108, 1.42 Ziegler, D. M., 225, 260, 261 Ziffer, H., 290, 338 Zileli, T., 34, 85 Zimmermann, H., 12, 82 Zipf, K., 36, 90 Zollman, P. E., 268, 272,275, 307, 310,348 Zuber, H., 18, 22, 51, 52, 8'7, 90 Zubrzychi, Z., 243, 244, 260 Zumoff, B., 133, 138 Zunin, C., 265, 294, 295, 298, 321, 346, 349 Zurod, C. G., 157, 205 Zutrauen, H. A., 200, 215 Zweifach, B. W., 32, 90, 174, 180, 217 Zweifler, A. J., 134, 148 Zylberszac, B., 13, 76
Subject Index 2-Amino-5-chlorobenzoxazole, see Zoxazolamine Acenocoumarin, 133 6-Aminonicotinamide, as teratogen, 298, Acetanilide, 331 effects on adrenals, 161 Aminophenaaone, hydroxylation in liver, 222 as antihistamine, 188-189 metabolism, inhibition, 238, 250 as antipyretic drug, 147-148, 172 toxic effects, 143 effects on adrenals, 161 Acetohexamide, uricosuric properties, Aminopterin, as teratogen, 265, 267, 295, 133 298, 323, 329-330 2-Acetylaminofluorene, hydroxylamine Aminopyrine, formation in liver, 226 anti-inflammatory action, 157 Acetyl p-aminophenol, 143 dealkylation in liver, 223 Acetylsalicylic acid, BS nonteratogen, metabolism, inhibition of, 238, 249317-318 250, 251 ACTH, as teratogen, 301 Amitriptyline, hepatic conversion to Actinomycin D, teratogenesis of, 268, amine, 224 293, 296-297, 321 Amphetamine, Adenine antagonists, as teratogens, 294, deamination in liver, 221 297 metabolism, inhibition of, 238 Adrenalin, as teratogen, 301 Anaesthesin, 150 Adrenals, depletion reactions of, 1 W Analgesia, as cause of anti-inflammatory 163 activity, 14S151 Agkistrodon contort& venom, 18 Analgesics, Agkistrodon halys blonihojji venom, 18 passage through placental barrier, 319 Agkistrodon piscivorus venom, 18 as teratogens, 317 Aldrin, epoxidation in liver, 222 Androgens, hepatic metabolism by enAlkaloids, as teratogens, 316 zymes, 230 Alkylaryl ethers, dealkylation in liver, Aniline, hydroxylamine formation in 221-222 liver, 226 Antibiotics, as possible teratogens, 318 Alkylating agents, as teratogens, 291296 Anticoagulants, embryopathology, 320 Alkylol derivatives, formation in liver, Antidepressants, as teratogens, 318 224-225 Antihistamines, as teratogens, 318 Anti-inflammato'Y drugs, 143-217 N-Alkylsulfamylbenaoic acids, as uricosuric drugs, 107-117 action, 144 antipyretic, 144 Alkylsulfonamitlobenzoic acids, as urion CNS, 148-156 cosuric drugs, 107-117 inflammatory focus and, 171-204 Allantoicase, 93 peripheral, 186-294 Allantoinase, 93 on phlogogenic polypeptides, 156Allopurinol, 92 171 as uric acid suppressnnt, 94, 101 pituitary-adrenal axis and, 190-193 mechanism of action, 98 on tissue cultures, 195-196 Alloxan, as teratogen, 313 analgesic component of, 194-195 Amines, hydroxylamines of, In liver, 226 antagonism with mediators, 187-193 381
A
382
SUBJECT INDEX
as antihistamines, 188-192 effect on mucopolysaccharide metabolism, 200-204 metabolic effects, 197-202 Antimetabolites, as teratogens, 294-295, 297-298 Antimitotic agents, as teratogens, 293295, 296-297 Antiphlogistics, see Anti-inflammatory drugs Antipyresis, inhibition of inflammation and, 151-152 Antipyretic-analgesic drugs, effects, early interpretation, 144-156 Antitumor drugs, as teratogens, 291-298 Aprobarbital, 156 Armadillo, use in teratogen screening, 273 Arteriosclerosis, kinin role in, 73 Arthritis, kinin role in, 58 Ascorbic acid, depletion in adrenals by salicylates, 1tXL163, 167 Aspirin, as kinin blocking agent, 42-44 as teratogen, 275 (See also Salicylates) Asthma, anti-inflammatory drug therapy of, 192-193 kinin role in, 58 Atrican, lack of teratogenicity, 318 toxicity, 275 Aturbane, embryotoxic effects, 317 Avitaminoses, tetratogenesis by, 265, 268, 276-277 Avitaminosis A, in teratogenesis, 265, 267 Azaserine, as teratogen, 294, 297, 321
B Bacillus subtilis N, Nagarse from, 19 Bacterial enzymes, hypotensive peptides and, 18-19 Barbiturates, hydroxylation in liver, 222 Bathocuproine sulfonate, 248 Benadryl, aa teratogen, 318 Benzothiadiazines, uricosuric properties, 132, 133-134 Bensoyl-L-arginine, kallikrein and, 10
Biguanides, as teratogens, 315 Bimez, as teratogen, 317 Blood transfusion, kinin role in, 58 Boguskinin, 22 Bothrops jararaca venom, 18 Bradykinin, 21 analogs, 24-26 anti-inflammatory drug action against, 190-193 blocking agents for, 42-44 discovery, 2 enzymic hydrolysis, 52 in inflammation, 178, 179 inhibitors for, 55 isotopic studies of, 5&51 metabolism, 45-47 inhibitors of, 48-49 physiology and pharmacology, 26-45 precursor, 1 retro-, 24-25 structure, 2, 2226 (See also Met-lys-bradykinin) Bradykininogen, probable identity with kallidinogen, 119 Burns, toxic substances in, 57, 58 Busulfan, as teratogen, 293, 296, 323 Butapyrin, anti-inflammatory action, 157
C Cancer, kallikrein in, 7 Carbon monoxide, as drug metabolism inhibitor, 238, 239, 240 Carboxypeptidase N, 3 4'-Carboxyphenylme thanesulfonalide, see Carinamide Carbutamide, as teratogen, 314, 315, 321 Carcinoid syndrome, kinin role in, 59 Carinamide, an uricosuric drug, l O & l l O limitations, 110-111 Cat, use in teratogen screening, 272 Central nervous system, role in inflammation, 149-151 control, 153-154 Chemoteratogenesis, 263-349 experimental, %E ' L291 drug administration in, 2%288 in vitro studies, 288-291 in vivo studies, 278-288 teratogenesis confirmation, 281 hypothesis for, 329-332
SUBJECT INDEX
interaction of factors in, 332-333 maternal stress and, 331-332 screening for, 269-278 administration routes, 275 doses for, 274-275 experimental precautions in, 276-278 number of animals, 274 species and strains, 269-273 selectivity of effects, 322 (See also Teratogenesis and individual compounds) Chemotherapeutic agents, as teratogens, 317-318 Chick, use in teratogen screening, 270272 Chickenpox virus, fetal effects, 277 Chlorambucil, as teratogen, 292, 296, 321, 323 Chlorinated hydrocarbons, epoxidation in liver, 222-223 Chlorocyclizine, a8 teratogen, 318 6-Chloropurine, as teratogen, 295, 298 Chloroquine diphosphate, effect on tissue culture, 196-197 Chlorothiaride, uricosuric properties, 134 Chlorpromazine, 153 a~ abortive, 316 hepatic oxidation of, 227 hydroxylation in liver, 222 metabolism inhibition, 249 Chlorpropamide, as teratogen, 314, 315 Chlorprothixene, uricosuric properties, 133 Cholesterol, cholic acid from, by liver enzymes, 22!&230 depletion in adrenals by salicylates, 161-163, 167 hepatic enzymes in synthesis of, 229 Cholic acid, from cholesterol, 229 Cinchophen, in gout therapy, 103-105 limitations, 104 Circulation, eledoisin effect on, 70-71 kinin effects on, 35-39 Cirrhosis (liver), kinin role in, 59 C l o s t d i u m histolyticum proteinase, hypotensive peptides and, 18-19 Clostripaine, 18-19 Codeine,
383
dealkylation in liver, 221 metabolism, inhibition, 238-239 Colchicine, 92 abortive action, 293, 331 in gout therapy, 97 Colostrokinin, 63-64 Colostrum, kinin in, 63-64 Connective tissue, in inflammation, 180, 182 Corticosteroids, anti-inflammatory drug effect on, 197-198 Corticotropin, uricosuric properties, 132 Cortisone, comparison with salicylates, 158 as teratogen, 268-269, 30M01, 303, 320, 322 Coumarins, uricosuric properties, 132 Crotolus atrox venom, 18 Cutscum, 238 Cyclirine, as teratogen, 318 Cyclophosphamide, as teratogen, 270, 271, 292, 296, 323, 333 Cytochrome c, inhibition of drug metabolism, 247
D Dalmatian coach hound, urate secretion in, 96, 99 N-Demethylases, 223 Deoxycholate, 238 Desacetylmethylcolchicine, as teratogen, 293 Diabetes, teratogenesis from, 313, 330 4,4’-Diaminodiphenyl sulfide, hepatic oxidation of, 227 6-Diazo-5-oxo-L-norleucine, see DON Dibenamine, 169 2,6-Dichloro-6-phenoxyethylamine,as microsomal drug enzyme inhibitor, 247-248 Dicoumarins, effect on fetus, 320, 323 Dicoumarol, uricosuric properties, 133 Diethylallylacetamide, 152 2-Diethylaminopropiophenone, lack of embryopathology, 319 Dihydrodiols, dehydrogenation in liver, 223 Dimethylaminoazobenzene, dealkylation in liver, 223 Dimethylbiguanide, as teratogen, 314
384
SUBJECT INDEX
N-Dimethylcarbamates, metabolism in liver, 224 1,2-Diphenyl-4- (Z’-phenylsulfinethyl) 3,5-pyrazolidinedione, see Sulfinpyrazone 1,2-Diphenyl-4-(phenylthioethyl)-3,5pyrazolidinedione, see G-25671 p-(Di-n-propylsulfamyl) benzoic acid, see Probenecid DK substance, see Kallidin Dog, use in teratogen screening, 272 Dominal, as teratogen, 318, 319 D.O.N., 92 as teratogen, 294, 297 DPEA, binding by microsomes, 251 Drug metabolism, “active oxygen” intermediate in, 235 hydroxylation mechanisms in, 235 inhibition, competitive, 249-250 kinetics, binding studies on, 250-251 limiting factors in, 254-255 microsomal, inhibition of, 244-252 Drugs, nonteratogenic, passage through placenta, 319-320 teratogenic, 291-319 mechanism of action, 320-333 structural aspects, 322-323
E Edema, kinin role in, 58, 60 permeability factor in, 17 Eledoisin, activity, compared to kinins, 28-29 discovery, 2, 64 in inflammation, 128 metabolism, 64-67 pharmacology, 67-72 structure, 64-67 Endoxan, see Cyclophosphamide Enzymes, bacterial, see Bacterial enzymes oxidative, in liver, 253-254 proteolytic, see Proteolytic enzymes releasing, see Kininogenases Epinephrine, effect on kinins, 41-42 as teratogen, 303, 321 Estradiol, aa teratogen, 300
Estradiol benzoate, as teratogen, 304 Estrogen(s), effect on fetus, 302 formation, from androgen, 229 hepatic enzyme metabolism of, 230 Estrone, as teratogen, 304 Ethacrynic acid, uricosuric properties, 134 17-Ethoxyacetanilide, dealkylation in liver, 221 Ethyl bis coumacetate, uricosuric properties, 132-133 Ethyl p-chlorophenoxyisobutyrate, uricosuric properties, 133 Ethyleneamines, in gout therapy, 100 Ethyl morphine, metabolism, inhibition of, 249 Ethyl urethane, 152
F Fatty acids, hepatic oxidation, 228-229 unsaturated, hepatic formation, 228 Fenton’s reagent, 243-244 p-Fluoroaniline, hepatic conversion to p-aminophenol, 228 Folic acid, antagonists, as teratogens, see individual compounds deficiency, teratogenesis from, 320, 321, 330 Folliculin, as teratogen, 304 Frog skin kinin, 64
G G-25671, as uricosuric drug, 122-124, 131 Galactoflavin, aa teratogen, 321 Gantrisin, as teratogen, 318 Glucocorticoids, metabolism by hepatic enzymes, 230-231 Glucose-6-phosphate dehydrogenase, 247 Glutamine antagonists, as teratogens, 294, 297 Gout, as inborn error of metabolism, 98 urate pool in, 93 uricosuric agents for, 91-141 nature of response to, 97 Gouty arthritis, kinin role in, 73 Guthion, hepatic oxidation, 227
SUBJECT INDEX
H Hageman factor, hypotensive pept,ides and, 15-16 kinin role in, 59 Halothane, hepatic dehalogcnnt ion, 227 Hepatic endothelium reticulum, amine oxidation by, 225-226 azo rctduction by, 231-232 dealkylation of metalloalkanes by, 228 0-dealkylation in, 221-222 N-dealkylation in, 223-224 deamination in, 221 dehalogenation by, 227-228 S-demethylation by, 226227 enzymatic oxidation and reduction mechanism in, 234-254 enzymatic reduction of drugs in, 219-263 enzymatic systems in, loralization, 220-221 epoxidation by, 222-223 formation of unsaturated fatty acids by, 228 hydroxylation in, 222 microsomal P-450 in, 236240 mixed oxygenase mechanism, 234-236 NADPH-dependent oxidative enzymes, substrates for, 228-231 nitro reduction by, 232-233 S-oxidation by, 227 a-oxidation by, 22S229 oxidative reactions in, 221-228 inhibitions of, 244-252 oxidative enzyme systems in, 253 N-oxide formation by, 225 phosphothionate oxidation by, 227 reductive enzyme systems, inhibition of, 244-252 Heptachlor, epoxidation in liver, 222223 Hexadimethrine, 3 Hexobarbital metabolism, inhibition of, 249 Histamine, in inflammatory response, 187-1 89 Hormones, in teratogenesis, 265, 298-304 HPC, as uricosuric drug, 104 H virus. in teratoaenesis. 267 HVJ virus, in teratogenesis, 267, 277
Hydrochlorothiazide, uricosuric properties, 134 Hjrdrocortisone, as teratogen, 301, 303, 320 Hpdroxylation mechanisms, in drug metabolism, 235 mechanism, 243-244 3-Hydroxy-2-phenylcinchononic acid, see H P C Hydroxyphenylethylamines, hydroxylation in liver, 222 4-Hydroxypyraaolo (3,4-d)pyrimidine, see Allopurinol p-Hydroxysulfinpurazone, from sulfinpyrazone, 123 Hyperglycemic agents, in teratogenesis, 313, 315 Hypertension, kinin role in, 59 Hgpervitaminosis A, teratogenesis by, 276, 277, 283, 320, 321 standards of reference for, 289 Hypnotics, effect on body temperature, 152 Hypocholesteremic agents, effect on embryo, 317 Hypoglycemic agents, as teratogens, 313-3 14 Hypopliyseal somatotropic hormone, as teratogen, 301, 303-304, 321 Hypotensive peptides, 1-90 anti-inflammatory drug effect on, 190194 (See also Bradvkinin, Eledoisin, Kallidin) Hypovitaminosis A, teratogenesis by, 288. 321
I Ileus, kinin effect on, 59 Imipramine, in binding studies on drug metabolism, 250 dimethylimipraniine from, in liver, 224 hydroxylation in liver, 222 lack of teratogenicity, 318 Immunoteratogenesis, 332 Imuran, as teratogen, 295, 299, 320, 321, 322 Indandiones, uricosuric properties, 132
386
SUBJECT INDEX
Indomethacine drugs, anti-inflammatory activity, 143 Infection, pathogenic, kinin role in, 59 Inflammation, CNS role in, 149-151, 153-156 focus, site of drug action and, 171204 kinin role in, 59 pituitary-adrenal axis and druginduced, 156-171 Inflammatory process, cause, 173 mechanism, 172-173 tissue alterations in, 175-181 chemical aspects, 175-181 kinins in, 178 physicochemical characteristics, 175 trauma and lesion in, 173-175 Inflammatory reaction, 181-186 metabolism in, 182-186 vascular system in, 181-186 Influenza virus, fetal effects of, 277 Inisindione, 133 Insulin, as teratogen, 268, 314, 315, 321, 333 Iodine, as teratogen, 299, 302 Iodopyracet, uricosuric properties, 132 Isodrin, epoxidation in liver, 22%223 Irgapyrin, see Butapyrin Irradiation, kinin effects on, 59
J JB-516, 248
K Kallidin, 4 analogs of, 24-26 anti-inflammatory drug antagonism to, 192 enzymic hydrolysis, 52 in inflammation, 178 physiology and pharmacology, 2645 precursor, 1 structure, 2, 22-23 Kallidinogen, kallidin from, 12 probable identity with bradykininogen, 19 Kallikrein(s), 3, 4-14 determination, 4 functions, 4-10
in inflammation, 178, 187 inhibitors of, 10-12 inhibition by salicylate, 193-194 in pathological conditions, 7 sources, 5 tachyphylaxis to, 30 unit of activity, 73-74 Kallikreinogen, 3, 5 Ketobemidon, anti-inflammatory activity of, 155-156 Kidney, eledoisin effect on, 71-72 kinin effect on, 39 Kininases, definition, 3 Kininogen, 19-21 definition, 3 Kininogenase(s1, 4-19, 74 definition, 3 snake venom, 18 Kinins, 21-64, 74 activity, compared to eledoisin, 28-29 analogs, 24-26 assay, 26-27 blocking agents for, 42-45 botanical type, 3 effect on, circulation, 35-39 CNS, 30-31 kidney, 39 permeability, 31-33 respiration, 40 smooth muscle, 26-27 enzymic hydrolysis, 52 formation, inhibition by antipyretics/ analgesics, 193-194 in inflammation, 178, 187 interaction with other agents, 40-42 metabolism of, 45-56 in vivo inhibition of, 54-56 proteolytic enzymes in, 51-54 as pain producers, 33-35 pathology of, 56-62, 73-74 liberation in, 61 physiology and pharmacology, 2645 potentiators for, 44-45 proteolytic enzyme effect on, 51-54 release, mechanism, 3 sources, 62-64 structure, 2, 22-26 tachyphylaxis to, 30
387
SUBJECT INDEX
1 Labor, kininogen depletion in, 21 Lithium, in early gout therapy, 99
M Mammals, in teratogenesis screening, 272-273 Meclizine, as possible terat’ogen, 318 Menadione, effect on fetus, 320 6-Mercaptopurine, w teratogen, 291, 294-295, 298, 323 Mersalyl, urixosuric properties, 132 Methionyl-lysyl-bradykinin, structure, 2 Methoxyfluorane, hepatic dehalogenation of, 227 Methylaniline, dealkylation in liver, 223 N-Methylbarbiturates, demethylation in liver, 224 N-Methylbutynamine metabolism, inhibition, 251 1-Methylguanosine, demethylation in liver, 223 6-Methylmercaptopurine, hepat>ic demethylation of, 226-227 3-Methyl-4-monometliylazobenzene metabolism, inhibition, 238 Met-lys-bradykinin, 23-24 enzymic hydrolysis, 52 Mice, use in teratogenesis screening, 272 Migraine, kinin effects on, 60 Mineral salts, teratogenic effects of, 316 Monkey, use in teratogenesis screening, 273 Monoamine oxidase inhibitors, effect on embryo, 317 Monomethyl-4-aminoantipyrine me t,abolism, inhibition, 238 Morphine, anti-inflammat.ory effect of, 153, 154, 155 Mucopolysaccharides, anti-inflammatory drug effect, on, 200204 in inflammation, 17g180, 182-184 Mumps virus, fetal effects of, 277
N NADPH-cytochrome c reductase, in microsornal oxidizing systems, 240242 Nagarse, hypotensive peptides and, 19
Naphthalene, hydroxylation in liver, 223 Narcotics, inflammation inhibition by, 153-154 Neocinrhophen, as uricosuric drug, 104 Seoplastic conditions, kinin effects in, 60 Neuroleptics, as teratogens, 316 Nialamide, in teratogenesis studies, 282 Nicotinamide antagonists, as teratogens, 298 Nicotine, cotinine from, by liver, 225 Nitrogen mustard, as teratogen, 292, 296, 321 Nit.ropheno1 derivatives, dealkylation in liver, 221 Nitro reduction, in liver, 232-233 Novonal, see Diethylallylacetamide Nucleotides, rise of in inflammation, 181
0 Ornithokallikrein, 5 Orthoform, 150 Oxygen, “active,” in drug metabolism, 235, 239 P-450 and, 238 transfer, mechanism, 242-243 Oxyphenbutazone, anti-inflammatory activity of, 171 as uricosuric drug, 120-122, 130
P P-450, in liver microsomes, 236-240 in activation of, 245, 248-249 as enzyme prosthetic group, 254 role in “active oxygen” system, 238 P-450 reductase, 240-242 inactivation, 245 inhibitors of, 247-248 Padutin, 10 Pain, bradykinin production of, 33-35 Pancreatitis, inin effects on, 60 Puntothenic acid deficiency, teratogenesis by, 321, 330 Papaverine, 152 Parathion, hepatic oxidation of, 227 Penicillin, as possible teratogen, 318 Pentylenetetrazol, 152, 153 Pepsin, hypotensive peptides and, 19 Pepsitocin, 19 Peptides, hypotensive, see Hypotensive peptides
388
SUBJECT INDEX
Permeability factor (PF), hypotensive peptides and, 16-17 Pesticides, as teratogens, 270 Phenacetin, metabolism, inhibition of, 252 toxic effects, 143 Phenazone, anti-inflammatory action, 157 Phenmetrazine, as possible teratogen, 318 Phenohulfonphthalein, uricosuric properties, 132 Phenothiazines, as teratogens, 316-317 Phenylasonaphtholsulfonates, hydroxylation in liver, 222 Phenylbutazone, antagonism against bradykinin, 192 antibacterial activity, 148 as antihistamine, 189 anti-inflammatory action, 157, 166 effect on tissue cultures, 196 metabolic effects, 197 on mucopolysaccharides, 200-204 metabolites, 120-122, 130 as uricosuric drug, 118-120 ZPhenylcinchonic acids, in gout therapy, 103-105 Phenylindanedione, effect on fetus, 320 Phenylquinolincarbonic acid, 151, 152 anti-inflammatory action, 157, 172 Phenylindandione, uricosuric properties, 133 Phocomelia, from thalidomide, 304-306, 321 Phosphorothionates, hepatic metabolism of, 227 Phthalimidines, uricosuric properties, 133-134 a-N-Phthalimidoglutarimide, see Thalidomide Phyllokin, 73 Physalaemin, 65-66, 67 Piperidine, in gout therapy, 99 Piperazine, in gout therapy, 99 Pituitary-adrenal axis, in drug-induced inhibition of inflammation, 156-171 Plasmin, pharmacology of, 14-15 Polycythemia Vera, uric acid secretion in, 93
Prednisone, effect on mucopolysaccharide metabolism, 202-205 Probenecid, analogs, structure-activity relationship of, 116-117 as uricosuric drug, 92, 95, 99, 107, 110-116 in gout therapy, 113-116 Procaine, 150 Prochlorpromasine, as teratogen, 323 Progesterone(s), deficiency, teratogenesis from, 302 synthetic, as teratogens, 302-303 Z-Proproxyphene, hepatic demethylation of, 225 Propylthiouracil, as fetal goitrogen, 302 Proteolysis, in inflammation, 176178 Proteolytic enzymes, effect on kinins, 51-54 Purine antagonists, as teratogens, 294295, 298 Puromycin aminonucleoside, demethylation in liver, 223 Pyrazinoic acid, 92 Pyrazoles, as anti-inflammatory drugs, 143 Pyrazolidinediones, as uricosuric drugs, 117-132 structure-activity relationships of, 128-132 Pyrazolidines, as anti-inflammatory drugs, 143 Pyroglutamic acid, 65 Pyrolazote, as teratogen, 318 Pyrophosphatase inhibition, in drug metabolism, 246
Q Quinazolines, uricosuric properties, 133134 Quinine, antipyretic action of, 147, 172 Quinoline derivatives, as anti-inflammatory drugs, 143
R Rabbits, use in teratogen screening, 272 Rat, use in teratogen screening, 272 Respiration, kinin effect on, 40 Rheumatism, history of treatment of, 144-148
389
SUBJECT INDEX Riboflavin deficiency, teratogenesis of, 320 Rickettsia1 virus, fetal sensitivity to, 277 Rodents, use in teratogen screening, 272 Rubella virus, in teratogenesis, 265, 268, 277-278 Rufocronionmycine, as teratogen, 297
Sulfadimethoxypyrimidine, as teratogen, 282 Sulfinpyrazone, as uricosuric drug, 92,95, 99, 117, 123, 124-128, 131 efficacy of, 127-128 low toxicity of, 128 Sulfonamides, as teratogens, 315, 317318, 323
S
T
Salicy lates, activity against bradykinin and kallidin, 192-193 as antihistamines, 189 as antipyretic-analgesic drugs, effect on edemas, 163-167 history, 145-148 hormonal action, 157-160, 161 mechanism, 170 pituitary-adrenal axis and, 157, 168 effect on tissue cultures, 196 metabolic effects, 197 on mucopolysaccharides, 2W204 as teratogens, stress and, 332 as uricosuric drug, 92, 99, 100-103, 159 dosage-dependent action in, 103 limitations of, 100-101 metabolism of, 101-102 Schradan, metabolism in liver, 224-225 Sedatives, effrct on embryo, 317 Serotonin, in inflammatory response, 187-189 Shock, kinin rolc in, 7, 57, 58, 59, 60 Shwartzman reaction, kinin role in, 60 Simmonds’ disease, salicylate action in, 160 SKF-525A, 248-249 binding by niicrosonies, 251 as enzyme inhibitor, 252, 253 Snake venoms, hypotensive peptides and, 17-18 Sodium barbital, anti-inflammatory cffrrt of, 153 Sodium snlicylate, as teratogcn, 317 Soluderadron, R R teratogen, 303 Steapsin, 238 Steroids, as teratogens, 322 Streptomycin, lack of tcratogenicity, 318 Sulfadiazine embryotoxicity of, 317
Te rat ogenesis, determining factors of, 267-269 dosage factor in, 268 environmental factors in, 277 genotype and, 268-269 glossary for, 334 history, 264-266 laws of, 266-269 sensitive period in, 267-268, 276 (See also Chemoteratogenesis) Testosterone, effect on fetus, 302 Teraethyl lead, hepatic conversion to triethyl lead, 228 Thalidomide, teratogenesis of, 265-266, 267, 272, 273, 282, 283, 291, 320, 321, 333 in animals, 270, 307-313 chemical structure and, 322-323 dosage factor in, 268, 275 as folic acid antagonist, 329-330 genotype in, 268, 269 as glutamic acid antagonist, 32W30 in humans, 304 mechanism of, 329-330 metabolism of, 324-328 sensitive period to, 267-268, 276 in tissue culture studies, 290 Thiadiazoles, substituted, 92 Thiobarbital, hepatic conversion to barbital, 227 Thiobarbiturntcs, hepatic conversion to oxybarbiturates, 227 Thiosemicarbazone derivatives, antiinflammatory action, 157 Thiouracil, as teratogen, 299 Thyroxin, as teratogcn, 269, 300, 303, 321, 333 Tolbntamide, as teratogen, 314, 315, 321 Toxoplasma, fetal sensitivity to, 277
390
SUBJECT INDEX
Trasylol, 3, IS14 therapy using, 12-13, 62, 73 Tremorine, metabolism in liver, 225 inhibition of, 252 Tribromoethyl alcohol, 154 Triethylenemelamine, as teratogen, 292, 296, 323 Trimersurius jlavoviridis venom, 238, 248 Trimethylaniline, N-oxide formation by liver, 225 2,6,8-Trioxypurine, see Uric acid Triton, as teratogen, 330 Trypan blue, as teratogen, 264, 283, 284, 320, 321, 330, 331, 332, 333 in various animals, 286-287 Trypsin pharmacology, 14
U Urate pool, in gout, 93-94 in man, 93 uricosuric drug effect on, 95 Urease, 93 Urethane, as teratogen, 293, 296 Uric acid, biochemistry of secretion, 9296 drug effect on, see Uricosuric drugs pathology, 93-95 species differences, 99 Uricase, 93 Uricosuric activity, definition, 91-92
Uricosuric drugs, 91-141 physiological basis for, 92-96 response to, 96-99 in gout, 97 search for, 99-117 (See also individual compounds) Urine, kinins in, 62-63
V Vasopressin, as teratogen, 303 Viruses, in teratogenesis, 265, 267, 277278 Vitamin imbalance, in teratogenesis, 276-277 Vitamin K, effect on fetus, 320
W Wasp venom kinin, 64
X Xanthine oxidase, in gout, 98 X-methylfolic acid, as teratogen, 298, 329-330
Z Zoxazolamine, metabolism, inhibition of, 250 as uricosuric drug, 91, 99, 1&107 hepatotoxicity, 107 metabolism, 106
