The role of leukocyte-generated reactive metabolites in the pathogenesis of idiosyncratic drug reactions.

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DRUG METABOLISM REVIEWS, 24(3), 299-366 (1992)

THE ROLE OF LEUKOCYTE-GENERATED REACTIVE METABOLITES IN THE PATHOGENESIS OF IDIOSYNCRATIC DRUG REACTIONS* J. p. UETRECHT~ Faculties of Pharmacy and Medicine Universify of Toronto and Sunnybrook Medical Centre Toronto, Canada

I.

INTRODUCTION.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301 A. Significance of Idiosyncratic Drug Reactions . . . . . . . . . . 301 B. Characteristics of Idiosyncratic Drug Reactions . . . . . . . . 302

11.

INVOLVEMENT OF THE IMMUNE SYSTEM IN IDIOSYNCRATIC DRUG REACTIONS . . . . . . . . . . . . . . . . 304 A. General Aspects of Immune-Mediated Reactions. . . . . . . . 304 B. Relevance of the Immune System to Idiosyncratic Drug Reactions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305

111.

INVOLVEMENT OF REACTIVE METABOLITES IN IDIOSYNCRATIC DRUG REACTIONS . . . . . . . . . . . . . . . . 306

IV.

FORMATION OF REACTIVE METABOLITES BY LEUKOCYTES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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*This paper was refereed by Mary F. Locniskar, Ph.D., Division of Graduate Nutrition, University of Texas, Austin, TX 78712-1097. 'Send correspondence to the author at the Faculty of Pharmacy, University of Toronto, 19 Russell Street, Toronto, Canada M5S 2S2. 299 Copyright 0 1992 by Marcel Dekker. Inc

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A . Enzyme Systems in Leukocytes That Might Metabolize 309 Drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B . Metabolism of Procainamide by Leukocytes . . . . . . . . . . .

System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E . Metabolism of Chlorpromazine by the Myeloperoxidase System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Metabolism of Amodiaquine and Acetaminophen by Leukocytes and the Myeloperoxidase System . . . . . . . . . . G . Metabolism of Vesnarinone by Leukocytes and the Myeloperoxidase System . . . . . . . . . . . . . . . . . . . . . . . . H . Metabolism of Clozapine by Leukocytes . . . . . . . . . . . . . I . Metabolism of Hydralazine and Isoniazid by Leukocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . J . Metabolism of Propylthiouracil and Thiols by Leukocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . K . Metabolism of Phenytoin by Leukocytes . . . . . . . . . . . . . . L . Metabolism of Carbamazepine by Leukocytes . . . . . . . . . . M . Metabolism of Phenylbutazone by Leukocytes . . . . . . . . . . N . Further Metabolism of Benzene Metabolites by Leukocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V.

314 315 315 316 316

316 318 318 319 320 321

AGRANULOCYTOSIS AND APLASTIC ANEMIA . . . . . . . . 321 A . General Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321 B . Aminopyrine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323 324 C . Procainamide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326 D. Dapsone. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Sulfonamides Sulfasalazine, and Trimethoprim. . . . . . . . . 326 F. Other Arylamines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328 G . Chloramphenicol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328 H . Chlorpromazine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329 1. Amodiaquine and Acetaminophen . . . . . . . . . . . . . . . . . . 330 J . Vesnarinone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331 K . Mianserin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332 L . Clozapine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333 M . Propylthiouracil and Methimazole . . . . . . . . . . . . . . . . . . 333 N . Captopril and Penicillamine . . . . . . . . . . . . . . . . . . . . . . 334 0. Carbamazepine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334 I? Phenylbutazone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335 Q . Benzene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335 R . General Conclusions Concerning Drug-Induced Agranulocytosis and Aplastic Anemia . . . . . . . . . . . . . . . 336

.


LEUKOCYTE-GENERATEDMETABOLITES

30 1

VI . DRUG-INDUCED LUPUS. . . . . . . . . . . . . . . . . . . . . . . . . . A . General Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B . Procainamide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . Sulfonamides and Sulfasalazine. . . . . . . . . . . . . . . . . . . .

338 338 339 339 340 340

D . Other Arylamines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E . Hydralazine, Isoniazid, and Hydrazine . . . . . . . . . . . . . . . F. Propylthiouracil, Methimazole Captopril. and Penicillamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . Carbamazepine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. Chlorpromazine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I . General Conclusions Concerning the Mechanism of Drug-Induced Lupus . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

341 341 341 341

VII . GENERALIZED IDIOSYNCRATIC DRUG REACTIONS . . . . A . General Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B . Arylamines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . Thiono Sulfur and Thiol Drugs . . . . . . . . . . . . . . . . . . . . D. Anticonvulsants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E . Nonsteroidal Anti-inflammatory Drugs. . . . . . . . . . . . . . . F. Gold Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . Conclusions Concerning the Mechanisms of Generalized Idiosyncratic Drug Reactions. . . . . . . . . . . . .

344 344 344 346 347 348 348

VIII . SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

349

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

351 351

348

.

I INTRODUCTION

.

A Significance of Idiosyncratic Drug Reactions Adverse drug reactions represent a major health problem . The majority of adverse drug reactions are due to known pharmacologic effects of a drug and would be expected to occur in most patients who take the drug if the dose of the drug is high . In principle it should be possible to prevent such reactions. However. because of interindividual differences in pharmacokinetics and pharmacodynamics. such reactions will continue to be a problem. A more difficult problem is the so-called idiosyncratic or hypersensitivity reaction . Idiosyncratic reactions are frequently life threatening. and at present they are almost totally unpredictable. Although idiosyncratic reactions are less common than predictable reactions. they are not rare. and they probably account for approximately 10% of all adverse drug reactions [l].


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The unpredictable nature of idiosyncratic drug reactions poses a difficult problem for the development of new drugs. Idiosyncratic reactions are not detected in standard toxicology testing, including animal testing. Therefore, the earliest that such reactions can be detected is during clinical trials, and they are often not detected until the drug has been released on the market. There are many drugs which were removed from the market shortly after they were introduced because of what was considered to be an unacceptable risk of idiosyncratic reactions [2], and there are probably many others that were never released. Since the cost of developing a new drug is more than $lOO,OOO,OOO,this represents a significant cost to the company as well as the nonmonetary costs to the patients who sustain serious reactions.

B. Characteristics of Idiosyncratic Drug Reactions The term idiosyncratic drug reaction is used in this review to indicate a reaction that does not occur in most patients, even at high doses, and one that does not represent an extension of the known pharmacological effects of the drug. Such reactions are also referred to as hypersensitivity reactions or type B reactions [3]. There are several good reviews of this type of reaction [4-71. The clinical characteristics of idiosyncratic drug reactions can vary considerably between different drugs and also for the same drug in different individuals. The most common type of idiosyncratic reaction involves the skin and can vary from a mild rash to life-threatening toxic epidermal necrolysis. Reactions are sometimes limited to specific organs such as the liver or bone marrow. Generalized reactions are also frequent in which there is fever, skin rash, and lymphadenopathy, along with involvement of specific organs. Despite this diversity, there are characteristics that are common to many idiosyncratic drug reactions including: A requirement for either prior exposure to the drug, or a delay of more than a week between starting the drug and the development of toxicity. A lack of delay in toxicity on reexposure of a patient to the offending drug. An apparent lack of correlation between dose and the risk of toxicity. Although these reactions must be dose dependent, in some cases the dosetoxicity curve is far to the left of that for the dose-response curve of the desired pharmacologic property. An unpredictable nature and lack of an animal model. The presence of a skin rash and peripheral blood eosinophilia. These characteristics are not invariable. For example, the delay tends to be different with different types of reactions. Agranulocytosis is most com-


LEUKOCYTE-GENERATED METABOLITES

303

mon in a window of time from 1 to 3 months after starting the drug. In contrast, drug-induced lupus usually requires at least a month of therapy, and it often requires a year or more before the development of clinically evident lupus. At the other extreme, reactions limited to a mild rash can occur shortly after starting therapy. Although reexposure to an offending drug usually leads to an immediate reaction, in some cases the delay is almost as long as that associated with the first adverse reaction [8]. Although there often appears to be a lack of correlation between dose and the risk of toxicity, the basic principles of pharmacology require that there should be a dose-response curve. In many cases the dose-response curve for toxicity may be shifted far to the left of the dose-response curve for the desired pharmacological effect. For example, the dose of penicillin that can cause anaphylaxis in patients who are allergic to penicillin is much lower than the clinically used dose; however, such patients do not have an anaphylactic reaction when they are exposed to very small quantities of penicillin such as are often found in milk. In other cases, the dose-response curve for an idiosyncratic reaction may be in the same range as that for the desired effect. For example, the risk of hydralazine-induced lupus is much lower if the dose is kept below 200 mg/day IS]. Another aspect that makes idiosyncratic reactions appear dose independent is that some patients (or animals) will not have an idiosyncratic reaction no matter how high the dose (although toxicity may occur by some other mechanism at a high dose), while patients who are at risk may demonstrate a clear dose-toxicity curve for the idiosyncratic reaction. As mentioned earlier, idiosyncratic reactions are rarely detected by animal tests, and there are very few animal models for idiosyncratic reactions that are similar to the reactions observed in humans. It is not that animals cannot have an idiosyncratic reaction; on the contrary, if a sufficient number of species were given a specific drug, it is likely that one would have a reaction similar to the reaction experienced by some patients. However, such reactions usually occur in humans with an incidence of 1 per loo0 or less. When animal toxicity testing is performed, even if a large number of animals are used, inbred strains are normally used, which is comparable to testing a single animal. However, it is known from observations by veterinarians that cats have a very high incidence of propylthiouracil-induced lupus [lo], and Doberman pinschers have a high incidence of an idiosyncratic reaction to sulfonamides similar to the reaction which occurs in some humans [ I l l . The presence of a rash is also not invariable, although the skin is probably the organ affected most frequently in idiosyncratic drug reactions. Eosinophilia is also considered evidence for an immune-mediated reaction, but the duration of eosinophilia is brief, and its absence should not be taken as evidence against an immune-mediated reaction. It is also not clear that there is a good correlation between the


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presence of eosinophilia in an adverse drug reaction and immune mediation of that reaction. Very little is known about the mechanism of idiosyncratic reactions; however, the preceding characteristics suggest involvement of the immune system. In a few cases there is direct evidence for involvement of the immune system, for example, penicillin-induced anaphylaxis [4], but such examples are uncommon. Inferring mechanism from clinical characteristics is hazardous because some reactions, such as radiocontrast-induced reactions, that have clinical characteristics of anaphylactic reactions do not appear to be antibody mediated [12]. However, it is a reasonable working hypothesis that many idiosyncratic reactions do involve the immune system.

11. INVOLVEMENT OF THE IMMUNE SYSTEM IN IDIOSYNCRATIC DRUG REACTIONS

A. General Aspects of Immune-Mediated Reactions In general, small molecules (molecular weight of less than IOOO) do not directly induce an immune response [4]. Therefore, for a small molecule such as a drug to induce an immune response, it must bind to a larger molecule, usually a biological macromolecule such as a protein. The nature of this binding must be essentially irreversible. The smaller molecule which binds to the macromolecule is referred to as a hapten. If the macromolecule is endogenous, that is, recognized by the immune system as a "self" protein, it also will not usually induce an immune response by itself, and only the macromolecule-hapten adduct will induce a response. For the hapten-macromolecule adduct to induce the formation of antibodies, further steps are necessary. Although a detailed account of these steps is beyond the scope of this review, a general overview of the process is important for understanding how drugs could induce a hypersensitivity reaction. First, the irnmunogen, in this case the hapten-macromolecule adduct, must be taken up by a macrophage or other antigen-presenting cell. The immunogen must then be processed. Processing appears to involve partial hydrolysis of the immunogen. The processed immunogen is then transported to the cell membrane and presented on the surface, along with the class I1 major histocompatibility antigen (MHC 11) [13,14]. It has been suggested that fragments of the immunogen that bind to the MHC 11 are protected from further hydrolysis, and it is the combination of this fragment with the MHC I1 that is transported to the cell membrane. If the combination of processed antigen and MHC I1 is recognized by specific CD4' T cells (commonly known as helper T cells), these cells produce lympho-



305

kines, such as interleukin 2, that stimulate clones of B cells that recognize the same immunogen. Although the T cells and B cells recognize the same immunogen, they recognize different parts of that immunogen; the T cells recognize processed immunogen together with MHC 11, and the B cells recognize the original immunogen. B cells that are stimulated by both helper T cells and the presence of immunogen proliferate and differentiate into mature antibody-producing plasma cells. The requirement for irreversible binding between hapten and macromolecule is presumably due to the several steps in this process. If the interaction between hapten and macromolecule were reversible, the hapten would diffuse away from the macromolecule before the process could be completed. Although a readily reversible noncovalent interaction is insufficient to induce an immune response, it does not appear that complete irreversibility is required, and haptens bound to protein through a covalent but reversible disulfide bond can be inimunogenic [ 151. Steps similar to these for an antibody-mediated response are also involved in the induction of a cell-mediated immune response [16]. One difference is that the immunogen must be associated with the class I major histocompatibility antigen (MHC I) rather than MHC 11. The other major difference is that the effector cell is the cytotoxic T cell rather than the plasma cell.

B. Relevance of the Immune System to Idiosyncratic Drug Reactions The terms idiosyncratic drug reaction and drug hypersensitivity reaction are often used interchangeably. However, to an immunologist the term hypersensitivity reaction has a more specific meaning. It indicates a reaction that involves the immune system, and such reactions have been classified by Gel1 and Coombs into four types [17] Type 1 'Qpe I1 'Qpe 111 Type IV

Mediated by specific IgE antibodies Mediated by antibodies against specific tissue antigens Involves immune complexes that activate complement Mediated by T cells

So little is known about the mechanism of most idiosyncratic drug reactions that it is usually unknown whether the immune system is involved at all. One exception is the type I hypersensitivity reactions such as penicillininduced anaphylactic reactions. Penicillin is chemically reactive due to the ring strain of the @-lactamring. Although the total amount of penicillin that becomes covalently bound to protein appears to be relatively small, it is sufficient to induce an immunologic reaction [18]. Thus penicillin can act as

UETR ECHT


306

a hapten, and there is compelling evidence that most of the antibodies in patients who are allergic to penicillin recognize penicillin bound to lysine 14, 191. In some patients the major type of antibody induced is IgE, and this can result in an anaphylactic reaction. Penicillin can also induce other types of allergic reactions such as serum sickness, but this is uncommon except with high doses. Even in the case of penicillin the mechanism is complicated, and many aspects are not understood. In some patients, breakdown products of penicillin represent the immunogen rather than the product formed by the reaction of the @-lactamring with lysine residues [4],and the cross-reactivity to different penicillins is not understood. Most important, it is not clear why some patients have a hypersensitivity reaction to penicillin while most patients do not. This idiosyncratic nature is common with immune-mediated reactions, and it is probably due to interindividual differences in the immune system which lead to very different responses in different individuals. Drug-related antibodies have been described in many idiosyncratic drug reactions; however, possibly due to the complexities of the immune system, in most cases of idiosyncratic reactions there is no direct evidence for involvement of the immune system, even when a search has been made for such evidence. Despite this lack of proof in the majority of cases, the idiosyncratic nature of such reactions has led many investigators to believe that most of these reactions are mediated by the immune system [4-6).Although the hypothesis that most idiosyncratic drug reactions are immune mediated is attractive because it provides a reasonable mechanism for their idiosyncratic nature and other characteristics, it is quite possible that many such reactions do not involve the immune system.

111. INVOLVEMENT OF REACTIVE METABOLITES IN

IDIOSYNCRATIC DRUG REACTIONS In general, chemically reactive agents tend to be toxic [20,211. They can be cytotoxic by reacting with critical biological molecules; for example, agents that react with DNA are associated with the induction of cancer. Reactive chemicals can also act as haptens and cause immune-mediated reactions as already mentioned [ 5 . 6, 221. An example of a reactive chemical that acts as a hapten is 2,4-dinitrofluorobenzene,which is a very effective sensitizing agent and is used by immunologists to study immune-mediated reactions. Most drugs are not chemically reactive; however, many (I would guess most) drugs can be metabolized to chemically reactive intermediates. A prevalent hypothesis in toxicology is that many types of toxicity are due to reactive metabolites rather than to the drug itself [23,241. Although in



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most cases this has not been rigorously proven, a great deal of evidence has accumulated over the iast 50 years to support this hypothesis. Even though there is less mechanistic information concerning idiosyncratic drug reactions, it is likely that many such reactions are also due to reactive metabolites [22]. For example, halothane is oxidized in the liver to the reactive metabolite trifluoroacetyl halide, and this metabolite reacts preferentially with lysine residues on hepatic protein [25, 261. It has been further demonstrated that patients with halothane-induced hepatitis have antibodies against specific trifluoroacetylated hepatic proteins. The association between the antibodies to trifluoroacetylated hepatic protein and halothaneinduced hepatitis is very strong, but it is still unclear how these antibodies cause hepatic damage. The amount of covalent binding is very important. In general, if a molecule is going to induce an immune-mediated reaction by acting as a hapten, more than one molecule of drug must be bound to a macromolecule ( 5 , 6, 221. It appears that the higher the hapten density, the more the antibodies recognize the drug portion of the epitope rather than the macromolecule [ 5 ] . If many idiosyncratic drug reactions involve a reactive metabolite acting as a hapten or similar mechanism, it might be expected that all of the drugs would cause the same type of reaction. In fact many drugs with similar functional groups are associated with a similar spectrum of idiosyncratic reactions. For example, essentially all drugs with a primary arylamine group are associated with a relatively high incidence of drug-induced agranulocytosis, and most have also been associated with an autoimmune syndrome termed lupus [7]. The physical characteristics of the reactive metabolite could play a role. It has been stated that the ability of the dinitrobenzene group to interact strongly with antibodies contributes significantly to the activity of 2,4-dinitrofluorobenzeneas a sensitizer; however, one would not expect the trifluoroacetyl group, which is involved in halothane-induced hepatitis, to make a very good hapten because it is small, and the trifluoromethyl group could be likened to Teflon, to which nothing should stick. Likewise, diazomethane is a methylating agent, and although the methyl group is very small and relatively inert, exposure to diazomethane appears to lead to sensitization [27]. Therefore it does not appear that there are major structural requirements involving size or charge for a hapten group to induce an immune-mediated reaction. In contrast, the chemical reactivity of the reactive metabolite appears to be a major factor in determining the degree and character of toxicity [22, 28, 291. If all reactive metabolites can cause idiosyncratic reactions, it is at first glance surprising that acetaminophen, whose quinone imine reactive metabolite has probably been studied more than any other reactive


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metabolite, is associated with a low incidence of idiosyncratic reactions. It appears that this low incidence of idiosyncratic reactions is due to the selectivity of its reactions. It is a soft electrophile and preferentially reacts with glutathione, which is a soft nucleophile. Even the reaction with glutathione is relatively slow in the absence of glutathione transferase 1301. Therefore, although a small amount of covalent binding to other sulfhydryl groups must occur, no binding to hepatic proteins was detected with an immunoassay except when a large dose of acetaminophen was given 131, 321. It also appears that very little of the reactive metabolite escapes the liver, and the drug protein found in the blood comes mostly from the liver. Furthermore, acetaminophen is associated with idiosyncratic reactions, and it has been estimated that 10% of agranulocytosis is due to acetaminophen 1331. In short, a reactive metabolite of relatively low reactivity can be selectively detoxified before binding to some critical molecule. At the other extreme, a very reactive intermediate may be so reactive that it reacts with water or the enzyme that formed it and has little chance to bind to other important structures. The reactivity and “softness” of the reactive metabolite also determine the amino acid residue to which it binds [34]. Cysteine and lysine are probably the two most common nucleophilic amino acids. The reactivity and site of reactive metabolite formation determine the intracellular site of greatest covalent binding. The reactivity of the intermediate can also determine the target organ for toxicity. Most reactive metabolites have a short biological half-life and would not be expected to reach significant concentrations at a site distant from their site of formation. There are exceptions to this. Penicillin, which is reactive without activation, and some metabolites, such as the acyl glucuronides, also reach high concentrations in the general circulation. However, if the intermediate is only formed in one organ and is too reactive to escape that organ, then the toxicity should be confined to that organ. Halothane is an example in which the metabolic activation involves oxidation of a carbon-hydrogen bond, and this metabolism appears to be confined almost exclusively to cytochrome P-450 in the liver. As would be expected, the idiosyncratic reactions associated with halothane are limited to the liver. Therefore, with the exception of long-lived reactive intermediates, most toxicity should be limited to the organ that formed the reactive intermediate. This reasoning leads to the prediction that most idiosyncratic reactions would involve the liver since the liver is the major site of drug metabolism. Although the liver is a major target organ of idiosyncratic drug reactions, many idiosyncratic reactions do not involve the liver. The skin and possibly the bone marrow are involved more frequently than the liver. One likely



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explanation is that the liver, along with a high potential to metabolize drugs, also has many systems, such as a high concentration of glutathione and glutathione transferases, for the inactivation of reactive metabolites. Organs also differ in their immune activity, in specific sensitivities to other toxic events, and in their ability to concentrate specific drugs. Furthermore, many organs other than the liver also have a significant ability to metabolize drugs, especially drugs which have a functional group that is easily oxidized. Such drugs can be metabolized by other enzyme systems such as the flavin-dependent oxidase prostaglandin synthase and by glutathione transferase. Therefore, it is logical to investigate the ability of the target organ of a specific idiosyncratic drug reaction to form reactive metabolites of that drug.

IV. FORMATION OF REACTIVE METABOLITES BY LEUKOCYTES A. Enzyme Systems in Leukocytes That Might Metabolize Drugs Many idiosyncratic drug reactions involve bone marrow toxicity. In addition, because of the role of leukocytes in the induction of an immune response, it is likely that interaction of reactive metabolites with leukocytes could induce an immunological reaction. Therefore metabolism of drugs by leukocytes could have major implications for adverse drug reactions [7]. Lymphocytes and monocytes are known to contain a small amount of cytochrome P-450,and because peripheral leukocytes are easier to obtain from patients than samples of liver or lung, attempts have been made to correlate the activity of cytochrome P-450 in lymphocytes or monocytes with the risk of disease such as lung cancer, which is thought to be due, at least in part, to reactive metabolites generated from xenobiotics found in cigarette smoke [35, 361. However, the concentration of cytochrome P-450 is very low. Lymphocytes, monocytes, and platelets are also known to contain relatively high concentrations of prostaglandin synthase [37].This system has been demonstrated to oxidize many different xenobiotics, several of which are drugs [38-401.Macrophages and neutrophils also contain an enzyme that converts arginine to nitric oxide [41,421. Nitric oxide can be converted to nitrite or can react with superoxide to form peroxynitrite, and all of these species are strong oxidants [43,441. A major function of neutrophils is to phagocytose infectious agents and other particulate matter [45].In addition to phagocytosis, another important


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function is the killing of infectious agents. Neutrophils contain several antimicrobial agents including myeloperoxidase [46]. The combination of myeloperoxidase with hydrogen peroxide produces a form of the enzyme called compound 1 that is a strong oxidant [47]. The hydrogen peroxide is generated by NADPH oxidase, which converts oxygen to superoxide, and superoxide is further converted to hydrogen peroxide, either spontaneously or catalyzed by superoxide dismutase. Compound I of myeloperoxidase can oxidize chloride ion to hypochlorous acid. Hypochlorous acid is the agent used to kill bacteria in municipal water supplies. In a resting cell the myeloperoxidase is packaged in granules known as primary or azurophilic granules and NADPH oxidase is inactive; however, when the cell is activated by bacteria, or by a variety of other factors including agents such as phorbol esters, the myeloperoxidase is released and the NADPH oxidase is activated [48]. This process utilizes a large amount of oxygen and is referred to as the respiratory burst. It has been estimated that an activated neutrophil generates enough hypochlorous acid in less than a second to kill 150 bacteria [46]. Hydrogen peroxide and superoxide are somewhat reactive; however, it appears that most of the superoxide that is generated is converted to hypochlorous acid, and hypochlorous acid is a much stronger oxidant than hydrogen peroxide or superoxide. Therefore the major oxidant produced by neutrophils is hypochlorous acid [46]. It has been postulated that neutrophils produce hydroxy radicals and singlet oxygen, but more recent evidence suggests that negligible quantities of these agents are actually produced under physiological conditions [49]. It is important to note that the myeloperoxidase is released outside the cell or into phagosomes, and that the enzyme system NADPH oxidase is also on the outside of the cell membrane. These systems are summarized in Fig. I . Hypochlorous acid, compound I of myeloperoxidase, prostaglandin synthase plus the hydroperoxides produced by this enzyme, and nitric oxide or related species would be expected to react with many drugs. In some cases this reaction is likely to result in the formation of a reactive intermediate. Of these leukocyte-associated oxidants, hypochlorous acid would be expected to play the quantitatively dominant role. These oxidants are unlikely to be able to oxidize substrates such as halothane to reactive intermediates. However, drugs which contain readily oxidizable functional groups are likely to be oxidized by leukocytes. In general, it should be possible to predict many of the metabolic pathways that occur with activated neutrophils from the reactions of hypochlorous acid with specific functional groups. Although metabolism by leukocytes is unlikely to make a quantitatively significant contribution to the metabolism of a drug, it may have important implications for its toxicity and even its therapeutic effects [SO, 511.



" NADPH oxldase

0 2

+ 202

CI

Neutrophil

31 I

superoxide dismutase

Leukocytes may also generate oxidants through the prostag land in s yn t h ase pathway and by generation of nitric oxide

0;

FIG. 1. Summary of pathways present in neutrophils that can lead to oxidation of drugs. B. Metabolism of Procainarnide by Leukocytes We found that the arylamine of procainamide was metabolized by activated human neutrophils to a hydroxylamine and further to a nitro group [52]. In the presence of added ascorbate, we found an increase in the quantity of the hydroxylamine and disappearance of the nitro metabolite. I n vitro the hydroxylamine was oxidized nonenzymatically by hydrogen peroxide to a nitroso derivative and further to the nitro derivative. Of these metabolites, the nitroso derivative was the most reactive and reacted with thiols to form sulfinamides [53].The same products were formed when procainamide was oxidized by the combination of myeloperoxidase and hydrogen peroxide. When chloride ion was added, the rate of the oxidation greatly increased, and the major product became N-chloroprocainamide. With time, the chlorine spontaneously rearranged to the ring ortho to the amino group [54]. Reaction of procainamide with hypochlorous acid also resulted in the formation of N-chloroprocainamide. Although no N-chloroprocainamide was observed in incubations of procainamide with activated neutrophils, this was presumably because it reacted very rapidly with the cells. These reactions are summarized in Fig. 2. Incubation of radiolabeled procainamide with activated neutrophils resulted in just under 1% of the drug becoming covalently bound to the cells [ 5 5 ] . Incubation of radiolabeled procainamide with myeloperoxidase,


3 12

UETR ECHT

(Hydroxylamine

I

MPO/H202/CIor NaOCl

FIG. 2. Metabolism of procainamide by activated neutrophils or mononuclear cells, or catalyzed by myeloperoxidase. Reprinted from Ref. 7 with permission. hydrogen peroxide, and albumin also led to covalent binding of the drug to albumin, and although addition of chloride ion increased the degree of covalent binding, the increase was small [54]. Although chlorination of procainamide is faster than the oxidation to the nitroso derivative, and Nchloroprocainamide is more reactive than the nitroso derivative, the major reaction of N-chloroprocainamide with protein appears to lead to chlorination of the protein rather than alkylation and may make less of a contribution to toxicity. Little metabolism of procainamide occurred in the absence of activation of the cells with phorbol ester or other activating agent, and the oxidation was inhibited by azide and catalase [52]. This suggested the involvement of myeloperoxidase and hydrogen peroxide. Inhibitors of prostaglandin synthase, such as aspirin and indomethacin. had no effect. Comparison of metabolism of procainamide by different leukocytes revealed that monocytes produced about one half the amount of hydroxylamine produced by neutrophils [55]. A small amount of hydroxylamine was observed in incubations with lymphocytes, but this appeared to be



3 13

due to contamination of the lymphocytes with monocytes because a good correlation was observed between the hydroxylamine and the degree of this contamination. No hydroxylamine was observed in incubations of procainamide with platelets that had been activated with collagen or fibrinogen. Likewise, comparison of the degree of covalent binding to different leukocytes revealed the highest degree of covalent binding to neutrophils, less to monocytes, and no significant binding to lymphocytes or platelets. In short, activated human neutrophils and monocytes oxidized procainamide to at least two reactive metabolites, and this was associated with covalent binding of the drug to the cells. The major enzyme involved in the formation of these reaciive metabolites appeared to be myeloperoxidase.

C. Metabolism of Other Arylamines by Leukocytes The metabolism of dapsone by activated neutrophils also led to the formation of the hydroxylamine and nitro metabolites [56]. No evidence for the formation of N-chlorodapsone was originally found in incubations of dapsone with myeloperoxidase/hydrogenperoxidekhloride. More recent experiments demonstrated the formation of N-chlorodapsone in incubations of dapsone with myeloperoxidase/hydrogen peroxidelchloride ion, although the amount formed is somewhat less than the N-chloroprocainamide formed under similar conditions (unpublished observation). Presumably because of the electron-withdrawing group in the para position, the rearrangement of N-chlorodapsone was much slower than that of N-chloroprocainamide, and therefore we also did not observe the rearranged product. No radiolabeled dapsone was available for study of covalent binding; however, it is likely that metabolism of dapsone by activated neutrophils results in some degree of covalent binding. We found that sulfadiazine is oxidized by activated neutrophils to its hydroxylamine [57], and likewise Cribb et al. found that sulfamethoxazole is metabolized to a hydroxylamine [58]. In addition, we found that the combination of myeloperoxidase/hydrogen peroxidekhloride converted the arylamine group of sulfamethoxazole to a chloramine (manuscript in preparation). We have also studied a derivative of chloramphenicol in which the nitro group is reduced to an arylamine. Several products are formed when it is incubated with myeloperoxidase/hydrogen peroxidekhloride ion (unpublished observation). Although the N-chloro derivative was not observed

3 I4

UETR ECHT


N-N

H3C‘

HOCl

i t

N ‘CH3

H3C‘

L

\CH3

radical cation

FIG. 3. Mechanism of aminopyrine oxidation proposed by Say0 and Saito [61]. directly, we did find a product with a chlorine ortho to the arylamine. Because this amine does not have an electron-withdrawing group in the para position, it is likely that the chloramine rearranges very rapidly to the ringsubstituted product. Diclofenac is a secondary arylamine that has been found to be oxidized by the myeloperoxidase system to dihydroxyazobenzene [ 59). From these observations, it appears that the metabolism of primary arylamines by neutrophils to hydroxylamines and chloramines is a general reaction. The major differences in the reaction of arylamines appear to be due to the electron withdrawing effect of groups in the para position. It is not surprising that metabolism involving myeloperoxidase would be less selective than that involving cytochrome P-450,especially those reactions that simply involve formation of hypochlorous acid and reaction of hypochlorous acid with drug.

D. Metabolism of Aminopyrine by the Myeloperoxidase System Aminopyrine had been shown to be oxidized to a radical cation by the prostaglandin synthase system [W]. More recently its oxidation by myeloperoxidase and hypochlorous acid has been studied by Sayo and Saito 1611. In the absence of chloride ion, oxidation was slow. In the presence of chloride ion, the oxidation was rapid, although the formation of hypochlorous acid was faster than its reaction with aminopyrine. In contrast, in the presence of bromide ion, the reaction with aminopyrine was not rate limiting. Even though hypochlorous acid is generally a two-electron oxidant, the product was the same radical cation as produced by other peroxidases. The same radical cation was also produced by hypochlorous acid itself. The suggested mechanism is shown in Fig. 3; however, it is possible that an iminium ion is the first product formed by loss of HCI rather than loss of a

3 I5



OH

0

FIG. 4. Oxidation of amodiaquine to a reactive intermediate. chlorine atom, and the imine could react with the parent drug to form two radical cations.

E. Metabolism of Chlorpromazine by the Myeloperoxidase System Kalyanaraman and Sohnle found that chlorpromazine, as well as aminopyrine and phenylhydrazine, were oxidized by hypochlorous acid or activated neutrophils to free radicals [62]. Likewise, van Zyl et al. found that chlorpromazine is oxidized to a relatively stable radical cation by the combination of myeloperoxidase and hydrogen peroxide [63]. In the presence of chloride ion, as with aminopyrine, it is not clear whether the reaction involves a direct one-electron oxidation or chlorination to form the twoelectron oxidation product followed by coproportionation with the parent drug to form two radical cations (the opposite of disproportionation).

F. Metabolism of Amodiaquine and Acetaminophen by Leukocytes and the Myeloperoxidase System Amodiaquine has been found to undergo autoxidation to a reactive quiCovalent none imine in the presence of oxygen, as shown in Fig. 4 [a]. binding was also observed to human liver microsomes and binding was decreased by NADPH, presumably because it reduced the quinone imine [65]. Binding was observed to albumin in the presence of horseradish peroxidase/ hydrogen peroxide or in the presence of chlorine. These systems probably led to the formation of the quinone imine. Oxidation by activated neutrophils led to a reactive intermediate which covalently bound to albumin, and this intermediate is presumably also the quinone imine (661. Acetaminophen was oxidized to the reactive N-acetylbenzoquinone imine by the myeloperoxidase system (671. This is a similar oxidation to that of

3 I6

UETRECHT

amodiaquine; however, although the conditions were different, it appears that the oxidation of acetaminophen was relatively slow.


G. Metabolism of Vesnarinone by Leukocytes and the Myeloperoxidase System We found that vesnarinone is metabolized to a reactive metabolite which covalently bound to activated neutrophils [68]. From a study of the metabolic pathway of vesnarinone by the myeloperoxidase system or by hypochlorous acid, we found that a reactive imidoiminium ion appears to be involved, as shown in Fig. 5 (manuscript in preparation). As with aminopyrine and chlorpromazine, it is unclear whether the formation of the iminium ion involves a radical cation intermediate or whether the iminium ion is formed by loss of HCI and this product is in equilibrium with the radical cation. The radical cation intermediate is less appealing because it involves loss of a chlorine atom rather than a chloride ion.

H. Metabolism of Clozapine by Leukocytes Fischer and Mason studied the metabolism of clozapine by activated leukocytes, horseradish peroxidase/hydrogen peroxide, and myeloperoxidase/ hydrogen peroxide [69]. They found evidence for a reactive metabolite which covalently bound to activated leukocytes and formed a glutathione conjugate. Although a free radical could not be observed directly, glutathione radicals were formed. We have good evidence for the formation of a relatively stable nitrenium ion from the reaction between clozapine and hypochlorous acid (unpublished observation).

I. Metabolism of Hydralazine and Isoniazid by Leukocytes Hydralazine is a hydrazine derivative that is rapidly metabolized by activated neutrophils or myeloperoxidase/hydrgen peroxide/chloride to phthalazinone and phthalazine, as shown in Fig. 6 [70]. In view of the general reactivity of hydrazines, it was somewhat surprising that the oxidation of hydralazine by hydrogen peroxide alone was very slow. Hydralazine breaks down in solution to phthalazine, but this reaction is much slower than that observed in the presence of activated neutrophils or the neutrophil oxidation system. As with most myeloperoxidase-mediated reactions, the rate is greatly increased by chloride ion, and the same products are produced by hypochlorous acid (although the ratio of phthalazine to phthalazinone ap-



3 17

veratrylpiperazinamide (OPC-MPO-1)

FIG. 5. Oxidation of vesnarinone by hypochlorous acid or by the myeloperoxidase system. pears to be different). Although myeloperoxidase/hydrogenperoxidekhloride can oxidize phthalazine to phthalazinone, the reaction is too slow to account for all of the phthalazinone that was produced. Incubation of radiolabeled hydralazine with activated neutrophils resulted in covalent binding of the drug to the cells to about same degree as that observed with procainamide. We postulated that the reactive intermediate could have been the diazonium salt; however, attempts to prove this by an alternate synthesis of the diazonium salt have been unsuccessful. Isoniazid is another hydrazine derivative which is metabolized by activated neutrophils [71]. The major product is isonicotinic acid. This could represent a simple hydrolysis reaction; however, the requirement for

3 18

UETRECHT


Phthalazinone

Phthalazine

FIG. 6. Oxidation of hydralazine by the myeloperoxidase system. activation of the cells and the duplication of the products with the myeloperoxidase system or simply hypochlorous acid indicates that this is an oxidation reaction. Although we have no direct evidence that a reactive intermediate is involved, the oxidation of hydrazines is likely to lead to reactive intermediates.

J. Metabolism of Propylthiouracil and Thiols by Leukocytes Propylthiouracil is metabolized by activated neutrophils or myeloperoxidase/hydrogen peroxidelchloride by a series of reactions, as shown in Fig. 7 172, 731. The first step is presumably the formation of the sulfenyl chloride; however, this intermediate would be expected to be reactive, and we did not observe it directly. The first product isolated was the disulfide. This is further oxidized all the way to a sulfonic acid. The sulfonic acid is also reactive and reacts with thiols such as glutathione to form a thioether as shown in Fig. 7 1731. Some of the other intermediates may also be reactive. Penicillamine appears to be oxidized by myeloperoxidase, even in the absence of chloride ion [74]. Although it does not appear as if the experiment has been done, there can be little doubt that hypochlorous acid would oxidize other thiol drugs, such as captopril. The reactions would presumably involve reactive sulfenyl chloride intermediates and lead to disulfides and other products.

K. Metabolism of Phenytoin by Leukocytes Phenytoin is N-chlorinated by the combination of myeloperoxidasel hydrogen peroxidekhloride to N,N’-dichlorophenytoin as shown in Fig. 8 175, 761. As with N-chloroprocainamide, this metabolite is not observed in

3 19


OH Propylthiouracil Drug Metabolism Reviews Downloaded from informahealthcare.com by McMaster University on 02/13/15 For personal use only.

OH

OH

Sulfenyl Chloride

Sulfinic Acid

Sulfonic Acid

Disulfide

Sulfhydryl Adduct

FIG. 7. Metabolism of propylthiouracil by activated leukocytes and reaction of the sulfonic acid metabolite with sulfhydryl groups. incubations of the drug with activated neutrophils, but this appears to be due to its rapid reaction with the cells. Also like N-chloroprocainamide, the major reaction of this metabolite with protein appears to involve chlorination of the protein rather than alkylation; however, there is a small degree of covalent binding of radiolabeled drug to activated neutrophils. Covalent binding to albumin in the presence of myeloperoxidase is increased by the presence of chloride ion, which suggests that it is the chlorinated metabolite that is responsible for the covalent binding.

L. Metabolism of Carbamazepine by Leukocytes Carbamazepine is metabolized by myeloperoxidase/hydrogen peroxide/ chloride ion to acridone by the unusual pathway shown in Fig. 9 [77]. Reaction of carbamazepine with hypochlorous acid forms the same products.

A Phenytoin

N,N’-Dichlorophenytoin

FIG. 8. Chlorination of phenytoin catalyzed by myeloperoxidase.

320

UETRECHT H H 'c=O


O"*NH,

OGC*NH2

Carbarnazepine

H 'CZO

FIG. 9. Oxidation of carbamazepine by the myeloperoxidase system. The reaction of carbamazepine with hypochlorous acid is slower than the reaction of most other drugs that we have studied. This may be due to the nature of the reaction, which involves reaction with a double bond rather than a heteroatom. Iminostilbene is oxidized to the same products, but the rate is much faster, and in the case of iminostilbene the initial reaction may involve chlorination of nitrogen rather than the double bond. The presumed carbonium ion intermediate in the carbamazepine reaction would be reactive, but it may rearrange too rapidly to result in significant covalent binding. Incubation of radiolabeled carbamazepine with activated neutrophils leads to covalent binding of the drug to the cells. The degree of covalent binding is less than that seen with procainamide, but greater than that observed with phenytoin.

M. Metabolism of Phenylbutazone by Leukocytes Phenylbutazone was shown by Ichihara to be metabolized by leukocytes enzyme to three metabolites, and all involved the 4 position of the drug [78]. The metabolites were 4-hydroxy-, 4-hydroperoxy-, and 4-chlorophenylbutazone. The 4-chloro metabolite presumably arises from myeloperoxidase-derived hypochlorous acid, and the 4hydropexoxyphenylbutazone from the radical formed by interaction by phenylbutazone with compound I of myeloperoxidase. Although the 4-


32 1

hydroxyphenylbutazone could be formed by the reduction of the hydroperoxy derivative, evidence suggested that it had a different source.


N. Further Metabolism of Benzene Metabolites by Leukocytes The majority of benzene metabolism occurs in the liver, and it is unlikely that significant metabolism of benzene can occur in the bone marrow or in peripheral leukocytes [79]. On the other hand, Eastmond et a]. have reported that the hepatic metabolites of benzene, such as phenol, are oxidized to reactive metabolites by the myeloperoxidase system [go]. The results of these experiments may not be a true reflection of the pathways that occur in vivo because it appears that chloride ion was not present in the incubations. The metabolism of xenobiotics by the myeloperoxidase system is, in general, very different in the presence of chloride ion, and there is a high concentration of chloride ion in vivo. It is likely that the addition of chloride ion to the incubations would have significantly increased metabolism of phenol. Metabolism and covalent binding of phenol was also observed with intact neutrophils [81].

V. AGRANULOCYTOSIS AND APLASTIC ANEMIA

A. General Aspects One frequent type of serious idiosyncratic drug reaction is agranulocytosis, which consists of an almost complete lack of granulocytic leukocytes in the circulation-usually defined as less than 500 cells compared to a normal count of 5,000-10,000 cells/pL of blood. There have been several reviews of this subject [33, 82-87]. The granulocytic leukocytes are neutrophils, eosinophils, and basophils; however, the predominate cell of this series is the neutrophil. Drug-induced agranulocytosis is usually reversible with an “overshoot” of the peripheral neutrophils approximately 1 week after the drug is stopped. The risk with agranulocytosis is the contraction of infections which can be fatal even with appropriate antibiotic therapy. The mortality rate from drug-induced agranulocytosis is about 10%;however, this can be reduced to some degree with early identification, discontinuation of the drug, and good supportive therapy. If the bone marrow of a patient with drug-induced agranulocytosis is examined, one of three different pictures is usually found.

322

U ETR ECHT


Depletion of almost all of the cells of the granulocyte series Normal or even increased population of the early cells of the granulocyte series, but an absence of the more mature cells Normal or increased population of the cells of the granulocyte series The picture depends on the time point in the course of the disease that the bone marrow is obtained, and one cause for a normal or increased population of all cells of the granulocytic series is that the bone marrow has already started to recover but normal numbers of cells have not yet reached the circulation. The same bone marrow picture would be expected if the bone marrow was unaffected, and the mechanism of agranulocytosis involves rapid peripheral destruction of cells. When the bone marrow has normal early precursors but none of the more mature cells, it has been referred to as maruralion arresr [86, 881. This name derives from a mechanistic hypothesis for which there is little evidence. This picture can also arise at a specific point in time from a bone marrow which is recovering from an insult to the immature cells. This picture characteristically appears to be due to depletion of cells at approximately the promyelocyte or myelocyte stage and beyond. Aplastic anemia results when all elements of the bone marrow are depleted [89]. Most cases of agranulocytosis are associated with drugs [ 8 5 ] ; in contrast, only about 50% of aplastic anemia is associated with drugs [89]. In general, the same drugs that cause agranulocytosis also cause aplastic anemia, although agranulocytosis is more common, and some drugs, such as chloramphenicol, have a higher relative incidence of aplastic anemia than others. The mortality rate of aplastic anemia is much higher than that of agranulocytosis, and the time to recovery is usually longer. In some cases of drug-induced bone marrow toxicity. two of the three elements are effected, especially the granulocytes and platelets [90, 911. The idiosyncratic nature of agranulocytosis and aplastic anemia suggests involvement of the immune system. There are many reports of antibodies against neutrophils in the sera of patients with drug-induced agranulocytosis, and specific examples are provided in the following sections. Yet in most cases investigators fail to find such antibodies, and negative findings are usually not published. There are several possible reasons why falsenegative results may be obtained. The antibody binds to the neutrophils and is rapidly cleared from the blood: Therefore free antibodies may only be present for a very brief period, if at all. There is one report where this appears to be the case [92]. In most investigations the availability of sera from different times during the progression of the illness is very limited.



323

The antibody is specific for neutrophils which have been haptenized by the drug: Although most investigators incubate the serum and neutrophils, both in the presence and in the absence of the drug, the drug itself will usually not be chemically reactive. Unless the cells have been activated, no reactive metabolite will be formed, although some degree of activation may occur during isolation of the cells. Now that the identity of several reactive metabolites formed by neutrophils is known, it should be possible to correct this problem by adding the appropriate metabolite to the neutrophils. It may also be possible to simply activate the neutrophils in the presence of the drug; however, activation leads to aggregation of the neutrophils, and this makes further investigation of the intact cells very difficult. Some investigators use urine as a source of metabolites; however, this is rather crude and any metabolite reactive enough to covalently bind to the neutrophils is unlikely to be present in the urine. Yet this has led to positive results in some cases [93]. The only way that this makes sense using accepted principles of immunology is that, although a drug must be covalently bound to a macromolecule to induce an antibody, once the antibody is present, in some cases a noncovalent interaction of drug and neutrophil can be recognized by the antibody. The antibody recognizes antigens on neutrophils which are genetically heterogeneous and it would be necessary to use the patient’s neutrophils or other neutrophils with the same antigens in order to detect the antibody. There are several examples of this type of antibody [94, 951. The antibody is specific for antigens on neutrophil precursors in the bone marrow and does not bind to antigens on mature neutrophils. This appears to be case in some patients [96, 971. The neutrophil is an innocent bystander, and the reaction occurs because of absorption of antigen-antibody complexes composed of some antigen not present on the neutrophils. The reaction is immune mediated but involves a cell-mediated reaction rather than an antibody-mediated reaction.

On the other hand, the presence of antineutrophil antibodies in the serum of a patient with drug-induced agranulocytosis does not prove that these antibodies are responsible for the agranulocytosis.

B. Aminopyrine One of the first drugs to be associated with a high incidence (0.1-1%, [98]) of agranulocytosis was aminopyrine [99]. The usual clinical picture is an acute onset of fever, sore throat, and various infections [IO]. The

UETRECHT


324

usual delay between starting the drug and the development of agranulocytosis is unclear. In some reports the delay often appeared short [loll. However, aminopyrine was readily available at that time and sold under many different trade names, as well as being a component of several different mixtures [102, 1031. Its use was often intermittent, much as aspirin or acetaminophen is used today. Therefore it was not clear to what extent there had been previous exposure to the drug. In other reports, the delay between starting the aminopyrine and the development of agranulocytosis was often on the order of I month, which is typical for other drug-induced agranulocytosis [98, 104, 1051. Whatever the delay before the first episode of agranulocytosis. on reexposure to aminopyrine, these patients invariably had a rapid onset of symptoms with fever, chills, and drop in peripheral neutrophils within a few hours. In several reports this occurred at a reduced dose or after dermal exposure [99, 100, 103, 106). Although the rapid drop in neutrophils on reexposure suggested peripheral destruction of neutrophils, the bone marrow in patients with aminopyrine-induced agranulocytosis usually revealed depletion of the granulocytic series, either all of the series or just the more mature cells [98, 102-104]. The incidence appears to be higher in females and in the elderly [103, 1071. The evidence for an immune-mediated reaction is strong, and a combination of acute serum from a patient incubated with normal neutrophils led to agglutination of the neutrophils [108, 1091. In addition, infusion of blood from a sensitive patient 3 h after a dose of aminopyrine into a normal subject matched for blood type led to granulocytopenia [108]. In another study, the serum from a patient with aminopyrine-induced agranulocytosis inhibited colonies cultured from a mononuclear fraction of the patient’s blood in the presence of drug, while serum from a normal control did not. Serum collected from the patient up to 1 year after the episode of agranulocytosis also inhibited colony-forming cells [ 1041.

C. Pmcainamide Pmcainamide is associated with a relatively high incidence of agranulocytosis, but the reported incidence varies significantly in different studies. In one study by Ellrodt et al. of patients who received procainamide after open heart surgery, the incidence of agranulocytosis and severe neutropenia was 4.4% [110]. This is a very high incidence, and the authors suggested that the high incidence was due to the fact that a sustained-release preparation of the drug was used. In another study by Meyers et al., a lower incidence of agranulocytosis was observed (0.55%), and there was no



325

difference in the incidence associated with the sustained-released preparation and that of the conventional preparation [Ill]. One difference in the populations studied that could have led to these disparate results is that the patients in the Ellrodt study had all recently undergone open heart surgery. The tissue damage associated with such major surgery would be expected to lead to a large increase in the number of activated neutrophils. If metabolism of drug by activated neutrophils plays a role in the pathogenesis of agranulocytosis, this could be the basis for the high incidence of severe neutropenia in the Ellrodt study. Even an incidence of 0.55% is high. The delay between starting the drug and the onset of agranulocytosis was typical of most drug-induced agranulocytosis-usually between 1 and 3 months; however, in one case the delay was 459 days [112]. The bone marrow of most of the patients in the Ellrodt study was abnormal, with either a “maturation arrest” at the promyelocyte or myelocyte stage, or a depletion of all granulocytic precursors. However, the bone marrow of one patient was hyperplastic, and granulomas were found in the bone marrow of one patient in another report [113]. Although the most common presentation was agranulocytosis, there are also reports of patients who had anemia and/ or thrombocytopenia [90,9 I , I 14- I 181. Immune-mediated hemolytic anemia has also been reported [119-1211. Drugs that cause both agranulocytosis and lupus have been placed in a separate category by Pisciotta [83]; however, possibly because both involve reactive metabolites generated by activated leukocytes, there are many drugs, such as procainamide, that cause both [7]. There is no higher incidence of ANA in patients with procainamide-induced agranulocytosis than in other patients treated with procainamide, and in fact, if different at all, the incidence appears to be lower [IlO]. This may be due to the fact that it usually takes a longer period of time for a drug to induce lupus than agranulocytosis. Although the mechanism of procainamide-induced agranulocytosis is unknown, and most attempts to find antineutrophil antibodies have been negative, there is one report by Azocar of lysis of HL-60 cells (a malignant cell line from promyelocytic leukemia) and K-562 cells (a cell line from a patient with myeloid leukemia in blast crisis) [97]. The cells were labeled with ”Cr, and acute phase serum from two patients with procainamideinduced agranulocytosis lysed cells from these cell lines (HL-60 > K-562) in the presence of complement. Serum from the same patients after recovery did not lyse these cells. There was no lysis of mature neutrophils or cells from a lymphocyte cell line. These results suggest that an antibody which recognized antigens present only on immature cells of the granulocytic series was responsible for agranulocytosis in these patients.

UETR ECHT

326


D. Dapsone The risk of dapsone-induced agranulocytosis was emphasized by experience in Vietnam where it was used for prophylaxis against malaria. This resulted in 16 cases of agranulocytosis and 8 deaths (1221. It was difficult to determine the incidence because the total number of troops treated was not known, but it appeared to be approximately 1 in 10,OOO. In a study of the incidence of dapsone-induced agranulocytosis in Sweden between 1972 and 1988, the incidence was estimated to be between 1 in 240 and 1 in 425 [123]. It was suggested that this higher incidence could be due to the patient population studied, which consisted of patients being treated with dapsone for dermatitis herpetiformis. It is possible that abnormalities that led to infiltration and activation of neutrophils in the skin of these patients also increased the metabolism of dapsone by neutrophils and the risk of agranulocytosis. The onset of agranulocytosis occurred from 3 weeks to 3 months after initiation of therapy in the Ognibene study [122] and from 4 weeks to 19 weeks in the Hornsten study [123]. The bone marrow in the Ognibene series was described as maturation arrest in 5 of the patients and as total absence of granulocytes in most of the others. In the Hornsten series, the bone marrow was described as either severe depression of granulopoiesis or no granulopoietic cells except in one case in which the bone marrow was not done until 10 days after admission. Recovery of the bone marrow after dapsone-induced agranulocytosis can be associated with a syndrome which mimics leukemia [124], and this also can occur during recovery of other drug-induced agranulocytosis. A case of dapsone-induced aplastic anemia has also been reported [ 1251. The mechanism of dapsone-induced agranulocytosis is unknown. The hydroxylamine metabolite of dapsone, but not dapsone itself, has been found to inhibit granulocytopoiesis [126].

E. Sulfonamides, Sulfasalazine, and Trimethoprim Sulfonamides were also among the first drugs to be associated with agranulocytosis [127]. In addition, they are currently associated with possibly the largest number of cases of agranulocytosis [84, 128-1301. Sulfonamides are frequently given in combination with trimethoprim, and trimethoprim can also cause neutropenia (131). The incidence is reported to be 1 case per 18,000 prescriptions of the combination [132]. The median treatment period was 13 days, but this shorter period may reflect the facts



327

that antibiotics are usually only given for 2 weeks, and many people probably have had prior exposure to sulfonamides. This combination must also cause some degree of toxic bone marrow suppression since 34% of children were reported to develop mild neutropenia when treated with trimethoprimsulfamethoxazole [ 1331. A positive lymphocyte transformation test was reported in a patient who was treated with topical sulfamylon and developed a rash, thrombacytopenia, and profound granulucytopenia [ 1341. Antineutrophil antibodies have also been detected [ 1351. Sulfasalazine is also associated with a high incidence of agranulocytosis, and it is reduced to two arylamines, 5-aminosalicylic acid and sulfapyridine, in the intestine [ 136, 1371. In both of these case reports a rash accompanied the agranulocytosis. A case report by Evans and Ford is very instructive [92]. A patient developed a rash 5 weeks after starting sulfasalazine for ulcerative colitis and fever. The symptoms continued for 2 weeks before he went to a physician. He was found to have agranulocytosis, and on the 7th day of hospitalization, examination of a bone marrow aspirate showed a large number of promyelocytes and metamyelocytes. On the 12th day of hospitalization he was given 0.5 g of sulfasalazine. This had no effect on the white blood cell count and was followed by 2 g of sulfasalazine, which also had no effect on the white blood cell count. On the 26th day of hospitalization he was started on sulfasalazine every 6 h. After the fourth dose he developed chills, malaise, and fever, and his white blood cell count jumped to 14,600 cells/pL with 90% neutrophils. The drug was continued, and the subjective symptoms resolved and the white blood cell count returned to normal. On the 4th day of this therapy the dosing interval was decreased to every 12 h because of a decrease in white blood cell count to 2,000 cells/pL. After 4 days at the reduced dosage, the white blood cell count was stable, and the drug was increased back to every 6 h. The following day (9 days after restarting the drug) the count fell to 360 cells/pL and the drug was discontinued. Examination of the bone marrow showed that the percentage of neutrophils had dropped from 13.8% 4 days earlier to 0.6%. Examination of the ability of sera collected at different times to cause neutrophil agglutination resulted in positive results only for serum obtained 4 h after the initial dose on the second course of therapy with sulfasalazine. The addition of sulfasalazine, sulfapyridine, or sodium salicylate had no effect on agglutination. These results, along with the rash, suggest an immune mediated reaction, and yet agranulocytosis was delayed for 9 days on reexposure to the drug. It is also interesting to note that the presence of leukoagglutinins could only be detected shortly after readministration of the drug, but not during agranulocytosis. Another report of sulfasalazine-induced agranulocytosis found a positive lymphocyte proliferation test as evidence for an immune-mediated mechanism [ 1381.

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F. Other Arylamines Not many drugs are primary arylamines, possibly because this functional group appears to be associated with a relatively high incidence of adverse reactions including agranulocytosis. Metoclopramide, which is a derivative of procainamide, has been reported to cause agranulocytosis even though it is usually given at a dose 1150th that of procainamide 1139, 1401. Paraaminosalicylic acid, an isomer of 5-aminosalicylic acid, which is released during the reduction of sulfasalazine, is also associated with agranulocytosis [141]. Aminoglutethimide is associated with a high incidence of idiosyncratic reactions including agranulocytosis [ 142- 1441. Aprindine, although it is a tertiary rather than a primary arylamine, is associated with a high incidence of agranulocytosis, which has severely limited its use as an antiarrhythmic agent (145-147). Diclofenac is also a secondary arylamine, and it has been reported to cause agranulocytosis and aplastic anemia (93, 1481. It is the only one of the newer nonsteroidal anti-inflammatory drugs to show a statistically significant association with aplastic anemia [149]. In addition, diclofenac and mefenamic acid and its analogs (which are also associated with agranulocytosis and hemolytic anemia [ 1501) are the only nonsteroidal antiinflammatory drugs that are arylamines.

G. Chloramphenicol Chloramphenicol is the classic drug associated with aplastic anemia, although the incidence is only approximately l in 20,000-40.000 [151. 1521, and to a lesser degree agranulocytosis. The risk of aplastic anemia does not appear to be related to dose and has even been reported after chloramphenicol eye drops [153-155]. Strangely, the mortality rate appears to increase with the delay between stopping the drug and the onset of aplastic anemia [151, 1521. Chloramphenicol is also associated with dose-related bone marrow depression. Evidence suggests that the mechanism of this dosedependent depression involves mitochondrial injury [1561. The mechanism of chloramphenicol-induced aplastic anemia is unknown. Thiamphenicol is a chloramphenicol analog in which the nitro group is replaced with a methylsulfone group. This drug is also associated with a dose-dependent suppression of bone marrow; however, despite widespread use, it has not been associated with aplastic anemia [156]. This suggests that the nitro group is necessary for the induction of chloramphenicolinduced aplastic anemia. Another analog of chloramphenicol in which the nitro group has been reduced to a nitroso group is much more toxic to bone



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marrow than chloramphenicol itself [157, 1581. The nitro group of chloramphenicol is reduced to an amino group by bacterial flora in the intestine [159], and this is, in turn, oxidized in the liver to a hydroxylamine and nitroso group [160]. However, in a study of the disposition of the nitroso derivative of chloramphenicol, Ascherl et al. found that it was too reactive to reach the bone marrow in significant concentration [160]. Although previous investigators found no evidence of oxidation of the amine by bone marrow cells, the cells were not activated [158]. Although there is no evidence that bone marrow cells can reduce chloramphenicol to the toxic nitroso derivative, another bacterial metabolite was found to be reduced to a toxic nitroso derivative by bone marrow cells [ 161- 1631. Another possible mechanism of chloramphenicol-induced aplastic anemia is the oxidation or chlorination of the amine derivative of chloramphenicol to a reactive metabolite in the bone marrow when the cells have been activated as described earlier. Whatever metabolite may be responsible for chloramphenicolinduced aplastic anemia, it is still unclear what mechanism is involved or the basis of its idiosyncratic nature.

H. Chlorpromazine Chlorpromazine is a significant cause of agranulocytosis. It is often used as an example of agranulocytosis that is mediated by direct toxicity to bone marrow. In an early report of 16 patients by Pisciotta et al., the average duration of therapy was 61 days, with a range of 13 to 318 [ l a ] . The decrease in peripheral neutrophil count was often more gradual than that seen with aminopyrine. Bone marrow examination demonstrated depletion of all elements of the granulocyte series. Only one fourth of the cases were males. Only two of the cases were associated with a rash, and one of these was pustular and may have been due to agranulocytosis rather than directly due to the drug. Reexposure of one patient at a lower dose for 1 year did not lead to agranulocytosis. In another patient reexposure did lead to granulocytopenia, but only after 121 days, and the onset was relatively gradual. This is in sharp contrast to the observations made in aminopyrine-induced agranulocytosis and suggests that the reaction is not immune mediated. Pisciotta also found that chlorpromazine was toxic to human leukocytes as measured by respiration and incorporation of thymidine. Cells from recovered patients were more sensitive to this toxicity than cells from normal controls; however, the concentrations used were 100- to 1000-fold greater than the therapeutic concentration [82, 1651. He also found that bone marrow cells from patients who had had chlorpromazine-induced agranulocytosis from 2 to 12 years prior to the study had significantly lower

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incorporation of radiolabeled thymidine than normal controls or patients who had recovered from phenylbutazone- or chloramphenicol-induced agranulocytosis [ 1661. This was taken as evidence that chlorpromazineinduced agranulocytosis involved direct toxicity to bone marrow, and the patients who were at risk were those that had a limited DNA synthetic ability, probably on a genetic basis. One investigator has found evidence of leukoagglutinins in chlorpromazine-induced agranulocytosis [ 1671, but it does not appear that other investigators have found evidence of an immune mediated mechanism.

I. Amodiaquine and Acetaminophen Amodiaquine is an antimalarial drug associated with a relatively high incidence of agranulocytosis. In one case report, 300 mg was given once a week for 2 months (1681. During this time the patient had a fever and felt unwell after the second dose; however, this resolved rapidly, and 1 week before the end of the amodiaquine prophylaxis she was immunized against smallpox, typhoid, and cholera. A few days after the last dose of amodiaquine she developed a fever and was found to have agranulocytosis. Bone marrow examination demonstrated a lack of all neutrophil precursors. Amodiaquine was found to inhibit bone marrow colony formation at a concentration of 0.5 pg/mL using cells from the patient but not using cells from a normal control. It is interesting to speculate that the immunization or brief febrile illness may have potentiated the development of agranulocytosis in this patient. A similar case was reported by Rhodes et al. in which a patient developed agranulocytosis after 3 months of weekly prophylactic treatment (1691. They also found that amodiaquine inhibited colony growth of bone marrow cells from the patient taken 5 months after recovery, but it did not inhibit cells from normal controls. They found that related drugs-chloroquine and sulfadoxine but not proguanil, pyrimethamine, and quinine-had similar effects. Aymard et al. found similar inhibition of colony growth in bone marrow cells from a patient treated with amodiaquine for 7 weeks (1701, yet this same group found evidence in two other patients of circulating amodiaquine-dependent IgG antineutrophil antibodies [ I7 1 1. This leaves the question of mechanism open. It may be that the mechanism is different in different patients, that one or the other observation is not related to agranulocytosis, or it may be that both direct marrow toxicity and antibodymediated toxicity contribute to the development of agranulocytosis in some patients. Winstanley et al. found that the quinone imine of amodiaquine was more toxic to peripheral blood mononuclear leukocytes than the parent drug or desethylamodiaquine, a major metabolite of amodiaquine [ 1721. No



33 1

difference between amodiaquine and the quinone imine was seen in toxicity to normal granulocytehonocyte colony-forming units; however, these incubations were much longer (10 to 14 days), and it is known that amodiaquine is spontaneously converted to the quinone imine, and the added quinone imine has a short half-life. Therefore, for such a long incubation, the total exposure of the cells to the quinone imine may be the same irrespective of whether amodiaquine or the quinone imine was added. Acetaminophen is considered to be associated with a low incidence of idiosyncratic drug reactions [ 1501. However, its oxidation by the myeloperoxidase system is similar to that of amodiaquine, and it has been estimated to be responsible for 10% of drug-induced agranulocytosis (33, 1731. Although one might question that acetaminophen-induced agranulocytosis is that prevalent, it does appear to cause a significant incidence of agranulocytosis. The formation of the reactive quinone imine in the liver does not lead to toxicity except in an overdose because almost all of this metabolite is detoxified by conjugation with glutathione. The same protective pathway is not present outside of cells where it would be generated by leukocytes, and although the production of the quinone imine by leukocytes is probably not sufficient to lead to direct toxicity, it may be enough to result in significant haptenization of cells.

J. Vesnarinone Vesnarinone (formerly known as OK-8212) is a new inotropic agent developed in Japan for the treatment of severe congestive heart failure. In clinical studies in Japan involving 259 patients, no significant toxicity was observed [68]. In early trials of the drug in the United States there were four cases of agranulocytosis in the first 28 patients. In addition to the obvious ethnic difference in the two populations, 7 of the US patients had received influenza vaccine, either just prior to or during vesnarinone treatment, and all of the cases of agranulocytosis were among these 7 patients. In contrast, the use of influenza vaccine is uncommon in Japan, and none of the Japanese study patients had received it. We found that, in v i m , opsonized influenza vaccine was almost as effective as phorbol ester for activation of neutrophils as measured by metabolism of procainamide to its hydroxylarnine. In addition, incubation of radiolabeled vesnarinone with neutrophils, activated by opsonized influenza vaccine, resulted in covalent binding of the drug to the cells. Although the degree of binding was less than observed when the cells were activated by phorbol ester, much of the difference could be due to binding to the serum protein added to the


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influenza vaccine to opsonize it, and only covalent binding of the drug to cells would be detected in the assay used. Since that time, the clinical studies with vesnarinone have been continued, but influenza vaccine is no longer given to the patients. New cases of agranulocytosis have occurred, but the incidence is far lower. The period of maximum risk appears to be between 1 and 3 months of therapy, and bone marrow reveals an absence of more mature cells such as myelocytes and promyelocytes. Vesnarinone has been released in Japan, and there have been cases of agranulocytosis in Japanese patients, although it appears that the incidence is still lower than that observed in US patients not receiving influenza vaccine. The sample size of 28 patients was too small to be certain that the high incidence of agranulocytosis in this population was due to the influenza vaccine and not a coincidence or some other factor; however, the in v i m results provide a mechanism by which influenza vaccine could modify the risk of agranulocytosis. Although the formation of a reactive metabolite by activated neutrophils or neutrophil precursors provides a possible initial step in the mechanism of vesnarinone-induced agranulocytosis, it does not provide information concerning whether the mechanism involves direct toxicity or an immune-mediated mechanism.

K. Mianserin Mianserin was found to be associated with a relatively high incidence of agranulocytosis soon after it was released [ 174-1761. The incidence appears to be about 1 in 2000, and the mean time of therapy before the onset of agranulocytosis was almost 14 weeks [176]. The number of females and average age for patients were slightly greater than for the cohorts in the study. Case studies show a depression of all granulocyte precursors in the bone marrow [177, 1781. The highest risk is reported to be at between 4 and 6 weeks of therapy [ 1791. Another study suggested that mianserin had a longer half-life and saturable kinetics in patients who developed agranulocytosis [180]. There is also a report of mianserin-associated aplastic anemia (1811.

Stricker et al. found that serum from patients with mianserin-induced agranulocytosis led to complement- and drug-dependent lysis of granulocytes [182]. The patient studied also had thrombocytopenia, and indirect immunofluorescence demonstrated mianserin-dependent antibodies against platelets. Although there is no report that mianserin is metabolized by leukocytes, Lambert et al. have shown it to be metabolized in the liver to a

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reactive metabolite. Although this metabolite was not positively identified, evidence strongly suggested that it was an iminium ion [183]. It is very likely that the myeloperoxidase system would also oxidize mianserin to the same iminium ion.

L. Clozapine Clozapine is an antipsychotic agent, and it is effective in patients who are refractory to other agents. However, within 6 months of the introduction of clozapine on the market in Finland, there were 17 cases of neutropenia or agranulocytosis, and there were 9 deaths among the approximately 3000 patients treated. This led to the withdrawal of the drug in Finland [184]. The incidence of agranulocytosis was reported to be about 2 4 , although it is probably closer to 1% [185]. The length of treatment before the development of symptoms in those patients who died was an average of 58 days, with a range of 16 to 107 days. Soon after there was a report of two more patients in Switzerland [186]. In most of the cases there was an almost complete absence of neutrophil precursors in the bone marrow [184, 1851. Lieberman et al. have found preliminary evidence for antibodies in the serum of the patients [ M I . They have also found evidence of an association with specific HLA antigens among Jewish subjects [187].

M. Propylthiouracil and Methimazole The most common serious adverse reaction to antithyroid medication is agranulocytosis. with an incidence of approximately 0.4% [188, 1891. The duration of therapy prior to agranulocytosis was reported to be an average of 37 days for methimazole and 18 days for propylthiouracil. The shorter duration of propylthiouracil therapy before the onset of agranulocytosis reported in this study may be due to prior therapy of 10 out of 12 of the patients with methimazole since there is a report of cross-reactivity between propylthiouracil and methimazole [190]. In another report the duration of propylthiouracil therapy before the development of agranulocytosis was 2 months, and the incidence of serious adverse reactions was not dependent on dose within the therapeutic dose range [191]. Of the 55 patients who developed agranulocytosis in the Tajiri study [189]. only one was a male while the ratio of females to males treated was 3:l. The elderly also appeared to be at increased risk. There are several reports which found evidence for involvement of the immune system in propylthiouracil-induced agranulocytosis [ 190. 192- 1961.


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All of these studies found evidence for antibodies to mature neutrophils. In addition, the study by Wall et al. [I901 and a study by Pacini et al. [I971 found evidence for an immune-mediated reaction with a positive lymphocyte transformation test. The study by Fibbe et al. [I951 found inhibition of bone marrow cells by the serum from a patient with propylthiouracilinduced agranulocytosis, and the inhibition was increased in the presence of the drug. This is consistent with the finding of depression of the granulocytic series in bone marrow of patients with propylthiouracil-induced agranulocytosis. The inhibition of bone marrow cells by the patient’s serum was complement dependent, and it affected not only the granulocytic colony-forming unit cells, but also the erythroid colony-forming unit and the erythroid burst-forming unit cells, even though the patient did not have anemia or a change in reticulocyte count. The authors interpreted the data as indicating that the antigen recognized by the antibodies was expressed to a greater degree on mature cells. Although the incidence is much lower, propylthiouracil and methimazole are also associated with aplastic anemia 1198, 1991.

N. Captopril and Penicillamine Captopril and penicillamine are about the only two common drugs that are thiols, and both are associated with bone marrow toxicity [200-2lO]. Both agranulocytosis and pancytopenia have been reported. The typical delay between starting therapy and the onset of neutropenia for captopril was 1 to 2 months, while it was longer for penicillamine, and the decline of the white blood cell count was usually, although not always, more gradual. Both drugs have been found to suppress in v i m granulocytopoiesis [211].

0. Carbamazepine Early studies with carbamazepine suggested that the incidence of agranulocytosis and aplastic anemia was high, and this led to the inclusion of a special warning with a black border in the Physicians Desk Reference [212]. It now appears that the incidence is quite low. approximately 1 in 20,000 [213]. Transient neutropenia is common with an incidence of approximately 10%. and persistent leukopenia has an incidence of approximately 2%. This often makes it difficult for a clinician to know when to stop the drug [2142161. The cases of aplastic anemia occurred after several months of therapy [213, 217, 2181.



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The mechanism of carbamazepine-induced agranulocytosis is unknown. The common mild leukopenia appears to be very dose dependent and is therefore thought to be “toxic” in nature. The more severe cases are often associated with a rash [217, 2181 and are thought to be immune-mediated. Gerson et al. reported a patient that developed aplastic anemia while on phenytoin, and again on the combination of carbamazepine and primidone 12191. This was thought to be due to an inherited defect in the ability to detoxify arene oxides. As described earlier, we have also found that the myeloperoxidase system of neutrophils can form a reactive metabolite.

!I Phenylbutazone Phenylbutazone is associated with a relatively high incidence of agranulocytosis and aplastic anemia, and it is rarely used at the present time [84, 220-2221. In the study by Inman, most of the cases of agranulocytosis or aplastic anemia were in older women, and the duration of therapy before the development of blood dyscrasia varied from less than 1 month to 7 years. In a review by Mauer, the point was made that most cases of agranulocytosis were preceded by a rash [223]. Phenylbutazone is chemically related to aminopyrine, and there may even be some cross-reactivity between the two drugs [109]. It is likely that the mechanism is immune mediated, and there are reports of both leukoagglutinins [ I 0 9 1 and a positive lymphocyte transformation test (2241.

Q. Benzene The toxicity of benzene to the bone marrow was recognized many years ago, and pancytopenia is probably the most common manifestation [225]. It is also associated with leukemia, especially acute myelogenous leukemia. There is overwhelming evidence that metabolism of benzene is necessary for its toxicity, and most of the initial metabolism occurs in the liver [79]. Several metabolites are produced [79], and it is likely that a combination of metabolites is involved in the toxicity [2261. Most of the toxicity of benzene involving bone marrow can be reproduced in animals; therefore, it is not really an idiosyncratic reaction and probably involves direct toxicity of reactive metabolites to the bone marrow. As indicated earlier, neutrophils, and presumably neutrophil precursors in the bone marrow, can form reactive metabolites from the hepatic metabolites of benzene, and this sequence of reactions may represent the early steps in benzene myelotoxicity.

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R. General Conclusions Concerning Drug-Induced Agranulocytosis and Aplastic Anemia In general the mechanism of drug-induced agranulocytosis is unknown. An effort was made in the preceding sections to compare the clinical and laboratory characteristics of agranulocytosis due to different drugs as it might reflect similarities or differences in the pathogenic mechanism. The evidence-that is, the discovery of a serum factor that agglutinates neutmphils in patients with aminopyrine-induced agranulocytosis and a rapid decline in neutrophils when blood from the patient was infused into normal subjects with the same blood type-strongly suggests that aminopyrineinduced agranulocytosis is mediated by an antibody. The rapid onset of agranulocytosis on reexposure of a patient to a small quantity of aminopyrine is considered typical of an immune-mediated reaction. Yet the major implication of this characteristic is that circulating neutrophils must be involved, since if the effects of the drug were confined to the bone marrow, it would take longer for the effect to be manifested in the circulation. However, the effect is not limited to circulating neutrophils since the bone marrows of most patients with aminopyrine-induced agranulocytosis show a depletion of neutrophil precursors. This could be due to the “stress” of peripheral destruction of neutmphils, but this seems unlikely. In contrast, nitrogen mustards cause direct bone marrow toxicity in all patients, and the granulocytopenia is apparent within a few days and lasts for 10 days to 3 weeks [227]. Chlorpromazine-induced agranulocytosis is considered an example of a direct bone marrow toxicity. One piece of evidence to support this is that the onset is delayed relative to aminopyrine, yet direct-acting agents such as nitrogen mustards act relatively rapidly, and in many cases of aminopyrine-induced agranulocytosis the onset is after a month or more of therapy. Where the onset of aminopyrine-induced agranulocytosis occurs after only a day or two, there is likely to have been prior exposure to the drug. The biggest difference is in the time course on reexposure to the drug. With aminopyrine the effect is seen within hours, while chlorpromazine agranulocytosis may not recur for months. To some degree this difference can reflect a difference in whether circulating cells are destroyed since only destruction of circulating cells will have an immediate effect on the white blood cell count. However, even if the effect was restricted to very early cells in the granulocyte line, agranulocytosis should be manifested in the circulation in 1 to 2 weeks. Is it therefore possible to conclude that chlorpromazine-induced agranulocytosis does not involve an immune mechanism? The case report of sulfasalazine-induced agranulocytosis by Evans and Ford described earlier is instructive in that on reexposure there were immediate clinical signs of an immune reaction as well as



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the detection of antibodies; however, agranulocytosis did not develop for over a week [92]. In a cat model of an autoimmune syndrome in which about 50% of mongrel cats develop fever, lymphadenopathy, hemolytic anemia, an elevated serum gamma globulin, and antinuclear antibodies after several weeks of treatment with propylthiouracil [lo], we find that it takes as long or almost as long for the syndrome to develop on reexposure to propylthiouracil as it did the first time (unpublished observation). This certainly must be an immune-mediated reaction, and yet there is still a delay on reexposure to the drug. The clinical picture does suggest that chlorpromazine-induced agranulocytosis does not involve a circulating antineutrophil antibody; however, it could involve another immune-mediated mechanism such as a type IV reaction. Another interesting aspect of the report by Evans and Ford [92] was the marked rise in the white blood cell count shortly before the development of agranulocytosis. This has been noticed with several cases of agranulocytosis due to clozapine and vesnarinone, although I am not aware that this observation has been reported in the literature. This spike in the white blood cell count is easy to miss because it only lasts for about 1 day. The mechanism and significance of this phenomenon are unknown. The clinical characteristics of drug-induced agranulocytosis associated with other drugs appear to fall somewhere between those of aminopyrine and chlorpromazine, with some characteristics suggesting more direct toxicity and others suggesting an immune-mediated reaction. For ethical reasons not many patients have been reexposed to the suspected drug, and so there is little data on the rate at which agranulocytosis recurs on second exposure with most drugs. Evidence strongly suggests that propylthiouracilinduced agranulocytosis involves the immune system, and yet the syndrome is somewhat different than that seen with aminopyrine. Many of the drugs that are associated with agranulocytosis or aplastic anemia appear to have some direct myelotoxicity, and it may be that the death and phagocytosis of a few cells contributes to the induction of an immune-mediated reaction. Unfortunately I know of no good animal model of drug-induced agranulocytosis that could be used for mechanistic studies, and because the mechanism may be different with different drugs and in different people, slich a model would also have limitations. Whether the mechanism of drug-induced agranulocytosis involves toxic or immune-mediated damage to neutrophil progenitors in the bone marrow, it is likely that in most cases the initiating step involves a reactive metabolite of the drug rather than the parent drug. Since most reactive metabolites have a short biological half-life, it is likely that the reactive metabolite responsible is formed in the bone marrow. Aromatic DNA adducts due to environmental exposure to chemicals have been detected in bone marrow,


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and the levels are higher than those found in peripheral cells [228]; therefore, although it is possible that haptenization of mature neutrophils leads to an immune response that also affects bone marrow cells, these data suggest that direct haptenization of bone marrow cells is likely. In addition to mature neutrophils, there are other cells present in the bone marrow that contain myeloperoxidase and that can generate hydrogen peroxide. Although myeloperoxidase is first seen at the promyelocyte stage of neutrophil development [229], it appears that the cells are not functionally capable of a respiratory burst until the metamyelocyte stage [230].

VI. DRUG-INDUCED LUPUS A. General Aspects A lupus-like syndrome is associated with the use of several drugs [2312341. Lirpus is an autoimmune disease in which antibodies are produced against "self" antigens (2351. Almost any organ can be affected, but the most serious disease occurs when the brain or kidneys are involved. The relationship between the production of autoantibodies and organ damage is not completely clear, but renal damage appears to be due to deposition of antigen-antibody complexes where the autoantigen involved is DNA. Several different autoantibodies are produced, but antinuclear antibodies (ANA) are almost always present. The most common antigens to be recognized by ANA are DNA and histone protein. When the cause of lupus is unknown it is referred to as idioparhic lupus, and when it is associated with the administration of a drug it is referred to as drug-induced lupus. It is estimated that about 10% of lupus is drug induced [236]. Drug-induced lupus tends to be less severe than idiopathic lupus, and renal and CNS involvement are uncommon. However, there is a large degree of overlap between the clinical presentation of drug-induced lupus and idiopathic lupus. It has even been proposed that idiopathic lupus is due to exposure to arylamines [237]. Toxic amino acids found in legumes such as alfalfa sprouts have been found to induce a lupus-like syndrome in monkeys [238], and four patients have been described with symptoms of lupus while taking alfalfa tablets (2391. Although antibodies against histone protein also occur in idiopathic lupus, there is evidence that they are more characteristic of drug-induced lupus [240]. The reason for this association is unknown. The exact specificity appears to be different for procainamide- and hydralazine-induced lupus 1241, 2421. Drugs commonly induce the production of ANA without any



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evidence of clinical disease. It is reported that there is an association between antibodies to a specific histone complex H2A-H2B and clinical lupus [243]. There is also evidence that leukocyte-specific ANA are characteristic of symptomatic drug-induced lupus [244]. The clinical characteristics of drug-induced lupus are more consistent than those of drug-induced agranulocytosis or aplastic anemia and have been described previously [231, 2451. In addition. there is little doubt that it involves the immune system; therefore, a clinical description of the syndrome is not provided for individual drugs. In general, drug-induced lupus is probably the slowest in onset of the idiosyncratic drug reactions. It requires at least 2 weeks and usually more than a month of drug exposure before the development of symptoms. It is common for patients to take drugs such as procainamide for more than a year before developing symptoms. Because of the length of time required to develop lupus, it is unlikely to occur with drugs that are not used for chronic therapy. After the drug is discontinued, symptoms usually resolve in about 1 week, but the ANA can persist for years. Symptoms usually occur within days on reexposure to the drug [232].

B. Procainamide Procainamide is associated with the highest incidence of drug-induced lupus of any known drug. The incidence of ANA associated with long-term use of procainamide is close to 90% and that of clinical lupus approaches 30% [246, 2471. The arylamine group appears to be involved in the pathogenesis of the syndrome because subjects who have genetically impaired acetylation of the arylamine require, on average, a longer period of treatment before they develop lupus [247]. In addition, N-acetylprocainamidecan be used as a drug, and it is not associated with lupus even in patients who have had procainamide-induced lupus [248, 2491.

C. Sulfonamides and Sulfasalazine Sulfonamides are another group of drugs that are arylamines and that are associated with drug-induced lupus. In fact, the first clinical reports of drug-induced lupus involved sulfonamides [250, 25 11. Sulfonamides are usually given for a relative short period of time, and therefore sulfasalazine,

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which is used chronically to treat ulcerative colitis, is probably responsible for more cases of lupus than simple sulfonamides (252, 2531. Sulfonamides have also been reported to cause exacerbation of idiopathic lupus [254].

D. Other Arylamines Although there are not many drugs that are primary arylamines, most of these drugs have been associated with the induction of lupus. This includes aminoglutethimide and nomifensine [255, 256). It also includes drugs that are metabolized to primary arylamines such as acebutolol and practolol [257-2601. The one exception is dapsone. Although it is used for chronic therapy, there is only one report of drug-induced lupus associated with dapsone, and it is not very convincing [261].

E. Hydralazine, Isoniazid, and Hydrazine Next to procainamide, hydralazine is associated with the highest incidence of drug-induced lupus with an overall incidence of about 7%; however, the incidence is very dose dependent [9]. The effect of acetylator phenotype is more dramatic for hydralazine-induced lupus than procainamide-induced lupus, and hydralazine-induced lupus is uncommon in patients of the rapid acetylator phenotype [262, 2631. Presumably the reason that acetylator phenotype has a greater impact on hydralazineinduced lupus is that, although the major metabolic pathway of procainamide is acetylation, the major route of elimination is urinary excretion of the parent drug [245]. The effect of acetylation also suggests that the hydrazine group is involved in the mechanism, and other hydrazine derivatives such as isoniazid and hydrazine itself are also associated with the induction of lupus [264-2661. The oxidative metabolite, phthalazinone, is found in higher concentrations in patients with hydralazine-induced lupus, and it has been suggested that an oxidative pathway may be involved in hydralazine-induced lupus [267-2691. This same metabolite is formed by activated neutrophils [70]. Hydralazine is a good nucleophile and could covalently bind without metabolism [270]; however, we have found that hydralazine is metabolized to a reactive metabolite by neutrophils and mononuclear leukocytes, and that binding is significantly higher if the cells are activated. This suggests that an oxidative metabolite is responsible for most of the covalent binding.


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F. Propylthiouracil, Methimazole, Captopril, and Penicillamine Drugs containing a thiono sulfur or sulfhydryl group are associated with a significant incidence of drug-induced lupus [271-2761. In the case reported by Takuwa et al. [272], the patient experienced an idiosyncratic reaction to both propylthiouracil and methimazole, which suggests that there can be cross-sensitivity.

C. Carbamazepine Lupus has been reported in association with the use of carbamazepine [277, 2781. Although the incidence of clinically evident lupus is probably relatively low, the incidence of carbamazepine-induced ANA is reported to be 78% [279].

H. Chlorpromazine Chlorpromazine and other phenothiazines have been associated with the induction of a lupus-like syndrome [280, 2811, but as with carbamazepine, the incidence of ANA and other immunological abnormalities is much higher than the incidence of clinically apparent lupus (282, 2831.

I. General Conclusions Concerning the Mechanism of Drug-Induced Lupus Many hypotheses have been proposed for the mechanism of drug-induced lupus [231, 284, 2851. One hypothesis is that the drug acts as a hapten to alter a nuclear antigen such as histone protein so that it is recognized as foreign. It has been demonstrated that chlorination increases the immunogenicity of chlorhexidine, presumably because it converts it into a reactive species [286]. Such chlorination could occur in vivo due to activated leukocytes. Buxman found that hydralazine and isoniazid react with nuclear protein, including histone protein, in the presence of transglutaminase [287]. We have found that incubation of the hydroxylamine of procainamide with histone protein leads to covalent binding [53]; however, the hydroxylarnine binds to many other proteins, and this does not provide an explanation for the consistent finding of antihistone antibodies. Rabbits immunized with hydralazine-albumin conjugates did develop ANA, but they were against DNA, and there was no evidence of clinical lupus [288]. Other


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attempts to induce an autoimmune syndrome by injecting drug-protein conjugates such as an adduct between procainamide and albumin into animals did not even induce ANA 12891. Gorsulowsky has found antinuclear antibodies in lupus that are specific for neutrophils, and to a lesser extent, other leukocytes [244], and Berkman et al. have found antineutrophil antibodies in propylthiouracil-induced lupus [290]. Monocytes appear to have histone protein on their surface which is recognized by ANA I29 11. Nassberger has found ant imyeloperoxidase antibodies in hydralazine-induced lupus [292]. We have found antibodies in the propylthiouracil-cat model for drug-induced lupus that bind to neutrophils and recognize myeloperoxidase [293]. Since myeloperoxidase is responsible for forming reactive metabolites of propylthiouracil, it is likely to be haptenized by the reactive metabolite. A similar induction of antibodies has been described for tienilic acid-induced hepatotoxicity in which antibodies are induced that recognize the specific cytochrome P-450 that is responsible for forming the reactive metabolite of the drug (2941. Both myeloperoxidase and histone protein are very basic proteins, and antibodies induced by altered myeloperoxidase could possibly cross-react with histone protein. In addition, antimyeloperoxidase antibodies may cause tissue damage because they have been shown to activate neutrophils [295]. However, the relationship between antimyeloperoxidase antibodies and lupus is speculative at present. Another general hypothesis is that the drugs could inhibit some critical function in leukocytes that disturbed control of the immune response. Hydralazine was found to inhibit chemiluminescence of phagocytes and other lymphocyte functions, and it was proposed that this was due to inhibition of transglutaminase [296]. However, most of the inhibition of leukocyte function required very high concentrations of hydralazine, and the inhibition of chemiluminescence is more likely due to conversion of compound I of myeloperoxidase to compound 11. This is a property of many drugs that are metabolized by the myeloperoxidase system and is probably responsible for anti-inflammatory effects of many of these drugs [Sl]. Hydralazine and procainamide were found to inhibit T-cell DNA methylation [297]. Inhibition of DNA methylation can cause the expression of genes and possibly a graft versus host reaction which is similar to lupus. It has been proposed that drug-induced lupus was due to inhibition of suppressor T-cell activity [298], but this was not found by other investigators [299]. We have found that the hydroxylamine of procainamide is toxic to lymphocytes at micromolar concentrations [300], but the significance of this observation is not clear. One observation that any hypothesis should attempt to explain is why drug-induced lupus appears to selectively induce antihistone antibodies. It



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appears that B cells that make autoantibodies are always present and the reason that only small amounts of low-affinity autoantibodies are normally produced is lack of autoreactive T cells (2851. A model which produces an autoimmune disease with a very similar pattern of antinuclear antibodies is the graft versus host reaction [301-3031. The graft versus host reaction that produces this spectrum of antibodies is produced by the injection of lymphocytes from parental strain mice into adult F, hybrid mice (i.e., semiallogeneic). The host T cells are genetically tolerant to parental strain donor cells, but the donor T cells react against the allogeneic host cells. This produces an autoimmune syndrome that is a very good model for lupus. What appears to be the major driving force for the autoimmune syndrome is the stimulation of the donor T cells by host cells with a different class I1 MHC antigen. The activation of helper T cells by the allogeneic cells leads to release of interleukins such as IL-2. This leads to nonspecific stimulation of B cells and bypass of the requirement for carrier-specific T cells [285]. Although this would be expected to lead to nonspecific induction of antibody synthesis, with the total antibody synthesis approximately doubled, the increase in some autoantibodies increases by a factor of 10 to 100 while other autoantibodies are not increased at all [304, 3051. The major increase is in specific antihistone antibodies (predominantly to H1, H2A, and H2B histones) and anti-DNA antibodies. As mentioned earlier, it is believed that B cells that produce antiself antibodies are normally present, and what usually prevents autoantibody production is lack of autoreactive helper T cells. With this type of nonspecific help, it is postulated that synthesis of high-affinity autoantibody may be limited to antigens with a repeating structure which can lead to crosslinking of Ig receptors on B cells [302, 3041. Histone protein and DNA in chromatin have just such repetitive structures. It has been further postulated that other autoimmune syndromes, such as idiopathic lupus, are due to alteration of class I1 MHC antigens by drugs or viruses, or altered recognition of normal self class I1 MHC antigens by abnormal T helper cells [285, 3061. Consistent with this hypothesis is the observation that anti-class I1 antibody inhibits activation of cells in patients with systemic lupus erythematosus [307]. Although B cells and other cells can express class 11 MHC antigens, a major cell type to express this antigen is the macrophage. Monocytes are macrophages, and since they can also form reactive metabolites via the myeloperoxidase system, it is likely that drugs that are metabolized to reactive intermediates by monocytes would bind to class I1 MHC antigens. We have demonstrated that drugs such as procainamide covalently bind to activated monocytes. Because the hydrogen peroxide is generated and myeloperoxidase is released into phagosomes and on the outside of the cell close to cell membrane, it is likely that much of

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the binding is to membrane proteins such as class I1 MHC. We postulate that these drug-altered class I1 MHC antigens could be responsible for drug-induced lupus, as shown in Fig. 10.

VII. GENERALIZED IDIOSYNCRATIC DRUG REACTIONS A. General Aspects Generalized idiosyncratic reactions are usually characterized by skin rash, fever, and lymphadenopathy. In addition, specific organs are often involved including the liver, bone marrow, kidneys, lungs, pancreas, etc. Drugs that are associated with agranulocytosis and drug-induced lupus are often associated with generalized idiosyncratic reactions, and certain functional groups, such as arylamines or thiols, are often present on the molecule. Since monocytes are involved in processing and presenting immunogens to helper T cells in the early steps of an immunological reaction, the formation of reactive metabolites by activated monocytes is likely to be a strong stimulus to the immune system and could also be involved in the initiating steps of generalized idiosyncratic drug reactions [7].

B. Arylamines Sulfonamides and sulfasalazine are associated with a high incidence of a variety of idiosyncratic adverse drug reactions including a generalized idiosyncratic reaction resembling serum sickness and cutaneous reactions such as urticaria, erythema multiforma, and toxic epidermal necrolysis. Patients of the slow acetylator phenotype appear to be at increased risk [308]. We have found that in v i m cytotoxicity of the hydroxylamine of sulfamethoxazole in the Spielberg assay correlated with toxicity in patients who have had a major reaction to sulfonamides [309, 3101. This suggests that the hydroxylamine or related metabolite is responsible for these idiosyncratic reactions. Cells from patients with a genetic defect in glutathione synthesis also have a higher sensitivity to the hydroxylamine metabolite [311], and the addition of glutathione to the incubation decreased the toxicity [309]. Patients with AIDS have an extraordinary incidence of generalized reactions to sulfonamides of about 50% [312-3151. This may be related to the low levels of glutathione and cysteine in AIDS patients [316, 3171, or it could be due to modification of the immune system by AIDS.


345

Aclivslion by Infection or Inflarnrnetion


MPO

HZOZ

-

Helper T Cell

Arornslic Arnine

ANA .Antinuclear Anlibdies

1 MHC II Class II Major Hislocompe1ebilily Antigen

YPO Myeloperoxidaae

'

Alteration of MHC II Lasds to nonspecific aclivalion of helper T cells and raleasa of lyrnphokines such me IL-2.

Cell specific lor hislone prolein

specific for Chrornslin containing repelalive hislona epilopes leading lo cross linking of Ig

Plasma

No Autoantibodies

i Antihistone

ANA

FIG. 10. Proposed mechanism of drug-induced lupus involving alteration of class I1 MMC by monocyte-generated reactive metabolites.

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346

Dapsone is also associated with a mononucleosis-like syndrome sometimes referred to as the dapsone syndrome [318-3201. Another arylamine, aminoglutethimide, is associated with a variety of idiosyncratic reactions 11441. Procainamide is associated with drug-induced fever, as well as lupus and agranulocytosis, but generalized reactions to procainamide are uncommon [321]. Another characteristic of procainamide is that its reactive metabolites do not circulate in significant concentration. A report by Wheeler et al. indicates that about 0.4%of procainamide is converted to nitro derivative of procainamide in rats [3221. However, it is likely that much of the reactive hydroxylamine and nitroso intermediates have an in vivo fate other than oxidation to the nitro derivative, and this is presumably an underestimation of the amount of reactive intermediate formed. Although it is likely that a significant amount of procainamide is converted to reactive intermediates, evidence suggests that very little of these intermediates is present in the circulation. Not only have we and others failed to find them in the circulation, but procainamide is not associated with methemoglobinemia [322]. In contrast, the hydroxylamines of dapsone and sulfonamides appear to exist at significant concentrations in the circulation, and the hydroxylamine of dapsone is found in urine [323]. Dapsone and sulfonamides are associated with a high incidence of methemoglobinemia (3241. There is good evidence that this methemoglobinemia is due to the hydroxylamine metabolites [325]. These data suggest that, in contrast to agranulocytosis and lupus, an arylamine will only cause a generalized idiosyncratic drug reaction if the reactive metabolites can reach the circulation in significant concentrations.

C. Thiono Sulfur and Thiol Drugs After agranulocytosis, the most common serious adverse reaction to propylthiouracil is hepatitis, and evidence suggests that it has an immunological basis [326]. Several target organs can be affected simultaneously, as in one report in which the patient had granulocytopenia, eosinophilia. skin reaction, and hepatitis [ 1971. Propylthiouracil and methimazole are also associated with vasculitis (327, 3281 and arthritis [329-3311. Thiol drugs such as captopril and penicillamine are associated with a broad range of idiosyncratic reactions (208, 332, 3331. The skin is the target organ for many of these reactions [334]. Other interesting autoimmune syndromes have also been reported such as myasthenia gravis [335].


347


D. Anticonvulsants All of the anticonvulsants are associated with a significant risk of severe idiosyncratic drug reactions. The reaction associated with phenytoin, carbamazepine, and phenobarbital usually includes fever and skin rash, and often includes lymphadenopathy, abnormal liver tests, and leukopenia. Spielberg has developed an assay that appears to detect a genetic defect in the ability to detoxify reactive metabolites of these drugs 1336, 3371. In this assay, reactive metabolites are generated by murine hepatic microsomes, and these metabolites are more toxic to lymphocytes from patients who have had a significant idiosyncratic reaction to the drug. Testing of relatives indicates that the abnormality is inherited [338]. Since the reactive metabolite is generated by the added microsomes, it suggests that the defect is in the ability of the cells to detoxify the reactive metabolite. The toxicity of phenytoin observed in this assay was increased by trichloropropylene oxide, which inhibits epoxide hydrolase, and that suggested that the toxicity was mediated by an arene oxide [339]. In addition, epoxide hydrolase abolished the toxicity (3371. Although an arene oxide of phenytoin has never been directly observed, the presence of an NIH shift has been used as evidence that it is formed as an intermediate in the dog [340]. However, there are other potential mechanisms that could lead to an NIH shift without an arene oxide intermediate [341]. Riley et al. have found similar results with phenytoin, carbamazepine, and structural analogs of these drugs which suggest the involvement of an arene oxide [342]. Although it seems unlikely that an arene oxide would have a sufficient half-life in blood to be formed in the liver and reach other target organs, at least one arene oxide, the arene oxide of bromobenzene, appears to have a surprisingly long half-life of about 13 sec [343]. Although the defect present in the cells appears to be a genetic defect in epoxide hydrolase, direct attempts to find such a defect have been unsuccessful. We have found phenytoin and carbamazepine to be metabolized by activated neutrophils to reactive metabolites, but those reactive intermediates are very unlikely to be arene oxides. The covalent binding of phenytoin reactive metabolites generated by both hepatic microsomes and neutrophils is rather low compared to that of other drugs [75, 3441. Other anticonvulsants, such as ethosuximide and trimethadione, which have a similar heterocyclic ring but without an aromatic ring, cause a similar spectrum of adverse reactions and cannot form an arene oxide [345]. However, these adverse reactions could involve a different mechanism. These conflicting observations leave the mechanism of phenytoin-induced idiosyncratic reactions unclear.

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E. Nonsteroidal Anti-inflammatory Drugs As a class, nonsteroidal anti-inflammatory drugs are associated with a high incidence of idiosyncratic drug reactions, and many of the drugs which have recently been removed from the market for reasons of toxicity belong to this class [2]. Some have functional groups, such as the secondary arylarnine of diclofenac, which could be oxidized to a reactive intermediate [59]. In addition, most nonsteroidal anti-inflammatory drugs are carboxylic acids and are metabolized to varying degrees to acyl glucuronides. Acyl glucuronides are chemically reactive and have been demonstrated to covalently bind to protein [ 346-35 1]. This covalent binding could lead to an immunogen and an immune response. Acyl glucuronides are relatively stable and circulate freely; therefore, adverse reactions are unlikely to be limited to the site of glucuronide formation and could easily be responsible for generalized idiosyncratic reactions.

F. Gold Compounds Gold compounds are used in the treatment of rheumatoid arthritis and are associated with a high incidence of idiosyncratic reactions [ 1501. Schuhmann et al. have found in an animal model that, although it is the lower oxidation state of gold [Au(I)] that is used as a therapeutic agent as well as in this model, it appears that oxidation to Au(II1) is required for toxicity, and it is this oxidation state that is recognized by the immune system (3521. The Au(ll1) oxidation state of gold is chemically reactive and it is likely that hypochlorous acid could oxidize Au(I) to Au(II1). It is also known that macrophages concentrate gold salts in phago-lysosomes. Therefore, monocytes could even play a role in the activation of, and immune response to, some inorganic compounds.

G. Conclusions Concerning the Mechanisms of Generalized Idiosyncratic Drug Reactions The characteristics of many generalized idiosyncratic reactions also suggest involvement of the immune system. The clinical assay of Spielberg for the anticonvulsants and sulfonarnides actually measures direct cytotoxicity. If the reactions associated with anticonvulsants are, in fact, immune mediated, it is curious that this assay should be so accurate at predicting those patients at risk. It suggests that genetic differences in detoxification rather than differences in the immune system are the major determinant of the risk of an idiosyncratic reaction. It may be that the reactions do not involve an



349

immune mechanism but rather some unknown cellular target. If this is the case, it is surprising that the reactions cannot be easily duplicated in animals at high doses of the drug, especially if the activation pathway is induced and detoxication pathways are inhibited. As suggested for agranulocytosis, the mechanism may involve a combination of mild cytotoxicity, which leads to cell necrosis, and the release of antigens required to induce an immune response.

VIII. SUMMARY Evidence strongly suggests that many adverse drug reactions, including idiosyncratic drug reactions, involve reactive metabolites. Furthermore, certain functional groups, which are readily oxidized to reactive metabolites, are associated with a high incidence of adverse reactions. Most drugs can probably form reactive metabolites, but a simple comparison of covalent binding in vitro is unlikely to provide an accurate indication of the relative risk of a drug causing an idiosyncratic reaction because it does not provide an indication of how efficiently the metabolite is detoxified in vivo. In addition, the incidence and nature of adverse reactions associated with a given drug is probably determined in large measure by the location of reactive metabolite formation, as well as the chemical reactivity of the reactive metabolite. Such factors will determine which macromolecules the metabolites will bind to, and it is known that covalent binding to some proteins, such as those in the leukocyte membrane, is much more likely to lead to an immune-mediated reaction [22, 3531 or other type of toxicity [24]. Some reactive metabolites, such as acyl glucuronides, circulate freely and could lead to adverse reactions in almost any organ; however, most reactive metabolites have a short biological half-life. and although small amounts may escape the organ where they are formed, these metabolites are unlikely to reach sufficient concentrations to cause toxicity in other organs. Many idiosyncratic drug reactions involve leukocytes, especially agranulocytosis and drug-induced lupus. We and others have demonstrated that drugs can be metabolized by activated neutrophils and monocytes to reactive metabolites. The major reaction appears to be reaction with leukocytegenerated hypochlorous acid. Hypochlorous acid is quite reactive, and therefore it is likely that many other drugs will be found that are metabolized by activated leukocytes. Some neutrophil precursors contain myeloperoxidase and the NADPH oxidase system, and it is likely that these cells can also oxidize drugs. Therefore, although there is no direct evidence, it is reasonable to speculate that reactive metabolites generated by activated leukocytes, or neutrophil precursors in the bone marrow, could be responsible


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for drug-induced agranulocytosis and aplastic anemia. This could involve direct toxicity or an immune-mediated reaction. These mechanisms are not mutually exclusive, and it may be that both mechanisms contribute to the toxicity, even in the same patient. In the case of drug-induced lupus, a prevalent hypothesis for lupus involves modification of class I1 MHC antigens. Since monocytes are a major cell that expresses class I1 MHC, and since they can also metabolize drugs to reactive metabolites, it is reasonable to speculate that the mechanism of drug-induced lupus involves modification of class I1 MHC antigens byreactive metabolites of the drug produced in close proximity to the surface of the monocyte. Although other types of idiosyncratic reactions could involve reactive metabolite formation by activated leukocytes, the circumstantial evidence to support their involvement is weaker. The mechanism could involve a combination of circulating reactive metabolite, or reactive metabolite formed at a peripheral site, and immune stimulation by reactive metabolite formed by activated monocytes. By definition, most patients given a drug will not have an idiosyncratic reaction. An understanding of the risk factors involved in such reactions is of major importance, both mechanistically and for the safe use of drugs. If an idiosyncratic drug reaction is immune mediated, it would provide an explanation for the idiosyncratic nature of the reaction since it is well known that genetic differences in the immune system can lead to very different responses to a given immunogen. The mode of exposure to a drug is also known to have a significant effect on the incidence and nature of an immune response. Repeated intermittent exposure also may lead to sensitization, and this appears to be a risk factor for halothane-induced hepatitis. Other genetic differences can alter the incidence of an idiosyncratic reaction. It is known that the risk of hydralazine-induced lupus and severe idiosyncratic sulfonamide reactions are lower in rapid acetylators. The studies by Spielberg et al. [336-3391 suggest that genetic differences in the ability to detoxify reactive metabolites also play a major role in the risk of an idiosyncratic reaction. In addition to genetic factors, other environmental factors also appear to be important. The risk of an idiosyncratic reaction to ampicillin is greatly increased in patients who also have mononucleosis [354],and the risk of aspirin-induced hepatotoxicity is increased in patients with lupus and juvenile rheumatoid arthritis [355. 3561. The risk of sulfonamide reactions is greatly increased in patients with AIDS. This could either be due to alterations in the immune system that occur with this disease, or it could be due to decreased levels of glutathione and other sulfhydryl groups found in the leukocytes and serum of these patients. A related observation is the apparent increase in the risk of agranulocytosis in subjects who received influ-



35 1

enza vaccine either just before or during therapy with vesnarinone. Such infections or other stress could change the ability of a drug to lead to an immune response by several mechanisms, including the expression of specific antigens such as class I1 MHC, heat shock proteins, or endoplasmin on cell membranes as well as levels of such agents as glucocorticoids and interferons [357]. In addition, if the adverse reaction is due to reactive metabolites generated by neutrophils, neutrophil precursors, or monocytes, an infection or inflammatory reaction would activate these cells and lead to formation of reactive metabolites.

Acknowledgments This research was supported by grants from the Medical Research Council of Canada (MA 9336 and MA 10036) and a grant from the Sunnybrook Trust for Medical Research.

REFERENCES [ l ] R. A. Goldstein and R. Patterson, J. Allergy Clin. Immunol., 7 4 , 643 ( 1984). [2] 0 . M. Bakke, W. M. Wardell, and L. Lasagna, Clin. Pharmacol. Ther., 35, 559 (1984). [3] D. M. Davies, Textbook of Adverse Drug Reactions, Oxford University Press, Oxford, 1985. (41 C. W. Parker, Pharmacol. Rev., 34, 85 (1982). [5] L. R. Pohl, H.Satoh, D. D. Christ, and J. G. Kenna. Ann. Rev. Pharmacol., 28, 367 (1988). [6] B. K. Park and J. W. Coleman, Br. J. Clin. Pharmacol., 26, 491 (1988). [7] J. I? Uetrecht, CRC Crir. Rev. Toxicol., 20, 213 (1990). [8] Y. €? Konttinen and L. lbominen. Lancet, 2 , 925 (1971). [9] H. A. Cameron and L. E. Ramsay, Br. Med. J., 289, 410 (1984). [lo] D. €? Aucoin, M. E. Peterson, A. I. Hurvitz, D. E. Drayer, R. G. Lahita, F. w . Quimby, and M. M. Reidenberg, J. Pharmacol. Exp. Ther., 234, 13 (1985). [ l l ] U. Giger, L. L. Werner, N. J. Millichamp, and N. T. Gorman, J. Am. Vet. Med. Assoc., 186, 479 (1985). [I21 €? A. Greenberger and R. Patterson, Prog. Cardiovas. Dis., 31, 239 ( 1988).


352

UETRECHT

(131 E. R. Unanue and I? M. Allen, Science, 236, 551 (1987). [14] B. M. Chain, I? M. Kaye, and M. Shaw, Immunol. Rev., 106, 33 ( 1988). [I51 J. H . K. Yeung, J. W. Coleman, and B. K. Park, Biochem. Pharmucol., 34, 4005 (1985). [I61 M. E. Kammiiller, N. Bloksma, and W. Seinen, Autoimmuniry and Toxicology, Elsevier, Amsterdam, 1989. 1171 R. R. A. Coombs and I? G. H. Gell, in Clinical Aspects of Immunology (I? G . H. Gell, and R. R. A. Coombs, eds.), Blackwell Scientific Publications. Oxford, 1963, p. 317. [I81 N. R. Kitteringham, G. Christie, J. W.Coleman, J. H.K. Yeung, and B. K. Park, Biochem. Pharmacol., 36. 601 (1987). [I91 A. L. de Weck, Curr. Opinion lmmunol., 2 , 548 (1990). 1201 F. I? Guengerich and D. C. Liebler, CRC Crit. Rev. Toxicol., 14, 259 (1985). [2I] M. W. Anders, Bioactivation of Foreign Compounds, Academic Press, Orlando, 1985. [22] B. K. Park and N. R. Kitteringham, Drug Metab. Rev., 22, 87 (1990).

[23] S . D. Nelson, J. Med. Chem., 25, 753 (1982). 124) S. D. Nelson and P.G. Pemon, Annu. Rev. Pharmacol. Toxicol., 30, 169 (1990). [25] D. Vergani, G. Mieli-Vergani, A. Alberti, J. Neuberger, A. Eddleston, M. Davis, and R. Williams, N . Engl. J. Med.. 303, 66 (1980). [26] J. G. Kenna, H. Satoh, D. D. Christ, and L. R. Pohl, J. Pharmacol. Exp. Ther., 245, 1 I03 (1988). [27] C. E. Lewis, J. Occup. Med., 6 , 91 (1964). [28] J. R. Gillette, Biochem. Pharmucol., 23, 2785 (1974). [29] J. R. Gillette, Biochem. Pharmacol., 23, 2927 (1974). [30] B. Coles, I. Wilson, F? Wardman, J. A. Hinson, S. D. Nelson, and B. Ketterer, Arch. Biochem. Biophys., 264, 253 (1988). [31] N. R. Pumford, J. A. Hinson, D. W. Potter, K. L. Rowland, R. W. Benson, and D. W. Roberts, J. Pharmacol. Exp. Ther., 248. 190 (1989). (321 D. W. Roberts, T. J. Bucci, R. W. Benson, A. R. Warbritton, T. A. McRae, N. R. Plumford, and J. A. Hinson, Am. J. Pathol., 138, 359 (1991). [33] G. Duhamel, A. Najman, N. C. Gorin, and J. Stachowiak, Ann. Med. Interne, 128, 303 (1977). 134) R. M. Carson, Environ. Health Prospect., 87, 227 (1990). (351 G. Kellermann, C. R. Shaw, and M. Luyten-Kellerman, N . Engl. J. Med., 289, 934 (1973).



353

1361 €? Okano, H. N. Miller, R. C. Robinson, and H. V. Gelboin, Cancer Res., 39, 3184 (1979). [37] C. K. Janniger and S. €? Racis, J. Med., 18, 69 (1987). [38] M. F. Hughes, R. F? Mason, and T. E. Eling, Mol. Pharmacol., 34, 186 (1988). [39] €? Moldeus, B. Anderson, A. Rahimtula, and M. Berggren, Biochem. Pharmacol., 31, 1363 (1982). [40] F. F. Kadlubar, C . B. Frederick, C. C. Weis, and T. V. Zenser, Biochem. Biophys. Res. Commun., 108, 253 (1982). 1411 M. A. Tayeh and M. A. Marletta, J. Biol. Chem., 264, 19654 (1989). [42] S. Moncada, R. M. J. Palmer, and E. A. Higgs. Pharmacol. Rev., 43, 109 (1991). [43] J. S . Beckman, T. W. Beckman, J. Chen, I? A. Marshall, and B. A. Freeman, Proc. Narl. Acad. Sci. USA, 87, 1620 (1990). [44] R. Radi, J. S. Beckman, K. M. Bush, and B. A. Freeman, J. Biol. Chem., 266, 4244 (1991). [45] S . J. Klebanoff and R. A. Clark, The Neutrophil: Function and Clinical Disorders, ElseviedNorth-Holland, Amsterdam, 1978. [46] S . J. Weiss, N. Engl. J. Med., 320, 365 (1989). [47] J. Harrison, T. Araiso, M. Palcic, and H. Dunford, Biochem. Biophys. Res. Commun., 94, 34 (1980). [48] R. R. Sandborg and J. E. Smolen, Lab. Invest., 59, 300 (1988). [49] S. Pou, M. S. Cohen, B. E. Britigan, and G. M. Rosen, J. Biol. Chem., 264, 12299 (1989). [50] J. Uetrecht, Trends Pharmacol. Sci., 10, 463 (1989). [51] A. J. Kettle and C. C. Winterbourn, Biochem Pharmacol., 41, 1485 (1991). [52] J. Uetrecht, N. W i d , and R. Rubin, Chem. Res. Toxicol., I , 74 (1988). [53] J. €? Uetrecht, J. Pharmacol. Exp. Ther., 232, 420 (1985). [54] J. F? Uetrecht and N. M i d , Chem. Res. Toxicof., 4 , 218 (1991). [ 5 5 ] J. Uetrecht and B. Sokoluk, Drug Merab. Dispos.. 20(1), (1992). [56] J. Uetrecht, N. Zahid, N. H. Shear, and W. D. Biggar, J. Pharmacol. Exp. Ther., 245, 274 (1988). 157) J. I? Uetrecht, N. Shear, and W. Biggar, Pharmacologisr. 28, 239 ( 1986). [58] A. E. Cribb, M. Miller, A. Tesoro, and S. F? Spielberg, Mol. Pharmacol., 38, 744 (1990). (591 K. W. M. Zuurbier, A. R. J. Bakkenist, R. H. Fokkens, N. M. M. Nibbering, R. Wever, and A. 0. Muijsers, Biochem. Pharmacol., 40, 1801 (1990). 1601 T. E. Eling, R. F? Mason, and K. Sivarajah, J. Biol. Chem., 260, 1601 (1985).

UETRECHT


354

[611 H. Sayo and M. Saito, Xenobiorica, 20, 957 (1990). 1621 B. Kalyanaraman and I? G. Sohnle, J. Clin. Invesr., 75, 1618 (1985). I631 J. M. van Zyl, K. Basson, A. Kriegler, and B. J. van der Walt, Biochem. Pharmacol., 40, 947 (1990). 1641 J. L. Maggs, N. R. Kitteringham, A. M. Breckenridge, and B. K. Park, Biochem. Pharmacol., 36, 2061 (1987). I651 J. L. Maggs, M. D. Tingle, N. R. Kitteringham, and B. K. Park, Biochem. Pharmacol., 37, 303 (1988). [66] J. B. Clarke, J. L. Maggs, N. R. Kitteringham, and B. K. Park, Inr. Arch. Allergy Appl. Immunol., 91, 335 (1990). [67] F! J. O’Brien, S. Khan, and S. D. Jatoe. in Biological Reactive Inrermediates IV: Molecular and Cellular Effects and Their Impact on Human Healrh (C. M. Witmer, R. R. Snyder, D. J. Jollow, G. F. Kalf, J. J. Kocsis, and I. G. Sipes, eds.), Plenum, N ~ York, N 1990, p. 51. [68] J. I? Uetrecht, N . Zahid, and S. F! Spielberg, Eur. J. Clin. Pharmacol., 36 (suppl), A53 (1989). I691 V. Fischer, J. A. Haar, L. Greiner, R. V. Loyd, and R. Mason, Mol. Pharmacol, 40, 846 (1991). (701 A. H. Hofstra, L. C. Matassa, and J. I? Uetrecht, J. Rheumarol. (in

press). [71] A. H. Hofstra, S. M. A. Li-Muller, and J. F? Uetrecht, Drug Merab.

Dispos., 20(2), in press. [72] E. Lee, Y. Miki, M. Hosokawa, H. Sayo, and K. Kariya, Xenobiotica, 18, 1 I35 (1988). (731 L. Waldhauser and J. Uetrecht, Drug Metab. Dispos., 19, 354 (1991). [74] B. E. Svensson, Biochem. J., 253, 441 (1988). [75] J. Uetrecht and N. Zahid, Chem. Res. Toxicol., I , 148 (1988). [76] C. A. Lipinski. J. Bordner, G. C. Butterfield, and L. C. Marinovic, J. Hererocyclic Chem., 27, 1793 (1990). [77] S . M. Furst and J. Uetrecht, presented at the Third North American ISSX Meeting, 1990. [78] S . Ichihara, H. Tomisawa, H. Fukazawa, M. Tateishi, R. Joly, and R. Heintz, Biochem. Pharmacol., 35, 3935 (1986). [79] G. F. Kalf, CRC Crir. Rev. Toxicol., 18. 141 (1987). [SO] D. A. Eastmond, M. T. Smith, L. 0. Ruzo, and D. Ross, Mol. Pharmacol., 30, 674 (1986). [Sl] D. A. Eastmond, R. C. French, D. Ross, and M. T. Smith, Chem.Biol. Interacrions, 63, 47 (1987). (821 A. V. Pisciotta, Semin. Hematol., 10, 279 (1973). (831 V. Pisciotta, Drugs, 15, 132 (1978). [84] G. C. de Gruchy, Drug-Induced Blood Disorders, Blackwell Scientific Publications, Oxford, 1975.



355

[ 8 5 ] H. Heimpel, Med. Toxicol., 3 , 449 (1988). [86] I? C. Vincent, Drugs, 31, 52 (1986). (871 F. H. J. Claas, Psychopharmacology, 99, S113 (1989). [88] W. Heit, H. Heimpel, A. Fischer, and N. Frickhofen, Scand. J. Haemarol., 35, 459 (1985). [89] D. M. Williams, R. E. Lynch, and G. E. Cartwright, Semin. Hemarol., 10, 195 (1973). [90] 1. K. Rothman and E. L. Amorosi, Arch. Intern. Med., 139, 246 (1979). 1911 D. J. Christensen, L. F. Palma, and K. Phelps, Ann. Intern. Med., 100, 918 (1984). [92] R. S . Evans and W. I? Ford, Arch. Inrern. Med., 101, 244 (1958). [93] A. Salama, B. Schiitz, V. Kiefel, H. Breithaupt, and C. MuellerEckhardt, Br. J. Haemarol., 72, 127 (1989). [94] F. H. J. Claas, J. Langerak, and J. J. van Rood, Immunol. Lerr., 2, 323 (I98 I ) . [95] F. H. J. Claas and J. J. van Rood, frog. Allergy, 36. 135 (1985). [96] J. G. Kelton, A. T. Huang, N. Mold, G. Logue, and W. F. Rosse, N . Engl. J. Med., 301, 621 (1979). [97] J. Azocar, Lancer, I , 1069 ( 1984). [98] J. Goudemand, J. Plouvier, F. Bauters, and M. Goudemand, Sem. Hhp. Paris. 52, 1513 (1976). [99]F. W. Madison and T. L. Squier, J. Am. Med. Assoc., 102,755 (1934). [loo] W. Dameshek and A. Colmes, J. Clin. Invest., 15, 85 (1936). [IOI] W. Hartl, Semin. Hemarol., 2, 313 (1965). [lo21 T. Fitz-Hugh, Ann. Intern. Med.. 8, 148 (1934). I1031 I? Plum, Lancet, I, 14 (1935). (1041 A. J. Barrett, E. Weller, N. Rozengurt, I? Longhurst, and J. G. Humble, Br. Med. J . , 2, 850 (1976). [I051 A. M. Hoffman, E. M. Butt, and N. G. Hickey, J. Am. Med. Assoc., 102, 1213 (1934). [I061 T. L. Squier and F. W. Madison, J. Allergy, 6 , 9 (1934). [I071 I. I? Palva and 0. 0. Mustala, Acra Med. Scand., 187, 109 (1970). [lo81 S. Moeschlin and K. Wagner, Acra Haemat., 8, 29 (1952). [ I 0 9 1 C. C. Magis, A. Barge, and 1. Dausset, Clin. Exp. Immunol., 3 , 989 (1968). [I101 A. G. Ellrodt, G. H. Murata, M. S. Reidinger, M. E. Stewart, C. Mochizuki, and R. Gray, Ann. Inrern. Med., 100, 197 (1984). [ I l l ] D. G. Meyers, E. R. Gonzalez, L. L. Peters, R. B. Davis, J. R. Feagler, J. D. Egan, and C . K. Nair, Am. Heart. J.. 109, 1393 (1985). [I121 J. F. Thompson, C. A. Robinson, and J. L. Segal. C w r . Ther. Res., 4 4 , 872 (1988).


356

UETRECHT

[I131 J. Riker, J. Baker, and M. Swanson. Arch. Intern. Med., 138, 1731 (1978). [I141 R. J. J. van Beek, R. Bieger, and G. J. den Ottolander, Scand. J. Huemutol., 2 1 , 150 (1978). (1151 K. S. Gill, 0. A. Hayne, and E. Zayed, Am. J. Hemurol., 3f, 298 ( 1989). I1161 R. Rosenstein, R. E. Kosfeld, L. Light, and Y.K. Liu, Am. J. Hemurol., 16, 181 (1984). [I171 A. Z. Bluming, D. Plotkin, I? Rosen, and R. Thiessen, J. Am. Med. Assoc., 236, 2520 (1976). [I181 A. F. Shields and J. A. Berenson, Am. J. Hematol.. 27, 299 (1988). [119] R. B. Schifman, H. Garewal, and D. Shillington, Am. J. Clin. Pathol., 80, 66 (1983). [I201 G. W. Jones, T. L. George, and R. D. Bradley, Transfusion, 18, 224 (1978). [121] S . Kleinman, R. Nelson, L. Smith, and D. Goldfinger, N. Engl. J. Med., 311, 809 (1984). (1221 A. J. Ognibene, Ann. Intern. Med., 72, 521 (1970). [I231 I? Hornsten, M. Keisu, and B. Wiholm. Arch. Dermurol., 126, 919 (1990). (1241 €? H. Levine and L. R. Weintraub, Ann. Intern. Med., 68, 1060 (1968). [125] J. Foucauld, W. Uphouse, and J. Berenberg, Ann. Intern. Med., 102, 139 (1985). [126] R. M. Weetman, L. A. Boxer, M. I? Brown, N. M. Mantich, and R. L. Baehner, Br. J. Huemurol., 45, 361 (1980). [127] S. S. Rinkoff and M. Spring, Ann. Intern. Med., 15, 89 (1941). (1281 I? Arneborn and J. Palmblad, Acru Med. Scand., 204, 283 (1978). [129] I? Arneborn and J. Palmblad, Acru Med. Scund., 206, 241 (1979). [130] L. E. Bottiger and B. Bottiger, Acru. Med. Scund., 210, 475 (1981). [131] J. Sheehan, Lancer, 2 , 692 (1981). [132] M. Keisu, B.-E. Wiholm, and J. Palmblad, J. Intern. Med., 228, 353 (1990). [133] B. I. Asmar, S. Maqbool, and A. S. Dajani. Am. J. Dis. Child., 135, 1100 (1981). [134] L. H. Maurer, I? Andrews, F. Rueckert, and 0. R. McIntyre, Plusric Reconstr. Surg., 46, 458 (1970). [135] S. A. Weitzman and T. I? Stossel, Lancet, I , 1068 (1978). [136] 1. M. Jacobson, I? B. Kelsey, G. T. Blyden, Z. N. Demirjian, and K. J. Isselbacher, Am. J. Gustroenterol., 80, 118 (1985). [137] M. I? Mitrane, A. Singh, and J. R. Seibold, J. Rheumurol., 13, 969 (1986).



357

[138] R. M. M. Victorino, V. A. J. Maria, and J. de Deus, Acra Haemarol., 84, 111 (1990). [139] R. L. Harvey and M. J. Luzar, Ann. Intern. Med., 108, 214 (1988). [140) A. Manoharan, Med. J. Ausr., 149, 508 (1988). [141] S. M. Rab and M. N. Alam, Br. J. Dis. Chest, 64, 164 (1970). [I421 B. Lawrence, R. J. Santen, A. Lipton, H. A. Harvey, R. Hamilton, and T. Mercurio, Catrcer Trear. Rep., 62, 1581 (1978). [143] E. Gez and A. Sulkes, Oncology, 41, 399 (1984). [144] R. C. Stuart-Harris and I. E. Smith, Cancer Treat. Rep., 11, 189 (1984). [145] I? Danilo, Am. Heart J., 97, 119 (1979). (1461 L. H. Opie, Lancet, 2 , 689 (1980). [147] I. Stoel and F. Hagemeijer, Eur. Heart J., 1 , 147 (1980). [148] S. Eustace, T. O'Neill, S. McHale, and J. Molony, Irish J. Med. Sci., 158, 217 (1989). [149] International Agranulocytosis and Aplastic Anemia Study. J. Am. Med. Assoc., 256, 1749 (1986). [150] M. N. G. Dukes, J. K. Aronson, B. Blackwell, S. Dittmann, I? I. Folb, R. Hoigne, E. F. Hvidberg, C. B. M. Tester-Dalderup. G. Tognoni, K. von Eickstedt, and B. Westerholm, Meyler's Side Effecrs of Drugs, Elsevier, Amsterdam, 1988. [151] R. 0. Wallerstein, I? K. Condit, C. K. Kasper, J. W. Brown, and E R. Morrison, J. Am. Med. Assoc., 208, 2045 (1969). [152] B. C. P. Polak, H. Wesseling, D. Schut, A. Herxheimer, and L. Meyler, Acra Med. Scand.. 192, 409 (1972). [153] R. L. Rosenthal and A. Blackman, J. Am. Med. Assoc., 191, 136 (1965). [154] G. Carpenter, Lancer. 2 , 326 (1975). [I551 S. M. Abrams, T.J. Degnan, and V. Vinceguerra, Arch. Intern. Med., 140, 576 (1980). [156] A. A. Yunis, Ann. Rev. Pharmacol. Toxicol., 28, 83 (1988). [157] A. A. Yunis, A. M. Miller, Z. Salem, M. D. Corbett, and G. K. Arimura, J. Lab. Clin. Med., 96, 36 (1980). [158] B. J. Gross, R. V. Branchflower, T. R. Burke, D. E. Lees, and L. R. Pohl. Toxicol. Appl. Pharmacol., 6 4 , 557 (1982). [159] R. R. Scheline, Pharmacol. Rev., 25, 451 (1973). [160] M. Ascherl, I? Eyer, and H. Kampffmeyer, Biochem. Pharmacol., 34, 3755 (1985). [161] M. Isildar, W. H. Abou-Khalil, J. J. Jimenez, A. Abou-Khalil, and A. A. Yunis, Toxicol. Appl. Pharmacol., 94, 305 (1988). [162] M. Isildar, J. J. Jimenez, G. K. Arimura, and A. A. Yunis, Am. J. Hemarol., 28, 40 (1988).


358

UETRECHT

(1631 J. J. Jimenez, G. K. Arimura, W. H. Abou-Khalil, M. Isildar, and A. A. Yunis, Blood, 70, 1180 (1987). [I641 A. V. Pisciotta, S. Ebbe. E. J. Lennon, G. 0. Metzger, and F. W. Madison, Am. J. Med., 25, 210 (1958). (1651 A. V. Pisciotta, J. Am. Med. Assoc., 208, 1862 (1969). I1661 A. V. Pisciotta, J. Lab. Clin. Med., 78, 435 (1971). (1671 G . C. Hoffman, J. S. Hewlett, and F. L. Garzon, J. Clin. Pathol., 16, 232 (1963). [I681 D. E. Lind, J. A. Levi, and F? C. Vincent, Br. Med. J., I , 458 (1973). (1691 E. G. H. Rhodes, J. Ball, and 1. M. Franklin, Br. Med. J., 292, 717 ( 1986). (1701 J. I? Aymard, B. Rouveix, R. Ferry, C. Janot, T. May, B. Legras, and F. Streiff, Acra Haemarol., 82, 40 (1989). 11711 B. Rouveix, L. Coulombel, I? Aymard, F. Chau, and L. Abel, Br. J. Haematol., 71, 7 (1989). [I721 I? A. Winstanley, J. W. Coleman, J. L. Maggs, A. M. Breckenridge, and B. K . Park, Br. J. Clin. Pharmacol., 29, 479 (1990). [173] J. P. Jouet, J. J. Huart, F. Bauters, and M.Goudemand, Now. Presse Med., 9, 1386 (1980). (1741 A. M. McHarg and J. F. McHarg, Br. Med. J., I , 623 (1979). [I751 D. A. Curson and A. S. Hale, Br. Med. J., 1, 378 (1979). [176] D. M. Coulter and I. R. Edwards, Lancet, 336, 785 (1990). [177] C. E. Page, Br. Med. J., 284, 1912 (1982). [178] K. N. Achar, Br. Med. J., 285, 208 (1982). (1791 H. M. Clink, Br. J. Clin. Pharmacol., IS, 291 (1983). (1801 J. L. O’Donnell, J. R. Sharman, E. J. Begg, B. M. Colls, and I? W. Moller, Br. Med. J., 291, 1375 (1985). [I811 S. Durrant and D. Read, Br. Med. J., 285, 437 (1982). [I821 B. H. C. Stricker, J. N. M. Barendregt, and F. H . J. Claas, Br. J. Clin. Pharmacol., 19, 102 (1985). [I831 C. Lambert, B. K. Park, and N. R. Kitteringham. Biochem. Pharmacol., 28, 2853 (1989). [I841 1. Idanpaan-Heikkila, E. Alhava, M. Olkinuora, and 1. I? Palva, Eur. J. Clin. Pharmacol., 11, 193 (1977). [I851 J. A. Lieberman, C. A. Johns, J. M. Kane, K. Rai, A. V. Pisciotta, B. L. Salz, and A. Howard, J. Clin. Psychiatry. 49. 271 (1988). (1861 H. J. Senn, W. E Jungi, H. Kunz, and W. Poldinger, Lancer, 1 , 547 ( 1977). [I871 J. A. Lieberman, J. Yunis, E. Egea, R. T. Canoso, J. M. Kane, and E. J. Yunis, Arch. Gen. Psychiurry, 47, 945 (1990).



359

(1881 D. S. Cooper, D. Goldminz, A. A. Levin, I? W. Ladenson, G.H. Daniels, M. E. Molitch, and E. C. Ridgeway, Ann. Intern. Med., 98, 26 (1983). [189] J. Tajiri. S. Noguchi, T. Murakami, and N. Murakami, Arch. Intern. Med., 150, 621 (1990). [190] J. R. Wall, S. L. Fang, T. Kuroki, S. H. Ingbar, and L. E. Braverman, J. Clin. Endocrinol. Metab., 5 8 , 868 (1984). [I911 M. C. Werner, J. H. Romaldini, N. Bromberg, R. S. Werner, and C. S. Farah, Am. J. Med. Sci., 297, 216 (1989). [192] R. A. Walzer and J. Einbinder, J. Am. Med. Assoc., 184, 743 (1963). [193] S. B. Bilezikian, Y. Laleli, M. Tsan, B. A. Hodkinson, S. Ice, and €? A. McIntyre, Johns Hopkins Med. J . , 138, 124 (1976). [194] M. M. Guffy, N. E. Goeken, and C. I? Burns, Arch. Intern. Med., 144, 1687 (1984). [195] W. E. Fibbe, F. H. J. Claas, W. Van der Star-Dijlstra, M. R. Schaafsma, R. H. B. Meyboom, and J. H. F. Falkenburg, Br. J. Huemutol., 64, 363 (1986). [196] E. L. Toth, M. J. Mant, S. Shamim, and J. Ginsberg, Am. J. Med., 85, 725 (1988). [197] F. Pacini, V. Sridama, and S. Refetoff, J. Endocrinol. Invest., 5 , 403 ( 1982). [198] 0. J. Martelo, R. B. Katims, and A. A. Yunis, Arch. Intern. Med., 120, 587 (1967). [199] J. Moreb, 0. Shemesh, S. Shilo, C. Manor, and S. Hershko, Acta. Haemat.. 69, 127 (1983). [200] I? van Brummelen, R. Willemze. W. D. Tan, and J. Thompson, Lancet, I , 150 (1980). [201] F. W. Amann, F. R. Buhler, D. Conen, F. Brunner, R. Ritz, and B. Speck, Lancer, 1 , 150 (1980). [202] J. Staessen, R. Fagard, I? Lijnen, and A. Amery, Lancet, I , 926 (1980). (2031 F. Elijovisch and L. R. Krakoff, Lancet. I , 927 (1980). [204] M. A. Watson, N. J. Radford. B. I? McGrath, G.W. Swinton, and J. W. M. Agar, Aust. N.Z. J. Med., 11, 79 (1981). [205] T. Forslund, H. Borgmastars, and F. Fyhrquist, Lancet, I , 166 (1981). [206] I. Gavras, L. G. Graff, B. D. Rose, J. M. McKenna, H. R. Brunner, and H. Gavras, Ann. Intern. Med., 94, 58 (1981). [207] A. G. L. Kay, Ann. Rheumat. Dis., 38, 232 (1979). [208] H. B. Stein, A. C. Patterson, R. C. Offer, C. J. Atkins, A. Teufel, and H. S. Robinson, Ann. Intern. Med., 92, 24 (1980). [209] I. A. Jaffe, Am. J. Med., 80, 471 (1986).


360

UETRECHT

[210] A. S. Weiss, J. A. Markenson, M. S. Weiss, and W. H. Kammerer, Am. J. Med., 64, 114 (1978). [211] W. F? Hammond, J. E. Miller, G. Starkebaum, H. J. Zweerink, A. S. Rosenthal, and D. C . Dale, Exp. Hematol., 16, 674 (1988). [212] B. B. Huff, Physicians Desk Reference, Medical Economics Company, Oradell, N.J., 1985. (2131 A. V. Pisciotta, in Antiepileptic Drugs (D. M. Woodbury, J. K. Penry, and C. E. Pippenger, eds.), Raven Press, New York, 1982, p. 533. [214] 1. M. Killian, Headache, 9, 58 (1969). [215] R. G. Hart and J. D. Easton, Ann. Neurol., 11, 309 (1982). [216] R. T. Joffe, R. M. Post, I? F? Roy-Byrne, and T. W. Uhde, Am. J. Psychiatry, 142, 11% (1985). [217] G. W. K. Donaldson and J. G. Graham, Br. J. Clin. Pract., 19, 699 (1965). (2181 M.Franceschi, G. Ciboddo, G. Truci, A. Borri, and N. Canal, Epilepsia, 29, 582 (1988). [219] W. T. Gerson, D. G. Fine, S. I? Spielberg, and L. L. Sensenbrenner, Blood, 61, 889 (1983). (220) G. Weissmann and E. D. Xefteris, Arch. Intern. Med., 103, 957 (1959). [221] M. F. Cuthbert, Curr. Med. Res. Opin., 2 , 600 (1974). [222] W. H. W. Inman, Br. Med. J., I , 1500 (1977). [223] E. F. Mauer, N . Engl. J. Med., 253, 404 (1955). [224] V. A. Maria, J. A. F? da Silva, and R. M. M. Victorino, J. Rheumatol., 16, 1484 (1989). [225] B. D. Goldstein, J. Toxicol. Environ. Health, Suppl. 2, 69 (1977). [226] D. A. Eastmond, M. T. Smith, and R. D. Irons, Toxicol. Appl. Pharmacol., 91, 85 (1987). [227] F? Calabresi and B. A. Chabner, in Goodman and Gilman's The Pharmacological Basis of Therapeutics (A. G . Gilman, T. W. Rall, A. S. Nies, and I? Taylor, eds.,) Pergarnon Press, New York, 1990, p. 1209. [228] D. H. Phillips, A. Hewer, and F? L. Grover, Carcinogenesis, 7 , 2071 (1986). [229] D. F. Bainton, J. L. Ullyot, and M. G. Farquhar, J. Exp. Med., 134. 907 (1971). 1230) B. Zakhireh and R. K. Root, Blood, 54, 429 (1979). [231] J. I? Uetrecht, Chem. Res. Taricol.. I , 133 (1988). [232] S. L. Lee and I? H. Chase, Semin. Arthritis Rheum., 5 , 83 (1975). [233] D. Alarcon-Segovia, Drugs, 12, 69 (1976). [234] S. E. Blomgren, Semin. Hematol., 10, 345 (1973).



36 1

[235] D. J. Wallace and E. L. Dubois, Dubois’ Lupus Eryrhemarosus, Lea & Febiger, Philadelphia, 1987. [236] S . L. Lee, I. Rivero, and M. Siege], Arch. Intern. Med., 117, 620 (1966). [237] M. M. Reidenberg, Am. J. Med., 75, 1037 (1983). [238] M. R. Malinow, E. J. Bardana, B. Pirofsky, S. Craig, and I? McLaughlin, Science, 216, 415 (1982). [239] I? E. Prete, Arthritis Rheum., 28, 1198 (1985). [240] M. J. Fritzler and E. M. Tan, J. Clin. Invest., 62, 560 (1978). [241] J. I? Portanova, R. L. Rubin, F. G. Joslin, V. D. Agnello, and E. M. Tan, Clin. Immunol. Immunopathol., 25, 67 (1982). [242] J. E. Craft, J. A. Radding, M. W. Harding, R. M. Bernstein, and J. A. Hardin, Arthritis Rheum., 30, 689 (1987). [243] M. C. Totoritis, E. M. Tan, E. M. McNally, and R. L. Rubin, N . Engl. J. Med., 318, 1431 (1988). [244] D. C. Gorsulowsky, I? W. Bank, A. D. Goldberg, T. G. Lee, R. H. Heinzerling, and T. K. Burnham, J. Am. Acad. Dermatol., 12, 245 ( 1985). [245] J. I? Uetrecht and R. L. Woosley, Clin. Pharmacokin., 6 , 118 (1981). [246] N. C. Henningsen, A. Cederberg. A. Hanson, and B. W. Johansson, Acta Med. S c a d . , 198, 475 (1975). [247] R. L. Woosley, D. E. Drayer, M. M. Reidenberg, A. S. Nies, K. Carr, and J. A. Oates, N . Engl. J. Med., 298, 1157 (1978). [248] G. €? Stec, J. J. L. Lertora, A. J. Atkinson, M. J. Nevin, W. Kush-

ner, C. Jones, F. R. Schmid, and J. Askenazi, Ann. Intern. Med., 90, 799 (1979). [249] D. M. Roden,

S. B. Reele, S. B. Higgins, G. R. Wilkinson, R. F. Smith, J. A. Oates, and R. L. Woosley, Am. J. Cardiol., 46, 463 (1980).

[250] [251] [252] [253] [254] [255]

B. J. Hoffman, Arch. Dermarol. Syph., 51, 190 (1945). M. Honey, Br. Med. J., I , 1272 (1956). I. D. Griffiths and S. I? Kane, Br. Med. J., 2 , 1188 (1977). B. A. Vanheule and F. Carswell, Eur. J. Pediatr., 140, 66 (1983). I? Cohen and F. H. Gardner, J. Am. Med. Assoc., 197, 163 (1966). M. McCraken, E. A. Benson, and €? Hickling, Br. Med. J., 281,

1254 (1980). (2561 0. Garcia-Morteo and J. A. Maldonado-Cocco, Arthritis Rheum., 26, 936 (1983). [257] R. J. Cody, L. H. Calabrese, J. D. Clough, R. C. Tarazi, and E. L. Bravo, Clin. Pharmacol. Ther., 25, 800 (1979). [258] R. J. Booth, J. D. Wilson, and J. Y. Bullock, Clin. Phurmacol. Ther., 31, 555 (1982).


362

UETRECHT

[2591 E. B. Raftery and A. M. Denman, Br. Med. J . , 2. 452 (1973). [260] M. W. Stewart and S. W. Clarke, Proc. Roy. SOC. Med., 69, 61 ( 1976). [261] F? R. Vandersteen and R. E. Jordon, Arch. Dermutol., 110, 95 (1974). [262] H. M. Perry, E. M. Tan, S. Cordody, and A. Sahamato, J. Lab. Clin. Med., 76. 114 (1970). [263] S. J. Harland, V. Facchini, and J. A. Timbrell. Br. Med. J . . 281, 273 (1980). I2641 D. Alarcon-Segovia, E. Fishbein, and H. Alcala, Arrhriris Rheum., 14, 748 (1971). [265] N. F. Rothfield, W. F. Bierer, and J. W. Garfield, Ann. Intern. Med., 88, 650 (1978). [2&] M. M. Reidenberg, l? J. Durant, R. A. Harris, G. De Boccardo, R. Lahita, and K. H. Stenzel, Am. J. Med.. 75, 365 (1983). (2671 J. A. Timbrell, V. Facchini, S. J. Harland, and R. Mansilla-Tinoco, Eur. J. Clin. Phurmucol., 27. 555 (1984). [268] A. J. S t a t e r and J. A. Timbrell, Drug Metub. Dispos., 13, 255 (1985). [269) L. B. LaCagnin, H. D. Colby, N. S. Dalal. and J. I? O’Donnell, Biochem. Phurmucol., 36, 2667 (1987). (2701 L. M. Dubroff and R. J. Reid Jr., Science, 208, 404 (1980). [271] J. A. Amrhein, F. M. Kenny, and D. Ross, J. Pediufr., 76, 54 (1970). [272] N. Takuwa, I. Kojima, and E. Ogata, Endocrinol. Jupun., 28, 663 (1981). [273] M. M. Reidenberg, D. B. Case, D. E. Drayer, S. Reis, and B. Lorenzo, Arthritis Rheum., 27, 579 (1984). [274] A. Chalmers, D. Thompson, H. E. Stein, G. Reid, and A. C. Patterson, Ann. Intern. Med., 97, 659 (1982). [275] T. M. Harkcom. D. L. Conn. and K. E. Holley, Ann. Intern. Med., 89, 1012 (1978). [276] R. J. Enzenauer, S. G. West, and R. L. Rubin, Arthritis Rheum., 33, 1582 (1990). [277] D. E. Bateman, Br. Med. J., 291, 632 (1985). [278] S. Alballa, M. Fritzler, and I? Davis, J. Rheumuful., 14, 599 (1987). (2791 D. Alarcon-Segovia, E. Fishbein, P. A. Reyes, H. Dies, and S. Shwadsky, Clin. Exp. Immunol., 12, 39 (1972). [280] E. L. Dubois, E. Tallman, and R. A. Wonka, J. Am. Med. Assoc., 221, 595 (1972). [281] M. S. Gold and D. R. Sweeney, J. Nerv. Mental Dis., 166, 442 (1978).



363

[282] F. F? Quismorio, D. F. Bjarnason, W. F. Kiely, E. L. Dubois, and G. J. Friou, Am. J. Psychiatry, 132, 1204 (1975). [283] M. H. Zarrabi, S. Zucker, F. Miller, R. M. Derman, G. S. Romano, J. A. Hartnett, and A. 0. Varma, Ann. Inrern. Med., 91, 194 (1979). [284] R. L. Rubin, in Autoimmunity and Toxicology (M. E. Kammiiller, N. Bloksma, and W. Seinen, eds.), Elsevier, Amsterdam, 1989, p. 119. [285] A. C. Allison, in Auroimmunity and Toxicology (M. E. Kammiiller, N. Bloksma, and W. Seinen, eds.), Elsevier, Amsterdam, 1989, p. 67. [286] G. T. Layton, D. R. Stanworth, and H. E. Amos, Immunology, 59, 459 (1986). [287] M. M. Buxman, J. Iwest. Dermarol., 73, 250 (1979). [288] Y. Yamauchi, A. Litwin. L. Adams, H. Zimmer, and E. V. Hess, J. Clin. Invesr., 56, 958 (1975). [289] E. F. Gold, S. Ben-Efraim, A. Faivisewitz, 2. Steiner, and A. Klajman, Clin. Immunol. Immunoparhol., 7, 176 (1977). [290] E. M. Berkman, J. B. Orlin, and J. Wolfsdorf, Transfusion. 23, 135 (1983). [291] V. M. Holers and B. L. Kotzin, J. Clin. Invesr., 76, 991 (1985). [292] L. Nassberger, A. G. Sjoholm. H. Jonsson, G. Sturfelt, and A. k e s s o n , Clin. Exp. Immunol., 81, 380 (1990). [293] L. Waldhauser and J. Uetrecht, presented at the Third International ISSX Meeting, 1991. [294] F? Beaune, I? M. Dansette, D. Mansuy, L. Kiffel, M. Finck, C. Amar, J. F? Leroux, and J. C. Homberg, Proc. Narl. Acad. Sci. USA, 84, 551 (1987). [295] R. J. Falk, R. S. Terrell, L. A. Charles, and J. C. Jennette, Proc. Narl. Acad. Sci. USA, 87, 4115 (1990). [296] R. E. Schopf, H. M. Hanauske-Abel, G. Tschank, H. SchulteWissermann, and V. Gunzler, J. Immunopharmacol., 7, 385 (1985). [297] E. Cornacchia, J. Golbus. J. Maybaum, J. Strahler, S. Hanash, and B. Richardson, J. Immunol., 140, 2197 (1988). [298] T. &hi, E. A. Goldings, I? E. Lipsky, and M. Ziff, J. Clin. Invest., 71, 36 (1983). [299] K. B. Miller and D. Salem, Am. J. Med., 73, 487 (1982). [300] R. L. Rubin, J. F? Uetrecht, and J. E. Jones, J. Pharmacol. Exp. Ther., 242. 833 (1987). [301] E. Gleichmann, E. H.Van Elven, and J. F? W. Van der Veen, Eur. J. Immunol., 12. 152 (1982). [302] E. Gleichmann, W. T. Pals, A. G. Rolink, T. Radaszkiewicz, and H. Gleichmann, Immunol. To&, 5 , 324 (1984).

364

UETR ECHT


[303] J. F? Portanova and B. L. Kotzin, in Cellular Aspects of Autoimmunify (J. M. Cruse and R. E. L. Jr., eds.), Karger, Basel, 1988, p. 119. [304] F. M. Van Rappard-Van der Veen, U. Kiesel, L. Poels, W. Schuler,

C. J. M. Melief, J. Landegent, and E. Gleichmann, J. Immunol., 132, 1814 (1984). [305] J. F? Portanova, H. N. Claman, and B. L. Kotzin, J. Immunol., 135, 3850 (1985). [306] R. A. Eisenberg and F? L. Cohen, Clin. Immunol. Immunopathol., 29. 1 (1983). [307] Y.Tanaka, K. Saito, H. Suzuki, S. Eto, and U. Yamashita, J. Immuno!., 143, 1584 (1989). [308] N. H. Shear, S. F? Spielberg, D. M. Grant, B. K. Tang, and W. Kalow, Ann. Intern. Med., 105, 179 (1986). [309] M. J. Rieder, J. Uetrecht, N. H. Shear, and S. F! Spielberg, J. Pharmucol. Exp. Ther., 244, 724 (1988). [310] M. J. Rieder, J. Uetrecht, N. H. Shear, M. Cannon, M. Miller, and S. F? Spielberg, Ann. Intern. Med., 110, 286 (1989). [311] N. H. Shear and S . F? Spielberg, Can. J. Physiol. Pharmacol., 63, 1370 (1985). [312] H. S. Jaffe, A. J. Ammann, D. I. Abrams, B. J. Lewis, and J. A. Goldin, Lancer, 2 , 1109 (1983). [313] F. M. Gordin, G. L. Simon, C. B. Wofsy, and J. Mills, Ann. Intern. Med., 100, 495 (1984). [314] M. A. Fischl, G. M. Dickinson, and L. La Voie, J. Am. Med. Assoc., 259, 1185 (1988). [315] K. Nahum, I? Saidi, H. C. Kim, and G. I. Karp, Am. J. Hemarol., 27, 144 (1988). [316] H. Eck, H. Gmunder, M. Hartmann, D. Petzoldt, V. Daniel, and W. Droge, Biol. Chem. Hoppe-Seyler, 370, 101 (1989). [317] R. Buhl, K. J. Holroyd, A. Mastrangeli, A. M. Cantin, H. A. Jaffe, F. B. Wells, C. Saltini, and R. G.Crystal, Lancer, 2 , 1294 (1989). [318] H. M. Frey, A. A. Gershon, W. Bordowsky, and W. E. Bullock, Ann. Intern. Med., 94, 777 (1981). [319] N. F? Kromann, R. Vilhelmsen, and D. Stahl, Arch. Dermurol., 118, 531 (1982). [320] R. C. Wille and J. D. Morrow, Am. J. Med. Sci., 2%. 270 (1988). [321) D. H. Lawson and H. Jick, Br. J. Clin. Phurmacol., 4 , 507 (1977). [322] J. F. Wheeler, L. E. Adams, A. Mongey, S. M. Roberts, W. R. Heineman, and E. V. Hess, Drug Metub. Dispos., 19, 691 (1991). [323] H. Uehleke and S . Tabarelli, Nuunyn-Schmiedeberg’s Arch. Pharmucol., 278, 55 (1973).



365

[324] J. Uetrecht, in Clinics in Dermarology (N. H. Shear, ed.),J. B. Lippincott, Philadelphia, 1989, p. 1 11. [325] S. J. Grossman and D. 1. Jollow, J. Pharmacol. Exp. Ther., 244, 118 (1988). [326] A. A. Mihas, €? Holley, R. S. Koff, and B. 1. Hirschowitz, Gasrroenrerology, 70, 770 (1976). [327] B. D. Houston, M. E. Crouch, J. E. Brick, and A. G. DiBartolomeo, Arrhriris Rheum., 22, 925 (1979). [328] M. D. Carrasco, C. Riera, B. Clotet, M. Grifol, and M. Foz, Arch. Intern. Med., 147, 1677 (1987). [329] M. D. Kurtz, NYS J. Med., 68, 2810 (1968). [330] K. Farbman, M. F. Wheeler, and S. M.Glick, NYS J. Med., 69, 826 ( 1969). [331] B. K. Oh, G. I? Overveld, and J. D. Macfarlane, Br. J. Rheumarol., 22, 106 (1983). [332] I? Netter, B. Bannwarth, G. Faure, I? Trechot, and R. J. Royer, J. Rheumarol., 15, 1730 (1988). [333] S. J. Hoorntje, J. J. Weening, C. G. M. Kallenberg, E. J. L. Prins, and A. J. M. Donker, Lancer, 2 , 1297 (1979). [334] L. Mallet, J. W. Cooper, and J. Thomas, DlCR 23, 63 (1989). [335] I. A. Jaffe, Springer Semin. Immunoparhol., 4 , 193 (1981). [336] S. I? Spielberg, G. B. Gordon, D. A. Blake, D. A. Goldstein, and H. F. Herlong, N . Engl. J. Med.. 305, 722 (1981). [337] S. I? Spielberg, Clin. Biochem., 19, 142 (1986). [338] N. H. Shear and S. I? Spielberg, J. Clin. lnvesr., 82, 1826 (1988). [339] S. €? Spielberg, G.B. Gordon, D. A. Blake, E. D. Mellits, and D. S. Bross, J. Pharmacol. Exp. Ther., 217, 386 (1981). [340] M.A. A. Moustafa, M. Claesen. J. Adline, D. Vandervorst, and J. H. Poupaert, Drug Merab. Dispos.. 11, 574 (1983). [341] F. I? Guengerich and T. L. Macdonald, Acc. Chem. Res., 17, 9 (1984). [342] R. J. Riley, N. R. Kitteringham, and B. K. Park, Br. J. Clin. Pharmacol., 28, 482 (1989). [343] S . S. Lau, T. J. Monks, K. E. Greene, and J. R. Gillette, Xenobiotica, 14, 539 (1984). [344] R. J. Riley, J. L. Maggs, C. Lambert, N. R. Kitteringham, and B. K. Park, Br. J. Clin. Phannacol., 26, 577 (1988). [345] R. H. Levy, F. E. Dreifuss, R. H. Mattson, B. S. Meldrum, and J. K. Penry, Anriepilepric Drugs, Raven Press, New York, 1989. [346] E. M. Faed, Drug Metab. Rev., 15, 1213 (1984). 13471 R. B. van Breemen and C. Fenselau, Drug Metub. Dispos., 13, 318 (1985).


366

UETRECHT

[348] M. L. Hyneck, €? C. Smith, A. Munafo, A. F. McDonagh, and L. Z . Benet, Clin. Phurmucol. Ther., 44, 107 (1988). [349] 1. A. Watt and R . G. Dickinson, Biochem. Phurmucol.. 39, 1067 (1990). [350] €? C. Smith, A. F. McDonagh, and L. Z. Benet, J. Phurmucol. Exp. Ther.. 252, 218 (1990). [351] A. Munafo, A. F. McDonagh, €? C. Smith, and L. Z. Benet, Phurmuceut. Res., 7 , 21 (1990). [352] D. Schuhmann, M. Kubicka-Muranyi, J. Mirtschewa, J. Gunter, l? Kind, and E. Gleichmann, J. fmmunol., 145, 2132 (1990). [353] B. K. Park, J. W. Coleman, and N. R. Kitteringham, Biochem. Phurmacol., 36, 581 (1987). [354) H. Pullen, N. Wright, and J. Murdoch, Lancet, 2, 1176 (1967). [355] W. E. Seaman, K. G. Ishak, and I? H. Plotz, Ann. Intern. Med., 80, 1 (1974). (3561 J. G. Schaller, Pediatrics, 62 (suppl), 916 (1978). [357] D. Thomassen, Drug Safety, 6, 235 (1991).

Drug metabolism by leukocytes and its role in drug-induced lupus and other idiosyncratic drug reactions.

Idiosyncratic drug reactions: a mechanistic evaluation of risk factors.

Pathogenesis of idiosyncratic drug-induced liver injury and clinical perspectives.

Idiosyncratic reactions to the antiepileptic drugs.

The pathogenesis of reactive axonal swellings: role of axonal transport.

Adverse reactions caused by drug metabolites.

In vitro testing for diagnosis of idiosyncratic adverse drug reactions: Implications for pathophysiology.

Role of reactive oxygen metabolites in experimental colitis.

Idiosyncratic reactions to gold salt preparations.

The role of reactive oxygen species in the pathogenesis of multiple sclerosis.

Current understanding of the mechanisms of idiosyncratic drug-induced agranulocytosis.

Role of metabolic lipases and lipolytic metabolites in the pathogenesis of NAFLD.

The idiosyncratic drug-induced gene expression changes in HepG2 cells.

Novel drug metabolites produced by functionalization reactions: chemistry and toxicology.

[Delayed-type cutaneous drug reactions. Pathogenesis, clinical features and histology].

The potential role of pharmacogenomics in the prevention of serious adverse drug reactions in multiple sclerosis.

Mini-dialysis tubes as tools to prepare drug-protein adducts of P450-dependent reactive drug metabolites.

Idiosyncratic drug hepatotoxicity: a 2008 update.

The role of mitochondria-derived reactive oxygen species in the pathogenesis of non-steroidal anti-inflammatory drug-induced small intestinal injury.

Idiosyncratic reactions to antidepressants: a review of the possible mechanisms and predisposing factors.

Two immunologic reactions in the pathogenesis of hepatitis?

Drug-drug interactions with oral anti-HCV agents and idiosyncratic hepatotoxicity in the liver transplant setting.

Increasing the number of adverse drug reactions reporting: the role of clinical pharmacy residents.

The Role of Gammaherpesviruses in Cancer Pathogenesis.