Researchin JULY/AUGUST 1988 VOLUME 1, NUMBER 4 @Copyright 1988 by the American Chemical Society
Persp e c tive Metabolic Activation of Valproic Acid and Drug-Mediated Hepatotoxicity. Role of the Terminal Olefin, 2-n -Propyl-4-pentenoic Acid Thomas A. Baillie Department of Medicinal Chemistry, School of Pharmacy, BG-20, University of Washington, Seattle, Washington 98195 Received July 1, 1988 Introduction Valproic acid (VPA', 1, Figure l),an anticonvulsant agent introduced in France in 1967 for the therapy of epilepsy and approved by the U.S.Food and Drug Administration in 1978, has now become a primary antiepileptic drug worldwide (1). In recent years, VPA has been shown to be effective against a broad spectrum of seizure types, and it has found use both as sole medication and as a component of polytherapy. The drug is unique within its therapeutic class, in terms of both its chemical structure and its mechanism of action, and as a consequence, it has been the focus of much basic and applied research (2). However, despite its numerous attributes as an antiepileptic drug, initial enthusiasm for valproate has been tempered by reports of a rare, but serious, liver injury associated with VPA therapy (3-5). Although sometimes reversible, this drug-mediated hepatotoxicity has led to instances of irreversible liver failure (usually characterized by hepatic steatosis with or without necrosis), and a t least 80 cases of fatal VPA hepatotoxicity had been reported to the manufacturer by the end of 1982 (6). Since the majority of these fatalities involved young children, the American Academy of Pediatrics issued, in the same year, Abbreviations: VPA,2-n-propylpentanoicacid or valproic acid; A2(A3 or A')-VPA, 2-n-propyl-2(3or 4)-pentenoic acid; A2v4-VPA,2-n-propyl3-n-propyl-5-(hy2,4-pentadienoic acid; 4,5-dihydroxy-VPA-y-lactone, droxymethyl)tetrahydro-2-furanone;3(4 or 5)-hydroxy-VPA, 2-npropyl-3(4 or 5)-hydroxypentanoic acid; 3-oxo-VPA, Z-n-propyl-3-0~0pentanoic acid; 3-hydroxy(3-oxo)-A'-VPA, 2-n-propyl-3-hydroxy(3oxo)-4-pentenoic acid; AIA, 2-isopropyl-4-pentenamideor allylisopropylacetamide; CoA, coenzyme A.
0893-228x/88/2701-0195$01.50/0
a recommendation that "...physicians adopt a conservative approach and utilize VPA only in carefully selected situations" (7). The biochemical mechanisms that underlie VPA-mediated liver injury are not understood, although a number of hypotheses for the toxicity have been advanced (8). The finding (9)that the incidence of VPA-related fatalities is strikingly higher in patients receiving VPA in polytherapy, rather than as sole agent, is noteworthy in that it is consistent with the view that metabolites of VPA (produced in increased amounts by an induced liver) may play a key role in the hepatotoxic response to this drug. In fact, this toxic metabolite theory was suggested by Gerber et al. (3), in one of the first published reports on fatal VPA hepatotoxicity, who drew attention to the similarity in structure between VPA and two known hepatotoxins, 4-pentenoic acid (4, Figure 1)and methylenecyclopropylaceticacid. On the basis of the fact that the latter compounds (both terminal olefins) produce a Reye-like syndrome in animals and cause hepatic steatosis as the primary tissue lesion, these authors proposed that a metabolite of VPA may well be responsible for the mitochondrial damage ( l o ) ,impairment of fatty acid @-oxidationactivity ( l l ) and , lipid accumulation ( 4 ) characteristically observed in cases of VPA hepatotoxicity. This line of reasoning was developed further by Zimmerman and Ishak (41, who proposed that the terminal olefin metabolite of VPA, 2-n-propyl-4-pentenoic acid (A4-VPA,2, Figure l), might be the responsible hepatotoxin. Interestingly, this unsaturated derivative of VPA, which was first detected as a minor metabolite in the plasma of epileptic children receiving valproate (12), 0 1988 American Chemical Society
196 Chem. Res. Toxicol., Vol. 1, No. 4, 1988
I
+ CONH,
3
Baillie
2
P-450
Lon
Heme Alkylation
02
\\
4
Figure 1. Structures of VPA (l), A4-VPA (2), AIA (3), and
4-pentenoic acid (4).
appears to be present a t higher levels in the serum of pediatric patients (the group most susceptible to VPAinduced liver damage) than in either youths or adults (13, 1 4 ) . Moreover, when biochemical studies with A4-VPA were performed, it was shown that this compound was indeed cytotoxic, in a dose-dependent fashion, to rat hepatocytes in culture (15),that it inhibited P-oxidation of medium-chain fatty acids in rat liver homogenates (16), and that, following administration to rats, pronounced elevations in blood urea and SGOT levels were obtained (I7). Subsequently, Kesterson, Granneman, and coworkers (1419) published the findings of a comprehensive study of the toxicity in rats of VPA and certain of its metabolites, in which A4-VPAand A2p4-VPA(the principal metabolite of A4-VPA) were found to be potent inducers of microvesicular steatosis and inhibitors of fatty acid @-oxidation. These authors noted that ”...the liver of animals dosed with 4-en-VPA was more severely affected than that in any other group, including 4-pentenoic acid treated rats” (18). The possible clinical relevance of these findings is suggested by the paper of Kochen et al. (20), which reported grossly elevated levels of A4-VPA and other unsaturated metabolites of VPA in plasma and urine of a 7-year-old boy who died of VPA-mediated liver failure. From the foregoing discussion, it is apparent that a considerable body of evidence has now accumulated which suggests that A4-VPAcould play a key role in mediating VPA hepatotoxicity. In an attempt to examine the validity of this hypothesis, and in order to elucidate the biochemical mechanism(s) by which A4-VPAexerts its hepatotoxic effects, a number of studies have been carried out in our laboratories to define the metabolic origin of this compound and to elucidate its biological fate. The approach adopted in these investigations has been to examine in detail the “A4” pathway of VPA metabolism in vitro and in vivo in order to assess the potential for toxic metabolite generation and resulting cellular injury. The outcome of these studies, which have focused largely on the role of hepatic cytochrome P-450 and P-oxidation enzymes in the formation and metabolic activation of A4-VPA, may be summarized as follows.
Metabolic Activation of A4-VPA Reactive Metabolites of A4-VPA Generated by Cytochrome P-450. Since liver microsomal cytochrome P-450enzymes are known to catalyze the oxidation of monosubstituted olefins to reactive free-radical and epoxide intermediates (21), it was of interest to examine the interaction of A4-VPAwith this mixed-function oxidase enzyme system. Studies conducted in vitro with rat liver microsomal preparations demonstrated that both the free acid and ethyl ester forms of A4-VPA underwent cytochrome P-450 dependent metabolism at the olefin functionality and yielded a common y-butyrolactone derivative, 4,5-dihydroxy-VPA-y-lactone (Figure 2) (22). That this process involved the intermediacy of a chemically reactive species was suggested by the observation that substrate turnover was accompanied by loss of spectrophotomet-
Intramolecular HO&
*
Rearrangement
j
rod
Figure 2. Proposed scheme for the cytochrome P-450 dependent olefin oxidation pathway of A4-VPAmetabolism. Initial oneelectron oxidation of the substrate yields a heme-bound freeradical intermediate, which partitions between heme alkylation (enzyme destruction) and epoxide formation. The unstable epoxide, in turn, undergoes intramolecular nucleophilic attack by the carboxylate group to give the final product, 4,5-dihydroxyVPA-y-lactone. This scheme, which is adapted from work by Ortiz de Montellano and Correia (23),is supported by the results of oxygen-18 labeling experiments (22).
rically detectable cytochrome P-450, in a fashion similar to that documented for a series of terminal olefin “suicide substrate” inhibitors of this enzyme, e.g., allylisopropylacetamide (AIA, 3, Figure 1) (23). Interestingly, oxygen-18 tracer experiments showed that three quite distinct ringclosure mechanisms were operative in the metabolism of A4-VPA, ethyl A4-VPA, and AIA to their respective ybutyrolactone derivatives but indicated that autocatalytic destruction of cytochrome P-450 in each case depended solely on the efficiency of the initial olefin oxidation step (23,24). This finding was consistent with earlier work by Oritiz de Montellano et al. (25),who showed that olefinmediated enzyme destruction is caused by free-radical species that alkylate the prosthetic heme but that are formed prior to the generation of epoxide intermediates. Hence, the fate of the epoxide in such systems, whether it be hydrolysis to a vicinal diol (as with ethyl A4-VPA) or intramolecular capture by a proximate nucleophilic center (as with A4-VPA and AIA), is of little consequence with respect to the phenomenon of drug-mediated cytochrome P-450 inhibition. A scheme consistent with these observations, and accounting for the formation of enzyme-bound (heme-alkylated) and y-lactone metabolites of A4-VPA, is depicted in Figure 2. When compared to AIA, A4-VPAwas found to be relatively weak in its ability to destroy microsomal cytochrome P-450 in vitro. However, the possibility that reactive metabolites of A4-VPA,generated by cytochrome P-450, might diffuse away from their site of formation in the endoplasmic reticulum and alkylate critical biomacromolecules to cause cellular damage remains to be explored. Reactive Metabolites of A4-VPA Generated by 8Oxidation. Bioactivation of A4-VPA by the enzymes of fatty acid P-oxidation is also suggested on structural grounds, since 4-pentenoic acid (of which A4-VPA is the 2-propyl derivative) is considered to be a mechanism-based irreversible inhibitor of this enzyme complex (26). In the case of 4-pentenoic acid, P-oxidation is believed to lead to 3-oxo-4-pentenoyl-CoA, a reactive, electrophilic species that is proposed to alkylate (and thereby inactivate) 3ketoacyl-CoA thiolase, the terminal enzyme of @oxidation. Thus, if the structural analogy between 4-pentenoic acid and A4-VPAextends to their respective routes of metabolism, it would be predicted that A4-VPAshould serve as an enzyme-activated inhibitor of fatty acid P-oxidation.
Chem. Res. Toxicol., Vol. I , No. 4, 1988 197
Perspective
L-RCOSCoA
I
G- +-
COSCoA
O
in vivo (18,19). A scheme depicting the proposed bioactivation pathway for A4-VPAin mitochondria is illustrated in Figure 3. While the above findings are strongly suggestive of a role for metabolic activation in the hepatotoxicity of A4-VPA, they are nevertheless indirect, and further studies are needed in order to both elucidate the mechanism(s) by which A4-VPA inhibits fatty acid metabolism and assess its contribution to VPA-mediated hepatic steatosis.
on
Alkylation of 3-KetoacylCoA Thiolase
Figure 3. Proposed metabolic activation pathway for A‘-VPA by the fatty acid @-oxidationcycle. Following conversion of A4-VPAto its coenzyme A derivative, sequential steps of @-oxidation lead to 2(E)-A2v4-VPA,3-hydroxy-A4-VPA,and 3-oxoA*-VPA. The latter species is believed to be a reactive, electrophilic metabolite that binds covalently to,and thereby inactivates irreversibly, 3-ketoacyl-CoA thiolase. The proposed diene and allylic alcohol intermediates have been identified as metabolites of A4-VPAin the isolated perfused rat liver (27) and in the rhesus monkey in vivo (29).
In order to address this intriguing mechanistic possibility, the metabolism of A4-VPA was examined in the isolated perfused rat liver, when products of @-oxidation were indeed observed. Not unexpectedly, metabolism occurred on both unsaturated and saturated side chains of A4-VPA to yield a variety of products, the most significant of which were the conjugated diene 2(E)-A2p4-VPA and the allylic alcohol 3-hydroxy- A4-VPA. These metabolites correspond to the first and second intermediates, respectively, of the proposed bioadivation pathway leading to 3-oxo-A4-VPA, the putative toxic alkylating species (Figure 3). Although the latter compound itself was not found in samples of either perfusate or bile, the chemical properties of synthetic 3-oxo-h4-VPA indicated that this a@-unsaturatedketone is indeed a highly reactive electrophile, which readily undergoes Michael-type addiion reactions through nucleophilic attack at the olefinic terminus (27). In subsequent studies performed in rhesus monkeys (considered to be a good animal model for man with respect to VPA metabolism), the pharmacokinetics and metabolism of an intravenous dose of A4-VPA were compared to those of a similar dose of VPA (28,29).Evidence was obtained that this nonhuman primate metabolizes both compounds via @-oxidation,although quantitative assessments of the various biotransformation products excreted into urine revealed interesting differences between the substrates. Thus, in the case of VPA, products of glucuronidation and cytochrome P-450 mediated reactions (both of which occur in the endoplasmic reticulum) accounted for -65% of the dose that was recovered in urine over 24 h, whereas metabolites formed by mitochondrial @-oxidationwere relatively minor and made up a further 6% (28). In contrast, when A4-VPAwas given, some 34% of the dose was recovered in the form of products of microsomal enzyme activity, while a full 22% appeared to derive from mitochondrial metabolism (29). A4-VPA, therefore, seems to exhibit a marked preference for metabolism by &oxidation as compared with the parent drug, a property that may contribute (through formation of 3-oxo-A4-VPA)to the high potency of this terminal olefin as an inhibitor of fatty acid @-oxidationin vitro (16)and
Metabolic Origin of A4-VPA Although the studies cited above provide considerable insight into the metabolic basis for the hepatotoxic properties of A4-VPA,details of the mechanism by which this olefin is generated during VPA biotransformation remained obscure until recently. Early experiments by Kochen and Scheffner (12)and by Granneman et al. (30) established that A4-VPA was not formed as an artifact (either in vitro or in vivo) by dehydration of 4- or 5hydroxy-VPA, and more recent studies on the metabolism of specifically deuterium labeled analogues of VPA in the rat indicated that this terminal olefin had a biochemical origin quite different from that of its nonhepatotoxic positional isomers, A2- and A3-VPA (31). Investigation of the products formed upon incubation of VPA with hepatic microsomes from phenobarbitalpretreated rats revealed that A4-VPAwas generated by this in vitro system and that its formation was both oxygenand NADPH-dependent (32). Moreover, since metyrapone, a relatively specific inhibitor of cytochrome P-450, blocked the desaturation reaction, a role for this hemoprotein in the metabolic process was indicated. Subsequent experiments with a purified and reconstituted form of cytochrome P-450 from rat liver (P-450 PB-4) served to verify the above conclusion, since VPA was metabolized by this enzyme to both 4- and 5-hydroxy-VPA and to A4-VPA. These results were of interest for two reasons: (i) They demonstrated that cytochrome P-450 enzymes can catalyze the oxidation of a nonactivated alkyl substituent to the corresponding olefin, a reaction not described previously. The underlying mechanism of this process is proposed to involve initial hydrogen atom abstraction by the enzyme to generate a transient free-radical intermediate, which partitions between recombination (alcohol formation) and elimination (olefin production) pathways. Recent deuterium isotope effect studies have lent support to this mechanistic interpretation and have indicated that the carbon-centered radical in question is located a t the C-4 (and not at the alternative C-5) position (33). (ii) The finding that phenobarbital pretreatment of animals (32, 33) induces metabolism of VPA to A4-VPA clearly has important clinical implications, especially with regard to the use of VPA in polytherapy. Thus, desaturation of the parent drug to yield the hepatotoxic A4 metabolite, catalyzed by isoenzymes of cytochrome P-450 that have been induced specifically by coadministration of antiepileptic drugs such as phenobarbital (32, 33), phenytoin (33),or carbamazepine (33),may prove to be a key step in the sequence of events leading to VPA-mediated liver injury. Indeed, preliminary results from clinical studies designed to assess the influence of polytherapy on the “A4” pathway of VPA metabolism in epileptic patients have indicated that classical inducers of cytochrome P-450 (phenytoin, carbamazepine) stimulate this pathway of biotransformation, whereas an inhibitor of the enzyme (stiripentol) reduces the formation of A4-VPA( 3 4 , s ) .In light of these observations, it appears that epileptic patients treated concomitantly with VPA and microsomal enzyme inducers
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may be at increased risk of liver injury and should be monitored carefully for early signs of hepatic dysfunction. Conversely, polytherapy with the newer generation of anticonvulsant drugs which act as inhibitors of cytochrome P-450 may, on theoretical grounds, prove to be a safer therapeutic strategy. Clearly, much more information is needed from controlled clinical studies before the validity of this prediction can be assessed accurately.
Conclusions On the basis of the findings reviewed above, it seems highly probable that the hepatotoxic effects of A4-VPAare a consequence of further biotransformation of this unsaturated VPA metabolite to chemically reactive intermediates that alkylate key cellular macromolecules. Enzymes of the fatty acid P-oxidation complex most likely play a pivotal role in the metabolic activation of A4-VPA (as they do with 4-pentenoic acid), although it is possible that products of cytochrome P-450 catalyzed olefin oxidation also contribute to the cytotoxic properties of this compound. Interestingly, both cytochrome P-450 and P-oxidation enzymes themselves appear to be targets for A4-VPA-mediated inhibition. However, in order to establish unequivocally that A4-VPAis a mechanism-based inhibitor of both cytochrome P-450 and 3-ketoacyl-CoA thiolase, it will be necessary, inter alia, to demonstrate that substrate turnover in each case is accompanied, on the one hand, by loss of catalytic activity and, on the other, by covalent modification of the enzyme through attachment of a A4-VPA residue a t the active site. Radiolabeled A4-VPAhas been synthesized recently for use in such work, and preliminary metabolic studies have shown that, following administration to rats by intraperitoneal injection, the compound does become covalently bound to proteins and that liver is the primary target organ for such protein alkylation (36). While the subcellular distribution of this binding and its role in the pathogenesis of A4-VPA-induced hepatotoxicity remain to be defined, the development of an in vitro model system, based on freshly isolated rat hepatocytes (36), should prove valuable in addressing such issues. Finally, it should be stressed that the studies discussed in this Perspective have dealt with only one mechanism by which VPA may cause liver injury, Le., via metabolism to, and subsequent activation of, the hepatotoxic olefin A4-VPA. Under normal conditions, the $‘A4’’ pathway represents a very minor route of VPA biotransformation, although it may assume quantitative significance in certain situations (20). However, other electrophilic metabolites of VPA are known to be formed in relatively large amounts, e.g., the acyl-linked glucuronide conjugate (37) and the coenzyme A derivative of VPA (38,391,either of which might play a role in VPA-mediated liver injury. Several other viable mechanisms of VPA hepatotoxicity, which have been the subject of a recent authoritative review (8), include competitive inhibition of 3-ketoacyl-CoA thiolase by 3-oxo-VPA (40), carnitine deficiency induced by VPA administration (41), depletion of mitochondrial pools of free coenzyme A by VPA (18,38, 42), and interference by VPA with processes of intermediacy metabolism in a liver whose function is already compromised by severe illness, inherited metabolic disorders, or exposure to multiple anticonvulsant drugs (9). Whatever the precise underlying factors, it is clear that a detailed knowledge of the metabolic fate of VPA, and of the properties of its hepatotoxic A* metabolite, will add greatly to our understanding of VPA-mediated hepatotoxicity and may also afford information of a fundamental nature on the complex
Baillie interplay between processes of foreign compound biotransformation and endogenous fatty acid metabolism (43). Acknowledgment. I acknowledge with thanks the contributions of the following colleagues at the University of Washington who participated in the studies cited in this review: A. W. Rettenmeier, R. H. Levy, K. S. Prickett, W. P. Gordon, A. E. Rettie, D. J. Porubek, M. Boberg, S. M. Bjorge, H. Barnes, M. P. Grillo, and W. N. Howald. I also thank Ms. S. West for assistance with manuscript preparation. Financial support for my work on VPA metabolism has been provided by the National Institutes of Health (Research Grants GM 32165, NS 17111, and DK 30699), which is gratefully acknowledged.
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