structural alerts, reactive metabolites, and protein covalent binding: how reliable are these...

REVIEW

Structural Alerts, Reactive Metabolites, and Protein Covalent Binding: HowReliable Are These Attributes as Predictors of Drug Toxicity?

by Amit S. Kalgutkar*a) and Mary T. Didiukb)

a) Pharmacokinetics, Dynamics and Metabolism Department, Pfizer Global Research & Development,Groton, CT 06340, USA (phone: þ18604468997; e-mail: [email protected])

b) Chemistry Department, Pfizer Global Research & Development, Groton, CT 06340, USA

In an increasing number of cases, a deeper understanding of the biochemical basis for idiosyncraticadverse drug reactions (IADRs) has aided to replace a vague perception of a chemical class effect with asharper picture of individual molecular peculiarity. Considering that IADRs are too complex to duplicatein a test tube, and their idiosyncratic nature precludes prospective clinical studies, it is currentlyimpossible to predict which new drugs will be associated with a significant incidence of toxicity. Becauseit is now widely appreciated that reactive metabolites, as opposed to the parent molecules from whichthey are derived, are responsible for the pathogenesis of some IADRs, the propensity of drug candidatesto form reactive metabolites is generally considered a liability. Procedures have been implemented tomonitor reactive-metabolite formation in discovery with the ultimate goal of eliminating or minimizingthe liability via rational structural modification of the problematic chemical series. While suchmechanistic studies have provided retrospective insight into the metabolic pathways which lead toreactive metabolite formation with toxic compounds, their ability to accurately predict the IADRpotential of new drug candidates has been challenged. There are several instances of drugs that formreactive metabolites, but only a fraction thereof cause toxicity. This review article will outline currentapproaches to evaluate bioactivation potential of new compounds with particular emphasis on theadvantages and limitation of these assays. Plausible reason(s) for the excellent safety record of certaindrugs susceptible to bioactivation will also be explored and should provide valuable guidance in the useof reactive-metabolite assessments when nominating drug candidates for development.

Introduction. – With an increasing number of commercial failures because ofblockbuster drug withdrawals and/or black box warnings due to safety concerns, thepharmaceutical industry faces an unprecedented challenge in bringing new and saferproducts into the marketplace that bring true value to the patient. Decreasedproductivity in terms of new drug approvals is perhaps the greatest challenge facing theindustry today despite a significant increase in research and development spending.The root causes of drug failure in the clinic over time have been reviewed by Kola andLandis [1]. In 1991, poor pharmacokinetics (high clearance, short half-life, and poororal bioavailability) resulting largely from sub-optimal physiochemical properties ofdrug candidates were the primary cause of attrition accounting for ca. 40% of allclinical failures. Throughout the 1990s, investments in state-of-the-art bioanalyticalmethodologies coupled with the development of high-throughput ADME assays tomonitor metabolic properties of new compounds dramatically reduced attrition ratesdue to adverse pharmacokinetics (<10% overall failure rates in 2000). However,

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009) 2115

� 2009 Verlag Helvetica Chimica Acta AG, Z�rich

together with gains in systemic exposures as a result of favorable pharmacokineticscame increased observations on unanticipated toxicities, which, by the year 2000,accounted for ca. 30% of all drug attrition (lack of efficacy accounted for ca. 30% offailures). Because much of the safety-related attrition (ca. 70%) occurs preclinically[2], it has been suggested that predictive tools to identify preclinical safety liabilitiesearlier in the process could lead to the design and/or selection of better candidates thathave increased probabilities of becoming marketed drugs. However, the solution is notthat simple; of a total of 548 drugs approved in the period from 1975 – 1999, 45 drugs(8.2%) acquired one or more black box warnings, and 16 (2.9%) were withdrawn fromthe market owing to adverse drug reactions (ADRs) that were not predicted fromanimal testing and/or clinical trials [3].

ADR Classification. – ADRs can be generally sub-divided into type A and type Breactions. Type-A reactions are associated with the primary pharmacology of the drug(e.g., risk of hypotension with antihypertensives) and are responsible for ca. 80% of allside effects. Type-A ADRs can be detected in animal models of pharmacology and/ortoxicology; they exhibit simple dose – response relationships and are usually avoided inthe clinic via dose adjustments. In contrast, type-B (bizarre or idiosyncratic ADRs –(IADRs)) are unrelated to known drug pharmacology, and are generally dose-independent. Because the frequency of occurrence of IADRs is very low (1 in 10,000 to1 in 100,000), these reactions are often not detected, until the drug has gained broadexposure in a large patient population. Importantly, standard regulatory animal toxicitystudies have traditionally shown a poor concordance with occurrence of IADRs inhumans [4]. Life-threatening IADRs noted for drugs include hepatotoxicity [5], severecutaneous reactions [6], aplastic anemia [7], and blood dyscrasias [8]. Amongst these,hepatotoxicity is the most frequent reason for drug withdrawal and is also the majorcause of attrition in drug discovery/development [9]. Manifestations of liver injury canrange from mild, asymptomatic changes in serum transaminases, which occur at highfrequency with a number of drugs, to fulminant liver failure, which, although rare, canbe potentially life-threatening and may necessitate a liver transplant. Considering thatthe liver is exposed to high concentrations of drug/metabolite(s) after oral admin-istration, it is not altogether surprising that the organ is particularly vulnerable todamage by xenobiotics including drugs.

Drug Metabolism and Toxicity. – Xenobiotics including drugs are usuallymetabolized via oxidative, reductive, and hydrolytic pathways (phase-I reactions),which lead to the introduction or exposure of a functional group (OH, SH, NH2, orCOOH) and to a modest increase in aqueous solubility; and with a few exceptions (e.g.,O-methylation and N-acetylation), phase-II conjugations modify the newly introducedfunctionality to form O- and N-glucuronides, sulfate and acetate esters, andglutathionyl adducts, all with increased hydrophilicity relative to the unconjugatedmetabolite. In most cases, metabolism results in the loss of biological activity of theparent drug, and such metabolic reactions are, therefore, regarded as detoxicationpathways. However, depending on the structural features present in some compounds,the same metabolic events on occasion can generate electrophilic, reactive metabolites,a process referred to as bioactivation. In the liver, most oxidative and/or reductive

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)2116

bioactivation reactions are mediated by cytochrome P450 enzymes (CYPs). There areseveral instances where bioactivation can also be catalyzed by non-CYP enzymes suchas monoamine oxidases, peroxidases, aldehyde oxidase, and/or alcohol dehydrogenases.Likewise, phase-II conjugation pathways including glucuronidation and sulfonation arealso known to transform latent functional groups such as carboxylic acids andhydroxylamines into reactive intermediates. In some cases, reactive metaboliteformation can involve a single enzymatic reaction, whereas in other instances, multipleenzymatic and/or chemical steps are required to form the electrophilic metabolites.Besides liver, drug-metabolizing enzymes in organs such as the lung, kidney, and skinare also known to bioactivate xenobiotics and drugs. Inadequate detoxication ofreactive metabolites is thought to represent a pathogenic mechanism for tissue necrosis,carcinogenicity, teratogenicity, and/or immune-mediated toxicities.

Consequences of Reactive Metabolite Formation. – Mechanism-Based Inactivationof CYP Enzymes. In many instances, CYP-mediated oxidative metabolism of drugsand/or constituents of herbal supplements leads to irreversible enzyme inactivation bythe reactive metabolite prior to its release from the active site [10]. Becausemetabolism precludes enzyme inactivation, these compounds fall into the category ofmechanism-based inactivators. Inactivation can occur via coordination of the reactivespecies with the heme prosthetic group (formation of a metabolite – inhibitor (MI)complex) or via covalent adduction of the reactive intermediate with heme and/or withan amino acid residue on the apoprotein. CYP Inactivation can translate into clinicaldrug – drug interactions (DDIs), some of which can be potentially deleterious, and canlead to the withdrawal of the perpetrator drug. For instance, co-administration of thecalcium-channel blocker and potent CYP3A4 inactivator mibefradil (Fig. 1) andsimvastatin in patients with hypertension has been associated with increased cases ofmyopathy including rhabdomyolysis. The biochemical mechanism for the clinical DDIsinvolves the mechanism-based inactivation of the CYP3A4-catalyzed simvastatinmetabolism process by mibefradil; a consequence which results in elevated plasmaconcentrations of the statin [11] [12]. Myopathy or rhabdomyolysis is a rare side-effectcommon to the statin class of compounds and is usually associated with high levels ofHMG-CoA reductase inhibitory activity in susceptible target tissue. Given thepotential for such life-threatening DDIs, the manufacturer of mibefradil announceda voluntary withdrawal of the drug from the market worldwide. An additional exampleof such DDI phenomenon is evident with the herbal medicine kava (Pipermethysticum), which has proven effective in treating anxiety and insomnia. Its


Fig. 1. Chemical structure of the calcium-channel blocker and potent CYP3A4 inactivator mibefradil

therapeutic benefits have been hampered by several reports of clinically significantherb – drug interactions upon concomitant use with benzodiazepines and barbiturates[13] [14], and several cases of idiosyncratic hepatotoxicity have also been reported [15].Consequently, over-the-counter sales of kava herbal preparations have been banned inseveral countries in the European Union. Studies on CYP inhibition with kavalactonederivatives methysticin and 7,8-dihydromethysticin (Scheme 1), the major constituentsof kava extract [16], have revealed potent mechanism-based inactivation of multiplehuman CYP enzymes [17]. The time- and NADPH-dependent formation of the 455-nmabsorbing MI complex is consistent with a bioactivation mechanism involving themetabolism of the 1,3-benzodioxole group to the corresponding carbene intermediatefollowed by its coordination with the heme iron (Scheme 1) [17]. Consistent with thisfinding, the kavalactone kawain (see Scheme 1), which differs from methysticin in thatit does not contain the 1,3-benzodioxole group is not a CYP inactivator [17].Glutathione (GSH) conjugates of electrophilic ortho-quinone intermediates obtainedvia the biotransformation sequence (1,3-benzodioxole!catechol!ortho-quinone) inmethysticin and 7,8-dihydromethysticin have also been identified in rat and human livermicrosomes (Scheme 1), and the involvement of these reactive quinonoid intermedi-ates in the immunoallergic hepatotoxic effects of kava extract has been speculated [18].


Scheme 1. Proposed Mechanisms of CYP Inactivation and Hepatotoxicity by Components of KavaExtract

Overall, given the potential for DDIs via enzyme inactivation, mechanism-basedinactivation of major human CYP enzymes by new compounds is routinely assessed in adrug-discovery paradigm. For compounds that exhibit CYP inactivation, follow-onstudies specifically designed to elucidate the mechanism of CYP inactivation are oftenpursued. Such studies often include metabolite-identification efforts involving charac-terization of downstream stable metabolites or conjugates of reactive metabolitesobtained via reaction with H2O or exogenously added trapping agents, respectively, aswill be discussed later. Elucidation of the structure(s) of the stable downstreammetabolites provides an insight into the mechanism of enzyme inactivation and on thebasis of which rational chemical manipulation strategies can be incorporated toeliminate CYP inactivation liabilities. Likewise, methodology to predict the magnitudeof in vivo metabolic DDIs using in vitro CYP inactivation data and predicted humanpharmacokinetics of the candidate drug have been established [19].

Mutagenicity via DNA-Adduct Formation. The concept of genotoxic/mutagenicresponse arising from metabolism was first proposed in the 1930s and the 1940s toaccount for the carcinogenicity of chemically inert polycyclic aromatic hydrocarbons,aminoazo dyes, and nitroso compounds [20] [21]. All pharmaceutical companies utilizea standard battery of genetic toxicology assays to test the mutagenic potential of drugcandidates [22]. These assays measure several different types of genetic damage in avariety of cell types to increase the probability of detecting a mutagenic response. Theendpoints routinely monitored include the induction of point mutations andchromosomal aberrations in bacteria and mammalian cells, respectively, as well asthe production of strand breaks, DNA intercalation, and covalent modification. Anariclor-1254-induced rat liver S-9/NADPH system has been adopted in these in vitrotests for detecting promutagens capable of forming DNA-reactive metabolites [23].

Genetic toxicology assessments have become an integral part of drug safetyevaluation and are required by regulatory agencies for drug approvals worldwide.Because a good correlation has been established between in vitro metabolism-dependent mutagenic response and the outcome of rodent carcinogenicity evaluations,drug candidates intended for non-life-threatening indications are generally discon-tinued from development, when they exhibit a positive response in the in vitro assays inthe presence of S-9/NADPH. An example of this phenomenon was recently highlightedwith our work on the anti-obesity agent and 5-hydroxytryptamine (5-HT)2C agonist 2-(3-chlorobenzyloxy)-6-(piperazin-1-yl)pyrazine (1; Scheme 2) [24]. The attractive invitro/in vivo pharmacology and pharmacokinetic attributes of 1 were offset by its S-9/NADPH-dependent genotoxic effects in the bacterial Salmonella Ames assay, whichled to its discontinuation from clinical development. Studies with [14C]-1 revealed theirreversible and concentration-dependent incorporation of radioactivity in calf thymusDNA in an S-9/NADPH-dependent fashion confirming that 1 was bioactivated to aDNA-reactive metabolite. Reactive-metabolite trapping studies in S-9/NADPHincubations containing exogenously added hard and soft nucleophilic trapping agentsmethoxylamine and GSH, respectively, led to the detection of conjugates of 1 and itsdownstream metabolites. Structural elucidation of these conjugates by mass spectrom-etry allowed an insight into the bioactivation pathways leading to the formation ofDNA-reactive metabolites. The mass spectrum of the methoxylamine conjugate of 1was consistent with condensation of amine with an electrophilic, aldehyde metabolite


derived from piperazine ring scission in 1 (Scheme 2, Pathway a), whereas, the massspectrum of the GSH conjugate suggested a bioactivation pathway involving initialaromatic ring hydroxylation on the 3-chlorobenzyl motif in 1, followed by b-eliminationto a quinone-methide species that reacted with GSH (Scheme 2, Pathway b). Theobservation that methoxylamine and GSH reduced mutagenicity suggested that thetrapping agents competed with DNA towards reaction with the reactive metabolites.Overall, the exercise provided indirect information on the structure of DNA-reactiveintermediates leading to mutagenic response with 1 and hence a rationale on which tobase subsequent chemical intervention strategy for designing non-genotoxic 5-HT2C

agonists.

Type-B ADRs. The relationship between drug metabolism and ADRs was firstdemonstrated with the anti-inflammatory agent paracetamol (acetaminophen). Para-cetamol is a major cause of drug-related morbidity and mortality in humans, capable ofproducing hepatic necrosis after a single toxic overdose [25]. The work by Brodie andco-workers, and others [26 – 30], which demonstrated CYP-mediated bioactivation ofparacetamol to a reactive quinone-imine intermediate (NAPQI), capable of depletingGSH levels and binding covalently to liver macromolecules (Scheme 3), has served as aparadigm for drug-toxicity assessment over the decades and also resulted in the clinicaluse of the orally available GSH analog N-acetylcysteine as an antidote for paracetamolpoisoning. N-Acetylcysteine replenishes hepatic anti-oxidant levels and preventsfurther damage to the liver.

Scheme 2. Postulated Bioactivation Pathways Which Explain the Mutagenicity of the Anti-Obesity Agent2-(3-chlorobenzyloxy)-6-(piperazin-1-yl)pyrazine (1) in the Salmonella Ames Assay


Following the studies with paracetamol, there have been myriad examples of drugsassociated with IADRs for which reactive-metabolite formation has been demon-strated [31]. These examples provide a circumstantial link between reactive-metaboliteformation and toxicity. It is very important to make a distinction here between drugsthat exhibit dose-dependent and dose-independent ADRs. The hepatotoxic effects ofparacetamol in humans can hardly be considered as idiosyncratic as they are dose-dependent and can be replicated in animals [26 –30]. In contrast, drugs like theantidiabetic agent troglitazone, which exhibits dose-independent hepatotoxicity in avery small segment of the population, is a true example of an idiosyncratic toxin, giventhat this compound received a �clean bill of health� in conventional animal toxicologicalassessments.

Downstream Consequences of Reactive-Metabolite Formation. –While the in vitrodetection of reactive metabolites can be relatively straightforward, the downstream invivo consequences relative to IADR (particularly immune-mediated ones) occurrenceare poorly understood, especially given the lack of predictive animal models andclinical safety biomarkers for these reactions. Several hypotheses, however, have beenproposed to explain these phenomena. The basic hypothesis that links the formation ofreactive metabolites with IADRs is the process of haptenization wherein low-molecular-weight (<1 kDa) reactive metabolites are converted to immunogens viabinding to high-molecular-weight proteins as is the case with penicillin-inducedanaphylactic reactions [32] [33]. It is clear that specific immunoglobulin (IgE)antibodies against penicillin mediate these reactions via an initial haptenizationprocess involving nonenzymatic b-lactam-ring scission by cysteinyl and/or terminallysine residue(s) in proteins, leading to the acylation of these amino acid nucleophiles[34] [35]. Immune-mediated IADRs can also result from drug-specific T-lymphocytesvia presentation of the hapten to T-cells as demonstrated in the course of b-lactam- andsulfamethoxazole-induced skin rash [33] [36]. In the case of the antibacterial agentsulfamethoxazole, the immune activation is thought to involve a CYP-catalyzedbioactivation of the aniline group to a reactive nitroso metabolite capable of covalentlybinding to cellular constituents. The finding that sulfamethoxazole can also be oxidizedin keratinocytes suggests that bioactivation and T-cell sensitization may occur directlyin the skin leading to cutaneous ADRs in susceptible population [37]. Additional

Scheme 3. CYP-Catalyzed Bioactivation of the Anti-Inflammatory Agent Paracetamol


examples of drugs associated with haptenization include halothane, tienilic acid, anddihydralazine, all of which are bioactivated to reactive metabolites and displaymechanism-based inactivation of the CYP isozymes responsible for their metabolism.Consistent with these observations, antibodies detected in sera of patients exposed tothese drugs specifically recognize CYP isozymes, responsible for their metabolism [38–42].

Perhaps the most important susceptibility factor for IADRs is genetic variability.Genetic polymorphisms have a strong influence on drug metabolism and may increaserisk of toxicity. For example, polymorphism of the N-acetyltransferase (NAT) 2 genedifferentiates fast from slow acetylators; the latter have increased susceptibility totoxicity of certain aniline-containing drugs such as isoniazid, sulfamethoxazole,dapsone, and procainamide [43] [44]. The major route of elimination of these drugsin humans involves N-acetylation of the aniline moiety by NAT2, resulting in theneutral amide metabolites. In a NAT2-deficient population, the aniline motif inisoniazid is hydrolyzed by amidases liberating hydrazine, which is toxic in its own right;likewise, sulfamethoxazole, dapsone, and procainamide are biotransformed by CYPenzymes to yield cytotoxic and protein-reactive metabolites that include N-hydroxy-aniline derivatives and the subsequent two-electron oxidation products, i.e., the nitrosointermediates. The reactivity of the nitroso metabolites of these drugs with GSH and/orproteins in target organs has also been demonstrated [45] [46]. Genetic polymorphismsin glutathione-S-transferase (GST) isozymes, which catalyze GSH conjugation toreactive metabolites, are also considered risk factors for hepatotoxicity caused byseveral drugs such as troglitazone and carbamazepine [47] [48]. It is possible thatpatients deficient in GST isozymes are most at risk towards liver injury by reactivemetabolites of troglitazone and carbamazepine because of ineffective scavenging of thereactive metabolites derived from the oxidative bioactivation of these drugs. There isalso a strong possibility that components of ingested foods including herbal supple-ments can modulate drug metabolism and, therefore, increase IADR risk. For example,chronic alcohol abuse increases the risk of paracetamol hepatotoxicity by inducingCYP2E1, which predominantly catalyzes paracetamol bioactivation to NAPQI [49].

Evaluating Bioactivation Potential of New Chemical Entities (NCEs) in DrugDiscovery. – Given the lack of availability of preclinical models as reliable predictors ofIADRs and the absence of relevant clinical safety biomarkers, it is currently impossibleto accurately predict which new drugs will be associated with a significant incidence ofIADRs. Under the assumption that reactive metabolites, as opposed to the parentmolecules from which they are derived, can be responsible for the pathogenesis ofcertain toxicities, most pharmaceutical companies have implemented assays to evaluatea compound�s potential to undergo bioactivation with the goal of eliminating orminimizing reactive-metabolite formation by rational structural modification of theproblematic chemical series.

Reactive-Metabolite Trapping. With the possible exception of acyl glucuronides andcyclic iminium ions, most reactive metabolites are generally short-lived and are notusually detectable in circulation. Their formation can often be inferred from stableconjugates obtained via reaction with the endogenous anti-oxidant GSH, which ispresent at ca. 10 mm concentration in mammalian liver. The presence of the soft


nucleophilic sulfhydryl group in glutathione ensures efficient conjugation with softelectrophilic centers on reactive species (e.g., Michael acceptors, epoxides, areneoxides, and alkyl halides) yielding stable sulfhydryl conjugates [31] [50]. Qualitative invitro assessment of reactive-metabolite formation usually involve �trapping� studiesconducted with NADPH-supplemented human liver microsomes and GSH; analysis ofthe resulting metabolites by mass spectrometry is employed to characterize thestructure of GSH conjugates, which, in turn, provides insight into the reactivemetabolite structure. Considering that drug-metabolizing enzymes other than cyto-chrome CYP (e.g., monoamine oxidases, aldehyde oxidase, alcohol dehydrogenases,myeloperoxidase, uridine 5’-diphosphoglucuronosyl transferase (UGT), and sulfo-transferases) are also capable of catalyzing bioactivation, due consideration must begiven to the use of alternate metabolism vectors (e.g., S-9 fractions, hepatocytes,neutrophils, monocytes, etc.), which support the activity of these enzymes. This isespecially important in cases where multiple enzymatic and/or chemical steps may beinvolved in the production of the reactive metabolite. It is noteworthy to point out thatnot all reactive metabolites can be trapped with GSH. Hard electrophiles includingDNA-reactive metabolites (e.g., electrophilic carbonyl compounds) will preferentiallyreact with hard nucleophiles such as amines (e.g., semicarbazide and methoxylamine),amino acids (e.g., lysine), and DNA bases (e.g., guanine and cytosine) affording thecorresponding Schiff bases [24]. Likewise, the cyanide anion is a �hard� nucleophile thatcan be used to trap electrophilic iminium species that are generated via metabolism oftertiary amines [51].

Covalent-Binding Studies. Assessment of the amount of in vitro metabolism-dependent covalent binding to biological tissue (e.g., microsomes, S-9, hepatocytes,neutrophils, etc.) is possible if radiolabeled drug is available [52]. The assay providesquantitative estimates of radioactivity irreversibly bound to tissue but does not provideinformation about the nature of covalently modified proteins. Covalent-binding studiescan be performed in vivo as well. Either tissue or blood/plasma can be examined for thedegree of covalent binding. However, covalent binding may require multiple dosing toestablish the true impact of the compound. Reactive metabolites formed after the firstdose may be efficiently trapped by GSH and eliminated from the body. Once GSH isdepleted, the extent of covalent binding with cellular macromolecules may increaserapidly, resulting in toxicity. This is certainly the case with paracetamol where itsreactive metabolite can cause direct hepatotoxicity upon an overdose, and yetparacetamol is rarely associated with IADRs. This is because, at usual therapeuticdoses, the phenolic group in paracetamol undergoes phase-II gluronidation andsulfonation, resulting in a small amount of NAPQI formed; most of which is scavengedby GSH before it binds to macromolecules. The hypothesis has been strengthenedbased on studies in mice which have shown that significant covalent binding does notoccur until over 60– 80% of the paracetamol overdose has been eliminated from theliver with concomitant reduction in GSH levels [26 – 30]. An example of this overallapproach is highlighted with studies on the potassium-channel opener, maxipost (BMS-204352) (Scheme 4), which undergoes CYP-mediated bioactivation in rats, dogs, andhumans to generate a reactive ortho-quinone-methide intermediate, which covalentlybinds to albumin in vivo in animals and human [53] [54]. Acidic hydrolysis of plasmacollected after intravenous administration of [14C]-BMS-204352 to rats and human led


to the characterization of a unique lysine conjugate of desfluoro de-O-methyl BMS-204352 (see Scheme 4). The relevance of the in vivo covalent-binding observation withregards to maxipost toxicity remains unknown.

Structure –Toxicity Relationships. – The availability of methodology to assessbioactivation potential of drugs has clearly aided to replace a vague perception of achemical-class effect with a sharper picture of individual molecular peculiarity.Information to qualify certain functional groups as structural alerts also has beeninferred from such studies based on numerous examples of drugs containing thesemotifs which are bioactivated to reactive metabolites and are associated with IADRs[55]. The presence or absence of a structural alert/toxicophore within a chemicalstructure can be inspected visually or via the use of the DEREK software. DEREK forWindows is a knowledge-based expert system that is often used to identify structuralalerts in a chemical. Its predictions are based in part on alerts that describe structuralfeatures or toxicophores associated with toxicity [56]. To date, there are no in silicotools that have been successfully utilized to predict the occurrence of a bioactivationpathway (and ensuing toxicity) with a drug candidate. While in silico tools such asMetaSite have been used to predict metabolic pathways of drug candidates with somesuccess [57], the utility of such software in predicting bioactivation has not beenexploited in drug discovery, partly due to lack of validation of such an approach.Electrochemical tools have been used to mimic oxidative drug metabolism, includingthe formation of reactive metabolites [58]. However, this approach requires experi-mental validation.

Of much interest within the overall context are examples of �successor� drugs whichare latent to bioactivation and toxicity because of the absence of the structural alertpresent in the toxic �predecessor�. While the evidence from such structure – toxicityexaminations is anecdotal at best, such analyses present a compelling case forchemotype-based liability as exemplified with the dibenzodiazepine derivatives andantipsychotic agents clozapine and quetiapine. While clozapine use is limited by a high

Scheme 4. Bioactivation of the Calcium-Channel Opener Maxipost in Rat and Human to a ReactiveQuinone-methide, Which Covalently Binds to Albumin in vivo


incidence of agranulocytosis, hepatotoxicity, and myocarditis, quetiapine does notcause these toxic events. Clozapine exhibits covalent binding to human neutrophilproteins in vitro, via a bioactivation pathway involving myeloperoxidase-mediatedoxidation of the dibenzodiazepine ring to a reactive iminium ion, which covalentlybinds to the target tissues and also reacts with GSH (Scheme 5) [59] [60]. Proteinscovalently modified with clozapine have been observed in neutrophils of patients beingtreated with clozapine, which reaffirms the relevance of the in vitro studies. In the caseof quetiapine, the bridging N-atom is replaced with a S-atom; consequently this drug isnot bioactivated to the reactive iminium species [61]. Despite administration at dosescomparable to clozapine, cases of agranulocytosis with quetiapine are extremely rare.

A second example of this phenomenon is evident when comparing the inhaledanesthetic and idiosyncratic hepatotoxin halothane with isofluorane and desfluorane.Bioactivation of the alkyl halide substituents in inhaled anesthetics to reactive acylatingagents is usually due to the availability of an extractable H-atom on the halogenatedalkyl C-atom. In susceptible patients, halothane, isoflurane, and desflurane can producesevere hepatic injury by an immune response directed against reactive acyl halidescovalently bound to hepatic biomacromolecules [38]. The relative incidence ofhepatotoxicity due to these agents appears to directly correlate with the extent of theirconversion to acyl halides by CYP, which in turn may be governed by the leaving-groupability of the respective substituents within these drugs. As is seen in Scheme 6,halothane, which exhibits the greatest incidence of hepatotoxicity in the clinic,undergoes the most conversion to reactive acyl chloride, a feature that can be attributed

Scheme 5. Bioactivation Pathway of the Antipsychotic Drug Clozapine – Structure–Toxicity Relation-ships


to the presence of Br substituent, which is a good leaving group. In contrast, isofluoraneand desfluorane also undergo oxidative metabolism resulting in the formation ofreactive acyl halides, but the degree to which these anesthetics are bioactivated issignificantly lower than halothane [62]. The lower yield of acyl halide formation withisofluorane may be traced back to changes in the electronic environment that reduceoverall affinity towards metabolism or to the relatively poor leaving-group ability of thedifluoromethoxy group compared to the Br.

A third example is highlighted with the CNS agents nefazodone and buspirone.Since its introduction in 1994, antidepressant therapy with nefazodone has beenassociated with several reported cases of idiosyncratic hepatotoxicity resulting in >20deaths [63]. In humans, para-hydroxynefazodone, a major circulating metabolite ofnefazodone [64], undergoes in vitro CYP-mediated bioactivation to electrophilicquinonoid intermediates, which are trapped as GSH conjugates (Scheme 7) [65]. Inaddition, reactive iminium ion intermediates arising from a-C oxidation of thepiperazine ring and its downstream metabolites have been characterized as stablecyanide conjugates (Scheme 7) [66]. Nefazodone bioactivation by CYP3A4 is alsoaccompanied by mechanism-based inactivation of the isozyme, which is consistent withDDIs between nefazodone and CYP3A4 substrates [67] [68]. As in the case ofnefazodone, para-hydroxybuspirone also represents a major circulating metabolite ofthe anxiolytic agent buspirone (Scheme 7) [69]. However, the failure to detect GSHconjugates in microsomal incubations of buspirone suggests that the drug is not proneto bioactivation [65]. The lack of bioactivation of buspirone in a manner similar tonefazodone is consistent with ab initio calculations that suggest that a weakerresonance stabilization of the oxidation products and the greater acidity make para-hydroxybuspirone less favorable for the two-electron oxidation (Scheme 7). Theseobservations appear to correlate with the lack of idiosyncratic hepatotoxicity withbuspirone despite decades of clinical use.

These examples imply that, by avoiding structural alerts in drug design, one wouldlessen the odds that a drug candidate will lead to toxicity due to reactive-metaboliteformation. However, it is noteworthy to point out that an exhaustive listing of structuralalerts also includes the phenyl ring which in the course of metabolism to a phenol forms

Scheme 6. CYP-Mediated Bioactivation of Inhaled Anesthetics


an electrophilic epoxide intermediate [55]. Certainly, this is the mechanism underlyingthe toxicity of the organic solvents benzene and bromobenzene [70] [71]. Likewise,avoiding structural alerts altogether can lead to missing out on potentially importantmedicines, ultimately beneficial to humanity. Certainly, this is the case withatorvastatin, which not only contains the acetanilide structural alert, but metabolismby CYP results in the formation of paracetamol-like metabolites (Fig. 2) [72]. Inaddition, glucuronidation of its carboxylic acid moiety results in the formation of thepotentially electrophilic acyl glucuronide, the likes of which, have been implicated inthe toxicity associated with the carboxylic acid-containing non-steroidal anti-inflam-matory drugs (cf. Fig. 3) [55] [73].

Therefore, rather than randomly applying such a conservative approach in drugdiscovery, it is pivotal to consider factors that could influence reactive metaboliteformation in any given molecule which contains a structural alert. These include: 1)


Scheme 7. Comparison of the Bioactivation Potential of Nefazodone (Hepatotoxin) and Buspirone(Non-Hepatotoxin)

does the molecule contain an alternate metabolic soft spot that competes withstructural alert metabolism, and 2) are there metabolic pathways that efficientlyscavenge the reactive metabolite and/or its precursor. In the latter case, bioactivationmay be discernible in standard in vitro systems, but the principal clearance mechanismin vivo may involve a distinctly different and perhaps more facile metabolic fate thatdoes not yield reactive metabolites. The importance of the first point becomes evidentupon comparison of the bioactivation potential of the catechol-O-methyltransferaseinhibitors tolcapone and entacapone. Tolcapone use has been associated withidiosyncratic hepatotoxicity (including death), which has resulted in its withdrawal inseveral countries, and the introduction of a black-box warning and intensive monitoringrequirements in the United States. Hepatotoxic IADRs, however, do not occur with theuse of the structurally related drug entacapone despite administration at doses similarto tolcapone (200 – 1000 mg QD). In humans, tolcapone is biotransformed viareduction of the nitrocatechol group to the aminocatechol derivative, which is thenconverted to the acetanilidocatechol metabolite by NAT (Scheme 8) [74]. Macdonaldand co-workers [75] showed that both metabolites are oxidized to reactive quinone-imine metabolites, which are trapped by GSH (Scheme 8). While entacapone alsopossesses the 3-nitrocatechol toxicophore, the primary metabolic pathway in humansdoes not involve reductive bioactivation; instead it involves N-deethylation of thetertiary amide, followed by glucuronidation of the catechol motif [76].

Concerning the second point on reactive metabolite detoxication, an examplewhich comes to mind is that of the selective estrogen-receptor modulator raloxifene.Raloxifene has been shown to undergo in vitro CYP3A4-catalyzed bioactivation on its


Fig. 2. Chemical structures of atorvastatin and its metabolites derived from oxidative and conjugationpathways

phenolic groups to yield reactive quinonoid species (Scheme 9), a phenomenonassociated with CYP3A4 irreversible inactivation, microsomal covalent binding, andformation of GSH conjugates [77]; however, in vivo, glucuronidation of the samephenolic groups in the gut and liver constitute the principal elimination mechanism ofraloxifene in humans (Scheme 9) [78]. Thus, the likelihood of raloxifene bioactivationin vivo is in question when compared with the phase-II glucuronidation process, aphenomenon that may provide an explanation for the extremely rare occurrence ofIADRs and lack of DDIs with CYP3A4 substrates.

Are Reactive Metabolite Trapping and Covalent-Binding Studies ReliablePredictors of IADR Potential of Drug Candidates? – The raloxifene examplechallenges the basic premise of using reactive metabolite-trapping/covalent-bindingstudies to predict idiosyncratic toxicity with drug candidates. While the detection ofGSH adducts and/or covalent binding to target tissue is indicative of the formation ofreactive metabolites, the data needs to be placed in proper context prior to making adecision on discarding a drug candidate associated with this liability. Our recent studiesexamining CYP-mediated covalent binding of 18 drugs (nine hepatotoxins and ninenon-hepatotoxins) to liver microsomes, S-9, and/or hepatocytes show no correlationbetween extent of covalent binding and toxicity. In fact, our work revealed that severalblockbuster drugs were false positives in these assays; i.e., they form GSH conjugatesand displayed a degree of protein covalent modification. The retrospective analysis ofselective serotonin reuptake inhibitor paroxetine serves as a notable example of thisphenomenon [79]. The NADPH-dependent covalent binding of [3H]paroxetine tohuman liver microsomes and liver S-9 fractions is consistent with a bioactivationpathway involving CYP2D6-mediated demethylenation of the 1,3-benzodioxozolegroup in paroxetine to a catechol metabolite, followed by a two-electron oxidationof this intermediate to reactive ortho-quinonoid intermediates. The characterizationof the GSH conjugates of paroxetine-catechol lends credence to this hypothesis


Scheme 8. Differential Metabolism of the Anti-Parkinsonian Drugs Tolcapone (Hepatotoxin) andEntacapone (Non-Hepatotoxin)

(Scheme 10). Thus, paroxetine fulfills all the obligatory requirements of a moleculeprone to bioactivation and, in the absence of any additional metabolism data, such anisolated finding could be interpreted as being a harbinger of a potential toxicologicalresponse in the clinic. However, despite decades of clinical use, life-threatening ADRs(e.g., hepatotoxicity) have been rarely noted with paroxetine. Plausible reason(s) forthis anomaly can be gauged from the additional experimental findings wherein additionof GSH and catechol-O-methyltransferase co-factor S-adenosylmethionine to themicrosomal and/or S-9 incubations results in a dramatic reduction of covalent binding.The finding that GSH drastically reduces covalent binding of paroxetine is analogous tothe case of paracetamol, where hepatotoxicity is only observed at supratherapeuticdoses (>4 g/day) believed to exceed the capacity of the liver to form GSH adducts withthe reactive NAPQI intermediate. The amount of ortho-quinone formed in vivo afteradministration of paroxetine at its daily dose of 20 mg/day may be readily handled bythe liver�s pool of GSH. In addition, reduction in covalent binding to S-9 in the presenceof S-adenosylmethionine is consistent with the known metabolic pathway of paroxetinein humans involving O-methylation of the paroxetine-catechol metabolite to thecorresponding guaiacol regioisomers (see Scheme 10). Overall, the results of thesestudies indicate that efficient scavenging of the catechol and quinone metabolites by S-adenosylmethionine and GSH, respectively, and its low daily dose serve as potentialexplanations for the excellent safety record of paroxetine despite undergoingbioactivation.


Scheme 9. Bioactivation and Competing Detoxication Pathways of the Selective Estrogen-ReceptorModulator Raloxifene

Effect of Daily Dosing Regimen. – Perhaps the most important factor in migratingIADR risks is the daily dose of the drug candidate. There are no examples of drugs thatare dosed at <20 mg/day that cause IADRs (whether or not these agents are prone tobioactivation). There are many examples of two structurally related drugs that possessidentical toxicophore susceptible to bioactivation, but the one administered at thelower dose is safer than the one given at a higher dose. It is likely that the improvedsafety of low-dose drugs arises from a marked reduction in the total body burden toreactive metabolite exposure and, therefore, unlikely to exceed the threshold neededfor toxicity. An illustration of this concept is evident with the antidiabetic thiazolidi-nedione drugs troglitazone, rosiglitazone, and pioglitazone. Troglitazone (Scheme 11)was withdrawn from the United States market after numerous reported cases of liver

Scheme 10. Parallel Detoxication Pathways that Compete with the CYP-Catalyzed Bioactivation Pathwayof the Anti-Depressant Paroxetine as Explanation for Its Wide Safety Margin


failures requiring immediate liver transplantation or leading to death. In contrast,rosiglitazone and pioglitazone are devoid of the hepatotoxicity associated withtroglitazone. In vitro, troglitazone is bioactivated by CYP3A4 in NADPH- and GSH-supplemented human liver microsomes generating several conjugates [80] [81]. Basedon the structures of these conjugates, the two proposed pathways for the bioactivationof troglitazone include metabolism of the chromane ring to the quinone or ortho-quinone-methide, and oxidative cleavage of the thiazolidenedione ring (Scheme 11).While rosiglitazone and pioglitazone do not contain the chromane ring system found introglitazone, they do contain the thiazolidinedione scaffold. And consistent with thefindings with troglitazone, both rosiglitazone and pioglitazone have been shown toundergo thiazolidinedione ring scission mediated by CYP enzyme(s) in humanmicrosomes resulting in reactive metabolites which are trapped by GSH [82]. Whilebioactivation of the thiazolidenedione ring is a common theme in these drugs, a keydifference lies in their daily doses – troglitazone (200 –400 mg/day) and rosiglitazone


Scheme 11. CYP-Mediated Bioactivation of the Thiazolidinedione Class of Anti-Diabetic Agents

and pioglitazone (<10 mg/day). This factor may offset the bioactivation liabilityresulting in an improved safety profile for the successor agents.

Likewise, the dibenzodiazepine derivative olanzapine (Fig. 3) forms a reactiveiminium metabolite very similar to the one observed with clozapine, yet olanzapine isnot associated with a significant incidence of agranulocytosis. One difference betweenthe two drugs is the daily dose; clozapine is given at a dose of >300 mg/day, while themaximum recommended daily dose of olanzapine is 10 mg/day. Additional examples ofthis phenomenon are illustrated with tadalafil, the antihypertensive prazosin and theestrogen ethinylestradiol (see Fig. 3). The 1,3-benzodioxole group in tadalafil under-goes CYP3A4-catalyzed bioactivation to an electrophilic catechol, a process that alsoleads to the suicide inactivation of CYP3A4 activity in vitro [83]. However, to datethere are no reports of IADRs or CYP3A4 drug – drug interactions associated withtadalafil use at the recommended dose of 10– 20 mg/day. Likewise, there are no reportsof IADRs with prazosin at the recommended daily dose of 1 mg/day, despite thebioactivation of the pendant furan ring to electrophilic intermediates, which have beentrapped with GSH and semicarbazide [84]. Ethinylestradiol, the major estrogeniccomponent of many oral contraceptives, undergoes CYP3A4-catalyzed bioactivation attwo different regions within its scaffold (phenol bioactivation to catechol and acetylenebioactivation to ketene), which results in the mechanism-based inactivation of CYPisozyme and a high degree of covalent binding in vitro [85], but does not lead to IADRsin the clinic. The extremely low dose of 35 mg must represent a significant mitigatingfactor. Finally, it is noteworthy to point out that the daily dose of atorvastatin, which isbiotransformed to potential precursors of reactive metabolites is 10 mg.

Concluding Remarks. – The ability to predict the potential of a drug candidate tocause IADRs is dependent on a better understanding of the pathophysiological

Fig. 3. Examples of low-daily-dose drugs devoid of IADRs despite bioactivation liability


mechanisms of such reactions. IADRs are too complex to duplicate in a test tube, andtheir idiosyncratic nature precludes prospective clinical studies. Genetic factors appearto have a crucial role in the induction of IADRs. A fruitful approach may, therefore, liein focused and well-controlled phenotype/genotype studies of the rare patients whohave survived this type of injury as has been shown with the HIV agent abacavir, whichis associated with hypersensitivity [86]. Until we develop a better understanding of therisk of toxicity arising from the formation of reactive metabolites, a strategy foridentifying and potentially minimizing their formation via rational and iterativemedicinal chemistry efforts seems logical in certain cases. It is noteworthy to point outthat reactive-metabolite formation is only one aspect of the overall risk/benefitassessment for advancing a drug candidate into development. Consequently, bioacti-vation data (reactive metabolite trapping and/or covalent binding) needs to be placedin a broader context with due consideration given to the following points:

1) Is the drug intended to address a previously unmet medical need or a life-threatening disease?

2) Is the drug candidate intended to provide proof-of-mechanism for a noveltarget?

3) Is the drug intended for acute or chronic use?4) Is the clinical dose predicted to be low?5) What is the intended patient population (e.g., would it be given to immune-

comprised patients or patients with impaired liver functions)?6) Are there alternate chemical series with comparable pharmacologic and

pharmacokinetic attributes, wherein bioactivation liability is minimized oreliminated?

7) Is there an alternative (higher affinity but innocuous) route of metabolismwithin the drug candidate that minimizes bioactivation liability associated withthe compound?

8) Is metabolism the exclusive route of elimination? What is the likelihood ofnonmetabolic elimination processes (e.g., renal and/or biliary excretion ofunchanged parent) in humans?

It is noteworthy to point out that similar �qualifying considerations� have also beenrecently presented by the Merck group [87].

REFERENCES

[1] I. Kola, J. Landis, Nat. Rev. Drug Discovery 2004, 3, 711.[2] J. A. Kramer, J. E. Sagartz, D. L. Morris, Nat. Rev. Drug Discovery 2007, 6, 636.[3] K. E. Lasser, P. D. Allen, S. J. Woolhandler, D. U. Himmelstein, S. M. Wolfe, D. H. Bor, J. Am. Med.

Assoc. 2002, 287, 2215.[4] H. Olson, G. Betton, D. Robinson, K. Thomas, A. Monro, G. Kolaja, P. Lilly, J. Sanders, G. Sipes, W.

Bracken, M. Dorato, K. Van Deun, P. Smith, B. Berger, A. Heller, Regul. Toxicol. Pharmacol. 2000,32, 56.

[5] M. Biour, R. Poupon, J. D. Grange, O. Chazouilleres, Gastroenterol. Clin. Biol. 2000, 24, 1052.[6] R. Wolf, E. Orion, B. Marcos, H. Matz, Clin. Dermatol. 2005, 23, 171.[7] K. B. Handoko, P. C. Souverein, T. P. van Staa, R. H. Meyboom, H. G. Leufkens, T. C. Egberts, P. M.

van den Bemt, Epilepsia 2006, 47, 1232.[8] E. Andres, L. Frederici, T. Weitten, T. Vogel, M. Alt, Expert Opin. Drug Saf. 2008, 7, 481.


[9] N. Kaplowitz, Clin. Infect. Dis. 2004, 38, S44.[10] A. S. Kalgutkar, R. S. Obach, T. S. Maurer, Curr. Drug Metab. 2007, 8, 407.[11] T. Prueksaritanont, B. Ma, C. Tang, Y. Meng, C. Assang, P. Lu, P. J. Reider, J. H. Lin, T. A. Baillie,

Br. J. Clin. Pharmacol. 1999, 47, 291.[12] D. Schmassmann-Suhijar, R. Bullingham, R. Gasser, J. Schmutz, W. E. Haefeli, Lancet 1998, 351,

1929.[13] D. L. Clouatre, Toxicol. Lett. 2004, 150, 85.[14] J. C. Almeda, E. W. Grimsley, Ann. Intern. Med. 1996, 125, 940.[15] A. A. Izzo, E. Ernst, Drugs 1996, 61, 2163.[16] Y. N. Singh, N. N. Singh, CNS Drugs 2002, 16, 731.[17] J. M. Mathews, A. S. Etheridge, S. R. Black, Drug Metab. Dispos. 2002, 30, 1153.[18] B. M. Johnson, S. X. Qiu, S. Zhang, F. Zhang, J. E. Burdette, L. Yu, J. L. Bolton, R. B. Van Breemen,

Chem. Res. Toxicol. 2003, 16, 733.[19] K. Venkatakrishnan, R. S. Obach, A. Rostami-Hodjegan, Xenobiotica 2007, 37, 1225.[20] L. F. Feiser, Am. J. Cancer 1938, 34, 37.[21] E. C. Miller, J. A. Miller, Cancer Res. 1947, 7, 468.[22] V. Thybaud, M. Aardema, J. Clements, K. Dearfield, S. Galloway, M. Hayashi, D. Jacobson-Kram,

D. Kirkland, J. T. Macgregor, D. Marzin, W. Ohyama, M. Schuler, H. Suzuki, E. Zeiger, Mutat. Res.2007, 627, 41.

[23] W. W. Ku, A. Bigger, G. Brambilla, H. Glatt, E. Gocke, P. J. Guzzie, A. Hakura, M. Honma, H. J.Martus, R. S. Obach, S. Roberts, Mutat. Res. 2007, 627, 59.

[24] A. S. Kalgutkar, D. K. Dalvie, J. Aubrecht, E. B. Smith, S. L. Coffing, J. R. Cheung, C. Vage, M. E.Lame, P. Chiang, K. F. McClure, T. S. Maurer, R. V. Coelho Jr., V. F. Soliman, K. Schildknegt, DrugMetab. Dispos. 2007, 35, 848.

[25] A. M. Larson, Clin. Liver Dis. 2007, 11, 525.[26] J. R. Mitchell, D. J. Jollow, W. Z. Potter, D. C. Davis, J. R. Gillette, B. B. Brodie, J. Pharmacol. Exp.

Ther. 1973, 187, 185.[27] D. J. Jollow, J. R. Mitchell, W. Z. Potter, D. C. Davis, J. R. Gillette, B. B. Brodie, J. Pharmacol. Exp.

Ther. 1973, 187, 195.[28] W. Z. Potter, D. C. Davis, J. R. Mitchell, D. J. Jollow, J. R. Gillette, B. B. Brodie, J. Pharmacol. Exp.

Ther. 1973, 187, 203.[29] J. R. Mitchell, D. J. Jollow, W. Z. Potter, J. R. Gillette, B. B. Brodie, J. Pharmacol. Exp. Ther. 1973,

187, 211.[30] D. C. Dahlin, G. T. Miwa, A. Y. Lu, S. D. Nelson, Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 1327.[31] A. S. Kalgutkar, J. R. Soglia, Expert Opin. Drug Metab. 2005, 1, 91.[32] E. Padovan, T. Bauer, M. M. Tongio, H. Kalbacher, H. U. Weltzein, Eur. J. Immunol. 1997, 27, 1303.[33] R. Rodriguez-Pena, C. Antunez, E. Martin, N. Blanca-Lopez, C. Mavorga, M. J. Torres, Expert

Opin. Drug Saf. 2006, 5, 31.[34] Z. Zhao, B. A. Baldo, J. Rimmer, Clin. Exp. Allergy 2002, 32, 1644.[35] E. S. Wagner, M. Gorman, J. Antibiot. (Tokyo) 1971, 24, 647.[36] D. A. Hess, M. E. Sisson, H. Suria, J. Wijsman, R. Puvanesasingham, J. Madrenas, M. J. Rieder,

FASEB J. 1999, 13, 1688.[37] P. Bhaiya, S. Roychowdhury, P. M. Vyas, P. M. Doll, D. W. Hein, C. K. Svensson, Toxicol. Appl.

Pharmacol. 2006, 215, 158.[38] H. Satoh, B. M. Martin, A. H. Schulick, D. D. Christ, J. G. Kenna, L. R. Pohl, Proc. Natl. Acad. Sci.

U.S.A. 1989, 86, 322.[39] S. Lecour, C. Andre, P. H. Beaune, Mol. Pharmacol. 1996, 50, 326.[40] M. Bourdi, J. C. Gautier, J. Mircheva, D. Larrey, A. Guillouzo, C. Andre, C. Belloc, P. H. Beaune,

Mol. Pharmacol. 1992, 42, 280.[41] M. Bourdi, W. Chen, R. M. Peter, J. L. Martin, J. T. Buters, S. D. Nelson, L. R. Pohl, Chem. Res.

Toxicol. 1996, 9, 1159.[42] M. Bourdi, D. Larrey, J. Nataf, J. Bernuau, D. Pessayre, M. Iwasaki, F. P. Guengerich, P. H. Beaune,

J. Clin. Invest. 1990, 85, 1967.


[43] P. Wolkenstein, V. Carriere, D. Charue, S. Bastuji-Garin, J. Revuz, J. C. Roujeau, P. Beaune, M.Bagot, Pharmacogenetics 1995, 5, 255.

[44] R. L. Woosley, D. E. Drayer, M. M. Reidenberg, A. S. Nies, K. Carr, J. A. Oates, N. Engl. J. Med.1978, 298, 1157.

[45] A. E. Cribb, M. Miller, J. S. Leeder, J. Hill, S. P. Spielberg, Drug Metab. Dispos. 1991, 19, 900.[46] J. P. Uetrecht, J. Pharmacol. Exp. Ther. 1985, 232, 420.[47] E. Y. Park, I. J. Cho, S. G. Kim, Cancer Res. 2004, 64, 3701.[48] K. Ueda, T. Ishitsu, T. Seo, N. Ueda, T. Murata, M. Hori, K. Nakagawa, Pharmacogenomics 2007, 8,

435.[49] N. A. Buckley, J. Srinivasan, Drug Saf. 2002, 25, 619.[50] T. A. Baillie, M. R. Davis, Biol. Mass Spectrom. 1993, 22, 319.[51] D. Argoti, L. Liang, A. Conteh, L. Chen, D. Berhas, C. P. Yu, P. Vouros, E. Yang, Chem. Res. Toxicol.

2005, 18, 1537.[52] D. C. Evans, A. P. Watt, D. A. Nicoll-Griffith, T. A. Baillie, Chem. Res. Toxicol. 2004, 17, 3.[53] D. Zhang, M. Ogan, R. Gedamke, V. Roongta, R. Dai, M, Zhu, J. K. Rinehart, L. Klunk, J. Mitroka,

Drug Metab. Dispos. 2003, 31, 837.[54] D. Zhang, R. Krishna, L. Wang, J. Zeng, J. Miroka, R. Dai, N. Narasimhan, R. A. Reeves, N. R.

Srinivas, K. J. Klunk, Drug Metab. Dispos. 2005, 33, 83.[55] A. S. Kalgutkar, I. Gardner, R. S. Obach, C. L. Shaffer, E. Callegari, K. R. Henne, A. E. Mutlib,

D. K. Dalvie, J. S. Lee, Y. Nakai, J. P. O�Donnell, J. Boer, Curr. Drug Metab. 2005, 6, 161.[56] J. C. Dearden, J. Comput.-Aided Mol. Des. 2003, 17, 119.[57] G. Cruciani, E. Carosati, B. De Boeck, K. Ethirajulu, C. Mackie, T. Howe, R. Vianello, J. Med.

Chem. 2005, 48, 6970.[58] K. G. Madsen, J. Olsen, C. Skonberg, S. H. Hansen, U. Jurva, Chem. Res. Toxicol. 2007, 20, 821.[59] Z. C. Liu, J. P. Uetrecht, J. Pharmacol. Exp. Ther. 1995, 275, 1476.[60] I. Gardner, J. S. Leeder, T. Chin, N. Zahid, J. P. Uetrecht, Mol. Pharmacol. 1998, 53, 999.[61] J. Uetrecht, N. Zahid, A. Tehim, J. M. Fu, S. Rakhit, Chem. Biol. Interact. 1997, 104, 117.[62] D. Njoko, M. J. Laster, D. H. Gong, E. I. Eger II, G. F. Reed, J. L. Martin, Anesth. Analg. 1997, 84,

173.[63] D. E. Stewart, Can. J. Psychiatry 2002, 47, 375.[64] R. F. Mayol, C. A. Cole, G. M. Luke, K. L. Colson, F. H. Kerns, Drug Metab. Dispos. 1994, 22,

304.[65] A. S. Kalgutkar, A. D. Vaz, M. E. Lame, K. R. Henne, J. Soglia, S. X. Zhao, Y. A. Abramov, F.

Lombardo, C. Collin, Z. S. Hendsch, C. E. Hop, Drug Metab. Dispos. 2005, 33, 243.[66] J. N. Bauman, K. S. Frederick, A. Sawant, R. L. Walsky, L. M. Cox, R. S. Obach, A. S. Kalgutkar,

Drug Metab. Dispos. 2008, 36, 1016.[67] D. S. Greene, D. E. Salazar, R. C. Dockens, P. Kroboth, R. H. Barbhaiya, J. Clin. Psychopharmacol.

1995, 15, 399.[68] D. S. Greene, R. H. Barbhaiya, Clin. Pharmacokinet. 1997, 33, 260.[69] H. K. Jajoo, R. F. Mayol, J. A. LaBudde, I. A. Blair, Drug Metab. Dispos. 1989, 17, 634.[70] A. Yardley-Jones, D. Anderson, D. V. Parke, Br. J. Ind. Med. 1991, 48, 437.[71] I. M. Rietjens, C. den Besten, R. P. Hanzlik, P. J. van Bladeren, Chem. Res. Toxicol. 1997, 10, 629.[72] W. Jacobsen, B. Kuhn, A. Soldner, G. Kirchner, K. F. Sewing, P. A. Kollman, L. Z. Benet, U.

Christians, Drug Metab. Dispos. 2000, 28, 343.[73] T. Prueksaritanont, R. Subramanian, X. Fang, B. Ma, Y. Qiu, J. H. Lin, P. G. Pearson, T. A. Baillie,

Drug Metab. Dispos. 2002, 30, 505.[74] K. Jorga, B. Fotteler, P. Heizmann, R. Gasser, Br. J. Clin. Pharmacol. 1999, 48, 513.[75] K. S. Smith, P. L. Smith, T. N. Heady, J. M. Trugman, W. D. Harman, T. L. Macdonald, Chem. Res.

Toxicol. 2003, 16, 123.[76] T. Wikberg, A. Vuorela, P. Ottoila, J. Taskinen, Drug Metab. Dispos. 1993, 21, 81.[77] Q. Chen, J. S. Ngui, G. A. Doss, R. W. Wang, X. Cai, F. P. DiNinno, T. A. Blizzard, M. L. Hammond,

R. A. Stearns, D. C. Evans, T. A. Baillie, W. Tang, Chem. Res. Toxicol. 2002, 15, 907.[78] D. C. Kemp, P. W. Fan, J. C. Stevens, Drug Metab. Dispos. 2002, 30, 694.


[79] S. X. Zhao, D. K. Dalvie, J. M. Kelly, J. R. Soglia, K. S. Frederick, E. B. Smith, R. S. Obach, A. S.Kalgutkar, Chem. Res. Toxicol. 2007, 20, 1649.

[80] K. Kassahun, P. G. Pearson, W. Tang, I. McIntosh, K. Leung, C. Elmore, D. Dean, R. Wang, G. Doss,T. A. Baillie, Chem. Res. Toxicol. 2001, 14, 62.

[81] K. He, R. E. Talaat, W. F. Pool, M. D. Reily, J. E. Reed, A. J. Bridges, T. F. Woolf, Drug Metab.Dispos. 2004, 32, 639.

[82] R. Alvarez-Sanchez, F. Montavon, T. Hartung, A. Pahler, Chem. Res. Toxicol. 2006, 19, 1106–1116.[83] B. J. Ring, B. E. Patterson, M. I. Mitchell, M. Vandenbranden, J. Gillespie, A. W. Bedding, H. Jewell,

C. D. Payne, S. T. Forgue, J. Eckstein, S. A. Wrighton, D. L. Phillips, Clin. Pharmacol. Ther. 2005, 77,63.

[84] J. C. Erve, S. C. Vashishtha, W. DeMaio, R. E. Talaat, Drug Metab. Dispos. 2007, 35, 908.[85] H. L. Lin, U. M. Kent, P. F. Hollenberg, J. Pharmacol. Exp. Ther. 2002, 301, 160.[86] S. Mallal, E. Phillips, G. Carosi, J. M. Molina, C. Workman, J. Tomazic, E. Jagel-Guedes, S. Rugina,

O. Kozyrev, J. F. Cid, P. Hay, D. Nolan, S. Hughes, A. Hughes, S. Ryan, N. Fitch, D. Thornborn, A.Benbow, N. Engl. J. Med. 2008, 358, 568.

[87] S. Kumar, K. Kassahun, R. A. Tschirret-Guth, K. Mitra, T. A. Baillie, Curr. Opin. Drug DiscoveryDev. 2008, 11, 43.

Received February 22, 2009


structural alerts, reactive metabolites, and protein covalent binding: how reliable are these...

Documents