Studies on the metabolism of troglitazone to reactive intermediates in vitro and in vivo. Evidence for novel biotransformation pathways involving quinone methide formation and thiazolidinedione ring scission

Therapy with the oral antidiabetic agent troglitazone (Rezulin) has been associated with cases of severe hepatotoxicity and drug-induced liver failure, which led to the recent withdrawal of the product from the U.S. market. While the mechanism of this toxicity remains unknown, it is possible that chemically reactive metabolites of the drug play a causative role. In an effort to address this possibility, this study was undertaken to determine whether troglitazone undergoes metabolism in human liver microsomal preparations to electrophilic intermediates. Following incubation of troglitazone with human liver microsomes and with cDNA-expressed cytochrome P450 isoforms in the presence of glutathione (GSH), a total of five GSH conjugates (M1-M5) were detected and identified tentatively by LC-MS/MS analysis. In two cases (M1 and M5), the structures of the adducts were confirmed by NMR spectroscopy and/or by comparison with an authentic standard prepared by synthesis. The formation of GSH conjugates M1-M5 revealed the operation of two distinct metabolic activation pathways for troglitazone, one of which involves oxidation of the substituted chromane ring system to a reactive o-quinone methide derivative, while the second involves a novel oxidative cleavage of the thiazolidinedione (TZD) ring, potentially generating highly electrophilic alpha-ketoisocyanate and sulfenic acid intermediates. When troglitazone was administered orally to a rat, samples of bile were found to contain GSH conjugates which reflected the operation of these same metabolic pathways in vivo. The finding that metabolism of the TZD ring of troglitazone was catalyzed selectively by P450 3A enzymes is significant in light of the recent report that troglitazone is an inducer of this isoform in human hepatocytes. The implications of these results are discussed in the context of the potential for troglitazone to covalently modify hepatic proteins and to cause oxidative stress through redox cycling processes, either of which may play a role in drug-induced liver injury.     

Roles of human hepatic cytochrome P450s 2C9 and 3A4 in the metabolic activation of diclofenac

Recently, it was shown that diclofenac was metabolized in rats to reactive benzoquinone imines via cytochrome P450-catalyzed oxidation. These metabolites also were detected in human hepatocyte cultures in the form of glutathione (GSH) adducts. This report describes the results of further studies aimed at characterizing the human hepatic P450-mediated bioactivation of diclofenac. The reactive metabolites formed in vitro were trapped by GSH and analyzed by LC/MS/MS. Thus, three GSH adducts, namely, 5-hydroxy-4-(glutathion-S-yl)diclofenac (M1), 4'-hydroxy-3'-(glutathion-S-yl)diclofenac (M2), and 5-hydroxy-6-(glutathion-S-yl)diclofenac (M3), were identified in incubations of diclofenac with human liver microsomes in the presence of NADPH and GSH. The formation of the adducts was taken to reflect the intermediacy of the corresponding putative benzoquinone imines. While M2 was the dominant metabolite over a substrate concentration range of 10-50 microM, M1 and M3 became equally important products at >/=100 microM diclofenac. The formation of M2 was inhibited by sulfaphenazole or an anti-P450 2C9 antibody (5-10% of control values). The formation of M1 and M3 was inhibited by troleandomycin, ketoconazole, or an anti-P450 3A4 antibody (30-50% of control values). In studies in which recombinant P450 isoforms were used, M2 was generated only by P450 2C9-catalyzed reaction, while M1 and M3 were produced by P450 3A4-catalyzed reaction. Good correlations were established between the extent of formation of M2 and P450 2C9 activities (r = 0.93, n = 10) and between the extent of formation of M1 and M3 and P450 3A4 activities (r = 0.98, n = 10) in human liver microsomal incubations. Taken together, the data suggest that the biotransformation of diclofenac to M2 is P450 2C9-dependent, whereas metabolism of the drug to M1 and M3 involves mainly P450 3A4. Although P450s 2C9 and 3A4 both catalyze the bioactivation of diclofenac, P450 2C9 is capable of producing the benzoquinone imine intermediate at lower drug concentrations which may be more clinically relevant.     

Studies on cytochrome P-450-mediated bioactivation of diclofenac in rats and in human hepatocytes: identification of glutathione conjugated metabolites.

The nonsteroidal anti-inflammatory drug diclofenac causes a rare but potentially fatal hepatotoxicity that may be associated with the formation of reactive metabolites. In this study, three glutathione (GSH) adducts, namely 5-hydroxy-4-(glutathion-S-yl)diclofenac (M1), 4'-hydroxy-3'-(glutathion-S-yl)diclofenac (M2), and 5-hydroxy-6-(glutathion-S-yl)diclofenac (M3), were identified by liquid chromatography-tandem mass spectrometry analysis of bile from Sprague-Dawley rats injected i.p. with a single dose of diclofenac (200 mg/kg). These adducts presumably were formed via hepatic cytochrome P-450 (CYP)-catalyzed oxidation of diclofenac to reactive benzoquinone imines that were trapped by GSH conjugation. In support of this hypothesis, M1, M2, and M3 were generated from diclofenac in incubations with rat liver microsomes in the presence of NADPH and GSH. Increases in adduct formation were observed when incubations were performed with liver microsomes from phenobarbital- or dexamethasone-treated rats. Adduct formation was inhibited by polyclonal antibodies against CYP2B, CYP2C, and CYP3A (40-50% inhibition at 5 mg of IgG/nmol of CYP) but not by an antibody against CYP1A. Maximal inhibition was obtained when the three inhibitory antibodies were used in a cocktail fashion (70-80% inhibition at 2.5 mg of each IgG/nmol of CYP). These data suggest that diclofenac undergoes biotransformation to reactive metabolites in rats and that CYP isoforms of the 2B, 2C, and 3A subfamilies are involved in this bioactivation process. With respect to CYP2C isoforms, rat hepatic CYP2C7 and CYP2C11 were implicated as mediators of the bioactivation based on immunoinhibition studies using antibodies specific to CYP2C7 and CYP2C11. Screening for GSH adducts also was carried out in human hepatocyte cultures containing diclofenac, and M1, M2, and M3 again were detected. It is possible, therefore, that reactive benzoquinone imines may be formed in vivo in humans and contribute to diclofenac-mediated hepatic injury.     

The role of physicochemical properties of entacapone and tolcapone on their efficacy during local intrastriatal administration

Aqueous solubility, apparent partition coefficient (log P_app) and catechol-O-methyltransferase (COMT, EC 2.1.1.6) inhibiting potency of entacapone and tolcapone were compared in vitro. Both drugs (at 10 and 100 μM) were also delivered directly into rat striatum via a microdialysis probe. Extracellular 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) concentrations were measured to evaluate the inhibition of striatal COMT in vivo. Although entacapone had 15-fold better aqueous solubility than tolcapone at pH 7.4, also tolcapone had sufficient aqueous solubility to remain in solution at 100 μM. The log P_app of tolcapone was higher than that reported for entacapone in the pH range from 5.0 to 7.4. Entacapone and tolcapone inhibited equally rat striatal COMT in vitro with K_i values of 1.86 and 2.50 nM, respectively. Both drugs had similar outflow from the microdialysis probe in vitro. Perfusion of 100 μM entacapone increased significantly extracellular DOPAC levels compared to the control group. Both entacapone and tolcapone (at 10 and 100 μM) decreased significantly HVA levels, but entacapone was significantly more effective than tolcapone at 100 μM. In conclusion, entacapone and tolcapone are equally potent COMT inhibitors against rat striatal COMT in vitro. After local intrastriatal administration, entacapone appeared to inhibit COMT faster and more effectively than the more lipophilic tolcapone. Thus, intrastriatal administration led to opposite results compared to those reported in the brain after systemic administration. The present results also suggest that the local distribution of entacapone and tolcapone differ when the drugs are delivered directly into the brain.     

Effects of entacapone and tolcapone on mitochondrial membrane potential

Catechol-O-methyl transferase (COMT) inhibitors, entacapone and tolcapone, are used as an adjunctive treatment to L-dopa in Parkinson's disease. Based on their catechol structure, both inhibitors are potential uncoupling agents, but only tolcapone shows this effect in vitro at clinically relevant concentrations. This study was designed to evaluate the direct uncoupling effects of the two COMT inhibitors in vitro and in vivo. In isolated rat liver mitochondria, entacapone had no effect on the membrane potential at therapeutical concentrations, but both tolcapone and the reference compound 2,4-dinitrophenol disrupted the potential at low microM concentrations. Since protein binding is speculated to decrease the uncoupling effects in vivo, the COMT inhibitory effect of entacapone and tolcapone as a surrogate for the overall activity of these inhibitors was evaluated in vitro with or without serum. The COMT inhibitory activity of entacapone was reduced to half, while tolcapone had only about 1/10 of its activity left in the presence of serum. Further, uncoupling is known to induce an increase in the body temperature in vivo, and these effects were evaluated in the rat by a possible hyperthermic response to the treatment with entacapone or tolcapone in combination with L-dopa (10 mg/kg) and carbidopa (20 mg/kg). This combination with entacapone (400 mg/kg) had no effect on the rectal body temperature. In contrast, tolcapone (50 mg/kg) caused an elevation in the body temperature together with L-dopa and carbidopa (P < 0.01). Both in vitro and in vivo results indicate that entacapone does not impair energy metabolism related to uncoupling of oxidative phosphorylation.     

Development and evaluation of an electrochemical method for studying reactive phase-I metabolites: correlation to in vitro drug metabolism

A reactive metabolite may react covalently with proteins or DNA to form adducts that ultimately may lead to a toxic response. Reactive metabolites can be formed via, for example, cytochrome P450-mediated phase 1 reactions, and in this study, we report the development and evaluation of an electrochemical method for generating reactive metabolites. Paracetamol was used as a test compound to develop the method. The stability of the electrochemically generated N-acetyl-p-benzoquinoneimine (NAPQI) from paracetamol was investigated at 37 degrees C at pH 5.0, 7.4, and 9.0. The highest stability of NAPQI was observed at pH 7.4. The reaction rate between NAPQI and glutathione (GSH) was studied with cyclic voltammetry. NAPQI reacted quantitatively with GSH within 130 ms. The reactivity of NAPQI toward other nucleophiles was investigated, and for the reaction with N-acetyltyrosine, a time-dependent formation of a conjugate with N-acetyltyrosine was observed from 0 to 4 min. The applicability of the method was evaluated with compounds that were able to form quinone imines (amodiaquine), quinones (3-tert-butyl-4-hydroxyanisole and p-cresol), imine methides (3-methylindole; trimethoprim), quinone methides (3,5-di-tert-butyl-4-hydroxytoluene), and nitrenium ions (clozapine). The compounds were oxidized in an analytical electrochemical cell, and the formed reactive metabolites were trapped with GSH. The samples were then analyzed by LC-MS and LC-MS/MS. For comparison, all compounds were incubated with GSH in rat and human liver microsomes, and the formation of GSH conjugates was compared with that observed by electrochemical oxidation. Furthermore, the electrochemical method was used to synthesize a GSH conjugate of clozapine, which made it possible to obtain structural information by NMR. In summary, a high degree of similarity was observed between the conjugates identified from electrochemical oxidation and GSH conjugates identified from incubation with liver microsomes. In conclusion, we have developed a method that is useful for studies on reactive metabolites and furthermore can be scaled up for the synthesis of GSH conjugates for NMR.     

Trimethoprim: novel reactive intermediates and bioactivation pathways by cytochrome p450s

Trimethoprim (TMP) is a widely used antibacterial agent that is usually considered as a safe drug. TMP has, however, been implicated in rare adverse drug reactions (ADRs) in humans. Bioactivation to a reactive iminoquinone methide intermediate has been proposed as a possible cause for the toxicity of the drug. However, little is known about the cytochrome P450s (P450s) involved in this bioactivation and in the metabolism of TMP in general. In this study, we have investigated the metabolism and bioactivation of TMP by human liver microsomes (HLM) and rat liver microsomes, by recombinant human cytochrome P450s, and by the bacterial P450 BM3 mutant M11(his). In addition to non GSH-dependent metabolites, five GSH adducts were identified in the HLM incubations. Next to two major GSH adducts probably originating from the iminoquinone methide intermediate described previously, three minor GSH adducts were also identified, indicating that other types of reactive intermediates are formed by HLM, such as ortho-quinones and para-quinone methide intermediates. The major GSH adducts were produced by P450 1A2 and P450 3A4, while the minor GSH adducts were mainly formed by P450 1A2, P450 3A4, and P450 2D6. Although preliminary, these results might implicate that genetic polymorphisms in P450 enzymes could play a role in the onset of TMP-related ADRs in humans.     

Metabolism of capsaicin by cytochrome P450 produces novel dehydrogenated metabolites and decreases cytotoxicity to lung and liver cells

Capsaicin is a common dietary constituent and a popular homeopathic treatment for chronic pain. Exposure to capsaicin has been shown to cause various dose-dependent acute physiological responses including the sensation of burning and pain, respiratory depression, and death. In this study, the P450-dependent metabolism of capsaicin by recombinant P450 enzymes and hepatic and lung microsomes from various species, including humans, was determined. A combination of LC/MS, LC/MS/MS, and LC/NMR was used to identify several metabolites of capsaicin that were generated by aromatic (M5 and M7) and alkyl hydroxylation (M2 and M3), O-demethylation (M6), N- (M9) and alkyl dehydrogenation (M1 and M4), and an additional ring oxygenation of M9 (M8). Dehydrogenation of capsaicin was a novel metabolic pathway and produced unique macrocyclic, diene, and imide metabolites. Metabolism of capsaicin by microsomes was inhibited by the nonselective P450 inhibitor 1-aminobenzotriazole (1-ABT). Metabolism was catalyzed by CYP1A1, 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4. Addition of GSH (2 mM) to microsomal incubations stimulated the metabolism of capsaicin and trapped several reactive electrophilic intermediates as their GSH adducts. These results suggested that reactive intermediates, which inactivated certain P450 enzymes, were produced during catalytic turnover. Comparison of the rate and types of metabolites produced from capsaicin and its analogue, nonivamide, demonstrated similar pathways in the P450-dependent metabolism of these two capsaicinoids. However, production of the dehydrogenated (M4), macrocyclic (M1), and omega-1-hydroxylated (M3) metabolites was not observed for nonivamide. These differences may be reflective of the mechanism of formation of these metabolites of capsaicin. The role of metabolism in the cytotoxicity of capsaicin and nonivamide was also assessed in cultured lung and liver cells. Lung cells were markedly more sensitive to cytotoxicity by capsaicin and nonivamide. Cytotoxicity was enhanced 5 and 40% for both compounds by 1-ABT in BEAS-2B and HepG2, respectively. These data suggested that metabolism of capsaicinoids by P450 in cells represented a detoxification mechanism (in contrast to bioactivation).     

The specificity of glucuronidation of entacapone and tolcapone by recombinant human UDP-glucuronosyltransferases.

The COMT inhibitors entacapone and tolcapone are rapidly metabolized in vivo, mainly by glucuronidation. In this work, the main UGT isoforms responsible for their glucuronidation in vitro were characterized by using a subset of representative cloned and expressed human UGT isoforms. Entacapone in particular was seen to be an exceptionally good substrate for UGT1A9 with an even higher reaction velocity value at 500 microM substrate concentration compared with that of the commonly used substrate, propofol (1.3 and 0.78 nmol min(-1) mg(-1), respectively). Neither entacapone nor tolcapone was glucuronidated by UGT1A6. Tolcapone was not detectably glucuronidated by UGT1A1, and the rate of glucuronidation of entacapone was also low by this isoform. However, UGT1A1 was the only UGT capable of catalyzing the formation of two glucuronides of the catecholic entacapone. Both COMT inhibitors were glucuronidated at low rates by the representative members of the UGT2B family, UGT2B7 and UGT2B15. Michaelis-Menten parameters were determined for entacapone and tolcapone using recombinant human UGT isoforms and human liver microsomes to compare the kinetic properties of the two COMT inhibitors. The kinetic data illustrates that UGT1A9 exhibited a much greater rate of glucuronidation and a far lower K(m) value for both entacapone and tolcapone than UGT2B15 and UGT2B7 whose contribution is minor by comparison. Entacapone showed a 3 to 4 times higher V(max) value and a 4 to 6 times lower K(m) value compared with those of tolcapone both in UGT1A9 cell lysates and in human liver microsomes.     

In vitro metabolism of a model cyclopropylamine to reactive intermediate: insights into trovafloxacin-induced hepatotoxicity

Trovafloxacin (Trovan) is a fluoroquinolone antibiotic drug with a long half-life and broad-spectrum activity. Since its entry into the market in 1998, trovafloxacin has been associated with numerous cases of hepatotoxicity, which has limited its clinical usefulness. Trovafloxacin possesses two substructural elements that have the potential to generate reactive intermediates: a cyclopropylamine moiety and a difluoroanilino system. The results presented here describe the in vitro metabolic activation of a synthetic drug model (DM) of trovafloxacin that contains the cyclopropylamine moiety. Cyclopropylamine can be oxidized to reactive ring-opened products-a carbon-centered radical and a subsequently oxidized alpha,beta-unsaturated aldehyde. Experiments with monoamine oxygenases, horseradish peroxidase, flavin monooxygenase 3, and cDNA-expressed P450 isoenzymes revealed that P450 1A2 oxidizes DM to a reactive alpha,beta-unsaturated aldehyde, M 1. Furthermore, myeloperoxidase (MPO) was also demonstrated to oxidize DM in the presence of chloride ion to produce M 1. DM proved to be a suicide inhibitor of MPO while showing no inhibition of P450 1A2. The structure of the reactive metabolite was confirmed by LC-MS/MS analysis by comparison with a synthetic standard. M 1 was further shown to react with glutathione and the related thiol nucleophile, 4-bromobenzyl mercaptan, suggesting the potential of this intermediate to react with protein nucleophiles. In summary, these data provide evidence that trovafloxacin-induced hepatotoxicity may be mediated through the oxidation of the cyclopropylamine substructure to reactive intermediates that may form covalent adducts to hepatic proteins, resulting in damage to liver tissue.     

In vitro metabolism of 2-acetylbenzothiophene: relevance to zileuton hepatotoxicity

Zileuton, an inhibitor of 5-lipooxygenase, the initial enzyme in the leukotriene pathway, was marketed as a new treatment for asthma. This drug has been associated with liver toxicity, which has limited its clinical usefulness. We provide evidence here that the liver toxicity likely involves a sequence of biotransformations leading to 2-acetylbenzothiophene (2-ABT), which is subsequently metabolized to give a reactive intermediate(s). In vitro experiments with the human lymphoblast MCL5 cell line demonstrated that 2-ABT is cytotoxic in a P450-dependent manner. Human liver microsome (HLM) incubations with 2-ABT revealed the formation of two short-lived oxidized species, "M + 16" and "M + 32". Both of these metabolites formed adducts in the presence of GSH or NAC. Singly oxidized M + 16 adducts, from either GSH or NAC, appeared to be unstable in acidic medium and eliminated water readily to form a new compound. Authentic synthetic standards demonstrated that 2-ABT-S-oxide M1 corresponded to the M + 16 metabolite and that the S-oxide underwent nucleophilic addition with GSH and NAC to produce the singly oxidized adducts observed in HLM. The S-oxide adducts readily eliminated water to form a rearomatized 2-ABT-GSH adduct or 2-ABT-NAC adduct. Coelution experiments with the synthetic standard confirmed the structure of the eliminated 2-ABT-NAC adduct C1. LC/MS analyses of urine samples collected from rats dosed with zileuton indicate that C1 is a metabolite of zileuton formed in vivo. The in vitro and in vivo data presented here demonstrate the formation of 2-ABT from zileuton and its further bioactivation to a potentially toxic metabolite.     

Pyrrole-protein adducts – A biomarker of pyrrolizidine alkaloid-induced hepatotoxicity

Pyrrolizidine alkaloids (PAs) are phytotoxins identified in over 6000 plant species worldwide. Approximately 600 toxic PAs and PA N-oxides have been identified in about 3% flowering plants. PAs can cause toxicities in different organs particularly in the liver. The metabolic activation of PAs is catalyzed by hepatic cytochrome P450 and generates reactive pyrrolic metabolites that bind to cellular proteins to form pyrrole-protein adducts leading to PA-induced hepatotoxicity. The mechanisms that pyrrole-protein adducts induce toxicities have not been fully characterized. Methods for qualitative and quantitative detection of pyrrole-protein adducts have been developed and applied for the clinical diagnosis of PA exposure and PA-induced liver injury. This mini-review addresses the mechanisms of PA-induced hepatotoxicity mediated by pyrrole-protein adducts, the analytical methods for the detection of pyrrole-protein adducts, and the development of pyrrole-protein adducts as the mechanism-based biomarker of PA exposure and PA-induced hepatotoxicity.     

Lack of interaction between tolcapone and tolbutamide in healthy volunteers

To assess the effect of tolcapone (an inhibitor of cytochrome P450 [CYP] 2C9 in vitro) on the pharmacokinetics and hypoglycemic effect of the CYP 2C9 substrate tolbutamide, 12 healthy male volunteers were randomized to receive a single dose of tolbutamide 500 mg plus either placebo or tolcapone 200 mg after an overnight fast and 30 minutes after the start of a 6.5-hour 5% glucose infusion (150 mL/h). The participants crossed over to receive the alternative regimen after a washout period of at least 7 days. Tolcapone had no effect on the pharmacokinetics of tolbutamide or its metabolites and did not influence the effect of tolbutamide on plasma glucose concentrations. No serious adverse events or abnormal laboratory results or vital signs were reported. In conclusion, clinically relevant drug-drug interactions between tolcapone and tolbutamide when given together in clinical practice appear unlikely.     

Identification of novel glutathione adducts of benzbromarone in human liver microsomes

Benzbromarone (BBR) is a potent uricosuric drug that can cause serious liver injury. Our recent study suggested that 1′-hydroxy BBR, one of major metabolites of BBR, is metabolized to a cytotoxic metabolite that could be detoxified by glutathione (GSH). The aim of this study was to clarify whether GSH adducts are formed from 1′-hydroxy BBR in human liver microsomes (HLM). Incubation of 1′-hydroxy BBR with GSH in HLM did not result in the formation of GSH adducts, but 1′,6-dihydroxy BBR was formed. In addition, incubation of 1′,6-dihydroxy BBR with GSH in HLM resulted in the formation of three novel GSH adducts (M1, M2 and M3). The structures of M1 and M2 were estimated to be GSH adducts in which the 1-hydroxyethyl group at the C-2 position and the hydroxyl group at the C-1′ position of 1′,6-dihydroxy BBR were substituted by GSH, respectively. We also found that the 6-hydroxylation of 1′-hydroxy BBR is mainly catalyzed by CYP2C9 and that several CYPs and/or non-enzymatic reaction are involved in the formation of GSH adducts from 1′,6-dihydroxy BBR. The results indicate that 1′-hydroxy BBR is metabolized to reactive metabolites via 1′,6-dihydroxy BBR formation, suggesting that these reactive metabolites are responsible for BBR-induced liver injury.     

Mechanism of inactivation of human cytochrome P450 2B6 by phencyclidine.

The mechanism behind the observed inactivation of human P450 2B6 by phencyclidine (PCP) has been evaluated over the past 2 decades. The scope of the current investigation was to contribute to the fundamental knowledge of PCP oxidation and perhaps the mechanism behind P450 inactivation. To study the chemistry of PCP oxidation, we subjected PCP to the Fenton reagent. Under Fenton chemistry conditions, oxidation on all three PCP rings was observed by liquid chromatography/tandem mass spectrometry (LC-MS/MS). When PCP was incubated with the Fenton system in the presence of glutathione (GSH), three GSH-PCP conjugates were identified. Subsequent LC-MS/MS analysis of these conjugates revealed two species that had GSH attached to the cyclohexane ring of PCP and a third conjugate in which GSH was adducted to the piperidine ring. When PCP was incubated across a panel of P450 enzymes, several enzymes, including P450s 2D6 and 3A4, were able to catalyze the formation of the PCP iminium ion, whereas P450s 2B6 and 2C19 were exclusively able to hydroxylate secondary carbons on the cyclohexane ring of PCP. Subsequent mechanistic experiments revealed that only P450s 2B6 and 2C19 demonstrated loss of catalytic activity after preincubation with 10 microM PCP. Finally, investigation of P450 2B6 inactivation using structural analogs of PCP revealed that blocking the para-carbon atom on the cyclohexane ring of PCP from oxidation protected the P450 2B6 from inactivation, which suggests that a reactive intermediate generated during the hydroxylation of the cyclohexane ring may be linked to the mechanism of inactivation of P450 2B6 by PCP.     

Acylation of protein lysines by trichloroethylene oxide

Stable lysine adducts were formed in proteins following reaction with trichloroethylene (TCE) oxide, the major reactive compound generated by the metabolism of TCE. The order of formation of these adducts is N(6)-formyllysine > N(6)-(dichloroacetyl)lysine > N(6)-glyoxyllysine, with the ratio being influenced by the particular protein. Protein lysine adducts were also analyzed following the enzymatic oxidation of TCE with several different cytochrome P450 (P450) enzyme systems. The ratio of formyl/dichloroacetyl lysine adducts was influenced by the enzyme system that was used. Chloral and TCE oxide formation was more extensive with rat liver microsomes isolated from phenobarbital-treated rats than with rat microsomes in which P450 2E1 was induced by treatment with isoniazid or in human P450 2E1 systems. Glutathione (GSH) and GSH transferase had inhibitory effects on the reaction of TCE oxide with albumin, with formylation being atteunated much more than the formation of dichloroacetyllysine. GSH is likely to react with the reactive acyl chloride intermediates formed from TCE oxide hydrolysis, instead of direct reaction with TCE oxide, as judged by the lack of an effect of GSH on the rate of decomposition of TCE oxide. Studies with the model enzymes aldolase and glucose-6-phosphate dehydrogenase, both known to have sensitive lysine groups, indicate that TCE oxide has effects similar to known acylating agents that form the same adducts; concentrations of TCE oxide (or the model acylating agents) in the low-millimolar range were needed for inhibition. The characterization of TCE-derived protein adducts can be used as a basis for consideration of the exposure and risk of TCE to humans. Human P450 2E1 was less likely to oxidize TCE to form TCE oxide and protein lysine adducts than rat P450 2B1, and the difference is rationalized in terms of the influence of the protein on chloride migration in an enzyme reaction intermediate.     

Metabolism and excretion of tolcapone, a novel inhibitor of catechol-O-methyltransferase

AIMS: To investigate the rate of excretion and routes of metabolism of tolcapone, a novel inhibitor of catechol-O-methyltransferase (COMT). METHODS: Six healthy male volunteers were given 200 mg [14C]-tolcapone (approximately 50 muCi) orally. To assess excretion balance and to identify metabolites, urine and faeces were collected before administration and until radioactivity fell below 75 d min-1 ml-1 (urine) and 100 d min-1 mg-1 (faeces). Blood samples were collected frequently before and after administration to determine plasma radioactivity, to identify tolcapone metabolites and to measure plasma tolcapone and its methylated derivative 3-O-methyltolcapone (3-OMT). RESULTS: The mean proportion of the dose excreted in urine was 57.3% and in faeces 40.5%. Excretion was almost complete (more than 95%) in all participants after 9 days. The major early metabolite present in plasma was the 3-O-beta, d-glucuronide conjugate, which was detectable within 2 h after dosing. The major late metabolite in plasma was 3-OMT. The 3-O-beta, d-glucuronide was also the most abundant metabolite in urine and faeces, accounting for 27% and 33%, respectively, of the total radioactivity excreted by these routes and for 26% of the original dose. Reduction of the nitro moiety yields an amine derivative, detected in both urine and faeces, with subsequent modifications, such as acetylation of the amine group and conjugation with glucuronic acid or sulphate, or both. Oxidative reactions due to cytochrome P450 enzymes are of small significance, as is 3-O-methylation by COMT. CONCLUSIONS: Tolcapone is almost completely metabolized and excreted in urine and faeces (only 0.5% of tolcapone was excreted unchanged); glucuronidation is the most important route of metabolism. The relatively long duration of excretion is caused by the long half-life of 3-OMT.     

Bioactivation of 2,3-diaminopyridine-containing bradykinin B1 receptor antagonists: irreversible binding to liver microsomal proteins and formation of glutathione conjugates

The 2,3-diaminopyridine (DAP) moiety was found to represent a core structure essential for the potency of a new series of human bradykinin B(1) receptor antagonists. However, incubation of (14)C-labeled 2,3-DAP derivatives with rat and human liver microsomes resulted in substantial irreversible binding of radioactive material to macromolecules by a process that was NADPH-dependent. Trapping the reactive species with GSH led to significant reduction of the irreversible binding of radioactivity, with concomitant formation of abundant GSH adducts. One type of thiol adducts (detected in both human and rat liver microsomes), resulting from addition of 305 Da to the parent compound, was observed with all 2,3-DAP compounds. These adducts also were detected in rat hepatocyte incubates, as well as in rat bile, following intravenous administration of 2,3-DAPs. Formation of the conjugates appeared to involve modification of the DAP ring, based upon mass spectral analysis of a number of representative GSH adducts; this was corroborated by detailed LC NMR analysis of one compound. Formation of this type of GSH conjugate was markedly reduced when the 2-amino methyl group linking the 2,3-DAP and the biphenyl moiety was replaced with an ether oxygen atom. It is postulated, therefore, that oxidation of the 2-amino group serves as a key step leading to the formation of reactive species associated with the DAP core. In addition, this step appears to be mediated primarily by P450 3A, as evidenced by the marked decrease in both the irreversible binding of radioactivity and the formation of the GSH adducts in human liver microsomes following treatment with ketoconazole and monoclonal antibodies against P450 3A. A mechanism for the bioactivation of 2,3-DAP is proposed wherein oxidation (dehydrogenation or N-hydroxylation followed by dehydration) of the 2-amino group, catalyzed by P450 3A, results in the formation of a highly electrophilic species, pyridine-2,3-diimine.     

Differences in toxicity of the catechol-O-methyl transferase inhibitors, tolcapone and entacapone to cultured human neuroblastoma cells

Tolcapone and entacapone are catechol-O-methyltransferase (COMT) inhibitors used as adjuncts to levodopa in the treatment of Parkinson's disease (PD). The use of tolcapone has been limited by its hepatotoxicity, the cause of which remains uncertain. Tolcapone compound is an uncoupler of mitochondrial respiration in isolated mitochondria and this action may be relevant to its effect on liver function. We have examined the actions of COMT inhibitors on cultured cells, comparing them with those of the classical uncoupler carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), in order to provide insight into their mechanism of potential toxicity. Tolcapone and FCCP were shown to be toxic to human neuroblastoma SH-SY5Y cells and caused a profound reduction in ATP synthesis. Entacapone was not toxic to SH-SY5Y. Tolcapone and FCCP were shown to be equally toxic to cells depleted of mtDNA and thus devoid of a functional respiratory chain. This study demonstrates that tolcapone markedly inhibits ATP synthesis in cultured cells mirroring the effects of a classical uncoupler. However its toxicity may also involve a mechanism independent of its effects upon oxidative phosphorylation.     

Characterization of novel glutathione adducts of a non-nucleoside reverse transcriptase inhibitor, (S)-6-chloro-4-(cyclopropylethynyl)-4-(trifluoromethyl)-3, 4-dihydro-2(1H)-quinazolinone (DPC 961), in rats. Possible formation of an oxirene metabolic intermediate from a disubstituted alkyne

The postulated formation of oxirene-derived metabolites from rats treated with a disubstituted alkyne, (S)-6-chloro-4-(cyclopropylethynyl)-4-(trifluoromethyl)-3, 4-dihydro-2(1H)-quinazolinone (DPC 961), is described. The reactivity of this postulated oxirene intermediate led to the formation of novel glutathione adducts whose structures were confirmed by LC/MS and by two-dimensional NMR experiments. These metabolites were either excreted in rat bile or degraded to mercapturic acid conjugates and eliminated in urine. To demonstrate the oxidation of the triple bond, an analogue of DPC 961 was synthesized, whereby the two carbons of the alkyne moiety were replaced with (13)C stable isotope labels. Rats were orally administered [(13)C]DPC 961 and glutathione adducts isolated from bile. The presence of an oxygen atom on one of the (13)C labels of the alkyne was demonstrated unequivocally by NMR experiments. Administration of (14)C-labeled DPC 961 showed that biliary elimination was the major route of excretion with the 8-OH glucuronide conjugate (M1) accounting for greater than 90% of the eliminated radioactivity. On the basis of radiochemical profiling, the glutathione-derived metabolites were minor in comparison to the glucuronide conjugate. Studies with cDNA-expressed rat enzymes, polyclonal antibodies, and chemical inhibitors pointed to the involvement of P450 3A1 and P450 1A2 in the formation of the postulated oxirene intermediate. The proposed mechanism shown in Scheme 1 begins with P450-catalyzed formation of an oxirene, rearrangement to a reactive cyclobutenyl ketone, and a 1,4-Michael addition with endogenous glutathione to produce two isomeric adducts, GS-1 and GS-2. The glutathione adducts were subsequently catabolized via the mercapturic acid pathway to cysteinylglycine, cysteine, and N-acetylcysteine adducts. The transient existence of the alpha,beta-unsaturated cyclobutenyl ketone was demonstrated by incubating the glutathione adduct in the presence of N-acetylcysteine and monitoring the formation of N-acetylcysteine adducts by LC/MS. Epimerization of GS-1 to GS-2 was also observed when N-acetylcysteine was omitted from the incubation.