Bioactivation of Lamotrigine in Vivo in Rat and in Vitro in Human Liver

Chem. Res. Toxicol. 2010, 23, 159–170

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Bioactivation of Lamotrigine in Vivo in Rat and in Vitro in Human Liver Microsomes, Hepatocytes, and Epidermal Keratinocytes: Characterization of Thioether Conjugates by Liquid Chromatography/Mass Spectrometry and High Field Nuclear Magnetic Resonance Spectroscopy Hao Chen,*,† Scott Grover,‡ Linning Yu,† Gregory Walker,§ and Abdul Mutlib† Department of Pharmacokinetics, Dynamics, and Metabolism, Pfizer Global Research and DeVelopment, 2800 Plymouth Road, Ann Arbor, Michigan 48105 ReceiVed September 9, 2009

Previous studies suggested that lamotrigene (LTG) underwent bioactivation to a reactive aryl epoxide intermediate in rats. Nevertheless, definitive structures of these thioether conjugates, which are often needed to substantiate the mechanism of bioactivation and identity of reactive intermediate(s), were not fully established. In the present study, GSH, cysteinylglycine, and N-acetyl cysteine conjugates of LTG were isolated from bile of rats orally dosed with LTG (100 mg/kg), and their structures were fully elucidated by LC/MS and NMR. The definitive structural characterization of these metabolites provided evidence for the existence of a reactive aryl epoxide that was trapped as a GSH adduct. In vitro studies using various hepatic cellular and subcellular fractions obtained from human and rat were performed to demonstrate that LTG underwent bioactivation to form a GSH conjugate that was identical to the one initially characterized from in vivo studies. Human P450 2A6 and rat P450 2C11 appeared to be the primary enzymes activating LTG in human and rat liver microsomes, respectively. Interindividual variation in the bioactivation of LTG was demonstrated with 20 individual human liver microsomes. Furthermore, it was shown that human epidermal keratinocytes were capable of forming the same GSH conjugate, suggesting that LTG could be bioactivated in skin cells. The results from these studies suggest that LTG has the potential to undergo hepatic and nonhepatic bioactivation, leading to a reactive aryl epoxide intermediate in human. The bioactivation of LTG in epidermal cells provides a possible explanation for the idiosyncratic cutaneous reactions associated with LTG therapy. Introduction Lamotrigine (LTG)1 [Lamictal, 3,5-diamino-6-(2, 3-dichlorophenyl)-1,2,4-triazine, Figure 1] is a widely used, broadspectrum anticonvulsant with efficacy comparable to other major antiepilepic drugs. Treatment with LTG has been associated with a relatively high incidence (5-10%) of allergic skin rashes in patients (1–4), although the drug is generally well-tolerated. The idiosyncratic nature of LTG-induced cutaneous reactions as part of a generalized syndrome that is termed as drug hypersensitivity (5) is not fully understood. The cutaneous reactions associated with LTG have also been reported for other aromatic antiepileptics such as phenytoin and carbamazepine (6). These adverse * To whom correspondence should be addressed. Tel: 484-865-2385. Fax: 484-865-9404. E-mail: [email protected]. † Present address: Drug Safety and Metabolism, Pfizer, 500 Arcola Rd., Collegeville, PA 19426. ‡ Present address: Pfizer Animal Health, Portage St., Kalamazoo, MI 49007. § Present address: Pfizer Global Research and Development, Eastern Point Rd., Groton, CT 06340. 1 Abbreviations: LTG, lamotrigine; Cys-Gly, cysteinylglycine; NAC, N-acetylcysteine; FAL, furafylline; CTD, cimetidine; COUM, coumarin; TCP, tranylcypromine; SPA, sulfaphenazole; MEPH, S-methylphenytoin; QND, quinidine; DDC, diethyldithiocarbamate; TAO, troleandomycin; KTO, ketoconazole; mAb, monoclonal antibody; pmol, picomole; ng, nanogram; ESI, electrospray ionization; CID, collision-induced dissociation; MS/MS, tandem mass spectrometry; LC/MS/MS, liquid chromatography tandem mass spectrometry; LC/MRM, liquid chromatography tandem mass spectrometry operated in the multiple reaction monitoring mode.

Figure 1. Chemical structure of LTG.

reactions have been speculated to be immune-mediated (3, 7), although the exact mechanism of drug hypersensitivity is not fully understood. A large amount of circumstantial evidence (8–14) suggests that chemically reactive metabolites of drugs, rather than the parent compound, are responsible for such idiosyncratic drug adverse reactions including hypersensitivity. It has been proposed that the covalent interaction of reactive metabolites with cellular macromolecules is a critical step in the manifestation of adverse effects. As suggested by the hapten and danger theories (15–19), a reactive metabolite may not only have to modify protein, it may also have to injure or stress cells to induce an immune response and produce consequential idiosyncratic drug reactions (IDRs). The metabolism of LTG was previously investigated in humans as well as in a number of preclinical species including rat, dog, rabbit, and cynomolgus monkey (20, 21). It was found that the primary metabolite excreted in urine was N-2-glucuronide for man, monkey, and

10.1021/tx9003243  2010 American Chemical Society Published on Web 12/04/2009

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rabbit, respectively, while an N-2-oxide was the primary urinary metabolite for rat. The major metabolite in dog urine was the N-2-methylated metabolite (22). Thioether conjugates that indicated bioactivation of LTG to reactive metabolite(s) were not reported in humans and animals until recently (23). In that study, bile duct-cannulated male Wistar rats were intravenously administered [14C] LTG (20 mg/kg). Several GSH-related conjugates were found in bile, suggesting the formation of chemically reactive intermediate(s) during the metabolism of LTG. These thioether metabolites were postulated to be derived from an aryl epoxide intermediate based on mass spectral analysis of GSH-related conjugates present in rat bile. Further studies demonstrated that the production of these thioether conjugates was significantly reduced by the pretreatment of rats with ketoconazole (KTO), indicating the role of P450 enzymes in mediating the bioactivation of LTG to the aryl epoxide intermediate. Nevertheless, the structures of LTG-derived thioether conjugates were not fully established. To further understand the bioactivation of LTG, in vivo metabolism study was conducted in rats. The bile and urine of rats dosed with LTG were analyzed by LC/MS to characterize the thioether conjugates of LTG, some of which were reported previously (23). Subsequently, the thioether conjugates were isolated from bile for further structural elucidation by NMR. In this article, we report the structures of thioether conjugates of LTG, which included the GSH, cysteinylglycine (Cys-Gly), and N-acetylcysteine (NAC) conjugates. Once the definitive structures of thioether conjugate(s) were established, the mechanism of bioactivation and nature of reactive intermediate(s) were postulated. Upon the availability of relatively pure thioether conjugates, further investigative studies were designed to better understand the mechanism and relevance of LTG bioactivation in humans. The bioactivation of LTG was investigated in vitro in rat and human liver microsomes and hepatocytes, in which the activation of LTG to the reactive intermediate was demonstrated by characterizing the GSH-related conjugates. After the bioactivation of LTG was established in rat and human liver microsomes, the identity of P450 enzymes responsible for bioactivating LTG was determined. In addition, the kinetics of P450-mediated activation of LTG to the reactive intermediate was indirectly studied by monitoring the formation of the corresponding GSH conjugate. These studies were performed in male rat and human liver microsomes to demonstrate species and interindividual differences or similarities in the bioactivation process. Finally, the potential bioactivation of LTG was investigated in human epidermal keratinocytes that have recently been demonstrated to have metabolic capabilities (24, 25). The bioactivation of LTG in local tissues such as skin may be more important than the hepatic formation of reactive metabolites, as it is unlikely that these intermediates, once formed in the liver, would be stable enough to be transported to a distant site, such as epidermal keratinocytes.

Materials and Methods Chemicals and Reagents. LTG, S-(4-nitrobenzene) glutathione conjugate (SNBG), and NADP(H) were obtained from SigmaAldrich (Milwaukee, WI). LTG glutathione conjugate (M2) was isolated and purified from bile of rats dosed with LTG. The purity of M2 was >95% based on HPLC analysis. Acetaminophen (APAP), its phenolic glucuronide, and the reactive intermediate, N-acetyl p-quinone imine (NAPQI), were also obtained from SigmaAldrich. The glutathione conjugate of APAP, 3-(glutathione-S-yl) acetaminophen, was obtained by reacting GSH with NAPQI using a previously described procedure (26). The GSH conjugate was subsequently characterized by LC/MS and NMR, and the structure

Chen et al. Table 1. Tandem Mass Spectral Fragment Ions of Thioether Conjugates of LTG thioether conjugate

parent ion ([M + H]+, 35Cl)

M1 M2 M3 M4 M5

579 561 432 375 417

characteristic fragment ionsa 561, 486, 375, 358, 375,

504, 450,b 432,b 375, 329, 288 432,b 375, 329, 288 329,b 288 329,b 288 329, 288b

a Postulated structures of the fragment ions are provided in the Supporting Information. b Base peak in the MS/MS spectrum.

of the conjugate was found to be consistent to what was reported previously (26). Selective P450 inhibitors including coumarin (COUM), tranylcypromine (TCP), sulfaphenazole (SPA), cimetidine (CTD), S-methylphenytoin (MEPH), quinidine (QND), diethyldithiocarbamate (DDC), and troleandomycin (TAO) were obtained from Sigma-Aldrich Chemical Co., while furafylline (FAL) was purchased from Research Biochemicals International (Natick, MA). Ketoconazole (KTO) was obtained from ICN Biomedicals Inc. (Aurora, OH). Waters Symmetry C18 columns (2.1 mm × 150 mm, 4.6 × 250 mm, 5 µm) were obtained from Waters Corp. (Milford, MA). Luna phenyl-hexyl columns (10 mm × 150 mm, 5 µm) and Gemini C18 columns (2.0 mm × 50 mm, 5 µm) were purchased from Phenomenex (Torrance, CA). Solid-phase extraction C18 cartridges (Bond Elut, 10 g/60 mL) were purchased from Varian (Harbor, CA). All solvents and reagents were of the highest grade commercially available. Male Sprague-Dawley rat liver microsomes (pooled from 162 animals), human liver microsomes (pooled from 10 individuals), cDNA-expressed rat P450 enzyme (1A1, 1A2, 2A2, 2B1, 2C6, 2C11, 2C12, 2C13, 2D1, 2D2, 3A1, and 3A2), cDNA-expressed human P450 enzyme (1A1, 1A2, 2A6, 2B6, 2C9*1, 2C19, 2D6*1, 2E1 3A4, and 3A5), monoclonal antibody (mAb) against human P450 1A2, 2A6, 2B6, 2C9, 2D6, 2E1, and 3A4, and the preimmune IgG were purchased from BD Biosciences (Woburn, MA). Twenty individual human liver microsomes designated HLA through HLT were obtained from Tissue Transformation Technologies (Edison, NJ). Cryopreserved hepatocytes obtained from rats (male SpragueDawley, pool of three livers) and humans (male, pool of three livers) were purchased from Xenotech (Kansas City, MO). Clonetics normal human epidermal keratinocytes (NHEK) were obtained from Cambrex Biosciences (Walkersville, MD). In Vivo Metabolism Study of LTG. The animal study was conducted in compliance with the Animal Welfare Act Regulation (9 CFR, parts 1, 2, and 3) as well as an approved Animal Use Protocol (AUP) that was reviewed and approved by the Pfizer Institutional Animal Care and Use Committee (IACUC). Three male Sprague-Dawley rats (250-300 g) fitted with catheters in the bile duct were purchased from Charles River Breeding Laboratories (Wilmington, NC). Upon arrival, animals were acclimated following a 12 h light/dark cycle in a humidity- and temperature-controlled environment for 4 days in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. After acclimation, surgically implanted biliary catheters were connected for the collection of bile under isoflurane anesthesia, and the rats were kept unrestrained in metabolic cages. After overnight recovery, the rats were given an oral dose of LTG (100 mg/kg), which was prepared in methylcellucose and polyethylene glycol (95:5, v:v). Predose bile and urine samples were collected in test tubes. Postdose bile and urine samples were collected at 0-4 and 4-24 h time intervals in test tubes. The volumes of bile and urine samples were recorded, and all samples were stored at -20 °C until analysis. Prior to LC/MS analysis, urine and bile samples were thawed at room temperature, vortexed, and centrifuged at 1600g for 5 min. The supernatants were removed, and aliquots were submitted for LC/MS analysis. Isolation and Purification of LTG Thioether Conjugates. The thioether conjugates of LTG including M1-M5 (Table 1) were isolated and purified by liquid chromatographic methods. Rat bile samples were pooled (∼30 mL), diluted with 30 mL of water, and

BioactiVation of Lamotrigine to an Aryl Epoxide Intermediate concentrated using a Bond-Elut C18 solid-phase extraction cartridges (10 g/60 mL) that were preconditioned with methanol and water. After the bile samples were loaded under gravity, the cartridge was dried and subsequently washed with 10 mL of water followed by gradient elution with 20 mL aliquots of solvent containing different proportions of methanol (5-100%) in water. LC/MS analysis of each sample showed that the thioether conjugates (M1-M5) were present in the 80% methanol fraction, which was subsequently concentrated for further separation and purification by Agilent 1100 HPLC system (Agilent Technologies, Palo Alto, CA) using a Phenomenex semipreparative C18 column (10 mm × 150 mm, 5 µm). The solvent system consisted of acetonitrile and 1 mM ammonium formate (pH 4). The percentage of acetonitrile was increased from 17 to 38% over 10 min with a flow rate set at 3.5 mL/min. Four individual fractions were collected based on the UV absorption at 254 nm. Each fraction was analyzed by LC/MS to confirm the presence of the thioether conjugates. These fractions were pooled separately after multiple injections and concentrated under vacuum. Further HPLC purification and separation of LTG thioether conjugates were carried out using a Waters Symmetry C18 column (4.6 mm × 250 mm, 5 µm) that was eluted with the same solvent system used during semipreparative purification step. To purify M1, the column was eluted with a gradient solvent system, in which the percentage of acetonitrile was increased from 5 to 40% over 8 min with a flow rate set at 1.0 mL/min. The fraction collected at 5.4 min was shown to be M1 by LC/MS analysis, and the fraction collected at 6.0 min corresponded to M2. An isocratic solvent system consisting of 15% acetonitrile was utilized to purify M2, with a flow rate set at 1.25 mL/min. The fraction collected at 5.6 min was determined to be M2 by LC/MS analysis. To separate M3 and M4, the column was eluted with an isocratic solvent system containing 13% acetonitrile with the flow rate set at 1.25 mL/min. M4 and M3 eluted at 5.4 and 6.1 min, respectively. An isocratic solvent system consisting of 22% acetonitrile was applied to the mixture of M3 and M5 with the flow rate set at 1.25 mL/min. The peak at 2.5 min was shown to be M3 by LC/MS analysis, and the fraction collected at 3.7 min consisted of M5. The fractions containing the same metabolite were pooled after multiple injections and dried under vacuum to yield white solids. Subsequently, NMR characterization of these isolated LTG thioether conjugates was performed. In Vitro Studies To Demonstrate LTG Metabolic Activation. Liver Microsomal Incubation. LTG (10-500 µM) was incubated with pooled rat and human liver microsomes (0.5 µM of P450) in the presence of GSH (2 mM) and NADPH (1 mM). All incubations were carried out in 0.1 M phosphate buffer (pH 7.4) containing MgCl2 (3 mM) with the final volume adjusted to 0.5 mL. The incubations were conducted at 37 °C for 30 min in a water bath agitated at a constant speed. The reaction was terminated with 2 mL of ice-cold acetonitrile. A positive control study employing APAP (50 µM) as the substrate was incubated under the same experimental conditions. The formation of GSH conjugate of NAPQI was determined to assess the metabolic viability of the microsomes. After termination, the incubation mixture was vortexed and centrifuged at 1600g for 10 min. The supernatant was removed and concentrated under a stream of nitrogen at room temperature. The dried samples were reconstituted in 200 µL of the HPLC mobile phase (50% acetonitrile in 10 mM ammonium acetate, v/v), and aliquots were analyzed by LC/MS. Hepatocyte Incubations. Rat and human hepatocytes were thawed and suspended in Williams’ E media supplemented with 24 mM NaHCO3 and 10% fetal bovine serum at a concentration of 1 × 106 cells/mL. The viability of the cells, determined by trypan blue exclusion, was g85%. LTG at 10-1000 µM was incubated with 1 × 106 rat and human hepatocyte cells in a final volume of 1 mL at 37 °C for 2 h with gentle agitation. A positive control study using APAP (100 µM) was performed under the same experimental conditions. The formation of GSH conjugate of NAPQI and phenolic glucuronide was determined to assess the metabolic viability of hepatocytes. After the reaction was terminated by adding 3 mL of ice-cold acetonitrile, the incubation mixture

Chem. Res. Toxicol., Vol. 23, No. 1, 2010 161 was vortexed and centrifuged at 1600g for 10 min. The supernatant was separated and dried under nitrogen. The dried samples were reconstituted in 200 µL of the HPLC mobile phase (50% acetonitrile in 10 mM ammonium acetate, v/v), and aliquots were analyzed by LC/MS. Human Epidermal Keratinocyte Culture and Incubation of LTG. The cell culture conditions for adult NHEK in preparation for incubation of LTG were based on those previously published (24). Clonetics adult NHEK were used as cryopreserved first passage cells. The cells were grown in T-75 cm2 flasks using keratinocyte basal medium-2 (KBM-2, 0.15 mM Ca2+, Cambrex Biosciences) with supplements (Clonetics KBM-2 Bullet Kit, Cambrex Biosciences) at 37 °C in the presence of 5% CO2. The media were replaced every 2-3 days. NHEK were subcultured when the cells reached 70-80% confluency. The cells were washed with HEPES-BSS and covered with approximately 2 mL of trypsin/ EDTA until the cells were rounded. Trypsinization was neutralized with approximately 4 mL of trypsin neutralizing solution followed by the addition of 4 mL of KBM-2, and the cells were transferred to a 50 mL conical tube. The cells were then centrifuged at 220g for 5 min to pellet the cells, and the supernatant was discarded. The cells were resuspended in 5 mL of KBM-2 and counted with a Cedex automated cell culture analyzer (Innovatis, Malvern, PA). The cells were dispensed into a culture flask containing an appropriate volume of KBM-2 to achieve a final cell seeding density of 3500 viable cells/cm2. The cells were subsequently dispensed into T-25 cm2 flask for further passage. The incubations of LTG were performed using third or forth passage cells. For incubation of LTG, the cells in T-25 cm2 flask were allowed to reach 80-90% confluency in KBM-2. The media were then replaced with KBM-2 containing 1.25 mM Ca2+, and the cells were allowed to grow to confluency for 48-72 h. Incubations were performed in the T-25 flask with a confluent monolayer in approximately 1 mL of KBM-2 containing 1.25 mM Ca2+. LTG in methanol was added to achieve a final concentration ranging from 100 to 1000 µM, and the incubations were performed at 37 °C in the presence of 5% CO2 and 95% O2 for 24 h. To terminate the metabolic reaction, 3 mL of ice-cold acetonitrile was added, and the entire content was transferred into test tubes for vortexing and centrifuging at 1600g for 10 min. The supernatant was dried under nitrogen and reconstituted in 200 µL of the HPLC mobile phase (50% acetonitrile in aqueous, v/v), and aliquots were analyzed by LC/MS analysis. Characterization of the P450 Enzyme(s) Responsible for LTG Activation. The P450 enzyme(s) responsible for the activation of LTG to an aryl epoxide intermediate was investigated by monitoring the formation of the GSH conjugate (M2). The incubations of LTG were conducted in microcentrifuge tubes (1.5 mL) placed in a water bath that was agitated at a constant speed. The final volume of incubation was adjusted to 200 µL with 0.1 M phosphate buffer (pH 7.4) containing MgCl2 (3 mM). The incubation was conducted at 37 °C for 30 min. The reaction was terminated by addition of 50 µL of ice-cold acetonitrile containing SNBG (500 ng/mL) as the internal standard. The incubation mixture was centrifuged at 1600g for 5 min. Aliquots (10 µL) of supernatants were analyzed for the presence of M2 by liquid chromatography tandem mass spectrometry operated in the multiple reaction monitoring mode (LC/MRM) as described below. The concentrations of M2 present in the matrix were calculated from the calibration curve prepared in the range 1-400 ng/mL of M2. Incubations with cDNA-Expressed P450 Enzymes. A panel of cDNA-expressed rat P450 (1A1, 1A2, 2A2, 2B1, 2C6, 2C11, 2C12, 2C13, 2D1, 2D2, 3A1, and 3A2) and human P450 (1A1, 1A2, 2A6, 2B6, 2C9*1, 2C19, 2D6*1, 2E1, 3A4, and 3A5) was examined for the formation of M2 following incubation with LTG. The incubations (in duplicates) consisted of the P450 (20 pmol), GSH (0.5 mM), and NADPH (1 mM). The concentration of LTG was 50 and 500 µM for rat and human P450 enzymes, respectively. The incubation mixture was preincubated at 37 °C for 5 min in the phosphate buffer before adding NADPH to initiate the reaction.

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Experiments with Selective P450 Inhibitiors. Incubations with male rat liver microsomes (in triplicate) containing microsomes (protein 0.2 mg), LTG (50 µM), GSH (0.5 mM), and NADPH (1 mM) were conducted in the presence of selected inhibitors. The following selective inhibitors were examined for the effect on M2 formation: FAL (10 µM), CTD (50 µM), TAO (50 µM), and KTO (10 µM). The stock solutions of inhibitors and LTG were prepared in dimethyl sulfoxide and subsequently diluted with methanol to appropriate concentrations prior to the incubations. The final concentrations of methanol and dimethyl sulfoxide in the incubation were less than 1.0 (v/v) and 0.1% (v/v), respectively. Each inhibitor, except for KTO, was preincubated with the microsomes and NADPH in the phosphate buffer at 37 °C for 20 min before adding LTG and GSH. KTO was coincubated with the microsomes, LTG, and other components including NADPH in the phosphate buffer. Two individual human liver microsomes, HLG and HLT, were chosen randomly from a panel of 20 human liver microsomes (Table S1 of the Supporting Information) for the inhibition experiments. The P450 content of both liver microsomes, HLG and HLT, was determined by the vendor (Table S1 of the Supporting Information) and found to be 0.33 and 0.47 nmol/mg, respectively. Both HLG and HLT showed no significant deviation in any of their P450 activities, as compared to each other and to the expected range of previously published values from pooled human liver microsomes (27). The incubations, done in triplicate, consisted of the microsomes (0.4 mg of protein), LTG (500 µM), GSH (0.5 mM), and NADPH (1 mM) in the presence of the inhibitor. The following inhibitors at respective concentrations were examined for their effect on M2 formation: FAL (20 µM), COUM (100 µM), TCP (2 µM), SPA (10 µM), MEPH (100 µM), QND (10 µM), DDC (100 µM), TAO (50 µM), and KTO (1 µM). The stock solutions of the inhibitors and LTG were prepared as described above. FAL, DDC, and TAO were each preincubated at 37 °C for 20 min with the microsomes and NADPH in the phosphate buffer before LTG and GSH were added. The chemical inhibitors, COUM, TCP, SPA, MEPH, QND, and KTO, were each coincubated with the microsomes, LTG, and other components in the presence of NADPH in the phosphate buffer. Experiments with the Inhibitory P450 Antibodies. The ability to inhibit the formation of M2 in human liver microsomes (HLG and HLT) in the presence of antibodies directed against P450 1A2, 2A6, 2B6, 2C9, 2D6, 2E1, and 3A4 was studied. Incubations were performed in triplicate consisting of human liver microsomal protein (0.2 mg, P450 0.7 µM), mAb (0.2 mg, 20 µL), LTG (500 µM), GSH (0.5 mM), and NADPH (1 mM). Microsomes were preincubated with individual antibodies or preimmune IgG (as the control) in the phosphate buffer for 20 min at room temperature, followed by the addition of other components. Enzyme Kinetics of LTG Activation. The kinetics of LTG bioactivation to an aryl epoxide intermediate in rat liver microsomes, human liver microsomes, and cDNA-expressed P450 enzymes were studied by determining the relationship between the rates of the GSH conjugate (M2) formation and various concentrations of LTG. It was assumed that initial P450-mediated epoxidation of LTG was the rate-limiting step during the overall activation process leading to M2 formation. The subsequent process involving the conjugation of aryl epoxide intermediate with GSH to form unstable M1 that rapidly decomposed to M2 was assumed to be nonrate limiting chemical reactions and hence considered not to contribute to the kinetics of the activation process. This assumption was supported by the lack of the presence of M1 in incubation mixtures. A similar approach that was used to study the kinetics of P450-mediated bioactivation to reactive intermediates has been described previously (28, 29). In preliminary experiments, the incubation times and the concentrations of microsomal proteins were studied to establish the linearity of M2 formation. It was found that the formation of M2 from LTG in the presence of rat and human liver microsomes increased linearly with the concentrations of microsomal protein or cDNA-expressed P450 enzymes up to 4 mg/mL and with incubation time up to 30 min. Incubations (in triplicate) consisted of the microsomal proteins or cDNA-expressed P450 and various

Chen et al. concentrations of LTG, GSH (0.5 mM), and NADPH (1 mM) in a final volume adjusted to 0.2 mL with 0.1 M phosphate buffer (pH 7.4) containing Mg2Cl2 (3 mM). The reaction was terminated by the addition of 50 µL of ice-cold acetonitrile containing SNBG (500 ng/mL). The incubation mixture was vortexed and centrifuged at 1600g for 5 min. The supernatants were removed, and aliquots (10 µL) were analyzed by LC/MRM. The concentrations of M2 were calculated from the calibration curve prepared in the range 1-400 ng/mL of M2 in the matrix. The kinetic parameters were estimated by fitting the data (the rates of M2 formation versus LTG concentrations) to the Michaelis-Menten kinetic models using nonlinear regression analysis (SigmaPlot 7.1, SPSS, Inc., Chicago, IL). To study the activation kinetics of LTG in male rat liver microsomes, 0.2 mg of microsomal proteins was used, and the rates of M2 formation were determined at LTG concentration ranging from 20 to 800 µM. The reaction was carried out for 10 min at 37 °C. In the case of human liver microsomes, 0.4 mg of microsomal proteins of the pooled human liver microsomes and individual human liver microsomes including HLA, HLB, HLG, HLN, and HLT were used. The rates of M2 formation were determined at a LTG concentration ranging from 50 to 2000 µM. In addition, the kinetic behaviors were also studied in the presence of the inhibitors such as TCP (2 µM) and QND (10 µM) for the pooled human liver microsomes. The reaction was carried out for 10 min at 37 °C. For comparison purposes, the kinetics of LTG activation in the cDNAexpressed P450 2A6 and P450 2D6*1 was investigated. The concentration of P450 2A6 was 0.25 µM, and the rates of M2 formation were determined at a LTG concentration ranging from 50 to 2000 µM. In contrast, 0.05 µM P450 2D6*1 was used, and the rates of M2 formation were determined at LTG concentration ranging from 5 to 400 µM. Incubations with either P450 2A6 or P450 2D6*1 were performed for 10 min at 37 °C. High Field NMR. All NMR spectra were acquired using a Varian 600 MHz Inova NMR spectrometer (Palo Alto, CA) at 298 K with a 3 mm Nalorac probe. LTG and its isolated thioether conjugates were dissolved in 0.3 mL of fully deuterated dimethyl sulfoxide (Cambridge Isotope Laboratories, Andover, MA). Onedimensional spectra were recorded using a sweep width of approximately 8000 Hz and a total recycle time of 3 s. The resulting time-averaged free induction decays were transformed using an exponential line broadening of 0.3 Hz to enhance the ratio of signalto-noise. The two-dimensional spectra, total correlated spectroscopy (TOCSY), were recorded and transformed using the standard pulse sequence provided by Varian. A 2K × 256 data matrix was acquired using a minimum of 32 scans and 16 dummy scans with a spectral width of 8000 Hz in the f2 dimension. A mixing time of 75 ms was used. The data were zero-filled to a size of 2K × 1K. A relaxation delay of 2 s was used between transients. Proton chemical shifts were referenced to dimethyl sulfoxide at δ 2.49. Signal multiplicities are reported as follows: s (singlet), d (doublet), t (triplet), b (broad), bs (broad singlet), dd (doublet of doublet), and cm (complex multiplet). LC/MS. LC/MS analysis of bile and urine samples of rats dosed with LTG was performed on a ThermoFinnigan LCQ Deca XP+ quadrupole ion-trap mass spectrometer (San Jose, CA) coupled to an Agilent 1100 HPLC system (Agilent Technologies). Data were processed using Xcalibur v.1.3 (ThermoFinnigan). The mass spectrometer equipped with an electrospray ionization (ESI) interface was operated in the positive ion mode for LC/MS and LC/MSn analysis. The metabolites were separated on a Waters Symmetry C18 column (2.1 mm × 150 mm, 5 µm) eluted with a gradient solvent system consisting of acetonitrile and 10 mM ammonium acetate buffer (pH 4). The percentage of acetonitrile was increased from 10 to 60% over 16 min with the solvent flow rate set at 0.4 mL/min. After 16 min, the percentage of acetonitrile was increased to 75% within 2 min before re-equilibrating with the initial mobile phase. Aliquots (10-40 µL) of rat bile and urine samples were injected directly onto the column for metabolite profiling and identification. To quantitate the GSH conjugate M2 formed from the incubation of LTG, an API 4000 triple quadrupole

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Figure 2. Tandem mass spectrometric analysis of GSH conjugate M1 ([M + H]+ at m/z 579, 35Cl) present in the bile of rats: (A) mass spectrum of the parent ion at m/z 579 and (B) MS/MS spectrum of the fragment ion at m/z 432.

mass spectrometer (PE-Sciex, Toronto, Ontario) operated in the positive ESI mode was used. The mass spectrometer was coupled to a LC-20AD Shimadzu HPLC system (Shimadzu, Columbia, MD). Data were acquired and processed using Analyst 1.4 software (PE-Sciex) for peak integration and analysis. The HPLC effluent was introduced into the source using a turbo ionspray interface. The mass spectrometer was operated in the multiple reaction monitoring (MRM) mode with a dwell time of 200 ms. The mass transitions of m/z 561 f 432 and m/z 443 f 297 set up for M2 and SNBG (the internal standard), respectively, were monitored during the analysis. The limit of detection (LOD) of M2 was achieved at 0.5 ng/mL. A calibration curve of M2 at 1, 2, 5, 10, 50, 100, and 400 ng/mL spiked in the respective matrix with SNBG (500 ng/mL) at each concentration was established for quantitative analyses. The linearity of calibration curve was exhibited by plotting the peak area ratios of M2 to SNBG versus M2 concentrations. A weighted (1/x2) linear least-squares regression of the concentrations of M2 and measured peak area ratios was used to construct the calibration curves. The HPLC analyses were achieved on a Gemini C18 column (2.0 mm × 50 mm, 5 µm) by a gradient solvent system consisting of acetonitrile and 10 mM ammonium acetate (pH 4). The percentage of organic was increased linearly from 5 to 40% in 3 min followed by a linear ramp to 60% in 1 min. The column was brought back to the initial conditions within the next 30 s before it was re-equilibrated with the initial mobile phase for 2 min. The solvent flow rate was set at 0.25 mL/min. Aliquots (10 µL) of incubation samples were injected directly onto the column.

Results Identification and Characterization of LTG Thioether Conjugates. Bile samples from rats dosed with LTG were analyzed by LC/MS for the presence of GSH-related conjugates. A number of thioether conjugates including M1-M5 (Table 1) were found in rat bile. These conjugates were readily identified by the unique dichlorine isotope pattern of the parent ion ([M + H]+) for each metabolite. The parent ion ([M + H]+, 35Cl) of M1 was found to be at m/z 579 (data not shown), 323 Da higher than the parent ion of LTG ([M + H]+ at m/z 256, 35Cl).

The two-chlorine isotope peak (MH+ + 2) was observed at m/z 581 (37Cl, ∼60% of the MH+). The tandem mass spectra (MS/ MS, Figure 2A) of parent ion at m/z 579 obtained by collisioninduced dissociation (CID) showed two product ions at m/z 450 and m/z 504, resulting from the neutral loss of γ-glutamate (129 Da) and glycine (75 Da), respectively. The formation of these characteristic fragment ions confirmed the presence of a GSH moiety in M1 (30). The fragment ion at m/z 561 was produced by the loss of a water molecule from the parent ion at m/z 579. Similarly, the fragment ion at m/z 432 was formed by the loss of a water molecule from the ion at m/z 450. Alternatively, the ion at m/z 432 could be due to the neutral loss of γ-glutamate (129 Da) from the ion at m/z 561. Further fragmentation of the product ion at m/z 432 yielded a number of major fragment ions at m/z 414, 375, 329, and 288 (Figure 2B). In particular, the ion at m/z 288 was formed through the cleavage of the cysteinyl S-C bond with the retention of the sulfur on LTG moiety (Figure 2B). The origin of major fragment ions is postulated in Figure 2A,B. The mass spectral data of M1 were consistent with what was previously demonstrated for the postulated dihydrohydroxy thioether conjugate found in rat bile (23). An attempt to isolate M1 for NMR characterization was not successful. It was found that M1 was not stable and decomposed to M2 when either the bile sample or the HPLC fraction containing M1 was processed for isolation and purification, consistent with previous findings (23). MS/MS analysis was also performed for other thioether conjugates (M2-M5). The characteristic fragment ions of each thioether conjugate and plausible structures are listed in Table S1 (Supporting Information). M2, with the parent ion [M + H]+ at m/z 561 (35Cl), exhibited a similar MS/MS spectrum as M1, except for the absence of the fragment ions at m/z 504 and 450 (Figure 2A) (Figure S1 of the Supporting Information). Further fragmentation of the ion at m/z 432, a major fragment ion resulting from the neutral loss of γ-glutamate (129 Da), produced an identical spectrum as shown by M1 (Figure 2B). To fully elucidate the

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Table 2. 1H NMR Chemical Shifts (δ), Multiplicity, and Coupling Constants (J) of Thioether Conjugates of LTGa

cysteineb compound

R

LTG

H

M2

GS

M3

Cys-Gly

M5

NAC

4′-H

5′-H

6′-H

δ 7.63 dd J ) 8.1 δ 7.70 s

δ 7.39 t J ) 7.7

δ 7.31 dd J ) 7.7 δ 7.32 s

δ 7.67 s δ 7.63 s

-

δ 7.32 s δ 7.25 s

1′′-H

glutamateb 2′′-H

3′′-H

-

-

-

δ 3.44/3.18 d J ) 11.2 δ 3.09 bs δ 3.43/3.26 b

δ 4.40 b δ 3.37 bs δ 4.01 cm

5′′-H

6′′-H

N-acetyl

-

-

-

-

δ 2.33 bs

δ 1.91/2.33 bs

δ 3.28 cm

δ 3.34 bs

-

-

-

-

δ 3.40 bs

-

-

δ 1.79 s

-

4′′-H

glycineb

-

-

a Key: -, no signal found; s, singlet; d, doublet; t, triplet; dd, doublet of doublet; b, broad; bs, broad singlet; cm, complex multiplet; and GS, glutathionyl. b Refer to Figure S2 for signal assignments of the thioether moiety.

structure of M2, this conjugate was isolated from rat bile and characterized by NMR. The proton spectrum (1H NMR, Figure S2 of the Supporting Information) of M2 contained the characteristic aliphatic proton signals for the GSH moiety (Table 2). The spectrum consisted of one less aromatic resonance as compared to LTG (Table 2). The two existing aromatic resonances are singlets at δ 7.70 and 7.32, respectively. In contrast, the corresponding aromatic resonances of LTG at δ 7.63 and 7.31 are doublets of doublets (Table 2), due to the presence of H-5′ between H-4′ and H-6′. On the basis of the integration, coupling patterns, and chemical shifts, the two aromatic resonances of M2 were assigned as H-4′ and H-6′ of the dichlorobenzene ring. The NMR data of M2 demonstrated the absence of H-5′ of the dichlorobenzene ring, clearly suggesting that GSH adduction was at C-5′ of the dichlorophenyl ring. M3, M4, and M5 were identified to be the Cys-Gly, cysteine, and NAC conjugate of LTG, respectively. The major mass spectral fragment ions of these thioether conjugates are listed in Table 1. M3 and M5 were isolated and characterized by NMR analysis, but an attempt to isolate M4 for NMR analysis was not successful. The 1H NMR spectrum of M3 demonstrated the characteristic aliphatic proton signals for the Cys-Gly moiety (Table 2). As with M2, the spectra presented two aromatic singlet resonances at δ 7.67 and 7.32. In addition, these aromatic resonances exhibited cross-peaks in the TOCSY, indicating a weak coupling between them. Consequently, these two resonances were assigned as H-4′ and H-6′ of the dichlorobenzene ring. The absence of aromatic H-5′ in M3 was demonstrated by NMR. As a result, the site of Cys-Gly adduction for M3 was assigned to be at C-5′ of the dichlorophenyl ring. As with the other thioether conjugates described above, the 1H NMR of M5 also showed two singlet aromatic resonances at δ 7.63 and 7.25 (Table 2). The presence of the cysteine moiety in M5 was confirmed by the resonances at δ 1.79, 3.26, 3.43, and 4.01 (Table 2). On the basis of the integration, coupling patterns, and chemical shifts, the two aromatic resonances found with M5 were assigned to H-4′ and H-6′ of the dichlorobenzene ring. Again, the presence of aromatic hydrogen at C-5′ was not observed by NMR analysis of M5. As a result, the site of NAC adduction was assigned at C-5′ of the dichlorophenyl ring. Of the five LTG thioether

conjugates identified in rat bile by LC/MS, three conjugates were isolated and characterized by NMR analysis. The collective NMR data for metabolites M2, M3, and M5 suggest that metabolites M3 and M5 were derived from the GSH adduct, M2, consistent with breakdown products expected from catabolism of a GSH adduct. Formation of LTG GSH Conjugate with Rat and Human Liver Microsomes and Hepatocytes. To demonstrate the activation of LTG to an aryl epoxide intermediate in both rat and human liver microsomes, a wide range of LTG concentrations (10-500 µM) were tested. The metabolic viability of these microsomes was assessed by monitoring the formation of APAP metabolites. It was found that the GSH conjugate M2 was produced in both rat and human liver microsomes when GSH was included in the incubation. The production of M2 was shown to be NADPH-dependent in both microsomes (data not shown). The confirmation of M2 formation was carried out by the comparison of LC/MS retention times and mass spectral data (MS/MS) with characterized M2 isolated from rat bile. It was found that the concentration of LTG as low as 10 µM produced detectable levels of M2 in male rat liver microsomes. The formation of M2 from LTG (50 µM) in the presence of rat liver microsomes was shown (Figure S3 of the Supporting Information). In contrast, the LTG concentration up to 200 µM was required to generate detectable levels of M2 using pooled human liver microsomes (Figure 3). Bioactivation of LTG to an aryl epoxide intermediate was further studied in rat and human hepatocytes. The metabolic viability of these hepatocytes was confirmed by monitoring the formation of APAP metabolites. Various concentrations of LTG ranging from 10 to 1000 µM were used to study the production of M2. The presence of M2 was clearly demonstrated by LC/MRM analysis when the concentrations of LTG were 50 µM in rat hepatocytes (Figure S3 of the Supporting Information) and 500 µM in human hepatocytes (Figure 3). It was apparent that detectable levels of M2 could be produced only at substrate concentrations equal or greater than 10 and 500 µM in rat and human hepatocytes, respectively. Formation of LTG GSH Conjugate with Human Epidermal Keratinocytes. The potential of LTG activation to an aryl epoxide intermediate in human skin was investigated in



Figure 5. Formation of M2 from LTG (500 µM) in the presence of various human cDNA-expressed P450 enzymes.

Figure 3. Formation of M2 from LTG in the presence of pooled human liver microsomes and cryopreserved human hepatocytes: (A) LC/MRM analysis showing the presence of M2 when LTG (200 µM) was incubated with liver microsomes, (B) LC/MRM analysis showing the presence of M2 when LTG (500 µM) was incubated with hepatocytes, and (C) LC/MRM analysis of M2 (5 ng/mL) spiked in control incubation medium.

Figure 4. Formation of M2 from LTG (500 µM) in the presence of human epidermal keratinocytes: (A) LC/MRM analysis demonstrating the absence of M2 when LTG was omitted, (B) LC/MRM analysis demonstrating the presence of M2 when LTG was incubated with the keratinocytes, and (C) LC/MRM analysis of M2 (1 ng/mL) spiked in control incubation medium.

human epidermal keratinocytes. A wide range of LTG concentrations (100-1000 µM) was tested. As shown in Figure 4, the presence of M2 was unequivocally demonstrated after 24 h of incubation with LTG at a concentration of 500 µM. It was estimated that the concentration of M2 was between 1 and 2

ng/mL as compared with standard M2 spiked in the medium. A minimum concentration of 500 µM of LTG was required to demonstrate the formation of M2 in human epidermal keratinocytes. Characterization of the Rat P450 Enzymes Responsible for LTG Activation. Incubation of LTG with a panel of cDNAexpressed rat P450 enzymes demonstrated that P450 2C11 generated the highest amount of M2 at an enzyme concentration of 100 pmol/mL (Figure S4 of the Supporting Information). Lesser quantities of M2 were produced from LTG in the presence of P450 1A1, 2C6, 2D2, and 3A1 under the same experimental conditions (Figure S4 of the Supporting Information). Undetectable levels of M2 were produced from LTG when it was incubated with P450 1A2, 2A2, 2B1, 2C13, 2D1, and 3A2. From the experiments conducted using selective P450 inhibitors, the formation of M2 was reduced approximately 30% (Table S2 of the Supporting Information) when rat liver microsomes were pretreated with CTD, a mechanism-based inactivator of P450 2C11 (31), as compared to the control. In contrast, the inhibitory effect on M2 formation was less than 10% by FAL, TAO, and KTO, respectively (Table S2 of the Supporting Information). As a result, P450 2C11 appeared to be the enzyme responsible for the bioactivation of LTG to an aryl epoxide intermediate in male rat liver microsomes. Characterization of the Human P450 Enzymes Responsible for LTG Activation. The initial screening using a panel of cDNA-expressed human P450 enzymes revealed that the highest yield of M2 was produced by P450 2D6*1 among ten P450 enzymes (100 pmol/mL per enzyme) (Figure 4). Lesser quantities of M2 were generated from LTG in the presence of P450 2A6, 2B6, 2E1, and 3A4 (Figure 4). M2 was not formed from LTG in the presence of P450 1A1, 1A2, 2C9*1, 2C19, and 3A5 under the same experimental conditions (Figure 5). The effect of selective P450 inhibitors on the formation of M2 in the presence of two individual human liver microsomal samples HLG and HLT was studied. The effective concentrations of inhibitors were chosen based on IC50 values reported in the literature (32, 33). As listed in Table 3, coincubation with COUM, a substrate of P450 2A6, caused 63 and 56% reduction in M2 formation in HLG and HLT, respectively. In addition, TCP, a potent competitive inhibitor of P450 2A6 (34), led to approximately 40% reduction in M2 formation in both HLG and HLT (Table 3). DDC, a mechanism-based inactivator of P450 2E1, resulted in approximately 50 and 40% reduction in M2 formation in HLG and HLT (Table 3), respectively. On the other hand, a minimum inhibitory effect on M2 formation was observed with other inhibitors including FAL, SPA, MEPH, QND, TAO, and KTO (Table 3). Furthermore, a study using mAb against the human P450 enzymes demonstrated that the

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Table 3. Effect of Selective P450 Inhibitors on the Formation of M2 in Individual Human Liver Microsomes (HLG and HLT) M2 formationa inhibitor

HLG

HLT

control FAL (1A) COUM (2A6) TCP (2A6) SPA (2C9) MEPH (2C19) QND (2D6) DDC (2E1) TAO (3A4) KTO (3A4)

100 ( 11 111 ( 2 37 ( 11 57 ( 12 91 ( 10 95 ( 8 97 ( 10 51 ( 6 88 ( 10 98 ( 6

100 ( 4 89 ( 9 44 ( 8 54 ( 10 94 ( 8 97 ( 4 97 ( 8 61 ( 8 97 ( 10 93 ( 4

a Values are the average of triplicates and expressed as percentage (mean ( SD) relative to the control without the inhibitor.

Table 4. Apparent Kinetic Parameter Estimates of M2 Formation in Rat Liver Microsomes, Individual and Pooled Human Liver Microsomes, and cDNA-Expressed P450 2A6 and 2D6*1a microsomes rat male female human HLA HLB HLG HLN HLT mean ((SD) pooled pooled with TCP pooled with QND recombinant P450 P450 2A6 P450 2D6*1

b Km (µM)

494 776

c Vmax (pmol/min/mg)

65 15

turnover numberd (min-1) 0.132 0.019

706 225 358 227 662 436 ((209) 460 433 458

1.4 0.4 4.3 1.1 3.2 2.1 ((1.4) 3.2 1.6 2.8

0.002 0.002 0.012 0.005 0.005 0.005 ((0.004) 0.007 0.004 0.006

205 23

1.45 6.35

0.007 0.276

a Kinetic parameters were obtained using the Michaelis-Menten equation for rat and human liver microsomes and recombinant P450 2A6 and 2D6*1. b Michaelis-Menten constant. c Maximum velocity in pmol/min/mg protein. d Vmax/Km in min-1.

Figure 6. Inhibition of M2 formation from LTG (500 µM) in the presence of mAb against various human P450 enzymes (values are the mean of triplicates and expressed as percentage relative to the preimmune IgG control).

formation of M2 was reduced by approximately 60% when either HLG or HLT was preincubated with mAb against P450 2A6 (Figure 6). Other mAb against P450 1A2, 2B6, 2C9, 2D6, 2E1, and 3A4 showed no significant inhibitory effect on the formation of M2 (Figure 5). These results indicated the role of P450 2A6 in metabolizing LTG to an aryl epoxide intermediate in human liver microsomes. Kinetics of LTG Activation. The apparent kinetic parameters (Km and Vmax) of the metabolic activation of LTG to an aryl epoxide intermediate in rat and human liver microsomes were obtained based on the rates of M2 formation at various concentrations of LTG. The substrate-velocity curve of M2 formation versus LTG concentrations in male rat liver microsomes was exhibited to be hyperbolic (data not shown). For comparison purposes, the kinetics of M2 formation was also studied in female rat liver microsomes, where a hyperbolic curve was also obtained. Eadie-Hofstee transformations of data (rates of M2 formation vs LTG concentrations) were shown to be linear for both male and female rat liver microsomes. The values of Km and Vmax obtained through fitting the data to the Michaelis-Menten equation are listed in Table 4. The calculated turnover number (Vmax/Km) obtained from male rat liver microsomes was 7-fold greater than that of female rat liver microsomes (Table 4). This observation supported the role of P450 2C11 in the bioactivation of LTG in male rat liver microsomes as demonstrated by the inhibitory studies. It should be noted that P450 2C11 is expressed in naı¨ve male rats but not in female rats (35). The kinetics of M2 formation was investigated in five individual human liver microsomes (HLA, HLB, HLG, HLN,

Figure 7. Representative substrate-velocity curve of M2 formation in human liver microsomes (insert: Eadie-Hofstee plot).

and HLT) as well as in the pooled human liver microsomes. It was found that the relationship between the rates of M2 formation and concentrations of LTG in these human liver microsomes was hyperbolic when the data (the rates of M2 formationvsLTGconcentrations)werefittedtotheMichaelis-Menten equation. Figure 7 shows a representative substrate-velocity curve of M2 formation obtained with HLB. The values of Km and Vmax obtained from these human liver microsomal samples are listed in Table 4. The average value of Km from five individual human liver microsomes was 436 ( 209 µM (mean ( SD), which appears to be similar to the value obtained from the pooled human liver microsomes (Table 4). A large variation (10-fold) on the rates of M2 formation was observed. The calculated turnover number (Vmax/Km) from human liver microsomes was substantially lower than the value obtained from male rat liver microsomes (Table 4). Additional kinetics studies were conducted to further examine the participation of P450 2A6 and P450 2D6 in the activation of LTG. As shown in Table 4, when 2 µM of TCP was coincubated with LTG in pooled human liver microsomes, a significant reduction (50%) in Vmax for M2 formation was demonstrated as compared to that obtained in the absence of TCP, whereas the Km remained unchanged. On the other hand, insignificant change in the Vmax was demonstrated when 10 µM of QND was coincubated with



Scheme 1. Postulated Metabolic Bioactivation of LTG to an Aryl Epoxide That Forms the Glutathione Adduct, M1a

a

Subsequent decomposition of M1 leads to M2 and various thioether conjugates M3-M5.

LTG in pooled human liver microsomes. Kinetic studies of M2 formation were also conducted in the recombinant P50 2A6 and P450 2D6*1. The apparent Km of P450 2A6 was found to be approximately 9-fold larger than that of P450 2D6*1 and similar to the values obtained from two individual human liver microsomes HLB and HLN (Table 4).

Discussion The structures of LTG thioether conjugates including M2, M3, and M5 were fully elucidated by LC/MS and NMR analyses. It was shown conclusively that the thiol moiety was covalently linked to C-5′ of LTG in each thioether conjugate. This structural information supported the postulated existence of an aryl epoxide intermediate of LTG. The proposed metabolic pathways leading to bioactivation of LTG are depicted in Scheme 1. The P450-mediated formation of either 4′,5′- or 5′,6′epoxide is postulated to be the initial intermediate step that subsequently leads to the GSH adduct, M1. The structure of M1 could not be established due to its instability. Hence, two possible dihydrohydroxy GSH conjugates are proposed; in both cases, the sulfhydryl of GSH has to be attached to C-5′ based on the definitive structure of M2 (Scheme 1). Although the structure of M1 would have revealed the exact position of epoxidation, the structures of M2 and other thioether conjugates provided sufficient evidence in support of an aryl epoxide metabolic intermediate. It appears that hydrolysis of the aryl epoxide of LTG to a dihydrodiol metabolite (mediated by epoxide hydrolases), a competing detoxification pathway, did not take place to an appreciable extent as evidenced by absence of this metabolite and its downstream products such as phenol glucuronide and sulfate conjugates. This observation is consistent with what was reported before by other investigators (23). In vitro studies conducted with human and rat liver microsomes and hepatocytes confirmed the formation of M2, suggesting that an aryl epoxide intermediate was produced by these hepatic tissues from both species. An attempt to understand the kinetics

of bioactivation of LTG was made by monitoring the formation of a downstream product, M2, since the formation of the epoxide intermediate could not be monitored directly. For kinetic analysis of the activation, the initial P450-mediated epoxidation was assumed to be the rate-limiting step. The subsequent process involving the conjugation of aryl epoxide with GSH to form M1 that subsequently decomposed to give rise to M2 was assumed to be nonenzymatic and hence was not considered to be rate-limiting. These two consecutive reactions were assumed to be principally chemical in nature following first-order kinetics with respect to the concentrations of aryl epoxide intermediate and M1, respectively. The results indicated that humans appeared to be less efficient than rats in forming the epoxide intermediate based on the kinetic parameters of M2 formation studied in human and rat liver microsomes. The average metabolic turnover of LTG to the aryl epoxide intermediate in pooled male rat liver microsomes was approximately 20-fold greater than in pooled human liver microsomes (Table 4), indicating that rats are perhaps better capable of producing the reactive intermediate than humans. The Km for the aryl epoxide formation was estimated to be approximately 460 µM in pooled human liver microsomes (Table 4). This apparent high Km value suggests the capacity of human liver microsomes to bioactivate LTG at increasingly higher substrate concentrations; hence, it is expected that this metabolic pathway will not be saturated at clinically relevant drug concentrations. It was also observed that significant variation in bioactivation of LTG to the epoxide intermediate existed among the 20 individual human liver microsomes (the rates of M2 formation ranging from 0.64 to 5.80 pmol/min/mg; mean ( SD, 1.98 ( 1.28 pmol/min/mg, data not shown). In addition, a high degree of variation in the value of Km (225-706 µM; mean ( SD, 436 ( 209 µM) for the activation leading to M2 formation was observed among five individual human liver microsomes (Table 4). The study with cDNA-expressed P450 enzymes showed that in addition to P450 2A6, other enzymes such as P450 2B6, 2D6, 2E1, and 3A4 were also capable of bioactivating LTG (Figure 5). It is well-

168


known that various P450 enzymes are expressed at significantly different levels in human livers; hence, it is difficult to identify the major bioactivating enzyme(s) for LTG based solely on the studies conducted with equivalent amounts of various recombinant P450 enzymes. A strategy based on selective inhibition of this metabolic reaction (epoxide formation) in human liver microsomes, where the levels of individual P450 enzymes are naturally expressed, assisted us in confirming the P450 (or set of P450s) enzymes involved in this metabolic pathway. The P450 2A6 selective inhibitors COUM and TCP as well as mAb directed against P450 2A6 led to significant reduction in the formation of M2 in human liver microsomes (Table 3 and Figure 6). It should be pointed out that the concentration of LTG used in these experiments was 500 µM, which was similar to the Km obtained from the studies conducted with pooled human liver microsomes. The kinetics of LTG bioactivation conducted with human liver microsomes in the presence of TCP further confirmed the role of P450 2A6 in bioactivating LTG. A significant reduction (50%) in Vmax for M2 formation was observed, while the Km remained unchanged (Table 4). P450 2A6 is expressed at relatively low levels, accounting for 4% of total hepatic P450 (27), and the genetic polymorphism of this P450 enzyme has been reported (36, 37). In addition, it is also suggested that the activities of P450 2A6 could be altered by some dietary components or drugs (38). Studies have demonstrated that P450 2A6 has a small binding site and a major conformational change is required to open and close the enzyme and allow the substrate (e.g., COUM) to enter and leave (39). Furthermore, low catalytic efficiency due to the inherent difficulty of chemistry of substrate oxidation and the lack of proclivity toward a linear pathway leading to product formation have been demonstrated for P450 2A6 (40). Consequently, these factors could potentially explain the interindividual differences and low catalytic efficiency in the bioactivation of LTG observed in this study (Table 4). Although cDNA-expressed P450 2D6 showed a marked ability to form the epoxide of LTG (Figure 5), its role in the LTG bioactivation in human liver microsomes could not be further demonstrated by inhibition studies conducted with 500 µM of LTG in the presence of QND and mAb against P450 2D6 in human liver microsomes (Table 3 and Figure 6). Furthermore, competitive kinetic studies of bioactivation conducted with human liver microsomes in the presence of QND showed no changes in Km and Vmax (Table 4). It is interesting to note that the Km value (23 µM) of recombinant P450 2D6 in bioactivating LTG was substantially lower than that exhibited by recombinant P450 2A6 (205 µM), which was similar to the Km values of liver microsomes obtained from some subjects (Table 4). Also, it is noteworthy to point out that bioactivation of LTG was observed in the in vitro systems only at high substrate concentrations (g200 µM), suggesting the primary role of P450 2A6. P450 2D6-mediated bioactivation of LTG was likely subject to saturation (low Km); however, the Vmax for P450 2D6 was greater than that observed for P450 2A6. Hence, the contribution of P450 2D6 in bioactivating LTG was not very clear. High free plasma concentrations of LTG (5-12 µM) have been observed in clinic, although the therapeutic or toxic (skin rash) exposure levels remain unknown. The relatively high concentrations of LTG would be favorable for both P450 2A6- and 2D6-mediated bioactivation of LTG. However, in our study, the bioactivation of LTG by P450 2D6 present in human liver microsomes at these clinically relevant concentrations could not be demonstrated. The relative contributions from P450 2D6 and 2A6 in bioactivating LTG in vivo warrant further investigation as both enzymes are polymorphic with potential implica-

Chen et al.

tions for the IDR observed with LTG treatment. The role of either P450 2B6 or 3A4 in the bioactivation of LTG in human liver microsomes also could not be established based on the inhibition studies (Table 3 and Figure 6), although cDNAexpressed P450 2B6 and 3A4 formed comparable levels of M2 produced by P450 2A6 (Figure 5). P450 2E1 appeared to be another enzyme capable of activating LTG, as suggested by results from studies with cDNA-expressed enzymes (Figure 5) and with the chemical inhibitor, DDC (Table 3). It should be noted that DDC also inhibits P450 2A6 (41). The results from the study with mAb directed against P450 2E1 showed little effect on the formation of M2 in human liver microsomes (Figure 6). Consequently, the participation of P450 2E1 in bioactivation of LTG remains undetermined. Further studies, such as the comparison of LTG binding to P450 enzymes, correlation of LTG bioactivation in human liver microsomes to individual P450 activities, and in silico P450 active site docking study, are proposed to confirm the roles of P450 2A6 and 2D6 (and other P450 enzymes). There is a considerable debate regarding the probable cause of IDR associated with LTG treatment, particularly the skin rash. Whether LTG or its reactive metabolite(s) induces the IDR remains unresolved. N-Glucuronidation of LTG appears to be the predominant metabolic pathway for LTG in humans as demonstrated by a previously conducted 14C-ADME study (42). Inhibition of N-glucuronidation of LTG by coadministration of valproic acid has been associated with an increased risk of skin reaction in patients (43). While that study ruled out the possibility of N-glucuronide as a causative agent for the IDRs, it did not provide any conclusive evidence on the identity of the offending agent. It can be argued that the inhibition of N-glucuronidation pathway for LTG could result in a higher systemic exposure of the drug and hence predispose some patients to the IDRs due to potential higher levels of reactive epoxide metabolite. As demonstrated in the current study, the Km value for activating LTG to an aryl epoxide intermediate in human liver microsomes was relatively high. The same scenario could also be applicable in human skin cells, where the bioactivation of LTG has not been explored until the present study. The possibility of metabolic activation of LTG in human skins was investigated in epidermal keratinocytes that are the most abundant cell type in the skin and considered to be a target of the immune-mediated damage during cutaneous reactions. The formation of M2 was clearly demonstrated (Figure 4), indicating the formation of aryl epoxide intermediate during the metabolism of LTG in the skin cells, although the role of particular P450 enzyme(s) in these cells remains to be investigated. The metabolic capacities of keratinocytes have become increasingly recognized, and the expression and interindividual variation of genes encoding the P450 enzymes have been reported (44, 45). Of particular interest related to LTG activation, the expression of mRNA for P450 2A6 in human skin keratinocytes was reported and the polymorphic expression of P450 2A6 mRNA has been demonstrated (46). These studies as well as findings from our current work are significant in understanding idiosyncratic cutaneous immune reactions associated with LTG. If the formation of reactive species is indeed a necessary initiating event in LTG-induced hypersensitivity, then local bioactivation (i.e., the skin) may be expected to be more important than hepatic formation of such metabolites. In conclusion, the bioactivation of LTG to the reactive metabolite(s) was demonstrated in vivo in rat and in vitro in human. The formation of an aryl epoxide intermediate was substantiated by elucidating the structures of thioether conjugates


derived from the reactive species. Human P450 2A6 appeared to be the primary enzyme in mediating the LTG bioactivation to an aryl epoxide intermediate in human liver microsomes; however, the role of other P450 enzymes, especially P450 2D6, needs to be further investigated. Human epidermal keratinocytes was demonstrated, for the first time, to be capable of bioactivating LTG to an aryl epoxide intermediate, which could potentially play a role in adverse skin reactions. Overall, the results from our studies provide important mechanistic insight with regard to the bioactivation of LTG and its potential role in eliciting idiosyncratic cutaneous hypersensitivity reaction associated with LTG treatment. Supporting Information Available: Characteristics of human liver microsomes (Table S1), tandem mass spectral fragment ions of thioether conjugates of LTG (M1-M5) (Table S2), and effect of selective P450 inhibitors on the formation of M2 in male rat liver microsomes (Table S3). Tandem mass spectrometric analysis of GSH conjugate M2 ([M + H]+ at m/z 561, 35Cl) present in the bile of rats: top panel, mass spectrum of the parent ion at m/z 561; bottom panel, MS/MS spectrum of the fragment ion at m/z 432 (Figure S1), 1H NMR spectrum of M2 isolated from the bile of rats orally dosed with LTG (Figure S2), the formation of M2 from LTG (50 µM) in the presence of male rat liver microsomes and cryopreserved hepatocytes; top panel, LC/ MRM analysis showing the presence of M2 when LTG was incubated with liver microsomes; middle panel, LC/MRM analysis showing the presence of M2 when LTG was incubated with hepatocytes; top and bottom panel, LC/MRM analysis of M2 (5 ng/mL) spiked in control incubation medium (Figure S3), and the formation of M2 from LTG (50 µM) in the presence of various rat cDNA-expressed P450 enzymes (Figure S4). This material is available free of charge via the Internet at http://pubs.acs.org.

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Bioactivation of Lamotrigine in Vivo in Rat and in Vitro in Human Liver

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