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SPECIES DIFFERENTIAL STEREOSELECTIVE OXIDATION OF A METHYLSULFIDE METABOLITE OF MK-0767 [(±)-5-[(2,4-DIOXOTHIAZOLIDIN-5-YL)METHYL]-2-METHOXY-N-[[(4-TRIFLUOROMETHYL)PHENYL]METHYL]BENZAMIDE], A PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR DUAL AGONIST

Departments of Drug Metabolism (B.V.K., V.G.R., S.V.) and Process Research (C.J.W., J.C., M.B.), Merck Research Laboratories, Rahway, New Jersey

(Received April 9, 2004; accepted June 24, 2004)

	Abstract

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MK-0767 [(±)-5-[(2,4-dioxothiazolidin-5-yl)methyl]-2-methoxy-N-[[(4-trifluoromethyl)phenyl]methyl]benzamide], a thiazolidinedione (TZD)-containing peroxisome proliferator-activated receptor agonist, is a rapidly interconverting racemate that possesses a chiral center at the five position of the TZD ring. M25 is a methyl sulfide metabolite generated from MK-0767 following CYP3A4-mediated TZD ring opening and subsequent methylation of the sulfide intermediate M22. M25, a major in vitro and in vivo metabolite, was further metabolized in liver microsomes to the methyl sulfoxide amide (M16) with two chiral centers and the methyl sulfone amide (M20) with one chiral center. Previous studies demonstrated that both CYP3A4 and flavin monooxygenase-3 (FMO3) catalyzed the formation of M16, whereas M20 was formed exclusively by CYP3A4. The relative contribution of CYP3A4 and FMO3 in the formation of M16 in human and preclinical species was evaluated by chiral analysis using supercritical fluid chromatography. No stereoselectivity was observed in incubations of M25 with human and rhesus liver and recombinant CYP3A4 microsomes, whereas a high degree of stereoselectivity (63 to >99% enantiomeric excess) was observed in rat and dog liver and human recombinant FMO3 microsomes. Also, polyclonal anti-rat CYP3A2 antibody and cytochrome P450 (P450) chemical inhibitors did not inhibit the oxidation of M25 in rat liver microsomes. Furthermore, M25 oxidation was more sensitive to heat inactivation at pH 8 and 8.7 in rat and dog liver microsomes than in human and monkey liver microsomes, consistent with the species difference in involvement of FMOs. Collectively, these results indicated thatS-oxidation of M25 was catalyzed primarily by P450 enzymes in human and monkey liver microsomes and by FMO enzymes in rat and dog liver microsomes.

View larger version (22K): [in this window] [in a new window]   FIG. 1. Partial biotransformation pathway of MK-0767.

The metabolism of MK-0767 in liver microsomes and hepatocytes from humans and preclinical species has been reported elsewhere (Karanam et al., 2004; Liu et al., 2004). The major site of metabolism is the TZD ring, which undergoes CYP3A-mediated opening to give the sulfide derivative M22 (Fig. 1). This conclusion was based on studies conducted with P450-specific antibodies and chemical inhibitors and investigating the metabolism of MK-0767 with recombinant P450 microsomes. Fortification of microsomal incubations with the methyl donor S-adenosyl methionine in addition to NADPH resulted in the generation of several S-methylated metabolites, such as the methyl sulfide M25 and two S-oxidized products: M16, the sulfoxide amide, and M20, the sulfone amide. M25, M16, and M20 were also the major metabolites observed in hepatocyte incubations (Karanam et al., 2004) and in vivo in preclinical species and humans (S. Vincent, M. Creighton, C. Kochansky, B. Karanam, and R. Franklin, unpublished data). MK-0767 is a racemic mixture of the two enantiomers with respect to the five position of the TZD ring. Sulfur oxidation leads to the creation of a second stereocenter and the possibility of two pairs of diastereomers (four possible stereoisomers) for M16.

Both P450 and flavin-containing monooxygenases (FMOs) catalyze NADPH- and oxygen-dependent oxidation of xenobiotics with shared substrate specificities. Unlike the cytochromes P450, there are only a few FMO isozymes present in the liver. Among the identified isozymes, FMO3 is the predominant form in adult human liver, with FMO2 and FMO4 present at very low levels (Cashman, 1995). Studies have shown that the FMO3 form is immunologically and catalytically quite distinct from FMO1, the major FMO in rat and dog liver and human kidney (Lemoine et al., 1990; Lattard et al., 2002), but related to the macaque hepatic FMO (Sadeque et al., 1993).

The metabolic products from drugs mediated by FMOs are relatively simple and mainly involve NADPH-dependent oxygenation of nucleophilic nitrogen, sulfur, phosphorus, and selenium in a structurally diverse range of compounds (Ziegler, 1993). Various drugs were reported to be substrates for FMOs, including cimetidine (Cashman et al., 1993a), ranitidine (Overby et al., 1997; Chung et al., 2000), imipramine (Lemoine et al., 1990), tamoxifen (Mani et al., 1993), chlorpromazine (Cashman et al., 1993b), and clozapine (Tugnait et al., 1997).

Since both P450s and FMOs catalyze the oxidation of N- and S-containing compounds, a hallmark feature of these enzymes is to oxidize prochiral substrates to products of distinct stereochemistry (Cashman, 1998). Determination of the stereochemistry of these oxidized products can help identify the major monooxygenase responsible for the biotransformation. The objective of this study was to compare the stereoselectivity of S-oxidation in M25, a major metabolite, to generate the corresponding sulfoxide amide, M16, by CYP3A and FMO enzymes and furthermore to determine their relative contribution in the formation of M16 in various species of interest. In addition to chiral chromatography, a combination of several other approaches was used, namely temperature and pH dependence as well as P450 isoform-specific chemical inhibitors, immunoinhibition, and metabolism by recombinant enzymes.

	Materials and Methods

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Chemicals and Reagents. MK-0767 and synthetic standards of its major metabolites (Fig. 1), including the sulfide derivative (M22), the methyl sulfide (M25), the methyl sulfoxide amide (M16), and the methyl sulfone amide (M20) were synthesized at Kyorin Pharmaceutical Co., Ltd. (Tokyo, Japan). All other chemicals and reagents were obtained from commercial sources and were of analytical grade.

Liver Preparations, Recombinant and Purified Enzymes, and P450 Inhibitory Antibodies. Human liver microsomal preparations were prepared from individual livers by differential centrifugation. Aliquots from each preparation were pooled on the basis of equivalent protein concentrations. Liver microsomes were prepared from fresh or frozen control tissue obtained from adult rat, dog, and rhesus monkeys. Microsomes containing recombinant human CYP3A4 with coexpressed P450 reductase were obtained from Dr. Tom Rushmore (Merck Research Laboratories, West Point, PA) and from BD Gentest (Woburn, MA). Microsomes containing recombinant rat P450s, FMO1, FMO3, and FMO5 were obtained from BD Gentest. Ascites fluid containing monoclonal antibodies to P450 isoforms were obtained from Dr. M. Shou (Merck Research Laboratories, West Point, PA). Anti-rat CYP3A2 and anti-rat CYP2C11 serum containing a polyclonal antibody produced in rabbits were obtained from BD Gentest.

Liver Microsomal Incubations. M25 (10 µM) was incubated with human, rat, dog, and rhesus liver microsomes at a protein concentration of 1 to 2 mg/ml in 0.1 M potassium phosphate buffer, pH 7.4, containing 1 mM EDTA and 5 mM MgCl₂ in a total volume of 200 µl (n = 2 or 3). M25 was added as an acetonitrile solution at a final organic content <1%. Incubations were initiated with the addition of an NADPH-regenerating system consisting of 10 mM glucose 6-phosphate, 10 mM NADP, and 1.4 units/ml glucose 6-phosphate dehydrogenase. The reactions were carried out for 20 to 30 min at 37°C in a shaking water bath. Incubation mixtures were quenched with 1 volume of acetonitrile, and samples were centrifuged at 10,000 rpm for 10 min at 4°C. The resulting supernatants were analyzed by HPLC.

Metabolism of M25 by Heterologously Expressed P450 Isoforms and FMOs. M25 was incubated at a concentration of 10 µM with microsomes from Baculovirus insect cells expressing recombinant human CYP3A4, FMO1, FMO3, and FMO5 and rat CYP2A2, CYP2B1, CYP2C6, CYP2C11, CYP2C12, CYP2C13, CYP2D1, CYP3A1, and CYP3A2 (20 pmol in a 200-µl incubation). Reactions (n = 2 or 3) were carried out in the presence of an NADPH-regenerating system at 37°C for 30 min and quenched with 1 volume of acetonitrile, and samples were centrifuged at 10,000 rpm for 10 min at 4°C. The resulting supernatants were analyzed by HPLC.

Effect of P450 Inhibitory Antibodies on Metabolism of M25 in Rat Liver Microsomes. To determine the contribution of P450 isozymes in the metabolism of M25, rat liver microsomes (0.3 mg/ml) were preincubated at room temperature for 10 min with anti-rat CYP3A2 or anti-rat CYP2C11 polyclonal antibody (80-250 µl of serum per milligram of microsomal protein). Reactions (n = 2 or 3) were initiated with the addition of 10 µM substrate and an NADPH-regenerating system and carried out for 30 min at 37°C. A similar protocol was followed to determine the effect of anti-rat CYP3A2 serum on 6ß-hydroxylation of testosterone in rat liver microsomes. The concentration of testosterone was 50 µM, and incubations were carried out for 20 min at 37°C.

Inhibition Studies in Rat Liver Microsomes. P450 isoform-specific inhibitors were used to investigate the role of CYP3A enzymes in the metabolism of M25 in rat. Solutions of the inhibitors were prepared in acetonitrile, and final concentrations in the incubations were 100 µM troleandomycin (TAO), 1 and 5 µM ketoconazole, and 25, 50, and 100 µM cimetidine. The final concentration of the organic solvent was less than 2%. Microsomes (0.3 mg/ml), suspended in 0.1 M potassium phosphate buffer, pH 7.4, with 1 mM EDTA and 5 mM MgCl₂, were first preincubated (n = 2 or 3) for 30 min with TAO or cimetidine and an NADPH-regenerating system. At the end of 30 min, M25 (20 µM) was added, and the reaction was continued for an additional 30 min. To study the effect of ketoconazole, microsomes were coincubated with M25 and ketoconazole in the presence of an NADPH-regenerating system for 30 min. The effects of TAO (100 µM) and ketoconazole (1 and 5 µM) on testosterone 6ß-hydroxylation in rat liver microsomes were studied in a similar manner. The concentration of testosterone was 50 µM, and the reactions were carried out for 20 min. The internal standard used was corticosterone. Reactions were quenched with acetonitrile, the denatured microsomal proteins were pelleted by centrifugation (10,000 rpm, 10 min, 4°C), and the supernatant was analyzed by reverse phase-HPLC.

Incubations of M25 in Heat-Treated Human, Monkey, Dog, and Rat Liver Microsomes. To determine the contribution of FMOs in the metabolism of M25, microsomes were suspended in potassium phosphate buffer, pH 7.4 (optimal for P450 reactions), or in sodium pyrophosphate buffer, pH 8 and 8.7 (both optimal for FMO reactions). Additionally, the microsomal suspensions were subjected to heating at 55°C for 55 s in the absence of NADPH to denature the FMOs. Reactions (n = 2 or 3) were initiated by the addition of M25 (10 µM) and an NADPH-regenerating system.

Analytical Methods. HPLC. Acetonitrile supernatants of in vitro incubations were analyzed on a Zorbax XDB-C8 column (3 x 150 mm, 3.5 µm; MAC-MOD Analytical, Chadds Ford, PA). The column was eluted at 0.5 ml/min, with a 30-min linear gradient from 27% A (10 mM ammonium acetate in water) to 50% B (1 mM ammonium acetate in 93% acetonitrile and 7% methanol). The wavelength for UV detection was 220 nm. Acetonitrile supernatants of incubations of testosterone were analyzed on a Zorbax C8 RX column (4.6 x 25 cm, 5 µm; MAC-MOD Analytical) eluted at a flow rate of 1 ml/min for 7 min with 60% A (0.1% trifluoroacetic acid in water) followed by a linear gradient for 8 min to 85% B (acetonitrile with 0.1% trifluoroacetic acid). Testosterone, 6ß-hydroxy testosterone, and the internal standard corticosterone were monitored by UV detection at a wavelength of 254 nm. The HPLC system consisted of a Shimadzu 10A system controller, two LC-10 AD pumps, an SIL 10A autosampler, an SPD 10A UV-VIS spectrophotometric detector, and an online radiometric detector (PerkinElmer Life and Analytical Sciences, Boston, MA).

Chiral analysis. To separate the stereoisomers of the sulfoxide (M16), methanol extracts from rat, dog, monkey, and human liver microsomal and recombinant P450 and FMO microsomal incubations of the methyl sulfide (M25) were analyzed by chiral supercritical fluid chromatography (SFC). Chiral SFC screening was carried out using a pair of analytical supercritical fluid systems (Berger Instruments, Newark, DE) fitted with six-position column selection valves and Agilent model 1100 diode array UV-visible detectors (Agilent Technologies, Palo Alto, CA). Chiral stationary phases evaluated in the study included Chiralpak AD and AS, Chiralcel OD, OJ, OF, and OB (Chiral Technologies, Inc., Exton, PA), Whelko (Regis Technologies, Inc., Morton Grove, IL), Chirobiotic V, R, and T (Astec, Whippany, NJ), and TBB (Kromasil chiral-O-O'-bis(4-tert-butylbenzoyl)-N,N'-diallyl-L-tartar diamide) (Eka Nobel, Bohus, Sweden). An achiral silica column (Kromasil; Eka Nobel) was also included in the SFC screening system as a means of identifying the presence of diastereomers or achiral impurities. All screening columns were of a standard 25-cm length and 4.6-mm inner diameter. The two systems were run in parallel and employed a standard gradient method with a flow rate of 1.5 ml/min, an outlet pressure of 200 bar, an oven temperature of 35°C, and a UV detection at 215 nm. The mobile phase consisted of 4% methanol in CO₂ for 4 min that increased to 40% methanol over 18 min with a hold at 40% methanol for 3 min and a 5-min wash. Using these standard gradient conditions, baseline separation of the enantiomers of the methyl sulfide M25 and the sulfone M20 was obtained on Chiralpak AD and Chiralpak AS columns. Baseline separation of all four stereoisomers of the sulfoxide M16 was obtained on the Chiralpak AD, Chiralpak AS, and Chiralcel OF columns; however, considerable problems with peak overlap were observed between the different analytes on the AD and AS columns. Therefore, the Chiralcel OF column was used since it afforded baseline separation of the four stereoisomers of M16, but it did not completely separate the enantiomers of M25 or M20.

Liquid chromatography/mass spectometry. Liquid chromatography/mass spectometry analysis was carried out using an Agilent 1100 HPLC MSD single quadrupole instrument using electrospray ionization in the positive ionization mode with variable fragmentor voltage (60-240 V) and scan (100-1200 atomic mass units). A Zorbax Extend C18 column (3 x 50 mm, 3 µm) eluted at 0.75 ml/min with a linear gradient from 95% A (2 mM aqueous ammonium hydroxide, pH 10.5) to 95% B (2 mM ammonium hydroxide, pH 10.5, in 90:10 v/v acetonitrile/water) over 3 min was used. Typically, 2-µl aliquots were analyzed. Under these conditions, the sulfide M25 was observed at 3.56 min ([M + H]⁺¹ at m/z 427), the M16 sulfoxide stereoisomers were eluted as a single peak at 3.22 min ([M + H]⁺¹ at m/z 443), and the sulfone M20 was eluted at 3.42 min ([M + H]⁺¹ at m/z 459).

Semipreparative SFC purification of M16 stereoisomers and analysis by circular dichroism (CD). To unambiguously determine the relationship between the four stereoisomers of M16, a 40-mg sample of the mixture of four stereoisomers was purified by semipreparative SFC, and the resulting fractions were analyzed by CD spectroscopy. Semipreparative SFC purification was carried out on a Multigram instrument (Berger Instruments). The column used was a Chiralpak AD (2 x 25 cm), which was eluted with a mobile phase consisting of 24% methanol/CO₂ at a flow rate of 50 ml/min and an outlet pressure of 100 bar. The column was maintained at 35°C, and the analytes were monitored at 220 nm. A methanolic solution of the M16 diastereomers at a concentration of 13 mg/ml (total volume, 1.5 ml) was injected onto the column. The isolated stereoisomers were evaluated by CD spectroscopy at a concentration of 0.05 mg/ml (in methanol) using a Jasco J 810 spectropolarimeter (Jasco, Inc., Easton, MD).

	Results

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Metabolism of M25 in NADPH-Enriched Liver Microsomes. Incubations of M25, the methyl sulfide metabolite of MK-0767 with NADPH-enriched rat, dog, rhesus monkey, and human liver microsomes, yielded qualitatively similar metabolite profiles (data not shown). In all species, M25 was converted to two major metabolites, the sulfoxide amide, M16, and the sulfone amide, M20 (Fig. 1). These metabolites were identified by comparison of their HPLC retention times to those of synthetic standards.

Metabolism of M25 by Heterologously Expressed Rat P450 and Human FMO Isoforms. To identify the cytochrome P450 isoform involved in its metabolism in the rat, M25 was incubated with microsomes containing expressed rat P450 isoforms: CYP2A2, CYP2B1, CYP2C6, CYP2C11, CYP2C12, CYP2C13, CYP2D1, CYP3A1, and CYP3A2. As shown in Fig. 2 (panel A), rat CYP3A1, CYP3A2, and CYP2C11 were the only isoforms that metabolized M25 to its oxidized products, the methyl sulfoxide M16 and the methyl sulfone M20. When incubated with recombinant FMOs, M25 was metabolized to M16 by FMO1, 3, and 5. Small amounts of M20, on the other hand, were generated only in recombinant FMO1 incubations (Fig. 2, panel B).

View larger version (26K): [in this window] [in a new window]   FIG. 2. Formation of M16 and M20 in recombinant rat P450 (A) and human FMO (B) microsomal incubations using M25 (10 µM) as a substrate.

Immunoinhibition of Rat Liver Microsomal Metabolism of M25. The effect of anti-CYP3A2 polyclonal antibody on the metabolism of M25 in rat liver microsomes was evaluated to determine the contribution of CYP3A isozymes. As shown in Fig. 3 (panel A), the formation of M16 was only minimally affected by the presence of the anti-rat CYP3A2 serum at amounts ranging from 80 to 250 µl/mg microsomal protein (0 to 10% inhibition in the formation of M16). On the other hand, the formation of M20 was decreased significantly in the presence of increasing amounts of antiserum by 60 to 75% at 80 to 250 µl/mg protein. Under similar conditions, the anti-rat CYP3A2 serum inhibited 6ß-hydroxylation of the CYP3A probe substrate testosterone in rat liver microsomes by >90% (data not shown). Similarly, when rat liver microsomal incubations of M25 were carried out in the presence of anti-rat CYP2C11 serum, there was no appreciable inhibition in the formation of the sulfoxide (M16) or sulfone (M20) metabolites (Fig. 3, panel B).

View larger version (14K): [in this window] [in a new window]   FIG. 3. Effect of anti-rat CYP3A2 and 2C11 antiserum on the metabolism of M25 (10 µM) to M16 and M20 in rat liver microsomes.

Effect of CYP3A and CYP2C11 Inhibitors on the Metabolism of M25 in Rat Liver Microsomes. The effects of two P450 3A1/2 inhibitors, TAO (Pessayre et al., 1982) and ketoconazole (Maurice et al., 1992), were examined on the oxidation of M25 to the sulfoxide amide, M16, and the sulfone amide, M20, in rat liver microsomes. As shown in Table 1, at a concentration of 100 µM TAO, the formation of M16 was not significantly affected (25% inhibition). Similarly, at a concentration of 1 µM ketoconazole, there was marginal (15%) inhibition of sulfoxide formation (Table 1). In comparison, the 6ß-hydroxylation of testosterone in rat liver microsomes was inhibited by 1 µM ketoconazole (82%, data not shown). Since recombinant CYP2C11 (Fig. 2) was the only other rat P450 isozyme that was observed to oxidize M25, the effect of cimetidine, a mechanism-based inhibitor of CYP2C11 (Levine and Bellward, 1995), was studied on the metabolism of M25 in rat liver microsomes. At concentrations of 25 and 50 µM, cimetidine had no effect on the formation of M16 (Table 1); at 100 µM, 37% inhibition was observed.

Incubations of M25 with Heat-Treated Rat, Dog, Monkey, and Human Liver Microsomes. To determine the contribution of liver FMOs, incubations of the methyl sulfide (M25) were conducted with rat, dog, monkey, and human liver microsomes (0.1-0.3 mg/ml protein) at pH values of 7.4 (optimum for P450 reactions) and 8 and 8.7 (optimum for FMO reactions). Furthermore, the effect of heating microsomal suspensions at 55°C for 55 s (to denature FMOs) on M16 formation was also evaluated. The results are shown in Table 2. In rat liver microsomal incubations, the conversion of M25 to M16 was 50, 45, and 26%, respectively, at pH 7.4, 8, and 8.7. When microsomes were heat-inactivated, these values decreased to 24, 7, and 7%, respectively, that is, an inhibition of 52, 84, and 73%, respectively. In dog liver microsomes, there was activation in the percentage of conversion of M25 to M16 from 40% at pH 7.4 to 59% at both pH values of 8 and 8.7. Furthermore, when microsomes were heat-inactivated, the conversion of M25 to M16 decreased considerably to 12, 4, and 0% at pH 7.4, 8, and 8.7, respectively, corresponding to a decrease in conversion by 70, 93, and 100%, respectively. In contrast, in the monkey, there was a decrease in the formation of M16 at pH 8 (32%) and 8.7 (16%) compared with pH 7.4 (53%). Heat inactivation of monkey microsomes resulted in a modest inhibition in the formation of M16 (27 and 50%) only at pH 8 and 8.7, respectively, with no inhibition at pH 7.4. Similarly, in human liver microsomes, the formation of M16 decreased from 44% conversion at pH 7.4 to 17% at pH 8 and 10% at pH 8.7, and these values decreased by 23, 18, and 40%, respectively, in heat-inactivated microsomal incubations.

Chiral Analysis and Circular Dichroism of M16 Diastereoisomers. To unambiguously determine the relationship between the four stereoisomers of M16, a 40-mg sample of the mixture of four stereoisomers was purified using semipreparative SFC using a Chiralpak AD column (Fig. 4). Four purified fractions obtained from this method (fractions A, B, C, and D) were each shown to be >99% pure by analytical SFC, with the exception of fraction D, which had poor recovery and reduced purity owing to a leaking collection line.

View larger version (16K): [in this window] [in a new window]   FIG. 4. Preparative SFC purification of four M16 stereoisomers (fractions A, B, C, and D) using a Chiralpak AD column. See Materials and Methods for experimental conditions.

Circular dichroism studies of methanol solutions of the four stereoisomers clearly showed fractions A and D, and B and C to be enantiomers (Fig. 5). Independent injection of the isolated stereoisomers onto an analytical Chiralcel OF column (the column used in the analysis of microsomal samples, vide infra) showed that fraction A eluted first, B fourth, C third, and D second. Thus, on the chiral SFC assay employing the Chiralcel OF column used in the metabolism studies (Fig. 6), M16 peaks 1 and 2, and 3 and 4 were enantiomers (vide infra). Attempts to assign absolute configuration to each of the stereoisomers were inconclusive; therefore, only their relative configuration was established.

View larger version (14K): [in this window] [in a new window]   FIG. 5. Circular dichroism analysis of isolated fractions from preparative SFC (see Fig. 4) on a Chiralpak AD column showing that fractions A and D and B and C are enantiomers.

View larger version (18K): [in this window] [in a new window]   FIG. 6. UV Chromatogram of SFC chiral analysis of human (A) and monkey (B) liver microsomal incubations of M25, analyzed on a Chiralcel OF column, showing a lack of stereoselectivity in the formation of M16 stereoisomers.

Chiral Analysis of Human, Monkey, Rat, and Dog Liver Microsomal Incubations. To determine the stereoselectivity of sulfoxide (M16) formation in liver microsomes, methanol extracts of human, monkey, rat, and dog liver microsomal incubations of M25 were subjected to chiral SFC analysis. Comparison of chromatograms in Fig. 6 from human (panel A) and monkey liver (panel B) indicated that there was little stereoselectivity in the formation of the stereoisomers of M16, with equal proportions of the four stereoisomers being formed in these species. In contrast, as shown in Fig. 7 (panel A), incubations of M25 with rat liver microsomes showed highly enantioselective formation of both diastereomers of sulfoxide (M16). An enantioselectivity excess (ee) of 94% was observed for the first pair of diastereomers (peaks 1 and 2), with a 69% ee observed for the formation of the second pair of diastereomers (peaks 3 and 4). Similarly, in dog liver microsomes, enantiomeric excesses of >99% and 88% were observed for the two pairs of enantiomers (Fig. 7, panel B), the pattern resembling that obtained from rat liver microsomes, showing enantioselective formation of the sulfoxides.

View larger version (16K): [in this window] [in a new window]   FIG. 7. UV Chromatogram of SFC chiral analysis of rat (A) and dog (B) liver microsomal incubations of M25, analyzed on a Chiralcel OF column, showing the stereoselective formation of M16 isomers.

Chiral Analysis of Incubations of M25 with Recombinant Human CYP3A4, Rat CYP3A1, 3A2, and 2C11 and Human FMO3 and FMO1 Microsomes. The stereoselectivity of M16 formation was further investigated in recombinant human CYP3A4 incubations of M25. Results of the chiral analysis shown in Fig. 8 indicated that, similar to human liver microsomes (see Fig. 6), recombinant human CYP3A4 did not exhibit any stereoselectivity in the sulfoxidation, with all four diastereoisomers being formed in equal amounts. Similarly, incubations of M25 conducted in recombinant rat CYP3A2 and 2C11 microsomes showed no stereoselectivity in the formation of the sulfoxide, with all four diastereoisomers being formed (data not shown).

View larger version (9K): [in this window] [in a new window]   FIG. 8. UV chromatogram of SFC chiral analysis of recombinant CYP3A4 microsomal incubations of M25, analyzed on an Chiralcel OF column, showing a lack of stereoselectivity in the formation of M16 stereoisomers.

Incubations of M25 with recombinant FMO3, the major FMO present in human liver, revealed a high degree of stereoselectivity, with a predominant formation of 2 of the 4 possible stereoisomers of M16 (Fig. 9). An enantioenrichment of 63% ee was observed for the first pair of diastereomers (peaks 1 and 2, panel A), with 83% ee being observed for the second pair of diastereomers (Fig. 9, panel A, peaks 3 and 4). Similarly, chiral analysis of microsomal incubations of M25 with recombinant human FMO1 (the form orthologous to the one present in rat liver) (Itoh et al., 1993) showed a highly stereoselective profile of M16 (>99% ee for the first eluted diastereomers and 98% ee for the second eluted diastereomers. (Fig. 9, panel B).

View larger version (16K): [in this window] [in a new window]   FIG. 9. UV chromatogram of SFC chiral analysis of recombinant human FMO3 (A) and FMO1 (B) microsomal incubations of M25, analyzed on a Chiralcel OF column, showing the stereoselective formation of M16 isomers.

	Discussion

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MK-0767 is a thiazolidinedione-containing PPAR agonist that is undergoing investigation for the control of diabetes and hyperlipidosis. Detailed in vitro studies demonstrated that the metabolism of MK-0767 was mediated primarily by CYP3A4, with the microsomal methyltransferases playing a major role in its secondary metabolism (Karanam et al., 2004). Additionally, there was evidence for the involvement of FMOs and esterases in the metabolism of the intermediate metabolites of MK-0767 (Karanam et al., 2004). A major in vitro and in vivo metabolite was M25, a methylated sulfide intermediate that underwent S-oxidation to the methyl sulfoxide amide, M16, and the methyl sulfone amide, M20 (Fig. 1). Earlier studies showed that both CYP3A4 and FMO3 mediated the S-oxidation of M25 to M16 in human liver microsomes (Karanam et al., 2004). The present study was undertaken to identify the enzyme(s) involved in the oxidation of M25 to M16 and M20 in rat, dog, and monkey liver microsomes. Additionally, the differential stereoselective oxidation of M25 to M16 was investigated in these species, as well as in humans, to help determine the relative contribution of the two monooxygenases (P450 and FMOs).

Metabolite profiles of the sulfide metabolite M25 in rat, dog, rhesus monkey, and human liver microsomes were qualitatively similar. In all these species, M25 was oxidized to M16 and M20. Studies with recombinant rat P450 microsomes indicated that CYP3A1, CYP3A2, and CYP2C11 were capable of catalyzing the S-oxidation of M25 to M16. However, incubating M25 with rat liver microsomes in the presence of rabbit antiserum raised against CYP3A1/2 failed to inhibit the oxidation of M25. Similarly, antiserum against CYP2C11 did not inhibit the oxidation of M25. The formation of the sulfone M20, however, was inhibited in these incubations by anti-CYP3A1 antibody. Also, the CYP3A inhibitors TAO (100 µM) and ketoconazole (1 µM) did not inhibit the formation of M16 from M25 in rat liver microsomal incubations. Similarly, the CYP2C11 inhibitor cimetidine (Levine and Bellward, 1995) did not inhibit the formation of M16 at concentrations of 25 or 50 µM, but some inhibition (36%) was observed at the highest concentration used (100 µM). These results collectively indicated that in rat liver microsomes, CYP3A1/2 or CYP2C11 may not play a role in the sulfoxidation of M25.

Further studies in rat liver microsomes involved an investigation in the role of FMOs in the S-oxidation of M25 by modulating incubation conditions to selectively enhance or inhibit FMO activity. FMOs are reported to have a catalytic optimum at pH values of 8, at which point there is minimum participation by P450s (Atta-Asafo-Adjei et al., 1993; Itagaki et al., 1996; Kawaji et al., 1997). Following incubation of M25 in rat liver microsomal incubations, there was a significant conversion of M25 to the methyl sulfoxide M16 at pH 8 and 8.7, 90 and 52%, respectively, of the extent formed at pH 7.4. Results from dog liver microsomal incubations showed a more pronounced involvement of FMOs in the sulfoxidation of M25 than in the rat; the extent of sulfoxidation of M25 was higher (59%) at pH 8 and 8.7 than at pH 7.4 (40%). Similar increases in the rate of FMO-mediated reactions at higher pH values (8 to 9) compared with pH 7.4 has been reported for the N-oxidation of nicotine (Cashman et al., 1992), xanomeline (Ring et al., 1999), and S-oxidation of SNI-2011 (Washio et al., 2001). On the other hand, in the case of monkey and human liver microsomes, the conversion of M16 to M25 was lower at pH 8 and 8.7 (10 to 32%) compared with pH 7.4 (44 to 53%). Collectively, these data suggest a higher involvement of FMOs in sulfoxidation of M25 in rat and dog than in monkey and human liver microsomes.

Another distinct property of FMO-mediated reactions that distinguishes them from P450-mediated reactions is the thermal lability of FMOs in the absence of NADPH (Uehleke, 1973; McManus et al., 1987; Grothusen et al., 1996). When rat liver microsomes were heated to 55°C (for 55 s) to denature FMOs, there was a significant inhibition in the formation of M16 at pH 8 (84%) and 8.7 (73%). Similarly, in dog liver microsomes, heat inactivation resulted in a 93 and 100% inhibition at pH 8 and 8.7, respectively. Inhibition at pH 7.4 ranged from 52 to 70% in these species, most likely because of significant FMO contribution at this pH. In monkey and human liver microsomes, on the other hand, the extent of inhibition in heat-inactivated microsomal incubations was less pronounced (18 to 50% at pH 8 and 8.7 and 0 to 23% at pH 7.4), consistent with a lower involvement of FMOs than in rat and dog.

A hallmark feature of both P450 and FMO enzymes is the oxidation of prochiral substrates to products of distinct stereochemistry, especially those of heteroatom (N- and S-)-containing compounds (Cashman, 1998). Determining the stereochemistry of these oxidized products can serve as a guide to the identification of the major enzyme responsible for the metabolism of a xenobiotic. Since the oxidation step in M25 led to the generation of a second stereogenic center in M16, the stereoselectivity of the diastereoisomers of M16 generated in liver and recombinant microsomes in the species of interest were analyzed by chiral SFC. Both human and monkey liver microsomes yielded an approximately equal mixture of all four of the possible M16 stereoisomers, thus revealing essentially no stereoselectivity. The human microsome profile matched that of the recombinant CYP3A4 profile, which also showed no stereoselectivity. A distinctly different profile, however, was obtained with the recombinant FMO3 and FMO1, which were more stereoselective in generating, predominantly, 2 of the 4 stereoisomers. The enantiomers of the initially eluted diastereomer (peaks 1 and 2) were formed with 63% ee, whereas the enantiomers of the later eluting diastereomer (peaks 3 and 4) were formed with 83% ee (Fig. 9). These data collectively suggested that the role of FMO3 in the S-oxidation of M25 in human liver microsomes was minor compared with that of CYP3A4.

Comparative chiral analysis was conducted also with products of M25 following incubations with rat and dog liver and rat recombinant CYP3A2 and CYP2C11 microsomes. Rat and dog liver microsomes exhibited a predominantly stereoselective S-oxidation of M25 generating 2 of the possible 4 stereoisomers of M16 (rat, 94 and 69% ee; dog >99% and 88% ee; Fig. 7). In addition, recombinant human FMO1, the ortholog of the major FMO in rat liver, was highly stereoselective. In contrast, recombinant rat CYP3A2 and CYP2C11 displayed essentially no stereoselectivity in the formation of M16 stereoisomers (data not shown), indicating that in the rat liver, the major monooxygenase responsible for the oxidation of M25 to M16 was most likely FMO1 and not P450s. The formation of M20 from M16, on the other hand, was mediated exclusively by P450s.

In conclusion, a distinct species difference was detected in the monooxygenases involved in the S-oxidation of M25, the methyl sulfide metabolite of MK-0767 in rats, dogs, rhesus monkeys, and humans. Although both P450 and FMOs were found to be capable of catalyzing this reaction in these species, the collective results from several techniques employed here, which included immunoinhibition, heat treatment, and pH modulation of microsomal incubations and stereochemistry of the sulfoxide metabolite, demonstrated that the major enzymes involved in the S-oxidation of the methyl sulfide M25 to the sulfoxide amide, M16, were the P450s in the primates and FMOs in rats and dogs.

	Acknowledgments

 
We thank the following individuals, all of Merck Research Laboratories: J. Pang for isolation of hepatocytes; R. Wang; D. Newton; V. Didolkar for preparing microsomes; Drs. T. Rushmore and M. Shou for the supply of recombinant P450 microsomes and monoclonal P450 antibodies; M. Wallace; C. Raab and D. Dean for the synthesis of C-14 tracer; Y. Jakubowski; H, Jenkins and Dr. A. Jones for the analysis of C-14 tracer; and Drs. T. Baillie, P. Pearson, R. Franklin, and S. H. Lee Chiu for helpful discussions. We also thank W. Gruber for technical assistance.

	Footnotes

 
doi:10.1124/dmd.104.000224.

ABBREVIATIONS: MK-0767, (±)-5-[(2,4-dioxothiazolidin-5-yl)methyl]-2-methoxy-N-[[(4-trifluoromethyl)phenyl]methyl]benzamide; TZD, thiazolidinedione; PPAR, peroxisome proliferator-activated receptor; P450, cytochrome P450; FMO, flavin monooxygenase; HPLC, high-performance liquid chromatography; TAO, troleandomycin; SFC, supercritical fluid chromatography; CD, circular dichroism; ee, enantioselectivity excess.

Address correspondence to: Bindhu V. Karanam, RY 80L-109, Merck Research Laboratories, P.O. Box 2000, Rahway, NJ 07065. E-mail: bindhu_karanam{at}merck.com

	References

Top Abstract Materials and Methods Results Discussion References

 


Atta-Asafo-Adjei E, Lawton M, and Philpot R (1993) Cloning, sequencing, distribution and expression in Escherichia coli of flavin-containing monooxygenase 1C1. J Biol Chem 268: 9681-9689.[Abstract/Free Full Text]

Cashman JR (1995) Structural and catalytic properties of the mammalian flavin-containing monooxygenases. Chem Res Toxicol 8: 165-181.

Cashman JR (1998) Stereoselectivity in S- and N-oxygenation by the mammalian flavin-containing and cytochrome P-450 monooxygenases. Drug Metab Rev 30: 675-707.[Medline]

Cashman JR, Park SB, Yang ZC, Washington CB, Gomez DY, Giacomini KM, and Brett CM (1993a) Chemical, enzymatic and human enantioselective S-oxygenation of cimetidine. Drug Metab Dispos 21: 587-597.[Abstract]

Cashman JR, Park SB, Yang ZC, Wrighton SA, Peyton J, and Benowitz NL (1992) Metabolism of nicotine by human liver microsomes: Stereoselective formation of trans-nicotine N'-oxide. Chem Res Toxicol 5: 639-646.[CrossRef][Medline]

Cashman JR, Yang Z, Yang L, and Wrighton SA (1993b) Stereo- and regioselective N- and S-oxidation of tertiary amines and sulfides in the presence of adult human liver microsomes. Drug Metab Dispos 21: 492-501.[Abstract]

Chung WG, Park CS, Roh HK, Lee WK, and Cha YN (2000) Oxidation of ranitidine by isozymes of flavin-containing monooxygenase and cytochrome P450. Jpn J Pharmacol 84: 213-220.[CrossRef][Medline]

Grothusen A, Hardt J, Brautigam L, Lang D, and Bocker R (1996) A convenient method to discriminate between cytochrome P450 enzymes and flavin-containing monooxygenases in human liver microsomes. Arch Toxicol 71: 64-71.[CrossRef][Medline]

Itagaki K, Carver GT, and Philpot RM (1996) Expression and characterization of a modified flavin-containing monooxygenase 4 from humans. J Biol Chem 271: 20102-20107.[Abstract/Free Full Text]

Itoh K, Kimura T, Yokoi T, Itoh S, and Kamataki T (1993) Rat liver flavin-containing monooxygenase (FMO): cDNA cloning and expression in yeast. Biochem Biophys Acta 1173: 165-171.[Medline]

Karanam BV, Hop CECA, Liu DQ, Wallace M, Dean D, Satoh H, Komuro M, Awano K, and Vincent SH (2004) In vitro metabolism of MK-0767 [(±)-5-[(2,4-dioxothiazolidin-5-yl)methyl]-2-methoxy-N-[[(4-trifluoromethyl)phenyl]methyl]benzamide], a peroxisome proliferator-activated receptor / agonist. I. Role of cytochrome P450, methyltransferases, flavin monooxygenases, and esterases. Drug Metab Dispos 32: 1015-1022.[Abstract/Free Full Text]

Kawaji A, Isobe M, and Takabatake E (1997) Differences in enzymatic properties of flavin-containing monooxygenase in brain microsomes of rat, mouse, hamster, guinea pig and rabbit. Biol Pharm Bull 20: 917-919.[Medline]

Kersten S, Desvergne B, and Wahli W (2000) Roles of PPAR in health and disease. Nature (Lond) 405: 421-424.[CrossRef][Medline]

Lattard V, Longin-Sauvageon C, Lachuer J, Delatour P, and Benoit E (2002) Cloning, sequencing, and tissue-dependent expression of flavin-containing monooxygenase (FMO1) and FMO3 in the dog. Drug Metab Dispos 30: 119-128.[Abstract/Free Full Text]

Lemoine A, Johann N, and Cresteil TG (1990) Evidence for the presence of distinct flavin-containing monooxygenases in human tissue. Arch Biochem Biophys 276: 336-342.[CrossRef][Medline]

Levine M and Bellward GD (1995) Effect of cimetidine on hepatic cytochrome P450: evidence for formation of a metabolite-intermediate complex. Drug Metab Dispos 23: 1407-1411.[Abstract]

Liu DQ, Karanam BV, Doss G, Sidler R, Kusajima H, Vincent SH, and Hop CE (2004) In vitro metabolism of MK-0767 [(±)5-[2,4-dioxothiazolidin-5-yl)methyl]-2-methoxy-N-[[(4-trifluoromethyl)phenyl]methyl]benzamide], a peroxisome proliferator-activated receptor dual agonist. II. Identification of metabolites by liquid chromatography-tandem mass spectrometry. Drug Metab Dispos, in press.

Mani C, Hodgson E, and Kupfer D (1993) Metabolism of the antimammary cancer antiestrogenic agent tamoxifen II: flavin-containing monooxygenase-mediated N-oxidation. Drug Metab Dispos 21: 657-661.[Abstract]

Maurice M, Pichard L, Daujat M, Fabre I, Joyeux H, Domerigue J, and Maurel P (1992) Effects of imidazole derivatives on cytochrome P450 from human hepatocytes in primary culture. FASEB J 6: 752-758.[Abstract]

McManus ME, Stopans I, Burgess W, Koenig JA, Hall PM, and Birkett DJ (1987) Flavin-containing monooxygenase activity in human liver microsomes. Drug Metab Dispos 15: 256-261.[Abstract]

Murakami K, Tobe K, Ide T, Mochizuki T, Ohashi M, Akanuma Y, Yazaki Y, and Kadowaki T (1998) A novel insulin sensitizer acts as a coligand for peroxisome proliferator-activated receptor- (PPAR-) and PPAR-. Diabetes 47: 1841-1847.[Abstract]

Overby LH, Carver GC, and Philpot RM (1997) Quantitation and kinetic properties of hepatic microsomal and recombinant flavin-containing monooxygenases 3 and 5 from humans. Chem Biol Int 106: 29-45.[CrossRef][Medline]

Pessayre D, Larrey D, Vitaux J, Breil P, Belghiti J, and Benhamou JP (1982) Formation of an inactive cytochrome P-450 Fe(II)-metabolite complex after administration of troleandomycin in humans. Biochem Pharmacol 31: 1699-1704.[CrossRef][Medline]

Ring BJ, Wrighton SA, Aldridge SL, Hansen K, Haehner B, and Shipley LA (1999) Flavin-containing monooxygenase-mediated N-oxidation of the M1-muscarinic agonist xanomeline. Drug Metab Dispos 27: 1099-1103.[Abstract/Free Full Text]

Sadeque AJ, Thummel KE, and Rettie AE (1993) Purification of macaque liver flavin-containing monooxygenase: a form of the enzyme related immunochemically to an isozyme expresses selectively in adult human liver. Biochem Biophys Acta 1162: 127-134.[CrossRef][Medline]

Tugnait M, Hawes EM, Mckay G, Rettie AE, Haining RL, and Midha KK (1997) N-Oxygenation of clozapine by flavin-containing monooxygenase. Drug Metab Dispos 25: 524-527.[Abstract/Free Full Text]

Uehleke H (1973) The role of cytochrome P450 in the N-oxidation of individual amines. Drug Metab Dispos 1: 299-313.[Medline]

Washio T, Arisawa H, Kohsaka K, and Yasuda H (2001) Identification of human drug-metabolizing enzymes involved in the metabolism of SNI-2011. Biol Pharm Bull 24: 1263-1266.[Medline]

Ziegler DM (1993) Recent studies on the structure and function of multisubstrate flavin-containing monooxygenases. Annu Rev Pharmacol Toxicol 33: 179-199.[CrossRef][Medline]

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