Drug Metabolism and Disposition Fast Forward
First published on September 7, 2007; DOI: 10.1124/dmd.107.017517
0090-9556/07/3512-2186-2195$20.00
DMD 35:2186-2195, 2007
Identification of Human Liver Cytochrome P450 Enzymes Involved in Biotransformation of Vicriviroc, a CCR5 Receptor Antagonist
Anima Ghosal,
Ragu Ramanathan,
Yuan Yuan,
Neil Hapangama,
Swapan K. Chowdhury,
Narendra S. Kishnani, and
Kevin B. Alton
Drug Metabolism and Pharmacokinetics, Schering-Plough Research Institute, Kenilworth, New Jersey
(Received July 3, 2007;
Accepted September 7, 2007)
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Abstract
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Vicriviroc (SCH 417690), a CCR5 receptor antagonist, is currently under investigation for the treatment of human immunodeficiency virus infection. The objective of this study was to identify human liver cytochrome P450 enzyme(s) responsible for the metabolism of vicriviroc. Human liver microsomes metabolized vicriviroc via N-oxidation (M2/M3), O-demethylation (M15), N,N-dealkylation (M16), N-dealkylation (M41), and oxidation to a carboxylic acid metabolite (M35b/M37a). Recombinant human CYP3A4 catalyzed the formation of all these metabolites, whereas CYP3A5 catalyzed the formation of M2/M3 and M41. CYP2C9 only catalyzed the formation of M15. There was a high correlation between the rates of formation of M2/M3, M15, and M41, which was determined using 10 human liver microsomal samples and testosterone 6ß-hydroxylation catalyzed by CYP3A4/5 (r 
0.91). Ketoconazole and azamulin (inhibitors of CYP3A4) were potent inhibitors of the formation of M2/M3, M15, M41, and M35b/M37a from human liver microsomes. A CYP3A4/5-specific monoclonal antibody (1 µg/µg of protein) inhibited the formation of all metabolites from human liver microsomes by 86 to 100%. The results of this study suggest that formation of the major vicriviroc metabolites in human liver microsomes is primarily mediated via CYP3A4. CYP2C9 and CYP3A5 most likely play a minor role in the biotransformation of this compound. These enzymology data will provide guidance to design clinical studies to address any potential drug-drug interactions.
Cytochrome P450 (P450) enzymes are a group of heme-containing enzymes embedded primarily in the lipid bilayer of the endoplasmic reticulum of liver cells. P450 isoenzymes are most predominant in the liver but can also be found in the intestines, lungs, and other organs. They are responsible for the oxidative, peroxidative, and reductive metabolism of a diverse group of compounds, including xenobiotics, therapeutic drugs, environmental pollutants, and endobiotics such as steroids, bile acids, fatty acids, prostaglandins, and leukotrienes (Nelson et al., 1996
).
Chemokines constitute a class of cytokines that regulate migration of leukocytes to sites of infection. The CCR5 chemokine receptor is expressed on a wide range of immune cell types and binding to this receptor mediates cellular entry by the majority of human immunodeficiency virus (HIV) isolates. Blocking viral entry via this receptor reduces the viral load in patients infected with HIV, suggesting that a CCR5 antagonist could become a key component in the treatment of HIV-compromised patients (Barber, 2004). In addition, CCR5 is the main coreceptor used by macrophage-tropic strains of HIV type 1and type 2, which are responsible for viral transmission. CCR5 therefore plays an essential role in HIV pathogenesis (Blanpain et al., 2002).
Vicriviroc (SCH 417690), a CCR5 antagonist, is currently under clinical investigation for the treatment of HIV infection. The identification of the enzyme(s) responsible for the oxidative metabolism of a drug allows one to predict and/or explain interindividual differences in the effects of the drug that are due to differences in its metabolic clearance. Knowledge of the P450 enzymes(s) responsible for metabolism also helps in designing drug-drug interaction studies in the clinic. The objective of this study was to identify the predominant in vitro biotransformation pathway(s) of vicriviroc.
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Materials and Methods
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Chemicals. Glucose-6-phosphate dehydrogenase, monosodium D-glucose 6-phosphate, NADP, magnesium chloride, Trizma base, ammonium acetate, and quinidine were purchased from Sigma-Aldrich (St. Louis, MO). Ketoconazole was purchased from Oxford Biomedical Research (Oxford, MI). HPLC grade acetonitrile, acetic acid, and methanol (Optima) were from Fisher Scientific (Fair Lawn, NJ). Distilled water was prepared using a Milli-Q water purification system from Millipore (Bedford, MA). Unlabeled vicriviroc was obtained from Schering-Plough (Kenilworth, NJ). Radiolabeled vicriviroc (14C, radiochemical purity >97%, specific activity 104 µCi/mg) (Fig. 1) was prepared by the Radiochemistry Group at Schering-Plough Research Institute (Kenilworth, NJ). Pooled human liver microsomes (n = 50) were purchased from XenoTech, LLC (Lenexa, KS). P450 Supersomes and CYP3A4 monoclonal antibodies were purchased from BD Biosciences (Woburn, MA), and a HepatoScreen Test kit was obtained from Human Biologics (Scottsdale, AZ).
Characterization of Metabolites. Metabolites were characterized using a Waters Alliance HPLC system (Alliance model 2690; Waters, Milford, MA), equipped with a model 996 photodiode array detector (Waters), model 500TR flow scintillation analyzer (FSA) (PerkinElmer Life and Analytical Sciences, Torrance, CA), and a 5-µm Luna phenyl-hexyl 250 x 4.6 mm column (Phenomenex, Torrance, CA). The column was maintained at room temperature for all HPLC experiments. The mobile phase consisted of 95% 10 mM ammonium acetate with 5% acetonitrile, adjusted to pH 6.0 with acetic acid (A) and 95% acetonitrile with 5% water (B). A linear gradient was programmed as defined in Table 1.
A constant flow rate (1 ml/min) was maintained, and the eluted drug-derived material was detected at 254 nm. All LC-MS and LC-MS/MS experiments were performed by using a TSQ Quantum mass spectrometer (Thermo Electron Corporation, San Jose, CA). The column effluent was split such that most (
80%) of the effluent was directed into the FSA, and the remainder was diverted to the mass spectrometer. This simultaneous detection of all drug-derived material by a mass spectrometer and an FSA provided confirmation of the molecular weight and structure of all radioactive peaks in a simple experiment.
LC-MS and LC-MS/MS Analysis. The mass spectrometer was nominally operated under the conditions listed in Table 2 with Q1 or Q3 resolution (full width at half-maximum) set at 0.7 Th for all LC-MS and LC-MS/MS experiments.
In LC-MS/MS experiments, ions were activated in Q2 with 25 to 30 eV collision energy while maintaining the collision gas (argon) pressure at 1.2 mtorr. After LC-MS/FSA analysis, the area of each detected radioactive peak in the FSA was expressed as a percentage of the total chromatographic radioactivity (TCR). Percent values for characterized metabolites, when provided, are therefore estimates and were not derived from a validated quantitative procedure.
Enzyme Assays. Incubation with pooled human liver microsomes. To establish the optimal conditions for the initial velocity measurement, the linearity of vicriviroc metabolite formation was determined with respect to time (15–120 min) and microsomal P450 concentration (0.25–2 nmol/ml). Substrate concentrations of 0.1 to 50 µM were used to determine kinetic parameters. All incubations (final volume 500 µl) contained microsomes, 3 mM magnesium chloride, 1 mM ß-NADP, 5 mM glucose 6-phosphate, 1.5 units/ml glucose-6-phosphate dehydrogenase, and [14C]vicriviroc in 0.5 ml of 50 mM potassium phosphate buffer, pH 7.4 (Ghosal et al., 2005). The incubation mixtures were prewarmed for 2 to 3 min at 37°C, and reactions were initiated by the addition of substrate and then terminated with ice-cold methanol. After centrifugation (10,000g) at 4°C for 10 min, each incubation supernatant was directly analyzed by HPLC. Incubations without NADPH and boiled human liver microsomes served as negative controls. After LC analysis, metabolite concentrations were calculated on the basis of the peak areas after FSA detection and a five-point standard curve produced by linear regression. For LC-MS analysis, supernatants were concentrated under nitrogen at room temperature.
Screening of 17 human P450 Supersomes. In vitro screening of 17 human P450 Supersomes (CYP1A1, CYP1A2, CYP2A6, CYP1B1, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, CYP4A11, CYP4F2, CYP4F3A, and CYP4F3B) was performed using a constant amount of cytochrome P450 (0.2 nmol/ml) and [14C]vicriviroc (1 and 10 µM) for 120 min. All incubations with Supersomes were performed as described earlier (Ghosal et al., 2005). Insect microsomes without cDNA of human P450 were used as controls. For CYP2C9 and CYP2A6, incubations were performed in Tris buffer (supplier's recommendation). These samples were also analyzed by LC-MS. Incubations of various concentrations of vicriviroc (1–100 µM) with CYP3A4, CYP2C9, and CYP3A5 were performed to calculate kinetic parameters.
Inhibition with chemical inhibitors and CYP3A4-inhibitory monoclonal antibody. Inhibition of vicriviroc metabolism was evaluated using both chemical inhibitors (ketoconazole, quinidine, and sulfaphenazole) and inhibitory antibodies specific for CYP3A4. Human liver microsomes (0.5 nmol/ml) were preincubated with various concentrations of inhibitors/antibodies for 15 min at room temperature followed by the addition of buffer, cofactor, and substrate (10 µM[14C]vicriviroc). The final concentration of the organic solvents in the incubation system was <1%. Incubations were performed, and samples were analyzed by HPLC coupled with a radioactivity detector.
Correlation study. The HepatoScreen Test Kit consisted of 10 individual human liver microsomal preparations from 10 individual donors. The ability of human liver microsomes from each donor to metabolize vicriviroc to its metabolites was correlated with the P450-specific enzyme activities for each sample from each kit. The assays were performed as described previously (under human liver microsomal incubations) with 10 µM substrate and incubated for 120 min.
Analysis of Kinetic Data. Untransformed enzyme kinetic data were analyzed by a nonlinear regression data analysis program (GraFit 5.0.1; Erithacus Software Limited, Staines, UK), assuming Michaelis-Menten kinetics over the substrate range studied.
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Results
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Optimization and Incubation with Pooled Human Liver Microsomes. Incubation of [14C]vicriviroc (Fig. 1) with human liver microsomes yielded a variety of metabolites that could be separated by HPLC. When pooled human liver microsomes were incubated with 10 µM[14C]vicriviroc over a range of concentrations of cytochrome P450 (0.25–2 nmol/ml) for various time periods (15–120 min), a P450 concentration of 0.5 nmol/ml and incubation time of 120 min were found to be optimal (not shown). Radiochromatographic profiles of metabolites after incubation of vicriviroc (1 and 10 µM) with human liver microsomes are presented in Fig. 2. No metabolite formation was observed in the absence of the NADPH-generating system (not shown) or with boiled microsomes (Fig. 2). Kinetic parameters for the production of various metabolites are shown in Table 3. Intrinsic clearance (Vmax/Km) data suggest that the formation of M41 (SCH 496903) and M2/M3 (SCH 643188) may be the preferred pathway for in vitro biotransformation of vicriviroc (Table 3). A substrate concentration of 10 µM was chosen for further experiments considering the linearity, percentage of conversion, and sensitivity of detection of M41.
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FIG. 2. Radiometric profile of metabolites after a 120-min incubation of [14C]vicriviroc (1 and 10 µM) with human liver microsomes (1 µM, top and middle) and 10 µM (bottom) Supplemented with an NADPH-generating system.
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TABLE 3 Kinetic parameters for the formation of metabolites from [14C]vicriviroc with pooled human liver microsomes
Data are means ± S.E.
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Screening with Human P450 Supersomes. In vitro incubation of 1 µM vicriviroc with 17 different recombinant human P450 Supersomes showed that CYP3A4 exhibited the most activity followed by markedly less substrate conversion with CYP3A5 and CYP2C9 (Figs. 3 and 4). The major metabolite formed by CYP2C9 was M15 (SCH 495415), whereas CYP3A5 yielded M2/M3 (detected by LC-MS only) and M41 (Fig. 4). CYP3A4 alone yielded the acid metabolite M35b/M37a (SCH 727870) at 10 µM (Fig. 4). The formation of metabolites with recombinant CYP3A4, CYP3A5, and CYP2C9 suggested possible involvement of these enzymes in the metabolism of vicriviroc. Kinetic parameters for metabolites formed after incubation of various concentrations of vicriviroc (ranging from 0.1 to 100 µM) with CYP3A4 and CYP3A5 Supersomes are presented in Table 4. Intrinsic clearance data from CYP3A4 (Vmax/Km = 25.5) and CYP3A5 (Vmax/Km = 1.22) suggest that the formation of M41 is the preferred in vitro biotransformation pathway. However, incubation of vicriviroc (ranging from 0.1 to 50 µM) with CYP2C9 Supersomes demonstrated atypical (biphasic) kinetics; the apparent Km and Vmax values for M15 metabolite were determined to be 672.7 µM and 1735 pmol/nmol of P450/min, respectively (not shown). Kinetic parameters of other metabolites were not calculated because of a lack of detection sensitivity over a range of concentrations necessary to determine Km and Vmax.
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FIG. 4. Radiometric profile of metabolites after a 120-min incubation of [14C]vicriviroc (10 µM) with CYP3A4 (top), CYP2C9 (middle), and CYP3A5 (bottom)
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TABLE 4 Kinetic parameters for the formation of metabolites from [14C]vicriviroc with human P450 Supersomes
Data are means ± S.E.
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The activities of P450 Supersomes (CYP1A1, CYP1A2, CYP2A6, CYP1B1, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP3A4, and CYP3A5) obtained from BD Biosciences and human liver microsomes were determined in assays using fluorometric substrates as described previously (Ghosal et al., 2003). The results of the activity determinations of 13 human P450 Supersomes and human liver microsomes demonstrated that the Supersomes and microsomes were active (not shown). The activities of CYP4A11, CYP4F2, CYP4F3A, and CYP4F3B were not determined.
Metabolite Identification. After incubation of [14C]vicriviroc (10 µM) with CYP3A4 for 120 min, five major radioactive components, each representing >5% of the TCR, and several minor to trace level drug-derived components were detected. The five major components included unchanged drug (vicriviroc), the rotameric pair M2/M3, M7, M15, and M41. LC-MS spectra of unchanged drug and the four major metabolites are shown in Fig. 5. The unchanged drug eluted at 31.1 min and exhibited a protonated ion at m/z 534. The MS/MS spectrum and the corresponding assignment of fragment ions of vicriviroc are provided in Fig. 6. HPLC retention times, LC-MS detected m/z values, and fragment ions observed in MS/MS experiments for prominent metabolites are listed in Table 5. The major CYP3A4-mediated metabolite, M41, had a HPLC retention time of 6.4 min and the corresponding LC-MS spectrum showed a molecular ion at m/z 332. Under the MS/MS conditions used, precursor ions at m/z 332 fragmented to give ions at m/z 101 and 135 (Table 5). Ions of m/z 135 and 101, respectively, confirmed that the 4,6-dimethyl-5-pyrimidinyl-carbonyl and methyl-1-piperazinyl moieties remained intact. Thus, M41 is most likely formed through N-dealkylation of vicriviroc. M41 was unambiguously confirmed using a synthetic reference standard (SCH 496903).
Similarly, M2/M3, M7, and M15 were characterized as vicriviroc-N-oxide, vicriviroc-hydroxylamine, and O-desmethyl-vicriviroc, respectively. Under the LC conditions used, the rotameric metabolites M2/M3 eluted at 27.5/27.9 min and often were not separable. The LC-MS spectrum of M2/M3 showed protonated ions at m/z 550. An increase in molecular mass of 16 Da over that of vicriviroc suggests that M2/M3 is associated with monooxidation of vicriviroc. With MS/MS conditions, precursor ions of m/z 550 fragmented to give ions of m/z 101, 151, 203, 248, 271, 303, and 315 (Table 5). A 16-Da shift of the fragment ion of m/z 135 to 151 suggests that oxygenation is most likely occurring on the 4,6-dimethyl-5-pyrimidinyl-carbonyl moiety of vicriviroc. By using the atmospheric pressure chemical ionization (APCI)-mass spectrometry method described previously (Ramanathan et al., 2000; Tong et al., 2001), M2/M3 was confirmed to be an N-oxide metabolite. The structure of M2/M3 was unambiguously confirmed using a synthetic reference standard (SCH 643188).
Another significant metabolite, M15, eluted at 24.9 min and yielded protonated ions at m/z 520. The molecular mass at 519, corresponding to a decrease of 14 Da over that of vicriviroc, suggests that M15 most likely results from demethylation of vicriviroc. Under MS/MS conditions, precursor ions at m/z 520 fragmented to give ions at m/z 101, 135, 189, 232, 289, and 301 (Table 5). Ions of m/z 101 and 135, respectively, confirmed that methyl-1-piperazinyl and 4,6-dimethyl-5-pyrimidinyl-carbonyl-4-methylpiperidine moieties are unchanged. The absence of fragment ions at m/z 203 and 303 Th and detection of ions at m/z 189 and 289 confirmed the fact that the ethoxy-4-trifluromethyl-phenyl-ethyl moiety has been modified by desmethylation. Furthermore, the HPLC retention time and the MS/MS spectrum of M15 matched those of a synthetic reference standard (SCH 495415).
M7, a minor in vivo and in vitro metabolite, had an HPLC retention time of 24.8 min, and [M + H]+ ions were observed at m/z 538. This metabolite was 5% of the radioactivity in the CYP3A4 incubation. In incubation with human liver microsomes this metabolite was below the quantification limit and was only detected by LC-MS. An increase of 4 Da in M7 over the molecular mass of vicriviroc suggested an uncommon metabolic modification. Precursor ions of m/z 538 fragmented to give ions at m/z 101, 139, 203, 236, 271, 303, and 315 Th (Table 5) under MS/MS condition. Ions at m/z 101 and 203, respectively confirmed that methyl-1-piperazinyl and ethoxy-4-trifluromethyl-phenyl-ethyl moieties are unchanged. A 4-Da shift of the fragment ion of m/z 135 to 139 suggests that an alteration is most likely occurring on the 4,6-dimethyl-5-pyrimidinyl-carbonyl moiety of vicriviroc. Modification in this region was further confirmed by a 4-Da shift of the fragment ion at m/z 232 to 236 Th. Using previously described APCI-MS and hydrogen-deuterium exchange mass spectrometry (Ramanathan et al., 2005a) and accurate mass measurement, M7 was characterized as vicriviroc hydroxylamine. Unambiguous identification of M7 involved matching the HPLC retention time and the MS/MS spectrum with those of the synthetic reference standard (SCH 727390). To our knowledge, the biotransformation of the pyrimidine moiety to a pyrazyl-hydroxylamine has not been previously reported. The mechanism of this intriguing metabolic process is currently being investigated.
Other minor CYP3A4-mediated metabolites M16, M35b/M37a, and M45-M47 were characterized as N,N-desalkyl-vicriviroc, vicriviroc-carboxylic acid, and a series of monoxy-N-desalkyl-vicriviroc isomers. Molecular ions for M16 (SCH 496903) and M35b/M37a (SCH 727870) were observed at m/z 494 and 534 and were unambiguously confirmed using LC-MS/MS, hydrogen/deuterium exchange mass spectrometry, and synthetic reference standards. Metabolites M45, M46, and M47 eluted between 4 and 6 min and exhibited [M + H]+ ions at m/z 348. Although the exact position(s) of oxidation are not known, M45, M46, and M47 were characterized as monooxy metabolites of M41 (monooxy-N-desalkyl-vicriviroc) using MS/MS data.
After incubation of [14C]vicriviroc with CYP2C9 and CYP3A5, on average, the unchanged drug accounted for 96% of TCR. CYP2C9-mediated metabolites with detectable radioactivity included M15 and M18/M19. M15 is a minor metabolite formed via O-demethylation of vicriviroc, and M18/M19 are trace level metabolites formed by oxidation of O-desmethyl-vicriviroc. CYP3A5-mediated minor metabolites included M2/M3 and M41. Trace levels of M18/M19 were also detected in CYP3A5 incubates. The biotransformation pathway of vicriviroc is presented in Fig. 7.
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FIG. 7. Biotransformation of vicriviroc in human liver microsomes and cDNA-expressed human P450 enzymes (only major routes are shown)
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Correlation Analysis. The formation rates of [14C]vicriviroc metabolites (M2/M3, M15, and M41) were measured in each of the 10 human liver microsomal samples provided in the HepatoScreen Test Kit. These values were then correlated with the biochemical activity data provided by the manufacturer. Because the biochemical activity data were mediated by specific P450 enzymes, high correlation would suggest that similar enzymes were involved in the formation of metabolites from vicriviroc.
The highest correlation between the HepatoScreen Test Kit assay data (n = 10) and the formation of M2/M3, M15, and M41 was noted for dextromethorphan N-demethylation (r 0.89), which is catalyzed by CYP3A4 (Table 6). Correlations among M2/M3 (r = 0.91, p = 0.0003), M15 (r = 0.93, p = 0.0001), M41 (r = 0.97, p < 0.0001), and testosterone 6ß-hydroxylation catalyzed by CYP3A4/5 were also significant (Table 6). A representative correlation plot of M41 is provided in Fig. 8. There was a high correlation between the formation of metabolites and CYP2B6 activity. Interestingly, no vicriviroc metabolites were formed in vitro with CYP2B6 Supersomes. Nonetheless, the findings are consistent with a significant correlation between the activity of CYP2B6 Supersomes and CYP3A4 observed by other investigators (Heyn et al., 1996). There was no significant correlation between tolbutamide methyl hydroxylation (CYP2C9) and M15 formation. Overall, correlation analysis between the enzyme activities and metabolite formation suggested that vicriviroc is metabolized primarily by CYP3A4 in human liver microsomes.
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TABLE 6 Correlation (r) values between metabolite formation rates and P450 enzyme specific activities at 10 µM vicriviroc
Correlation between CYP2B6 and CYP3A4 activity is high (r = 0.98); similar observation was reported by Heyn et al (1996).
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FIG. 8. Regression analysis of M41 formation rate from [14C]vicriviroc to CYP3A4/5 catalyzed testosterone 6ß-hydroxylase activity in 10 individual human liver microsomes
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Inhibition Studies. All inhibition studies were performed with pooled human liver microsomes at a drug concentration of 10 µM. Ketoconazole was shown to be a potent inhibitor of vicriviroc metabolism (all metabolites) by human liver microsomes (Table 7). The mean IC50 values of ketoconazole for M2/M3, M15, and M41 formation from human liver microsomes were 0.84, 1.1, and 0.79 µM, respectively (Table 7). Azamulin, a specific CYP3A4/5-inhibitor, inhibited M2/M3, M15, and M41 formation from human liver microsomes by 90 to 100% (Table 7) at a 5 µM concentration. Neither quinidine (CYP2D6 inhibitor) at 5 µM nor sulfaphenazole (CYP2C9-specific inhibitor) at 0.5 and 3 µM had a significant effect on the metabolism of vicriviroc (Table 7).
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TABLE 7 Effect of P450-specific chemical inhibitors on vicriviroc metabolism with human liver microsomes
Activity remaining is calculated as a percentage of vehicle control.
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Studies with a CYP3A4/5-specific inhibitory monoclonal antibody showed a significant inhibitory effect on the metabolism of vicriviroc (Fig. 9). CYP3A4/5-specific inhibitory monoclonal antibody (1 µg/µg protein) inhibited the formation of M41, M2/M3, M15, and M35b/M37a from human liver microsomes by 86, 83, 78, and 100%, respectively, whereas control experiments showed no inhibition.
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Discussion
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After administration of a single oral dose of [14C]SCH 417690 (50 mg) to humans, M41, M37, M35, M15, and M2/M3 were major circulating metabolites representing at least 5% of the circulating drug-derived radioactivity in 4-, 8-, or 24-h plasma samples (Ramanathan et al., 2005b). The level of M35b/M37a, a carboxylic acid metabolite detected at a trace level after a single dose to humans, was found to increase after multiple dose administration and represented a major metabolite at steady state. All major in vivo human metabolites were identified after incubation of 10 µM[14C]SCH 417690 with human liver microsomes (Fig. 2) with the exception of M35, which is a glucuronide conjugate of M15. M35 was not expected to be formed in this incubation with human liver microsomes without the cofactor UDP-glucuronic acid required to form the glucuronide conjugate. The same metabolites were also detected after incubation of [14C[SCH 417690 with cDNA-expressed CYP3A4. Only M15 was detected upon incubation with CYP2C9, and M41 and M2/M3 upon incubation with CYP3A5 (Fig. 4). M7, a minor metabolite in both human liver microsomal and CYP3A4 incubations, was present at trace levels in human plasma after administration of [14C]SCH 417690. Therefore, the identification of human cytochrome P450 enzymes involved in the in vitro metabolism of vicriviroc represented true in vivo human metabolites.
A multistep approach was used to identify the P450 isoform(s) responsible for vicriviroc metabolism. This "reaction phenotyping" included correlation analysis with a panel of characterized microsomal preparations, chemical and antibody inhibition, and the use of cDNA-expressed human P450 isoforms. Incubation of vicriviroc with human liver microsomes showed that M2/M3, M15, M16, M35b/M37a, and M41 were primary in vitro metabolites. Formation of all metabolites was mediated via CYP3A4, based on inhibition by ketoconazole and the production of these metabolites from Supersomes overexpressing CYP3A4. Biotransformation of vicriviroc in human liver microsomes and cDNA-expressed human P450 enzymes is shown in Fig. 7.
In vitro incubation with 17 different recombinant human P450 Supersomes showed that CYP3A4 exhibited the most activity followed by CYP2C9 and CYP3A5. The formation of metabolites with recombinant CYP2C9, CYP3A4, and CYP3A5 suggested minimal involvement of CYP2C9 and CYP3A5 in the metabolism of vicriviroc. In human liver microsomes, the apparent Km (8.92 µM) for M41 is lower than the Km values in CYP3A4 and CYP3A5 (25.6 and 51 µM). In the present study, formation of M41 from CYP3A5 is very low; only 2% converted to M41. In addition CYP3A5 plays a minor role in the metabolism of vicriviroc; therefore, its Km does not reflect the Km from human liver microsomes. Differences in Km values in human liver microsomes and in recombinant systems are also reported in the literature for other compounds. Yamazaki et al. (1999) showed that the apparent Km (28 µM) for troglitazone metabolite-3 formation in human liver microsomes is lower than the Km in CYP3A4 (120 µM) but higher than the Km of other P450s (CYP2C8, CYP2C9, and CYP2C19) involved. For diltiazem N-demethylation, the Km in human liver microsomes is 53 µM, whereas that in CYP3A4 is lower (16 µM) and that in CYP3A5 is higher (81 µM) (Jones et al., 1999). Kumar et al. (2006) also reported lower Km values for (S)-warfarin and (S)-flurbiprofen as 3.7 and 1.9 µM in human liver microsomes compared with those for CYP2C9 (13 and 21.6 µM), respectively. Therefore, there are several indications for the differences in Km values between human liver microsomes and recombinant P450s in the literature. The ratio of P450 reductase to P450 or that of cytochrome b5 to P450 in the recombinant systems and their specific P50 content may be responsible for the apparent difference in Km values observed in human liver microsomes and recombinant P450s.
Incubation of vicriviroc with CYP2C9 Supersomes exhibited biphasic kinetics for the formation of M15. The profile eventually became linear with increasing substrate concentration (not shown); however, saturation was not obtained up to 100 µM. This reaction profile for production of M15 did not obey classic Michaelis-Menten kinetics. A biphasic kinetic profile is generally characterized by an initial Michaelis-Menten-like increase in velocity with increasing substrate concentration. However, the profile does not become asymptotic and eventually becomes linear with increasing substrate concentration. This behavior results in the inability to predict an apparent Vmax and Km and has previously been reported for this and other recombinant P450s. For example, the saturation of naproxen demethylation by CYP2C9 is not achieved up to 1800 µM (Korzekwa et al., 1998; Hutzler et al., 2001), and CYP3A4-mediated naphthalene metabolism to 1-naphthol continues up to 400 µM (Korzekwa et al., 1998). Examples of biphasic kinetics are becoming more prevalent with several P450 isoforms (CYP3A4, CYP2C9, and others) apparently exhibiting this type of behavior (Hutzler and Tracy, 2002). Hutzler et al. (2001) suggested that activation of CYP2C9-mediated dapsone metabolism and its biphasic profile may be explained by a two-site binding mode. Interestingly, incubations of vicriviroc with human liver microsomes do not show biphasic behavior for the formation of M15, as M15 is largely mediated by CYP3A4 and it also plays a minor role in the metabolism of vicriviroc.
Ketoconazole (Wrighton and Ring, 1994; Newton et al., 1995; Ghosal et al., 1996; Desai et al., 1998; Masimirembwa et al., 1999) and azamulin (Stresser et al., 2004) (both CYP3A4-selective inhibitors) were shown to be potent inhibitors of vicriviroc metabolism by human liver microsomes, suggesting the involvement of CYP3A4 in its metabolism. Stresser et al. (2004) reported that azamulin is a highly potent and selective inhibitor of CYP3A. However, sulfaphenazole (CYP2C9-selective inhibitor) (Newton et al., 1995) had no significant effect on the metabolism of vicriviroc, suggesting that CYP2C9 plays a minor role in the metabolism of vicriviroc. Studies with CYP3A4/5 inhibitory antibody demonstrated that it inhibited >80% of vicriviroc metabolism in human liver microsomes. In addition, there was a significant correlation between the formation of M2/M3, M15, M41, and dextromethorphan N-demethylation or testosterone 6ß-hydroxylation, known to be mediated by CYP3A4 (Gorski et al., 1994; Newton et al., 1995). These inhibition and correlation studies suggest that CYP3A4 is primarily responsible for the biotransformation of vicriviroc.
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Footnotes
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.107.017517.
ABBREVIATIONS: P450, cytochrome P450; HIV, human immunodeficiency virus; SCH 417690, vicriviroc; HPLC, high-performance liquid chromatography; FSA, flow scintillation analyzer; LC, liquid chromatography; MS, mass spectrometry; MS/MS, tandem mass spectrometry; TCR, total chromatographic radioactivity; SCH 496903, N-desalkyl-vicriviroc; SCH643188, vicriviroc-N-oxide; SCH 495415, O-desmethyl-vicriviroc; APCI, atmospheric pressure chemical ionization; SCH 727390, vicriviroc-hydroxylamine.
Address correspondence to: Dr. Anima Ghosal, Drug Metabolism and Pharmacokinetics, Schering-Plough Research Institute, 2015 Galloping Hill Rd., K-15-1945, Kenilworth, NJ 07033. E-mail: anima.ghosal{at}spcorp.com
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Abstract
Materials and Methods
, M41; 
, M2/M3; 
, M15; 
, M35b/M37a.