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IDENTIFICATION OF HUMAN LIVER CYTOCHROME P450 ENZYMES RESPONSIBLE FOR THE METABOLISM OF LONAFARNIB (SARASAR)

Drug Metabolism and Pharmacokinetics, Schering-Plough Research Institute, Kenilworth, New Jersey

(Received October 17, 2005; accepted January 25, 2006)

	Abstract

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Lonafarnib (Sarasar), a farnesyl transferase inhibitor, is currently under development for the treatment of solid tumors. Incubation of lonafarnib with human liver microsomes resulted in the formation of four oxidative metabolites (M1, M2, M3, and M4). Minor to trace levels of these metabolites were detected in humans after multiple-dose administration of lonafarnib. Liquid chromatography-mass spectrometry analyses exhibited a mass to charge ratio (m/z) for the (M+H)⁺ ion of M1, M2, M3, and M4 at 653, 635, 669, and 653 Th, respectively. These metabolites, respectively, resulted from changes of +O, –2H, +2O, and +O relative to lonafarnib. Recombinant human CYP3A4 and CYP3A5 exhibited catalytic activity with respect to the formation of M1, M2, and M3, whereas CYP2C8 exhibited catalytic activity with respect to the formation of M4. There was a high correlation between the formation of M1, determined in 10 human liver microsomal samples, and 6ß-hydroxylation of testosterone catalyzed by CYP3A4/5 (r = 0.93). The IC₅₀ values of ketoconazole for inhibition of M1 and M2 were 0.61 and 0.92 µM, respectively. The formation of M4 by human liver microsomes was inhibited 72% by 50 µM quercetin, suggesting that the formation of M4 was mediated via CYP2C8. A CYP3A4/5-specific inhibitory monoclonal antibody inhibited the formation of M1, M2, and M3 by 85, 75, and 100%, respectively. In conclusion, the formation of metabolites M1, M2, and M3 from lonafarnib was mediated via CYP3A4 and CYP3A5.

Protein farnesylation catalyzed by the enzyme farnesyl protein transferase involves the addition of a 15-carbon farnesyl group to conserved amino acid residues at the carboxy terminus of certain proteins (Haluska et al., 2002). Protein substrates of farnesyl transferase include several G proteins, which are critical intermediates of cell signaling and cytoskeletal organization, such as Ras, Rho, PxF, and lamins A and B. Activated Ras proteins trigger a cascade of phosphorylation events through sequential activation of the phosphatidylinositol 3 kinase/AKT pathway, which is critical for cell survival, and the Raf/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase kinase pathway, which has been implicated in cell proliferation. Ras mutations, which encode for constitutively activated proteins, are found in 30% of human cancers. Because farnesylation of Ras is required for its transforming and proliferative activity, the farnesyl protein transferase inhibitors were designed as anticancer agents to abrogate Ras function (Haluska et al., 2002).

Lonafarnib [1-piperidinecarboxamide, 4-[2-[4-(3,10-dibromo-8-chloro-6,11-dihydro-5H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-yl)-1-piperidinyl]-2-oxoethyl] (Fig. 1), a selective inhibitor of farnesyl protein transferase, is currently being clinically evaluated as a chemotherapeutic agent for the treatment of a variety of solid tumors. Lonafarnib, specific for its target enzyme, is rapidly absorbed after oral administration and has linear pharmacokinetics (Caponigro, 2002). It undergoes phase I metabolism in vivo and in vitro. Preliminary evidence suggests that lonafarnib can be converted to hydroxy and dihydroxy metabolites when incubated with rat, mouse, dog, and human liver hepatocytes (unpublished). The major in vivo human metabolites after single-dose administration are M1 and M2, which are potentially mediated by oxidation. However, these became minor metabolites after multiple-dose administration. The objective of this study was to identify the predominant in vitro biotransformation pathway(s) for lonafarnib oxidation. These enzymology data were critical in designing clinical studies to address potential drug interactions and in support of claims in the product label.

View larger version (14K): [in this window] [in a new window]   FIG. 1. Chemical structure of lonafarnib (SCH 66336); asterisk denotes site of 14C label.

	Materials and Methods

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Analysis of Lonafarnib Metabolites. Analyses were performed on an HPLC system (Alliance model 2690; Waters Corp., Milford, MA) equipped with a model 996 photodiode array detector (Waters Corp.) and a model 500TR (PerkinElmer Life and Analytical Sciences Inc., Boston, MA) radioactivity detector. The analytical column (5-µm Luna Phenyl-Hexyl 250 x 4.6-mm column from Phenomenex, Torrance, CA) was maintained at 40°C for all HPLC experiments. The mobile phase, consisting of 20 mM ammonium acetate adjusted to pH 6.0 with acetic acid (A) and acetonitrile (B), was operated at a constant flow rate (1 ml/min). UV detection of metabolites was performed at 254 nm. The elution gradient was 100% A for 10 min, followed by a rapid linear decrease to 60% A in 10 min. At 65 min, the gradient was adjusted to achieve 10% A in 1 min and then reverted back to 100% A until the system was re-equilibrated (6 min). After LC analysis, the concentrations of metabolites were calculated based on radiometric detection and a five-point standard curve.

LC-MS Analysis. All LC-MS and LC-MS/MS experiments were performed using a Finnigan LCQ mass spectrometer (Finnigan MAT, San Jose, CA) equipped with an electrospray ionization source (positive ionization mode). The spray needle voltage was set at 4.5 kV, capillary temperature at 260°C, and sheath gas at 60 to 70 psi, and auxiliary gas was set at 5 to 20 psi.

After UV detection, the HPLC column effluent was split so that 19 to 23% of the flow was analyzed by the LCQ mass spectrometer and 77 to 81% diverted to the radiometric detector. Analog outputs from the UV and the radiometric detectors were collected using both the LCQ data acquisition software and a separate computer controlling the radiometric detector. A Shimadzu HPLC system (Shimadzu Corporation, Kyoto, Japan) coupled with a PE Biosystem QSTAR/Pulsar LC-MS (QTOF) mass spectrometer (PE Biosystem, Concord, ON, Canada) was also used for selected high-mass accuracy LC-MS and LC-MS/MS experiments (Tong et al., 2005).

Enzyme Assays. Incubation of Lonafarnib with Pooled Human Liver Microsomes. Substrate concentrations of 1 to 500 µM, microsomal P450 concentrations of 0.1 to 2 nmol/ml, and incubation times of 5 to 120 min were used to optimize assay conditions. All microsomal incubations contained microsomes, 3 mM magnesium chloride, 0.5 mM NADP, 5 mM glucose 6-phosphate, 1.5 units/ml glucose 6-phosphate dehydrogenase, and [¹⁴C]lonafarnib in 0.5 ml of 50 mM potassium phosphate buffer, pH 7.4 (Ghosal et al., 1996). Before the addition of substrate, the incubation mixtures were prewarmed for 3 min at 37°C. The final concentration of the organic solvent in the incubation system was 1%. Reactions were initiated by the addition of substrate, allowed to proceed at 37°C, and terminated with ice-cold methanol or acetonitrile. The incubation mixtures were centrifuged (10,000g) at 4°C for 10 min, and the supernatants were analyzed by HPLC. Incubations without NADPH and boiled human liver microsomes served as negative controls. For LC-MS analyses, the reactions were either terminated as described above or terminated by cooling in ice water followed by solid phase extraction (SPE). For SPE, each tC18 Sep-Pak cartridge was preconditioned by sequential washing with methanol and water. The incubation material was then loaded onto the cartridge and washed with water, and the drug-derived material was eluted with acetonitrile.

View larger version (24K): [in this window] [in a new window]   FIG. 2. Radiometric profile after 30-min incubation of [14C]lonafarnib (25 µM) with active (top) or boiled (bottom) human liver microsomes together with an NADPH-generating system. Note that the CPM scales were amplified to show the minor metabolites.

Inhibition with Chemical Inhibitors and Inhibitory Monoclonal Antibody. Inhibition of lonafarnib metabolism was evaluated using both chemical inhibitors and cytochrome P450 inhibitory antibodies. Human liver microsomes (1 nmol/ml) or human P450 Supersomes (0.2 nmol/ml) were preincubated with various concentrations of inhibitors/antibodies for 15 min at room temperature, followed by the addition of buffer, the cofactor, and the substrate (25 µM [¹⁴C]lonafarnib). The final concentration of organic solvent in the incubation system was 1%. The K_i values were determined by coincubating various concentrations of the inhibitor (0–2 µM ketoconazole or 0–100 µM quercetin) with various concentrations of lonafarnib (5–50 µM) in the presence of pooled human liver microsomes and an NADPH-generating system. All incubations were carried out as described before. Before the addition of substrate, the incubation mixture was preincubated for 3 min at 37°C, then allowed to proceed for 30 min, and then terminated as described earlier. K_i values were calculated by traditional graphical method.

Correlation Study. Individual human liver microsomal preparations from 10 donors (includes data for P450-specific enzyme activities) were obtained from HBI. Correlation studies were performed with individual human liver microsomes under steady-state condition of 25 µM lonafarnib (near K_m value). The isoform-specific marker activities were obtained from HBI. The ability of human liver microsomes from each donor to metabolize lonafarnib to its metabolites was correlated with the P450-specific enzyme activities from each kit (Ghosal et al., 2005). The assays were performed as described previously with 25 µM substrate and incubated for 30 min. The correlation parameter used was the linear regression coefficient (r). The GraphPad Prism program (GraphPad Software Inc., San Diego, CA) was used to calculate P values.

View larger version (23K): [in this window] [in a new window]   FIG. 3. The mass spectra of lonafarnib and its metabolites after incubation of [14C]lonafarnib (25 µM) with active human liver microsomes together with an NADPH-generating system.

	Results

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Optimization and Incubation with Pooled Human Liver Microsomes. When [¹⁴C]lonafarnib (Fig. 1) was incubated with pooled human liver microsomes and a NADPH-generating system, two major metabolites (M1 and M2) and two minor metabolites (M3 and M4) were observed by radiometric detection (Fig. 2). LC-MS analyses showed that the mass to charge ratio (m/z) for the (M+H)⁺ ions of M1, M2, M3, and M4 was 653, 635, 669, and 653 Th, respectively. Therefore, the monoisotopic molecular weights of these metabolites were 652, 634, 668, and 652, respectively. Relative to lonafarnib, biotransformation to these metabolites resulted in changes of +O, –2H, +2O, and +O, respectively. The peak eluting after M4 in Fig. 2 was identified to have an m/z of 626, whereas the peak eluting just before the parent drug was detected at an m/z of 651 (M+O-2H). Four metabolites were chosen based on the major in vitro metabolites formed in human liver microsomes and in recombinant P450s. The mass spectra of lonafarnib and the two major metabolites M1 and M2 are shown in Fig. 3. Note that lonafarnib (C₂₇H₃₁N₄O₂Br₂Cl) contains two bromine atoms and one chlorine atom. Therefore, the mass spectra of lonafarnib and its metabolites consist of multiple ion clusters resulting primarily from the natural isotope distribution of chlorine and bromine; the contributions of other elements to the clusters are less pronounced. The first ion peak for M1 was observed at m/z 653 Th. This value is 16 higher than that for lonafarnib and thus resulted from the addition of a single O atom. LC-MS/MS and NMR analyses revealed that the hydroxylation had occurred in the pendant piperidine ring (Chowdhury et al., 2004; Tong et al., 2005). Likewise, M2 resulted from dehydrogenation (–2H) in the pendant piperidine ring (Fig. 4). Because M1 and M2 are major human metabolites, a detailed study of enzyme kinetics was only performed on these two metabolites.

View larger version (14K): [in this window] [in a new window]   FIG. 4. Possible metabolic pathway for the formation of M1 (M + 16) and M2 (M-2H) metabolites from lonafarnib. MW, monoisotopic molecular weight.

Screening with Human P450 Supersomes. Incubation of the drug with 14 human P450 Supersomes (CYP1A1, CYP1A2, CYP2A6, CYP1B1, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, and CYP4A11) revealed that only CYP3A4 and CYP3A5 metabolized lonafarnib to M1 and M2, which were major human metabolites (Fig. 5). Incubations were also conducted at 2, 25, and 100 µM lonafarnib concentrations, but data were presented for 25 µM (near K_m value).

View larger version (13K): [in this window] [in a new window]   FIG. 5. Screening of lonafarnib with 14 P450 Supersomes for the formation of metabolites.

View larger version (19K): [in this window] [in a new window]   FIG. 6. Radiometric profile of metabolites after 30-min incubation of [14C]lonafarnib (25 µM) with CYP3A4 (top) or CYP3A5 (bottom) Supersomes in the presence of an NADPH-generating system.

Correlation studies were conducted only with major human metabolites M1 and M2. The highest correlation between the HepatoScreen Test kit assay data (n = 10) and the formation of M1 was noted for dextromethorphan N-demethylation (r = 0.86) and testosterone 6ß-hydroxylation (r = 0.93) catalyzed by CYP3A4 and CYP3A4/5, respectively, at 25 µM lonafarnib (Table 2). Correlation between M2 and CYP3A4/5 is moderate (r = 0.57). In addition, correlation studies performed with M2 and M3 with a separate set of human liver microsomes and at 5 µM lonafarnib concentrations showed significant correlation with testosterone 6ß-hydroxylation (r = 0.73 and r = 0.81) (not shown). Correlation data were not calculated for M4 because of a lack of CYP2C8 activity data from the supplier. There was also no significant correlation of M4 with any other P450 activities in this study (not shown). There was high correlation between CYP2A6 and the formation of M1 and M2 and also between CYP2B6 and M1, but there was no detectable lonafarnib-metabolite formed with these Supersomes in vitro. The correlation value between CYP2A6 and CYP3A4 enzyme activity was 0.82 and between CYP2B6 and CYP3A4 enzyme activity was 0.99 (values calculated from supplier's activity data sheet). There was no significant correlation between S-mephenytoin 4'-hydroxylation (CYP2C19) or dextromethorphan O-demethylation (CYP2D6) and M1 or M2 formation (Table 2). The results of correlation analysis between the enzyme activities and metabolite formation suggested that lonafarnib is metabolized to M1 and M2 exclusively by CYP3A4/5 in human liver microsomes.

Inhibition Study. All inhibition studies were performed with pooled human liver microsomes at a drug concentration of 25 µM except in case of quercetin, where the drug concentration was 5 µM and a single human liver microsome was used (donor with high CYP2C8 level obtained from HBI.

The effect of incubations with ketoconazole is presented in Tables 3 and 4. Ketoconazole (2 µM) was shown to be a potent inhibitor of M1 and M2 formation from human liver microsomes (82 and 73% inhibition), and it completely inhibited M3 formation at 2 µM concentration. The mean IC₅₀ values of ketoconazole for M1 and M2 formation from human liver microsomes were 0.61 and 0.92 µM, respectively (Table 3). The IC₅₀ values of ketoconazole for M1 and M2 formation from CYP3A4 and CYP3A5 Supersomes are provided in Table 3. The inhibition constant (K_i) of M1 and M2 formation from human liver microsomes was 0.32 and 0.7 µM, respectively (Table 3). CYP1A1, an extrahepatic P450, is usually not expressed in human liver. -Naphthoflavone (an inhibitor of CYP1A1/2 and activator of CYP3A4) did not inhibit oxidative metabolite formation (M1 and M2) with human liver microsomes; instead, it stimulated the formation of M3 at the low concentration of 1 µM (Table 4). At 50 µM, quercetin, a selective inhibitor of CYP2C8, inhibited M4 formation from human liver microsomes by 72% (Fig. 7A); its IC₅₀ was determined to be 32 µM (not shown). Under identical conditions, M4 formation from CYP2C8 Supersomes was inhibited 100% by quercetin (positive control; Fig. 7A).

View larger version (14K): [in this window] [in a new window]   FIG. 7. A, effect of quercetin on the formation of M4 from lonafarnib in human liver microsomes (HLM) and CYP2C8. B, immunoinhibition of lonafarnib metabolism with CYP2C8-specific inhibitory monoclonal antibody (MAb). C, immunoinhibition of lonafarnib metabolism with CYP3A4/CYP3A5-specific inhibitory MAb. Human liver microsomes were incubated with control IgG or CYP3A4/5 antibody and lonafarnib. Formation of M1 from lonafarnib is represented by and formation of M2 by ; and are their respective controls with control IgG.

Immunoinhibition with CYP2C8-monoclonal antibody showed that there was slight inhibition of M4 formation from human liver microsomes (30%; Fig. 7B) as well as from recombinant CYP2C8 enzymes (not shown). The results with CYP2C8 antibody suggested that the antibody was not very specific for CYP2C8. Studies with CYP3A4/5-specific inhibitory monoclonal antibody showed that 85% of M1, 75% of M2, and 100% of M3 formation were inhibited when [¹⁴C]lonafarnib was incubated with human liver microsomes at 2 µg of IgG/µg microsomal protein (Table 4 and Fig. 7C). Mouse IgG was used as control because monoclonal antibody was raised in mice. The results of immunoinhibition studies with CYP3A4/5 antibody also confirmed that CYP3A4 and CYP3A5 were major enzymes involved in the metabolism of lonafarnib.

	Discussion

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P450-reaction phenotyping of lonafarnib was performed using a combination of three basic approaches as described by Bjornsson et al. (2003). The first approach was to examine the metabolic reaction in the absence and presence of P450-specific chemical inhibitors and/or inhibitory antibodies. The second approach was to determine whether heterologously expressed recombinant human P450s were capable of metabolizing lonafarnib. The third approach was to correlate the rate of the reaction with a P450-specific marker activity across a panel of well characterized liver microsomal samples from individual donors. Incubation of lonafarnib with human liver microsomes showed that M1 and M2 were the primary metabolites formed in vitro. The metabolites M1 and M2 were also major human metabolites in vivo. LC-MS analyses revealed that M1 contains a hydroxyl group (+O) and M2 contains a double bond (–2H) in the pendant piperidine ring (Chowdhury et al., 2004; Tong et al., 2005). A possible metabolic pathway for the formation of the two metabolites from lonafarnib is presented in Fig. 4. In addition, M4 and M3 were minor metabolites formed because of the mono- and dioxidation of lonafarnib.

Formation of M1, M2, and M3 were mediated via CYP3A4/CYP3A5 based on a high correlation with testosterone 6ß-hydroxylation, inhibition by ketoconazole, inhibition by monoclonal antibody raised against CYP3A4/CYP3A5, and the formation of the metabolite from Supersomes overexpressing CYP3A4 and CYP3A5. Intrinsic clearance (V_max/K_m) for M1 and M2 was estimated to be 10.52 and 3.02 µl/nmol P450/min, respectively. Intrinsic clearance data suggested that M1 is the preferred biotransformation pathway in human liver microsomes.

Incubation with 14 different recombinant human P450 Supersomes showed that CYP3A4 and CYP3A5 exhibited the most activity (Fig. 6), followed by markedly less substrate conversion with CYP2C8 and CYP1A1. Ketoconazole is a known selective inhibitor of CYP3A4 (Wrighton and Ring, 1994; Ghosal et al., 1996; Desai et al., 1998; Masimirembwa et al., 1999). Production of M1 and M2 by human liver microsomes was inhibited 82 and 73% by a CYP3A4-selective inhibitor, ketoconazole, with IC₅₀ values of approximately 0.61 and 0.92 µM, respectively. However, IC₅₀ values for M1 and M2 formation from CYP3A4 were 1.12 and 1.45 µM, respectively. The high IC₅₀ for ketoconazole against CYP3A4 has been reported by several investigators [Eagling et al., 2002 (IC₅₀ = 0.55 µM); Wang et al., 1999 (IC₅₀ = 8.5 µM); and Kumar et al., 1997 (IC₅₀ = 2.9 µM)]. Nomeir et al. (2001) stated that for certain compounds, including ketoconazole, there were substrate-dependent differences in the inhibition, and there was a discrepancy in the IC₅₀ between the substrates. Nomeir et al. (2001) also reported that increased concentration of protein in CYP3A4 Supersomes did not significantly affect IC₅₀ values, indicating that protein concentration was not a significant factor in this discrepancy. Likewise, in the present study, nonspecific binding did not account for the high IC₅₀ value of ketoconazole. In addition, there was a highly significant correlation between the formation of M1 and dextromethorphan N-demethylation or testosterone 6ß-hydroxylation, known to be mediated by CYP3A4 (Table 3) (Wrighton and Ring, 1994). Correlation studies performed with M2 and M3 at 5 µM lonafarnib concentration also showed significant correlation with testosterone 6ß-hydroxylation (r = 0.73 and 0.81), suggesting the involvement of CYP3A4 in their biotransformation. However, correlation study was not conducted with M4 because of lack of CYP2C8 activity data from the supplier. The correlation values between CYP2A6 and CYP3A4 and CYP2B6 and CYP3A4 were generated from their activities provided by the supplier (HBI data sheet). Correlation value between CYP3A4 and CYP2A6 activity was r = 0.82 (p = 0.004, significant), and between CYP3A4 and CYP2B6 activity was r = 0.99 (p < 0.0001, significant). This high correlation with CYP2B6 may be due to significant correlation (r = 0.98) between the activities of CYP2B6 Supersomes and CYP3A4 Supersomes, a phenomena previously reported by Heyn et al. (1996). A poor correlation was observed between microsomal caffeine N3-demethylase (CYP1A2), tolbutamide methyl-hydroxylase (CYP2C9), S-mephenytoin 4'-hydroxylase (CYP2C19), dextromethorphan O-demethylase (CYP2D6), chlorozoxazone 6-hydroxylase (CYP2E1), and lauric acid 12-hydroxylase (CYP4A11) and lonafarnib metabolism. The formation of M2 with recombinant CYP1A1 and M4 with CYP2C8 suggested minor involvement of these enzymes in the metabolism of lonafarnib. We also observed that the CYP1A1/2-specific inhibitor -naphthoflavone (Ghosal et al., 1996; Masimirembwa et al., 1999) caused no substantial inhibition of the metabolite M2. Thus, the contribution of CYP1A was judged to be minor, at least in human liver microsomes. However, there was slight stimulation of M3 formation at 1.0 µM concentration of -naphthoflavone. This activation of CYP3A activity by -naphthoflavone has been previously observed by many investigators (Guengerich et al., 1994; Ghosal et al., 1996; Koley et al., 1997; Ueng et al., 1997). -Naphthoflavone was reported not only as an inhibitor of CYP1A (IC₅₀ = 0.05–0.5 µM; Sai et al., 2000) but also an in vitro activator of CYP3A4 (Lee et al., 1994). This observation also provides additional evidence for the involvement of CYP3A in the metabolism of lonafarnib. In 1994, Shou et al. suggested that the activation of CYP3A4 by -naphthoflavone was due to the simultaneous binding of two substrates in the active site of CYP3A4. Quercetin, a CYP2C8 inhibitor (Desai et al., 1998; Masimirembwa et al., 1999), showed an inhibitory effect toward M4 (72%), suggesting the involvement of CYP2C8 in the formation of M4 (a minor metabolite). In our study, the IC₅₀ was found to be 32 µM. Likewise, previous studies have shown that the IC₅₀ value of quercetin was between 1.87 to 75.6 µM in rCYP2C8 (Walsky et al., 2005). Of the 209 drugs investigated, 48 were found to inhibit CYP2C8 by greater than 50% at 30 µM. In contrast, anti-CYP2C8 antibody was found to inhibit the formation of M4 by only 30%, showing that the involvement of CYP2C8 is minor in the formation of M4 in human liver microsomes. In a parallel incubation, CYP2C8 antibody did not inhibit metabolite formation significantly when incubated with recombinant CYP2C8. There was no inhibition of lonafarnib metabolism with the CYP2A6 inhibitor coumarin or with antibody against CYP2D6 and CYP2B6 (not shown). However, the formation of M1 and M2 was inhibited 85 and 75% by a CYP3A4/5-specific inhibitory monoclonal antibody. It was interesting to note that 2 µM ketoconazole (82 and 73%) and CYP3A4 monoclonal antibody (85 and 75%) inhibited M1 and M2 to a similar extent, thereby confirming the involvement of CYP3A4 and CYP3A5 in the oxidation of lonafarnib.

	Footnotes

 
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.105.007906.

ABBREVIATIONS: P450, cytochrome P450; HPLC, high-performance liquid chromatography; LC, liquid chromatography; MS, mass spectrometry; MS/MS, mass spectrometry/mass spectrometry or fragmentation mass spectrometry; SPE, solid phase extraction; Th, Thompson.

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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