Introduction

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Cysteinyl leukotrienes (Cys-LTs¹), including LTC₄, LTD₄, and LTE₄ are products of arachidonic acid metabolism resulting from the 5-lipoxygenase pathway (Samuelsson, 1983). These arachidonate-derived metabolites mediate effects on cell membranes that are characteristic of asthma (i.e., bronchoconstriction, increased endothelial membrane permeability leading to airway edema, and enhanced secretion of mucus) (Dahlen et al., 1980; Wenzel, 1997). In human tissues, activation of Cys-LT₁ receptors appears to initiate asthmatic reactions. Several Cys-LT₁ receptor antagonists, including zafirlukast (accolate), pranlukast, and montelukast (Singulair) have shown clinical efficacy against asthma, thus validating intervention at the Cys-LT₁ receptor as an attractive therapeutic target for the treatment of asthma (Jones et al., 1995; Reiss et al., 1996; Delepeleire et al., 1997).

Recently, we described the discovery of CP-85,958 (R,R-4-[6-(5-fluoro-benzothiazol-2-ylmethoxy)-4-hydroxy-chroman-3-ylmethyl]-benzoic acid) (Fig. 1), a potent Cys-LT₁ receptor antagonist in which clinical evaluation was discontinued due to unacceptable hepatotoxicity in monkeys (Andrews et al., 1995). Analysis of monkey bile after dosing with CP-89,958 revealed the presence of significant quantities of a lactol metabolite 1, presumably generated from a CYP-mediated hydroxylation  to the oxygen atom in the chromanol ring. It is quite likely that the toxicity in the monkey is mediated by this lactol derivative by ring opening to a reactive hydroxyaldehyde intermediate 2 that covalently binds to cellular proteins (Fig. 1). To avoid the metabolic bioactivation of CP-85,898, extensive structure-activity relationship studies were undertaken to prevent hydroxylation on the chromanol ring (Masamune et al., 1995; Chambers et al., 1998a). Efforts consisted of blocking the hydroxylation site or introducing substituents prone to metabolism at an alternate site on the molecule (Chambers et al., 1998b). This undertaking led to the identification of CP-199,331 (R,R-trifluoro-N-{3-[6-(5-fluorobenzothiazol-2-ylmethoxy)-4-hydroxy-chroman-3-ylmethyl]-4-methoxyphenyl}methane sulfonamide) as an optimized Cys-LT₁ receptor antagonist in the series with an overall increase in antagonist potency compared with its predecessor (Chambers et al., 1999). Preliminary metabolism studies revealed that the major metabolite of CP-199,331 in rat and human liver microsomes was O-desmethyl derivative 3. Lack of formation of lactol 4 correlated well with the absence of hepatotoxic effects in monkeys and rats after dosing with CP-199,331 at concentrations well exceeding those predicted for clinical efficacy. Overall, these observations indicated the potential utility of CP-199,331 as an agent in the treatment of asthma.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Chemical structures of CP-85,958, lactol 1, hydroxyaldehyde 2, CP-199,331, and its metabolites.

In the present report, in vitro metabolism studies were undertaken in male and female rat and human liver microsomes and male rat and human hepatocytes for elucidation of hepatic metabolism and quantitative prediction of in vivo plasma clearance (CL_p). The projected rat hepatic clearance (CL_h) determined in microsomes and hepatocytes was compared with the in vivo CL_p in this species. The observation of sexual dimorphism in rats led us to conduct additional mechanistic studies, including the identification of the specific CYP isoform(s) responsible for the metabolism of CP-199,331 in male and female rats as well as in human liver microsomes.

	Experimental Procedures

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Reagents. CP-199,331-sodium salt and the O-desmethyl metabolite 3 were synthesized as described previously (Chambers et al., 1999). Isoform-specific CYP inhibitors were purchased from Sigma (St. Louis, MO) or GENTEST (Woburn, MA). All other chemicals and solvents (reagent grade or better) were obtained from Sigma. Microsomal fractions were prepared from male and female Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) and human livers (IIAM, Exton, PA) using standard procedures. Protein concentrations were determined using the bicinchoninic acid assay method (Pierce Chemical, Rockford, IL). Total CYP content was measured according to published protocols (Omura and Sato, 1964), and human microsomes were characterized using CYP-specific marker substrate activities. Male Sprague-Dawley rat and human cryopreserved hepatocytes were obtained from IVT (Baltimore, MD). Rat or human rCYPs and anti-rat CYP3A2 and 2C11 antibodies were obtained from GENTEST.

Microsomal and Hepatocyte Incubations.

Microsomes CP-199,331 (1-100 µM) was incubated with rat (pool of ~10-15 livers) or human (individual livers with low, average, and high CYP3A4 activities) liver microsomes at CYP concentrations of 0.5 to 1.5 µM in the presence of an NADPH-generating system consisting of NADP (0.54 mM), DL-isocitric acid (6.2 mM), isocitric dehydrogenase (0.5 U/ml), and MgCL₂ (11 mM). All incubations were carried out in 0.1 M potassium phosphate buffer, pH 7.4, at 37°C. Reactions were terminated by addition of ice-cold methyl-tert-butyl ether (5:1, v/v) containing internal standard. Samples were analyzed by HPLC-UV by comparing the retention times of the analytes and their synthetic standards.

Hepatocytes. Rat (male Sprague-Dawley, pool of 15-20 livers) or human (pool of three livers, one female and two males) cryopreserved hepatocytes were thawed and suspended in Williams' E media supplemented with 24 mM NaHCO₃ and 10% fetal bovine serum at either 0.5 × 10⁶ viable cells/ml for clearance prediction or 2 × 10⁶ viable cells for metabolite identification. CP-199,331 (1 µM for clearance predictions or 20 µM for metabolite identification) was incubated with hepatocytes at 37°C for 2 to 4 h with gentle agitation. A gas mixture of O₂/CO₂ (95:5) maintained at ~2.5 kPa for ~5 s was passed through this mixture at every hour of incubation. Flasks were corked immediately after gassing. Reactions were stopped by freezing the incubation aliquots in liquid nitrogen.

Kinetic Analysis for O-Demethylation and Prediction of Hepatic Clearance. To estimate the rat and human hepatic intrinsic clearance (CL'_int) for O-demethylation of CP-199,331 in liver microsomes, kinetic parameters (V_max and K_m) of the O-desmethyl product formation were determined at substrate concentrations of 1 to 100 µM for a time period associated with reaction linearity. Estimates of V_max and K_m were calculated using Eadie-Hofstee linearization. The CL'_int was estimated as V_max/K_m and was expressed initially in the unit of volume per time per amount of CYP. The estimated CL'_int, was then scaled up to a CL'_int expressed in a typical clearance unit such as milliliters per minute per kilogram. This was done using a scale-up factor based on CYP concentration in the liver and the amount of protein represented by 1 kg of body weight of rat or human (Obach et al., 1997). The CL_h was estimated using the nonrestricted well stirred model (CL_h = Q × CL'_int/Q + CL'_int, where Q is hepatic blood flow) under the assumption that protein binding of CP-199,331 was similar in blood and microsomal incubation (Pang and Rowland, 1977).

Identification of Human and Rat CYP Isoforms Responsible for O-Demethylation of CP-199,331.

Human inhibition studies For mechanism-based CYP inhibition studies, human liver microsomes (0.5 µM) (from a lot with high CYP3A4, 2D6, 2C9, 2C19, and 1A2 activities) were preincubated with an NADPH-generating system at 37°C in the presence of triacetyloleandomycin (20 µM) (a CYP3A4 inactivator) or furafylline (10 µM) (a CYP1A2 inactivator) for 30 and 10 min, respectively. CP-199,331 (10 µM) was then added and these mixtures were further incubated for 30 min at 37°C. For competitive CYP inhibition studies, liver microsomes from the same lot were incubated with CP-199,331 (10 µM), NADPH-generating system, and a CYP inhibitor, quinidine at 1 µM (CYP2D6), sulfaphenazole at 10 µM (CYP2C9), and omeprazole at 10 µM (CYP2C19) for 30 min at 37°C. Incubations were conducted in duplicate. Workup and sample analysis was conducted as described previously.

Correlation analysis. CP-199,331 (10 µM) was incubated with human liver microsomes from six individual donors in the presence of an NADPH-generating system. Incubations were conducted in duplicate. The individual microsomal lots included in the study were previously characterized for specific CYP activities using standard marker substrates (e.g., CYP3A4 activity was exemplified by testosterone 6-hydroxylase activity). Formation rate of 3 was then correlated with CYP-specific activities (3A4, 2D6, 2C9, 2C19, and 1A2).

Metabolism by heterologously expressed CYP isoforms. CP-199,331 (20 µM) was incubated with microsomes from cells containing human rCYPs 3A4, 2D6, 2C9, 2C19, and 1A2 (CYP concentration of 0.05 µM) or rat rCYPs 3A1, 3A2, 2C11, 2C12, and 2C13 (CYP concentration of 0.05 µM) in the presence of an NADPH-generating system at 37°C. Incubations with human isoforms were conducted for 45 min, incubations with male rat-specific rCYP3A1, 3A2, 2C11, and 2C13, for 10 min, and incubation with female rat-specific rCYP2C12, for 40 min. Reactions were terminated by addition of ice-cold acetonitrile (2:1, v/v). The formation of 3 was assessed by liquid chromatography/mass spectrometry, as described here.

Antibody inhibition studies in rats. For the immunoinhibition study, male rat liver microsomes (200 µg) were mixed with various amounts of anti-rat CYP3A1 or anti-rat CYP2C11 antibodies (0-50 µl) and incubated at room temperature for 30 min followed by addition of CP-199,331 (10 µM) in phosphate buffer. After preincubation at 37°C for 2 min, the reaction was initiated by adding NADPH (1 mM) and terminated after a 40-min incubation by adding cold acetonitrile (2:1, v/v).

Pharmacokinetic Analysis in Rats. Two sets (n = 4/sex) of fasted male and female Sprague-Dawley rats (~200-220 g) with jugular vein catheters were administered CP-199,331-sodium salt in saline as an i.v. bolus solution (5 mg/kg). Rats received food (standard rodent diet) at 8 h after administration and were allowed full access to water throughout the study. Blood samples were collected from the jugular vein at appropriate time intervals and plasma samples were separated immediately by centrifugation and stored at 20°C until HPLC-UV analysis. Pharmacokinetic calculations were performed using noncompartmental analysis.

Analysis of CP-199,331 and O-Desmethyl Metabolite 3 by HPLC-UV. Sample analysis was performed using an HPLC-UV system consisting of an LDC Analytical ConstaMetric 4100 gradient pump (Riviera Beach, FL) and SpectroMonitor 3200 variable wavelength UV detector, HPLC membrane degasser (PerkinElmer Instruments, Norwalk, CT), and Waters 717^plus autoinjector (Waters, Milford, MA).

Microsomes. After centrifugation of the quenched reaction mixture, the supernatant was dried under a steady nitrogen stream and reconstituted in 150 µl of HPLC mobile phase. An autosampler was programmed to inject 70 µl on a Waters 3.9- × 150-mm Nova-Pak C₁₈ 4-µm HPLC analytical column preceded by an in-line Supelco 4.6- × 20-mm Pelliguard C₁₈ 40-µm guard column. The mobile phase was a binary mixture of acetonitrile (65%) and an aqueous solution of 0.1% glacial acetic acid, 0.05% phosphoric acid, and 0.1% triethylamine (35%). A gradient pump maintained an isocratic condition at a continuous flow rate of 1.2 ml/min. A variable wavelength UV detector was operated at 293 nm. Under these conditions, CP-199,331 and 3 eluted at 3.51 and 2.11 min, respectively. HPLC data were processed by Multichrom-2 integrating software operating in the peak height mode.

Rat plasma. Acetonitrile (300 µl) containing internal standard was added to 100 µl of plasma. After centrifugation, the supernatant was evaporated to dryness under nitrogen, reconstituted in HPLC mobile phase (125 µl), and analyzed as described above. The dynamic range of the assay was 0.05 to 20.0 µg/ml.

Analysis of CP-199,331 and O-Desmethyl Metabolite 3 by LC/MS/MS. The qualitative formation of 3 in rat and human rCYPs and hepatocytes was assessed using a Sciex API model 2000 LC/MS/MS triple quadrupole mass spectrometer (Thornhill, ON, Canada) in conjunction with an LDC Analytical SpectroMonitor 3200 variable wavelength UV detector. Analytes were chromatographically separated using a Hewlett Packard series 1100 HPLC system (Palo Alto, CA). After workup, the supernatant was evaporated to dryness and reconstituted in HPLC mobile phase (150 µl). An autosampler was programmed to inject 50 µl on a Zorbax Rx-C₈ 4.6 × 150 mm column using a binary gradient consisting of a mixture of 10 mM ammonium formate, 0.1% formic acid (solvent A), and acetonitrile (solvent B) at a flow rate of 1 ml/min. The LC gradient was programmed as follows: solvent A to solvent B ratio was held at 100:0 (v/v) for 3 min and then adjusted from 100:0 (v/v) to 10:90 (v/v) for 20 min and from 10:90 (v/v) to 100:0 (v/v) from 20 to 25 min. The column was reequilibrated for 5 min prior to the next analytical run. Postcolumn flow was split such that mobile phase was introduced into the mass spectrometer via an ion spray interface at a rate of 50 µl/min. The remaining flow was diverted to the UV detector positioned in line so as to provide simultaneous UV detection and total ion chromatogram. Ionization was conducted in the positive ion mode at the ion spray interface temperature of 150°C and using nitrogen for nebulizing and heating gas. Ion spray voltage was 4.5 kV and the orifice voltage was optimized at 40 eV.

	Results

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Pharmacokinetic and Metabolism Studies on CP-199,331 in Rats: in Vitro-in Vivo Correlations, Metabolite, and Gender-Specific Isoform Identification. Dramatic sex-related differences in the pharmacokinetic profiles of CP-199,331 were observed in male and female rats. When CP-199,331 (5 mg/kg) was administered intravenously, the compound was cleared ~42-fold faster in male rats than in female rats (Table 1; Fig. 2). Consistent with the in vivo observations of sexual dimorphism in CL_p, liver microsomes from male rats catalyzed the oxidative O-demethylation of CP-199,331 at a faster rate than those from female rats (Fig. 3). Estimates of V_max and K_m were calculated from the initial rate data shown in Fig. 3 using Eadie-Hofstee linearization (Table 1). The kinetic parameters (low K_m and high V_max) indicated that CP-199,331 displays a better affinity toward oxidation by male rat liver microsomes, resulting in a greater intrinsic clearance. Scale-up of the in vitro microsomal data, assuming that rat protein binding is similar in blood and microsomes, yielded a prediction of high CL_h in male rat and low CL_h in female rat (Table 1). These results were consistent with the actual CL_p values in male and female rats after intravenous administration. Furthermore, scale-up of the CL'_int, reflecting depletion of CP-199,331 in male rat hepatocytes, resulted in a projected CL_h that was in good agreement with the projected CL_h using male rat liver microsomes and the actual CL_p in male rats (Table 1). The excellent correlation between the projected CL_h and the determined in vivo CL_p suggested that in vivo CL_p of CP-199,331 in male and female rats was due entirely to hepatic metabolism. Consistent with this hypothesis was the observation that the concentration of the circulating O-desmethyl metabolite 3 of CP-199,331 was significantly higher in male than in female rat plasma after intravenous administration of the drug (Fig. 4).

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Plasma concentration of CP-199,331 in male () and female () rats after an intravenous dose of 5 mg/kg (mean ± S.D.; n = 4).

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Product formation plots for the metabolism of CP-199,331 to O-desmethyl metabolite 3 by male (1 µM CP-199,331) (A) and female (10 µM CP-199,331) (B) rat liver microsomes. Each point represents the average of duplicate determination.

View larger version (32K): [in this window] [in a new window]   Fig. 4.   HPLC chromatograms of male (A) and female (B) rat plasma at 1 h after intravenous administration of CP-199,331 (5 mg/kg).

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Immunoinhibition of CP-199,331 O-demethylation in male rat liver microsomes by anti-rat CYP2C11 () and 3A1 () antibodies.

Metabolism of CP-199,331 in Human Hepatic Tissue. Biotransformation studies on CP-199,331 (20 µM) were also conducted in human hepatocytes. As observed in rat hepatocytes, the primary route of metabolism in human hepatocytes involved O-demethylation to 3; however, O-glucuronidation of 3 to 5 was not observed in human hepatocytes (Fig. 1). Scale-up of the CL'_int, reflecting depletion of CP-199,331 (1 µM) in human hepatocytes, resulted in a projected moderate CL_h of 6.7 ml/min/kg (Table 1).

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Correlation between formation rates of O-desmethyl CP-199,331 (3) and 6-hydroxytestosterone in individual human livers. Each point represents the average of duplicate determination.

View larger version (21K): [in this window] [in a new window]   Fig. 7.   Vmax and Km determination for the O-demethylation of CP-199,331 in human liver microsomes with low (), average (), and high (×) CYP3A4 activities. Each point represents the average of duplicate determination.

	Discussion

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The superfamily of CYP isoforms has been extensively characterized in rats (for review, see Mugford and Kedderis, 1998). There are ~40 genes coding for specific isoforms in the rat genome with four major subfamilies of CYP isoforms in the rat liver (for review, see Wrighton and Stevens, 1992; Guengerich, 1994). Female rats have ~10 to 30% less total CYP compared with male rats, and male rats generally exhibit distinctly higher activities than females. There are instances, however, when female rats have higher activities than males (Skett, 1989). This gender-related discrepancy stems from the fact that CYP isoforms can be expressed specifically or preferentially in either males or females. It is now well established that sexual dimorphism in rat drug metabolism is due to the differential expression of various gender-dependent CYP isoforms mediated by hormonal regulation (Waxman et al., 1985; Legraverend et al., 1992a; Lin et al., 1996; Thompson et al., 1997). For example, CYP2C11 is expressed only in male rats, whereas CYP2C12 is limited to female rats (Bandiera, 1990; Waxman et al., 1990; Kobliakov et al., 1991; Legraverend et al., 1992b). In contrast, no sex-dependent differences have been reported in any of the CYP isoforms expressed in human liver (Wolff and Strecker, 1992). Other key differences between rat and human CYP isozymes include the species-specific expression of CYP isoforms. For instance, the CYP2C subfamily, in particular CYP2C11 and CYP2C12, the major constitutively expressed isoforms in rat liver is not found in human liver (Nelson et al., 1996). Similarly, CYP3A4, the major CYP isoform detected in human liver is in relatively low concentration in rat liver.

In the present study, significant sex-related differences were observed in the metabolism of a cysteinyl receptor antagonist, CP-199,331, in rats. The observation that male rats metabolized CP-199,331 to O-desmethyl CP-199,331 more rapidly than females, was consistent with the results that CP-199,331 was preferentially metabolized by the male rat-specific CYP2C11 and to a lesser extent by the male rat-predominant CYP3A isoforms. Overall, these results strongly suggest that the sexual dimorphism observed in CP-199,331 metabolism may result from the gender-related differential expression of CYP2C and/or CYP3A isoforms in rats. Consistent with the in vitro microsomal data that predicted significantly higher CL_h of CP-199,331 in male versus female rat, males demonstrated a greater CL_p than females. In addition, a good correlation also was discernible in the predicted high CL_h of CP-199,331 in male rat hepatocytes and the in vivo CL_p in male rats. These results strongly suggest that CL_p of CP-199,331 in rats is mediated primarily by hepatic metabolism. If so, then these results also implicate that gender-related differences in hepatic metabolism in vivo can be predicted accurately by in vitro metabolic data using liver microsomes and/or hepatocytes.

Also of particular interest is the in vivo finding that CP-199,331 was cleared ~42-fold faster in male rats than in female rats. Although several literature studies have addressed gender-dependent differences in pharmacokinetics of drug candidates, few demonstrate gender differences in CL_p as dramatic as that observed with CP-199,331. For instance, the anti-acquired immunodeficiency virus drug indinavir is cleared ~2-fold faster in male rats than in female rats (Lin et al., 1996). Such a dramatic difference in CL_p between male and female rats can be attributed to the predominant contribution of male rat-specific CYP2C11 in the metabolism of CP-199,331. Overall, these observations suggest that CP-199,331 and related analogs may represent a useful class of selective CYP2C11 substrates that could be used in probing the active site of the enzyme.

Although gender-specific CYP2C and to lesser extent 3A were responsible for metabolism of CP-199,331 in rats, subsequent studies in human liver microsomes revealed that CYP3A4 was the principal isoform responsible for metabolism of CP-199,331 in this species. This finding was further substantiated when a high correlation (r² = 0.95) between CYP3A4 activities of individual human livers for CP-199,331 O-demethylation, and 6-hydroxytestosterone formation was observed. In contrast, CYP1A2, CYP2C9, CYP2C19, and CYP2D6 activities of individual human livers did not correlate with rates of CP-199,331 O-demethylation. Although there are some gender-dependent metabolic differences between men and women, it is noteworthy to point out that these differences are not related to differential expression of CYP isoforms (e.g., CYP3A4), and intraindividual differences in metabolism can outweigh any gender-dependent differences in metabolism (Wolff and Strecker, 1992). Metabolic studies in human liver microsomes revealed that the projected human CL_h of CP-199,331 varied ~6-fold from low to moderate, depending on CYP3A4 activity (Table 1). Assuming that O-demethylation is the rate-limiting step in the elimination of CP-199,331 in human, the in vivo CL_p of CP-199,331 may exhibit moderate variability, depending on the abundance of CYP3A4 in individual livers. Studies are currently underway to determine whether CP-199,331 exhibits gender-dependent hepatic metabolism/pharmacokinetics in other preclinical species relevant for prephase I toxicological assessments.