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Cytochrome P450 3A-Dependent Metabolism of a Potent and Selective -Aminobutyric Acid_A2/3 Receptor Agonist in Vitro: Involvement of Cytochrome P450 3A5 Displaying Biphasic Kinetics

Department of Drug Metabolism, Merck Research Laboratories, West Point, Pennsylvania

(Received January 9, 2007; accepted April 23, 2007)

	Abstract

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In vitro metabolism studies were conducted to determine the human cytochrome P450 enzyme(s) involved in the biotransformation of 7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3b]pyridazine (TPA023), a selective agonist of human -aminobutyric acid_A receptor 2 and 3 subunits. Incubation of TPA023 with NADPH-fortified human liver microsomes resulted in the formation of t-butyl hydroxy TPA023, N-desethyl TPA023, and three minor metabolites. Both t-butyl hydroxylation and N-deethylation reactions were greatly inhibited (>85%) in the presence of CYP3A-selective inhibitory antibodies and chemical inhibitors, indicating that members of the CYP3A subfamily play an important role in TPA023 metabolism. Eadie-Hofstee plots of t-butyl hydroxylation and N-deethylation in pooled CYP3A5-rich human liver microsomes revealed a low K_m (3.4 and 4.5 µM, respectively) and a high K_m (12.7 and 40.0 µM, respectively) component. For both metabolites, the high K_m component was not observed with a pool of microsomal preparations containing minimal levels of CYP3A5. Preincubation of liver microsomes with mifepristone (selectivity for CYP3A4 > CYP3A5) greatly inhibited both t-butyl hydroxylation and N-deethylation (>75%); however, the residual activities were significantly higher in the pooled CYP3A5-rich liver microsomes (p < 0.0005). In addition, elevated levels of residual t-butyl hydroxylase and N-deethylase activities were observed in the presence of both CYP3A5-rich and CYP3A5-deficient preparations when the substrate concentration increased from 4 to 40 µM. In agreement, metabolite formation catalyzed by recombinant CYP3A5 was described by a biphasic model. It is concluded that CYP3A4 plays a major role in TPA023 metabolism, and CYP3A5 may also contribute at higher concentrations of the compound.

View larger version (11K): [in this window] [in a new window]   FIG. 1. Structures of TPA023 and its metabolites. Asterisk indicates the position of 14C label.

	Materials and Methods

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Chemicals and Biologicals. [¹⁴C]TPA023 (>99% radiochemical purity), (–)-N-3-benzyl-phenobarbital, montelukast, and authentic standards of the five TPA023 metabolites listed were synthesized in-house (Merck Research Laboratories, Rahway, NJ). NADPH, sulfaphenazole, quinidine, troleandomycin, furafylline, and mifepristone were obtained from Sigma-Aldrich (St. Louis, MO). Pooled human liver microsomes (n = 20 livers) used in chemical and immunoinhibition studies were purchased from XenoTech, LLC (Lenexa, KS). Individual human liver microsomes (HH9, HH48, HH89, HG42, HG89, and HK23) were obtained from BD Biosciences Discovery Labware (Bedford, MA). Expression levels of CYP3A4 and CYP3A5 (picomole of immunodetected protein/milligram microsomal protein) were provided by the vendor and are listed in Table 1. Microsomes prepared from insect cells infected with baculovirus-coexpressing individual human P450 isoforms (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, and CYP3A7) and P450 reductase (OR) were obtained in-house (Mei et al., 1999; Shou et al., 1999; Mei et al., 2002). Human cytochrome b₅ (b₅) and human CYP3A5 + OR + b₅ supersomes were purchased from Invitrogen (Carlsbad, CA) and BD Biosciences Discovery Labware, respectively. Inhibitory mouse anti-human P450 antibodies selective for CYP1A2, CYP2A6, CYP2C, CYP2D6, and CYP3A were prepared inhouse, whereas anti-human CYP2B6 and CYP2E1 antibodies were purchased from BD Biosciences Discovery Labware. All the other reagents were of analytical grade or higher.

Incubations with Human Liver Microsomes and Recombinant Human P450 Isoforms. Metabolite-profiling experiments were conducted by incubating ¹⁴C-labeled TPA023 (10 µM final concentration in 1-ml final volume) with potassium phosphate (100 mM, pH 7.4), magnesium chloride (1 mM), pooled human liver microsomes (0.5 mg/ml), and NADPH (1 mM). The reaction was started by the addition of TPA023 after a 3-min preincubation at 37°C in a shaking water bath. An aliquot of equal volume of acetonitrile was added at 2 h to terminate the reaction. After centrifugation at 2000g for 10 min, supernatant fractions were transferred to clean tubes and dried under nitrogen. Dried samples were reconstituted in 0.30 ml of mobile phase for high-performance liquid chromatography (HPLC) analysis. Measurement of the formation rates of metabolites M1 and M2 (described below) was performed in 0.2-ml incubation mixtures. Human liver microsomes (0.2 mg/ml) or recombinant human P450 (25–100 pmol/ml) was incubated with TPA023 (0.1–150 µM) for 20 min. TPA023 was dissolved in acetonitrile, with a final concentration of 1% v/v. In experiments conducted to evaluate the effects of b₅ in CYP3A5-catalyzed reactions, the molar ratio of supplemented b₅ to CYP3A5 was adjusted to mirror the b₅/CYP3A5 ratio of purchased CYP3A5 + OR + b₅ supersomes. For kinetics and inhibition studies, the formation rates of metabolites M1 and M2 were linear with respect to protein and P450 concentrations, as well as time of incubation.

Inhibition Studies. Immunoinhibition studies were conducted in a similar manner as published previously (Ma et al., 2004). Pooled human liver microsomes (0.04 mg of protein) were preincubated on ice (10–15 min) with the anti-P450 antibody preparation (2 µl) or control sera prepared from mouse ascites. The mixture was then diluted with potassium phosphate buffer and other assay components as described above. Chemical inhibition was performed using known P450 isoform-selective inhibitors (Newton et al., 1995; Khan et al., 2002; Suzuki et al., 2002; Walsky et al., 2005). Concentrations of furafylline (50 µM), montelukast (5 µM), sulfaphenazole (10 µM), (–)-N-3-benzyl-phenobarbital (5 µM), quinidine (5 µM), mifepristone (20 µM), ketoconazole (1 µM), and troleandomycin (25 µM) were chosen to give a maximal inhibitory effect as reported. Stock solutions of chemical inhibitors were prepared in 50% acetonitrile/water (v/v). The final concentration of acetonitrile in the incubation mixture was 1.5% (v/v). In the experiments with furafylline, mifepristone, and troleandomycin, inhibitors were preincubated with liver microsomes in the presence of NADPH for 30 min at 37°C before addition of substrate. All the other inhibitors were coincubated with the substrate.

Liquid Chromatography/Mass Spectrometry Analysis. Chromatographic separation was performed using a PerkinElmer LC system (Boston, MA) equipped with a Zorbax RXC8 column (4.6 x 250 mm, 5 µm; Agilent Technologies, Palo Alto, CA). The mobile phase consisted of 25 mM ammonium formate in water, pH 3.0 (solvent A), and acetonitrile (solvent B) and was delivered at a constant flow rate of 1 ml/min. The solvent gradient initiated at 10% B for 10 min and then increased linearly to 50% B in 30 min. After isocratic flow (50% B) for 10 min, the gradient returned to 10% B. The column was re-equilibrated at the initial condition for 5 min before injection of the next sample. Postcolumn effluent was analyzed by both an IN/US ß-ram radiochemical detector (Tampa, FL) and a Thermo Electron TSQ 7000 tandem mass spectrometer (San Jose, CA) interfaced to the HPLC. Mass spectrometric analysis was carried out with electrospray ionization (ESI) in the positive ion mode. The capillary temperature was 230°C, and the ESI ionizing voltage was maintained at 5.0 kV. Collision-induced dissociation was performed with a collision gas flow of 1.7 mtorr and a collision energy range of 25 to 45 eV. Metabolite identification was based on the matching retention time and fragmentation pattern of each metabolite obtained from microsomal incubations with those of authentic standards (Polsky-Fisher et al., 2006). Quantitation of metabolites was also achieved using fluorescence detection, with excitation and emission wavelengths set at 250 and 430 nm, respectively. The lower limit of quantitation was 5 nM, and the interday and intraday variation was within 10% CV.

View larger version (13K): [in this window] [in a new window]   FIG. 2. Representative radiochromatogram of TPA023 and its oxidative metabolites after incubation with NADPH-fortified human liver microsomes.

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	Results

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Metabolism of TPA023 in Human Liver Microsomes. After incubation of ¹⁴C-TPA023 with NADPH-fortified pooled human liver microsomes, two major metabolites (M1 and M2) and three minor metabolites (M3, M4, and M5) were observed by HPLC-radiochemical detection (Fig. 2). Under positive ion ESI, the molecular ions (MH⁺) of TPA023, M1, M2, M3, M4, and M5 were at m/z 396, 412, 368, 384, 426, and 410, respectively. Based on the matching column elution time and tandem mass spectra obtained from the metabolite standards, M1 and M2 were identified as t-butyl hydroxy TPA023 and N-desethyl TPA023, respectively (Fig. 1). The minor metabolites (M3, M4, and M5) were subsequently identified as t-butyl hydroxy/N-desethyl TPA023, t-butyl carboxylic acid TPA023, and t-butyl aldehyde TPA023, respectively. With the exception of M5, all the metabolites identified in the current study were previously identified as in vivo metabolites in humans (Polsky-Fisher et al., 2006). No metabolites were observed in incubations that do not contain NADPH.

Inhibition Studies. Immunoinhibition and chemical inhibition experiments were conducted to determine the P450 isoform(s) contributing to the biotransformation of TPA023 (Fig. 3). Because M3, M4, and M5 were subsequent metabolites of M1 and/or M2, the formation rates for these three metabolites were not monitored. Preincubation of inhibitory anti-CYP3A monoclonal antibody with pooled human liver microsomes abolished (>90%) the formation of both M1 and M2, indicating that CYP3A4/5 plays an important role in the formation of these two metabolites. Although antibodies selective for CYP1A2, CYP2A6, CYP2C, and CYP2D6 did not significantly inhibit TPA023 metabolism, some inhibition (20%) was observed with antibodies against CYP2B6 and CYP2E1. The formation of M1 and M2 was also examined using known P450 isoform-selective chemical inhibitors. Consistent with the immunoinhibition data, the t-butyl hydroxylation and N-deethylation were greatly inhibited (>85%) by chemical inhibitors selective for CYP3A (ketoconazole and troleandomycin). Relatively little inhibition (10%) was observed in the presence of chemical inhibitors selective for the other human P450 forms (Fig. 3). Collectively, the results described herein indicate that human liver microsomal CYP3A subfamily members play a major role in the metabolism of TPA023.

View larger version (25K): [in this window] [in a new window]   FIG. 3. Inhibition of TPA023 metabolism in human liver microsomes (pool of n = 20 livers) by isoform-selective antibodies (A) and chemical inhibitors (B). Experiments were conducted using 10 µM TPA023. M1 and M2 represent t-butyl hydroxyl TPA023 and N-desethyl TPA023, respectively. Chemical inhibitors selective for CYP1A2 (furafylline, FUR), CYP2C8 (montelukast, MNT), CYP2C9 (sulfaphenazole, SUL), CYP2C19 [(–)-N-3-benzyl-phenobarbital, BPB], CYP2D6 (quinidine, QND), and CYP3A (troleandomycin, TAO, and ketoconazole, KTZ) were used. Each data point represents the mean ± S.D. of triplicates.

View larger version (10K): [in this window] [in a new window]   FIG. 4. TPA023 metabolism in the presence of recombinant human P450 enzymes. Incubations were performed using a substrate concentration of 10 µM in the presence of 100 pmol/ml of human P450 enzymes coexpressed with P450 reductase. Asterisk indicates that the preparation of CYP3A5 was coexpressed P450 reductase and cytochrome b5. Each data point represents the mean of duplicates. M1 and M2 represent t-butyl hydroxyl TPA023 and N-desethyl TPA023, respectively.

View larger version (18K): [in this window] [in a new window]   FIG. 5. Kinetic plots of TPA023 metabolism after incubation with pooled CYP3A5-rich human liver microsomes (n = 3) (A and B), pooled CYP3A5-deficient human liver microsomes (n = 3) (C and D), recombinant CYP3A4 (E and F), and CYP3A5 (G and H). Eadie-Hofstee plots are shown for both t-butyl hydroxylation (A, C, E, and G) and N-deethylation (B, D, F, and H). Lines represent linear regressions of Eadie-Hofstee transformed data (A and B) or best-fits to a hyperbolic model (D and F), a substrate inhibition model (C and E), or a biphasic model (G and H). Insets, velocity-substrate concentration plots.

Recombinant CYP3A4-mediated t-butyl hydroxylation and N-deethylation displayed substrate inhibition and hyperbolic kinetics, respectively. The corresponding K_m and K_si values determined in CYP3A4-catalyzed reactions were within a 2-fold range of those obtained in CYP3A5-deficient human liver microsomes (Table 2). Metabolite formation catalyzed by CYP3A5 conformed to biphasic kinetics described by K_m1 values of 1.3 µM (M1) and 0.6 µM (M2) and by K_m2 values of 18.9 µM (M1) and 82.3 µM (M2). The K_m2 values determined in CYP3A5-rich liver microsomes and recombinant CYP3A5 were within 2-fold of each other for both t-butyl hydroxylation (12.7 versus 18.9 µM) and N-deethylation (40.0 versus 82.3 µM) reactions.

Inactivation by Mifepristone. Mifepristone (RU486), a previously reported selective mechanism-based inactivator of CYP3A4 (He et al., 1999; Khan et al., 2002), was used to evaluate the involvement of CYP3A5 in TPA023 metabolism. Metabolite formation was monitored at two concentrations of TPA023 (4 and 40 µM), using pools of CYP3A5-rich and CYP3A5-deficient human liver microsomes preincubated with mifepristone (Fig. 6). As the substrate concentration increased from 4 to 40 µM, elevated levels of residual t-butyl hydroxylase and N-deethylase activities were observed for both sets of microsomal preparations, indicating an increasing involvement of CYP3A5 at higher substrate concentrations. In addition, metabolite formation in mifepristone-treated CYP3A5-rich liver microsomes was significantly higher (2- to 3-fold; p < 0.0005) than that obtained from the CYP3A5-deficient pool, although the contribution of CYP3A5 appeared to be minor (versus CYP3A4).

View larger version (15K): [in this window] [in a new window]   FIG. 6. Inhibition of TPA023 metabolism in pooled CYP3A5-rich human liver microsomes (A) and pooled CYP3A5-deficient human liver microsomes (B) preincubated with 20 µM mifepristone. Each data point represents the mean ± S.D. of triplicates. M1 and M2 represent t-butyl hydroxyl TPA023 and N-desethyl TPA023, respectively.

	Discussion

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The results of the present study have shown that t-butyl hydroxylation and N-deethylation are major oxidative biotransformation pathways of TPA023 in the presence of NADPH-fortified human liver microsomes. These results are consistent with the results of an earlier human radiolabeled study (Polsky-Fisher et al., 2006). After the administration of ¹⁴C-TPA023 to healthy male volunteers, the two pathways together gave rise to 10 metabolites and accounted for more than 77% of the radioactivity recovered. At the same time, less than 1% of the dose was recovered as unchanged drug. Analysis of the excreta revealed that two minor metabolic pathways of TPA023, fluorophenyl hydroxylation and O-dealkylation, were operative but contributed to a minor fraction (<5% combined) of TPA023 metabolism. In accord with this observation, the corresponding metabolites, fluorophenyl hydroxyl TPA023 and O-dealkyl TPA023, were not detected in the microsomal incubates described herein.

Several lines of evidence strongly indicated that CYP3A was the major P450 subfamily involved in TPA023 metabolism. Inhibitory antibody and chemical inhibitors selective for CYP3A significantly inhibited (>85%) the formation of M1 and M2. In addition, recombinant CYP3A4 and CYP3A5 were the only P450s that metabolized TPA023. Immunoinhibition data indicated that CYP2B6 and CYP2E1 may play a minor role in the metabolism of TPA023 in human liver microsomes. However, the lack of metabolism by recombinant CYP2B6 and CYP2E1 suggested that the involvement of these two enzymes may be limited. It is possible that the minor inhibitory effect of the anti-CYP2E1 antibody may be caused by minor cross-reactivity with CYP3A, a phenomenon that has been previously observed in simvastatin metabolism (Prueksaritanont et al., 1997).

CYP3A4 and CYP3A5 share 84% identity in amino acid sequence and have substantial overlapping substrate specificities (Wrighton and Stevens, 1992). Although CYP3A4 and CYP3A5 often display similar kinetics, differential kinetic profiles have been observed (Williams et al., 2002; Galetin et al., 2004; Huang et al., 2004; Shen et al., 2004). Based on the reported K_m (or K_m1) values, CYP3A4 often displays comparable or lower K_m values (versus CYP3A5) for a given biotransformation regardless of the type of kinetics. The best-fit kinetic models for t-butyl hydroxylation and N-deethylation of TPA023 catalyzed by recombinant CYP3A4 differed from those describing CYP3A5 (Table 2). Interestingly, the estimated K_m1 values of CYP3A5-mediated TPA023 metabolism were more than 2-fold lower than those of CYP3A4. Similar observations have been reported for lidocaine, etoposide, and S-(–)-verapamil (Huang et al., 2004; Shen et al., 2004; Zhuo et al., 2004). In addition, to our knowledge this is the first report of a substrate exhibiting biphasic kinetics with CYP3A5 but not CYP3A4.

In recent years, several in vitro approaches have been used to estimate the clinical relevance of CYP3A5, including the use of inhibitors displaying isoform selectivity (Khan et al., 2002; Patki et al., 2003; Galetin et al., 2004; Huang et al., 2004). Although differential inhibition of CYP3A4 and CYP3A5 has been observed for many compounds (Khan et al., 2002; Patki et al., 2003; McConn et al., 2004; Wang et al., 2005; Granfors et al., 2006), only mifepristone appears to exhibit suitable isoform selectivity for reaction-phenotyping experiments. On pretreatment of mifepristone, the residual activity of t-butyl hydroxylation and N-deethylation was indicative of the expression level of CYP3A5 in microsomal preparations used. These results, along with those obtained from kinetics studies, suggested that the contribution of CYP3A5 is more prominent at higher substrate concentrations as a result of the biphasic kinetics displayed by the enzyme. However, the clinical relevance of CYP3A5 in TPA023 metabolism may be very limited, given that the plasma concentration of TPA023 observed in clinical trials was well below the micromolar range (de Hass et al., 2007).

In summary, the current study showed that TPA023 underwent extensive oxidative metabolism in the presence of NADPH-fortified human liver microsomes, confirming the results of a human radiolabeled study (Polsky-Fisher et al., 2006). Inhibition data were consistent and showed that CYP3A isoforms were the major P450 enzymes catalyzing t-butyl hydroxylation and N-deethylation of TPA023. In agreement with this hypothesis, itraconazole elicits a clinically significant effect (5-fold increase) on the oral plasma area under the plasma concentration versus time curve of TPA023 (Polsky-Fisher et al., 2006). However, the in vitro reaction phenotype is complicated by CYP3A5, the contribution of which is dependent on the expression level of the enzyme and the concentration of substrate under consideration. At least in vitro, CYP3A4 appeared to be the major enzyme involved in the biotransformation of TPA023, especially over the concentration range that is more relevant clinically. Further confirmation will require pharmacokinetic studies with subjects who have been genotyped for various CYP3A5 alleles.

	Footnotes

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

doi:10.1124/dmd.107.014753.

ABBREVIATIONS: TPA023, 7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine; P450, cytochrome P450; OR, P450 reductase; b₅, cytochrome b₅; HPLC, high-performance liquid chromatography; ESI, electrospray ionization.

¹ Current affiliation: Metabolism and Pharmacokinetics, Bristol-Myers Squibb, Princeton, New Jersey.

Address correspondence to: Bennett Ma, Department of Drug Metabolism, WP75B-200, Merck Research Laboratories, West Point, PA 19486. E-mail: bennett_ma{at}merck.com

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This Article Abstract Full Text (PDF) All Versions of this Article: dmd.107.014753v1 35/8/1301    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Ma, B. Articles by Rodrigues, A. D. Search for Related Content PubMed PubMed Citation Articles by Ma, B. Articles by Rodrigues, A. D.