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METABOLISM, TRANSPORT, AND PHARMACOGENOMICS
Departments of Drug Metabolism and Pharmacokinetics (C.T., B.A.C., B.M., S.L.P.-F., Y.K., K.S.-B., A.N., K.R., R.E., E.J.C., N.X.Y., C.E.R., T.R., T.P.), Medicinal Chemistry (C.N.D.M., S.D.K., M.G.B.), and Safety Assessment (C.B.F.), Merck Research Laboratories, West Point, Pennsylvania; and Laboratories Merck Sharp & Dohme-Chibret, Merck Research Laboratories, Clermont-Ferrand, France (F.P.)
Received January 2, 2008; accepted February 28, 2008.
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Abstract |
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2-fold greater than the value for a control group. Studies with recombinant rhesus P450s and monoclonal antibodies against human P450 enzymes suggested that CYP2C75 played an important role in the formation of M11 and M13. The induction of this enzyme by MK-0686 was further confirmed by a concentration-dependent increase of its mRNA in rhesus hepatocytes, and, more convincingly, the enhanced CYP2C proteins and catalytic activities toward CYP2C75 probe substrates in liver microsomes from MK-0686-treated animals. Furthermore, a good correlation was observed between the rates of M11 and M13 formation and hydroxylase activities toward probe substrates determined in a panel of liver microsomal preparations from control and MK-0686-treated animals. Therefore, MK-0686, both a substrate and inducer for CYP2C75, caused autoinduction of its own metabolism in rhesus monkeys by increasing the expression of this enzyme.
). Altered catalytic activity or capacity of the enzymes, by inhibition or induction, is the major source of metabolism-based drug-drug interactions (Tanaka, 1998). The past decades have witnessed the advance in prediction of drug-drug interactions due to P450 inhibition (Brown et al., 2006), but prediction of P450 induction is still evolving. Unlike inhibition, which is an instantaneous response, induction is relatively slow, and it involves transcriptional activation of the gene or stabilization of mRNA or protein. The complexity of enzyme induction has hampered the efforts in extrapolating in vitro human data to clinical outcomes (Lin, 2006). Thus, in vivo studies using preclinical species continue to provide critical information, in conjunction with in vitro human and animal data, to estimate the potential of drug interaction mediated via enzyme induction in clinical settings.
However, marked species differences in P450 induction exist (Lin 2006; Martignoni et al., 2006). For example, rifampicin is a potent inducer for CYP3A enzymes in rabbits and humans, whereas it has little inductive effect on CYP3A enzymes in rats (Nebert and Gonzalez 1987; Kocarek et al., 1995). Thus, choosing the right animal models for extrapolating to humans is very important. In this regard, nonhuman primates, which are genetically closer to humans than rodents, are considered to be better species in predicting drug interactions in humans. Rhesus monkeys (Macaca mulatta) and cynomolgus monkeys (Macaca fascicularis) are commonly used throughout the pharmaceutical industry as preclinical safety species. They have successfully mimicked drug interactions by inhibitors or inducers verified in humans, although mainly for CYP3A (Tang et al., 2000; Jin et al., 2003; Kumar et al., 2003; Kanazu et al., 2004; Prueksaritanont et al., 2006). In a recent study, African green monkeys (Cercopithecus aethiops) have been reported to be a good model for studying induction of brain and liver CYP2E1 by nicotine (Joshi and Tyndale, 2006; Lee et al., 2006b) and CYP2B6 by phenobarbital (Lee et al., 2006a). However, similar cases with monkey CYP2C enzymes are lacking.
The CYP2C subfamily is the most complex subfamily of P450s found in human and animal species (Martignoni et al., 2006), and the monkey CYP2Cs seem to be least studied compared with human and rodents. Only recently have efforts been reported in expression, isolation and characterization of some monkey CYP2C members, including CYP2C20, 2C43, 2C75, and 2C76 (Matsunaga et al., 2002; Uno et al., 2006; Tang et al., 2007). It has been revealed that monkey CYP2C members basically share substrate specificity with their human orthologs, but they behave differently in enzyme kinetics and response to chemical inhibitors (Mitsuda et al., 2006; Tang et al., 2007). The lack of selective chemical inhibitors for monkey CYP2C family members complicates reaction phenotyping, which is a key tool for characterizing drug interactions. It seems then that studies on drug interactions in monkeys would rely heavily on anti-human antibodies with potent cross-reactivity and recombinantly expressed monkey CYP2Cs (Tang et al., 2007). In this communication, we disclose our efforts in elucidating the mechanism of reduced systemic exposure of MK-0686 in rhesus monkeys following repeated oral administration with a series of in vitro and in vivo studies. MK-0686 is a potent and selective antagonist of the human bradykinin B1 receptor under investigation for the treatment of neuropathic pain (Kuduk et al., 2007). We found that autoinduction of MK-0686 metabolism was responsible for the reduction in exposure and the incidence involved the induction of CYP2C75, an important enzyme that mediated MK-0686 oxidative metabolism. To our knowledge, this is the first report of a CYP2C enzyme-involved autoinduction in monkeys.
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Materials and Methods |
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). Rhesus CYP2C74 (GenBank accession no. AY635462
[GenBank]
) and CYP2C75 (GenBank accession no. AY635463
[GenBank]
) have been cloned and expressed in Sf21 cells, and the respective microsomes have been prepared by a method described previously (Mei et al., 1999). Preparations of control male rhesus liver microsomes were obtained from Xenotech LLC (Lenexa, KS). Mouse ascites containing monoclonal antibodies raised against human CYP2C9 (mAb 30-12-1) and CYP3A4 (mAb 10-1-1) were prepared in-house. The antibodies have been characterized with respect to their P450 selectivity (Mei et al., 1999). Maximal inhibition of reactions catalyzed by CYP2C and CYP3A enzymes (80 and >90%, respectively) in rhesus monkey liver microsomes is achieved in the presence of 50 µl of respective antibody preparations for 1 mg of liver microsomal proteins (Tang et al., 2007). High-performance liquid chromatography grade solvents were purchased from Fisher Scientific (Pittsburgh, PA).
Animal Experiments
All experimental procedures described in this manuscript were conducted in accordance with guidelines established and reviewed by the Institutional Animal Care and Use Committee.
In a 4-week safety assessment study, 16 female and 16 male rhesus monkeys in total, approximately 2 to 3 years old and weighing 2.5 to 3.3 and 2.9 to 4.1 kg, respectively, were individually housed in stainless steel cages in environmentally controlled, high-efficiency particulate air-filtered rooms with a 12-h light cycle. The monkeys received approximately 150 g of SAFE/UAR 107C Certified Primate Diet (Scientific Animal Food & Engineering, Augy, France) at least 1 h before dosing. Water was available ad libitum. Three groups of four female and four male monkeys were given oral doses of 30, 60, or 150 mg/kg/day MK-0686 dissolved in Imwitor 742/Tween 80 [1:1 (w/w); Imwitor 742; Sasol Corp., Houston, TX; Tween 80; Fisher Scientific, Pittsburgh, PA]. The control animals (four females and four males) received Imwitor 742/Tween 80 [1:1 (w/w)] only. The dosing volume for all animals was 1 ml/kg. To determine toxicokinetics of MK-0686 at drug day (DD)1 and DD28, blood samples (heparinized) were collected from all monkeys at approximately 0.5, 1, 2, 4, 6, 8, 12, and 24 h after dose after the first dose and the last dose. The blood samples were centrifuged at 4°C. Plasma was removed, and it was stored at -10°C until analysis.
At the end of the study, animals were euthanized under anesthesia, and they were exsanguinated before necropsy. Liver samples were taken from each animal, snap-frozen in liquid nitrogen, and then used for the preparation of liver microsomes by a method described previously (Tang et al., 2007).
For a mass balance and excretion study, two bile duct-cannulated male rhesus monkeys (5.7 and 8.1 kg) were orally dosed with 3 mg/kg [14C]MK-0686 in 0.5% methyl cellulose with 0.05% SDS (5 ml/kg); bile, urine, and feces were collected over 72 h. To prepare samples for metabolite profiling and identification, urine and bile were mixed with acetonitrile (3 volumes), vortexed, and centrifuged. The resulting supernatants were transferred to clean test tubes and evaporated under a stream of nitrogen. The residues were reconstituted in mobile phase, and then they were analyzed by radiochemical LC-MS analysis.
In Vitro Metabolism Studies
All in vitro metabolism studies were carried out with liver microsomes from untreated, vehicle- or MK-0686-treated rhesus monkeys. In some studies, recombinant rhesus CYP3A64 and CYP2C75 were used.
Formation rate of M11 and M13 in microsomes was determined in incubations (0.50 ml final volume) consisting of liver microsomes (0.5–1.0 mg protein/ml) or recombinant P450s (100 pmol/ml), [14C]-MK-0686 (1 or 10 µM), MgCl2 (10 mM), and NADPH (1 mM) in potassium phosphate buffer (100 mM, pH 7.4). In the immunoinhibition studies, rhesus liver microsomes were pretreated with mouse ascites (fluid containing antibodies against human CYP2C and CYP3A) at a fixed ratio of liver microsomal proteins (25 µg) to antibodies (1.0 µl of ascites). The ratio was determined from a pilot titration experiment for an optimal inhibition. The reactions were initiated by the addition of NADPH, and they were terminated with 250 µl of acetonitrile after 60-min incubation at 37°C. Before radiochromatography, the samples were centrifuged to remove the precipitated pellets.
Diclofenac and testosterone were used as the substrates to determine the activities of CYP2C75 and CYP3A64 in liver microsomes from vehicle control and MK-0686 treated monkeys. The rates of the specific reactions, namely, diclofenac 4'-hydroxylation and testosterone 6β-hydroxylation, were measured according to previously described methods (Carr et al., 2006; Tang et al., 2007).
Sample Analysis
LC-MS/MS Analysis for Unchanged MK-0686 in Plasma Samples. An electrospray ionization (ESI) LC-MS/MS method was developed and validated to quantify MK-0686 in rhesus plasma ranging from 2.5 to 2000 ng/ml. A constant known quantity of internal standard (labetalol) was added to plasma samples, standards, and quality control samples. MK-0686 and the internal standard were isolated from plasma by acetonitrile precipitation. An aliquot of the supernatant was injected onto a Chromolith SpeedROD RP-18e column (4.6 x 50 mm; Merck, Darmstadt, Germany). Analytes were eluted isocratically from the column using TSP P4000 pump (Thermo Electron Corporation, Waltham, MA) that delivered a mobile phase of acetonitrile/0.1% formic acid (60:40) at a rate of 2 ml/min. One tenth of the elute was directed into the mass analyzer (Finnigan MAT model 7000 mass spectrometer; Thermo Electron Corporation), which operated at a positive scan mode with the capillary temperature set at 350°C.
Radiochemical LC-MS Analysis. [14C]MK-0686 and metabolites in monkey bile and urine samples were separated on a Synergi Hydro-RP column (4.6 x 250 mm, 4 µm; Phenomenex, Torrance, CA) using an Agilent 1100 system (Agilent Technologies, Palo Alto, CA) equipped with a binary pump and an autosampler. The aqueous mobile phase (solvent A) consisted of 25 mM ammonium formate in water, pH 7.0, and the organic phase (solvent B) consisted of acetonitrile. The gradient was maintained at 5% B at 1 ml/min, increased to 60% B in 39 min, and then further increased to 90% B in 2 min. After maintaining 90% B for 3 min, the gradient was returned to 5% B in 5 min and equilibrated for 5 min at 5% B before injection of the next sample. Postcolumn radiochemical detection and mass spectrometry were performed with a Packard 525TR (PerkinElmer Life and Analytical Sciences, Boston, MA) radiochemical detector and a Finnigan TSQ Quantum system (Thermo Electron Corporation), respectively. The postcolumn flow rate of 1 ml/min was split 1:4, with 200 µl/min directed to the mass spectrometer and 800 µl/min directed to the radiochemical detector. Radiochemical detection was performed using a liquid flow cell (500 µl) with Packard scintillation cocktail (Ultima-FloM; PerkinElmer Life and Analytical Sciences) running at 3 ml/min. All mass spectrometry data were collected using a TSQ Quantum system equipped with an ESI source. ESI was operated in a positive ion mode, with a spray voltage of 4.2 kV, a sheath gas flow of 33, an auxiliary gas flow of 6, and a capillary temperature of 350°C.
Quantitation of CYP2C75 and CYP3A64 Proteins by ELISA
CYP2C75 and CYP3A64 protein levels in the microsomes prepared from the liver tissues of vehicle- and MK-0686-treated monkeys were determined by ELISA using monoclonal antibodies developed in-house against human CYP2C9 (mAb99) and CYP3A4 (mAb10-1-1) (Mei et al., 1999). In brief, 96-well high binding plates were incubated overnight with 0.02 to 2.5 µg of microsomes from each animal at 4°C followed by washing with phosphate-buffered saline containing 0.2% Tween 20 (PBST). Wells were blocked with PBST containing 1% BSA at room temperature for 2 h, and then they were washed with PBST and incubated with 1:1000 of the primary antibody (mAb99 or mAb10-1-1) in PBST containing 0.1% BSA at 37°C for 1 h. The unbound primary antibody was thoroughly rinsed from wells before adding the second antibody (anti-mouse) linked to horseradish peroxidase in PBST containing 0.1% BSA. After incubation at 37°C for 1 h, wells were finally rinsed again three times with PBST before addition of tetramethyl benzidine substrate. Color development was quenched by addition of 2 M H2SO4, and it was quantitated at 450 nm. Relative -fold change compared with vehicle control was determined by dividing the slopes calculated from absorbance values of the range of concentrations tested.
In Vitro Induction Studies with Rhesus PXR and Primary Hepatocytes
MK-0686 was evaluated in a rhesus pregnane X receptor (PXR) transactivation assay. In brief, HepG2 cells (American Type Culture Collection, Manassas, VA) grown to 90% confluence in 150-cm2 flasks were transfected with Lipofectamine 2000 (100 µl/flask; Invitrogen, Carlsbad, CA), 10 µg/flask of the rhesus PXR-GAL4, and 10 µg/flask of the pFR-UASLUC reporter plasmid, all in 15 ml of Opti-MEM I (Invitrogen). After a 3-h incubation, additional Opti-MEM I medium (15 ml/flask) was added to each transfection, and the cells were allowed to incubate overnight. Transfected cells were split onto 24-well plates (150,000 cells/well) on the following day. Immediately after plating, cells were treated with 10 µM rifampicin, MK-0686 (0.5–25 µM), or dimethyl sulfoxide (0.1%) vehicle control. Forty-eight hours after treatment, the experiment was terminated by aspiration of the media and addition of Glo-Lysis buffer (100 µl; Promega, Madison, WI). An equal volume of cell lysate from each sample was combined with luciferase assay reagent (Promega), and luminescence was assessed in a Microbeta 1450 liquid scintillation/luminescence plate reader (PerkinElmer Life and Analytical Sciences–Wallac Oy, Turku, Finland).
Rhesus hepatocytes were prepared in-house by a similar method described previously (Gibson et al., 2005). Cells were plated and cultured for 48 h in InVitroGRO CP plating medium (Celsis In Vitro Technologies, Baltimore, MD), with media replaced every 24 h. Cultured hepatocytes were maintained at 37°C, 95% humidity, and 5% CO2 on collagen type I-coated plates. Forty-eight hours after plating, the hepatocytes were treated for an additional 48 h with vehicle control 0.1% (v/v) DMSO, MK-0686 (0.1–20 µM), and the positive control inducer rifampicin (10 µM) prepared in InVitroGRO HI incubation medium. The dosing solutions were replaced every 24 h. At the end of the 48-h incubation, changes in CYP3A64 and CYP2C75 gene expression were determined by measuring mRNA relative to the internal reference 18S ribosomal RNA. Changes in mRNA expression after treatment with MK-0686 were reported as -fold DMSO control, and they were compared with the changes observed with the positive control rifampicin. Plates for mRNA quantitation were aspirated, and then they were stored at -70°C until RNA isolation.
Cell cultures frozen at -70°C were thawed to room temperature and total RNA was isolated using an RNeasy 96 kit (QIAGEN, Valencia, CA) according to the manufacturer's protocol on a BioRobot 3000 (QIAGEN). Samples were treated with RNase-free DNase, as described in the protocol. Samples were eluted with 110 µl of water, and total RNA concentration was measured using a RiboGreen RNA Quantitation Reagent (Invitrogen).
A two-step reverse transcription-PCR reaction was conducted by reverse transcribing an aliquot of total RNA (
50 ng) to cDNA using TaqMan reverse transcription reagents and random hexamer primers according to the TaqMan Universal PCR Master Mix protocol (Applied Biosystems, Foster City, CA). PCR reactions were prepared by adding an aliquot of cDNA (2 µl) to a reaction mixture containing TaqMan Universal PCR Master Mix solution, the internal reference 18S ribosomal RNA (Hs99999901_s1; Applied Biosystems) primers and probe (50 and 200 nM, respectively), and CYP3A64 [RhCYP3A-For: 5'-CACAAACCGGAGGCCTTTT-3' (position 306–324); RhCYP3A-Rev: 5'-TCTTCCATTCTTCATCCTCAGCTA-3' (position 359–382); RhCYP3A-Probe: 5'-5-carboxyfluorescein-TCCAGTGGGATTTATGAAAAATGCCATCTCT-TAM-3' (position 327–357)] or CYP2C75 [RhCYP2C75-For: 5'-AGATCCGGCGTTTTTCCC-3' (position 365–382); RhCYP2C75-Rev: 5'-CACGATCCTCAATGCTCCTCTT-3' (position 412–433); RhCYP2C75-Probe: 5'-5-carboxyfluorescein-CCCATCCCAAAATTCCGCAGTGTCAT-TAM-3' (position 385–410)] primers and probes (300 and 200 nM, respectively). The TaqMan primers and probes for CYP3A64 (GenBank accession no. NM_001040414) and CYP2C75 (GenBank accession no. NM_001040211) were designed using Primer Express software version 1.0 (Applied Biosystems). The binding position, length, and specificity of each primer and probe were optimized using the BLAST program on the National Center for Biotechnology Information homepage (http://www.ncbi.nlm.nih.gov/blast). PCR-amplified cDNAs were detected by real-time fluorescence on an ABI Prism 7700 Sequence Detection System (PerkinElmer Life and Analytical Sciences). The relevant change of the target cDNA in treated samples versus DMSO control samples was calculated using the 2-
Ct method, following normalization of the 18S ribosomal RNA in each sample (Livak and Schmittgen, 2001).
Pharmacokinetic Parameter Calculation and Statistic Analysis
Cmax and Tmax were read directly from the concentration-time profiles. Areas under the concentration-time curve (AUC)0-t were calculated by the trapezoidal rule. AUC0-
values for the first dose were compared with AUC0-24h values for the last dose. When it was necessary to compare the means between the treatments, an unpaired t test was used. For this study, p < 0.05 was accepted as denoting statistical significance. Data were expressed as the mean ± S.D.
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2 h at 30 and 60 mg/kg/day and 4 h at 150 mg/kg/day). The reduced systemic exposure following repeated oral administration is suggestive of autoinduction of MK-0686 metabolism.
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MK-0686 Disposition in Rhesus Monkeys. The hypothesis of autoinduction was supported by the finding that MK-0686 was cleared primarily by metabolism in rhesus monkeys as indicated by the negligible level of unchanged form excreted in bile and urine samples following an oral dose (3 mg/kg). The resultant metabolites were excreted to bile and urine (62 and 14% of administered dose), whereas 18% of radioactivity remained in feces. Thus, approximately 80 and 20% of the absorbed dose was eliminated to bile and urine, respectively. LC-MS/MS analysis of such bile samples revealed a very complex metabolite profile (Fig. 2) with more than 15 metabolites being detected (Fig. 3). The structures of some metabolites (M1, M5, M13, and M14) were confirmed by comparison with authentic standards according to molecular weight, retention time, and product ion spectrum; others (M2, M7, M9, and M11) were characterized by NMR analysis, and the remaining metabolites were tentatively elucidated by MS/MS spectral interpretation. Efforts of metabolite identification will be a subject of a separate manuscript. The biliary metabolite profile revealed methyl ester hydrolysis and oxidation on the terminal phenyl moiety as the two primary metabolic pathways for MK-0686 in rhesus monkeys. Combination of these two pathways, coupled with amide hydrolysis and conjugation, converted the primary products to various secondary metabolites that dominated MK-0686-related components in rhesus monkey bile. Products originally derived from oxidation (M2, M4, M6, M7, M9, M10, M11, M12, and M13) approximated 50% of the total biliary metabolites. Because only hydrolytic products ended up in urine (Fig. 2), oxidation seemed to contribute to 40% of total metabolism of MK-0686 in rhesus monkeys. Due to the complexity of the end products in excreta, identifying the primary metabolites became a priority to facilitate the further investigation of autoinduction of MK-0686 metabolism.
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Reaction Phenotyping. Further efforts were devoted to identify the enzymes responsible for MK-0686 oxidation in rhesus monkey liver microsomes. The dependence of NADPH and thermal insensitivity (50°C for 2 min) for MK-0686 oxidation in liver microsomes pointed endeavors to P450 enzymes (data not shown). However, unlike human P450 phenotyping where a set of established tools is available (Rodrigues, 1999), the means for monkey P450 phenotyping is limited. Taking advantage of the cross-reactivity of human monoclonal antibodies against rhesus CYP3A (Mei et al., 1999) and CYP2C (Tang et al., 2007), we assessed the relative contribution of CYP3A and CYP2C families by means of immunoinhibition. Treatment with anti-human CYP3A antibodies offered no significant change in total turnover of MK-0686 and formation of M11 and M13 in rhesus monkey liver microsomes, but formation of a minor metabolite M16 (derived from the opening of aminocyclopropyl ring) decreased appreciably (Fig. 4B). However, treatment with anti-human CYP2C strongly inhibited M11 and M13 production and concomitantly reduced MK-0686 total turnover from 20 to 10% at 20 µM MK-0686 (Fig. 4C). These results clearly indicate the key contribution of CYP2C enzymes to the oxidative metabolism of MK-0686 in rhesus monkeys.
The immunoinhibition results were substantiated by MK-0686 metabolism mediated by recombinant rhesus P450 isoforms. Rhesus monkey CYP3A64 (orthologous to human CYP3A4), CYP2C74 (orthologous to human CYP2C8), and CYP2C75 (orthologous to human CYP2C9) have recently been cloned, expressed, and characterized (Carr et al., 2006; Tang et al., 2007). As expected, CYP3A64 did not catalyze MK-0686 efficiently. Metabolites at negligible levels were derived from further metabolism of M17, an aminocyclopropyl derivative from the trifluoroacetyl amide hydrolysis (data not shown). No metabolites were detected after incubation with CYP2C74. In contrast, CYP2C75 efficiently turned over MK-0686, giving rise to a metabolic profile similar to that from microsomal incubation except for the negligible formation of the product of the methyl ester hydrolysis M5 (Fig. 5A). The reaction by CYP2C75 was significantly inhibited by the treatment of anti-human CYP2C antibodies (Fig. 5B), but not by anti-human CYP3A antibodies (data not shown). It is obvious that rhesus CYP2C75 is the principal enzyme responsible for MK-0686 oxidative metabolism in this species.
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), the levels of both CYP2C75 and CYP3A64 mRNA were determined in rhesus hepatocytes treated with MK-0686 for 48 h. As shown in Fig. 7, MK-0686 treatment significantly enhanced mRNA levels for both CYP2C75 and CYP3A64. Its effects were comparable with rifampicin response at 10 µM. The mean EC50 values were approximately 3 µM. In agreement, CYP2C75 activity (measured as diclofenac 4'-hydroxylase) was increased by 2-fold after incubation with 10 µM MK-0686, even slightly higher than the effect of rifampicin at same concentration (data not shown). However, MK-0686 treatment failed to cause a corresponding increase in CYP3A activity (measured as testosterone 6β-hydroxylase). Instead, the enzyme activity was only 10% of the solvent control after treatment with 10 µM MK-0686 (data not shown), in contrast to the effect of the positive control (2-fold increase in CYP3A activity with 10 µM rifampicin). These results indicate that MK-0686 possesses a high potential to induce rhesus CYP2C75 and CYP3A64, but the inductive effect on CYP3A64 may be offset by its inhibition of the enzyme.
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; Singh 2006). It has important implications in drug discovery and development due to possibly altered toxicity associated with the enhanced metabolism as well as the problem achieving adequate exposure for efficacy and safety evaluation. Autoinduction occurs when the administered compound induces the enzymes that mediate its own metabolism, resulting in decreased steady-state exposure. Because autoinduction is time- and dose-dependent (Lin, 2006), it is therefore frequently observed in the studies of safety assessment in which test compounds are repeatedly administered at a wide range of doses. Such findings usually elicit concerns about the likelihood of autoinduction in humans. Evaluation of this possibility has to be based on a good understanding of species difference in elimination mechanisms, metabolic pathways, important metabolizing enzymes (most commonly P450s), and induction potency of the test compound for the enzymes. Although information about the first two parameters is well precedented, tools for identifying important P450s are not readily available for preclinical species. Only limited efforts have been reported to investigate the P450 isozymes involved in autoinduction in dogs (Gibson et al., 2005) and rats (Kitamura et al., 2007). The remarkable reduction of systemic exposure of MK-0686 in rhesus monkeys after repeated oral administration presents the first report of autoinduction attributed to rhesus CYP2C75. The potent cross-inhibition of rhesus P450s activity by anti-human P450 monoclonal antibodies (Mei et al., 1999) and recombinant rhesus P450 isozymes recently expressed in-house (Carr et al., 2006; Tang et al., 2007) have greatly facilitated our efforts in elucidating the mechanism of multiple-dose pharmacokinetics of MK-0686.
Several lines of evidence point to autoinduction of MK-0686 metabolism as the mechanism responsible for the reduced systemic exposure after repeated oral administration to rhesus monkeys. First, MK-0686 was primarily eliminated via metabolism with negligible amount of dosed MK-0686 excreted intact in bile and urine. The majority of the dosed radioactivity was recovered in the bile as metabolites. Second, oxidation accounted for a significant portion of MK-0686 metabolism, because approximately 40% of metabolites were derived from oxidation on the terminal phenyl ring. Thus, oxidative reactions played an important role in MK-0686 metabolism in rhesus monkeys. Third, the formation rate of the primary oxidative metabolites M11 and M13 was significantly increased in liver microsomal preparations from MK-0686 treated rhesus monkeys in a dose-dependent manner. Taken together, these results support the argument that autoinduction accounts for the reduced systemic exposure after repeated administration to rhesus monkeys.
The subsequent task was to address the identification of the enzyme(s) responsible for the observed autoinduction. Not only should such enzymes be subject to induction by MK-0686, but they should also be the significant contributor to the metabolism of the inducer. Involvement of the CYP2C family was implicated because the formation of M11 and M13 in rhesus liver microsomes was significantly inhibited in the presence of monoclonal antibodies selective for human CYP2C enzymes, which have been demonstrated to have a strong cross-reactivity against monkey CYP2C enzymes (Tang et al., 2007). In contrast, antibodies selective for human CYP3A enzymes showed no significant effect on overall MK-0686 oxidative metabolism, whereas the formation of a few minor metabolites was inhibited. With the recombinant rhesus P450 enzymes, we further identified CYP2C75 as the enzyme that was primarily responsible for MK-0686 oxidation in rhesus monkeys. This enzyme efficiently catalyzed the formation of M11 and M13, and it gave rise to a metabolite profile similar to that obtained with rhesus liver microsomes, except for the generation of the methyl ester hydrolytic metabolite M5, whereas CYP2C74 was not involved. In agreement with the findings from the immunoinhibition study, CYP3A64 did not contribute to the formation of M11 and M13, and it showed a low catalytic rate for MK-0686 oxidation. The unavailability of other rhesus P450 enzymes prevented us from evaluating the possibility of their involvement in MK-0686 metabolism. However, the contributions of other enzymes is suspected to be minor, given the virtually complete eradication of MK-0686 oxidation in liver microsomes in the presence both anti-CYP2C and anti-CYP3A antibodies (data not shown). Hence, it is evident that CYP2C75 is the principal enzyme responsible for MK-0686 oxidation in rhesus monkeys.
The induction of CYP2C75 by MK-0686 in rhesus monkeys was further confirmed with in vitro studies. Rhesus PXR reporter assays and primary rhesus hepatocyte induction studies revealed the potential of MK-0686 as a rhesus CYP2C75 inducer. Its intrinsic induction potency seemed to be similar to rifampicin at concentrations up to 10 µM. This potential was realized in the 4-week safety study, as evidenced by the increased CYP2C75 activity and expression level in liver microsomes from MK-0686 treated animals. However, it remains unclear whether other nuclear or steroid receptors participated in CYP2C75 induction by MK-0686. It is speculated that the human constitutive androstane receptor works in concert with PXR and other receptors to enhance CYP2C9 enzyme gene expression (Pascussi et al., 2003). For example, Goldstein's group has demonstrated that the constitutive androstane receptor and PXR cross-talk with hepatic nuclear factor 4
to synergistically activate the human CYP2C9 promoter (Chen et al., 2005). Although the relevant information in monkeys is lacking, similar networks could conceivably be operational because cross-talk has also observed in rodents (Slatter et al., 2006).
The magnitude of induction achieved under in vitro and in vivo conditions is noteworthy. As measured by CYP2C75 mRNA levels, the Emax in rhesus hepatocytes fell into the range of 3-to 5-fold. In comparison, CYP2C75 amount and activity increased by 3-fold in liver microsomes from the 150 mg/kg/day group (Table 2), reasonably reflecting the Emax determined with CYP2C75 in rhesus hepatocytes. It was unknown whether MK-0686 in those monkey livers reached a level required for the maximal induction, but the unbound concentration therein was probably in excess of the estimated EC50 (2–4 µM). Supporting this hypothesis is the finding that differences in MK-0686 concentration ranging from 7- to 10-fold in liver and plasma was observed in rats following an oral dose (data not shown). This case is not an exception because many drugs have shown high liver-to-plasma ratios after oral administration, largely due to the higher concentrations in portal vein than in plasma, even in the absence of an uptake mechanism (Hoffman et al., 1995). Midazolam, for example, shows a liver-to-plasma ratio of 13.8 in rats (Yamano et al., 2001). Assuming a similar liver-to-plasma partition in rhesus monkeys, and given the unbound fraction in rhesus plasma (0.05) and the mean total plasma concentration (20 µM), we could reasonably consider a mean unbound concentration greater than the estimated EC50 (2–4 µM, derived from the data in Fig. 7) in rhesus livers. Therefore, the in vitro findings were reasonably reflected in the in vivo situation regarding rhesus CYP2C75 induction by MK-0686.
However, a discrepancy was observed for CYP3A64 induction. Although MK-0686 activated rhesus PXR and subsequently increased CYP3A64 gene expression, the enzyme activity in rhesus hepatocytes was decreased. Likewise, the enzyme activity in liver microsomes from MK-0686-treated animals was lower than that in the control group, although the protein level was not significantly altered (Table 3). This conflicting outcome can be accounted for, at least in part, by the fact that MK-0686 was hydrolyzed to M17, a cyclopropylamine derivative. Cyclopropylamine analogs are known to exert mechanism-based inhibition of monoamine oxidase (Silverman 1991) and P450s (Kalgutkar et al., 2007). M17 time-dependently inhibited testosterone 6β-hydroxylase, but not diclofenac 4'-hydroxylase, in rhesus liver microsomes (data not shown). With the potential of two opposing effects on CYP3A64 activity attributed to its oxidation, MK-0686 highlights the uncertainty in predicting in vivo consequences associated with CYP3A64. Because this enzyme did not significantly contribute to the overall MK-0686 oxidation, the net effect could only be demonstrated by the pharmacokinetics of a potential victim drug, not through autoinduction.
Unlike several reported autoinduction cases where the induced P450 enzymes dominated the total clearance of the compounds in either human or preclinical species (Gibson et al., 2005; Shimizu et al., 2006; Kitamura et al., 2007), MK-0686 was eliminated via both hydrolytic reactions and CYP2C75-mediated oxidation in rhesus monkeys. The contribution of the former pathways seemed to be equal to or greater than the latter for the total clearance of MK-0686 (Fig. 2). The comparable formation of M5 (the product of methyl ester hydrolysis, the predominant hydrolytic reaction both in vivo and in vitro in rhesus monkeys) in liver microsomes from vehicle-control and MK-0686-treated monkeys suggests negligible alteration of esterase activity during MK-0686 treatment (Table 2). Thus, MK-0686 was sensitive to CYP2C75 induction, although the enzyme only partially contributed to its total metabolism. The magnitude of AUC change in this case (decreased by 45–70% at the dose range tested) seemed to exceed what would be expected under the first order conditions. A simulation method described in the literature (Zhang et al., 2007) indicated an AUC decrease by 30 and 45%, with the fraction of CYP2C75-mediated oxidation equal to 0.4, and intrinsic clearance increasing 2- and 3-fold, respectively. The magnitude of observed MK-0686 AUC decrease suggested that CYP2C75 induction offset the mitigating effect of hydrolytic reactions. This could happen if metabolism was saturated, because once additional CYP2C75 became available, MK-0686 being in large excess would be primarily metabolized by the induced enzyme, leading to an increased total clearance.
In the present study, composed of a series of in vitro and ex vivo experiments, we identified autoinduction as the primary mechanism responsible for the reduced systemic exposure of MK-0686 in rhesus monkeys that received a daily oral dose for 4 weeks. The enzyme involved in the autoinduction was CYP2C75, not CYP3A64, although the compound was capable of increasing the transcriptional expression of both enzymes. MK-0686 acted as both the substrate and inducer of CYP2C75. As a result, induction of the enzyme led to an accelerated turnover of the compound itself, leading to a significant decline of its systemic exposure. The good agreement between in vivo and in vitro results in this case indicates that rhesus monkey hepatocytes can be used to predict the in vivo outcome from CYP2C75 induction even though this enzyme only partially contributed to the total metabolism.
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ABBREVIATIONS: P450, cytochrome P450; MK-0686, methyl 3-chloro-3'-fluoro-4'-{(1R)-1-[({1-[(trifluoroacetyl)amino]cyclopropyl}carbonyl)-amino]ethyl}-1,1'-biphenyl-2-carboxylate; M, metabolite; mAb, monoclonal antibody; ELISA, enzyme-linked immunosorbent assay; PBST, phosphate-buffered saline containing 0.2% Tween 20; BSA, bovine serum albumin; PXR, pregnane X receptor; DMSO, dimethyl sulfoxide; PCR, polymerase chain reaction; For, forward; Rev, reverse; ESI, electrospray ionization; LC, liquid chromatography; MS/MS, tandem mass spectrometry; DD, drug treatment day; AUC, area(s) under the concentration-time curve.
1 Current affiliation: Department of Chemistry, Novartis Institutes for Bio-Medical Research, Inc., Cambridge, Massachusetts. 
Address correspondence to: Dr. Cuyue Tang, Department of Drug Metabolism and Pharmacokinetics, Merck Research Laboratories, WP75A-203, Merck and Co., Inc., West Point, PA 19486. E-mail: cuyue_tang{at}merck.com
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