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INDUCTION OF CYP1A IN THE BEAGLE DOG BY AN INHIBITOR OF KINASE INSERT DOMAIN-CONTAINING RECEPTOR: DIFFERENTIAL EFFECTS IN VITRO AND IN VIVO ON MRNA AND FUNCTIONAL ACTIVITY

Department of Drug Metabolism, Merck Research Laboratories, West Point, Pennsylvania (C.R.G., C.L., R.Si., C.M.B., K.R., J.B., K.M., J.A., E.C., C.B.-G., M.L., A.M., M.P., P.C., R.Su., P.K., A.D.R., T.A.B., T.H.R.); and Department of Drug Metabolism, Merck Research Laboratories, Rahway, New Jersey (C.R.)

(Received January 28, 2005; accepted April 12, 2005)

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Compound I [3-[5-(4-methanesulfonyl-piperazin-1-ylmethyl)-1H-indol-2-yl]-1H-quinolin-2-one] is a potent inhibitor of human kinase insert domain-containing receptor (KDR kinase), which is under investigation for the treatment of cancer. Bile duct-cannulated male beagle dogs were administered 6 mg/kg compound I q.d. for 14 days. There was an approximately 2.5-fold decrease in the mean plasma area under the curve of I on days 7 and 14 (11.3 µM · h), relative to day 1 (28.2 µM · h). In the dog, compound I was eliminated by metabolism, with a major pathway being aromatic hydroxylation and subsequent sulfation to form the metabolite M3. Metabolic profiling suggested that the pathway leading to the formation of the sulfated conjugate M3 was induced upon multiple dosing of I. Studies conducted in vitro suggested that CYP1A1/2 was responsible for the formation of the hydroxylated metabolite, which is sulfated to yield M3. Additional studies confirmed induction of CYP1A protein and activity in the livers of dogs treated with I. However, studies in a dog hepatocyte model of induction showed a surprising decrease both in CYP1A mRNA and enzymatic activity in the presence of I, emphasizing the need to consider the results from a variety of in vitro and in vivo studies in deriving an understanding of the metabolic fate of a drug candidate. It is concluded that the autoinduction observed after multiple treatments with compound I occurs since compound I is both an inducer and a substrate for dog CYP1A.

In the present communication, we report on a series of in vitro and in vivo studies aimed at elucidating the mechanism of the multiple-dose pharmacokinetics of an experimental kinase insert domain-containing receptor (KDR kinase) inhibitor in the dog. The compound 3-[5-(4-methanesulfonyl-piperazin-1-ylmethyl)-1H-indol-2-yl]-1H-quinolin-2-one (referred to as compound I) is a potent and relatively selective inhibitor of human KDR kinase which is under investigation for the treatment of cancer. It was found during a 14-week safety assessment study in beagle dogs that the steady-state exposure of compound I was much lower than anticipated based on single-dose data, raising the possibility that I was subject to autoinduction of metabolism. The goals of the present study, therefore, were to identify the source of the apparent autoinduction and to explore the utility of in vitro canine systems as a predictor of the pharmacokinetic behavior of I in vivo.

View larger version (16K): [in this window] [in a new window]   FIG. 1. Synthesis of [14C]3-[5-(4-methanesulfonyl-piperazin-1-ylmethyl)-1H-indol-2-yl]-1H-quinolin-2-one, compound I. THF, tetrahydrofuran; MeOH, methanol; DMF, dimethylformamide; DDQ, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone; RP, reverse phase.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

[¹⁴C]I (Fig. 1) was synthesized at Merck Research Laboratories with the carbon-14 label incorporated at the benzylic 5' position of the indole moiety (Merck Labeled Compound Synthesis Group, Rahway, NJ). Briefly, [¹⁴C]carbonylation precursor, triflate 3, was prepared by reaction of 5-hydroxyindole derivative 2 with trifluoromethanesulfonic anhydride and lutidine. Using a procedure adapted from the literature, compound 3 was carbonylated with [¹⁴C]carbon monoxide, generated from [¹⁴C]CO₂ (500 mCi), to give [¹⁴C]-methyl ester 4 (Elmore et al., 2000). After purification by silica gel chromatography, compound 4 was reduced with LiAlH₄ to give [¹⁴C]alcohol 5, which was oxidized with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone to generate [¹⁴C]aldehyde 6. Reductive amination of 6 with piperazine derivative 7 and sodium triacetoxyborohydride gave [¹⁴C]Boc-protected chloroquinoline 8, which was converted to the desired product, quinolone I, by refluxing in acetic acid. Purification by reversed-phase HPLC yielded 5.9 mCi (58 mCi/mmol) of I with 98.7% radiochemical purity.

Instrumentation and Analytical Methods. The levels of radioactivity in urine and bile were determined using a PerkinElmer Tri-Carb 2900TR (PerkinElmer Life and Analytical Sciences, Boston, MA) liquid scintillation counter. A 500-µl aliquot was removed from each urine sample and a 100-µl aliquot was taken from each bile sample for total radioactivity analysis. Liquid scintillation cocktail (15 ml; PerkinElmer Ultima-Gold) was added to plastic scintillation vials containing the sample aliquots, and the mixture was vortexmixed and counted for 10 min.

For the purpose of metabolite profiling, 100-µl aliquots of bile from each dog were diluted (1:1) in plastic test tubes using 20% aqueous acetonitrile. Aliquots of the supernatant (100 µl) then were analyzed by high-pressure liquid chromatography with with radiochemical detection (LC-RAM). The LC-RAM system consisted of a PerkinElmer Series 200 HPLC interfaced with a Series 500TR flow scintillation analyzer. The diluted bile samples were injected onto a Hypersil BDS C18 column (5 µm, 4.6 x 250 mm) (Thermo Electron, Waltham, MA). The mobile phase consisted of a 0.1% aqueous formic acid and acetonitrile gradient and was operated at a flow rate of 1 ml/min. The column effluent was monitored by radiochemical detection using PerkinElmer Ultima-Flo M scintillation cocktail with a ratio of scintillation cocktail to mobile phase of 3:1 (v/v).

Relative quantitation of the metabolites of I was performed using radiochemical methods. The following equation was used to calculate the percentage of the administered dose accounted for by a given metabolite: % of dose = · f_p, where represents the percentage of the total radioactive dose that was accounted for in a sample (as determined by liquid scintillation counting) and the term f_p was the fraction of radioactivity in the sample, which was accounted for in the area of a given metabolite radiochromatographic peak.

Metabolite identification was performed using high-pressure liquid chromatography coupled with tandem radiochemical and mass spectrometric analysis (LC-RAM-MS/MS). The LC-RAM-MS/MS system consisted of a Thermo Electron Surveyor LC system coupled to a Thermo Electron LCQ Deca XP ion trap mass spectrometer and a PerkinElmer Series A-500 radiochemical detector. The chromatography conditions were identical to those described above. The LC effluent was split postcolumn using a stainless steel T-splitter with 100 µl/min being diverted to the mass spectrometer, while the remaining flow was sent to the radiochemical detector. Radiochromatograms were obtained using a PerkinElmer Series A-500 radiochemical detector (the parameters were identical to those described above). Mass spectral analyses were performed using electrospray ionization (ESI) in the positive ion mode. The ESI source parameters were as follows: capillary temperature 250°C, capillary voltage 35 V, and ESI voltage approximately 4.5 kV. Collision-induced dissociation of parent ions (MH⁺) of I and its metabolites was performed in the data-dependent mode. The identification of the proposed metabolite structures was confirmed, when possible, by comparison of HPLC and MS/MS characteristics with those of authentic standards.

NMR Studies of M1-OH and M3. Both M1 and M3 were isolated from dog bile using multistep preparative HPLC. Briefly, aliquots of dog bile were injected onto a Thermo Electron Hypersil BDS C18 semipreparative column (5 µm, 150 x 10 mm) using a 0.1% aqueous formic acid and acetonitrile gradient at a flow rate of 9 ml/min. The fractions containing the metabolites were collected and lyophilized. After lyophilization, the samples were reconstituted in mobile phase and purified further using a Phenomenex (Torrance, CA) Prodigy C8 (250 x 10 mm) semipreparative HPLC column with a 0.1% aqueous formic acid and methanol gradient (9 ml/min flow rate). The fractions containing the metabolites again were collected and lyophilized prior to NMR analysis. Due to its instability upon being isolated, the metabolite M1 was treated with sulfatase (as per the manufacturer's instructions) to yield the de-sulfated product (M1-OH), which then was repurified using the Prodigy C8 column prior to NMR analysis. NMR studies on the M1-OH and M3 HPLC isolates were performed at 25°C on a 500-MHz spectrometer (Varian, Inc., Palo Alto, CA) equipped with a 3-mm inverse detection probe (MIDG; Varian Inc.). The metabolites and I were dissolved in deuterated methanol and deuterated methanol with 10% DMSO-d₆, respectively, and transferred to 3-mm tubes.

Animal Experiments. All experimental procedures described in this report were conducted in accordance with guidelines established and reviewed by the Institutional Animal Care and Use Committee. Three male beagle dogs, weighing 9 to 11.3 kg, were surgically implanted with cannulas to allow for the sampling of bile and blood. The dogs were administered a daily 6 mg/kg p.o. dose of I (suspension in 0.5% aqueous methylcellulose) for 14 days. Radiolabeled [¹⁴C]I was coadministered on days 1 and 14 for the purpose of metabolite profiling. Blood was collected from the jugular vein into heparinized tubes at various times ranging 24 h postdose on days 1, 7, and 14, and the resultant plasma samples were stored at –20°C until analyzed by LC-MS/MS. Urine and bile samples were collected on days 1 and 14 at intervals over 24 h postdose and also were stored at –20°C until analyzed.

A separate study was conducted, using a liver biopsy approach, to further characterize the mechanism of the apparent autoinduction. A total of eight male beagle dogs were anesthetized and a 200- to 500-mg liver wedge biopsy was removed from each animal using a minimally invasive laproscopic technique. Once removed, the liver samples were immediately placed on ice and frozen (–70°C) until used for the preparation of microsomal fractions. Each animal then was allowed to recover for 1 week. The dogs then were randomized and were administered daily oral doses of either vehicle (0.5% aqueous methylcellulose) (n = 4) or 6 mg/kg I (n = 4) for 14 days. Following the treatment period, each dog was again anesthetized and the livers were biopsied using the same procedure as described above.

Quantitation of I in Dog Plasma Using LC-MS/MS. Stock standard and quality control solutions of I and labetalol (internal standard) were prepared by dissolving appropriate amounts of the compounds in DMSO to give final free base concentrations of 1 mg/ml. Calibration standards of I were prepared in dog plasma over a concentration range of 0.01 to 100 µg/ml. A working internal standard solution was prepared by diluting the 1 mg/ml labetalol stock solution to a final concentration of 3000 ng/ml, and 50 µl of this solution was added to all samples, standards, and quality controls. Aliquots of each plasma sample (100 µl) were removed for analysis and were added to 400 µl of acetonitrile. The samples then were vortex-mixed and centrifuged for 15 min at 11,000 rpm. The supernatants were transferred into glass culture tubes and evaporated to dryness under nitrogen at 50°C in a Turbovac LV evaporator (Caliper Life Sciences, Hopkinton, MA). The sample residues were reconstituted in 100 µl of 20% aqueous acetonitrile, vortex-mixed, and transferred into a Chromacol 96-well plate with amber glass inserts (Chromacol Ltd., Welwyn Garden City, Hertsfordshire, UK).

For the purposes of quantitation, an LC-MS/MS system consisting of a PerkinElmer Series 200 Micro Pump and a CTC PAL Autosampler (LEAP Technologies, Carrboro, NC) was coupled to an API 2000 triple-stage quadrupole mass spectrometer (Applied Biosystems/MDS Sciex, Foster City, CA) equipped with a heated nebulizer ionization source. Nitrogen was used at a pressure of 40 psi for curtain gas and 4 psi for collision gas. The source block temperature was set at 475°C, and the nebulizer current was set at 2 V. Data were acquired in the selected reaction monitoring mode using the following transitions: I m/z 437 (MH⁺) 273 (collision energy 57 V, dwell time 50 ms) and labetalol m/z 329 (MH⁺) 91 (collision energy 21 V, dwell time 50 ms). Reversed phase HPLC separation of I, the N-oxide metabolite of I (M5), and internal standard was accomplished using a Phenomenex Prodigy C18 column (5 µm, 100 x 2.0 mm). The mobile phase consisted of a 0.1% aqueous formic acid and 0.1% formic acid in acetonitrile gradient delivered at a flow rate of 0.7 ml/min. The analytical procedure was validated and shown to have intraday and interday accuracy (percentage difference from the nominal concentration) and precision (coefficient of variation as a percentage value – %CV) of no greater than 15% (data not shown). The lower limit of quantitation was determined to be 10 ng/ml, at which the intraday %CV did not exceed 15% (data not shown).

Pharmacokinetics. Noncompartmental pharmacokinetic analyses were conducted on the plasma concentration-time data using WinNonlin v.3.2 (Pharsight, Mountain View, CA). The apparent metabolic clearances (CL_M/F) for the sulfate conjugates M1 and M3 were calculated on days 1 and 14 using the following equation: (CL_M/F) = f_M · (CL_T/F), where f_M is the fraction of the dose of I metabolized to M1 or M3 within 24 h of dosing and Cl_T/F is the apparent total body plasma clearance following p.o. administration.

Cloning and Expression of Beagle Dog CYP1A2. Beagle dog RNA was isolated from liver using an RNEasy Kit (QIAGEN, Valencia, CA) and used for cDNA synthesis with a TimeSaver cDNA Synthesis Kit (GE Healthcare, Piscataway, NJ). First-stand synthesis was primed using oligo(dT)_12-18 primers. Amplification of dog CYP1A2 was achieved using forward and reverse primers having the sequences (5'3') ATGGCATTGTCCCAGT and TCACTTGATGGAGAAGCG, respectively. The polymerase chain reaction products were analyzed by agarose gel electrophoresis, purified, and inserted into pCR2.1 (Invitrogen). The sequence was analyzed using an Applied Biosystems (Foster City, CA) 310 automated sequencer. Dog CYP1A2 was subcloned into the pFASTBAC1 vector (Invitrogen) in the EcoRI-Hind III multicloning site. Recombinant dog CYP1A2 baculovirus then was generated using the Bac-to-Bac expression system (Invitrogen). Briefly, DH10BAC cells were transformed with pFASTBAC1-dogCYP1A2 plasmid to generate a dog CYP1A2 bacmid. Generation of the recombinant bacmid was confirmed using polymerase chain reaction amplification. Sf9 insect cells were transfected with the dog CYP1A2 recombinant bacmid, and the resulting recombinant virus was amplified after four consecutive rounds of infection. Microsomes isolated from Sf9 cells coinfected with recombinant dog CYP1A2 and rabbit oxidoreductase were evaluated for P450 expression by CO difference spectra and immunoblot analysis. The function of dog CYP1A2 was determined using 7-ethoxyresorufin O-deethylase (EROD) activity as described previously (Shou et al., 2003).

In Vitro Microsomal and Recombinant Dog P450 Studies. Liver microsomes from dogs treated with either rifampin, BNF, phenobarbital, or clofibric acid were purchased from XenoTech LLC (Lenexa, KS) and were used to study the change in metabolic profiles compared with control microsomes obtained from saline/corn oil-treated animals. The incubates (1 ml total volume) contained 100 mM potassium phosphate buffer (pH 7.4), 1 mg/ml liver microsomal protein (induced or control), and I at a concentration of 2 µM. These incubation conditions have been shown in our laboratory to be linear with respect to the formation of the oxidative metabolites of I (data not shown). The reactions were initiated by the addition of NADPH (1 mM) and were allowed to proceed for 60 min at 37°C in a shaking water bath. After incubation, the reactions were terminated by the addition of 3 volumes of acetonitrile. The samples then were vortex-mixed and centrifuged (4000 rpm, at 4°C) for 10 min. The supernatants were removed, placed in separate plastic tubes, and dried under a nitrogen stream at 30°C. The residues were reconstituted in 120 µl of 20% aqueous acetonitrile, containing labetalol (0.8 µg/ml) as internal standard, and were assayed by LC-MS/MS for the formation of the phenolic metabolite M3-OH.

Quantitative LC-MS/MS analysis of M3-OH from the in vitro samples was performed using a Thermo Electron Surveyor HPLC system coupled to a Thermo Electron LCQ Deca XP mass spectrometer using positive ESI. The samples were injected (30 µl) onto a Thermo Electron Hypersil BDS C18 column (5 µm, 2 x 150 mm) and analyzed using a 0.1% aqueous formic acid and acetonitrile gradient operated at a flow rate of 0.2 ml/min. The capillary temperature was 275°C, the capillary voltage was 35 V, and the ESI voltage was maintained at approximately 4.5 kV. Quantitative analyses were based on selected reaction monitoring of the following transitions: m/z 453 (MH⁺ of M3-OH) 289 and m/z 329 (MH⁺ of labetalol) 207.

A panel of previously characterized cDNA-expressed dog P450s (CYP 1A1, 3A12, 3A26, 2B11, 2C21, 2C41, and 2D15) was obtained from Merck Research Laboratories (West Point, PA) and were used to examine the metabolism of I in vitro (Shou et al., 2003). Recombinant dog CYP1A2, cloned and expressed as described above, also was evaluated as a catalyst for the turnover of I. Experiments were performed by incubating 100 pmol of each recombinant P450 enzyme with 2 µM I in 100 mM potassium phosphate buffer (pH 7.4) fortified with 1 mM NADPH (1 ml total volume). The samples were incubated for 60 min at 37°C in a shaking water bath, and the reactions were terminated with acetonitrile and prepared for LC-MS/MS analysis of M3-OH as described above.

ANF (0.01–1 µM) was used to assess the contribution of CYP1A to the formation of M3-OH in dog liver microsomes. The incubates (1 ml total volume) contained 100 mM potassium phosphate buffer (pH 7.4), 1 mg/ml liver microsomal protein, 2 µM compound I, and varying concentrations of ANF. The reactions were initiated by the addition of NADPH (1 mM) and were allowed to proceed for 120 min at 37°C in a shaking water bath. Incubations without inhibitor served as control. The reactions were terminated with acetonitrile and prepared for LC-MS/MS analysis of M3-OH as described above.

Microsomes were prepared from both the pre- and post-treatment dog liver biopsy samples using standard laboratory procedures (homogenization in buffer followed by differential centrifugation). Marker activities of dog CYP1A (EROD) and 3A12 (testosterone 6ß-hydroxylase) were determined using methods described previously (Shou et al., 2003). Briefly, 7-ethoxyresorufin (5 µM) or testosterone (320 µM) was incubated with the liver microsomes (0.1 mg/ml protein) in NADPH-fortified (1 mM) 100 mM phosphate buffer (pH 7.2) in a shaking water bath at 37°C for 5 to 10 min. Incubations containing solvent alone served as controls. After the incubations, the reactions were terminated by the addition of acetonitrile, and product formation rates were determined for either resorufin (fluorometric detection) or 6ß-hydroxytestosterone (LC-MS/MS).

Dog Cytochromes P450 ELISA. The microsomes prepared from the liver biopsies of either control dogs or dogs treated with I were evaluated for levels of CYP1A, CYP2B11, CYP2C21, and CYP3A12 proteins using a commercially available ELISA kit (Daiichi Pure Chemicals Co., Tokyo, Japan). Briefly, microtiter plates were coated (n = 3 for each sample) with microsomal protein (0.003–3.0 µg/100 µl) in phosphate-buffered saline (PBS) overnight at 4°C. The plates then were blocked with PBS containing 1% bovine serum albumin for 2 h at room temperature. Each plate then was incubated with anti-dog CYP1A, anti-dog CYP2B11, anti-dog CYP2C21, or anti-dog CYP3A12 in PBS containing 0.1% bovine serum albumin for 1 h at 37°C. After washing with PBS containing 0.2% Tween 20, the plates were incubated with horseradish peroxidase-labeled anti-rabbit IgG for 1 h at 37°C. Subsequently, the plates were incubated for 10 min with 150 µg/ml 3,3',5,5'-tetramethylbenzidine in 0.2 M Na₂HPO₄ buffer (pH 4.5) containing 0.1 M citric acid. The colorimetric reaction was terminated by the addition of 2 N H₂SO₄, and the plates were analyzed by UV-visible spectrophotometry at 450 nm. The amount of each enzyme was calculated by comparison to microsomes of known enzyme levels as per the manufacturer's instructions.

In Vitro Induction Studies in Primary Dog Hepatocytes. A small lobe (35–70 g) was removed from fresh male dog (beagle) liver acquired from Laboratory Animal Resources within Merck and Co., Inc., and hepatocytes were isolated using a two-step hepatocyte isolation procedure described by Strom et al. (1982). Hepatocytes were dissociated from undigested tissue with hepatocyte wash medium, and the cell suspension was centrifuged (500g) for 5 min. The resulting cell pellet was resuspended in hepatocyte wash medium containing 30% Percoll (90% isotonic) and centrifuged (1000g) for 5 min to remove nonparenchymal and nonviable cells. The hepatocytes were suspended in hepatocyte attachment medium supplemented with 5% fetal bovine serum and plated onto 24-well Collagen I-coated dishes at a concentration of 0.25 x 10⁶ cells/well. Cultures were maintained in a humidified incubator at 37°C with a 95:5 air to CO₂ mixture. After a 2- to 4-h period, the attachment medium was replaced with hepatocyte culture medium (Williams' Medium E) supplemented with insulin/transferrin/selenium premix, dexamethasone (1 µM), nonessential amino acids, penicillin, streptomycin, and glutamine. After 24 h in Williams' Medium E, the cells were rinsed with fresh medium containing either compound I (0.2, 2, and 20 µM) or BNF (25 µM). After 24 h of exposure, the medium was replaced with fresh medium containing the test compounds. The medium was removed after a second 24 h of exposure, and the cells were washed two times with PBS and either immediately frozen at –70°C until processed for total RNA or assayed for EROD activity. The EROD activity was evaluated by incubating the cells for 15 min at 37°C in the presence of 7-ethoxyresorufin (8 µM), followed by measurement using a fluorometer. Total RNA was isolated using an RNEasy 96 kit with a BioRobot 3000 (QIAGEN). The RNA was treated with DNase I and eluted with 110 µl of deionized water, and then was assayed using a RiboGreen RNA Quantitiation Reagent kit (Molecular Probes, Eugene, OR). The RNA for specific genes (CYP1A1 and 1A2) was quantified using TaqMan technology according to the manufacturer's guidelines (Applied Biosystems). The primers and probes were designed using Primer Express Software v.1.0 (Applied Biosystems) and GenBank accession numbers L-77459 and CQ840744 [GenBank] , respectively. The primers and probes were optimized and validated using cDNA templates for each specific enzyme.

In addition, a lactate dehydrogenase (LDH) assay was performed on the hepatocytes exposed to compound I. An LD Kinetic kit was purchased from Thermo Electron and was used according to the manufacturer's instructions. In addition to the solvent control group, hepatocytes were treated with CellLytic Express (Sigma-Aldrich) to demonstrate the maximum degree of LDH release by cell death under the experimental conditions.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Pharmacokinetics. The plasma pharmacokinetics of I on days 1, 7, and 14 are summarized in Table 1, and the mean plasma concentration-time profiles are shown in Fig. 2. There was an approximately 2.5-fold decrease in the mean AUC₀ of I on days 7 and 14, relative to day 1. Consequently, assuming that there were no changes, over this period, in the oral bioavailability, the mean apparent total body plasma clearance was increased on days 7 and 14, relative to day 1. There was an approximately 33% reduction in the apparent harmonic mean terminal half-life on days 7 and 14, relative to day 1.

View larger version (15K): [in this window] [in a new window]   FIG. 2. Mean (±S.D.) plasma concentration-time profile of compound I on days 1, 7, and 14 after daily 6 mg/kg oral administration to male bile duct-cannulated beagle dogs (n = 3). denotes n = 2 for those time points.

Metabolism. Representative radiochromatograms from bile, collected from 0 to 6 h after administration on days 1 and 14, are shown in Fig. 3. Two of the metabolites resulted from oxidation of the piperazine ring: M5 was the product of N-oxidation and M4A was the result of piperazine ring cleavage (Fig. 4). The identification of M5 and M4A was confirmed by comparing the chromatographic retention times and collision-induced dissociation spectra of authentic standards to those of the metabolites. Metabolites M1 and M3 were sulfate conjugates of the hydroxylated metabolites M1-OH and M3-OH, respectively (Fig. 4). The phenol precursors to M1 (M1-OH) and M3 (M3-OH) were not observed in the excreta of the dogs, indicating rapid and efficient sulfation in vivo. NMR studies, using material that had been isolated and purified from the bile of dogs treated with [¹⁴C]I, established the sites of hydroxylation of M1-OH and M3-OH as being the 6- and 8-positions, respectively, of the quinolone ring. The H4 singlet appeared at 8.40 ppm in the metabolite M1-OH. Four resonances with appropriate scalar couplings were observed for H5, H6, H7, and H8 spin systems at 7.81, 7.34, 7.58, and 7.43 ppm, respectively in the one-dimensional spectrum of I. In the M1-OH spectra, only three resonances were observed: 7.13 (d, 2.5 Hz), 7.10 (dd, 9.0, 2.5 Hz), and 7.28 (d, 9.0 Hz). The nuclear Overhauser enhancement difference spectrum obtained by irradiating H4 (at 8.40 ppm) showed positive nuclear Overhauser enhancement peaks at 7.30 ppm (singlet, H3' of indole ring) and 7.13 ppm (d, 2.5 Hz). In M3, the NMR data indicated that the sulfate was at carbon 8 of the quinolone ring. As in M1-OH, H4 appeared practically unchanged at 8.51 ppm. Only three additional resonances were observed for the quinolone ring: 7.28 (dd, both 8.2 Hz), 7.60 (d, 8.2Hz), and 7.65 (d, 8.2Hz). Metabolite M3 rotating-frame Overhauser enhancement spectroscopy (data not shown) revealed cross peaks from H4 (8.51 ppm) to 7.28 ppm (H3' of indole) and the doublet at 7.60 ppm (H5).

View larger version (17K): [in this window] [in a new window]   FIG. 3. Representative radiochromatograms of dog bile (0–6 h postdose) on days 1 and 14 after daily 6 mg/kg oral administration of compound I, showing an increase in the formation of M3.

View larger version (14K): [in this window] [in a new window]   FIG. 4. Metabolic pathways of compound I in the dog.

The radiochromatograms shown in Fig. 3 suggested that the sulfate conjugate M3 was present in the dog bile in higher relative amounts after multiple dosing. Values for the biliary metabolic clearance of the sulfate conjugates M1 and M3 were calculated for the 24-h time interval following administration of I (Table 2). There was a statistically significant (P < 0.05) increase (4- to 24-fold) in the metabolic clearance of M3 on day 14, relative to day 1, whereas the corresponding increase for M1 was not significant (2-fold), suggesting that the clearance of compound I to M3-OH had been induced. There was insufficient radioactivity recovered in the urine of dogs administered [¹⁴C]I to allow for a metabolite analysis. Therefore, the bile was considered to be the only matrix available to evaluate changes in the metabolic profile of I following multiple administration. Moreover, the parent drug accounted for the vast majority of the radioactivity in the plasma following administration of [¹⁴C]I, which suggested that the presence of significant levels of circulating metabolites was unlikely.

In Vitro Reaction Phenotyping. All of the oxidative metabolites of compound I (M1-OH, M3-OH, M4A, M5, and M7) were observed in the dog liver microsomal and recombinant P450 incubates; however, only the formation of M3-OH was studied extensively (data not shown). Of the recombinant dog P450 enzymes studied, only four enzymes (CYP1A1, CYP1A2, CYP2D15, and CYP3A12) catalyzed the turnover of I (data not shown). Furthermore, only CYP1A1 and CYP1A2 led to the formation of detectable amounts of M3-OH (Fig. 5). The involvement of CYP1A1/2 in the metabolism of I to M3-OH was supported further by experiments using chemical inhibitors in dog liver microsomal incubates in which the formation of M3-OH was inhibited using ANF (CYP1A-selective) (Fig. 6). Furthermore, following incubation of I with BNF-induced dog liver microsomes, the formation of M3-OH demonstrated a statistically significant (P < 0.05) 8-fold increase relative to that in liver microsomes obtained from control dogs (Fig. 7). In contrast, microsomal preparations from dogs treated with rifampin, clofibrate, or phenobarbital did not afford significantly greater amounts of M3-OH relative to their respective controls.

View larger version (12K): [in this window] [in a new window]   FIG. 5. Formation of M3-OH from compound I after a 60-min incubation in the presence of various recombinant dog P450 enzymes. The data are expressed as mean peak area ratio (analyte/internal standard) ± standard deviation (n = 4).

View larger version (11K): [in this window] [in a new window]   FIG. 6. Formation of M3-OH from compound I after a 2-h incubation in dog liver microsomes (1 mg/ml) in the presence of varying concentrations of BNF. The data are expressed as mean values (n = 2) relative to control incubations lacking inhibitor.

View larger version (11K): [in this window] [in a new window]   FIG. 7. Formation of M3-OH from compound I in the presence of liver microsomes (1 mg/ml) prepared from dogs treated with prototypical P450 inducers [BNF, rifampin, phenobarbital (PB), clofibric acid]. The data are expressed as mean peak area ratios (analyte/internal standard) ± standard deviation (n = 3). , statistically significant increase relative to control (P < 0.05).

In Vivo Enzyme Induction Study. Marker activities of CYP1A (EROD) and CYP3A12 (testosterone 6ß-hydroxylase) were evaluated using microsomes prepared from the dog liver biopsy samples. There was a significant increase (approximately 6.5-fold) in the EROD activity ratio (day 14 activity versus predose) in the microsomes from all the dogs treated with I, consistent with induction of CYP1A (Fig. 8). In contrast, there was no apparent change in the testosterone 6ß-hydroxylase activity ratio (day 14 versus predose), which suggested that compound I did not affect the activity of CYP3A12 in vivo. Similarly, there was no apparent change in either marker activity ratio from the microsomes of vehicle-treated dogs.

View larger version (13K): [in this window] [in a new window]   FIG. 8. Day 14 versus predose marker activities (EROD and testosterone 6ß-hydroxylase) in liver microsomes prepared from dogs receiving daily oral doses of either 6 mg/kg compound I or vehicle. , statistically significant increase relative to vehicle control (P < 0.05).

The microsomes prepared from the biopsy samples also were assayed for protein levels of CYP1A, CYP2B11, CYP2C21, and CYP3A12 using a dog-specific ELISA. There was a significant (P < 0.05) increase (approximately 3.5-fold) in the CYP1A protein ratio (day 14 versus predose) in the microsomes of dogs treated with I when compared with vehicle-treated animals (Fig. 9). In contrast, no significant change in the protein level ratios of CYP2B11, CYP2C21, or CYP3A12 in the microsomes of dogs treated with I was observed.

View larger version (15K): [in this window] [in a new window]   FIG. 9. ELISA results showing day 14 versus predose levels of CYP1A1/2, 2B11, 2C21, and 3A12 protein in liver microsomes prepared from dogs receiving daily oral doses of either 6 mg/kg compound I or vehicle. , statistically significant increase relative to vehicle control (P < 0.05).

	Discussion

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The multiple-dose pharmacokinetics of I in the dog show a trend for decreasing systemic exposure to parent drug as a function of time (Table 1). There also was a decrease in the apparent terminal half-life of I on days 7 and 14 relative to day 1, which suggested that there was a higher rate of elimination following multiple dosing. The changes in the multiple-dose pharmacokinetics of I were consistent with what would be anticipated from a compound that was inducing an enzyme(s) that was responsible for its clearance.

Studies of [¹⁴C]I in dogs established that the compound was extensively eliminated by metabolism, with less than 5% of the administered dose being excreted unchanged into the urine and bile (data not shown). The compound was primarily metabolized by aromatic hydroxylation followed by sulfation to form two metabolites (M1 and M3). Since compound I was predominantly eliminated by metabolism, it could be anticipated that if it was an autoinducer, the fraction of the dose being metabolized by different pathways might change and could provide some insight as to what enzyme(s) was being affected. Radiochromatographic mass-spectrometric analysis of the bile suggested that the sulfate conjugate M3 was present in higher amounts, relative to day 1, after 14 days of dosing. The biliary metabolic clearance of the sulfate conjugates M1 and M3 were calculated for the 0- to 24-h interval after administration on both days 1 and 14. Assuming that the formation of the phenol M3-OH was the rate-limiting step in this pathway, changes in the metabolic clearance of the sulfate conjugates would represent changes in the clearance of parent drug (I) to the phenol metabolites. There was marked increase in the metabolic clearance of M3 on day 14, relative to day 1, which indicated that the enzyme(s) responsible for formation of M3-OH in the dog may have been induced.

A series of in vitro experiments then was conducted to determine what enzyme(s) was responsible for the formation of M3-OH from I in the dog. The formation of M3-OH was significantly inhibited in dog liver microsomal incubates of I in the presence of the prototypical CYP1A inhibitor BNF, suggesting that CYP1A may play a role in the formation of M3-OH. Likewise, there was a significant (8-fold) increase in the formation of M3-OH, relative to control, when compound I was incubated with microsomes from dogs treated with BNF, which further indicated the involvement of CYP1A1/2. Furthermore, when compound I was incubated with various dog recombinant P450 enzymes, only CYP1A1 and 1A2 were capable of forming M3-OH. It was therefore concluded, based on the results of in vitro studies, that CYP1A enzymes were responsible for the formation of M3-OH in the dog.

Once the P450 enzymes responsible for the formation of M3-OH had been identified, in vitro studies were conducted using dog primary hepatocyte models to confirm the induction of CYP1A1/2. The use of primary hepatocytes has shown great utility in the ability to model the induction of P450 enzymes in vitro (Silva et al., 1998; Silva and Nicoll-Griffith, 2002). Both CYP1A1 and CYP1A2 mRNAs were detectable in the livers of untreated dogs, albeit at low levels, suggesting some degree of constitutive expression, and both enzymes appear to be highly inducible by polychlorinated biphenyls and BNF (Uchida et al., 1990; Graham et al., 2002). However, when varying concentrations of compound I were incubated in the presence of dog primary hepatocytes for 48 h, no functional or transcriptional induction of CYP1A1/2 was observed. In fact, there was a dose-dependent suppression of CYP1A1 and CYP1A2 mRNA in the hepatocytes treated with compound I. As anticipated, control incubations containing BNF induced both mRNA expression (CYP1A1 and CYP1A2) and EROD activity. A limited data set, which examined enzyme induction following both 24 and 48 h of treatment, demonstrated that the suppression of CYP1A1/2 mRNA was observable after 24 h, suggesting a relatively rapid and sustained effect (data not shown). The results from an LDH assay on the hepatocytes suggested that there was no significant cytotoxic effect of compound I, indicating that the reason for the dose-dependent decrease in CYP1A1/2 transcription was not due to cell death.

Since experiments using primary hepatocyte models of enzyme induction did not correlate with the results obtained from other studies, an additional confirmatory in vivo study was conducted utilizing a liver biopsy approach. Previous reports, using heterologously expressed dog P450 enzymes and dog hepatocyte models, have suggested the use of EROD and testosterone 6ß-hydroxylase activities as a marker for CYP1A1 and CYP3A12, respectively (Graham et al., 2002; Shou et al., 2003). Additionally, dog CYP1A2 was heterologously expressed in our laboratory and has been shown to catalyze EROD activity (data not shown). There was a significant increase (6.5-fold relative to predose) in microsomal EROD activity in all dogs treated with I, indicating induction of CYP1A1/2. However, assessment of microsomal testosterone 6ß-hydroxylase activity from the same dogs suggested that CYP3A12 was not affected. Furthermore, ELISA analysis of the microsomes from the dogs treated with I showed a significant increase in CYP1A1/2 protein, relative to vehicle controls. ELISA analysis of recombinantly expressed dog CYP1A1 and CYP1A2 indicated that the antibody used for the protein determination was nonspecific and cross-reacted with both enzymes. None of the protein levels for the other enzymes examined showed an increase relative to vehicle control.

Compound I was an autoinducer in the dog since it was both a substrate and inducer of CYP1A. Surprisingly, we were not able to verify the induction of CYP1A either by mRNA or enzyme activity in the dog primary hepatocyte model. In fact, there was a troubling suppression of CYP1A1 and CYP1A2 mRNA and a trend for loss of EROD activity when compound I was incubated with dog primary hepatocytes. The reason for the suppression of CYP1A expression in vitro is unclear but is under investigation and may involve an off-target kinase inhibition. It should be noted that although in vitro models can be extremely useful in screening compounds for their ability to induce drug-metabolizing enzymes, false-negative results remain a clear possibility and may require further in vivo investigation. These studies highlight the need to consider the results from a variety of in vitro and in vivo experimental systems in deriving a comprehensive understanding of the metabolic fate of a drug candidate.

	Acknowledgments

 
We thank Dr. Brian Carr for assistance in preparing the microsomes from the in vivo dog enzyme induction study. We also thank Dr. David Gilberto and the Merck West Point Laboratory Animal Research staff for their efforts during the in vivo dog enzyme induction study.

	Footnotes

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

doi:10.1124/dmd.105.003913.

ABBREVIATIONS: KDR, kinase insert domain-containing receptor; ANF, -naphthoflavone; BNF, ß-naphthoflavone; DMSO, dimethyl sulfoxide; LC-RAM, liquid chromatography with radiochemical detection; LC-RAM-MS/MS, liquid chromatography with tandem radiochemical and mass spectrometry; HPLC, high-pressure liquid chromatography; ESI, electrospray ionization; EROD, ethoxyresorufin O-deethylase; ELISA, enzyme-linked immunosorbent assay; CL_M, apparent metabolic clearance; CL_T, apparent total body plasma clearance; PBS, phosphate-buffered saline; LDH, lactate dehydrogenase; AUC, area under the curve.

¹ Current address: Regulatory Affairs International, Merck Research Laboratories, Blue Bell, PA.

² Current address: Barrier Operations, Merck Manufacturing, West Point, PA.

³ Current address: RAS Analytical, Merck Research Laboratories, West Point, PA.

⁴ Current address: Drug Metabolism, Amgen, Thousand Oaks, CA.

⁵ Current address: MAPK, Bristol Myers Squibb, Princeton, NJ.

Address correspondence to: Dr. Christopher R. Gibson, Department of Drug Metabolism, Merck Research Laboratories, WP75A-203, P.O. Box 4, West Point PA, 19486. E-mail: Christopher_Gibson{at}merck.com

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