Abstract
Compound I (1-(3-chlorophenyl)-4-[(1-(4-cyanobenzyl)-1H-imidazol-5-yl)methyl]piperazin-2-one) is a potent and selective inhibitor of farnesyl-protein transferase ( FPTase ). The pharmacokinetics and metabolism of compound I displayed species differences in rats and dogs. After oral administration, the drug was well absorbed in dogs but less so in rats. Following i.v. administration, compound I was cleared rapidly in rats in a polyphasic manner with a terminalt1/2 of 41 min. The plasma clearance (CLp) and volume of distribution (Vdss) were 41.2 ml/min/kg and 1.2 l/kg, respectively. About 1% of the dose was excreted in rat bile and urine as unchanged drug over a period of 24 h, suggesting that biotransformation is the major route of elimination of compound I . Using liquid chromatography (LC)-tandem mass spectometry, nineteen metabolites of compound I were identified in urine and bile from dogs and rats. Structures of two major metabolites were confirmed by LC-NMR. N-Dealkylation and phase II metabolism were the major metabolic pathways. Animal and human liver microsomal intrinsic clearance values were scaled to predict hepatic clearance and half-life in humans, and the predicted values were in good agreement to the in vivo data.
CompoundI1 is a selective and potent inhibitor of farnesyl-protein transferase (IC50 of 2 nM) that has been evaluated clinically for the treatment of cancer. FPTase is believed to be an appropriate target in cancer therapy because several mammalian proteins that contribute to the regulation of cell replication require post-translation modification by FPTase to achieve biological activity (Gibbs et al., 1997; Gibbs and Oliff, 1997;Kohl et al., 1998; Lobell and Kohl, 1998). Farnesyl transferase inhibitors as anticancer agents were developed based on the assumption that cellular transformation by Ras oncoproteins requires posttranslational modification of farnesylation catalyzed by FPTase (Gibbs, 2000).
This report describes the pharmacokinetics and metabolic fate of14C-labeled compound I in bile duct-cannulated rats and dogs, the two species used in the toxicological evaluation of this compound. In addition, data from animal and human liver microsomal intrinsic clearance values were scaled to predict hepatic clearance and half-life in humans and compared with the in vivo data.
Materials and Methods
Chemicals.
All chemicals and solvents were of HPLC or analytical grade. Water was distilled and purified through a Milli-Q reagent system (Millipore Corp., Bedford, MA). Ultima Gold (liquid scintillation counting), Carbo-sorb E, and Permaflour E+ (for combustion of samples) were obtained from Packard Instrument Co. (Meriden, CT). β-Glucuronidase (from Helix pomatia, type H-1 with sulfatase activity) was obtained from Sigma Chemical Co. (St. Louis, MO).14C-labeled I and some of its metabolites (M4, M8, M10,M11, and M14) were prepared at Merck Research Labs. The 14C label was incorporated at the cyano-moiety (Fig. 1), having a specific activity of 22.4 μCi/mmol and a radiochemical purity of >98% as determined by radio-HPLC. All other chemicals were purchased from commercial sources and were of the highest purity available.
Animal Studies.
Rats
Male Sprague-Dawley rats (n = 3), weighing approximately 300 g, had an indwelling cannula implanted in the right jugular vein for drug administration and blood sampling. The surgery was performed under light pentobarbital anesthesia (40 mg/kg i.p.) 1 day before the experiment. During the study, all animals were housed in individual metabolism cages and were unrestrained throughout the experiment. Following an overnight fast, a 10-mg/kg dose was given by oral gavage and a 5-mg/kg dose by bolus intravenous injection. Oral bioavailability was determined using a solution of free base in 0.05 M citric acid, and the intravenous dose was administered as a saline solution. Blood samples were obtained serially at selected time points after drug administration via the jugular vein cannula. After centrifugation, the resultant plasma was stored frozen at −20°C.
The biliary and renal excretion of 14C-labeled compound I and its metabolites were assessed in bile duct-cannulated rats (n = 3). Following i.v. administration of a 10-mg/kg dose (solution in saline), bile and urine were collected over ice for 72 h and were stored frozen at −20°C.
Dogs.
Oral bioavailability of a 10-mg/kg oral dose was assessed in fasted beagle dogs (n = 3) using a solution formulation in 0.05 M citric acid. Dose proportionality studies were conducted by administering compound I i.v. at doses of 2-, 5-, and 10-mg/kg doses of compound I as a solution in saline. For assessment of excretion and metabolic pathways, bile and urine were collected from fasted, male, bile duct-cannulated beagle dogs (n = 3). An i.v. dose was administered at 10 mg/kg in saline containing compound I14C radiotracer at a final specific activity of 0.89 μCi/mg. Bile and urine were collected up to 72 h postdose and were kept at −20°C until analysis.
Pharmacokinetic Calculations.
Area under plasma concentration-time curve (AUC) was determined by linear-log trapezoidal interpolation. The apparent half-life was estimated from the log-linear portion of the slope of time-concentration profiles. The apparent volume of distribution (Vdss) of the drug was calculated by the following equation:
Bioanalytical.
Quantitation of compound I in plasma by LC-MS/MS
Concentrations of compound I in plasma were determined by a LC-MS/MS method following a protein precipitation procedure. An aliquot of the extract was analyzed by LC-MS/MS in the positive ion mode using an SCIEX API III triple quadrupole mass spectrometer (PE SCIEX, Concord, ON, Canada) equipped with a heated nebulizer interface using a discharge current of 3 μA and an orifice potential of 35 V. The heated nebulizer was maintained at 450°C. The instrument was operated in the selected reaction monitoring mode. The precursor and product ions of compound I were 406 to 195. The linear range for the assay was 10 to 2500 ng/ml.
Radiochemical analysis.
Quantitation of radioactivity was achieved by liquid scintillation counting of samples in a Beckman LS5601 liquid scintillation counter (Beckman Coulter, Inc., Fullerton, CA) with external quench correction. Aliquots of urine and bile were assayed by direct addition to 15 ml of Ready-Safe (Beckman) scintillation cocktail.
LC-MS-radiometric analysis.
Metabolite profiles in urine and bile were determined using a Finnigan LCQ mass spectrometer (Thermo Finnigan, San Jose, CA) coupled to an inline radioactivity flow detector that allowed simultaneous mass spectral and radioactive detection. The instrument was operated in the electrospray ionization mode with a 5-kV potential, and the transfer capillary temperature was maintained at approximately 200°C. The chromatographic column was a BDS Hypersil C18column (4.6 × 150 mm, 5-μm particle size; Keystone Scientific, Bellefonte, PA). A linear gradient commenced with an initial concentration of 95% solvent A (5 mM ammonium acetate, pH 4.5) and 5% solvent B (acetonitrile) and increased to 10% A over a period of 18 min at a flow rate of 1 ml/min. The samples were centrifuged (4000 rpm × 10 min), filtered through a 0.22-μm filter and injected directly on the HPLC system. The column effluent was split such that approximately 0.1 ml/min was introduced into the ion trap mass spectrometer equipped with an electrospray ionization source, and the remainder (0.9 ml/min) was mixed with 3 flow volumes of the Ultima M scintillation cocktail before detection by a Packard Radiometric Detector (515 TR; Packard Instruments Co.).
LC-NMR.
A linear gradient consisted of 95% solvent A (D2O), 5% solvent B (acetonitrile), and increased to 25% A in over a period of 25 min and then to 5% A from 25 to 35 min at a flow rate of 1 ml/min using a BDS Hypersil C18 column (4.6 × 150 mm). The eluent was monitored by UV at 235 nm with a Varian 9065 photodiode array UV detector (Varian, Palo Alto, CA).
A Varian Inova 500-MHz NMR instrument equipped with a1H and a 13C pulse field gradient indirect detection microflow LC-NMR probe (flow cell 60 μl; 3-mm diameter) was used. Reverse-phase HPLC of the samples was carried out on a Varian modular HPLC system comprising of a Varian 9012 pump and a Varian 9065 photodiode array UV detector. The2H resonance of the D2O was used for field-frequency lock, and the spectra were centered on the acetonitrile methyl resonance (1.94 ppm). Suppression of resonances from HOD (partially deuterated water), methyl of acetonitrile, and its two 13C satellites was accomplished using a train of four selective water suppression enhanced through T1 effect pulses, each followed by a Bo gradient pulse and a composite 90-degree read pulse was used (Smallcombe et al., 1995).1H NMR spectra were acquired in a stop-flow mode using the UV signal to trigger peak detection. After peak detection and a time delay of about 33 s, the HPLC pump was stopped, trapping the peak of interest in the LC-NMR microprobe. 1H NMR stop-flow spectra were acquired using an acquisition time of 1.5 s, a spectral width of 9000 Hz, and 32,768 time-domain data points. Nuclear Overhauser effect (NOE) experiments were conducted on a Varian Unity 400 MHz NMR instrument.
In Vitro Metabolism.
To determine the intrinsic clearance required for the prediction of hepatic clearance, varying concentrations of compound I were incubated with liver microsomes (2 mg/ml protein) from rats, dogs, monkeys, and humans at 37°C for up to 60 min. Reactions were stopped by the addition of 2 volumes of acetonitrile, and the disappearance of parent drug was determined by LC-MS/MS. Intrinsic clearance was calculated from the ratio ofVmax/Km (well stirred hepatic model), scaled up to whole liver, and this value was applied, in conjunction with the experimentally determined unbound fraction in blood, to calculate hepatic clearance using the equations below (Wilkinson and Shand, 1975; Lin et al., 1996; Lin, 1997).
Plasma protein binding and blood to plasma distribution ratio.
The binding of 14C-labeled compound Ito human, dog, rat, and monkey plasma protein was determined by an ultrafiltration method. Compound I was added to plasma to yield a final concentration of 40 μg/ml. The drug solution was added in a volume equal to or less than 1% of the plasma volume. Following incubation of the plasma samples at 37°C for 15 min, an aliquot of plasma (1 ml) was immediately transferred to a centrifuge tube (Amicon Co., Danvers, MA). The tube was then centrifuged at 1500gfor 30 min at 37°C; the ultrafiltrates were removed and analyzed by liquid scintillation counting. The free drug fraction was determined by dividing the amount (dpm) of drug in the ultrafiltrate by the amount in the original plasma sample. The percentage of protein bound values were not corrected for nonspecific binding (<5%). The equilibrium blood to plasma distribution ratio of14C-labeled compound I (2 μM) was determined at 37°C in freshly drawn rat, dog, monkey, and human blood after incubation for 15 min. Total radioactivity in whole blood and plasma was determined by combustion of the respective samples using a Packard 307 Oxidizer (Packard Instruments Co.).
Combined β-glucuronidase/sulfatase enzyme hydrolysis.
Urine and bile samples (0.1 ml) were added to 0.9 ml of sodium acetate buffer (pH 5, 0.1 M) and treated with 2000 units of β-glucuronidase/sulfatase, and the mixture was incubated at 37°C overnight (Shimada et al., 1997). The reaction was stopped by adding 2 ml of acetonitrile and centrifuged at 4000 rpm. The supernatant was evaporated under nitrogen and reconstituted in 0.2 ml of the HPLC mobile phase (95% ammonium acetate and 5% acetonitrile). Incubation of urine and bile samples without the enzymes served as controls.
Results
Absorption and Pharmacokinetics.
Following i.v. administration (5 mg/kg), compound I was cleared rapidly in rats in a polyphasic manner with a terminal half-life of 41 min. The plasma clearance (CLp) and Vdss were 41.2 ml/min/kg and 1.2 l/kg, respectively (Table 1). Less than 2% of the dose was excreted in rat bile and urine as unchanged drug over a period of 24 h, indicating that biotransformation was the major route of elimination of compound I. When the drug was administered orally as a solution in 0.05 M citric acid at a dose of 10 mg/kg, the absorption was rapid; the plasma concentration of compoundI (Cmax, 7.36 μM) was reached in about 16 min, and the bioavailability was 45% (Table 1).
Following i.v. dosing to dogs, pharmacokinetic parameters of compoundI appeared to be independent of the dose over the dose range of 2 to 10 mg/kg. The plasma clearance (10 ml/min/kg) was low relative to the hepatic blood flow in dogs, and the i.v. terminal half-life was about 35 min. Following an oral administration to dogs, the absorption of compound I was complete. In dogs, the time to reach peak plasma concentration (Tmax) was 33 min, and the mean Cmax, AUC, and bioavailability were 22.8 μM, 40 μM · h, and 100%, respectively (Table 1).
Excretion.
Following i.v. administration of the radiolabeled I, most of the radioactivity was recovered within 72 h postdose in rats and dogs (Table 3). In rats, about two-thirds of the dose was excreted in bile and about a third of the dose in urine, whereas in the case of dogs, about 60% of the dose was excreted in urine and 29% was recovered in bile. In both species, the parent drug accounted for less than 2% of the recovered dose.
Plasma Protein Binding and Blood to Plasma Partition Ratio.
The fraction unbound was 33.6, 32.5, and 30.9% in the rat, dog, and human plasma, respectively, indicating that I was not bound extensively to plasma proteins. The mean equilibrium blood-to-plasma concentration ratio in rats, dogs, and humans at the therapeutically relevant concentration of 2 μM was 0.83, 0.53, and 0.68. This modest extent of distribution into red blood cells indicated that plasma clearance underestimates slightly the blood clearance.
Prediction of Human Hepatic Clearance.
Using the well stirred model (Wilkinson and Shand, 1975; Lin et al., 1996; Lin, 1997), hepatic clearance was calculated for laboratory animals and humans from in vitro metabolic data and the experimentally determined unbound fraction in plasma. The predicted clearance values based on the in vitro data were in good agreement with the observed in vivo plasma clearance in dogs and monkeys, whereas it was lower in case of rats (Table 2). Using the same approach with three different pools of human liver microsomes, the clearance of compound I in humans was predicted to be low, with a mean value of 3 ml/min/kg. In humans, provided that compoundI is cleared primarily by hepatic metabolism exhibiting monoexponential pharmacokinetic behavior, and assuming that the distribution volume in humans to be within the range estimated in rats, dogs, and monkeys (0.6–1.2 l/kg), the corresponding half-life was predicted to be in the range of 2.3 to 4.6 h.
Metabolism.
Radiolabeled components of urine and bile were analyzed for unchanged compound I and its metabolites using tandem LC-MS-radiometric detection. Representative radiochromatograms from rat and dog urine and bile are shown in Fig.2. The radiochromatograms indicate presence of minor amounts of unchanged I (<2%) and about 19 metabolites in urine and bile (Table3).
Detailed LC-MS/MS and LC-NMR spectroscopic analyses of the parent compound I was carried out to facilitate the identification of the structures of its metabolites. Full scan mass spectrum ofI showed a prominent protonated molecular ion atm/z 406 Da, which upon CAD resulted in a base ion at m/z 326 (Fig.3). The ion at m/z326 was proposed to arise via an unusual exclusion of the imidazole moiety, and the mechanism of this rearrangement is rationalized in Fig.3. Further fragmentation of the ion at m/z 326 yielded a base peak at m/z 298, which arises via loss of CO molecule, which upon further fragmentation resulted in dissociation between the 4-cyanobenzyl and the modified piperazin-2-one moiety. The product ion spectrum of m/z 408 (containing 37Cl isotope) resulted in a base ion at m/z 328, which corroborates the proposed fragmentation pattern.
NMR analysis was conducted on the synthetic standard of compoundI (Table 4). The aromatic region of the 1H NMR spectrum of Iindicated four protons having an AB system (δ 7.64 and 7.20), suggesting a p-substituted aromatic ring. The two broad aromatic singlets (δ 7.79 and 6.96) were assigned to the imidazole ring. The chemical shift and splitting pattern of the remaining four aromatic protons were indicative of the 1,3-disubstituted aromatic ring with chlorine as one of the substituents (δ 7.35 t, 7.28bs, 7.15 d, and 7.04 d).
Metabolite M1 had a retention time of about 4.5 min and was found in urine and bile of rats and dogs. The electrospray ionization negative ion spectrum resulted in a deprotonated molecular ion atm/z 203. A product ion spectrum of the ion atm/z 203 showed fragment ions atm/z 159 (loss of methylcarboxylic acid) and atm/z 131 (loss of glycine). The structure ofM1 was assigned as the glycine conjugate of 4-cyanobenzoic acid (Table 4).
Metabolites M2 and M3 have retention times of 5.1 and 7.4 min on HPLC and were detected in rat and dog bile, whereasM2 was detected only in dog urine (Fig. 2). MetabolitesM2 and M3 yielded protonated molecular ions atm/z 390 and 404, respectively, and CAD of these ions resulted in base ions at m/z 214 and 228 Da, respectively, which arise from loss of 176 Da (loss of dehydroglucuronic acid). Subsequent fragmentation of the base-ions on m/z 214 and 228 yielded spectra similar to those of M4 and M8, suggesting thatM2 and M3 are glucuronide conjugates ofM4 and M8, respectively.
Metabolites M4 and M8 had retention times of 5.7 min and 13.9 min, respectively, and were detected in rat and dog urine and bile. Metabolites M4 and M8 had protonated molecular ions at m/z 228 and 214, respectively. Upon CAD of the ions at m/z 228 and 214, the product ion spectrum of M4 showed a prominent ion atm/z 184 (—CO2), suggesting the presence of a carboxyl group. Additional ions were observed at m/z 113 and 116, resulting from cleavage between benzylic position and imidazole ring, and subsequent loss of water from m/z 113 resulted in ion atm/z 95. The CAD spectrum of ion atm/z 214 (M8) resulted in a base ion atm/z 196 (M18), suggesting a presence of a hydroxy group. The LC-MS/MS fragmentation pattern and retention times of the metabolites M4 and M8 were compared with those of the synthetic standard 1-[(4-cyanophenyl)methyl]imidazole-5-carboxylic acid and 4-{[5-(hydroxymethyl)imidazolyl]methyl}benzenecarbonitrile derivatives, respectively, and were found to be identical.
Metabolite M17 had a retention time of about 28.9 min and was found in urine and bile of rats and dogs (Fig. 2). A full scan mass spectrum of M17 yielded a protonated molecular ion atm/z 422, which is 16 Da higher than that of the parent, suggesting monooxygenation of compound I. CAD of the ion at m/z 422 resulted in a base fragment ion atm/z 342, suggesting addition of oxygen to the 1-(3-chlorophenyl)piperazin-2-one moiety. Subsequent CAD fragmentation of the ion at m/z 342 resulted in a loss of 28 Da (−CO) from the piperazinone moiety, similar to the parent, suggesting that no biotransformation occurred on the piperazinone ring and that oxidation most likely had occurred on the chlorophenyl portion of the molecule.
Metabolites M9 and M15 had HPLC retention times of 16.5 and 22.3 min, respectively. M9 was found only in rat and dog bile, whereas M15 was found in both the urine and bile of dogs and rats (Fig. 2). Mass spectrometric analysis ofM9 and M15 yielded protonated molecular ions atm/z 598 and 502, respectively. The product ion spectrum of both the metabolites resulted in formation of a base ion atm/z 422. This product ion corresponds to a loss of 176 Da (loss of dehydroglucuronic acid) and 80 Da (loss of SO3) from M9 and M15, respectively. Subsequent CAD of the ion at m/z422 resulted in a base ion at m/z 342, suggesting that the addition of oxygen occurred on the chlorophenyl moiety. LC-NMR (1H NMR spectrum) of peak at retention time of ∼16 min revealed the presence of a 1,2,4-trisubstituted aromatic ring (δ 7.22 d, 7.15 d, and 6.93 dd) (Table 4). The glucuronic acid conjugate was shown to be attached at C4 by an NOE experiment. Irradiating the i-methylene elicited NOE signals from H2 and H6, thus eliminating the possibility of substitution at C6 position (Fig. 1).
Treatment of the bile and urine samples with β-glucuronidase/sulfatase resulted in the disappearance of both the peaks and appearance of a new peak having the same retention time and molecular weight (MH+ = 422) as M17. The CAD product ion spectrum of m/z 422 was identical to that observed for metabolite M17. Thus, it was concluded that M9 and M15 were the glucuronide and sulfate conjugates of M17, respectively. Also, through inference, it was concluded that the oxidation of M17occurred at position 4 of the chlorophenyl ring.
Metabolite M10 had a HPLC retention time of 18.3 min and was found in the urine and bile of rats and in the bile of dogs. By LC-MS, the protonated molecular weight was determined to be 211 Da, and the product ion spectrum of ion at m/z 211 resulted in an intense base ion at m/z 183 (−CO). The retention time and fragmentation pattern of metabolite M10were compared with a synthetic standard of 1-(3-chlorophenyl)piperazin-2-one and were found to be identical.
LC-MS analysis of metabolite appearing at a retention time of 19.6 min has a protonated molecular weight of 225 Da and was found in the urine and bile of rats and dogs. Based on the ion intensities of the reconstructed ion chromatogram from dog and rat urine, it appears thatM11 is one of the major metabolites. A full scan mass spectrum yielded a protonated molecular ion atm/z 225, and CAD spectrum of the ion atm/z 225 resulted in a base ion atm/z 197 (−CO) and also an ion atm/z 154 (−HNCO), indicating that additional oxidation occurred on the piperazinone ring. The protonated molecular ion of M11 is 14 Da higher than M10. LC-NMR indicated the presence of only four aromatic protons in the 1,3-disubstituted aromatic ring, which indicated that the cleavage of the parent drug occurred between the piperazinone and the imidazole ring. There were only two triplets present on the aliphatic region for both metabolites (δ 3.57 and 3.91), and the absence of the isolated methylene signal indicated oxidation α to the keto-group on the piperazinone 32 ring. In addition, the HPLC retention time and mass spectral fragmentation pattern of M11 was found to be identical to the synthetic standard (1-(3-chlorophenyl)piperazine-2,3-dione).
M16 had an HPLC retention time of 25.1 min and was found mostly in rat urine and bile, albeit to a smaller extent in dog bile. Full scan MS was carried out to yield a protonated molecular ion atm/z 582, which is 176 Da higher than the parent compound, suggesting that it is a glucuronide conjugate of the parent (Hawes, 1998). Product ion spectrum of m/z 582 resulted in a very intense fragment ion at m/z406 (loss of dehydroglucuronic acid). Further CAD of the ion atm/z 406 revealed mass spectrum identical to compound I. Treatment of urine and bile samples with β-glucuronidase had no affect on M16. In addition to the data described here, the structures of other minor metabolites shown in Fig. 4 were assigned based on the proposed LC-MS/MS fragmentation pattern rationalized in Table5.
Discussion
The pharmacokinetics and metabolism of compound Idisplayed species differences in rats compared with dogs. CompoundI had a lower plasma clearance in dogs (10.6 ml/min/kg) compared with that in rats (41 ml/min/kg), but the half-life was similar in both the species (∼40 min). The similar half-life, yet different clearance values, could partly be explained by the difference in Vdss (about half in dog compared to rat; Table 1). Moreover, the mean equilibrium blood-to-plasma concentration ratio in rats and dogs was 0.83 and 0.53, respectively, indicating that the plasma CLb > CLp in both the species. Oral bioavailability was complete in dogs but somewhat less in rats. Since the rat CLb (∼49 ml/min/kg) is moderate relative to hepatic blood flow, the incomplete bioavailability may be due in part to hepatic first-pass metabolism.
The use of in vitro systems affords the opportunity to produce quantitative data on human clearance before initiation of pharmacokinetics studies in vivo (Houston, 1994; Ito et al., 1998; Lin, 1998; Obach, 1999). Hence, these in vitro-in vivo correlations have become very useful in helping select drug candidates with desirable pharmacokinetic properties in humans. In the present study, using the well stirred model (Wilkinson and Shand, 1975; Lin et al., 1996; Lin, 1997), hepatic clearance of compound I was estimated from in vitro CLint values using liver microsomes from rats, dogs, monkeys, and humans. With the exception of rats, the predicted clearance values in dogs and monkeys were in good agreement to those observed in vivo (Table 2). The predicted clearance values in rats were about 10-fold lower than the observed values, and one possible explanation is a large extrahepatic metabolism component of clearance. Using the predicted human clearance value of 3 ml/min/kg, at initial phase I dose of 35 mg/m2/day (administered by continuous infusion), the projected steady-state concentration was calculated as 0.5 μM.
The predicted pharmacokinetic values were in good agreement with results obtained in subsequent clinical studies in patients with solid tumors. Following administration of a dose of 35 mg/m2/day, the mean steady state concentration was 0.8 μM (range, 0.3–1.3; n = 3), the plasma clearance range was 2.3 to 4.3 ml/min/kg, and the half-lives for α- and β-phases were 0.6 and 3.3 h, respectively (Soignet et al., 1999). As expected, based on the preclinical studies, metabolism was the major route of clearance with less than 1% of the parent drug detected in the urine.
Metabolism of compound I was extensive with only a small percentage of the unchanged drug (<2% of the dose) excreted into the urine and bile of dogs and rats (Table 3). Biliary excretion was the major route of elimination in rats, whereas the reverse was true in dogs. N-Dealkylation at two different positions of the molecule was the most important biotransformation pathway, resulting in formation of about a dozen products, including subsequent phase II conjugation products. Metabolism of compound I was extensive in rat, dog, monkey, and human liver microsomes, and qualitatively the metabolic pathways were similar in all species (unpublished results, Merck Research Labs). The dealkylated metabolites that contain the imidazole portion of the molecule, but lack the radiotracer (M5, M12 and M14), were not quantified. Metabolite M12 was formed via either oxidation of M14 or N-dealkylation of M17 orM19. Based on LC-MS/MS data, the site of oxidation was identified to be on the chlorophenyl ring, but the regio-chemistry was not determined. Another major biotransformation observed in rats and dogs is the hydroxylation on the C-4 atom of the chlorophenyl ring (M17). The corresponding phase II metabolism was also different between rats and dogs. In case of rats, M17underwent mostly sulfation (M15), whereas glucuronidation was the preferred pathway in dogs (M9).
Both dog and rat showed extensive dealkylation and oxidation of compound I, but there are a few differences in metabolite profiles. The process of dealkylation between the imidazole and piperazinone rings most likely goes through an aldehyde intermediate, which either undergoes reduction to M8 or oxidation toM4 (Testa and Caldwell, 1995). There was some species difference between these two pathways in rats and dogs. In the case of rats, the reductive pathway to M8 and its glucuronide accounted for about 27% of the dose, whereas about 15% was accounted for in the dog. Conversely, the oxidative pathway leading toM4 and its glucuronide M3 accounted for about 30% of the dose in dogs and about 11% in rats.
In conclusion, compound I exhibited some differences in pharmacokinetics and metabolic behavior of the laboratory animals. It had a relatively short half-life and was cleared primarily by phase I and II metabolism in rats and dogs. In this study, reasonably accurate human clearance and half-life was predicted using human and animal in vitro metabolism data. Such predicted clearance data could be useful in selecting the best candidate for progression into development and also to assist in determination of the optimum first dose in man.
Acknowledgments
We thank F. A. deLuna, J. Brunner, K. Michel, and the late S. White for technical support with the animal studies, T. Marks and A. Jones for preparation of the radiolabeled compound I, D. McLoughlin for LC-MS/MS analysis, and C. Dinsmore and T. Williams for providing the synthetic standards. We also thank K. M. Baillie for helpful scientific discussions and assistance in preparation of this manuscript.
Footnotes
- Abbreviations used are::
- compound I
- (1-(3-chlorophenyl)-4-[(1-(4-cyanobenzyl)-[1H]-imidazol-5-yl)methyl]piperazin-2-one)
- FPTase
- farnesyl-protein transferase
- HPLC
- high-pressure liquid chromatography
- AUC
- area under plasma concentration-time curve
- Vdss
- volume of distribution
- CL
- clearance
- LC-MS/MS
- liquid chromatography-tandem mass spectrometry
- CAD
- collisionally activated dissociation
- NOE
- nuclear Overhauser effect
- Received May 23, 2001.
- Accepted September 14, 2001.
- The American Society for Pharmacology and Experimental Therapeutics