Research ArticleArticle

Species Differences in the Biotransformation of an α4β2 Nicotinic Acetylcholine Receptor Partial Agonist: The Effects of Distinct Glucuronide Metabolites on Overall Compound Disposition

Christopher L. Shaffer, Mithat Gunduz, Tim F. Ryder and Thomas N. O'Connell
Drug Metabolism and Disposition February 2010, 38 (2) 292-301; DOI: https://doi.org/10.1124/dmd.109.030171

Abstract

The metabolism and disposition of (1R,5S)-2,3,4,5-tetrahydro-7-(trifluoromethyl)-1,5-methano-1H-3-benzazepine (1), an α4β2 nicotinic acetylcholine receptor partial agonist, was investigated in Sprague-Dawley rats and cynomolgus monkeys receiving (1R,5S)-2,3,4,5-tetrahydro-7-(trifluoromethyl)-1,5-methano-1H-4[14C]-3- benzazepine hydrochloride ([14C]1) orally. Although both species chiefly (≥62%) cleared 1 metabolically, species-specific dispositional profiles were observed for both 1 and total radioactivity. Radioactivity was excreted equally in the urine and feces of intact rats but largely (72%) in bile in bile duct-cannulated animals. In monkeys, radioactivity recoveries were 50-fold greater in urine than feces and minimal (<5%) in bile. Both species metabolized 1 similarly: four-electron oxidation to one of four amino acids or two lactams (minor) and glucuronide formation (major). In rats, the latter pathway predominantly formed an N-carbamoyl glucuronide (M6), exclusively present in bile (69% of dose), whereas in monkeys it afforded an N-O-glucuronide (M5), a minor biliary component (4%) but the major plasma (62%) and urinary (42%) entity. In rats, first-pass hepatic conversion of 1 to M6, which was confirmed in rat hepatocytes, and its biliary secretion resulted in the indirect enterohepatic cycling of 1 via M6 and manifested in double-humped plasma concentration-time curves and long t1/2 for both 1 and total radioactivity. In monkeys, in which only M5 was formed, double-humped plasma concentration-time curves were absent, and moderate t1/2 for both 1 and total radioactivity were observed. A seemingly subtle, yet critical, difference in the chemical structures of these two glucuronide metabolites considerably affected the overall disposition of 1 in rats versus monkeys.

Species differences in the metabolism and pharmacokinetics of xenobiotics is a well known phenomenon (Hucker, 1970; Kato, 1979), and such metabolic variations have been observed for both phase I (Smith, 1991) and phase II (Chiu and Huskey, 1998) biotransformations. From a metabolism perspective, the preference of one species versus another for forming a specific metabolite may be attributable to species dissimilarities in the expression of the enzyme responsible for metabolizing that substrate [e.g., nitrogen acetylation may occur in rats but not dogs because of the lack of N-acetyltransferase expression in canines (Parkinson, 2001)] or in the metabolic capacities and/or rates of the common culprit enzyme (or isozyme) [e.g., cytochrome P450 (P450)-mediated hexobarbitone clearance in mice versus humans (Quinn et al., 1958)]. Furthermore, the physicochemical properties of the metabolite(s) arising from a molecule administered to an animal dictate the in vivo disposition of total compound equivalents. Hence, even if a molecule undergoes the same extent of metabolic clearance in two species yet the major metabolite within each species is structurally distinct, the overall disposition of total compound-related material can be drastically different within each species because of each metabolite's unique physicochemical properties, which ultimately dictate important dispositional properties such as volume of distribution, protein binding, and transporter susceptibility.

Herein, we report the absorption, metabolism, and excretion of (1R,5S)-2,3,4,5-tetrahydro-7-(trifluoromethyl)-1,5-methano-1H-3- benzazepine (1), an α4β2 nicotinic acetylcholine receptor partial agonist (Coe et al., 2005b) potentially useful for smoking cessation (Coe et al., 2005a) or other diseases such as depression (Rollema et al., 2009), in Sprague-Dawley rats and cynomolgus monkeys after a single oral dose of [14C]1 (Fig. 1). Although extensive metabolism of 1 was observed in both species, two completely distinct dispositional profiles were observed for both 1 and its total radioactivity. These species divergences in pharmacokinetics for both 1 and total compound equivalents were directly attributable to the differing physicochemical properties of species-specific secondary glucuronide conjugates, each of which was the major (i.e., ≥42% of total matrix radioactivity) metabolite for each species. A seemingly subtle variance in the chemical structures of these two glucuronide metabolites caused considerable differences in the overall disposition of 1 in rats versus monkeys.

Fig. 1.
Fig. 1.

Chemical structure of [14C]1 (∗, denotes 14C).

Materials and Methods

Chemicals and Reagents.

Compounds 1, 2, 3, and 4 were prepared by the Synthesis Group at Pfizer Global Research and Development [(PGRD) Groton, CT]; [14C]1 (45.8 mCi/mmol, >99% radiochemical purity, >99% enantiomeric excess) was synthesized by the Radiochemical Synthesis Group at PGRD (Groton, CT). The chemical purity of all the synthetic compounds was >99%. Male Sprague-Dawley rat liver microsomes (RLMs; 21 mg of protein/ml, 0.61 nmol of P450/mg protein), male cynomolgus monkey liver microsomes (MLMs; 20 mg of protein/ml, 1.2 nmol of P450/mg protein), and male Sprague-Dawley rat hepatocytes were prepared by the Candidate Enhancement Group at PGRD (Groton, CT). Cryopreserved male cynomolgus monkey hepatocytes were purchased from In Vitro Technologies Inc. (Baltimore, MD). β-Glucuronidase (EC 3.2.1.31, Type IX-A, 1–5 × 106 units/g protein) was obtained from Sigma-Aldrich (St. Louis, MO). Chemicals and solvents of reagent or high-performance liquid chromatography (HPLC) grade were supplied by Aldrich Chemical Co. (Milwaukee, WI), Thermo Fisher Scientific (Waltham, MA), and Mallinckrodt Baker, Inc. (Phillipsburg, NJ). Deuterated solvents were procured from Cambridge Isotope Laboratories, Inc. (Cambridge, MA). All the excreta, bile, plasma, whole brains, and cage debris/wash were collected gravimetrically and stored at −20°C until analysis.

In Vivo Studies in Sprague-Dawley Rats.

The in-life portion of the study was conducted at XenoBiotic Laboratories, Inc. (Plainsboro, NJ) in accordance with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, 1996). A single dose (3 mg/kg, approximately 40 μCi) of [14C]1 in deionized H2O (1.5 mg/ml) was administered via oral gavage to each rat, which was fasted from 6 h predose to 2 h postdose. Individual animal doses were calculated based on respective pretreatment body weights and a dose volume of 2 ml/kg. The actual amount of dose solution administered to each animal was determined by weighing the loaded dosing syringe before and after it was dispensed. The study included four groups of rats.

Group 1 (3/Sex).

From intact (i.e., noncannulated) animals, urine, feces, and cage rinse were collected separately into preweighed metabolism cage containers surrounded by dry ice predose and in 24-h intervals from 0 to 168 h postdose for the assessment of cumulative excretion of total radioactivity, mass balance, and metabolite profiling and identification.

Group 2 (2/Sex).

From bile duct-cannulated animals, which received an infusion of a bile salt replacement solution (44 mmol of cholic acid and 13 mmol of NaHCO3/l of 0.9% NaCl, pH 7.0–7.4) during the collection period, bile was collected into preweighed containers surrounded by cold packs predose and from 0 to 24 and 24 to 48 h postdose for metabolite profiling and identification.

Group 3 (3/Sex).

From jugular vein-cannulated rats, blood samples (0.5 ml) were collected just before dosing and at 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 h postdose for the pharmacokinetic evaluation of 1 and total radioactivity. After collection of the 1- and 8-h samples, whole blood (2 ml) from control donor animals was administered to each rat via the jugular vein cannula to maintain blood volume.

Group 4 (2/Sex/Time Point).

From intact animals, blood samples were collected by cardiac puncture after animal euthanasia by CO2 asphyxiation at 1, 4, 8, and 24 h postdose for metabolite profiling and identification. For identical purposes, the brain was extracted subsequently from each animal, rinsed of excess blood with saline, placed into a preweighed vial, weighed, and snap-frozen in liquid nitrogen.

All the blood samples were collected into heparinized tubes and processed immediately to obtain plasma. Control plasma was harvested from blood collected from untreated animals that were not sacrificed.

In Vivo Studies in Cynomolgus Monkeys.

The in-life portion of the study was conducted at Charles River Laboratories, Inc. (Wilmington, MA) in accordance with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, 1996). A single dose (1.5 mg/kg, approximately 150 μCi) of [14C]1 in deionized H2O (3.0 mg/ml) was administered via oral gavage to each monkey, which was fasted from 6 h predose to 4 h postdose. Individual animal doses were calculated based on respective pretreatment body weights and a dose volume of 0.5 ml/kg. The actual amount of dose solution administered to each animal was determined by weighing the loaded dosing syringe before and after it was dispensed. The study included two groups of monkeys.

Group 1 (2/Sex).

From intact animals, urine, feces, and cage rinse (mostly composed of residual food and hair debris) were collected separately into preweighed metabolism containers surrounded by dry ice predose and in 24-h intervals from 0 to 192 h postdose for the assessment of cumulative excretion of total radioactivity, mass balance, and metabolite profiling and identification. In addition, cage wash was collected after the final excreta time point (i.e., 192 h postdose). Blood samples (3 ml) were collected via venipuncture of a peripheral vessel just before dosing and at 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 h postdose for the pharmacokinetic evaluation of 1 and total radioactivity, as well as metabolite profiling and identification. All the blood samples were collected into EDTA-containing tubes and processed immediately to obtain plasma. Control plasma was harvested from blood collected from untreated animals that were not sacrificed.

Group 2 (two males).

From bile duct-cannulated animals, which received an infusion (23 ml/kg/day) of a bile salt replacement solution (44 mmol of cholic acid and 13 mmol of NaHCO3/l 0.9% NaCl, pH 7.4–7.8) during the collection period, bile was collected into preweighed containers surrounded by cold packs predose and from 0 to 24 and 24 to 48 h postdose for metabolite profiling and identification. Urine, feces, and cage rinse were also collected as described previously over the same intervals as bile, with cage wash collected at 48 h postdose.

Determination of Radioactivity within Biological Matrices.

For rat samples, triplicate gravimetric aliquots of urine (0.1 g), bile (0.025 g), plasma (0.05 g), and cage wash (1 g) were mixed with liquid scintillation mixture (10 ml for bile, 5 ml for all the other matrices) and counted for 2 min in a model LS 6000 or LS 6500 liquid scintillation counter (Beckman Coulter, Fullerton, CA). Monkey samples were analyzed identically in triplicate by combining gravimetric aliquots of urine (0.2–0.5 g), bile (0.05 g), plasma (0.05–0.1 g), and cage rinse (0.4–0.5 g) with liquid scintillation mixture (15 ml). All the fecal samples were homogenized with deionized H2O (5 ml of H2O/g of feces) using a probe-type homogenizer. All the cage debris/rinse samples (collected in 50% reagent alcohol in H2O) were homogenized directly with a probe-type homogenizer. Triplicate gravimetric aliquots (0.5 g for either fecal homogenate or cage debris/rinse homogenate) were transferred into tared cones and pads, weighed, and then dried for a minimum of 8 h at ambient temperature before combustion by a Packard Instruments model A0387 sample oxidizer (Canberra Industries, Meriden, CT). Combustion efficiency using a [14C] standard was determined daily before the combustion of study samples, and the measured radioactivity content in feces and cage debris/rinse was adjusted using daily combustion efficiency values. Liberated [14C]CO2 was trapped in Carbo-Sorb E (PerkinElmer Life and Analytical Sciences, Waltham, MA) and mixed with Perma-Fluor scintillation fluid (Canberra Industries), and the samples were analyzed for total radioactivity in a model LS 6000 or LS 6500 liquid scintillation counter for 2 min. Scintillation counter data were automatically corrected for counting efficiency using an external standardization technique and an instrument-stored quench curve generated from a series of sealed quench standards. For rat brains, thawed whole brain was weighed, diluted with H2O (3 ml of H2O/g of brain) and homogenized with a probe-type homogenizer. After liquefaction, aliquots (1 g) of brain homogenate in scintillation mixture (20 ml) were analyzed by liquid scintillation counting (LSC) for 5 min as described previously.

Quantitative Analysis of 1 in Plasma.

Concentrations of 1 in either rat or monkey plasma were quantified using a characterized liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay. To each plasma sample (100 μl) contained in a deep-well 96-well plate was added MeCN (200 μl) containing an internal standard, and the samples were vortex-mixed and centrifuged [832 relative centrifugal force (RCF) for 10 min] to sediment precipitated proteins. The respective supernatants (280 μl) were transferred to a second deep-well 96-well plate, concentrated under N2 at 40°C, and the resulting residues were reconstituted in 40% MeCN in 10 mM ammonium acetate, pH 4.0 (100 μl, Solvent A). Samples were analyzed by an LC/MS/MS composed of a PE Sciex API-3000 tandem quadrupole mass spectrometer with a TurboIonSpray interface (PerkinElmer Life and Analytical Sciences), two Shimadzu LC-10A HPLC pumps (Shimadzu USA, Columbia, MD), and a CTC PAL autosampler (LEAP Technologies, Carrboro, NC). Analytes within sample aliquots (10 μl) were eluted isocratically on a Waters Symmetry C18 analytical column (5 μm, 2.1 × 150 mm; Waters, Milford, MA) over a 4-min period at 0.2 ml/min with Solvent A. Mass spectral data were collected in positive ionization mode using multiple-reaction monitoring (MRM) of the m/z 228.2→191.2 fragmentation for 1. Instrument settings and potentials were adjusted to provide optimal data. The assay dynamic range was 2 to 2000 ng/ml for 1.

Pharmacokinetic Calculations.

Pharmacokinetic parameters were calculated for each animal by noncompartmental analyses using WinNonlin Professional Version 3.2 (Pharsight, Mountain View, CA). Values used to determine the pharmacokinetic parameters of total radioactivity were calculated by converting the raw data generated by LSC to concentrations (ng-Eq/ml) using the specific activity (13 mCi/mmol for rats, 5.7 mCi/mmol for monkeys) of administered [14C]1. The area under the concentration-time curve (AUC)0–24 was calculated using the linear trapezoidal method; the elimination rate constant (kel) was determined by linear regression of the log concentration versus time data during the last observable elimination phase; half-life (t1/2) was calculated as 0.693/kel; and AUC0–∞ was calculated as the sum of AUC0–24 and AUC24–∞, which was determined by dividing the plasma concentration at 24 h postdose by kel. Both maximal plasma concentration (Cmax) and its first time of occurrence (Tmax) were taken directly from the plasma concentration versus time data. Means and S.D. were calculated when half or greater of the values exceeded the lower limit of quantification for 1 (2 ng/ml) or total radioactivity (2× background radioactivity, which equated to 5 and 11 ng-Eq/ml in rat and monkey plasma, respectively). A value of 0 was used when a measured value was less than the lower limit of quantification.

Sample Preparation for Metabolite Profiling and Identification.

At each step during the sample preparation of all the biological matrices, total radioactivity levels were determined by LSC for recovery calculations. After preparation, all the samples were analyzed as described later by LC/ MS/MS with radiometric detection. Predose and blank samples served as controls for determining background radioactivity and endogenous, noncompound-related ions observed within respective matrices or their extracts by LC/MS/MS.

Urine.

Urine samples from each rat (collected 0–72 h postdose representing >92% of total urinary radioactivity) and monkey (collected 0–48 h postdose representing ≥95% of total urinary radioactivity) were pooled proportional to the amount of urine in each sampling period. Pooled rat samples were centrifuged (3300 RCF for 5 min) to remove particulate materials, and the resulting supernatants were analyzed directly. Pooled monkey samples were concentrated to near dryness by an N2 stream at 35°C, reconstituted in 10% MeCN in 10 mM ammonium formate, pH 3.4 (2 ml, Solvent B), and vortex-mixed and centrifuged (3300 RCF for 5 min) to sediment any solids to yield the analytical supernatant.

Bile.

Aliquots (1 ml) of bile collected from 0 to 24 h postdose from each bile duct-cannulated rat (representing >99% of total biliary radioactivity) and monkey (representing ≥87% of total biliary radioactivity) were centrifuged (3300 RCF for 5 min) to sediment particulate materials to provide the analytical supernatant.

Feces.

As a result of inconsequential amounts of fecal radioactivity recovered from monkeys (<2% of dose), only rat feces were profiled. Fecal homogenates (approximately 3.3–4.6 g) from each rat collected 0 to 72 h postdose, representing ≥90% of total fecal radioactivity, were pooled proportional to the amount of feces in each sampling period. Pooled homogenates were diluted with H2O (1 ml/g homogenate) and shaken at 37°C for 16 h in a reciprocal water bath. The softened samples were diluted with MeCN (2 ml/g homogenate), vortex-mixed, and centrifuged (1811 RCF for 10 min), and the resulting supernatants were isolated. If necessary, the remaining fecal pellets were extracted further with 33% H2O in MeCN (9 ml) until ≥90% of the radioactivity, as determined by LSC analysis of the combined supernatants, from each pooled sample was extracted. The supernatants were concentrated to dryness by an N2 stream at 35°C and reconstituted in Solvent B (1 ml) for analysis.

Rat Plasma.

Plasma from blood samples (approximately 5 ml) collected by terminal bleeding of rats at 1, 4, 8, and 24 h postdose were used for circulatory metabolite profiling and identification. Plasma samples were pooled within gender according to the method of Hamilton et al. (1981); i.e., 0.5, 0.9, 2.5, and 2.0 ml, respectively, of plasma from each time point sample per animal was combined to afford 11.8 ml of pooled plasma for each gender profile. This pooling procedure afforded one composite sample encompassing the entire AUC comprising multiple individual time point samples, allowing one sample injection to provide a representative of total exposures (AUC) to metabolites relative to each other. Proteins were precipitated from the plasma pools by the addition of MeCN (24 ml), and the samples were vortex-mixed and centrifuged (3661 RCF for 10 min). The remaining plasma protein pellets were extracted an additional time with MeCN (4 ml) to ensure that >90% of radioactivity, as determined by LSC analysis of the pooled supernatants, from each pooled plasma sample was extracted. The combined supernatants were concentrated to near dryness by an N2 stream at 35°C, and the resulting residues were reconstituted in Solvent B (0.6 ml) to afford the analytical sample.

Monkey Plasma.

Plasma from blood samples (approximately 3 ml) collected via venipuncture of a peripheral vessel at 0.5, 1, 4, 8, 12, and 24 h postdose were used for circulatory metabolite profiling and identification. Plasma samples were pooled (Hamilton et al., 1981) within gender and processed identically as rat plasma to afford the analytical sample (0.35 ml). For both genders, >90% of radioactivity was extracted from respective pooled plasma samples.

Rat Brain.

Thawed brains (2/time point/gender), isolated from the same rats sacrificed at 1, 4, 8, and 24 h postdose for the collection of blood for circulatory metabolite profiling and identification, were processed as described previously (Shaffer et al., 2009) with >92% of total brain homogenate radioactivity extracted. The resulting residues were reconstituted in Solvent B (1 ml) for analysis.

Cage Rinse.

A significant amount of radioactivity was detected within the cage rinse of both rats and monkeys necessitating its profiling. Cage rinse samples collected 0 to 72 h postdose from each rat (representing >68% of total cage rinse radioactivity) or monkey (representing ≥89% of total cage rinse radioactivity) were pooled proportionally to the amount of cage rinse in each sampling period. Pooled samples were concentrated to near dryness by an N2 stream at 35°C, reconstituted in Solvent B (1 ml), and vortex-mixed and centrifuged (3300 RCF for 5 min) to provide the analytical supernatant.

Metabolite Profiling, Identification, and Quantification.

Samples were analyzed by an LC/MS/MS (described previously) equipped with a Column Engineering Monitor C18 analytical column (5 μm, 4.6 × 150 mm) in series with a β-RAM radiometric detector (IN/US Systems, Tampa, FL) containing a liquid scintillant cell (250 μl). Analytes within sample aliquots (20–100 μl) were eluted at 1 ml/min with 10 mM ammonium formate, pH 3.4 (Solvent C), and MeCN (Solvent D). The following gradient was used: 0 to 20 min, 10% solvent D in solvent C; 20 to 55 min, 10 to 50% D in C; 55 to 56 min, 50 to 80% D in C; and 56 to 60 min, 80% D in C. After the elution of 1 and its metabolites, the column was returned over 1 min to 10% D in C where it remained for 5 min before the next injection. For each matrix, >98% of the radioactivity injected onto the column eluted within the first 55 min of analysis. HPLC effluent was split 1:9 between the mass spectrometer and the radiometric flow detector; liquid scintillation mixture flowed at 3 ml/min to the radiometric detector. Mass spectral data were collected using electrospray ionization in the positive ion mode and in full, precursor ion, neutral loss, product ion, and MRM scanning modes. Instrument settings and potentials were adjusted optimally in each mode. Analyst version 1.3 (PerkinElmer Life and Analytical Sciences) and Laura version 3.1.3.39 (LabLogic Systems Ltd., Sheffield, UK) software were used for the acquisition and processing of mass spectral and radiochromatographic data, respectively. Metabolites were quantified by peak integration within generated radiochromatograms.

Because the radioactivity in all the reconstituted monkey plasma samples was too low for quantification by radiometric flow detection, these samples were profiled by LC/MS/MS using MRM and fraction collection. During MRM analysis of the plasma sample, HPLC effluent was isolated in 30-s intervals by a Gilson FC 204 fraction collector (Gilson, Inc., Middleton, WI), and each respective fraction was mixed with TruCount (IN/US Systems) scintillation fluid (7 ml) and subjected to LSC for 5 min. Individual plasma radiochromatograms were generated from respective liquid scintillation data using Microsoft Excel (Microsoft Office 2000 9.0.7619 SP-3; Microsoft, Redmond, WA) and paired with their respective MRM chromatograms for the quantification of 1 and its metabolites.

Isolation of [14C]M6 from Rat Bile.

An aliquot (1 ml) of the 0- to 24-h bile collected from one bile duct-cannulated rat, which only contained one radioactive peak (i.e., M6), was subjected to solid-phase extraction using a Waters Sep-Pak 5 g C18 cartridge. After column conditioning and sample loading, the cartridge was washed with H2O (3 × 5 ml) to remove biliary salts, and M6 was eluted with MeCN (3 × 5 ml). The organic effluent (approximately 6 μg of M6/ml per LSC analysis of an effluent aliquot) was concentrated to near dryness by an N2 stream at 35°C to afford approximately 30 μg (132 nmol, 13 nCi/nmol) of [14C]M6.

Treatment of Isolated [14C]M6 with β-Glucuronidase.

Isolated [14C]M6 (24 nmol, 13 nCi/nmol) was dissolved in KH2PO4 (0.1 M, pH 7.4) buffer (0.6 ml), and equal aliquots (275 μl) were transferred to two separate vials. To the first vial was added more buffer (275 μl), and to the second vial was added β-glucuronidase (2750 units in 275 μl of buffer). Each solution was briefly vortex-mixed and then incubated at 37°C for 2 h in a shaking water bath. Aliquots (250 μl) were removed from each vial after 0 and 2 h of incubation, quenched with MeCN (250 μl), vortex-mixed, centrifuged (1509 RCF for 5 min), and analyzed directly (100-μl analytical aliquots) by LC/MS/MS with radiometric detection.

Isolation of M5 from Monkey Urine for NMR Analysis.

In a separate study in which a male monkey was orally administered nonradiolabeled 1 (1 mg/kg), urine (125 ml) was collected 0 to 24 h postdose for the isolation of M5 for liquid chromatography/mass spectrometry/nuclear magnetic resonance (LC/MS/NMR) analysis. Before preparatory workup of the matrix, HPLC/MS/MS analysis of the collected monkey urine confirmed the presence of M5 and determined its approximate concentration by comparing the intensity of its neutral loss of 176 scan signal with that observed for [14C]M5 in the urine from a group 1 monkey that had been dosed [14C]1. Subsequently, a portion (40 ml) of the 0- to 24-h nonradiolabeled urine sample was subjected to solid-phase extraction using a Waters Sep-Pak 5 g C18 cartridge. After column conditioning and sample loading, the cartridge was washed with H2O (3 × 10 ml) to remove urinary salts, and the metabolite(s) was eluted with MeCN (3 × 10 ml). The organic effluent (approximately 3 μg M5/ml) was concentrated to near dryness by an N2 stream at 35°C to afford a viscous amber oil [150 μl, approximately 80 μg (approximately 191 nmol) M5], which underwent LC/MS/NMR analysis as described in the supplemental data.

In Vitro Incubations with [14C]1 or 1.

All the samples from incubations using [14C]1 or 1 were analyzed by the LC/MS/MS systems already described; for incubations with [14C]1, in-line radiodetection quantified 1 and its metabolites, whose identification was confirmed by previously observed LC tRs and collision-induced dissociation (CID) spectra.

RLMs and MLMs.

Incubations (1 ml) were performed in duplicate with and without NADPH (1.3 μmol) in round-bottom glass test tubes open to air at 37°C in a shaking water bath. Each incubation contained RLMs or MLMs (1 mg of protein/ml 0.1 M KH2PO4 buffer, pH 7.4), MgCl2 (3 μmol), and [14C]1 (10 nmol, 13 nCi/nmol). Sample aliquots (0.4 ml) were removed by micropipette at 0 and 60 min after NADPH (or buffer) addition, quenched with MeCN (1 ml), and centrifuged (1509 RCF for 5 min), and the resulting supernatant was concentrated and reconstituted in Solvent A (0.4 ml) for analysis.

Rat Hepatocytes.

Incubations (5 ml), which were gassed every hour with 5% CO2 in O2, were performed in duplicate in capped 15-ml Erlenmeyer flasks at 37°C in a shaking water bath. Each incubation contained thawed cryopreserved Sprague-Dawley rat hepatocytes (1 × 106 viable cells/ml bicarbonate-based Williams' E medium with 10% fetal bovine serum) and [14C]1 (5 nmol, 13 nCi/nmol). A sample aliquot (2 ml) was removed by micropipette 0 and 4 h after the addition of [14C]1, quenched with MeCN (4 ml), vortex-mixed, and centrifuged (1509 RCF for 5 min). The resulting supernatant was evaporated under N2 at 35°C, and the residue was reconstituted in Solvent A (0.4 ml) for analysis.

Monkey Hepatocytes.

Incubations (5 ml) were performed as described for rat hepatocytes with each incubation containing thawed cryopreserved male cynomolgus monkey hepatocytes (0.75 × 106 viable cells/ml bicarbonate-based Williams' E medium with 10% fetal bovine serum) and 1 (5 nmol). A sample aliquot (2 ml) was removed by micropipette at 0 and 4 h after the addition of 1, quenched with MeCN (6 ml), vortex-mixed, and centrifuged (1800 RCF for 5 min). The resulting supernatant was evaporated under N2 at 37°C, and the residue was reconstituted in Solvent A (0.2 ml) for analysis.

Results

Excretion of Total Radioactivity in Rats.

Because of no readily apparent gender-related differences in overall excretory routes or mass recoveries in either intact or bile duct-cannulated animals, combined male and female radioactivity recovery values (mean ± S.D.) for rats receiving [14C]1 are listed in Table 1. The excretion of total compound-related material was fairly rapid in rats; >79% of the administered radioactivity was recovered within the first 48 h. Over the entire study, the mean recovery of compound-related material within excreta and cage rinse from intact rats was 89.8 ± 1.9%, with nearly equal amounts of radioactivity in urine as in feces. Because of a significant (and atypical) amount (18.5 ± 7.8%) of dosed radioactivity detected within the cage rinse over the collection period, the mass balance portion (i.e., group 1 animals) of this study was repeated. It is interesting to note that quantitatively similar (i.e., ±5% of the original group 1 recovery values for urine, feces, and cage rinse) excretion patterns and consistent high amounts of radioactivity within cage rinses were observed in the repeat mass balance study. Although we are unable to explain readily these consistently high recoveries of radioactivity in cage rinse, which we did not observe in rat radiolabeled mass balance studies with the enantiomer of 1 (Shaffer et al., 2009), we believe they are attributed to residual excreta that accumulated within the metabolism cage during the study period. Hence, the excreta and cage rinse collected from the original group 1 rats were used for the quantitative profiling and identification of metabolites. On average in bile duct-cannulated rats, 71.7 ± 7.1% of the dose was detected in bile from 0 to 48 h postdose, with >99% of the total biliary radioactivity within the 0- to 24-h sample.

View this table:
TABLE 1

Cross-species comparison of excretory routes and metabolite profiles after oral administration of [14C]1

Pharmacokinetics of 1 and Total Radioactivity in Rats.

As with total radioactivity excretion patterns, no obvious gender-related differences in systemic exposure to 1 or total radioactivity were observed. For both 1 and total radioactivity, mean plasma concentration-time curves are found in Fig. 2, and pharmacokinetic parameters (mean ± S.D.) are listed in Table 2. On average, plasma concentrations of 1 increased to a mean Cmax of 116 ± 37 ng/ml at a mean Tmax of 2.1 ± 2.4 h postdose with a mean t1/2 of 9.8 ± 1.8 h. For total radioactivity, mean Cmax and Tmax values were 183 ± 29 ng-Eq/ml and 4.8 ± 2.6 h, respectively, whereas the mean t1/2 was 18.9 ± 5.7 h. On average, the AUC0–∞ for 1 represented 36% of the AUC0–∞ for total compound-related material. It is intriguing that plasma concentration-time curves for both 1 and total radioactivity had a double-humped profile with the second upward leg occurring between 2 and 4 h postdose, indicative of the enterohepatic cycling of 1 in rats.

Fig. 2.
Fig. 2.

Mean plasma concentrations of 1 (solid symbols) and total radioactivity (open symbols) in rats (top) and monkeys (bottom) after oral administration of [14C]1.

View this table:
TABLE 2

Cross-species pharmacokinetics summary for 1 and total radioactivity after oral administration of [14C]1

Excretion of Total Radioactivity in Monkeys.

Because of no readily apparent gender-related differences in overall excretory routes or mass recoveries in intact animals, combined male and female radioactivity recoveries (mean ± S.D.) for monkeys receiving [14C]1 are listed in Table 1. The excretion of total compound-related material was rapid in monkeys; >77% of the administered radioactivity was recovered within 24 h postdose. During the study period, the mean recovery of radioactivity within excreta and cage rinse from intact monkeys was 90.5 ± 4.7%, with the vast majority of excreta radioactivity detected in urine. Because of a considerable amount (13.0 ± 4.7%) of dosed radioactivity detected within the cage rinse over the entire collection interval, it too underwent metabolite identification and quantitative profiling. As for rats, we cannot readily rationalize these higher-than-usual cage rinse recoveries for monkeys, other than this radioactivity was caused by accumulated excreta within the cage during the study period. On average in bile duct-cannulated male monkeys, 5.6% of the dose was detected in bile from 0 to 48 h postdose, with ≥87% of the total biliary radioactivity within the 0- to 24-h sample, whereas 66.1, 0.5, and 16.1% of the dose was observed in urine, feces, and cage rinse, respectively.

Pharmacokinetics of 1 and Total Radioactivity in Monkeys.

As with total radioactivity excretion patterns, no obvious gender-related differences in systemic exposure to 1 or total compound-related material were observed. For both 1 and total radioactivity, average plasma concentration-time curves are found in Fig. 2, and pharmacokinetic parameters (mean ± S.D.) are listed in Table 2. On average, plasma concentrations of 1 increased to a mean Cmax of 96.2 ± 21.6 ng/ml at a mean Tmax of 1.0 ± 0.0 h postdose with a mean t1/2 of 4.7 ± 0.9 h. For total radioactivity, mean Cmax and Tmax values were 580 ± 146 ng-Eq/ml and 1.0 ± 0.0 h, respectively, whereas the mean t1/2 was 5.3 ± 1.0 h. On average, the AUC0–∞ for 1 represented 23% of the AUC0–∞ for total compound-related material. In contrast to rats, monkey plasma concentration-time curves for both 1 and total radioactivity lacked a double-humped profile.

Structural Rationalization of 1 and Its Metabolites.

Compound 1 had a protonated molecular ion of m/z 228 and an LC tR of approximately 32.3 min; its CID product ion spectrum contained fragment ions with m/z 211, 199, 196, 191, 179, 177, 159, 141, and 44 (Table 3). Both the mechanistically rationalized structures for the majority of the cited fragment ions and the MS scan strategy for metabolite identification have been described previously (Shaffer et al., 2009) for the enantiomer of 1. A summary of all the metabolite LC/MS/MS data is found in Table 3. The identification of a metabolite as a fully characterized standard was determined by the compounds' indistinguishable CID spectra and LC tRs, as well as by an increase in metabolite MS peak height on addition of the authentic standard to the analytical sample. As for the regioisomeric lactam metabolites of the enantiomer of 1 (Shaffer et al., 2009), base-catalyzed hydrolysis of authentic standards 2 and 3 (4 μM in 1 M NaOH, 42 h, 25°C) each afforded two compounds with LC tRs and CID spectra identical to M1 and M3, and M2 and M4, respectively.

View this table:
TABLE 3

Chromatographic and mass spectral data for 1 and its metabolites

The primary focus of the metabolite identification work was the characterization of the suspected N-O-glucuronide (M5) and N-carbamoyl glucuronide (M6) metabolites. Previous rat radiolabeled biotransformation work (Shaffer et al., 2009) with the enantiomer of 1 also identified these two glucuronide metabolites, with the enantiomer of M6 definitively identified by NMR as an N-carbamoyl glucuronide. When treated with β-glucuronidase, the N-carbamoyl glucuronide was converted rapidly and quantitatively to the enantiomer of 1, whereas the N-O-glucuronide was deconjugated more slowly to two compounds: N-hydroxyl-1 and its nitrone. This identical enzyme-mediated hydrolytic behavior was observed in this study with M5 and M6. Because of the minor nature of M5 in rats receiving the enantiomer of 1, M5 never underwent definitive structural elucidation like its N-carbamoyl glucuronide counterpart. However, because of the significance of M5 in monkeys reported herein, LC/MS/NMR analyses of nonradiolabeled M5 purified from the urine of a monkey administered 1 unequivocally identified M5 as an N-O-glucuronide of 1, consistent with the mechanistic structural rationalization of fragment ions within its product ion spectrum (Fig. 3) and previously characterized alicyclic N-O-glucuronides (Straub et al., 1988; Delbressine et al., 1992; Edlund and Baranczewski, 2004; Miller et al., 2004). In particular, the 1H spectrum of M5 clearly showed the anomeric proton signal (4.37 ppm) of the glucuronide moiety, whereas the correlation spectroscopy spectrum indicated that this was the most downfield resonance in a five-proton spin system that terminated with a doublet at 3.59 ppm. Together, this pattern is consistent with a glucuronide conjugate of 1. Both the three aromatic proton resonances and all the alkyl proton resonances of 1 are present in the 1H and correlation spectroscopy spectra of M5, suggesting cumulatively that the glucuronide moiety must be projecting from its nitrogen. In addition, in LC/MS/NMR experiments, the [M+D]+ detected by mass spectrometry for M5 in deuterated solvents was 5 atomic mass units greater than its [M+H]+ in nondeuterated solvents, consistent with four exchangeable protons within M5. A summary of all the NMR data with full spectral assignments for both 1 and M5 is found in the supplemental data.

Fig. 3.
Fig. 3.

CID spectrum of protonated M5 (m/z 420).

Quantitative Profile of [14C]1 and Its Metabolites in Excreta and Cage Rinse.

Urine.

In addition to 1, the same five metabolites were observed in both rat and monkey urine (Table 1). Four metabolites (M1, M2, M3, and M4) were tentatively identified as amino acids, whereas the fifth (M5) was an N-O-glucuronide of 1. Metabolites M3 and M4 were quantified as one radioactive entity because they were resolved by mass spectrometry but not by radiochromatography. On average in rats and monkeys, 22.4 ± 5.4% and 3.4 ± 1.5% of administered 1, respectively, was excreted unchanged in urine equating to a renal clearance of 2.2× and 0.6× glomerular filtration rate (Davies and Morris, 1993), respectively, suggesting no net active renal transport of 1 in either species.

Feces.

In addition to 1, which comprised 15.5 ± 4.2% of the administered dose, only the urinary amino acid metabolites were also observed in rat feces (Table 1). Monkey feces was not profiled because it contained an inconsequential amount (<2%) of dose.

Cage Rinse.

Because of the unusually high amounts of dosed radioactivity detected in the cage rinses of both rats (18.5 ± 7.8%) and monkeys (13.0 ± 4.7%), this matrix underwent quantitative profiling. In rats, 1 and each amino acid metabolite were detected. Because these compounds were common to both urine and feces, they could not be assigned to either excreta exclusively for mass balance purposes. For monkeys, cage rinse profiles were identical to urine but were once again not added to urinary quantitative metabolite profiles because it was unknown if this radioactivity came from uncollected urine and/or feces contaminating the cage.

Bile.

In rats, major quantities of M6 were detected solely in bile with only very minor amounts of 1 (Table 1). In contrast, only minimal amounts of both 1 and M5 were observed in monkey bile (Table 1).

Quantitative Profile of [14C]1 and Its Metabolites in Plasma and Rat Brain.

In addition to 1, the same eight metabolites were identified in both rat and monkey plasma: amino acids M1, M2, M3, and M4, N-O-glucuronide M5, regioisomeric lactams 2 and 3, and formamide 4 (Table 4). However, although qualitative profiles were identical across species, quantitative profiles were significantly different. In particular, 1 and 3 comprised much greater amounts of total circulating radioactivity in rats versus monkeys, whereas monkeys had lower contributions of 1 with much greater composition by M5. On average in rats, 1 comprised 85.7% of total brain radioactivity with minor amounts of 2, 3, and 4 (Table 4).

View this table:
TABLE 4

Cross-species comparison of circulatory and brain metabolite profiles after oral administration of [14C]1

In Vitro Incubations with [14C]1 or 1.

During the in-life portion of the radiolabeled studies, the in vitro metabolism of [14C]1 was only evaluated in rat and monkey liver microsomes using conventional procedures (i.e., not optimized for N-carbamoyl glucuronidation) (Schaefer, 1992). For both species, no consumption of [14C]1 was observed during 1 h of incubation irrespective of NADPH. Cross-species microsomal viability was confirmed by their NADPH-dependent metabolism of a proprietary positive control compound. After the cross-species identification of the in vivo metabolites of 1, incubations were conducted with [14C]1 in rat hepatocytes and with 1 in monkey hepatocytes. After 4 h of incubation, rat hepatocytes converted approximately 96% of 1 to the four amino acids, M6 and 3, with M6 comprising 80% of the initial radioactivity within the incubation (Table 5). The nearly exclusive conversion of 1 to M6 in rat hepatocytes is consistent with the biliary metabolite profiles observed for bile duct-cannulated rats in which M6 comprised approximately 70% of the administered dose (Table 1). In monkey hepatocytes, approximately 21% of 1 was metabolized after 4 h of incubation, and all the in vivo metabolites were detected qualitatively by MRM scanning mode with M5 having the greatest extracted ion intensity; M6 was observed, but its ion intensity was 16-fold lower than that of M5.

View this table:
TABLE 5

Metabolite profiles in rat hepatic in vitro systems using [14C]1

Discussion

No clear gender-related differences were observed for the pharmacokinetics, excretion patterns, or metabolic profiles in Sprague- Dawley rats or cynomolgus monkeys ingesting [14C]1. In intact rats, 89.8% of administered radioactivity was recovered during the study with 40.4% in urine, 31.0% in feces, and 18.5% in cage rinse. In bile duct-cannulated rats, 71.7% of the dose was excreted in bile over 48 h postdose. A relatively short plasma Tmax and large amounts of radioactivity excreted in bile and urine from 0 to 24 h postdose suggest [14C]1 was readily and substantially absorbed in rats. In rats, 1 underwent metabolic (≥62%) and net passive renal (22%) clearance, had a long t1/2, and showed significant central penetration (brain-to-plasma AUC0–24 ratio of 30). In intact monkeys, 90.5% of administered radioactivity was recovered during the study period with 76.0% in urine, 1.5% in feces, and 13.0% in cage rinse. In bile duct-cannulated monkeys, only 5.6% of the dose was excreted in bile over 48 h postdose, with 66.1, 0.5, and 16.1% detected in urine, feces, and cage rinse, respectively. A short plasma Tmax, significant quantities of radioactivity renally excreted from 0 to 24 h postdose, and essentially no fecal radioactivity during the entire study period suggest [14C]1 was rapidly and completely absorbed in monkeys. In monkeys, 1 underwent primarily (>95%) metabolic clearance, with only a very minor (approximately 3%) net passive renal contribution, to afford a moderate t1/2.

A schematic overview of the metabolism of 1 in Sprague-Dawley rats and cynomolgus monkeys is presented in Fig. 4. Based on the identification of all the significant metabolites in excreta, bile, and plasma, the sole metabolically susceptible site within 1 exploited by both species was its nitrogen, resulting in either its direct modification (to M5, M6, or 4) or ultimate oxygenation of an adjacent carbon (to one of four amino acids or two lactams). In both species, the latter four-electron oxidations yielded identical amino acids and lactams, whose formation from the enantiomer of 1 in rats is rationalized elsewhere (Shaffer et al., 2009). In rats, the glucuronidation pathway for 1 predominantly generated an N-carbamoyl glucuronide (M6), exclusively present in bile (69% of dose), and minimally produced a suspected N-O-glucuronide (M5), absent in bile but detected in both plasma (9% of total circulatory radioactivity) and urine (<2%). Contrastingly in monkeys, the only detected glucuronide of 1 was M5, which, although a minor biliary component (4%), was the major entity within plasma (62%) and urine (42%).

Fig. 4.
Fig. 4.

An overview of the proposed metabolic pathways of 1 in rats and monkeys. Bold numerical designations are for observed metabolites; all the other structures are putative metabolite intermediates.

We recently reported (Shaffer et al., 2009) rat radiolabeled biotransformation work for the enantiomer of 1 in which its N-carbamoyl glucuronide was purified from bile for structural confirmation by NMR analyses. Based on indistinguishable MS fragmentation spectra, nearly equivalent LC tRs (under slightly different chromatographic conditions), and identical in vitro behavior in the presence of β-glucuronidase, M6 was confidently deemed the same (albeit enantiomeric) N-carbamoyl glucuronide of 1. Subsequently, the significance of M5, the putative N-O-glucuronide of 1, in monkeys prompted its isolation from monkey urine for structural confirmation by NMR. The definitive identification of M5 and M6 has allowed an important mechanistic understanding of how each of these distinct glucuronides uniquely affects the overall disposition of 1 (and total radioactivity) in rats versus monkeys.

In rats, first-pass hepatic conversion of 1 to M6, which was confirmed in rat hepatocytes (Table 5), and its biliary secretion results in the indirect enterohepatic cycling (EHC) of 1 via M6 as elucidated in rats for the enantiomer of 1 (Shaffer et al., 2009). This indirect EHC depends on M6 being innately susceptible to rapid intestinal deconjugation by β-glucuronidase and decarboxylation to 1, and manifested in double-humped plasma concentration-time curves in rats, with the upward leg to the second peak occurring between 2 and 4 h postdose (Fig. 2). This M6-dependent dispositional process for 1 in rats resulted in a long t1/2 and much greater amounts of unchanged 1 in urine (and most likely in feces) than predicted by rat hepatocytes or observed in monkeys, in which EHC of 1 did not occur.

In monkeys, M6 was conspicuously absent, whereas M5, also observed minimally in rat urine (2%) and plasma (9%), was the major entity in all the matrices. The detection of M6 in rat bile and the documented stability of N-carbamoyl glucuronides in urine (Elvin et al., 1980; Straub et al., 1988) and plasma (Tremaine et al., 1989) suggest the absence of M6 in monkeys was not because of its degradation before matrix analyses. These data, combined with the lack of double-humped plasma-concentration time curves for both 1 and total radioactivity (Fig. 2), suggest that in monkeys 1 is primarily metabolized (presumably hepatically) to M5, which underwent limited biliary transport (unlike M6) resulting instead in its substantial systemic exposure and urinary excretion. This dispositional behavior of M5 in monkeys paralleled that in rats. Thus, the exclusive formation of M5, and not M6, in monkeys afforded distinct pharmacokinetic properties for 1 and total radioactivity versus rats (Table 2). Furthermore, based on β-glucuronidase treatment data, the molecular nature of M5, unlike M6, prohibits the formation of 1 from its aglycone. Instead, M5 deconjugation by β-glucuronidase in vitro, a much slower process than that for M6, afforded N-hydroxyl-1, which is readily converted to its nitrone and not 1. It is interesting to note that this nitrone was not detected in any in vivo-derived matrix in these studies, but its enantiomer was detected minimally in plasma from rats administered the enantiomer of 1 (Shaffer et al., 2009). Hence, even if M5 underwent primarily biliary clearance once formed hepatically (like M6), it would most likely not reform 1 intestinally, thus preventing the indirect EHC of 1 observed via M6 in rats.

The distinct dispositional characteristics of the two discussed glucuronides of 1 prompt two important questions: 1) why is M6 predominantly formed in rats and M5 is exclusively formed in monkeys; and 2) if M5 is formed hepatically, why does it not undergo exclusive biliary clearance like M6? The first question implicates potential differences in the enzyme(s), or even possibly tissue CO2 concentrations, dictating the biotransformation of 1 to M5 versus M6. Previous work (Shaffer et al., 2009) reported the enantiomer of 1 was converted to the enantiomer of M6 in RLMs optimized for N-carbamoyl glucuronidation; no metabolism of [14C]1 (or its enantiomer) was observed in RLMs (±NADPH). For this study, rat hepatocytes primarily (80%) metabolized [14C]1 to [14C]M6 with no detectable formation of M5 (Table 5). Together these data suggest both tested rat hepatic systems contain the enzyme(s) necessary for M6 formation. In MLMs (±NADPH), [14C]1 was also inert. Although subjecting [14C]1 to MLMs optimized for N-carbamoyl glucuronidation was not conducted, MLMs are typically capable of producing RLM-observed N-carbamoyl glucuronides (Schaefer, 2006). To explore further qualitatively the selective formation of M5 in monkeys, 1 (1 μM) was incubated in monkey hepatocytes. Over 4 h, in which 21% of 1 was consumed, all the in vivo metabolites were detected in hepatocytes by LC/MS/MS with M5 having the greatest extracted ion intensity; M6 was detected, but its ion intensity was 16-fold lower than that of M5. Based on these in vitro data, if one assumes the liver predominantly forms M5 in vivo, then possibly the species difference in major glucuronide metabolites is the hepatic presence/activity of the enzyme(s) required to generate the N-hydroxylamine of 1, the requisite precursor of M5. If this N-hydroxylamine was formed by FAD-containing monooxygenase 3 (FMO3), an FMO isozyme expressed minimally in rat liver but abundantly in primate liver (Sadeque et al., 1993; Rettie and Fisher, 1999), then the dearth of rat hepatic FMO3 might shunt 1 toward sequential carbamate and M6 formation, whereas significant FMO3 activity in monkey liver would readily yield N-hydroxylamine from 1, precluding the M6 pathway.

For the second question, the combination of M6 only detected in bile (approximately 70%) and its major (approximately 80%) generation in rat hepatocytes suggests that once formed hepatically M6 undergoes exclusive biliary clearance, possibly via a hepatic organic anion transporter such as the canalicular transporter multidrug resistance-associated protein 2 (Keppler and Arias, 1997; Xiong et al., 2000). Alternatively, M5 underwent no biliary clearance in rats and only a minimal amount (approximately 4%) in monkeys but instead reached systemic circulation and was ultimately excreted renally in both species. These data may suggest that M5 undergoes preferential transport into blood, like that mediated by multidrug resistance-associated protein 3 (Soroka et al., 2001; Villaneuva et al., 2008). Species differences exist in hepatic transporter expression (Bleasby et al., 2006) and activities (Klaassen and Watkins, 1984; Li et al., 2008); thus, we are currently investigating the significance of such considerations on the observed interspecies dispositional disparities for M6 and M5.

An understanding of the species difference in the metabolism-mediated conversion of 1 to M5 versus M6 is key to its preclinical safety assessment and to the accurate projection of its human clearance pathways, as the major preclinical clearance mechanism believed to be most relevant to humans dictates the optimal human clearance-scaling methodology. Future clinical studies with 1 should provide greater insight into the preclinical species most predictive of its human metabolism and disposition.

Acknowledgments.

We thank Dr. Klaas Schildknegt (Radiosynthesis Group at PGRD, Groton, CT) for synthesis and purification of [14C]1, Dr. Jotham Coe for providing 1, Jason McKinley and Brett Lillie for synthesizing 2 and 3, and Nga Do for supplying 4.

Footnotes

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

    doi:10.1124/dmd.109.030171

  • ↵Embedded Image The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material.

  • P450
    cytochrome P450
    1
    (1R,5S)-2,3,4,5-tetrahydro-7-(trifluoromethyl)-1,5-methano-1H-3-benzazepine hydrochloride
    [14C]1
    (1R,5S)-2,3,4,5-tetrahydro-7-(trifluoromethyl)-1,5-methano-1H-4[14C]-3-benzazepine hydrochloride
    2
    (1R,5S)-2,3,4,5-tetrahydro-7-(trifluoromethyl)-1,5-methano-4-oxo-1H-3-benzazepine
    3
    (1R,5S)-2,3,4,5-tetrahydro-7-(trifluoromethyl)-1,5-methano-2-oxo-1H-3-benzazepine
    4
    (1R,5S)-2,3,4,5-tetrahydro-7-(trifluoromethyl)-1,5-methano-1H-3-benzazepine-3-carboxaldehyde
    PGRD
    Pfizer Global Research and Development
    RLM
    rat liver microsome
    MLM
    monkey liver microsome
    HPLC
    high-performance liquid chromatography
    LSC
    liquid scintillation counting
    LC/MS/MS
    liquid chromatography/tandem mass spectrometry
    RCF
    relative centrifugal force
    MRM
    multiple-reaction monitoring
    AUC
    area under the concentration-time curve
    kel
    elimination rate constant
    LC/MS/NMR
    liquid chromatography/mass spectrometry/nuclear magnetic resonance
    CID
    collision-induced dissociation
    EHC
    enterohepatic cycling
    FMO
    FAD-containing monooxygenase.

    • Received September 8, 2009.
    • Accepted November 6, 2009.

References

  1. ↵
    1. Bleasby K,
    2. Castle JC,
    3. Roberts CJ,
    4. Cheng C,
    5. Bailey WJ,
    6. Sina JF,
    7. Kulkarni AV,
    8. Hafey MJ,
    9. Evers R,
    10. Johnson JM,
    11. et al.
    (2006) Expression profiles of 50 xenobiotic transporter genes in humans and pre-clinical species: a resource for investigations into drug disposition. Xenobiotica 36: 963–988.
    OpenUrlCrossRefPubMed
  2. ↵
    1. Chiu SH,
    2. Huskey SW
    (1998) Species differences in N-glucuronidation. Drug Metab Dispos 26: 838–847.
    OpenUrlAbstract/FREE Full Text
  3. ↵
    1. Coe JW,
    2. Brooks PR,
    3. Vetelino MG,
    4. Wirtz MC,
    5. Arnold EP,
    6. Huang J,
    7. Sands SB,
    8. Davis TI,
    9. Lebel LA,
    10. Fox CB,
    11. et al.
    (2005a) Varenicline: an α4β2 nicotinic receptor partial agonist for smoking cessation. J Med Chem 48: 3474–3477.
    OpenUrlCrossRefPubMed
  4. ↵
    1. Coe JW,
    2. Brooks PR,
    3. Wirtz MC,
    4. Bashore CG,
    5. Bianco KE,
    6. Vetelino MG,
    7. Arnold EP,
    8. Lebel LA,
    9. Fox CB,
    10. Tingley FD 3rd,
    11. et al.
    (2005b) 3,5-Bicyclic aryl piperidines: a novel class of α4β2 neuronal nicotinic receptor partial agonists for smoking cessation. Bioorg Med Chem Lett 15: 4889–4897.
    OpenUrlCrossRefPubMed
  5. ↵
    1. Davies B,
    2. Morris T
    (1993) Physiological parameters in laboratory animals and humans. Pharm Res 10: 1093–1095.
    OpenUrlCrossRefPubMed
  6. ↵
    1. Delbressine LP,
    2. Moonen ME,
    3. Kaspersen FM,
    4. Jacobs PL,
    5. Wagenaars GL
    (1992) Biotransformation of mianserin in laboratory animals and man. Xenobiotica 22: 227–236.
    OpenUrlPubMed
  7. ↵
    1. Edlund PO,
    2. Baranczewski P
    (2004) Identification of BVT.2938 metabolites by LC/MS and LC/MS/MS after in vitro incubations with liver microsomes and hepatocytes. J Pharm Biomed Anal 34: 1079–1090.
    OpenUrlCrossRefPubMed
  8. ↵
    1. Elvin AT,
    2. Keenaghan JB,
    3. Byrnes EW,
    4. Tenthorey PA,
    5. McMaster PD,
    6. Takman BH,
    7. Lalka D,
    8. Manion CV,
    9. Baer DT,
    10. Wolshin EM,
    11. et al.
    (1980) Tocainide conjugation in humans: novel biotransformation pathway for a primary amine. J Pharm Sci 69: 47–49.
    OpenUrlCrossRefPubMed
  9. ↵
    1. Hamilton RA,
    2. Garnett WR,
    3. Kline BJ
    (1981) Determination of mean valproic acid serum level by assay of a single pooled sample. Clin Pharmacol Ther 29: 408–413.
    OpenUrlCrossRefPubMed
  10. ↵
    1. Hucker HB
    (1970) Species differences in drug metabolism. Annu Rev Pharmacol 10: 99–118.
    OpenUrlCrossRefPubMed
  11. ↵
    Institute of Laboratory Animal Resources (1996) Guide for the Care and Use of Laboratory Animals, 7th ed. Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council, Washington DC.
  12. ↵
    1. Kato R
    (1979) Characteristics and differences in the hepatic mixed function oxidases of different species. Pharmacol Ther 6: 41–98.
    OpenUrlCrossRefPubMed
  13. ↵
    1. Keppler D,
    2. Arias IM
    (1997) Hepatic canalicular membrane. Introduction: transport across the hepatocyte canalicular membrane. FASEB J 11: 15–18.
    OpenUrlPubMed
  14. ↵
    1. Klaassen CD,
    2. Watkins JB 3rd
    (1984) Mechanisms of bile formation, hepatic uptake, and biliary excretion. Pharmacol Rev 36: 1–67.
    OpenUrlPubMed
  15. ↵
    1. Li M,
    2. Yuan H,
    3. Li N,
    4. Song G,
    5. Zheng Y,
    6. Baratta M,
    7. Hua F,
    8. Thurston A,
    9. Wang J,
    10. Lai Y
    (2008) Identification of interspecies difference in efflux transporters of hepatocytes from dog, rat, monkey and human. Eur J Pharm Sci 35: 114–126.
    OpenUrlCrossRefPubMed
  16. ↵
    1. Miller RR,
    2. Doss GA,
    3. Stearns RA
    (2004) Identification of a hydroxylamine glucuronide metabolite of an oral hypoglycemic agent. Drug Metab Dispos 32: 178–185.
    OpenUrlAbstract/FREE Full Text
  17. ↵
    1. Klaassen CD
    1. Parkinson A
    (2001) Biotransformation of xenobiotics, in Casarett and Doul's Toxicology. The Basic Science of Poisons ( Klaassen CDed) pp 133–224, McGraw-Hill, New York.
  18. ↵
    1. Quinn GP,
    2. Axelrod J,
    3. Brodie BB
    (1958) Species, strain and sex differences in metabolism of hexobarbitone, amidopyrine, antipyrine and aniline. Biochem Pharmacol 1: 152–159.
    OpenUrlCrossRef
  19. ↵
    1. Rettie AE,
    2. Fisher MB
    (1999) Transformation enzymes: oxidative; non-P450, in Handbook of Drug Metabolism (Woolf TF ed) Marcel Dekker, New York.
  20. ↵
    1. Rollema H,
    2. Guanowsky V,
    3. Mineur YS,
    4. Shrikhande A,
    5. Coe JW,
    6. Seymour PA,
    7. Picciotto MR
    (2009) Varenicline has antidepressant-like activity in the forced swim test and augments sertraline's effect. Eur J Pharmacol 605: 114–116.
    OpenUrlCrossRefPubMed
  21. ↵
    1. Sadeque AJ,
    2. Thummel KE,
    3. Rettie AE
    (1993) Purification of macaque liver flavin-containing monooxygenase: a form of the enzyme related immunochemically to an isozyme expressed selectively in adult human liver. Biochim Biophys Acta 1162: 127–134.
    OpenUrlCrossRefPubMed
  22. ↵
    1. Schaefer WH
    (1992) Formation of a carbamoyl glucuronide conjugate of carvedilol in vitro using dog and rat liver microsomes. Drug Metab Dispos 20: 130–133.
    OpenUrlPubMed
  23. ↵
    1. Schaefer WH
    (2006) Reaction of primary and secondary amines to form carbamic acid glucuronides. Curr Drug Metab 7: 873–881.
    OpenUrlCrossRefPubMed
  24. ↵
    1. Shaffer CL,
    2. Ryder TF,
    3. Venkatakrishnan K,
    4. Henne IK,
    5. O'Connell TN
    (2009) Biotransformation of an α4β2 nicotinic acetylcholine receptor partial agonist in Sprague-Dawley rats and the dispositional characterization of its N-carbamoyl glucuronide metabolite. Drug Metab Dispos 37: 1480–1489.
    OpenUrlAbstract/FREE Full Text
  25. ↵
    1. Smith DA
    (1991) Species differences in metabolism and pharmacokinetics: are we close to an understanding? Drug Metab Rev 23: 355–373.
    OpenUrlPubMed
  26. ↵
    1. Soroka CJ,
    2. Lee JM,
    3. Azzaroli F,
    4. Boyer JL
    (2001) Cellular localization and up-regulation of multidrug resistance-associated protein 3 in hepatocytes and cholangiocytes during obstructive cholestasis in rat liver. Hepatology 33: 783–791.
    OpenUrlCrossRefPubMed
  27. ↵
    1. Straub K,
    2. Davis M,
    3. Hwang B
    (1988) Benzazepine metabolism revisited. Evidence for the formation of novel amine conjugates. Drug Metab Dispos 16: 359–366.
    OpenUrlAbstract
  28. ↵
    1. Tremaine LM,
    2. Stroh JG,
    3. Ronfeld RA
    (1989) Characterization of a carbamic acid ester glucuronide of the secondary amine sertraline. Drug Metab Dispos 17: 58–63.
    OpenUrlAbstract
    1. Villanueva SS,
    2. Ruiz ML,
    3. Ghanem CI,
    4. Luquita MG,
    5. Catania VA,
    6. Mottino AD
    (2008) Hepatic synthesis and urinary elimination of acetaminophen glucuronide are exacerbated in bile duct-ligated rats. Drug Metab Dispos 36: 475–480.
    OpenUrlAbstract/FREE Full Text
  30. ↵
    1. Xiong H,
    2. Turner KC,
    3. Ward ES,
    4. Jansen PL,
    5. Brouwer KL
    (2000) Altered hepatobiliary disposition of acetaminophen glucuronide in isolated perfused livers from multidrug resistance-associated protein 2-deficient TR(−) rats. J Pharmacol Exp Ther 295: 512–518.
    OpenUrlAbstract/FREE Full Text
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Research ArticleArticle

Species Differences in the Biotransformation of an α4β2 Nicotinic Acetylcholine Receptor Partial Agonist: The Effects of Distinct Glucuronide Metabolites on Overall Compound Disposition

Christopher L. Shaffer, Mithat Gunduz, Tim F. Ryder and Thomas N. O'Connell
Drug Metabolism and Disposition February 1, 2010, 38 (2) 292-301; DOI: https://doi.org/10.1124/dmd.109.030171
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