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METABOLISM, PHARMACOKINETICS, TISSUE DISTRIBUTION, AND EXCRETION OF [¹⁴C]CP-424391 IN RATS

Drug Metabolism and Pharmacokinetics, Genentech, Inc., South San Francisco, California (S.C.K.-B.); and Pfizer Global Research and Development, Groton, Connecticut (J.P.O., M.J.P.)

(Received June 18, 2004; accepted October 1, 2004)

	Abstract

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CP-424391, 2-amino-N-[3aR-benzyl-2-methyl-3-oxo-2,3,3a,4,6,7-hexahydro-pyrazolo[4,3-c]pyridin-5-yl)-1R-benzyloxymethyl-2-oxoethyl]-isobutyramide, is an orally active growth hormone secretagogue currently being developed. In this study, we investigated the metabolic fate and disposition of radiolabeled CP-424391 in rats. Following 15 mg/kg single oral administration to Sprague-Dawley rats, 91% of the radiolabeled dose was recovered. Feces was the major route of excretion: 77% of the dose recovered in feces of the female rat and 84% in the male. Excretion in the urine was 15% in the female rat compared with 7% in the male. Both fecal and urinary metabolic profiles were consistent in both genders. The metabolic pathways of CP-424391 were oxidation at the benzyl group of the O-benzylserine moiety, N-demethylation of pyrazolidine, and/or O-debenzylation. In circulation, CP-424391 was absorbed within the first hour to an average apparent C_max of 1.44 µg/ml. CP-424391 accounts for about 40% of radioactivity area under the plasma concentration-time curve and C_max in circulation. The plasma terminal elimination half-life of CP-424391 was 2.4 h and for total radioactivity was 2.8 h. The radioactivity was widely distributed in all tissues except for the central nervous system. [¹⁴C]CP-424391 radioactivity was eliminated from most tissues by 9 h with the exception of liver, skin, and uvea. By 168 h, [¹⁴C]CP-424391 radioactivity remained localized only in the uvea.

CP-424391 belongs to the pyrazolidine-piperidine class of compounds currently being developed as an orally active GH secretagogue for the treatment of growth hormone-compromised states. Its pharmacological mechanism of action is the elevation of circulating GH levels by stimulation of GH release from the pituitary gland (Pan, 2000). The objective of the present study was to investigate the metabolic fate and disposition of CP-424391 in rats.

	Materials and Methods

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Chemicals. CP-424391 and radiolabel, [¹⁴C]CP-424391, were synthesized at Pfizer (Groton, CT) (Fig. 1). [¹⁴C]CP-424391 had a specific activity of 4.97 mCi/mmol with radiochemical purity >99%.

View larger version (15K): [in this window] [in a new window]   FIG. 1. The structure of CP-424391.

Animal Studies. Sprague-Dawley rats were used for metabolism, plasma pharmacokinetics, and excretion studies, and Long-Evans rats were used for the whole-body autoradioluminography study. Study animals were acclimated to standard housing and environmental conditions in communal cages and rooms where light cycles, temperature, and humidity were documented daily for 2 days before experimentation. The rats were fasted overnight before dosing and were given free access to food and water throughout the studies.

Metabolism, Pharmacokinetics, and Excretion Studies. Dose Preparation and Administration. The dose was prepared by combining a solid uniform powder of [¹⁴C]CP-424391 tartrate salt with unlabeled CP-424391 tartrate salt in a solution of 100% water. The concentration of the dose was 1.5 mg/ml free base (Fig. 2; Table 1). For material balance studies, six rats (three per sex) were administered single 15 mg/kg (free base equivalent; 20 µCi/animal) oral doses of [¹⁴C]CP-424391 by gavage. The animals were placed in separate metabolism cages. For circulation studies, six rats (three per sex) equipped with jugular vein catheters were used.

View larger version (17K): [in this window] [in a new window]   FIG. 2. The mean plasma concentrations of radioactivity and CP-424391 in plasma following oral administration of [14C]CP-424391 at 15 mg/kg (free base).

Sample Collection. The day before dosing, urine and feces were collected for the background count in these matrices. Following oral administration of [¹⁴C]CP-424391, urine and feces were quantitatively collected into preweighed sample jars. Urine and feces were collected at 0 to 24, 24 to 48, 48 to 72, 72 to 96, 96 to 120, 120 to 144, and 144 to 168 h postdose (HPD). All matrices were stored at -20°C prior to analysis. In the circulation study, blood samples (0.7 ml) were obtained and processed for plasma at 0.25, 0.5, 1, 2, 4, 8, 12, and 24 HPD. Aliquots (50 µl) of plasma were used for circulating radioactivity determination and another 50-µl aliquot was used for CP-424391 concentration determination.

Quantitation of Radioactivity. The radioactivity at each time point in urine and plasma was determined by quantifying aliquots of these matrices in triplicate by liquid scintillation counting. Samples were combined with Eco-Lite scintillation cocktail (5 ml; Valeant Pharmaceuticals, Costa Mesa, CA) and counted using a Wallac Liquid Scintillation Counter Model 1409 (PerkinElmer Wallac, Turku, Finland). Radioactivity in feces was determined by combusting the samples. Feces collected at each sampling time were placed directly into tarred stomacher bags, hydrated with water, and homogenized. Triplicate aliquots (50-100 mg) of each slurry were weighed into oxidizer sample cups and dried overnight before combustion. The samples were oxidized using a model 306 oxidizer (PerkinElmer Life and Analytical Sciences, Boston, MA), and the liberated [¹⁴C]CO₂ was trapped by Carbosorb E (PerkinElmer Life and Analytical Sciences). Scintillation cocktail Permafluor V (PerkinElmer Life and Analytical Sciences) was added to the samples and counted. All samples were counted on the PerkinElmer Wallac Liquid Scintillation Counter using an internal quench curve and a 2- value of 4 (95% confidence). Counting efficiencies for carbon-14 in urine and fecal samples were generally 93% and 73%, respectively. Burning efficiencies were >95% throughout the sample oxidation analysis. Predose urine, feces, and plasma samples were counted for background count rate for each matrix. The amount of radioactivity in urine and feces was expressed as a percentage of the total amount of radioactivity administered to each animal. The amount of radioactivity in the plasma was expressed as µg-Eq of CP-424391/ml and was calculated by using the specific activity of the dose administered. The lower limit of quantification (LLOQ) was considered to be twice the background rate.

Profiling, Identification, and Quantitation of Metabolites. Radiolabeled material in urine, feces, and plasma was analyzed by reverse phase HPLC. The HPLC system consisted of a gradient pump (Hewlett Packard Analytical Direct, Wilmington, DE) on-line with a ß-radioactivity detector (ß-RAM; IN/US Systems Inc., Pine Brook, NJ) equipped with a 500-µl flow cell. Chromatography was carried out on a reverse phase Zorbax Rx C-18 column (4.6 x 150 mm, 3 µm; Hewlett-Packard Analytical Direct) utilizing a binary gradient of a mobile phase consisting of a mixture of ammonium formate (10 mM plus 1% formic acid) (solvent A) and acetonitrile (solvent B). The flow rate was 1.0 ml/min and the separation was achieved at ambient temperature. The gradient for the separation of metabolites in all of the matrices was programmed as follows. The gradient was programmed to increase at a constant rate from 10% to 70% B in 30 min. The column was re-equilibrated to 10% B for 10 min before the next injection. Under the gradient conditions, the retention time of the unchanged drug was 13.0 min. Recovery off the column was >95% for all the matrices.

Urine and fecal homogenate samples were pooled relative to excreted volume/mass at each time point so that greater than 90% of excreted radioactivity was accounted for in each matrix. Pooled urine was analyzed directly after centrifugation at 2100g for 10 min. Pooled fecal homogenates were extracted with two acetonitrile washing steps (3 ml/g) and concentrated overnight under nitrogen gas at 40°C. Plasma samples were pooled from 0.25- to 4-h time points to account for >90% radioactivity. Acetonitrile (4x volume) was added to pooled plasma samples, followed by isolation and evaporation of the supernatant. Recoveries of radioactivity from the pooled plasma samples were greater than 88%. Concentrated plasma and fecal extracts were reconstituted in 100 µl of 90:10 ammonium formate (10 mM plus 1% formic acid)/acetonitrile before analysis. A radiochromatogram was obtained for each pooled matrix using on-line radioactivity detection (ß-RAM). Integration of the radioactive peaks provided quantitative assessment of each metabolite as a percentage of total radioactivity in each sample.

Characterization of Metabolites. Metabolites in the urine, feces, and plasma were characterized using a SCIEX API 2000 mass spectrometer (PerkinElmer-Sciex Instruments, Boston, MA) using the same gradient system as described above. Postcolumn effluent was split 95:5 and introduced into the atmospheric ionization source via an ion spray interface at a rate of 50 µl/min. The remaining effluent was directed to a ß-RAM detector allowing simultaneous detection of radioactivity. The mass spectrometer was operated in the positive ion mode and the ion-spray interface was operated at 4500 V. Collision-induced dissociation (CID) studies were performed using nitrogen gas at a collision energy of 40 electron volts. The parameters for the SCIEX API 2000 triple quadrupole mass spectrometer were optimized using a synthetic standard of CP-424391 (1 µg/ml solution) in the positive ion mode.

CP-424391 Plasma Concentrations. Plasma samples were analyzed for CP-424391 using HPLC/tandem mass spectrometry. The internal standard (an analog of CP-424391; 50 µl of 100 ng/ml solution in water) and sodium hydroxide solution (25 µl of 1.0 M solution) were added to 50 µl of plasma sample and mixed thoroughly. Analytes were extracted from the samples using 1.5 ml of ethyl acetate. The samples were vortexed for 2 min and centrifuged at 2100g for 3 min. The organic layers were removed and evaporated to dryness under nitrogen gas at 50°C. The sample residues were reconstituted in 100 µl of 50:50 acetonitrile/water prior to analysis. Chromatography was carried out on a Monitor C18 column (30 x 4.6 mm; 3 µm) under an isocratic condition consisting of 50:50 ammonium acetate (10 mM)/acetonitrile. The flow rate was 0.5 ml/min and the run time was 2 min. Under these conditions CP-424391 and internal standard eluted at 0.9 and 1.0 min, respectively. CP-424391 and the internal standard were detected by single reaction monitoring of the reactions m/z 506 to 244 and m/z 611 to 313, respectively. These single reaction monitoring reactions were based on the collision-induced dissociation spectra of CP-424391 and the internal standard. The mass spectrometer was operated using the same conditions as previously described. CP-424391 was quantitated by weighted (1/x²) linear regression of drug to the internal standard peak area ratio. The lower and upper limits of quantitation were 5 ng/ml and 1000 ng/ml, respectively.

Pharmacokinetic analysis. Pharmacokinetic parameters were calculated from the plasma concentration data. The apparent maximum plasma concentration (C_max) and the time at which this concentration was achieved (T_max) were taken directly from the concentration data. The area under the plasma concentration-time curve, AUC_0-tlast, was calculated from time 0 to the last quantifiable time point, using linear trapezoidal approximation. The plasma terminal elimination rate constant (K_el) was estimated by regression of the plasma concentration data from the apparent beginning of the elimination phase to the last sample point. Terminal elimination half-life (t_1/2) was calculated by ln2/K_el relationship. The area from t_last to infinity was estimated as C_est(tlast)/K_el, where C_est(tlast) represents the estimated concentration at the last time point in which drug was quantitated based on the regressional analysis. The total area under the curve (AUC_0-) was estimated as the sum of AUC_0-tlast and AUC_tlast-. For the purpose of calculating mean drug concentrations at each sampling time, concentrations less than the LLOQ were considered to be 0 ng/ml; a mean was not calculated when 50% or more of the concentrations were <LLOQ.

Whole-Body Autoradioluminography (WBAL). Tissue distribution of [¹⁴C]CP-424391 was investigated in female and male Long-Evans rats (Charles River Laboratories, Raleigh, NC) following oral administration. [¹⁴C]CP-424391 was dissolved in sterile water at concentrations of 1.5 mg/ml and 13.29 µCi/ml. Specific activity of this [¹⁴C]CP-424391 dose formulation was 4.97 mCi/mmol.

Rats were individually housed in standard rodent cages with ad libitum access to water and feed. [¹⁴C]CP-424391 was orally administered to female rats (177 ± 4 g body weight) at a dose of 15.2 ± 0.03 mg/kg and 135 ± 3 µCi/kg. [¹⁴C]CP-424391 was orally administered to male rats (182 ± 12 g body weight) at a dose of 14.7 ± 0.4 mg/kg and 131 ± 4 µCi/kg. One rat of each gender was euthanized by CO₂ asphyxiation at 0.5, 1, 3, 9, and 168 h post dose. Immediately following euthanasia each rat was prepared for WBAL by immersion into a freezing chamber (-75°C) containing dry ice and hexanes for 10 min.

The whole-body cryosectioning technique developed by Ullberg (1977) was used to acquire whole-body cryosections for autoradioluminography. Each rat was individually embedded onto a microtome stage with 3% carxboxymethyl cellulose (Sigma, St. Louis, MO). Cryosection quality control samples (CQCS) of four known radioactivity concentrations prepared with rat blood were embedded in triplicate into each specimen block of frozen carxboxymethyl cellulose (Potchoiba et al., 1998). CQCS are essential for quantitative measurement of sensitivity and the foundation for determining the error associated with storage phosphor technology and quantification of this WBAL analytical method. After equilibration to -18°C, each specimen block containing CQCS was cryosectioned at 25 µm using a heavy-duty microtome housed in a CM3600 Cryomacrocut (Leica Microsystems, Deerfield, IL). Sagittal whole-body cryosections were acquired directly onto Type 810 Scotch tape (3M, St. Paul, MN). Seven cryosections were acquired from each sectioning level (5-7 levels) until the acquisition of all tissues and organs presented in Table 2. Typically, cryosections from three rats can be hung within the Cryomacrocut for dehydration. The duration of the dehydration cycle for 168 cryosections is approximately 90 h. After dehydration, each cryosections is identified with radioactive ink and covered with 0.5 mil Mylar (Fralock Inc., Canoga Park, CA).

Four cryosections from each sectioning level for each rat along with a 48-point (16-standards in triplicate) calibration curve were apposed to background erased storage phosphor screens (Molecular Dynamics, Sunnyvale, CA). Storage phosphor screens were placed in a lead and copper lined cabinet for 4-days to minimize background signals resulting from environmental radiation. Storage phosphor screens were scanned with a STORM PhosphorImager (Molecular Dynamics, Sunnyvale, CA) for the acquisition of electronic images. The MicroComputer Imaging Device (MCID; Imaging Research Inc., St. Catharines, Ontario, Canada) was used to quantify the concentration of radioactivity in calibration curve standards, CQCS, and tissues of whole-body cryosections as described by Potchoiba et al. (1995). The lower (LLOQ) and upper limits of quantification were previously determined (Potchoiba et al., 1995, 1998) using quality control samples across a radioactivity concentration range similar to the calibration curve calibrations. Calibration curves for each storage phosphor screen were generated by weighted (1/x) linear regression analysis. The linear regression curve was then utilized to assess the radioactivity concentration in the CQCS and to determine the concentration (nCi/g) of unknown radioactivity in tissues and organs of whole-body cryosection electronic images. Calibration curves were linear over the range of 5.7 to 22,950 nCi/g. The mean (± S.D.) correlation coefficient for 55 calibration curves was 0.9997 ± 0.0002. The precision (coefficient of variation) for CQCS did not vary by more than 11% (7 ± 1%) from nominal concentrations. CQCS accuracy (relative error) ranged from 97 to 108% (102 ± 2%) of nominals. Tissue concentrations of radioactivity (nCi/g) were converted to µg equivalents/g (µg Eq/g) by using the [¹⁴C]CP-424391 specific activity of dose formulation. Whole-body images obtained from Long-Evans rats presented in Figs. 3 to 6 were generated using ImageQuant Software (Molecular Dynamics, Sunnyvale, CA). Color and increasing intensity of a particular color (gray<red<orange<yellow<green<blue<black) corresponds to increasing concentrations of drug radioequivalents.

View larger version (52K): [in this window] [in a new window]   FIG. 3. Distribution of [14C]CP-424391 radioequivalents at 0.5 h after an oral dose of 15.2 mg/kg [14C]CP-424391 to a female Long-Evans rat. Note the high concentrations of radioequivalents in GIT, liver, lung, salivary gland, spleen, and uvea. Note the low concentrations of radioequivalents in adipose, blood, and cerebrum.

View larger version (148K): [in this window] [in a new window]   FIG. 6. Distribution of [14C]CP-424391 radioequivalents at 168 h after an oral dose of 14.7 mg/kg [14C]CP-424391 to a male Long-Evans rat. Note the high concentrations of radioequivalents in the uvea and the lack of radioequivalents in all other tissues.

	Results

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Pharmacokinetics. Pharmacokinetic parameters of plasma demonstrate no significant gender differences. The mean apparent C_max of total radioactivity and CP-424391 was 3.6 µg-equivalent/ml and 1.4 µg/ml, respectively. The C_max, of total radioactivity and unchanged drug were achieved within the first hour post dose. The AUC_0- of the total radioactivity and CP-424391 was 6.6 µg-Eq·h/ml and 2.5 µg·h/ml, respectively. The plasma terminal elimination half-life of total radioactivity and CP-424391 were 2.8 and 2.4 h, respectively.

Metabolism and Excretion Study. Following an oral administration of [¹⁴C]CP-424391, a mean of 91% of the radioactive dose were recovered (Table 3). Most of the radioactivity was excreted within the first 48 HPD with the feces being the major route of elimination. In feces, a mean of 84.1% was recovered in the male rat and 76.6% in the female rat. In urine, the male rat excreted 6.6% and the female 14.8%.

Identification of Metabolites. Under the chromatographic conditions described, CP-424391 has a retention time of 13.0 min. In feces, CP-424391 represents 19% of the dose in the female rat and 31% in the male (Table 4). The unchanged drug in urine of the female rat was 5.3% to 1.9% in the male. In a positive ion mode, CP-424391 generated a parent ion signal at m/z 506 ([M + H]⁺). A CID mass spectrum of the m/z 506 generated product ions at m/z 215, 235, 244, 263, and 421 (Fig. 7). Product m/z 421 is formed from the neutral loss of -methylalanine from the parent ion. The ion at m/z 263 is rationalized by cleavage of the amide bond containing -methylalanyl-O-benzylserine moiety. Further loss of a carbonyl group from the ion at m/z 263 resulted in m/z 235 generation. The product ion at m/z 244 corresponds to the pyrazolidine-piperidine part of the molecule cleaved at the amide bond. Further loss of NH = CH₂ by reverse alder reaction generates m/z 215.

View larger version (11K): [in this window] [in a new window]   FIG. 7. Collision-induced dissociation mass spectrum of CP-424391 in Sprague-Dawley rats at m/z 506 [M + H]+.

Metabolites in feces, urine and plasma were screened by precursor ion scans of m/z 235 and m/z 244. These ions were not only the most abundant ions in CP-424391, but also represent opposite sides of the compound (Fig. 7). Precursor ion scans of m/z 230 were run as well to account for possible N-demethylated metabolites.

Metabolite M1 had a retention time of 3.5 min. It accounted for 0.5% of the dose in both feces and urine (Table 4). In precursor ion scans of m/z 230, a molecular ion at m/z 402 ([M + H]⁺) was detected. The CID spectrum of m/z 402 generated major product ions at m/z 173, 230, and 317 (Table 5). The product ion m/z 230 was 14 amu less than the unchanged drug ion at m/z 244 and was rationalized as N-demethylation of the pyrazolidine. The product ions m/z 173 and 317 were the neutral loss of 90 amu from the unchanged drug m/z 263 and 407, respectively. This was consistent with the O-debenzylation of O-benzylserine. Based on these findings, it is proposed that M1 was formed by N-demethylation and O-debenzylation (Fig. 8).

View larger version (14K): [in this window] [in a new window]   FIG. 8. The metabolic scheme of CP-424391 in Sprague-Dawley rats.

Metabolite M2 had a retention time of 5.8 min. In urine, it accounted for 3.6% of the dose and in feces 14% (Table 4). In circulation, this metabolite accounted for 19% of the plasma radioactivity profile in the male rat as compared with 10% in the female (Table 6). In the precursor ion scan of m/z 244, a molecular ion at m/z 416 ([M + H]⁺) was detected. This was 90 amu less than the unchanged drug. The CID spectrum of m/z 416 generated major ions at m/z 215, 244 and 331 (Table 5). The product ions at m/z 244 and 215 suggested no modification to the pyrazolidine-piperidine half of the compound. The product ion at m/z 331 corresponded to the neutral loss of unchanged -methylalanine. This was consistent with the loss of benzyl group from O-benzylserine. Based on these finding, it is proposed that M2 was formed by O-debenzylation (Fig. 8).

Metabolite M3 had a retention time of 7.9 min. It accounted for 2.6% of the dose in feces (Table 4). In a precursor ion scan of m/z 230, the molecular ion at m/z 508 ([M + H]⁺) was detected. This was 2 amu higher than the unchanged drug. The CID spectrum of m/z 508 generated product ions at m/z 173, 230, and 402 (Table 5). Similar to M1, M3 generated the product ion m/z 230, which suggested N-demethylation of the pyrazolidine. The product ion m/z 402 was a neutral loss 106 amu from the molecular ion. This was rationalized by a loss of hydroxylated benzyl group. The product ion at m/z 173 was the neutral loss of pyrazolidinepiperidine from m/z 402. Based on these finding, it was proposed that M3 was formed by N-demethylation and hydroxylation of O-benzylserine (Fig. 8).

Metabolite M4 had a retention time of 8.5 min. It accounted for 2.4% of the dose in feces. In a precursor ion scan of m/z 244, a molecular ion at m/z 538 ([M + H]⁺) was detected. The increase of 32 amu to unchanged drug could be rationalized by two hydroxylations of the compound. The CID spectrum of m/z 538 generated major ions at m/z 123, 173, 244 and 331 (Table 5). The product ions at m/z 244, 331, and 173 isolated the modification to benzyl group of the O-benzylserine moiety. The tropylium product ion at m/z 123 was consistent with two hydroxylations as well. Based on these finding, it was proposed that this metabolite was formed by two hydroxylation on O-benzylserine moiety (Fig. 8).

Metabolite M5 had a retention time of 9.9 min. It accounted for 0.3% of the dose in urine and 16% in the feces (Table 4). In a precursor ion scan of m/z 244, the molecular ion was detected at m/z 522 ([M + H]⁺). The molecular ion was consistent with a single hydroxylation to unchanged drug. A CID spectrum of m/z 522 generated major ions at m/z 107, 145, 173, 244, 279, 331, and 416 (Table 5). The product ion at m/z 244 suggested no modifications to the pyrazolidine-piperidine half of the molecule. The product ion at m/z 279 was 16 amu higher than the unchanged drug m/z 263. The product ion at m/z 416 was 14 amu higher than m/z 402 from M3 and it positions the hydroxylation site on the benzene group of O-benzylserine. This was confirmed by the formation of tropolium ion m/z 107. M5 has a common CID ion m/z 173, which corresponds to unchanged -methylalanine and serine. Based on these finding, it was proposed that this metabolite was formed by hydroxylation on O-benzylserine moiety (Fig. 8).

Metabolite M6 had a retention time of 10.2 min. It accounted for 1.8% of the dose in feces (Table 4). In a precursor ion scan of m/z 244, the molecular ion at m/z 552 ([M + H]⁺) was detected. The CID spectrum of m/z 552 generated major product ions at m/z 137, 173, 215, 244, and 331 (Table 5). Similar to metabolite M4, the product ions at m/z 244 and 173 isolated the modification to benzyl group of the O-benzylserine moiety. The tropylium ion at m/z 137 was consistent with two hydroxylations followed by methylation of the catechol, presumably by catechol O-methyl transferase. Based on these findings, it was proposed that this metabolite was formed by two hydroxylations on the O-benzylserine moiety followed by methylation of one of these hydroxyl groups (Fig. 8).

Metabolite M7 had a retention time of 11.3 min. It accounted for 0.3% of the dose in urine and 2.5% in feces (Table 4). In a precursor ion scan of m/z 230, a molecular ion at m/z 492 ([M + H]⁺) was detected. The CID spectrum of m/z 492 generated major ions at m/z 91, 201, 230, 235, and 407 (Table 5). Similar to metabolite M1, the product ions at m/z 230 and 201 suggested N-demethylation of unchanged drug. The product ion at m/z 235 suggests no modifications to the -methylalanyl-O-benzylserine moiety. The product ion observed at m/z 407 was also consistent with N-demethylation of the benzylpiperidine half of the compound as well. Based on these findings, it was proposed that this metabolite was formed by N-demethylation (Fig. 8).

Metabolite M8 had a retention time of 11.8 min. It accounts for 0.06% of the dose in urine and 1.9% in feces (Table 4). In a precursor ion scan of m/z 244, a molecular ion at m/z 432 ([M + H]⁺) was detected. The loss of 74 amu from unchanged drug could be rationalized as O-debenzylation and a single hydroxylation. The CID spectrum of m/z 432 generated major ions at m/z 244, 331, and 418 (Table 5). The product ion at m/z 244 suggested no modification the benzylpiperdine side of the compound. The product ion at m/z 331 localized the hydroxylation to the -methylalanine moiety of the compound and suggests O-debenzylation. Based on these findings, it was proposed that M8 was formed by O-debenzylation and hydroxylation of the methylalanine (Fig. 8).

WBAL. [¹⁴C]CP-424391 radioequivalents were absorbed and distributed into 24 tissues of the Long-Evans female and male rat at 0.5 h after a single, oral bolus dose (Table 2). Drug radioequivalents did not distribute into ocular tissues devoid of melanin or into tissues of the central nervous system except for the pituitary. The pituitary, which resides behind a leaky blood-brain barrier, was devoid of radioactivity by 9 h and 3 h in the female and male rat, respectively. In the female rat, maximum concentrations (C_max) of drug radioequivalents occurred at 0.5 h (Fig. 3) for all tissues except the uvea (1 h) and skin (9 h). C_max concentrations of [¹⁴C]CP-424391 radioequivalents occurred at 0.5 h (Fig. 4) for 11 tissues, 1 h for 12 tissues, and 3 h for skin in the male rat. Drug radioequivalents were sustained in blood for 1 h for both rat genders, but only for the male rat were hepatic blood concentrations of drug radioequivalents (0.68 µg Eq/g) slightly above the LLOQ of 0.57 µg Eq/g at 3 h. By 3 h, [¹⁴C]CP-424391 radioequivalents declined below the LLOQ for 9 and 15 of 24 tissues in the female and male rat, respectively. Drug radioequivalents were present in liver, skin, and uvea at 9 h in rats of both genders. By 168 h, [¹⁴C]CP-424391 radioequivalents were present in only the uvea of both rat genders, indicating an affinity for melanin (Figs. 5 and 6).

View larger version (49K): [in this window] [in a new window]   FIG. 4. Distribution of [14C]CP-424391 radioequivalents at 0.5 h after an oral dose of 14.7 mg/kg [14C]CP-424391 to a male Long-Evans rat. Note the high concentrations of radioequivalents in GIT, liver, salivary gland, spleen, and uvea. Note the low concentrations of radioequivalents in adipose, blood, cerebrum, and testis.

View larger version (143K): [in this window] [in a new window]   FIG. 5. Distribution of [14C]CP-424391 radioequivalents at 168 h after an oral dose of 15.2 mg/kg [14C]CP-424391 to a female Long-Evans rat. Note the high concentrations of radioequivalents in the uvea and the lack of radioequivalents in all other tissues.

The highest concentrations of [¹⁴C]CP-424391 radioequivalents at 1, 3, and 9 h were located in the contents of the gastrointestinal tract (GIT). This presence of radioequivalents in GIT contents probably represented unabsorbed drug as well as contents from the elimination of radioequivalents in bile or via intestinal secretion. For tissues in the female rat, the highest concentrations of [¹⁴C]CP-424391 radioequivalents occurred at 0.5 h in 14 tissues over the range of 11 to 23 µg Eq/g. Ten additional tissues in the female rat at 0.5 h had radioequivalent concentrations from 2.9 to 8.6 µg Eq/g. In the male rat, only the Harderian gland and liver had double-digit concentrations of 11 and 20 µg Eq/g, respectively. All other tissues in the male rat at 0.5 h had radioequivalent concentrations from 0.92 to 9.5 µg Eq/g. Except for blood, brown adipose, liver, pancreas, skin, and the whole body, all other tissues in the female rat at 0.5 h had 2 to 3 times higher concentrations of drug radioequivalents than those observed for male tissues. At 1 h, tissue concentrations of drug radioequivalents were similar between genders. The testis had the lowest concentration at all time points for any tissue, with measurable levels of drug radioequivalents. These observations may be indicative of a higher first-pass hepatic metabolism in the male rat compared with the female rat.

Exposure to [¹⁴C]CP-424391 radioactivity was greatest for the uvea, based on tissue AUC_(0-Tlast) values regardless of gender (Table 7). Tissue exposure to [¹⁴C]CP-424391 radioactivity was 2 to 5 times greater in adrenal gland, bone marrow, kidney, lymph node, muscle, myocardium, pancreas, pituitary, spleen, and thyroid of the female rat than in those observed for the male rat. The uvea had a projected total radioactivity t_1/2 of 135 and 92 h for the female and male rats, respectively. Testis had the second greatest t_1/2 of 17 h. Half-lives for whole-body total radioactivity were 3.3 and 4.3 h for the female and male rats, respectively. For all other tissues, where half-lives could be determined, t_1/2 ranged from 0.6 to 2.5 h.

	Discussion

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In the present study, the feces was the major route of excretion in Sprague-Dawley rats following a single 15 mg/kg oral dose of [¹⁴C]CP-424391. Mass balance was generally achieved within the first 48 h of the study (recovery ranging from 84 to 99%). Gender differences were observed in the quantity of radioactive dose excreted in each matrix, but not in the total amount recovered. Excretion in urine was higher in the female rat compared with the male.

CP-424391 was extensively metabolized. Only 25% of the radioactive dose in feces was unchanged drug. The females excrete a lesser amount of the dose in feces compared with the male. At the same time, of that recovered in feces, 19% is unchanged drug in females compared with 31% in the male. The difference could be the possible gender-dependent bioavailability differences of this compound in rats. The metabolic pathways for CP-424391 involved oxidation of the benzyl group of the O-benzylserine moiety and O-debenzylation to form metabolites M2 and M5. N-Demethylation of the pyrazolidine ring was the minor metabolic pathway, which generates metabolite M7. Further metabolism of these metabolites leads to secondary metabolites with combination of N-demethylation, O-debenzylation, and phenylic hydroxylation. Another metabolite is formed by oxidation of M5 to M4 by oxidation of the benzyl. Methylation of this metabolite led to M6, possibly by catechol-O-methyltransferase.

The [¹⁴C]CP-424391 radioequivalents distributed into most tissues of the female and male Long-Evans rats except for central nervous system tissues and ocular tissues lacking melanin. The absence of radioequivalents in central nervous system tissues (except the pituitary) illustrated a poor permeability of the blood-brain barrier for this compound and/or related metabolites. The distribution appeared to be rapid, with the maximal concentrations occurring in most tissues at 0.5 h for the female rat and at either 0.5 or 1 h for the male rat. In general, tissue drug radioequivalents at 0.5 h were 2 to 3 times higher in concentration and tissue exposures to [¹⁴C]CP-424391 radioactivity in 10 tissues were 2 to 5 times greater in the female rat compared with the male rat. These slight gender differences in tissue concentrations and exposures probably resulted from a higher first-pass hepatic metabolism in the male rat. The highest concentrations of radioequivalents were found in contents of the GIT resulting from unabsorbed drug following oral administration or from biliary elimination of drug radioequivalents. The majority of [¹⁴C]CP-424391 radioequivalents were excreted within the first 9 h, which was consistent with mass balance data obtained from Sprague-Dawley rats. By 168 h, only the uvea still had measurable concentrations of drug radioequivalents.

The presence of radioactivity in the uvea of rats of both genders at 168 h indicated a slow elimination of drug radioequivalents from this ocular structure. Association of [¹⁴C]CP-424391 radioequivalents with the uvea probably resulted from the affinity of melanin-rich tissues for organic amines and polycyclic aromatic hydrocarbons. The retention or accumulation of xenobiotics having cationic properties by ocular tissues impregnated with melanin appears to be common (Larsson and Tjalve, 1979). Ocular toxicity observed in rats after the administration of chloroquine and chlorpromazine was related to the affinity of melanin for these drugs (Potts, 1962). However, chloroquine- and chlorpromazine-induced oculotoxicities (cataracts and thickening of the lens) not only affected ocular tissues that were impregnated with melanin (uvea and retina), but also affected ocular tissues (cornea and lens) that were devoid of melanin (Koneru et al., 1986). Other xenobiotics such as adrenaline, dopamine, and nicotine have a strong in vivo affinity for ocular melanin, but have no associated oculotoxicities (Potts, 1974). Thus, the affinity of a xenobiotic for melanin-bearing tissues of the eye apparently does not necessarily result in oculotoxicity. In fact, the physiological function(s) of melanin binding is not fully understood, and it has been suggested that melanin might serve as a protective chemical filter for preventing potentially harmful substances from reaching susceptible receptor cells (Larsson, 1993). Therefore, it must be emphasized that a drug's affinity for melanin-rich tissues of the eye does not necessarily indicate a relationship to oculotoxicity.

In conclusion, CP-424391 is readily distributed to tissues. It is metabolically eliminated by oxidation on the benzene ring of O-benzylserine, O-debenzylation, and N-demethylation. In addition, there are minor pathways involving further oxidation of the benzene ring of O-benzylserine and methylation of one of these hydroxyl groups. The major route of elimination of CP-424391 and its metabolites is excretion in feces.

	Acknowledgments

 
We thank Beth Obach for conducting the animal experiments, Kathleen Zandi and Michael Nesler for the synthesis of [¹⁴C]CP-424391, and Dr. Deepak Dalvie for critical reading of the manuscript.

	Footnotes

 
doi:10.1124/dmd.104.001065.

ABBREVIATIONS: CP-424391, 2-amino-N-[3aR-benzyl-2-methyl-3-oxo-2,3,3a,4,6,7-hexahydro-pyrazolo[4,3-c]pyridin-5-yl)-1R-benzyloxymethyl-2-oxo-ethyl]-isobutyramide; amu, atomic mass unit(s); CID, collision-induced dissociation; CQCS, cryosection quality control sample(s); GH, growth hormone; GIT, gastrointestinal tract; HPD, hours postdose; HPLC, high performance liquid chromatography; LLOQ, lower limit of quantification; WBAL, whole-body autoradioluminography.

¹ Retired.

Address correspondence to: Michael J. Potchoiba, Pfizer Inc., Global Research and Development, PDM Development, MS 4096, Eastern Point Road, Groton, CT 06340. E-mail: michael_j_potchoiba{at}groton.pfizer.com

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