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Pfizer Global Research and Development, Groton, Connecticut
(Received December 8, 2006; accepted March 12, 2007)
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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was 6.7 h and 45.0 h for EM and PM subjects, respectively. The serum concentrations of CP-122,721 reached a peak of 7.4 and 69.8 ng/ml for extensive and poor metabolizers, respectively. The major metabolic pathways of CP-122,721 were due to O-demethylation, aromatic hydroxylation, and indirect glucuronidation. The minor metabolic pathways included aliphatic oxidation at the piperidine moiety, O-dealkylation of the trifluoromethoxy group, N-dealkylation, and oxidative deamination. In addition to the major human circulating metabolite 5-trifluoromethoxy salicylic acid (TFMSA), all other circulating metabolites of CP-122,721 were glucuronide conjugates of oxidized metabolites. TFMSA was identified using high pressure liquid chromatography/tandem mass spectrometry and NMR and mechanisms were proposed for its formation. There are no known circulating active metabolites of CP-122,721.
; Nakayama et al., 2004; Herpfer et al., 2005).
Metabolism and pharmacokinetic studies in rat (Kamel et al., 2006) indicated that CP-122,721 is extensively metabolized, resulting in low drug concentrations in the systemic circulation. Most of the metabolic pathways of CP-122,721 were similar in both rats and humans. However, one additional route of metabolism was detected only in humans (not in rats), and this led to the formation of the major circulating metabolite 5-trifluoromethoxy salicylic acid (TFMSA). The metabolism of CP-122,721 was investigated in beagle dogs, another toxicology species used for safety studies, and routes of metabolism were determined. The formation of TFMSA metabolite was detected in dog plasma at considerably lower levels compared with humans, and results may suggest that dog is the most relevant preclinical species in which metabolism of CP-122,721 could be studied for extrapolating results to humans (A. Kamel, Y. Du, K. Colizza, and C. Prakash, unpublished results). There are no known circulating active metabolites of CP-122,721 including TFMSA.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Radiolabeled Drug and Reference Compounds. [14C]CP-122,721 (HCl salt; Fig. 1), labeled at the benzylic position of the trifluoromethoxy anisole moiety, was synthesized by the Radiochemistry group at Pfizer Global Research and Development (Groton, CT). [14C]CP-122,721 has a specific activity of 1.27 mCi/mmol and a radiochemical purity of 
99% as determined by radio-HPLC. CP-114,621 [(2S,3S)-2-phenyl-piperidin-3-ylamine], CP-125,611 [(2-[((2S,3S)-2-phenyl-piperidin-3-ylamino)-methyl]-4-trifluoromethoxy-phenol], and CP-677,623 (2-methoxy-5-trifluoromethoxy-benzylamine) were synthesized by the Medicinal Chemistry group at Pfizer Global Research and Development and served as synthetic standards to confirm the structures of metabolic pathway intermediates, where applicable.
Urinary and Fecal Excretion Study. Mass balance and excretion study was conducted in six healthy male human subjects [four extensive metabolizers (EMs) of CYP2D6, subjects 1-4; and two poor metabolizers (PMs) of CYP2D6, subjects 5-6). Human subjects were administered a single 30-mg (free base) oral dose of CP-122,721 containing 100 µCi of [14C]CP-122,721. Urine and feces were collected from the subjects in 24-h intervals for a minimum of 7 days to a maximum of 13 days postdose. The first urine sample was collected at 0 to 12 h post dose. All biological samples were stored at -20°C until analysis. Sample analysis was carried out within days after the completion date of the study and metabolite profiles were obtained on pooled samples.
Pooling Methods to Obtain Quantitative and Qualitative Data. It is essential that quantitative metabolite profiling represent accurate metabolite percentages relevant to AUC (or Cavg) rather than an arbitrary plasma pool.
For quantitative profiling of metabolites in excreta (urine, feces), samples were pooled from each individual in proportion to the amount (weight or volume) of excreta in each sampling period. For subsequent metabolite identification (qualitative analysis), the most concentrated samples were used to avoid unnecessary dilution of metabolites.
For quantitative profiling of metabolites in circulation, pooling was done according to the method whereby samples are pooled in proportion to the time interval each sample represents (Hamilton et al., 1981; Riad et al., 1991; Hop et al., 1998). Serum samples were pooled such that at least 80% of total radiolabel AUC is captured. For subsequent metabolite identification, the most concentrated samples were used to avoid dilution of circulating drug-related materials.
Analysis of Serum Metabolites. For serum profiles and metabolite identification, blood samples were collected into nonheparinized Vacutainers (BD Diagnostics, Franklin Lakes, NJ) at the following time points: 1, 4, 8, 12, and 24 h postdose. The blood samples were centrifuged at 4°C and serum was transferred to clean tubes. Serum samples were pooled as described above by human subject and stored at -20°C until analysis.
Determination of Radioactivity. Quantification of total radioactivity in urine and serum was determined by counting sample aliquots (50-100 µl) using a 14C program on either a Beckman Coulter model LS 3801 or model LS 5000TD liquid scintillation counter (Beckman Coulter Canada Inc., Mississauga, ON, Canada). Ecolite (+) scintillation cocktail (5 ml; MP Biomedicals) was used for determination of the radioactivity in the samples. Quench curves were prepared separately for [14C]CP-122,721.
Fecal samples were homogenized with 50:50 ethanol/water, and the total weights of the fecal homogenate were recorded. Aliquots (100-300 mg, in triplicate) of the homogenates were air-dried and combusted in a Harvey OX-300 or OX-500 biological sample oxidizer (R. J. Harvey Instrument Corporation, Hillsdale, NJ). The liberated 14CO2 was trapped in scintillation Carbon-14 Cocktail (15 ml; R. J. Harvey Instrument Corporation) and the radioactivity in the trapped samples was determined by counting in a liquid scintillation counter. Combustion efficiencies were determined by combusting 14C-labeled standards (Spec-Chec; PerkinElmer Life and Analytical Sciences, Boston, MA) in an identical manner. The samples obtained at predose were used as controls and counted to obtain a background count rate. The amount of radioactivity in the dose was expressed as 100% and the radioactivity in urine and feces at each sampling time was expressed as the percentage of dose excreted in the respective matrices at that sampling time.
Concentrations of Total Radioactivity and CP-122,721. Blood samples (10 ml) from each subject (n = 6) were collected into nonheparinized Vacutainers at the following time points: 0.5, 1, 2, 3, 4, 6, 8, 12, 24, 36, 48, 72, 96, 144, 196, and 240 h postdose and were centrifuged. The resulting serum samples were placed into clean tubes for shipment and subsequent analysis. Total radioactivity in serum was determined by counting 50-µl aliquots from each sample in duplicate using a 14C program on a PerkinElmer Wallac (Gaithersburg, MD) #1409 liquid scintillation counter. Ecolite (+) scintillation cocktail (
7.0 ml; MP Biomedicals) was used for the determination of radioactivity in the samples. Total radioactivity in the serum samples was converted into ng-Eq/ml based on the dose received by each patient (30 mg of CP-122,721, 100 µCi of 14C per patient). Serum concentrations of the unchanged CP-122,721 (ng/ml) were determined by a validated HPLC/MS/MS assay (Kamel et al., 1997).
Pharmacokinetic Analysis. Pharmacokinetic parameters were calculated using the WinNonLin software (V02.1A; Pharsight Corp., Mountain View, CA). The areas under the CP-122,721 and total radioactivity concentration-time curves (AUC) were calculated from serum concentrations of CP-122,721 and total radioactivity using a trapezoidal approximation of area, using zero as the time 0 concentration. Tmax was the time of the first occurrence of the maximal serum concentration (Cmax). Noncompartmental model analysis was used to estimate the pharmacokinetics parameters.
Extraction of Metabolites from Biological Samples. Urine samples were pooled (0-72 h for EM subjects and 0-192 h for PM subjects) as described above. The pooled samples were vortex-mixed, and 10 ml from each pool was evaporated to dryness in a turbo evaporator (TurboVap; Zymark, Hopkinton, MA). The residue was reconstituted in 0.5 ml of 5:95 acetonitrile/water, and then transferred to 1.5-ml Eppendorf tubes and centrifuged at 14,000 rpm for approximately 2 min. Aliquots (10 µl) of the supernatants were directly injected without further purification onto the liquid chromatography-accurate radioisotope counting (LC-ARC) system, which stopped the flow every 10 s and counted for 60 s. The LC system is described below.
A fraction of the total fecal sample mass from each human time point was pooled from 0 to 96, 0 to 48, 0 to 120, 72 to 168, 24 to 120, and 12 to 168 h for subjects 1, 2, 3, 4, 5, and 6, respectively. From the pooled samples (100-200 g), aliquots (10 g) were suspended in 25 ml of acetonitrile. Suspensions were sonicated (20 min), vortexed, and centrifuged at approximately 3200 rpm for 6 min. After the measured supernatant transfer to clean, 15-ml conical tubes, duplicate aliquots (100 µl) from each extraction were counted in a liquid scintillation counter. The total recovery of radioactivity averaged 94%. The supernatants were evaporated to dryness under nitrogen in a TurboVap LV evaporator operated at 37°C. The residues were reconstituted in 2 ml of mobile phase. Aliquots (100 µl) of concentrated fecal extracts were injected onto the LC-ARC system. Because of the very low levels of radioactivity in the feces, highly concentrated samples were necessary to obtain radiochromatograms. This presented numerous background problems for radiochemical detection. The problem was resolved on the LC-ARC system by allowing the first 5 min of the analysis to be performed with nonstop flow and then performing stop-flow analysis from 5 to 45 min.
Serum samples were pooled by human subject separately from each sampling time, 1, 4, 8, 12, and 24 h postdose, and aliquots of 1.5 ml from each pool were extracted with 5 ml of acetonitrile. The mixtures were vortex-mixed for 5 min and centrifuged at 3500 rpm for 5 min to remove the precipitated proteins. Supernatants were combined and a small aliquot of supernatant was counted. Approximately 96% (average of all subjects) of the radioactivity was recovered in the supernatant. The supernatants were evaporated under nitrogen in a TurboVap at room temperature. The residues were reconstituted with mobile phase and aliquots of 100 µl for each subject were injected on the HPLC system for profiling and metabolite identification.
Quantitative Assessment of Metabolite Excretion. Quantification of the metabolites was carried out by measuring radioactivity in the individual HPLC-separated peaks using a ß-RAM instrument (IN/US, Tampa, FL). The ß-RAM provided an integrated printout in cpm and the percentage of the radiolabeled material, as well as peak representation. The ß-RAM was operated in the homogeneous liquid scintillation counting mode with addition of 4 ml/min Ecolite scintillation cocktail to the eluent after UV detection. Urinary, fecal, and serum profiles were quantitatively very similar over time for at least 12 months, indicating sample stability in these matrices.
Enzyme Hydrolysis. Pooled human urine samples (10 ml) were adjusted to pH 5 with sodium acetate buffer (0.1 M) and treated with 2500 units of ß-glucuronidase/sulfatase. The mixture was incubated in a shaking water bath at 37°C for 12 h and was diluted with acetonitrile (10 ml). The precipitated protein was removed by centrifugation. The pellet was washed with an additional 2 ml of acetonitrile and both supernatants were combined. The supernatant was concentrated and dissolved in 0.5 ml of mobile phase, and an aliquot (50 µl) was injected on the HPLC apparatus. Incubation of the urine samples without the enzyme served as a control.
Formation, HPLC Isolation, and Purification of TFMSA Metabolite from [14C]CP-122,721 Incubation with Human Liver S9 Fraction. [14C]CP-122,721 (27 µM) was incubated with human liver S9 fraction (HL-1123; 37 mg/ml protein) in the presence of 100 mM potassium phosphate buffer, pH 7.4, and cofactor solution (9 mM MgCl2, 0.54 mM NADP, 6.2 mM DL-isocitric acid, and 0.5 U/ml isocitric dehydrogenase) in a total volume of 10 ml. The reaction mixture was initiated by the addition of the S9 fraction (1.35 ml, total of 50 mg of protein), and incubation was carried out in a shaking water bath at 37°C. The reaction mixture was incubated for 16 h and stopped by the addition of acetonitrile (5 ml). The mixture was vortex-mixed for 5 min and centrifuged at 3500 rpm for 5 min to remove the precipitated proteins. The supernatant was evaporated under nitrogen in a TurboVap at room temperature. The residue was reconstituted with mobile phase and aliquots of 100 µl were injected onto the HPLC system for purification. The 5-trifluoromethoxy salicylic acid metabolite was isolated and purified using the HPLC system described below. The purified HPLC fraction (27-30 min) was dried under nitrogen stream and stored at -20°C until use.
Chemical synthesis of 5-trifluoromethoxy salicylic acid. A novel metabolite was synthesized chemically by carboxylation and demethylation of 2-bromo-1-methoxy-4-trifluoromethoxy-benzene to serve as a standard for HPLC, NMR, and mass spectral studies (Fig. 2).
Synthesis of 2-methoxy-5-trifluoromethoxy-benzoic acid. To the solution of 2-bromo-1-methoxy-4-trifluoromethoxy-benzene (1.0 g, 3.69 mmol) in anhydrous ether (50 ml), at -78°C, under a nitrogen atmosphere, was added n-butyl lithium (0.738 ml, 1.85 mmol, 2.5 M solution in hexane) over 5 min, while magnetically stirring. After 15 min, anhydrous carbon dioxide was bubbled through the reaction mixture for 10 min. The reaction mixture was allowed to warm up to room temperature. The ethereal solution was washed with a 5% solution of aqueous sodium hydroxide (three times, 50 ml). The combined aqueous solution was acidified witha1N solution of hydrochloric acid (pH 2) and then extracted with ether (three times, 200 ml). The combined ethereal solution was dried over anhydrous sodium sulfate and concentrated to give 0.26 g of an off-white solid. No further purification was attempted. A sample of the off-white solid product, approximately 15 mg, was taken for structure confirmation by NMR as described below.
Synthesis of TFMSA. To a solution of the off-white solid product, 2-methoxy-5-trifluoromethoxy-benzoic acid (23.6 mg, 0.1 mmol) in anhydrous methylene chloride (0.9 ml), was added boron tribromide (0.100 ml, 1 M solution in methylene chloride) at 0°C. The reaction was allowed to continue for 16 h. The solution was made acidic (pH 0) usinga1N solution of hydrochloric acid and was extracted with ethyl acetate (three times, 3 ml). The ethyl acetate solution was dried over anhydrous sodium sulfate, filtered, and concentrated to give 11 mg of a white solid. No further purification was attempted. The structure identification of the white solid was accomplished using mass spectral analysis and standard NMR proton-proton experiments. Complete 1H chemical shifts were obtained and are reported in ppm. The white solid was used for NMR and MS analyses as described below.
LC/MS/MS. LC/MS/MS was conducted with a SCIEX API IIIplus (PerkinElmer, Thornhill, ON, Canada) equipped with an ionspray source. The effluent from the HPLC column was split and approximately 50 µl/min was introduced into the atmospheric pressure ionization source. The remaining effluent was directed into the flow cell of the ß-RAM. The ß-RAM response was recorded in real time by the mass spectrometer data system, which provided simultaneous detection of radioactivity and mass spectrometry data. The delay in response between the two detectors was about 0.2 min with the mass spectrometric response recorded earlier. The ionspray interface was operated at 6000 V and the mass spectrometer was operated in the positive mode. CID studies were performed using argon gas at a collision energy of 25 to 28 eV and a collision gas thickness of 3.5 x 1014 molecules/cm2.
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-Max model 481 UV detector, a radioactive monitor (ß-RAM), and an SP 4200 computing integrator. Chromatography was carried out on a Phenomenex Luna HPLC column [4.6 x 150 mm, 5 µm particle diameter, C-18(2)] with a binary mixture of acetonitrile (solvent A) and 10 mM ammonium acetate (pH 5.0 with acetic acid, solvent B). A flow rate of 1.0 ml/min was used for all analyses. The mobile phase initially consisted of solvent A/solvent B (5:95) for a 10-min period. It was then linearly programmed to achieve a mixture of solvent A/solvent B (40:60) over a 20-min period and operated isocratically for 5 min under these conditions. The mobile phase was then linearly programmed to achieve a mixture of solvent A/solvent B (80:20) over a 5-min period and operated isocratically for 5 min under these conditions. It was then linearly programmed to return to the initial mixture of solvent A/solvent B (5:95) over a 5-min period. This gave a total run time of 50 min with an additional time of 10 min for column reequilibration between injections. The retention times of radioactive peaks were compared with those of the synthetic standards.
LC-ARC. The LC-ARC system maintained the same chromatography conditions as above and was equipped with advanced stop-flow controller and ARC data system (AIM Research Co., Newark, DE). The flow cell (2.2-ml volume) and cocktail were obtained from AIM Research Co. The flow was stopped in 10-s intervals and counted for 60 s. The 10 s of flow (0.17 ml) mixes with enough Tru-count scintillation cocktail to fill the cell for any given stop-flow period. The background counting threshold is 30 cpm.
LC-ARC provides several advantages over traditional detection systems. The typical limit of detection of LC-ARC is 5 to 20 dpm for 14C and 10 to 40 dpm for 3H. The sensitivity is 10- to 20-fold better than that of traditional flow-through detection systems. The LC-ARC method dramatically improves the radioactivity counting accuracy and reduces the radioactive wastes. LC-ARC eliminates the need of collecting fractions for off-line counting for low-level radioactivity, thus reducing the study cost (Nassar et el., 2003; Wang et al., 2005).
Nuclear Magnetic Resonance. Nuclear magnetic resonance experiments were performed at 400 MHz (1H) on a Varian-400 spectrometer (Varian Inc., Palo Alto, CA). The temperature was set to 298K throughout the analysis and samples were dissolved in DMSO-d6. Typical one-dimensional proton experiments were performed over the spectral range 0 to 8 or 0 to 13 ppm.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Pharmacokinetics. The mean serum concentration-time curves for CP-122,721 (ng/ml) and total radioactivity (ng-Eq/ml) for EM and PM human subjects are shown in Fig. 4. Absorption of CP-122,721 was rapid in both EM and PM subjects, as indicated by the rapid appearance of radioactivity in serum after the oral administration of [14C]CP-122,721. The serum concentrations of total radioactivity were always much greater than those of unchanged drug, indicating early formation of metabolites. As shown in Table 2, the terminal phase t values in EM and PM subjects were approximately 4 and 1.2 times higher, respectively, for total radioactivity than for unchanged CP-122,721 (CP-122,721 t was 6.7 and 45 h for EMs and PMs, respectively). The total radioactivity t was 23.7 and 55.2 h for EMs and PMs, respectively. The serum concentrations of CP-122,721 reached a peak of 7.4 and 69.8 ng/ml for EMs and PMs, respectively. The serum concentrations of total radioactivity reached a peak of 844 and 447 ng-Eq/ml for EMs and PMs, respectively. Based on AUC0-t last, 0.5% and 16% of the circulating radioactivity was attributable to unchanged drug for EM and PM subjects, respectively, and the balance, approximately 99.5 and 84% of the serum radioactivity, was due to metabolites.
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Metabolic Profiles in Biological Samples. Urine. Representative HPLC radiochromatograms of metabolites in urine from EM (subject 2, 0-72 h postdose pooled) and PM (subject 6, 0-192 h postdose pooled) subjects following oral administration of [14C]CP-122,721 with on-line radioactivity monitoring are depicted in Fig. 5. A total of 11 metabolites in EM subjects and 8 metabolites in PM subjects were identified. M4 was the major metabolite in EM subjects; however, the major metabolite in PM subjects was M19.
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Serum. Representative profiles of serum metabolites from EM and PM subjects (1, 4, 8, 12, and 24 h pooled) are depicted in Fig. 7. The average percentages of each metabolite recovered in serum of EM and PM male human subjects following oral administration of [14C]CP-122,721 are shown in Table 4. Qualitative and quantitative similarities were observed for all EM subjects. Similarly, qualitative and quantitative similarities were also observed for the two PM subjects. However, there are quantitative and some qualitative differences between EM and PM subjects. Unchanged drug was only detected in the radiochromatogram of one PM human subject. CP-122,721 and a total of four metabolites accounted for approximately 99% (average of EM and PM subjects) of the total radioactivity.
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Identification of Metabolites. The structures of metabolites were elucidated by ionspray LC/MS/MS using both positive and negative ion modes. Combined liquid chromatography/ionspray mass spectrometry (full-scan) and tandem mass spectrometry, such as precursor ion, product ion, single ion monitoring, and multiple reaction monitoring scanning, techniques were used for the identification of metabolites. Where possible, the identities of metabolites were confirmed by coelution on HPLC with synthetic standards. The word "tentative" was utilized when the exact sites of some structural modification could not be determined.
Synthetic standard of CP-122,721 had a retention time of 35.2 min on the HPLC system and showed a protonated molecular ion at m/z 381. The CID mass spectrum of CP-122,721 showed abundant fragment ions at m/z 160 and 205, which represent the phenyl piperidinyl moiety and the trifluoromethoxy anisole benzyl moiety, respectively (Fig. 8). The fragment ions at m/z 143, 132, and 117 were derived from the fragment ion at m/z 160. The fragment ion at m/z 177 represents the phenyl piperidinyl amine moiety. The fragment ion at m/z 91 represents the tropylium ion.
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CP-122,721 and a total of 16 metabolites were identified. Unchanged drug was found in both urinary and fecal samples of PM subjects and was only detected in the serum of one PM human subject. The structures of 11 of these metabolites were identical to those identified previously in rat (Kamel et al., 2006): M1, M2, M3, M4, M5, M7, M8, and M9, and dog (A. Kamel, Y. Du, K. Colizza, and C. Prakash, unpublished results): M12, M14, and M15. M15 was a novel major circulating metabolite and required further structural confirmation. Five additional metabolites were identified, namely, M16, M17, M18, M19, and M20.
Metabolite M15. M15 had a retention time of approximately 28.5 min and had a poor ionization efficiency. It was detected in all EM and PM subjects and accounted for approximately 56 and 29% of the total radioactivity for EM and PM subjects, respectively. M15 was also detected in the urine of EM and PM subjects.
Incubation of [14C]CP-122,721 with human liver S9 fraction resulted in the formation of a radioactive peak that had a retention time similar to that of M15 from a human subject. After further HPLC isolation and purification, spiking human serum from subject 2 (EM) with the HPLC-purified fraction (27-30 min) resulted in coelution of M15 with the HPLC-purified fraction (Fig. 9). Based on these data, the HPLC-purified fraction from incubation of [14C]CP-122,721 with human liver S9 was identified as M15 serum metabolite.
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The CID mass spectrum of M15 is depicted in Fig. 10 and showed a deprotonated molecular ion at m/z 221, suggesting that M15 was a cleaved product with zero or an even number of nitrogen atoms. M15 was detected in the 14C chromatogram, indicating that it contained the benzyl moiety of the parent drug, and further suggested that the phenyl piperidine moiety had been removed.
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The 1H NMR spectra of 5-trifluoromethoxy salicylic acid (Fig. 11, top) and an expansion of its aromatic region (Fig. 11, bottom) are shown in Fig. 11. The resonance at 3.81 ppm (s, 3H) corresponds to -OCH3 (impurity from the starting material 2-methoxy-5-trifluoromethoxy-benzoic acid). The two resonances at 2.47 ppm (m) and 1.95 ppm (s) correspond to DMSO (partially deuterated) and ethyl acetate, respectively. Other small resonances are due to residual solvents. The three aromatic resonances at 7.64 ppm (d, 1H, J = 2.9 Hz), 7.53 to 7.50 ppm (dd, 1H, J = 9.1, 2.9 Hz), and 7.05 ppm (d, 1H, J = 9.1 Hz) are consistent with a ring system containing three substitutions. The resonance at 13.03 ppm (broad s, 1 H) corresponds to -COOH. Based on the above data, the final product was identified as 5-trifluoromethoxy salicylic acid. Human serum M15 was coeluted with the synthetic standard on HPLC and its CID mass spectrum was identical to those from the synthetic standard and HPLC-purified fraction from human liver S9 (Fig. 12). Based on the above data, human serum M15 was unambiguously identified as 5-trifluoromethoxy salicylic acid
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9.1 min on the HPLC system and showed a protonated molecular ion at m/z 409, 28 mass units higher than unchanged drug. Accounting for 2.4% of the administered dose in urine, M16 was not detected in feces. The CID spectrum of M16 is depicted in Fig. 13 and shows major fragment ions at m/z 205, 188, and 146. The fragment ions at m/z 205 are the same as that found in the CID spectrum of CP-122,721, suggesting that the methoxy trifluoromethoxy benzyl moiety is unchanged. The abundant signal at m/z 188 corresponds to the CP-122,721 fragment m/z 160 plus 28 amu. The peak at m/z 146 suggests the loss of CH2CHO (42 amu) from the m/z 188 fragment. Based on the above data, M16 was tentatively identified as piperidine dione CP-122,721.
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Metabolite M17. Metabolite M17 had a retention time of 17.4 min on the HPLC system and accounted for 3.8% of the administered dose in urine (average of EM and PM subjects), M17 was not detected in feces. M17 showed a protonated molecular ion at m/z 384, suggesting that the molecule contains an odd number of nitrogen atoms. The CID product ion spectrum of M17 showed the prominent fragment ions at m/z 208, 191, and 85 (Fig. 14). The ion at m/z 208, 176 amu lower than the molecular ion, indicated that a glucuronide moiety was lost from a CP-122,721 cleaved product containing the radioisotope. The fragment at m/z 85 shows that the OCF3 moiety was present. The loss of 17 amu (NH3) from the fragment m/z 208 to form the m/z 191 fragment further suggested that the molecule has a primary amine functionality. Based on the above data, metabolite M17 was tentatively identified as the glucuronide conjugate of 2-aminomethyl-4-trifluoromethoxy phenol.
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21.3 min on the HPLC system and accounted for 10.2% of the administered dose in urine (average of EM and PM subjects), M18 was not detected in feces. Determination of the molecular ion was assisted by the full-scan spectra in both positive and negative ion modes. Metabolite M18 showed an intense ion at m/z 402. The negative ion full-scan mass spectrum showed a deprotonated molecular ion at m/z 383, suggesting that the ion at m/z 402 represents the ammonium adduct of a metabolite with a molecular weight of 384. The molecular weight of M18 further suggested that the molecule has zero or an even number of nitrogen atoms. The CID product ion spectrum of [M + NH4]+ at m/z 402 (Fig. 15) showed the loss of NH +4 to give rise to the protonated molecular ion at m/z 385. The CID spectrum also showed two characteristic fragment ions at m/z 209 (loss of 176 amu from the protonated molecular ion) and 191 (loss of 194 amu from the protonated molecular ion), suggesting that M18 was a glucuronic acid conjugate of a cleaved product, which had undergone O-demethylation and oxidative deamination. A distinction between aromatic and aliphatic O-glucuronidation can be made by observing the differences in the fragment ion intensity and the pattern of loss of the glucuronyl moiety under MS/MS conditions (Billets et al., 1973; Mutlib and Abbott, 1992).
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Metabolite M19. Metabolite M19 had a retention time of 24.6 min on the HPLC system and accounted for 6.3 and 17.2% of the administered dose in urine for EM and PM subjects, respectively. M19 was not detected in feces and showed a protonated molecular ion at m/z 557, 176 amu higher than the unchanged drug, suggesting a glucuronide conjugate. Product ion spectrum of M19 showed prominent fragment ions at m/z 381, 364, 191, and 174 (data not shown). The fragments at m/z 174 and 191 (14 amu higher than the drug CID fragments of m/z 160 and 177) suggest oxidation, most likely 
to the piperidine nitrogen followed by the loss of two hydrogen atoms to form a lactam. The ion at m/z 381 resulted from the loss of 176 amu, a glucuronide conjugate. This suggests the formation of an O-desmethyl product followed by glucuronidation of the exposed phenolic oxygen. Based on these data, M19 was tentatively identified as the glucuronide conjugate of the O-desmethyl CP-122,721 lactam.
Metabolite M20. Metabolite M20 had a retention time of 26.6 min on the HPLC system and accounted for 7.3 and 2.7% of the administered dose in urine for EM and PM subjects, respectively. M20 was not detected in feces and showed a protonated molecular ion at m/z 573, 192 amu higher than the parent drug, suggesting conjugation. Product ion spectrum of M20 showed fragment ions at m/z 488, 397, 380, 207, 190, and 160 (data not shown). The ions at m/z 397 and 380 resulted from the loss of a glucuronide (-176 amu) and the subsequent loss of ammonia (-17 amu). The prominent ions at m/z 190 and 207 (30 amu higher than the drug CID fragments of m/z 160 and 177) indicate the addition of two oxygen atoms and subsequent loss of two hydrogen atoms from the phenyl piperidine moiety. The lack of any H2O losses suggests that one oxidation occurs on the phenyl ring, whereas the other is an oxidation most likely to the piperidine nitrogen followed by the loss of two hydrogens to form a lactam. The site of glucuronidation could not be determined from mass spectral information. Based on these data, M20 was identified as the glucuronide conjugate of 2-phenol-5-one-piperidine CP-122,721.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Approximately 84% of the administered radioactivity was recovered from the urine and feces of the subjects over a period of 312 h. Approximately 80% of the dose for EM subjects was recovered within 48 h. PM subjects excreted only about 45% of the dose in 48 h and required the full 312 h to achieve nearly 80% recovery.
CP-122,721 was extensively metabolized in humans, since only a small amount of unchanged drug was detected in the excreta. The profiles of metabolites before and after treatment with ß-glucuronidase were different, indicating that the elimination of the drug was due to phase I metabolism and subsequent phase II metabolic pathways. A total of 4 metabolites in feces, 12 metabolites in urine, and 4 metabolites in serum were identified. In all subjects, total radioactivity recovered from the feces was between 2% and 10% of the dose and most of the recovery was obtained from the urine (65-85%).
Qualitative and quantitative similarities were observed for all EM subjects. Similarly, qualitative and quantitative similarities were also observed for the two PM subjects. However, there are quantitative and qualitative differences between EM and PM subjects.
For the EM subjects, the major component of drug-related material in the excreta was tentatively identified as the glucuronide-conjugated O-desmethyl metabolite M4 (27.5% of the administered dose). The PM subjects contained no detectable levels of M4 but, rather, the glucuronide conjugate of the O-desmethylated lactam, M19, was the major component of drug-related material in the excreta (17.2% of the administered dose).
Excretion of radioactivity in the feces of all subjects was quantitatively similar, but the metabolites were different. Most importantly, the EM subject feces did not contain CP-122,721, however, this was the major fecal component for the PM subjects.
The serum concentrations of total radioactivity were always much greater than those of unchanged drug, indicating early formation of metabolites. CP-122,721 t was 6.7 and 45 h for extensive and poor metabolizers, respectively. The serum concentrations of CP-122,721 reached a peak of 7.4 and 69.8 ng/ml for extensive and poor metabolizers, respectively. Based on AUC0-t last, approximately 0.5% (EM) and 16% (PM) of the circulating radioactivity was attributable to unchanged drug. The balance, approximately 99.5% (EM) and 84% (PM) of the serum radioactivity, was due to metabolites. Of the total radioactivity extracted from the serum, the drug and metabolites (average of all human subjects) accounted for approximately 99%. M3 was detected in the radiochromatograms of all EM and PM subjects and accounted for approximately 25 and 62% of the total radioactivity for EM and PM subjects, respectively. M4 was only detected in the radiochromatograms of all EM subjects and accounted for approximately 14% of the total radioactivity. M5 (isomeric structure of M3) was detected in the radiochromatograms of all EM subjects and one PM subject and accounted for approximately 5 and 2% of the total radioactivity for EM and PM subjects, respectively. M15 (TFMSA) was detected in the radiochromatograms of all EM and PM subjects and accounted for approximately 56 and 29% of the total radioactivity for EM and PM subjects, respectively. Unchanged drug was only detected in the radiochromatogram of one PM human subject and accounted for approximately 6% of the total radioactivity. There are no known circulating active metabolites of CP-122,721.
A proposed scheme for the biotransformation pathways of CP-122,721 in humans is shown in Fig. 16. Based on the structures of these metabolites, three major and three minor routes of metabolism of CP-122,721 were identified. The major routes were due to O-demethylation, aromatic hydroxylation, and indirect glucuronidation. The minor routes included aliphatic oxidation at the piperidine moiety, aliphatic oxidation at the benzylic position of the trifluoromethoxy anisole moiety, O-dealkylation of the trifluoromethoxy group, and N-dealkylation and oxidative deamination to form TFMSA (M15) metabolite.
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The formation of M15 could be derived from two different metabolic pathways: O-demethylation and direct oxidative deamination (by -carbon hydroxylation) to yield 2-hydroxy-5-trifluoromethoxy-benzaldehyde intermediate and the phenyl-piperidine amine metabolite (CP-114,621), which has been identified in rat plasma (A. Kamel and C. Prakash, unpublished results). Subsequent oxidation of 2-hydroxy-5-trifluoromethoxy-benzaldehyde intermediate would result in the formation of M15 (Fig. 17a). The other metabolic pathway responsible for the formation of M15 is O-demethylation and N-dealkylation of the secondary amine, which proceeds by carbinolamine pathway (Fig. 17b) to give rise to the corresponding primary amine metabolite (CP-677,623), which was detected in rat plasma (A. Kamel and J. Davis, unpublished results) and is susceptible to oxidative deamination. Therefore, oxidative deamination of the primary amine metabolite (CP-677,623) and subsequent oxidation would result in the formation of M15.
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) that the extent of glycine conjugation of benzoic acids with the halogen group decreased in the order F > Cl > Br > I. The conjugation of 2-OH-substituent (salicylic acid) with glycine took place only in the kidney. The 2-CH3O-substituent, however, exhibited no glycine conjugation in the liver and kidney. In addition, the size and the electronegativity of the substituent had a far greater influence over glycine conjugation. Kasuya et al. (2000) also reported that it may be important that the substrates undergoing glycine conjugation contain a flat region coplanar to the carboxylate group. From these findings, it is reasonable to assume that the lack of glycine conjugation of TFMSA could be attributed to the expected profound effect of the bulky trifluoromethoxy (-OCF3) substituent in the meta-position.
In summary, the results of this study provide the first analysis of formation and excretion of metabolites of CP-122,721 in humans. Results were similar to those reported previously in rat (Kamel et al., 2006) and dog (A. Kamel, Y. Du, K. Colizza, and C. Prakash, unpublished results). CP-122,721 is very rapidly eliminated in humans, mainly by phase I oxidative metabolism and subsequent phase II metabolic pathways. We have reported a full characterization of a novel circulating metabolite, resulting from O-demethylation, oxidative deamination, and subsequent oxidation. The identification of these metabolic pathways will have relevance to understanding the metabolism of other trifluoromethoxy-benzylamine piperidinyl anisole drugs.
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Acknowledgments |
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Footnotes |
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This work was presented in part at the 52nd ASMS Conference on Mass Spectrometry and Allied Topics, Nashville, TN 2004.
ABBREVIATIONS: CP-122,721, (+)-(2S,3S)-3-(2-methoxy-5-trifluoromethoxybenyzlamino)-2-phenylpiperidine; SP, substance P; NK, neurokinin; TFMSA, 5-trifluoromethoxy salicylic acid; HPLC, high pressure liquid chromatography; radio-HPLC, HPLC with on-line radioactivity detection; MS/MS, tandem mass spectrometry; CP-114,621, (2S,3S)-2-phenyl-piperidin-3-ylamine; CP-125,611, (2-[((2S,3S)-2-phenyl-piperidin-3-ylamino)-methyl]-4-trifluoromethoxy-phenol; CP-677,623, 2-methoxy-5-trifluoromethoxy-benzylamine; ß-RAM, radioactivity monitor; EM, extensive metabolizer; PM, poor metabolizer; LC-ARC, liquid chromatography-accurate radioactivity counting; Cmax, peak plasma concentration; Tmax, time at which Cmax occurred; AUC, area under the plasma concentration-time curve; CID, collision-induced dissociation; DMSO, dimethyl sulfoxide; amu, atomic mass unit(s).
Address correspondence to: Dr. Amin Kamel, Exploratory Medicinal Sciences, Pfizer Global Research and Development, Groton Laboratories, Groton, CT 06340. E-mail. Amin.m.kamel{at}pfizer.com
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, urine; 
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