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Metabolism, Pharmacokinetics, and Excretion of a Nonpeptidic Substance P Receptor Antagonist, Ezlopitant, in Normal Healthy Male Volunteers: Characterization of Polar Metabolites by Chemical Derivatization with Dansyl Chloride

Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer Global Research and Development, Groton, Connecticut

(Received February 23, 2007; accepted April 10, 2007)

	Abstract

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The excretion, biotransformation, and pharmacokinetics of ezlopitant [(2-benzhydryl-1-aza-bicyclo[2.2.2]oct-3-yl)-(5-isopropyl-2-methoxy-benzyl)-amine], a substance P receptor antagonist, were investigated in healthy male volunteers after oral administration of a single 200-mg (93 µCi/subject) dose of [¹⁴C]ezlopitant. The total recovery of administered radioactive dose was 82.8 ± 5.1, with 32.0 ± 4.2% in the urine and 50.8 ± 1.4% in the feces. Mean observed maximal serum concentrations for ezlopitant and total radioactivity were achieved at 2 h after oral administration; thus, ezlopitant was rapidly absorbed. Ezlopitant was extensively metabolized in humans, since no unchanged drug was detected in urine and feces. The major pathway of ezlopitant in humans was the result of the oxidation of the isopropyl side chain to form the -hydroxy and -1-hydroxy (M16) metabolites. M16 and ,-1-dihydroxy (1,2-dihydroxy, M12) were identified as the major circulating metabolites accounting for 64.6 and 15.4% of total circulating radioactivity, respectively. In feces, the major metabolite M14 was characterized as the propionic acid metabolite and formed by further oxidation of the -hydroxy metabolite. The urinary metabolites were the result of cleaved metabolites caused by oxidative dealkylation of the 2-benzhydryl-1-aza-bicyclo[2.2.2]oct-3-yl moiety. The metabolites (M1A, M1B, and M4), approximately 34% of the total radioactivity in urine, were identified as benzyl amine derivatives. These were polar metabolites that were further characterized using the reaction with dansyl chloride to derivatize the primary amines and phenol moieties to less polar analytes. The other metabolites were the result of O-demethylation, dehydrogenation of the isopropyl group, and oxidation on the quinuclidine moiety.

Ezlopitant, (2-benzhydryl-1-aza-bicyclo[2.2.2]oct-3-yl)-(5-isopropyl-2-methoxy-benzyl)-amine (Fig. 1), is a highly potent and selective NK1 receptor antagonist with a K_i of 0.4 nM (Evangelista, 2001). It acts on the central nervous system to exert the excellent antiemetic activity for the control of both acute and delayed emesis induced by cisplatin in ferrets at a dose of 3 mg/kg (Tsuchiya et al., 2002, 2005). It also inhibited SP-induced contraction of guinea pig trachea with a pA₂ value of 7.8, but had no effects on the baseline tension and maximum contractile response. Recent clinical studies have shown that ezlopitant is effective for the treatment of either the acute or delayed phase of emesis associated with antineoplastic treatment using cisplatin, suggesting a role of NK1 receptor antagonists in the clinic for the control of emesis to improve the quality of life of cancer patients undergoing chemotherapy (Hesketh et al., 1999).

View larger version (26K): [in this window] [in a new window]   FIG. 1. Proposed routes for biotransformation of ezlopitant in humans.

	Materials and Methods

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General Chemicals. Commercially obtained chemicals and solvents were of HPLC or analytical grade. An Ultrasphere C-18 HPLC column was obtained from Beckman Coulter (Fullerton, CA). Ecolite (+) scintillation cocktail was obtained from MP Biomedicals (Irvine, CA). Carbosorb and Permafluor E⁺ scintillation cocktails were purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). HPLC-grade acetonitrile, methanol, and water were obtained from J. T. Baker (Phillipsburg, NJ). Certified American Chemical Society grade ammonium acetate and acetic acid were obtained from Fisher Scientific Company (Pittsburgh, PA). Dansyl chloride and 1-methyl-3-nitro-1-nitrosoguanidine were purchased from Sigma-Aldrich Co. (St. Louis, MO). Diazomethane was generated just before use from 1-methyl-3-nitro-1-nitrosoguanidine.

Radiolabeled Drug and Reference Compounds. [¹⁴C]Ezlopitant (HCl salt) was synthesized by the Radiosynthesis Group at Pfizer Global Research and Development (Groton, CT) as described previously (Zandi et al., 2000). [¹⁴C]Ezlopitant showed a specific activity of 212.2 µCi/mmol and a radiochemical purity of >99.8%, as determined by HPLC using an in-line radioactivity detector. The synthetic standards, CJ-12,764 and CJ-12,458 were synthesized by the Medicinal Chemistry Group at Pfizer Global Research and Development (Nagoya, Japan).

Subjects and Dose Administration. Four normal, healthy male subjects between the ages of 18 and 45 years participated in the study. The study protocol was reviewed and approved by the Institutional Review Board at the Clinical Research Facility of PPD Pharmaco (Austin, TX). After being informed of the purpose, design, and potential risks of the study, the volunteers gave written consent. Subjects entered the Clinical Research Facility approximately 12 h before dosing and remained there for 264 h (11 days) after dosing under continuous medical observation. All subjects had fasted for at least 8 h and were given a single oral 200-mg free base equivalent dose of [¹⁴C]ezlopitant (93 µCi/subject). The dose was administered in an open fashion in the morning. A standard meal was provided 4 h later. The dosing formulation was prepared by dissolving the radiolabeled material in water. Total volume of water given was 240 ml. Subjects were required to refrain from lying down and from eating or drinking caffeinated and carbonated beverages during the first 4 h after drug administration.

Sample Collection. After dosing, urine samples were collected for 11 days at 0 (predose), 0 to 12, 12 to 24, 24 to 48, 48 to 72, 72 to 96, 96 to 120, 120 to 144, 144 to 168, 168 to 192, 192 to 216, 216 to 240, and 240 to 264 h postdose. One of the subjects (#1) was removed from the study after 48 h because of personal reasons. The total volumes of urine samples were recorded after each collection. Feces were collected, as passed, from time of dosing until 264 h after dosing.

Blood sufficient to provide a minimum of 4 ml of serum was collected, in evacuated tubes containing no anticoagulants, phase separator, or preservative, from each subject at 0 (predose), 0.5, 1, 2, 4, 8, 12, 24, 36, 48, 72, 96, 120, 144, 168, 192, 216, and 240 h after the dose. In addition, blood sufficient to yield 5 ml of serum was collected at 2, 5, and 12 h after dosing for metabolite identification. The serum was allowed to clot at room temperature and was separated within 1 h from the whole blood in a refrigerated centrifuge, transferred into labeled plastic tubes, and immediately frozen.

Determination of Radioactivity. Radioactivity in urine, feces, and serum was measured by liquid scintillation counting. Aliquots of serum and urine (0.2–1.0 ml, in triplicate) for each sampling time were mixed with 5 ml of Ecolite (+) scintillation cocktail and counted in a Wallac 1409 (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK) or a PerkinElmer 2500 liquid scintillation counter (PerkinElmer Life and Analytical Sciences, Boston, MA).

Fecal samples were placed into Stomacher 400 bags and homogenized in equal amounts of water to a thick slurry, using a Stomacher homogenizer from Cooke Laboratory Products (Alexandria, VA). Aliquots (30–200 mg) of the fecal homogenates were air-dried before combustion in an automatic sample oxidizer (PerkinElmer oxidizer model 307). Radioactivity in the combustion products was determined by trapping the liberated CO₂ in Carbosorb followed by liquid scintillation counting using Permafluor E+ as a scintillation cocktail. Combustion efficiency of the oxidizer was determined by combustion of the [¹⁴C]methyl methacrylate standard in an identical manner. Radioactivity less than twice the background value was considered to be below the limit of determination.

Samples collected before dosing were used as controls and counted to obtain a background count rate. Radioactivity in the dose was expressed as 100% and the radioactivity in urine and feces at each sampling time was defined as the percentage of dose excreted in the respective matrices at that sampling time. The amount of radioactivity in serum was expressed as nanogram-equivalents of parent drug per mililiter and was calculated by using the specific activity of the dose administered.

Pharmacokinetic Analysis. Serum concentrations of ezlopitant were quantitated by an LC/MS/MS assay (Kamel et al., 2000). Pharmacokinetic parameters were determined using the WinNonlin-Pro Ver. 1.5 program (Pharsight, Mountain View, CA) by noncompartmental analysis.

Extraction of Metabolites from Biological Samples. Urine samples obtained at 0 to 12, 12 to 24, 24 to 48, and 48 to 72 h postdose were pooled on the basis of collected weight for each subject, which accounts for greater than 90% of the urine radioactive excreta. The pooled urine samples (8 ml) were lyophilized overnight. The residues were reconstituted in 0.25 ml of acetonitrile/20 mM ammonium acetate (20:80) and aliquots (200 µl) were injected onto the HPLC column.

Fecal homogenates containing the highest levels of excreted radioactivity (0–96 h) were pooled, and the pooled samples (10 g) were extracted twice with 20 ml of acetonitrile. The two supernatants were combined and aliquots of 0.5 ml were counted in duplicate to determine recovery of the radioactivity. The combined supernatants were evaporated to dryness in a nitrogen Turbo Vap LV evaporator (Zymark, Hopkinton, MA). The residues were reconstituted in 250 µl of acetonitrile/20 mM ammonium acetate (20:80) and aliquots (200 µl) were injected onto the HPLC column.

Serum samples (3 ml, 1 ml from each at 2-, 5-, and 12-h time points) were extracted twice with 6 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 90% of the radioactivity was recovered in the supernatant. The supernatants were concentrated to dryness in a nitrogen Turbo Vap LV evaporator at a room temperature. The residues were reconstituted in 150 µl of acetonitrile/20 mM ammonium acetate (20:80) and centrifuged to remove insoluble matter; then, 100-µl aliquots were injected onto the HPLC column.

Reaction with Dansyl Chloride. The metabolites M1A, M1B, and M4 from human urine were isolated by HPLC. The HPLC eluent from regions for metabolites M1A, M1B, and M4 were collected and evaporated to dryness. The residues were treated with 100 µl of 100 mM sodium bicarbonate solution. A 50-µl aliquot of each reconstituted fraction was reacted with 25 µl of dansyl chloride solution (1 mg/ml in acetone). The mixture was allowed to stand at room temperature for 10 min and then evaporated to dryness. The residue was reconstituted in 150 µl of acetonitrile/20 mM ammonium acetate (20:80) and centrifuged to remove insoluble matter, and a 100-µl aliquot was injected onto the HPLC column.

Reaction with Methanol/Sulfuric Acid. The dried fecal extract was treated with 0.25 ml of 10% sulfuric acid in methanol. The mixture was allowed to stand at room temperature for 4 h. The solvents were evaporated to dryness and the residue was reconstituted in 150 µl of acetonitrile/20 mM ammonium acetate (20:80). The mixture was centrifuged to remove insoluble matter, and a 100-µl aliquot was injected onto the HPLC column.

Chromatography. The HPLC system consisted of an HP-1050 solvent delivery system, an HP-1050 membrane degasser, and an HP-1050 autoinjector from Hewlett Packard (Palo Alto, CA), a Thermo Separations spectromonitor 3200 UV (Thermo Fisher Scientific, Waltham, MA), and an IN/US ß-RAM radioactive monitor (IN/US, Tampa, FL). Chromatography was performed on a Beckman Coulter Ultrasphere C-18 HPLC column (4.6 mm x 250 mm, 5 µm) with a mobile phase containing a mixture of 20 mM ammonium acetate (solvent A) and acetonitrile (solvent B). The mobile phase was initially composed of solvent A/solvent B (90:10); it was then linearly programmed to solvent A/solvent B (30:70) over 30 min followed by a gradient to solvent A/solvent B (10:90) over 10 min. The mobile phase composition was returned to the starting solvent mixture over 5 min. The system was allowed to equilibrate for approximately 15 min before making the next injection. A flow rate of 1.0 ml/min was used for all analyses.

Quantitative Assessment of Metabolite Excretion. Quantification of metabolites in urine and feces was carried out by measuring radioactivity in the individual HPLC-separated peaks using a ß-RAM. The ß-RAM provided an integrated printout in cpm and the percentage of the radiolabeled material, as well as the peak representation. The ß-RAM was operated in the homogeneous liquid scintillation counting mode with the addition of 3 ml/min Tru-Count scintillation cocktail to the effluent post-UV detection. The radiochromatograms of metabolites in serum were generated by collecting fractions at 0.33-min intervals and counting the fractions in a PerkinElmer 2500CA liquid scintillation counter. The retention times of the radioactive peaks, where possible, were compared with those of synthetic standards and/or metabolites.

Mass Spectrometry Analysis. Identification of metabolites was performed on a PE-Sciex API 2000 LC/MS/MS apparatus (PerkinElmerSciex Instruments, Boston, MA) using ionspray. The effluent from the HPLC column was split and 50 µl/min was introduced into the mass spectrometer. 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, which provided simultaneous detection of radioactivity and mass spectrometry data. The interface was operated at 4500 V and the mass spectrometer was operated in the positive mode. CID studies were performed using argon gas at a collision energy of 30 to 40 eV.

	Results

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Mass Balance. After a single oral dose of [¹⁴C]ezlopitant to human subjects, a major portion of the administered radioactivity was excreted in the feces. By 264 h after the dose, the mean cumulative excretion amounted to 32.0 ± 4.2% in the urine and 50.8 ± 1.4% in the feces (Table 1). In total, 82.8% of the radioactive dose was recovered in urine and feces (Table 1). Of the entire radioactivity recovered in urine, approximately 83% was excreted in the first 48 h. Because feces was collected after natural defecation, the fecal elimination of radioactivity was delayed, compared with that in urine. The major portion of radioactivity (88%) in feces appeared during the 24- to 144-h time period after the dose. Although the reason for the low recovery of radioactivity is not entirely clear, a possible reason could be the noncompliance of subjects in complete collection of excreta.

View larger version (10K): [in this window] [in a new window]   FIG. 2. Mean serum concentration-time curves for ezlopitant, CJ-12,764, and CJ-12,458, and total radioactivity in humans following a 200-mg oral dose of [14C]ezlopitant.

The absorption of ezlopitant was rapid, as indicated by the early appearance of radioactivity in serum after oral administration. Serum concentrations for ezlopitant and total radioactivity peaked at 2 h after oral administration (Table 2). C_max values for the parent drug ranged from 193 to 370 ng/ml with a mean value of 271 ng/ml. C_max values for the total radioactivity ranged from 938 to 2024 ng Eq/ml with a mean value of 1460 ng/ml. These values were five times higher than those of the parent drug, suggesting that a major portion of the circulating radioactivity was attributable to metabolites. The mean terminal phase t_1/2 values were 13.0 and 108.9 h for parent drug and total radioactivity, respectively. Mean AUC_(0-t) values for parent drug and total radioactivity were 3260 ng Eq · h/ml and 34,000 ng Eq · h/ml, respectively.

Metabolic Profiles in Biological Samples. Urine. A representative HPLC-radiochromatogram for metabolites in urine is shown in Fig. 3. A total of 12 metabolites were detected in the radiochromatogram. The percentages of metabolites in relation to the administered dose are presented in Table 3. The major metabolites in urine were the result of cleaved products, which were caused by oxidative dealkylation of the 2-benzhydryl-1-aza-bicyclo[2.2.2]oct-3-yl moiety. The metabolites (M1A, M1B, and M4), approximately 34% of the total radioactivity in urine, were identified as benzyl amine derivatives.

View larger version (28K): [in this window] [in a new window]   FIG. 3. HPLC-radiochromatograms for metabolites of ezlopitant in human urine (0–72 h) (A) and feces (0–96 h) (B).

Feces. On average, 96% of the radioactivity was recovered after extraction of fecal samples. A representative HPLC-radiochromatogram for fecal metabolites is shown in Fig. 3. A total of eight radioactive peaks were detected in the radiochromatogram. The percentages of fecal metabolites in relation to the administered dose are presented in Table 3. The major metabolites in feces were the result of hydroxylation at the isopropyl moiety. The metabolites M12 and M14, corresponding to 67% of the total radioactivity in feces, were identified as 1,2-propanediol and -carboxyl metabolites.

Circulating Metabolites. A representative HPLC-radiochromatogram of circulating metabolites is shown in Fig. 4. Ezlopitant and a total of five metabolites were detected. The relative percentages of circulating metabolites are presented in Table 4. The major circulating metabolites were the result of oxidation at the isopropyl moiety. On the basis of the pooled samples, metabolites M12 and M16 (CJ-12,764), accounting for approximately 84% of the total circulating radioactivity between 2 and 12, were identified as 1,2-propanediol and propan-2-ol (-1-hydroxy metabolite). Unchanged ezlopitant and its alkene metabolite accounted for only <8% of the total radioactivity.

View larger version (12K): [in this window] [in a new window]   FIG. 4. HPLC-radiochromatogram for circulating metabolites of ezlopitant in human plasma (0–12 h).

Mass Spectral Fragmentation of Ezlopitant. Ezlopitant had a retention time of 38.7 min on the HPLC system. Full-scan MS of ezlopitant produced a protonated molecule (MH⁺) of m/z 455. Its CID spectrum gave product ions at m/z 276, 167, 163, 133, 121, and 91 (Fig. 5; Table 5). The structure assignments of major fragment ions are proposed in Fig. 5 (bottom).

View larger version (24K): [in this window] [in a new window]   FIG. 5. CID product ion spectrum of ezlopitant, m/z 455 (top), and proposed structural assignment of major fragment ions (bottom).

Identification of Metabolites. Metabolites M1A, M1B, and M4. M1A, M1B, and M4 had retention times of 3.1, 5.7, and 8.3 min, respectively, on HPLC and they were found only in urine. The full-scan MS of M1A, M1B, and M4 produced protonated molecules of m/z 198, 182, and 196, respectively, suggesting that they all were cleaved products. Furthermore, the even mass of protonated molecular ions suggested that they had an odd number of nitrogen atoms.

The fragment ion at m/z 165, a loss of 17 Da (NH₃), indicated the presence of a primary amine, loss of a methyl group, and addition of an oxygen atom (Fig. 6A). The fragment ion at m/z 147, a loss of H₂O from the ion at m/z 165, suggested the presence of an aliphatic hydroxy group. Treatment of M1B with dansyl chloride resulted in the formation of a product with a protonated molecule of m/z 648, 466 Da higher than the protonated molecule of M1B, suggesting the addition of two molecules of dansyl chloride. The CID product ion spectrum of m/z 648 showed fragment ions at m/z 398 (loss of dansyl amide), 380 (loss of dansyl amide and H₂O), and 171 (dimethylaminonaphthalene) (Fig. 6B). On the basis of these data, M1B was identified as 2-aminomethyl-4-(1-hydroxy-1-methyl-ethyl)-phenol.

View larger version (20K): [in this window] [in a new window]   FIG. 6. CID product ion spectra of metabolite M1B, m/z 182 (A), and its dansyl derivative, m/z 648 (B).

View larger version (20K): [in this window] [in a new window]   FIG. 7. CID product ion spectra of metabolite M1A, m/z 198 (A), and its dansyl derivative, m/z 664 (B).

View larger version (19K): [in this window] [in a new window]   FIG. 8. CID product ion spectra of metabolite M4, m/z 196 (A), and its dansyl derivative, m/z 429 (B).

Metabolites M7A, M7B, and M7C. M7A and M7B had retention times of 15.4 and 18.3 min and were found both in urine and feces. M7A and M7B produced protonated molecules of m/z 473, 18 Da higher than that of the parent compound, indicating the addition of two oxygen atoms and a loss of the methyl group. The CID spectrum of M7A showed characteristic fragment ions at m/z 167, 276, and 293, similar to parent molecule, suggesting that the modification had occurred remote from the 2-benzhydryl-1-aza-bicyclo[2.2.2]oct-3-ylamino moiety (Table 5). The fragment ion at m/z 181, 18 Da higher than that of the parent compound, further suggested that the modification had occurred at the isopropyl-2-methoxy-benzyl moiety. The ion at m/z 106 supported the presence of a hydroxy-phenyl moiety and supported the notion that the addition of two oxygen atoms had occurred at the isopropyl moiety. Based on these data, M7A was tentatively identified as 2-{3-[(2-benzhydryl-1-aza-bicyclo[2.2.2]oct-3-ylamino)-methyl]-4-hydroxy-phenyl}-propane-1,2-diol.

The CID spectrum of protonated M7B showed fragment ions at m/z 309 and 292, 16 Da higher than those of M7A, suggesting that an addition of one oxygen atom had occurred at the 2-benzhydryl-1-azabicyclo[2.2.2]oct-3-ylamino moiety (Table 5). The fragment ion at m/z 167, similar to parent compound, suggested that the hydroxylation had occurred at the quinuclidine moiety and remote from the benzhydryl moiety. The fragment ion at m/z 165, 2 Da higher than that of the parent molecule, suggested that an addition of one oxygen atom and a loss of the methyl group from the isopropyl-2-methoxy-benzyl moiety. The ion at m/z 147, loss of H₂O, suggested the presence of an aliphatic hydroxyl group. Based on these data, M7B was tentatively identified as 2-[(2-benzhydryl-1-aza-bicyclo[2.2.2]oct-3-ylamino)-methyl]-4-(1-hydroxy-1-methyl-ethyl)-phenol with hydroxylation at the quinuclidine moiety.

M7C produced a protonated molecule of m/z 457, 2 Da higher than that of the parent compound, suggesting the addition of one oxygen atom and a loss of the methyl group. Its CID spectrum showed fragment ions at m/z 293, 276, and 167, suggesting that the modification had occurred remote from the 2-benzhydryl-1-aza-bicyclo[2.2.2]oct-3-ylamino moiety (Table 5). The fragment ion at m/z 165, 2 Da higher than that of the parent drug, suggested that the molecule had undergone modification at the isopropyl-2-methoxybenzyl moiety. The ions at m/z 147 (loss of H₂O), and 59 strongly indicated the hydroxylation of the isopropyl moiety. Based on these data, M7C was tentatively identified as 2-[(2-benzhydryl-1-aza-bicyclo[2.2.2]oct-3-ylamino)-methyl]-4-(1-hydroxy-1-methyl-ethyl)-phenol.

Metabolites M12 and M15. M12 and M15 had HPLC retention times of 19.2 and 22.0 min, respectively, and were found in all matrices. Both M12 and M15 produced protonated molecules of m/z 487, 32 Da higher than that of the parent compound, suggesting that these compounds were di-hydroxylated derivatives of ezlopitant. The fragment ion at m/z 195, 32 Da higher than that of the parent compound, further suggested that the modification had occurred at the isopropyl-2-methoxy-benzyl moiety. The ion at m/z 177, a loss of H₂O, suggested at least one aliphatic hydroxylation (Fig. 9A; Table 5). On the basis of these data, M12 was tentatively identified as 2-{3-[(2-benzhydryl-1-aza-bicyclo[2.2.2]oct-3-ylamino)-methyl]-4-methoxy-phenyl}-propanol.

View larger version (24K): [in this window] [in a new window]   FIG. 9. CID product ion spectrum of metabolites M12, m/z 487 (A), and M15, m/z 487 (B).

Metabolite M14. M14 had a retention time of 16.1 min on HPLC and was found in urine and feces. It produced a protonated molecule of m/z 485, 30 Da higher than that of the parent compound. The 30-Da increase in molecular mass is in accordance with oxidation of the methyl group to a carboxyl group. The fragment ion at m/z 193, 30 Da higher than that of the parent compound, suggested that the modification had occurred at the isopropyl-2-methoxy-benzyl moiety. The ion at m/z 276 further indicated that the modification had occurred remote from the 2-benzhydryl-1-aza-bicyclo[2.2.2]oct-3-ylamino moiety (Fig. 10A; Table 5).

View larger version (18K): [in this window] [in a new window]   FIG. 10. CID product ion spectra of metabolite M14, m/z 485 (A). and its methyl ester; m/z 499.

Treatment of fecal concentrate with 10% sulfuric acid in methanol resulted in the disappearance of peak corresponding to M14 and gave a product that produced a protonated molecule of m/z 499. The ion at m/z 499 was 14 Da higher than the MH⁺ of M14, suggesting the addition of one methyl group. The CID product ion spectrum of m/z 499 showed fragment ions at m/z 207 and 177, 14 Da higher than those of M14, suggesting the addition of a methyl group to the anisole moiety (Fig. 10B). On the basis of these data, M14 was identified as 2-{3-[(2-benzhydryl-1-aza-bicyclo[2.2.2]oct-3-ylamino)-methyl]-4-methoxy-phenyl}-propionic acid.

Metabolite M16 (CJ-12,764). M16 had a retention time of 24.5 min and was found in all matrices. It produced a protonated molecule of m/z 471, 16 Da higher than that of the parent compound, suggesting that the molecule had undergone monohydroxylation. Its CID spectrum showed fragment ions at m/z 276, 179, 167, 161, 131, and 121 (Table 5). The fragment ion at m/z 179, 16 Da higher than the fragment ion at m/z 163 of the parent, suggested that hydroxylation had occurred at the isopropyl-2-methoxy-benzyl moiety. The fragment ion at m/z 161, a loss of H₂O from the ion at m/z 179, suggested an aliphatic hydroxylation. M16 had a CID spectrum identical to that of synthetic standard of CJ-12,764. On the basis of these data, M16 was identified as 2-{3-[(2-benzhydryl-1-aza-bicyclo[2.2.2]oct-3-ylamino)-methyl]-4-methoxy-phenyl}-propan-2-ol.

Metabolite M17 (CJ-12,458). M17 had a retention time of 38.7 min and was detected only in serum. It produced a protonated molecule of m/z 453, 2 Da lower than the parent molecule, suggesting that the molecule had undergone dehydrogenation. Its CID spectrum showed fragment ions at m/z 167 and 276, indicating that the modification had occurred remote from the 2-benzhydryl-1-aza-bicyclo[2.2.2]oct-3-ylamino moiety (Table 5). The fragment ion at m/z 161, 2 Da lower than the fragment ion at m/z 163 of the parent compound, further suggested that dehydrogenation had occurred at the isopropyl moiety. M17 had a CID spectrum identical to that of an authentic standard. On the basis of these data, M17 was identified as (2-benzhydryl-1-aza-bicyclo[2.2.2]oct-3-yl)-(5-isopropenyl-2-methoxy-benzyl)-amine.

Metabolite M19. M19 had a retention time of 14.8 min and was found only in feces. It produced a protonated molecular ion at m/z 501, 46 Da higher than that of the parent molecule, suggesting that the M19 had the addition of three oxygen atoms and a dehydrogenation. Its CID spectrum showed fragment ions at m/z 455, 439, 193, 167, 163, 147, 123, and 110 (Table 5). The fragment ion at m/z 193, 30 Da higher than the fragment ion at m/z 163 of the parent compound, was indicative of the presence of a carboxyl group. The ion at m/z 167 suggested that the benzhydryl moiety was unchanged.

Treatment of fecal concentrate with 10% sulfuric acid in methanol resulted in the disappearance of peak corresponding to M19 and gave a product that produced a protonated molecule of m/z 515. The ion at m/z 515 was 14 Da higher than the protonated molecular ion of M19, suggesting the addition of one methyl group. The CID product ion spectrum of m/z 515 showed fragment ions at m/z 207 and 177, 14 Da higher than those of M19, suggesting the addition of a methyl group to the anisole moiety. On the basis of these data, M19 was tentatively identified as 2-{3-[(2-benzhydryl-1-aza-bicyclo[2.2.2]oct-3-ylamino)-methyl]-4-methoxy-phenyl}-propionic acid with hydroxylation at the quinuclidine moiety.

Metabolite M20. M20 had a retention time of 18.0 min and was found only in feces. It produced a protonated molecule of m/z 503, 48 Da higher than that of the parent drug, suggesting that the molecule had undergone three hydroxylations. Its CID spectrum showed fragment ions at m/z 195, 177, 167, 123, and 110 (Table 5). The fragment ion at m/z 195, 32 Da higher than that of the parent compound, suggested the addition of two oxygen atoms at the isopropyl-2-methoxy-benzyl moiety. The ion at m/z 177, a loss of H₂O, suggested at least one aliphatic hydroxylation. The exact site for the second hydroxylation was, however, not established by the mass spectral data. The ion at m/z 167 suggested that the benzhydryl moiety was unchanged. On the basis of these data, M20 was tentatively identified as 2-{3-[(2-benzhydryl-1-aza-bicyclo[2.2.2]oct-3-yl-amino)-methyl]-4-methoxy-phenyl}-propane-1,2-diol with hydroxylation at the quinuclidine moiety.

	Discussion

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We here report the metabolic fate and disposition of ezlopitant following oral administration of a 200-mg dose of [¹⁴C]ezlopitant, labeled at the benzylic carbon of the isopropyl anisole ring, to four healthy male volunteers. On average, total radioactivity recovered from subjects was 83%, not including one of the subjects who did not continue with the study after 48 h postdose. The majority of the radioactivity was excreted in the feces, suggesting that fecal/biliary excretion was the major route of elimination of ezlopitant and its metabolites. The radioactivity recovered in urine accounts for 32% of the dose.

After oral administration, the serum concentrations of total radioactivity were greater than that of the parent compound at all time points. This finding suggested the early formation of metabolites. Unchanged ezlopitant accounted for only 9.5% of the total radioactivity in serum pooled from the 0- to 12-h period. M16 and ,-1-dihydroxy (1,2-dihydroxy, M12) were identified as the major circulating metabolites, accounting for approximately 64.6 and 15.4% of total circulating radioactivity pooled from the 0- to 12-h period, respectively. The half-life for total radioactivity was higher than that of the unchanged drug, suggesting that the elimination of metabolites was much slower than that of parent compound in humans.

Unchanged elimination of ezlopitant was an insignificant portion of the total elimination such that there was no unchanged drug detected in urine and feces. A total of 13 metabolites were identified. The metabolites were characterized by LC/MS/MS with simultaneous online radioactivity monitoring, which is clearly a technique of choice for the rapid structural characterization of metabolites from biological fluids (Johnson and Prakash, 2005; Kamel and Prakash, 2006). The structures of two metabolites, M16 and M17, were confirmed by comparisons of their HPLC retention times and CID product ion spectra to those of the synthetic standards. The structures of other metabolites were proposed on the basis of molecular ions and fragmentation patterns and further supported by derivatization with acidic methanol or dansyl chloride. All the metabolites were formed from oxidative metabolism. On the basis of the structures of metabolites, a plausible scheme for the biotransformation pathways of ezlopitant in humans is depicted in Fig. 1.

The major metabolic pathway of ezlopitant was the result of the oxidation of the isopropyl side chain to form the -1-hydroxy (M16; 6% in urine and 1% in feces) and ,-1-dihydroxy (M12; 8.12% in feces and 3.81% in urine) metabolites and the -carboxyl (M14; 20.67% in feces and 1.65% in urine) metabolite. The derivatization of M14 with acidic methanol supported the presence of a carboxyl group, although the -hydroxy metabolite was not identified either in urine or feces, but it could be envisioned that M14 metabolite was formed by further oxidation of the -hydroxy metabolite. Hydroxylation at the tert-butyl- or isopropyl-side chains to form -hydroxy metabolites is not uncommon and, for example, was observed for several compounds containing these side chains (Ishida et al., 1998; Johnson and Prakash, 2005). M12 is a 1,2-dihydroxy metabolite that is most likely formed because of further oxidation of the -1-hydroxy metabolite (M16) at the isopropyl group. Alternatively, it could also be formed by further metabolism of the alkene metabolite (M17) (Obach, 2000). Metabolites M12 and M15 produced the same protonated molecule of m/z 487, but the CID mass spectra of these metabolites were able to distinguish oxidative sites characterized by distinct product ions.

The major metabolites in urine were the result of cleaved products caused by oxidative dealkylation of the 2-benzhydryl-1-aza-bicyclo[2.2.2]oct-3-yl moiety. These polar metabolites (M1A, M1B, and M4) had to be derivatized to increase the HPLC retention time away from the solvent front to minimize the interference from the chemical background found in the lower mass range of a conventional mass spectrum. Dansyl chloride proved to be a suitable agent for this purpose that confirmed the presence of the amine and phenol functional groups in these metabolites. Derivatization of M1A and M1B with dansyl chloride resulted in the formation of new peaks with protonated molecules of m/z 664 and 648, respectively. These protonated molecules were 466 Da higher than the protonated molecules of underivatized M1A and M1B, respectively, suggesting the presence of both an amine and a phenolic functionality in these metabolites. On the other hand, the derivatization of M4 with dansyl chloride gave a protonated molecule of m/z 429, 233 Da higher than the protonated molecule of underivatized M4, corresponding to the presence of only one amino group. Further CID spectra of dansylated metabolites supported the proposed structures.

In vitro studies using hepatic microsomes form humans and recombinant human P450 suggested that ezlopitant is metabolized predominantly by CYP3A4/3A5 and, to a lesser extent, by CYP2D6 (Obach, 2000, 2001). This finding suggests that elimination of ezlopitant primarily relies on a single enzyme, which may contribute to patient-to-patient pharmacokinetic variability. The human CYP3A family, accounting for 50% of the total P450 in human liver, is clinically very important because it has been shown to metabolize a large number of drugs (Wrighton et al., 1996). The human CYP3A subfamily includes CYP3A4, CYP3A5, CYP3A7 (Li et al., 1995), and CYP3A43 (Domanski et.al., 2001) as subfamily members. CYP3A4 is the major human liver CYP3A enzyme, whereas CYP3A5 is present in only 10 to 30% of the human liver and CYP3A7 is only present in fetal liver. Although ezlopitant is not a potent inhibitor of CYP3A, but is a substrate of this enzyme, attention needs to be given when it is coadministered with CYP3A inhibitors.

In conclusion, the results of this study provide the first analysis of formation and excretion of metabolites of ezlopitant in humans. Ezlopitant is rapidly absorbed in humans. The majority of the total administered ezlopitant radioactivity was excreted in the feces. Ezlopitant was eliminated mainly by oxidative metabolism, followed by excretion of metabolites into urine and feces, since no ezlopitant excreted unchanged.

	Acknowledgments

 
We thank Dr. Kathy Zandi and Sandra Miller for the synthesis of radiolabeled ezlopitant, Dr. Scott Obach for providing the deuterated standards, and Beth Obach and Jana Eaton for technical assistance.

	Footnotes

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

doi:10.1124/dmd.107.015362.

ABBREVIATIONS: SP, substance P; NK1, neurokinin 1; ezlopitant, (2-benzhydryl-1-aza-bicyclo[2.2.2]oct-3-yl)-(5-isopropyl-2-methoxy-benzyl)amine; CJ-12,764, 2-{3-[(2-benzhydryl-1-aza-bicyclo[2.2.2]oct-3-ylamino)-methyl]-4-methoxy-phenyl}-propan-2-ol; CJ-12,458, (2-benzhydryl-1-aza-bicyclo[2.2.2]oct-3-yl)-(5-isopropenyl-2-methoxy-benzyl)-amine; P450, cytochrome P450; LC/MS/MS, liquid chromatography-tandem mass spectrometry; MS, mass spectrometry; CID, collisionally induced dissociation.

¹ Current affiliation: Drug Metabolism and Pharmacokinetics, Genentech, Inc., South San Francisco, California.

Address correspondence to: Dr. Chandra Prakash, Pharmacokinetics, Dynamics and Metabolism, Pfizer Global Research and Development, Groton, CT 06340. E-mail: Chandra.prakash{at}pfizer.com

	References

Top Abstract Materials and Methods Results Discussion References

 


Diemunsch P and Grelot L (2000) Potential of substance P antagonists as antiemetics. Drugs 60: 533–546.[CrossRef][Medline]

Domanski DL, Finta C, Halpert JR, and Zaphiropoulos PG, (2001) cDNA cloning and initial characterization of cYP3A43, a novel human cytochrome P450. Mol Pharmacol 59: 386–392.[Abstract/Free Full Text]

Evangelista S (2001) Ezlopitant. Pfizer. Curr Opin Investig Drugs 2: 1441–1443.[Medline]

Frakes LA, Brehm TL, Kosty MP, Miller WE, McMillan R, Mason J, and Meisenberg BR (1997) An all oral antiemetic regimen for patients undergoing high-dose chemotheraphy with peripheral blood stem transplant. Bone Marrow Transplant 20: 473–478.[CrossRef][Medline]

Gardner CJ, Armour DR, Beattie DT, Gale JD, Hawcock AB, Kilpatrick GJ, Twissell DJ, and Ward P (1996) GR205171: a novel antagonist with high affinity for the tachykinin NK1 receptor, and potent broad-spectrum anti-emetic activity. Eur J Pharmacol 65: 45–53.

Gonsalves SF, Watson JW, and Ashton CA (1996) Broad spectrum antiemetic effects of CP-122,721, a tachykinin NK1 receptor antagonist, in ferrets. Eur J Pharmacol 305: 181–185.[CrossRef][Medline]

Grélot L, Dapzol J, Esteve E, Frugiere A, Bianchi AL, Sheldrick RL, Gardner CJ, and Ward P (1998) Potent inhibition of both the acute and delayed emetic responses to cisplatin in piglets treated with GR205171, a novel highly selective tachykinin NK1 receptor antagonist. Br J Pharmacol 124: 1643–1650.[CrossRef][Medline]

Hesketh PJ, Gralla RJ, Webb RT, Ueno W, DelPrete S, Bachinsky ME, Dirlam NL, Stack CB, and Silberman SL (1999) Randomized phase II study of the neurokinin 1 receptor antagonist CJ-11,974, in the control of cisplatin-induced emesis. J Clin Oncol 17: 338–343.[Abstract/Free Full Text]

Ishida T, Matsumoto T, Caldwell J, and Drake A (1998) Enantioselective biotransformation of ary-isopropyl, -isopropenyl, and -propenyl groups by the rabbit or by the rat. Enantiomer 3: 133–147.[Medline]

Johnson K and Prakash C (2005) Metabolism, excretion and pharmacokinetics of (3-{[4-tertbutyl-benzyl)-(pyridine-3-sulfonyl)-amino]-methyl}-phenoxy)-acetic acid, an EP-2 receptor selective pge2 agonist, in the rat. Drug Metab Dispos 33: 1191–1201.[Abstract/Free Full Text]

Kamel A and Prakash C (2006) High performance liquid chromatography/atmospheric pressure ionization/tandem mass spectrometry (HPLC/API/MS/MS) in drug metabolism and toxicology studies. Curr Drug Metab 7: 837–852.[CrossRef][Medline]

Kamel A, Prakash C, Saverino C, Obach B, and Fouda H (2000) Narrow-bore high-performance liquid chromatography in combination with ionspray tandem mass spectrometry for the determination of the substance P receptor antagonist ezlopitant and its two active metabolites in plasma. J Chromatogr B Biomed Sci Appl 748: 179–188.[CrossRef][Medline]

Li AP, Kaminsky DL, and Rasmussen A (1995) Substrate of human hepaticcytochrome P450 3A4. Toxicology 104: 1–8.[CrossRef][Medline]

Obach RS (2000) Metabolism of ezlopitant, a nonpeptidic substance P receptor antagonist, in liver microsomes: enzyme kinetics, cytochrome P450 isoform identity, and in vitro-in vivo correlation. Drug Metab Dispos 28: 1069–1076.[Abstract/Free Full Text]

Obach RS (2001) Cytochrome P450-catalyzed metabolism of ezlopitant alkene (CJ-12,458), a pharmacologically active metabolite of ezlopitant: enzyme kinetics and mechanism of an alkene hydration reaction. Drug Metab Dispos 29: 1057–1067.[Abstract/Free Full Text]

Reed-Hagen AE, Tsuchiya M, Shimada K, Wentland J-A, and Obach RS (1999) Pharmacokinetics of ezlopitant, a novel non-peptidic neurokinin-1 receptor antagonist in preclinical species and metabolite kinetics of the pharmacologically active metabolites. Biopharm Drug Dispos 20: 429–439.[CrossRef][Medline]

Roila F (1996) Control of acute cisplatinum-induced emesis over repeat course of chemotherapy. Oncology 53 (Suppl 1): 65–72.[Medline]

Tsuchiya M, Fujiwara Y, Kanai Y, Mizutani M, Shimada K, Suga O, Ueda S, Watson JW, and Nagahisa A (2002) Anti-emetic activity of the novel nonpeptide tachykinin NK1 receptor antagonist ezlopitant (CJ-11,974) against acute and delayed cisplatin-induced emesis in the ferret. Pharmacology 66: 144–152.[CrossRef][Medline]

Tsuchiya M, Sakakibara A, and Yamamoto M (2005) a tachykinin NK1 receptor antagonist attenuates the 4 beta-phorbol-12-myristate-13-acetate-induced nociceptive behavior in the rat. Eur J Pharmacol 507(1–3): 29–34.[CrossRef][Medline]

Watson JW, Gonsalves SF, and Fossa AA (1995a) The anti-emetic effects of CP-99,994 in the ferret and the dog: role of the NK1 receptor. Br J Pharmacol 115: 84–94.[Medline]

Watson JW, Nagahisha A, and Lucot JB (1995b) The tachykinins and emesis: toward complete control, in Serotonin and the Scientific Basis of Anti-emetic Therapy (Reynolds DJM, Andrew PLR, and Davis CJ eds) pp 233–238. Oxford Clinical Communications, Oxford, UK.

Wrighton SA, VandenBranden M, and Ring, B. J., (1996), The human drug metabolizing cytochrome P450. J Pharmacokinet Biopharmacol 24: 461–473.[CrossRef][Medline]

Zandi KS, Miller SA, McCarthy KE, Massefski WW, and Kamel A (2000) Synthesis and stability of substance P antagonists [¹⁴C]CJ-11,974-01 and [¹⁴C]CJ-11,972-01, in Isotope Production and Applications in the 21st Century. Proceedings of the 3rd International Conference on Isotopes, Vancouver, BC, Canada, 6–10 September, 1999 (Stevenson NR ed), pp 400–402. TRIUMF, Vancouver.

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