Differentiation of regioisomeric glucuronides by LC/MS/MS
Abstract
The absorption and metabolism of a novel antianxiety drug candidate, CP-93,393, ((7S,9aS)-2-(pyrimidin-2-yl)-7-(succinimidomethyl)-octahydro-1H-pyrido[1,2-a]pyrazine) were investigated in bile-cannulated Long Evans rats after oral administration of a single 30 mg/kg base equivalent dose of14C-CP-93,393 (HCl salt). Urine, bile, plasma, and feces were collected and assayed for total radioactivity. Plasma samples were also analyzed for unchanged drug using an LC/MS/MS assay. Metabolic profiles of 14C-CP-93,393 were obtained in urine, bile, and plasma. Structural characterization of metabolites was carried out by LC/MS using a combination of full scan, product ion, constant neutral loss, and multiple reaction ion monitoring techniques. CP-93,393 was completely absorbed, as less than 1% of the administered radioactivity was recovered in the feces. The major portion of the radioactivity was recovered in bile, suggesting that the biliary route was the primary route of excretion. Total recovery of administered dose was 97.4 ± 3.3% from male rats and 85.3 ± 9.6% from female rats. Approximately 32% of the administered radioactive dose was recovered in urine of female rats, while only 20% was recovered in urine of male rats. In contrast, biliary recoveries of the radioactivity were higher for male rats (approximately 77% for males and 53% for females). MeanCmax values for the unchanged CP-93,393 and total radioactivity were significantly higher in the female rats than in the male rats. Similarly, mean AUC(0-t)values for the unchanged drug and radioactivity were also higher in female rats. These findings suggested that CP-93,393 was eliminated more rapidly in the male rats than in the female rats.
CP-93,393 and a total of 16 metabolites were identified in urine, bile, and plasma. Based on the structures of oxidative metabolites, four metabolic pathways of CP-93,393 were identified: hydroxylation at the pyrimidine ring, hydroxylation at the succinimide ring, hydroxylation alpha to the nitrogen of the piperazine ring, and hydrolysis of amide bond of the succinimide ring. The major oxidative metabolites were excreted as sulfate and/or glucuronide conjugates. The structures of regioisomeric glucuronides were established by a novel tandem mass spectrometric technique. The glucuronides were dissociated at the orifice, and the resulting aglycones were then analyzed by MS/MS studies. The identified metabolites accounted for >90% of the total radioactivity present in urine, bile, and plasma.
CP-93,393, (7S,9aS)-2-(pyrimidin-2-yl)-7-(succinimidomethyl)-octahydro-1H-pyrido[1,2-a]pyrazine (fig. 1), is a selective and potent 5-HT1 serotonin autoreceptor agonist currently being developed for the treatment of anxiety and depression (1-4). The anxiolytic effect of CP-93,393 is believed to result from decreased serotonergic neurotransmission in the brain, a consequence of stimulating the inhibitory serotonin autoreceptors that control pacemaker potential in 5-HT neurons. CP-93,393, therefore, differs in mechanism from the benzodiazepines, which are generally believed to act by potentiating the neural inhibition mediated by GABA (5, 6).
The pharmacokinetic studies of unchanged CP-93,393 have been carried out in rats and monkeys during early development phase. These studies suggested that CP-93,393 is rapidly eliminated with at1/2 of approximately 0.5 hr in rats and 0.3 hr in monkeys.2 In addition, extensive first-pass metabolism of CP-93,393 occurred after oral administration resulting in bioavailabilty ranging from 4 to 15%.2In vitro metabolism studies using rat, monkey, and human liver microsomes suggested that CP-93,393 is mainly metabolized by hydroxylation at the pyrimidine ring (7). We now report an exhaustive study of absorption and metabolism of CP-93,393 in rats dosed orally with 14C-CP-93,393 (HCl salt) labeled at C-2 and C-3 positions of the succinimide moiety (fig. 1). Because it has been found that there were gender-related differences in bioavailability and a large interanimal variability in systemic exposure in rats after oral dosing, the routes of excretion and metabolism were investigated in both male and female rats. Metabolic profiles of 14C-CP-93,393 were obtained in urine, bile, and plasma, and the structural characterization of metabolites was carried out by LC/MS using a combination of full scan, product ion, CNL, and MRM techniques. The differentiation of the regioisomeric structures of glucuronides was accomplished by a combination of up-front CID and tandem CID. The proposed structures of several metabolites were supported by comparison of their HPLC retention times and mass spectra with those of synthetic standards.
Materials and Methods
General Chemicals.
Commercially obtained chemicals and solvents were of HPLC or analytical grade. β-Glucuronidase (from Helix Pomatia, type H-1 with sulfatase activity) was obtained from Sigma Chemical Company (St. Louis, MO). YMC basic columns were obtained from YMC (Wilmington, NC). Ecolite (+) scintillation cocktail was obtained from ICN (Irvine, CA). Carbosorb and Permafluor V scintillation cocktails were purchased from Packard Instrument Company (Downers Grove, IL).
Radiolabeled Drug and Reference Compounds.
14C-CP-93,393 HCl salt, (labeled at C-2 and C-3 positions of the succinimide ring), 5-OH-CP-93,393, and CP-93,558 were synthesized at Pfizer Central Research (Groton, CT).14C-CP-93,393 showed a specific activity of 4.76 mCi/mmol (12.9 μCi/mg) and a radiochemical purity of ≥ 98% as determined by radio-HPLC. 2-OH-CP-93,393 was synthesized by the condensation of CP-93,558 with malic acid (8). NPMSA, 2-NPMHSA, 3-NPMHSA, and 5-NHPMSA were prepared using the published procedures (9).
Animals.
Long Evans (LE) rats were purchased from Charles River Laboratories (Stoneridge, NY). Animals were quarantined for a minimum of 7 days before treatment and maintained on a 12-hr light/dark cycle. Animals were fed food and water ad libitium. All studies were conducted in a research facility accredited by the American Association for the Accreditation of Laboratory Animal Care.
Urinary, biliary, and fecal excretion studies.
A group of four male and four female LE rats (240–260 g) were implanted with a PE-10 cannula into the common bile-duct under anesthesia (ketamine/acepromazine via ip injection). The animals were housed individually in stainless steel metabolic cages and were allowed to recover overnight before drug administration. The animals were orally administered a 30 mg/kg dose of14C-CP-93,393. The dose was prepared by dissolving the radiolabeled and unlabeled CP-93,393 (mono HCl salt) in de-ionized water at a concentration of 10 mg/ml. Each rat received ∼25 μCi of radiolabeled material. All rats were fed at 2.5 hr after the dose and received electrolyte Krebs-Ringer solution as drinking solution throughout the study. Urine, bile, and feces were collected from animals for 7 days at 0–8, 8–24, 24–48, 48–72, 72–96, 96–120, 120–144, and 144–168 hr after the dose. The first feces sample was collected at 0–24 hr after the dose. Bile and urine samples were collected in 50-ml tubes containing 1 ml of ammonium acetate (pH 5.5) to avoid the hydrolysis of drug and its metabolites. The volumes of urine and bile samples were recorded, and all of the biological samples were stored at −20°C until analyzed.
Plasma time course study.
Another group of LE rats (N = 18/gender, 270–320 g) were dosed by gavage a 30 mg/kg dosage of14C-CP-93,393. Blood was collected in heparinized tubes by decapitation of two male and two female rats at 0, 0.5, 1, 2, 4, 6, 8, 12, and 24 hr after the dose. The blood samples were centrifuged at 1000g for 10 min to obtain the plasma. Samples were transferred to clean tubes and stored at −20°C until analyzed.
Determination of Radioactivity.
The radioactivity in urine, bile, and plasma was determined by liquid scintillation counting. Aliquots of plasma (100 μl), urine (20 μl), and bile (20 μl) samples were mixed with 5 ml of Ecolite (+) scintillation cocktail and counted in a Packard 2500 TR liquid scintillation counter (Downers Grove, IL). Fecal samples (0.1–0.5 g) at each time point were suspended in 20–50 ml of water and then homogenized with a Brinkman polytron. Aliquots (50–200 mg) of the homogenates were air dried over night and combusted using a Packard Tricarb Oxidizer model 307. The liberated14CO2 was trapped in Carbosorb and Permafluor V, and the radioactivity in the trapped samples was determined by counting in the liquid scintillation counter. Combustion efficiencies were determined by combustion of14C standards in an identical manner. The samples obtained at 0 hr after the dose were used as controls and counted to obtain background count rate.
Pharmacokinetic Analysis.
Plasma concentrations of the unchanged CP-93,393 were determined by an HPLC/MS/MS assay (10). Pharmacokinetic parameters were determined by standard methods. The AUC(0-t) values were calculated up to the last detectable concentration time pointt using a trapezoidal rule. TheTmax value was the time of the first occurrence of the maximal plasma concentration (Cmax). The levels of radioactivity (dpm) were expressed as μg equiv/ml by using the specific activity of the dose administered.
Extraction of Metabolites from Biological Samples.
Urine (0–24 hr) from each animal was centrifuged, and small aliquots (50 μl) were injected into the HPLC system without further purification. Bile (0–8 hr) and plasma (0–12 hr) samples were diluted with 4 volumes of acetonitrile, and the precipitated material was removed by centrifugation. The pellet was washed with an additional 2 volumes of acetonitrile, and both supernatants were combined. Small aliquots of supernatant and pellet were counted. The supernatant was concentrated and dissolved in mobile phase, and an aliquot was injected into the HPLC system.
Enzymatic Hydrolysis.
Pooled rat bile and urine samples (0.5 ml each) were adjusted to pH 5 with sodium acetate buffer (0.1 M) and treated with 2,500 units of β-glucuronidase/sulfatase. The mixture was incubated in a shaking water bath at 37°C for 12 hr and was diluted with acetonitrile. The precipitated protein was removed by centrifugation. The pellet was washed with an additional 2 ml of acetonitrile, and the two supernatants were combined. The supernatant was concentrated and dissolved in 0.5 ml of mobile phase, and an aliquot (50 μl) was injected into the HPLC system. Incubation of bile and urine samples for 12 hr without the enzyme served as a control.
HPLC.
HPLC was conducted on a system that consisted of a Rheodyne injector for manual injections, a LDC/Milton Roy constametric CM4100 gradient pump (Riviera Beach, FL), a Waters Lambda-Max model 481 UV detector (Milford, MA), a radioactivity monitor (β-RAM, INUS, Tampa, FL), and a SP 4200 computing integrator (Riviera Beach, FL). Chromatography was performed on a YMC basic HPLC column (4.6 mm × 250 mm, 5 μm) with a binary mixture of 20 mM ammonium acetate (solvent A) and acetonitrile (solvent B) at a flow rate of 1 ml/min. The mobile phase was initially composed of solvent A/solvent B (95:5) for 10 min. It was then linearly programmed to solvent A/solvent B at 60:40 over 25 min. Chromatography was conducted under isocratic conditions for 10 min and then programmed back to the starting solvent mixture over 10 min. The system was allowed to equilibrate for approximately 10 min before the next injection was made. The retention times of the radioactive peaks were compared with those of the synthetic standards.
Quantitative Assessment of Metabolite Excretion.
Quantification of the metabolites was carried out by measuring radioactivity in the individual peaks that were separated on HPLC using a β-RAM. The β-RAM provided an integrated printout in cpm and percentage of radiolabeled material as well as peak representation. The β-RAM was operated in the homogeneous liquid scintillation counting mode with addition of 4 ml/min Ecolite scintillation cocktail to the effluent after UV detection.
LC/MS.
LC/MS was conducted on a Perkin-Elmer (PE) SCIEX API III triple-stage quadrupole instrument equipped with an ion spray interface (Toronto, Canada). The effluent from the HPLC column was split, and about 50 μl/min was introduced into the atmospheric ionization source. The remaining effluent was directed into the flow cell of the β-RAM. The β-RAM response was recorded in real time by the mass spectrometer, which provided simultaneous detection of radioactivity and MS data. The delay in response between the two detectors was about 0.2 min with the mass spectrometric response being recorded first. The ion spray 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 eV and a collision gas thickness of 2.5 × 1014molecules/cm2. For the up-front CID experiments, dissociation of the protonated molecular ions of the conjugates was induced by increasing the orifice voltage to 85 eV. The resulting aglycone fragment ions were then selected in first quadrupole (Q1) and subjected to CID studies in the collision cell (Q2). Data were processed with Qudra 950 Macintosh computer operating Mac-Spec software (PE SCIEX, Toronto, Canada).
Results
Excretion.
Table 1 shows the urinary, biliary, and fecal excretion of radioactivity after dosing with14C-CP-93,393. The mean recovery of radioactivity was 97.4 ± 3.3% in male rats and 85.4 ± 9.6% in female rats. There was a marked difference in urinary excretion between male and female rats. Female rats excreted approximately 32% of the administered radioactivity in urine while only 20% was excreted in urine of males. Biliary excretion showed the opposite sex difference, that is more than 77% of the radioactivity was excreted in the bile of males while only 53% was excreted in the bile of females after oral administration.
Circulation.
The mean plasma concentration time curves of CP-93,393 and total radioactivity for male and female rats are shown in fig.2. Absorption of CP-93,393 was rapid in both male and female rats, as indicated by the early appearance of radioactivity in plasma after oral administration. The plasma concentration of total radioactivity reached a peak of 17.67 μg equiv/ml and 9.17 μg equiv/ml for female and male rats, respectively, within 0.25–0.5 hr after the dose (table2). Mean Cmaxvalues for the unchanged CP-93,393 were 5.39 μg/ml and 1.62 μg/ml in female and male rats, respectively (table 2). On the basis of AUC(0-t) values, approximately 95 and 83% of the plasma radioactivity was attributable to metabolites in male and female rats, respectively.
Identification of Metabolites. Metabolite profile in urine.
Representative profile of urinary metabolites from a male rat after oral administration of 14C-CP-93,393 with on-line radioactivity monitoring is shown in fig.3A. A total of 16 radioactive peaks were detected in the chromatogram. The metabolites were quantified with on-line integration of the radio-chromatographic peaks. The quantitative estimation of metabolites (% of dose) in urine is shown in table 3. There were no sex-related qualitative differences in the profile of urinary metabolites. However, there were significant gender-related quantitative differences in the excretion of unchanged drug and metabolites in male and female rat urine. Approximately 60% of the total radioactivity (19.2% of the dose) and 9% of the radioactivity (1.3% of the dose) was attributable to unchanged drug in urine of female and male rats, respectively (table 3).
The molecular ions of conjugates were determined using the CNL scanning of 176 (glucuronides) and 80 (sulfate) with simultaneous radioactivity monitoring. The CNL scan of 176 and the response from the radioactivity detector from a urine sample revealed the protonated molecular ions (MH+) for four metabolites: M-1,M-2, M-7, and M-12. The molecular ions of remaining metabolites were determined by full scan LC/MS. The structures of all the metabolites were elucidated by interpreting their product ion spectra and, where possible, were supported by comparison of HPLC retention times with those of synthetic standards.
Metabolite M-1.
M-1 had a retention time of 9:18–10:14 min (min:sec) on HPLC. Its full scan MS showed a protonated molecular ion (MH+) at m/z 540, 210 Da higher than the parent drug, indicating that it was a conjugate. The CNL (176) scan also confirmed the protonated molecular ion at m/z 540. CID product ion spectrum of M-1 (m/z 540) gave an abundant ion at m/z 364; loss of 176 Da (glucuronic acid moiety) from the precursor ion indicated that it was a glucuronide. The ion at m/z 364, 34 Da higher than the parent drug, suggested that the molecule had undergone monooxygenation and an addition of water. The CID product ion spectrum of the ion at m/z 364 (generated at the orifice) gave fragment ions at m/z 318, 290, 248, 231, 136, and 122 (fig.4). The fragment ion at m/z 318 (loss of 46 Da) suggested the presence of a free carboxyl group. The other prominent fragment ions at m/z 248, 231, 136, and 122 indicated that the oxidation had occurred at the succinimide moiety. The characteristic fragment ion at m/z 290 (loss of.C(OH)COOH) suggested the presence of a hydroxy group alpha to the carboxyl moiety. Based on these data, M-1was identified as 2-NPMHSA glucuronide.
Metabolite M-2.
M-2 had a retention time of 12:48–12:59 min on HPLC. It also showed a protonated molecular ion at m/z 540, suggesting that it was a regioisomer of M-1. The CID product ion spectrum ofM-2 gave an abundant ion at m/z 364; loss of 176 Da from the precursor ion indicated that it was a glucuronide. The CID product ion spectrum of m/z 364 (generated from M-2) showed fragment ions at m/z 304, 248, 231, 136, and 122 (fig.5). The presence of prominent fragment ions at m/z 248, 231, 136, and 122 suggested that the oxidation had occurred at the succinimide moiety. The characteristic fragment ion at m/z 304 (loss of CH3COOH from m/z 364) suggested the presence of a hydroxy group beta to the carboxyl moiety. Based on these data, M-2 was identified as 3-NPMHSA glucuronide.
Metabolite M-4.
M-4 had a retention time of 17:19–17:32 min on HPLC. It displayed a protonated molecular ion at m/z 380, 50 Da higher than the parent drug, indicative of the addition of two oxygen atoms and a molecule of water. CID product ion spectrum of m/z 380 gave the characteristic fragment ions at m/z 138 and 152, suggesting that an oxygen atom had been added to the pyrimidine moiety. The fragment ions at m/z 247 and 264 suggested that the addition of another oxygen atom and water had occurred at the succinimide ring. The characteristic ion at m/z 306 (loss of .COHCOOH from the protonated molecular ion) indicated the presence of a hydroxyl group alpha to the carboxyl group. Based on these data, M-4 was tentatively identified as 2,5-(OH)2-NPMSA.
Metabolite M-6.
M-6 displayed a protonated molecular ion at m/z 364, 34 Da higher than the parent drug, indicative of the addition of an oxygen atom and a molecule of water. The CID product ion spectrum of m/z 364 gave prominent and significant ions at m/z 138 and 152, suggesting the addition of an oxygen atom on the pyrimidine ring. The fragment ion at m/z 247 indicated that the addition of water had occurred at the succinimide ring. It coeluted with synthetic 5-NHPMSA on HPLC and had an identical CID spectrum. M-6 was thus identified as 5-NHPMSA.
Metabolite M-7.
M-7 had a retention time of 18:28–19:02 min on HPLC. It showed a protonated molecular ion at m/z 522, 176 Da higher than the hydroxylated CP-93,393, indicating that it was a glucuronide conjugate of the oxidative metabolite. The CNL (176) scanning also confirmed the protonated molecular ion at m/z 522. The CID product ion spectrum of m/z 346 (generated from M-7) showed fragment ions at m/z 209, 195, 152, and 138 (fig. 6). The fragment ions at m/z 138 and 152 suggested the presence of a hydroxyl group on the pyrimidine ring. The presence of other ions at m/z 209 and 195 further suggested that the oxidation had occurred remote from the succinimide portion of the molecule. Treatment of the urine sample with β-glucuronidase resulted in the disappearance of M-7 and the increase in peak area of M-15. Based on these data,M-7 was identified as 5-OH-CP-93,393 glucuronide.
Metabolite M-8.
M-8 had a retention time of 19:39–20:12 min. It showed a protonated molecular ion at m/z 364, 34 Da higher than the parent drug, indicative of the addition of an oxygen atom and a molecule of water. The fragment ion at m/z 318, a loss of 46 Da in its CID product ion spectrum, suggested the presence of a free -COOH group. The presence of other fragment ions at m/z 231, 248, 136, and 122 suggested that the oxidation and hydrolysis had occurred at the succinimide moiety. The ion at m/z 290 (loss of CHOHCOOH from the protonated molecular ion) indicated the position of a hydroxyl group to be alpha to the carboxyl group. It coeluted with synthetic 2-NPMHSA on HPLC and had an identical CID product ion spectrum. M-8 was thus identified as 2-NPMHSA.
Metabolite M-9.
M-9 had a retention time of 20:05–20:19 min. It exhibited a protonated molecular ion at m/z 348, 18 Da higher than the parent drug, suggesting that the molecule had undergone hydrolysis. Its CID product ion spectrum showed fragment ions at m/z 122 and 136, indicating that the pyrimidine ring was unsubstituted. The presence of an ion at m/z 231 further suggested that hydrolysis had occurred remote from the pyrimidine portion of the molecule. M-9 coeluted with synthetic NPMSA standard on HPLC and had identical CID product ion spectrum. Based on these data, M-9 was identified as NPMSA.
Metabolite M-10.
M-10 had a retention time of 20:56–21:20 min. It showed a protonated molecular ion at m/z 346, 16 Da higher than the parent drug, suggesting that it was a monohydroxylated product. The CID product ion spectrum of m/z 346 gave fragment ions at m/z 328, 221, 201, 195, 120, and 108 (fig. 7). The fragment ion at m/z 328, with the loss of a water molecule, suggested the presence of a hydroxyl group. The prominent fragment ions at m/z 209, 195, and 108 indicated that that the pyrimidine and succinimide portions of the molecule were unsubstituted. The significant and distinct fragment ions at m/z 221 and 120 suggested that oxidation had occurred alpha to the nitrogen of the piperazine ring. Based on these data, M-10 was tentatively identified as (7S,9aS)-2-(pyrimidin-2-yl)-7-(succinimidomethyl)-octahydro-hydroxy-pyrido[1,2-a]pyrazine.
Metabolite M-11.
M-11 had a retention time of 21:50–22:31 min. Its full scan MS showed a protonated molecular ion at m/z 442, 112 Da higher than the parent drug, suggesting that it was a conjugate. CID product ion spectrum of M-11 (m/z 442) gave an abundant ion at m/z 362, a loss of 80 Da (SO3), indicating that it was a sulfate conjugate. The CID product ion spectrum of m/z 362 showed a fragment ion at m/z 138, indicating the presence of a hydroxyl group on the pyrimidine ring. The significant and distinct fragment ions at m/z 325 and 245 [loss of 117 Da (succinimide+H2O) from the ions at m/z 442 and 362, respectively] suggested that another oxidation had occurred at the pyrazinyl-methyl part of the molecule (fig. 8). Based on these data,M-11 was tentatively identified as a sulfate conjugate of hydroxy (7S,9aS)-2-(5-hydroxypyrimidin-2-yl)-7-(succinimido-methyl)-octahydro-1H-pyrido[1,2-a]pyrazine.
Metabolite M-12.
M-12 had a retention time of 22:42–23:09 min on HPLC. It showed a protonated molecular ion at m/z 522, 176 Da higher than the hydroxylated CP-93,393, indicating that it was a glucuronide conjugate. The CNL (176) scan also confirmed the protonated molecular ion at m/z 522. The CID product ion spectrum of m/z 346 (generated fromM-12) showed fragment ions at m/z 251, 225, 211, 136, and 122 (fig. 9). The fragment ions at m/z 251, 225, and 211 suggested that the oxidation had occurred at the succinimide portion of the molecule. The presence of other characteristic fragment ions at m/z 122 and 136 further suggested that the pyrimidine moiety was unchanged. Treatment of the urine sample with β-glucuronidase resulted in the disappearance of M-12 and the increase in peak area of M-16. Based on these data,M-12 was identified as 2-OH-CP-93,393 glucuronide.
Metabolite M-13.
M-13 had a retention time of 23:49–24:02 min on HPLC. Its full scan MS showed an ammoniated molecular ion at m/z 443 and a protonated molecular ion at m/z 426, 80 Da higher than the hydroxylated CP-93,393, indicating that it was a conjugate. The ion at m/z 346, a loss of 80 Da (SO3) from the protonated molecular 1ion, suggested that it was a sulfate conjugate. The CNL (80) scan also confirmed the protonated molecular ion at m/z 426. The MS/MS spectrum of m/z 346 (generated from M-13) showed fragment ions at m/z 235, 209, 195, 152, and 138. The fragment ions at m/z 138 and 152 suggested the presence of a hydroxyl group at the pyrimidine ring. The presence of other prominent ions at m/z 235, 209, and 195 further suggested that the oxidation had occurred remote from the succinimide portion of the molecule. Based on these data,M-13 was identified as 5-OH-CP-93,393 sulfate.
Metabolite M-14.
M-14 had a retention time of 24:51–25:52 min on HPLC. Its full scan MS showed a protonated molecular ion at m/z 362, 32 Da higher than the drug, suggesting that the two oxygen atoms had been added to the molecule. The fragment ions at m/z 138 and 152 in its CID spectrum suggested the presence of a hydroxyl group at the pyrimidine ring. The presence of other ions at m/z 251 and 225 further suggested that the oxidation had also occurred at the succinimide portion of the molecule. Based on these data, M-14 was identified as 2,5-(OH)2-CP-93,393.
Metabolite M-15.
M-15 had a retention time of 26:01–26:28 min on HPLC. Its full scan MS showed a protonated molecular ion at m/z 346, 16 Da higher than the parent drug, suggesting that it was a monooxygenation product of CP-93,393. The fragment ions at m/z 138 and 152 in its CID product ion spectrum suggested the presence of a hydroxyl group at the pyrimidine ring. The presence of other prominent ions at m/z 209 and 195 further suggested that the oxidation had occurred remote from the succinimide portion of the molecule. M-15 coeluted with synthetic 5-OH-CP-93,393 on HPLC and had an identical CID spectrum.M-15 thus was identified as 5-OH-CP-93,393.
Metabolite M-16.
M-16 also indicated a protonated molecular ion at m/z 346, 16 Da higher than the drug, suggesting that it was a regioisomer ofM-15. The fragment ions at m/z 251, 225, and 211 suggested that the oxidation had occurred at the succinimide portion of the molecule. The presence of characteristic ions at m/z 122 and 136 indicated that the pyrimidine moiety was unchanged. M-16coeluted with synthetic 2-OH-CP-93,393 on HPLC and had an identical CID product ion spectrum. Based on these data, M-16 was identified as 2-OH-CP-93,393.
Metabolite M-17 (unchanged drug).
M-17 had a retention time of 29:15–30:05 min. Its full scan MS showed a protonated molecular ion at m/z 330, the same as the parent drug. Its CID product ion spectrum showed the fragment ions at m/z 122 and 136, suggesting that the pyrimidine ring was unsubstituted. The presence of ions at m/z 235, 209, and 195 further suggested that the succinimide ring was intact. M-17 coeluted with synthetic CP-93,393 standard on HPLC. Based on these data, M-17 was identified as unchanged drug.
Metabolite profiles in bile.
Representative profile of biliary metabolites from a male rat after oral administration of 14C-CP-93,393 is shown in fig. 3B. The following metabolites that were identified from rat urine were also observed in the bile: M-1,M-2, M-4, and M-7 throughM-17. Two additional metabolites, M-3 andM-5, were characterized in rat bile. The quantitative estimation of metabolites (% of dose) excreted in the bile is presented in table 3. Like urine, there were no qualitative differences in the profile of biliary metabolites from male and female rats. However, there were significant sex-related quantitative differences in the excretion of unchanged drug and metabolites in male and female rat bile. Approximately 4.2% of the total radioactivity (2-3% of the dose) in the bile of females was due to unchanged drug while only <0.4% of the radioactivity (0.3% of the dose) was attributable to unchanged drug in males. The conjugated metabolites, sulfate (M-13) and glucuronides (M-1 and M-2), were higher in male rats than in the female rats.
Metabolite M-3.
M-3 had a retention time of 12:12–12:32 min on HPLC. It showed a protonated molecular ion at m/z 460. The CID product ion spectrum of M-3 gave an abundant ion at m/z 380, a loss of 80 Da from the precursor ion, indicating a sulfate conjugation. The other characteristic ion at m/z 138 indicated that the one oxygen atom had been added to the pyrimidine moiety. Furthermore, the fragment ions at m/z 247 and 264 suggested that the addition of another oxygen atom and water had occurred at the succinimide ring. The prominent ion at m/z 306 (loss of COHCOOH from the protonated molecular ion) indicated the presence of a hydroxyl group alpha to the carboxyl group. Based on these data, M-3 was tentatively identified as sulfate conjugate of 2,5-(OH)2-NPMSA.
Metabolite M-5.
M-5 had a retention time of 17:53–18:10 min on HPLC. It showed a protonated molecular ion at m/z 538, 208 Da higher than the parent drug, indicating that it was a conjugate. The CID product ion spectrum of M-5 gave an ion at m/z 362, a loss of 176 Da (glucuronic acid moiety) from the precursor ion, indicating that it was a glucuronide. The fragment ion at m/z 362, 32 Da higher than the drug, suggested that two oxygen atoms had been added to the molecule. The fragment ions at m/z 138 and 152 suggested that one oxygen atom had been added to the pyrimidine moiety. The significant and distinct fragment ions at m/z 421 and 245 [loss of 117 Da (succinimide +H2O) from the ions at m/z 538 and 362, respectively] indicated that another oxidation had occurred at the pyrazinyl-methyl part of the molecule. Based on these data,M-5 was tentatively identified as the glucuronide conjugate of hydroxy (7S,9aS)-2-(-5-hydroxy-pyrimidin-2-yl)-7-(succinimido-methyl)-octahydro-pyrido[1,2-a]pyrazine.
Metabolic profiles in plasma.
A total of 12 radioactive peaks were detected in the radio-HPLC chromatograms from both male and female rats (not shown). The metabolites were quantified by counting the radioactivity of individual peaks that were separated on HPLC. The mean percentages of circulating metabolites in relation to the total radioactivity observed in plasma are presented in table 4. The metabolites were identified by ion spray LC/MS/MS using MRM technique and by comparison of retention times on HPLC with synthetic standards and/or urine samples. MRM ions used to detect the metabolites in plasma were as follows: M-1 (m/z 540→364), M-2 (m/z 540→364), M-4 (m/z 380→138), M-6 (m/z 364→138), M-7 (m/z 522→346), M-9 (m/z 348→122), M-10 (m/z 346→221), M-11 (m/z 459→362), M-13 (m/z 443→346), M-15 (m/z 346→138), and M-17 (m/z 330→122). The identified metabolites accounted for >90% of the total radioactivity present in plasma of male and female rats.
Discussion
The present study has shown that the major excretion route of orally administered 14C-CP-93,393 in LE rats wasvia the bile. The administered radioactivity was quantitatively recovered in all rats except two females (table 1). A major portion of the total radioactivity (98%) was recovered during the first 24 hr after the dose, suggesting that CP-93,393 was very rapidly eliminated. There were marked sex-related differences in the excretion of CP-93,393. Urinary excretion of 14C label was about two times greater in females than in the male rats. In contrast, biliary excretion showed the opposite sex difference. The absorption of CP-93,393 was rapid in both male and female rats with maximal observed plasma concentrations for the total radioactivity occurring between 0.25 and 0.5 hr after the dose. TheCmax and AUC(0-t)values for unchanged drug and total radioactivity were significantly greater in the female rats than in the male rats (table 2). These findings suggested that CP-93,393 was eliminated more rapidly in the male rats than in the female rats. The sex-related differences in the elimination and pharmacokinetics of drugs, particularly in rats, have been well known and can be a result of the differences in hormones levels, plasma protein binding, and/or rate and extent of metabolism (11-19).
CP-93,393 was rapidly and extensively metabolized in both male and female rats. CP-93,393 and a total of 16 metabolites were identified by ion spray LC/MS/MS (20-24). Most of the major metabolites found in urine were also found in bile and plasma, although there were significant quantitative differences in the relative amounts (table 3). The plausible scheme for the biotransformation pathways of CP-93,393 in rat is shown in fig. 10. The metabolism of CP-93,393 occurred by four pathways: aromatic hydroxylation at the 5-position of the pyrimidine ring leading to the formation of 5-OH-CP-93,393 (M-15), hydroxylation alpha to the succinimide carbon at the 2-position of the succinimide ring to give 2-OH-CP-93,393 (M-16), hydroxylation alpha to the piperazinyl nitrogen attached to the pyrimidine ring to give 1-or 3-OH-CP-93,393 (M-10), and hydrolysis of the amide bond of the succinimide ring (M-9). Metabolites derived from these pathways were found to undergo subsequent metabolism by various combinations of the primary routes or by conjugation with glucuronic acid and/or sulfuric acid.
The first major route, hydroxylation on the pyrimidine ring was similar to other drugs containing a pyrimidinyl-piperazine ring structure (25-27). The metabolites (M-7, 35.8%; M-13, 20.5%; and M-15, 5.8%), derived from this pathway, comprised a major fraction (62%) of the biliary metabolites and a major percentage (40%) of dose. Quantitatively less radioactivity was excreted in the urine, and lesser amounts of 5-hydroxylated metabolites were present in the urine (6.3% of the dose, table 3). The exact position of the hydroxyl group was determined by comparing the HPLC retention time with synthetic standard.
Aliphatic hydroxylation at the succinimide ring leads to the formation of 2-OH-CP-93,393 (M-16). Metabolite M-16undergoes further metabolism by conjugation with glucuronic acid (M-12) and subsequent hydrolysis to form a mixture of two isomeric glucuronides (2-NPMHSA, M-1; and 3-NPMHSA,M-2). The CID product ion spectra of 2-NPMHSA and 3-NPMHSA glucuronides did not provide any significant fragment ions other than aglycones. The structures of these isomeric glucuronides were elucidated by a combination of up-front CID and tandem MS/MS technique. The glucuronide conjugates were dissociated at the orifice, and the resulting aglycones were subjected to CID studies. The metabolitesM-1 and M-2 were identified as glucuronides of 2-NPMHSA and 3-NPMHSA by interpretation of CID spectra of the aglycones. Hydroxysuccinimide metabolites and their hydrolysis products are known for other compounds, but the formation of glucuronide conjugates has not been reported (28-30). Based on the hydrolytic behavior of hydroxysuccinimides, detection of both 2-NPMHSA and 3-NPMHSA is not surprising (31). Furthermore, 2-NPMHSA-glucuronide was formed predominantly, and it could be assumed that these metabolites were the result of hydrolysis of 2-OH-CP-93,393 glucuronide (31). An alternative pathway for the formation of these metabolites by initial hydrolysis of CP-93,393 to NPMSA (M-9) followed by hydroxylation and subsequent glucuronidation, however, could not be ruled out at this time.
The other important findings in the present study were the oxidations alpha to the nitrogen of the piperazine ring (M-10) and succinimide ring (M-5 and M-11) to form the carbinolamine metabolites. The formation of carbinolamine intermediates is well established, but they are unstable and spontaneously decompose to the corresponding aldehyde and dealkylated amine (32, 33). However, substituents adjacent to the nitrogen atom, which de-localize the lone pair of nitrogen electrons, can stabilize the carbinolamines. Stable carbinolamine metabolites have been isolated for a number of drugs including hexamethylmelamine (34), benzamides (35), carbamates (36), the muscle relaxant xilobam (37), and the antitumor agent procarbazine (38). The stability of metabolites M-5, M-10, andM-11 could, therefore, be speculated by either de-localization of the lone pair of nitrogen electrons or the formation of a six member transition state. Metabolite M-5 was present only in bile while M-10 and M-11 were present both in bile and urine. Recently, hydroxylation alpha to the piperazinyl nitrogen has also been reported for a veterinary drug amperozide (39).
The major portion of radioactivity recovered was excreted in urine and bile as conjugates of hydroxylated metabolites. There were no sex-related qualitative differences in the profile of metabolites; however, there were significant gender-related differences in the excretion of unchanged drug and metabolites in male and female rat urine and bile. Approximately 21% of the dose was excreted as unchanged drug in female rats, while only 1.6% of the dose was attributable to unchanged drug in the male rats (table 3). Similarly, unchanged drug was significantly higher in the plasma of female rats than the male rats (table 4). These findings suggested that male rats metabolized CP-93,393 more rapidly than females. Many drugs and steroids have been shown to exhibit sex-dependent metabolism in rats (40, 41). Investigations with in vitro enzyme systems have suggested that these sex differences were mainly a result of the differential expression of various drug-metabolizing enzymes (CYP isoforms) mediated by hormonal regulations (40-43).
In conclusion, the present study has demonstrated that CP-93,393 was completely absorbed and extensively metabolized by a variety of routes yielding a large number of metabolites in rat after oral administration. The proposed structural identity of 10 metabolites was supported by co-elution on HPLC with synthetic standards, and six additional metabolites were tentatively identified. The combination of up-front CID and tandem CID information proved to be very helpful for differentiation of the structures of regioisomeric glucuronides. The identified metabolites accounted for >90% of the total radioactivity present in urine, bile, and plasma. The present study will contribute to our understanding the metabolism of CP-93,393 in human subjects.
Acknowledgments
We thank Drs. Keith McCarthy, Kathleen Zandi, and Michael Bright for providing radiolabeled CP-93,393 and synthetic standards; we also thank Drs. Jim Baxter, Hassan Fouda, and Robert Ronfeld for helpful suggestions.
Footnotes
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Send reprint requests to: Chandra Prakash, Ph. D., Department of Drug Metabolism, Central Research Division, Pfizer Inc., Groton, CT 06340.
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This work was presented in part at the 8th North American International Society for the Study of Xenobiotics meeting, San Diego, CA (1996).
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↵2 J. Baxter et al. Manuscript in preparation.
- Abbreviations used are::
- 5-HT
- 5-hydroxytryptamine
- GABA
- γ-aminobytyric acid
- 5-OH-CP-93
- 393, (7S,9aS)-2-(5-hydroxypyrimidin-2-yl)-7-(succinimidomethyl)-octahydro-1H-pyrido[1,2-a]pyrazine
- 2-OH-CP-93
- 393, (1S,9aS)-2-(pyrimidin-2-yl)-7-(2-hydroxysuccinimidomethyl)-octahydro-1H-pyrido[1,2-a]pyrazine
- CP-93
- 558, 2-(pyrimidin-2-yl)-octahydro-1H-pyrido[1,2-a]pyrazin-7-yl)methylamine
- NPMSA
- N-(2-pyrimidin-2-yl-octahydro-pyrido[1,2-a]pyrazin-7-ylmethyl)-succinamic acid
- 2-NPMHSA
- N-(2-pyrimidin-2-yl-octahydro-pyrido[1,2-a]pyrazin-7-yl-methyl)-2-hydroxysuccinamic acid
- 3-NPMHSA
- N-(2-pyrimidin-2-yl-octahydro-pyrido[1,2-a]pyrazin-7-yl-methyl)-3-hydroxysuccinamic acid
- 5-NHPMSA
- N-[2-(5-hydroxy-pyrimidin-2-yl)octahydro-pyrido[1,2-a]pyrazin-7-ylmethyl]succinamic acid
- LE
- Long Evans
- radio-HPLC
- HPLC with on-line radioactivity detector
- β-RAM
- radioactive monitor
- CID
- collisionally induced dissociation
- CNL
- constant neutral loss, MRM, multiple reaction monitoring
- CYP
- cytochrome P450
- Received April 3, 1997.
- Accepted July 15, 1997.
- The American Society for Pharmacology and Experimental Therapeutics