Introduction

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Vasopeptidase inhibitors inhibit two key enzymes involved in the regulation of cardiovascular function, neutral endopeptidase (NEP¹) and angiotensin-converting enzyme (ACE) (Robl et al., 1999). ACE and NEP are zinc metalloproteases that catalyze the conversion of angiotensin I to angiotensin II and degradation of atrial natriuretic peptide (ANP), respectively (Burnett, 1999). Angiotensin II is a potent vasoconstricter that triggers the release of aldosterone, a sodium retaining steroid. This effect leads to elevated blood pressure caused by increasing both vascular resistance and fluid volume (Trippodo et al., 1995). Atrial natriuretic peptide, secreted by the heart in response to atrial distention, by interaction with its receptor, promotes the generation of cyclic guanosine monophosphate resulting in vasodilation, natriuresis, diuresis, and possibly inhibition of aldosterone formation. Atrial natriuretic peptide has an opposite effect to that of angiotensin II (Seymour et al., 1995; Trippodo et al., 1995).

As a potent inhibitor of both ACE and NEP, omapatrilat offers several advantages over existing ACE inhibitors (Robl et al., 1997, 1999; Trippodo et al., 1998). Omapatrilat is currently under development as a new drug for the treatment of hypertension and is expected to be highly effective when given once daily (Ruddy et al., 1999; Waeber et al., 1999). In a previous study, biotransformation profiles of human urine from subjects dosed orally with single-labeled [¹⁴C]omapatrilat showed extensive metabolism in vivo (Iyer et al., 2001; Malhotra et al., 2001). Nine metabolites were identified, and together these metabolites accounted for 94% (60% dose) of the radioactivity excreted in the urine. Metabolic pathways identified for omapatrilat in humans include methylation and disulfide conjugation at the free thiol group, glucuronide conjugation of the carboxyl group, oxidation of the S-methyl metabolite, and hydrolysis of the exocyclic amide bond. However, in that study, no metabolites from the amine product of the hydrolysis of the exocyclic amide bond were determined due to the lack of the ¹⁴C-label on the amine side of the molecule. Therefore the study described here was specifically conducted with a mixture of [¹⁴C]ompatrilat, labeled at the exocyclic carbonyl carbon and at the ethyl carbons attached to the bicyclic ring (Fig. 1). The objective was to identify all the possible metabolites, including the potential amine side metabolites arising from hydrolysis of the exocyclic amide bond, in both urine and plasma of rats, dogs, and humans. Rats and dogs were the toxicological species used in the development of omapatrilat. [¹³C₂]Omapatrilat (Fig. 1) was also included in the dose to assist in metabolite identification based on the occurrence of a characteristic M + 2 isotope cluster in the mass spectrum. This study describes the comparative biotransformation of omapatrilat in rats, dogs, and humans.

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Structures of omapatrilat labeled with C-14 and C-13 labels. The dose was a mixture of [14C]omapatrilat and [13C2]omapatrilat. *, site of C-14 labels; every radiolabeled molecule contains only one C-14 atom at any one of the three possible positions indicated. #, site of C-13 labels; every stable-labeled molecule contains two C-13 atoms, one at each position indicated.

	Materials and Methods

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Chemicals. The reference standards for HPLC, namely, [4S-(4,7,10a)]-4-aminooctahydro-5-oxo-7H-pyrido[2,1-b][1,3]thiazepine-7-carboxylic acid (amine side hydrolysis product of omapatrilat), diastereomeric sulfoxides of (S)-2-thiomethyl-3-phenylpropionic acid, L-cysteine mixed disulfide of omapatrilat, diastereomeric sulfoxides of S-methyl omapatrilat, bicyclic ring sulfoxide of S-methyl omapatrilat, (S)-2-thio-3-phenylpropionic acid, (S)-2-thiomethyl-3-phenylpropionic acid, omapatrilat, S-methyl omapatrilat, and symmetrical disulfide of omapatrilat were obtained from Research Chemical Distribution, Bristol-Myers Squibb Pharmaceutical Institute. [¹⁴C]Omapatrilat with C-14 label on the exocyclic carbonyl carbon (10.4 µCi/mg, radiochemical purity 99.5%), [¹⁴C]omapatrilat with two C-14-labeled carbons on the bicyclic portion (10.3 µCi/mg, radiochemical purity 98.4%), and [¹³C₂]omapatrilat with C-13 labels on two carbons of the bicyclic portion (purity 99.4%) were provided by the Radiochemistry Group, Discovery Chemistry (Princeton, NJ). The site of the C-14 and C-13 labels on omapatrilat are shown in Fig. 1. Ecolite liquid scintillation cocktail was purchased from ICN Biomedicals, Inc. (Costa Mesa, CA). -Glucuronidase from Helix pomatia and D-saccharic acid 1,4-lactone were obtained from Sigma-Aldrich (St. Louis, MO). For solid phase extraction, Sep-Pak Vac 20.0 cc (C₁₈, 5.0 g) cartridges were obtained from Waters Corporation (Milford, MA). All other chemicals used were reagent grade or better.

Dosing and Sample Collection. Samples of blood (for plasma), urine, and feces were obtained from three studies in which single oral doses of [¹⁴C]omapatrilat (equal portions of side chain-labeled and bicyclic-labeled compound) and [¹³C₂]omapatrilat were administered to male rats (20 mg/kg, 2 µCi/mg), dogs (200 mg, 20 µCi), and healthy human subjects (50 mg, 100 µCi). In each case, [¹⁴C]/[¹³C₂]omapatrilat was administered as a freshly prepared solution in 2% sodium bicarbonate. Blood samples for the determination of plasma concentrations of radioactivity were collected serially up to 96 h postdose for rat and up to 168 h postdose for dog and human into vacutainer tubes containing EDTA as an anticoagulant. For biotransformation analysis, additional blood samples were collected at 1 and 6 h postdose. For the determination of unchanged omapatrilat in plasma, samples were collected in pharmacokinetic studies conducted separately with either unlabeled (rat) or single-labeled [¹⁴C]omapatrilat (dog and human) administered at the same dose level as in the biotransformation studies. The blood samples for pharmacokinetic determination were collected up to 24 h (rat), 96 h (dog), or 120 h (dog and human) into EDTA Vacutainer tubes that also contained methyl acrylate (10 µl per ml of blood). Methyl acrylate was used to form a Michael adduct with the sulfhydryl group of omapatrilat and its metabolites (Jemal and Hawthorne, 1997a,b) and to prevent disulfide formation. Plasma was prepared from the blood samples by centrifuging for 15 min at 1000g and 5°C. Urine and feces were collected from the same dogs and humans at 24-h intervals for 168 h after dosing and from three additional rats at 24-h intervals for 48 h after dosing. Urine samples for dog and human studies were collected into 2.5 ml of acetic acid. Urine samples for rat study were collected in 2.5 ml of 2% v/v acetic acid.

Analyses of Radioactivity. The radioactivity in plasma, urine, and fecal homogenate was quantified as disintegrations per min (DPM) with a Packard Tri-Carb-2750 liquid scintillation counter (LSC; PerkinElmer Life Sciences, Boston, MA). The samples were counted for 20 min and any net counts per minute value with a >20% counting error in determination was considered below the lower limit of quantitation. Plasma samples (0.2 ml) were mixed with 15 ml of Hionic-Fluor scintillation cocktail (PerkinElmer Life Sciences), and analyzed by LSC. The DPM value was converted to nanogram-equivalent of omapatrilat from the specific activity of the dosing solution. For analysis of urine samples, 0.2 ml of urine from each interval was mixed with 15 ml of Hionic-Fluor scintillation cocktail and analyzed by LSC. For analysis of fecal samples, about 0.2 g of the fecal homogenate from each interval was combusted to ¹⁴CO₂ with a model 307 sample oxidizer (PerkinElmer Life Sciences). The ¹⁴CO₂ was trapped in 8 ml of Carb-Sorb solvent. Perma-flour scintillation cocktail (9 ml) was added and radioactivity analyzed by LSC.

Analysis of Omapatrilat. Plasma sample preparation for analysis of omapatrilat methylacrylate adduct in rat, dog, and human was similar to the procedure used by Jemal and Hawthorne (1997a,b). The plasma concentrations of unchanged omapatrilat were determined by validated LC/MS or LC/MS/MS methods (Jemal and Hawthorne, 1997a,b; Jemal et al., 2001). The plasma volume used for analysis was 0.1 ml (rat), 0.5 ml (dog), and 1.0 ml (human).

Preparation of Samples for Biotransformation.

Plasma For radioactivity profiling, pooled 1-h plasma samples were prepared as described below. The plasma volume used for analysis for rat, dog, and human was 0.25, 1.0, and 1.0 ml, respectively. Pooled plasma was extracted by addition of two volumes of methanol to one volume of plasma, sonicated for 5 min and centrifuged on an IEC Centra-7R refrigerated centrifuge (Thermo IEC, Needham Heights, MA). The supernatant was removed and saved. The extraction was repeated three times and the supernatants combined. A mixture of reference standards in solution (25 µl) was added to the combined supernatant fractions, and the solution was evaporated to dryness under a stream of nitrogen. The residue was suspended in 150 µl of mobile phase (95% A and 5% B) used for biotransformation analysis and filtered through a Gelman Acrodisc LC PVDF 0.45-µm syringe filters before injecting onto the HPLC column (Pall Corp., Ann Arbor, MI).

Urine. Radioactivity profiling of pooled rat urine was done by directly injecting 50 µl of the sample onto the HPLC column after centrifugation on an IEC Centra-7R refrigerated centrifuge. For profiling of dog and human pooled 0- to 168-h urine samples, 10 ml of the urine was evaporated to dryness in a Savant Speed-Vac (Savant Instruments, Holbrook, NY). The residue was suspended in 1 ml of mobile phase (95% A and 5% B) used for biotransformation analysis, centrifuged on an IEC Centra-7R refrigerated centrifuge, and the supernatant (100 µl) injected onto the HPLC column.

-Glucuronidase incubation of urine and plasma. Pooled urine samples from dogs (0-168 h) and humans (0-168 h), concentrated for profiling as described above, were used for the -glucuronidase incubations. The detailed procedure for -glucuronidase hydrolysis has been described in a previous paper (Iyer et al., 2001). Pooled rat urine was not subjected to glucuronidase hydrolysis since no radioactive peaks corresponding to the retention times of glucuronide conjugates were observed in the rat urine.

HPLC for Biotransformation Analysis. The HPLC conditions, solvents, and collection of fractions for radioactivity profiling have been described in the previous paper (Iyer et al., 2001). A gradient was used in which solvent B was maintained at 0% for 5 min and then increased in a linear manner as follows: 25% (15 min), 30% (30 min), 33% (45 min), 55% (50 min), and 90% (60 min).

LC/MS and LC/MS/MS for Biotransformation Analysis. HPLC was done with a Shimadzu Class VP system (Shimadzu, Columbia, MD) described in the previous paper (Iyer et al., 2001). The solvent system consisted of solvent A, water containing 0.4% formic acid and 0.1% triethylamine (pH 2.6) and solvent B, acetonitrile/water (90:10) containing 0.4% formic acid and 0.1% triethylamine. HPLC analysis was done at 35°C at a flow rate of 0.4 ml/min with a Zorbax RX C₁₈ column (2.1 × 150 mm, 5-micron; Agilent Technologies Inc., Wilmington, DE). Solvent B was maintained at 0% for 5 min and then increased in a linear manner as follows: 25% (15 min), 30% (30 min), 33% (45 min), 55% (50 min), and 90% (60 min). The HPLC effluent was directed to waste through a divert valve for 4 min after sample injection and then redirected through a 1:1 splitter into a Finnigan LCQ mass spectrometer (Thermo Finnigan MAT, San Jose, CA). Mass spectral analysis of all standards and metabolites was performed on a Finnigan LCQ mass spectrometer with an electrospray ionization probe. Samples were analyzed in the negative ion mode for metabolites M1-M9 and in the positive ion mode for metabolite M1-a. For standard samples introduced by infusion, the flow rate was 3 to 5 µl/min, and the solvent was either acetonitrile/water (1:1) or acetonitrile/10 mM ammonium acetate buffer, pH 4.9 (1:1). For samples introduced by HPLC, the flow rate to the mass spectrometer was 200 µl/min. The capillary temperature used for analysis was between 220 to 230°C. The nitrogen gas flow rate, spray current, and voltages were adjusted for each metabolite to give the maximal sensitivity.

	Results

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Radioactivity Elimination Profile. The urinary and fecal excretion of radioactivity in rats, dogs, and humans after oral doses of [¹⁴C]omapatrilat is summarized in Table 1. Radioactivity excreted in the urine and feces for all three species was between 51 to 62% and 16 to 36%, respectively. Plasma concentration versus time profiles for omapatrilat and radioactivity are shown in Fig. 2. Omapatrilat doses were rapidly absorbed, and the concentrations of radioactivity were substantially higher than unchanged omapatrilat in all species. Following the completion of the absorption phase, concentrations of omapatrilat declined rapidly in all three species. Although the plasma concentrations of omapatrilat were measurable up to the last time point of pharmacokinetic sampling in dogs (96 h) and humans (120 h), the levels were below the limit of quantitation (10 ng/ml) in rats, likely due to the lower sensitivity of the assay method for rat plasma. The initial decline of radioactivity concentrations appeared to be slower than that for unchanged omapatrilat. Throughout the time course of pharmacokinetic observations postdose, the plasma concentrations of radioactivity were substantially higher compared with unchanged omapatrilat.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Plasma concentration versus time profiles for radioactivity and unchanged omapatrilat in rat, dog, and human. Unchanged omapatrilat, not bound to plasma thiols via disulfide bond, was measured as the methyl acrylate adduct. For the determination of unchanged omapatrilat in plasma, samples were collected in pharmacokinetic studies conducted separately with either unlabeled (rat) or single-labeled [14C]omapatrilat (dog and human) administered at the same dose level as in the biotransformation studies. The radioactivity, expressed as nanogram-equivalent of omapatrilat, was measured by liquid scintillation counting and corresponds to omapatrilat (both free thiol and disulfide forms) and its metabolites.

Biotransformation Profiles in Plasma. Biotransformation profiles of the methanol extracts of plasma from rat, dog, and human at 1 h after single oral doses of [¹⁴C]omapatrilat are summarized in Table 2. The recovery of plasma radioactivity into methanol after treatment with DTT was greater than 97% for all three species. The metabolites in the plasma were identified based on the retention times of the standards that were cochromatographed with the samples.

Rat. In rat 1-h plasma, the methanol extractable radioactivity was 73%. The prominent radioactive peak in 1-h plasma was M1-a, in both DTT- and non-DTT-reduced samples. Based on the retention time of the standard, M1-a was identified as the amine side hydrolysis product of omapatrilat. The other prominent peaks identified were the diastereomeric sulfoxides of (S)-2-thiomethyl-3-phenylpropionic acid, M1 and M2, and (S)-2-thiomethyl-3-phenylpropionic acid, M1-c. Two additional peaks, which were not present in the non-DTT-reduced sample, were observed after DTT reduction. These peaks were identified as (S)-3-phenyl-2-mercaptopropionic acid, M1-b, and omapatrilat. Together, the metabolites and omapatrilat accounted for 95% of the radioactivity in 1-h reduced plasma samples.

Dog. In dog 1-h plasma the methanol extractable radioactivity was 60%. The prominent metabolites in the dog plasma before DTT reduction were the amide hydrolysis products of omapatrilat M1-a, M1, M2 and M1-c. Two additional peaks, which were not present in the non-DTT-reduced sample, were observed after DTT reduction. These peaks were identified as (S)-3-phenyl-2-mercaptopropionic acid, M1-b, and omapatrilat. Together, the metabolites and omapatrilat accounted for 93% of the radioactivity in 1-h reduced plasma samples.

Human. In human 1-h plasma, the methanol extractable radioactivity was 47%. The prominent metabolites in 1-h human plasma before DTT reduction were the acyl glucuronide of S-methyl omapatrilat, M8 and S-methyl omapatrilat, M9. DTT reduction of 1-h plasma samples showed the appearance of (S)-3-phenyl-2-thiopropionic acid, M1-b, and omapatrilat. Together, the metabolites and ompatrilat accounted for 79% of the radioactivity in 1-h reduced plasma samples.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Biotransformation profiles of 1-h pooled human plasma before (A) and after (B) glucuronidase treatment. The profiles are a background subtracted reconstructed radiochromatogram of 30-s fractions collected from a HPLC run. Metabolite M8 was identified as the acyl glucuronide of S-methyl omapatrilat (M9).

Species Comparison of Biotransformation Profiles in Urine. Biotransformation profiles of pooled urine from rat (0-48 h), dog (0-168 h), and human (0-168 h) studies after single oral doses of [¹⁴C]omapatrilat are shown in Fig. 4 and summarized in Table 3. LC/MS/MS analysis of the urinary metabolites, M1-a, M3, and M5-M9, is shown in Figs. 5 and 6. LC/MS/MS analysis of metabolites M1, M2, and M4 had already been previously reported (Iyer et al., 2001) and therefore is not reported here. The structures of the metabolites identified are shown in Fig. 7.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Biotransformation profiles of pooled urine samples after oral administration of [14C]omapatrilat to rats (A), dogs (B), and humans (C). The profiles are a background subtracted reconstructed radiochromatogram of 30-s fractions collected from a HPLC run.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   LC/MS/MS analysis of metabolites M1-a, M3, M5, and M6 in pooled urine samples. Metabolite M1-a was analyzed in the positive ion mode, and metabolites M3, M5, and M6 were analyzed in the negative ion mode. Also, shown are the fragmentation of the corresponding 13C2 (M + 2) molecular ion confirming the identity of the metabolites. Fragmentation of metabolites M1, M2, and M4, which do not retain the 13C2 label, have been previously reported (Iyer et al., 2001).

View larger version (21K): [in this window] [in a new window]   Fig. 6.   LC/MS/MS analysis of metabolites M7, M8, and M9 in pooled urine samples. The metabolites were analyzed in the negative ion mode. Also, shown are the fragmentation of the corresponding 13C2 (M + 2) molecular ion confirming the identity of the metabolites.

View larger version (25K): [in this window] [in a new window]   Fig. 7.   Proposed biotransformation pathway for human metabolites of omapatrilat detected in plasma and urine samples.

Rat. Rats excreted 63% of the radioactive dose in urine. Profile A in Fig. 4 shows the distribution of radioactivity in rat urine and is summarized in Table 3. No parent compound was detected in the urine. Only four metabolites M1-a, M1, M2, and M1-c were detected. The prominent metabolites were M1-a and M2. Based on the retention times of the standards, M1-a was identified as the amine side hydrolysis product of omapatrilat, M1 and M2 were identified as the diastereomeric sulfoxides of (S)-2-thiomethyl-3-phenylpropionic acid, and M1-c was identified as (S)-2-thiomethyl-3-phenylpropionic acid. The identities of M1-a, M1, and M2 were further confirmed by LC/MS and LC/MS/MS analysis of urine. Metabolite M1-a showed a characteristic isotopic cluster of ¹²C-¹³C (1:1), which was two mass unit apart due to the presence of two C-13-labeled carbons in the molecule. The ratio of ¹²C-¹³C was 1:1 since [¹³C₂]omapatrilat constituted 50% of the dose mixture in rat. Metabolites M1 and M2 after hydrolysis do not retain the C-13 labels and as expected did not show the characteristic isotopic cluster. The fragmentation patterns seen in the LC/MS/MS analysis of M1-a (Fig. 5), M1, and M2 were the same as those of the synthetic standards. The four metabolites identified in the rat urine together accounted for 93% of the urinary radioactivity.

Dog. Dogs excreted 55% of the radioactive dose in urine. Profile B in Fig. 4 shows the distribution of radioactivity in dog urine. No parent compound was detected in the urine. The metabolites detected were M1-a, M1-M3, M5-M8, M9, and M10 (Table 3). The prominent metabolites were M1-a, M2, M3, M8, M9, and M10 that together accounted for 89% of the radioactivity. -Glucuronidase treatment of the urine led to the disappearance of metabolite peak M8 and a corresponding increase in radioactivity of peak M9 (data not shown). Incubation in the presence of 1,4-saccharolactone, a -glucuronidase inhibitor, prevented the hydrolysis of M8 showing that the metabolite is a -glucuronic acid conjugate. The identity of the metabolites was based on the retention time of the synthetic standards and was further confirmed by LC/MS and LC/MS/MS analysis of 0- to 24-h urine (Figs. 5 and 6). Metabolites M1-a, M3, M5, M6, M7, M8, and M9 showed the characteristic isotopic cluster of ¹²C-¹³C (4:1), which was two mass units apart due to the presence of two C-13-labeled carbons. The ratio of ¹²C-¹³C was 4:1 since [¹³C₂]omapatrilat constituted only 20% of the dose mixture in dog. The fragmentation patterns seen in the LC/MS/MS analysis of metabolites M1-a, M1-M3, M5 to M7, and M9 were the same as those of the respective synthetic standards. Metabolite M8, which was identified above as the glucuronic acid conjugate of M9, showed the loss of m/z 176 (-glucuronide) (Fig. 6). The 10 metabolites identified in dog urine together accounted for 93% of the urinary radioactivity.

Human. Humans excreted 50% of the radioactive dose in the urine. Profile C in Fig. 4 shows the distribution of radioactivity in human urine. No parent compound was detected in the urine, indicating that in vivo the compound is extensively metabolized. The metabolites detected were M1-a and M1-M9 (Table 3). The most prominent metabolite was M2, which accounted for 25% of the radioactivity.

	Discussion

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In this study, comparative metabolic profiles were generated to evaluate the systemic exposure of omapatrilat and its metabolites in rats, dogs, and humans after oral administration of omapatrilat. Rats and dogs were the species used in long term toxicity studies during the development of the compound. Also, this study was done with a mixture of radiolabeled [¹⁴C]omapatrilat (Fig. 1), labeled at the exocyclic carbonyl carbon and at the ethyl carbons attached to the bicyclic ring (Fig. 1), to evaluate the metabolic fate of the amine part of the molecule after hydrolysis in all three species. In addition to the ¹⁴C-label, the dose also contained ¹³C₂-labeled omapatrilat which helped in the rapid identification of the metabolites and obviated extensive purification of the sample.

In dog and human, the urinary and fecal recovery of radioactivity did not account for 100% of the administered radioactivity, when given either as multiple-labeled [¹⁴C]omapatrilat in this study or as single-labeled [¹⁴C]omapatrilat in other studies (Iyer et al., 2001; Malhotra et al., 2001). Most of the recovered radioactivity was collected within the first 72 h postdose, and the radioactivity recovery in the urine and feces collected on subsequent days were extremely low (less than 1% of the dose per day). On the day of discharge following the first dose, the recovery of radioactivity in urine and feces was less than 0.5% of the dose. These results indicate extremely slow increments in the cumulative recovery of radioactive doses of omapatrilat beyond 72 h postdose. Although the reason for the incomplete recovery of radioactivity is not clear, possible explanations, based on the biochemical and pharmacokinetic observations, are provided below.

Comparative Profiles of Radioactivity Versus Omapatrilat in Plasma. The thiol functional group of omapatrilat and its thiol-containing metabolites readily form disulfide conjugates with endogenous thiol groups, such as those on cysteine in the proteins present in biological media (Iyer et al., 2001; Malhotra et al., 2001). The radioactivity concentrations, representing omapatrilat (both free thiol and disulfide forms) and its metabolites, are much higher than unchanged omapatrilat (free thiol) (Fig. 2). The prolonged terminal-phase decline in plasma concentrations of radioactivity, and omapatrilat appeared to be parallel, evidenced in the profiles for dogs and humans. In rats, however, the assay for unchanged omapatrilat was not sensitive compared with the assay for radioactivity; therefore, plasma concentrations of unchanged omapatrilat could not be quantitated beyond 12 h postdose, and the terminal phase could only be completely characterized for radioactivity but not for parent drug.

Comparative Biotransformation Profile in Plasma. The metabolic pathways in vivo for omapatrilat are similar in rats, dogs, and humans. The major metabolites in human plasma were also present in dog plasma and, except for the acyl glucuronide of S-methyl omapatrilat (M8), in rat plasma. The metabolites identified in the extracted sample, except for the amine side hydrolysis product (M1-a) and the cysteine-disulfide adduct (M3), were all derived from S-methyl omapatrilat. The circulating metabolites identified are all expected to be inactive toward ACE and NEP because of the absence of the free sulfhydryl group, which is necessary for coordinating with the zinc atom in the active site of both the enzymes (Delaney et al., 1994).

Comparative Biotransformation Profile in Urine. The admixture of C-14-labeled compound with the cold material is routinely done in absorption, distribution, metabolism, and excretion studies to look at the metabolism of a drug in vivo. This is really useful since drug and its metabolites can be easily quantified with radioactive detection in complex biological matrices without interference from endogenous compounds. However, identifying metabolites by LC/MS from this complex mixture is still a challenge and requires extensive purification. This is especially true for compounds that are dosed at low levels where identification of metabolites formed in small amounts may not be trivial. In our previous study, to unequivocally identify the structure of human metabolites, extensive isolation and purification from human urine was performed to isolate microgram quantities of metabolites for NMR and LC/MS/MS analysis. In that study, nine metabolites, M1 to M9, were identified and extensively characterized from human urine (Iyer et al., 2001). For that reason, and the fact that reference compounds were available for the metabolites, isolation of the metabolites for NMR analysis was not deemed necessary for this study. Furthermore, the inclusion of [¹³C₂]omapatrilat in the dose facilitated the identification of metabolites. The admixture of a stable-labeled drug along with the radiolabeled drug is not routinely done in absorption, distribution, metabolism, and excretion studies, but it offers distinct advantages in the identification of metabolites. This is especially true for drugs that lack chlorine or bromine atoms, which show characteristic isotope pattern on the mass spectrometer. Since omapatrilat lacked chlorine or bromine atoms, stable-labeled ¹³C₂ omapatrilat was included in the dose to help in the identification of the metabolites by LC/MS and LC/MS/MS analysis. The metabolites that retained the C-13 labels, namely metabolites of intact omapatrilat (M3 and M5-M9) and the amine side hydrolysis product M1-a, showed characteristic ¹²C-¹³C isotope cluster in the full mass spectrum that were two mass units apart. LC/MS/MS analysis of the molecular ions (m/z and ¹³C₂m/z) further confirmed the identity of the metabolites (Figs. 5 and 6). The observation that the daughter ions retained or lost the ¹³C₂-label helped establish the fragmentation pattern for the metabolites. For example, the daughter ions generated from the fragmentation of the regioisomeric sulfoxides of S-methyl omapatrilat (M5-M7), m/z 437 and (¹³C₂)m/z 439, helped to identify the sites of sulfoxidation (Figs. 5 and 6).