Urinary metabolites of clomethiazole. Detection and structural analysis by gas chromatography-mass spectrometry

As the result of a renewed extensive investigation of clomethiazole (Distraneurin) metabolism five previously unknown metabolites could be isolated from human urine. Their structures were elucidated by mass spectrometry. Whereas previous investigations on the metabolism of clomethiazole had demonstrated changes only of the ethyl group, we now found metabolites attached to the methyl group, too. The newly isolated compounds 5-(1-hydroxy-2-chloroethyl)-4-methylthiazole (9) and 5-(2-hydroxyethyl)-4-thiazole carboxylic acid lactone (5) were found to be more abundant in human urine than 4-methyl-5-thiazole acetic acid previously considered as the main metabolite.     

Identification of major urinary metabolites of nafarelin acetate, a potent agonist of luteinizing hormone-releasing hormone, in the rhesus monkey

Nafarelin acetate (less than Glu-His-Trp-Ser-Tyr-3-(2-naphthyl)-D-Ala-Leu-Arg-Pro-Gly-NH2) is a potent agonistic analogue of luteinizing hormone-releasing hormone. After a single iv administration of nafarelin acetate (with 14C label at C-3 of 3-(2-naphthyl)-D-Ala) to female rhesus monkeys, about 80% of the radioactivity was eliminated in urine. Five major radioactive urinary metabolites were isolated and purified by reversed phase HPLC. Four of these metabolites, identified by amino acid analysis, were short peptides: the 5-10-hexapeptide amide, the 6-10-pentapeptide amide, the 5-7-tripeptide, and the 6-7-dipeptide. The fifth metabolite, which accounted for about 15% of the radioactivity administered, was shown by NMR and mass spectrometry to be 2-naphthylacetic acid. A possible pathway of its formation is by oxidative deamination of 3-(2-napthyl)-D-Ala to give the corresponding alpha-keto acid, followed by oxidative decarboxylation of the alpha-keto acid. These five metabolites together accounted for about 70% of the radioactivity recovered in the urine of rhesus monkeys, or more than half of the radioactivity in the administered dose. A minor metabolite, which was not isolated, coeluted with 3-(2-naphthyl)-D-Ala in two solvent systems on HPLC. Nafarelin acetate was also present in small amounts. Several of these metabolites were also present in plasma of the rhesus monkey.     

Identification of ginkgolic acid (15:1) metabolites in rats following oral administration by high-performance liquid chromatography coupled to tandem mass spectrometry

1.?In this article, metabolites of ginkgolic acid (GA) (15:1) in rats plasma, bile, urine and faeces after oral administration have been investigated for the first time by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) with the aid of on-line hydrogen/deuterium (H/D) exchange technique and β-glucuronidase hydrolysis experiments.

2.?After oral administration of GA (15:1, M0) to rats at a dose of 10?mg/kg, it was found that metabolites M1-M5 together with parent compound (M0) existed in rat plasma; parent compound (M0) and metabolites M2–M5 were observed in rat bile, and parent compound (M0) with metabolites M1 and M2 were discovered in rat faeces, and there was no parent compound and metabolite detectable in rat urine.

3.?Two oxidative metabolites of GA (15:1, M0) were identified as 2-hydroxy-6-(pentadec-8-enyl-10-hydroxy) benzoic acid (M1) and 2-hydroxy-6-(pentadec-8-enyl-11-hydroxy-13-carbonyl) benzoic acid (M2), respectively. Metabolites M3, M4 and M5 were identified as the mono-glucuronic acid conjugates of parent compound (M0), M1 and M2, respectively.

4.?The results indicated that M1 and M2 with parent compound (M0) were mainly eliminated in faeces and three glucuronide metabolites (M3, M4 and M5) excreted in bile as the predominant forms after oral administration of GA (15:1) to rats.     

Metabolism of nabumetone (BRL 14777) by various species including man

Radiotracer methodology was used to study the metabolic fate of 4-(6-methoxy-2-naphthyl)-butan-2-one (nabumetone) after oral administration to rats, mice, rabbits, dogs, rhesus monkeys and healthy human subjects. Parent compound was not detected in plasma and urine and the major circulating metabolite in all species was identified as 6-methoxy-2-naphthylacetic acid, a compound known to possess anti-inflammatory activity. Metabolites were mainly excreted in urine from which four principal metabolites were isolated and identified by mass spectrometry and independent synthesis. Pathways involving O-demethylation, reduction of the ketone group and oxidation of the butanone side-chain to acetic acid occurred in all species, but the ratios of the metabolic end-products tended to be species dependent. In the rat about half of the administered nabumetone was oxidized to the pharmacologically active acid metabolite.     

Identification of urinary metabolites of 2-isopropylnaphthalene in rats

Metabolism of 2-isopropylnaphthalene in rats was found to proceed through the oxidation of the isopropyl chain of the molecule. Four unconjugated metabolites were isolated from the urine and identified: 2-(2-naphthyl)propionic acid, 2-(2-naphthyl)-2-propanol, 2-(2-naphthyl)-1,2-propanediol, and 2-(2-naphthyl)-2-hydroxypropionic acid, together with a small amount of the unchanged compound. Identification was made by means of mass, infrared, and nuclear magnetic resonance spectroscopy. The presence of glucuronides of four metabolites described above was also suggested by thin-layer and gas-liquid chromatography of the extract that was obtained after enzymatic hydrolysis.     

Absorption, metabolism and excretion of [(14)C]mirabegron (YM178), a potent and selective β(3)-adrenoceptor agonist, after oral administration to healthy male volunteers

The mass balance and metabolite profiles of 2-(2-amino-1,3-thiazol-4-yl)-N-[4-(2-{[(2R)-2-hydroxy-2-phenylethyl]amino}ethyl)[U-(14)C]phenyl]acetamide ([(14)C]mirabegron, YM178), a β(3)-adrenoceptor agonist for the treatment of overactive bladder, were characterized in four young, healthy, fasted male subjects after a single oral dose of [(14)C]mirabegron (160 mg, 1.85 MBq) in a solution. [(14)C]Mirabegron was rapidly absorbed with a plasma t(max) for mirabegron and total radioactivity of 1.0 and 2.3 h postdose, respectively. Unchanged mirabegron was the most abundant component of radioactivity, accounting for approximately 22% of circulating radioactivity in plasma. Mean recovery in urine and feces amounted to 55 and 34%, respectively. No radioactivity was detected in expired air. The main component of radioactivity in urine was unchanged mirabegron, which accounted for 45% of the excreted radioactivity. A total of 10 metabolites were found in urine. On the basis of the metabolites found in urine, major primary metabolic reactions of mirabegron were estimated to be amide hydrolysis (M5, M16, and M17), accounting for 48% of the identified metabolites in urine, followed by glucuronidation (M11, M12, M13, and M14) and N-dealkylation or oxidation of the secondary amine (M8, M9, and M15), accounting for 34 and 18% of the identified metabolites, respectively. In feces, the radioactivity was recovered almost entirely as the unchanged form. Eight of the metabolites characterized in urine were also observed in plasma. These findings indicate that mirabegron, administered as a solution, is rapidly absorbed after oral administration, circulates in plasma as the unchanged form and metabolites, and is recovered in urine and feces mainly as the unchanged form.     

Metabolic fate of a new histamine H2-receptor antagonist, Z-300, in rat.

1. The metabolisms of (N-[3-[3-(piperidinomethyl)-phenoxy]-propyl]-2-(2-hydroxyethyl-1-t hio) acetamide.2-(4-hydroxy benzoyl) benzoate) (Z-300) in rat have been studied. 2. Five metabolites were identified, namely as sulphoxide (SO), sulphone (SO2), phenol (PH), propionic acid (PA) and thioglycolic acid (TH) derivatives. Furthermore, two metabolites, M1 and M2, were proposed as PH conjugated to sulphate and glucuronic acid respectively. 3. There were several metabolites in the plasma samples after oral administration of 14C-Z-300. The main metabolites were PH (mainly existing as the conjugates), PA and SO, and Z-300 was present as <10% of plasma radioactivity. SO2 and TH were barely detected in plasma. In the urine, the composition of metabolites was similar to that in the plasma. In the bile, the main metabolites were PH (mainly existing as the conjugates) and SO, and very little Z-300 was detected. In the faeces, the main compounds were Z-300 and TH, and very little SO. 4. During in vitro metabolism experiments, Z-300 was metabolized mainly in the liver, and a little in the kidney and lung. PH or its conjugates were produced little on the in vitro metabolism; this was different from that found in the in vivo experiment. The metabolism of Z-300 to SO appeared to be catalysed by cytochrome P450 rather than the flavin-containing monooxygenase. Both hepatic clearance calculated from the in vitro metabolism experiment (28.9 ml/min/kg) and from the liver perfusion metabolism experiment (33.2 ml/min/kg) were lower than the total clearance from the in vivo experiment (119 ml/min/kg). 5. After administration of 14C-SO into the caecum of rat, SO was converted to Z-300 and excreted in the faeces. 14C-SO was reduced by incubation for 4 h within the caecal content of rat under an anaerobic condition in vitro. The results suggest that SO was reduced to Z-300 by gut flora in the lower intestine.     

Analysis of doxorubicin, 4'-epidoxorubicin, and their metabolites by liquid chromatography

An analytical method based on isocratic HPLC separation and fluorescence detection was developed to allow for sensitive and specific analysis of anthracyclines and their metabolites in plasma and urine. The method is particularly advantageous when comparing the metabolism and/or pharmacokinetics of analogues, such as doxorubicin [(8S, 10S)-10-[(3-amino-2,3,6-trideoxy-alpha-L-lyxo-hexopyranosyl)- oxy]-8-glycolyl-7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-meth- oxy-5,12-naphthacenedione] (1) and 4'-epidoxorubicin (2), since both drugs and their metabolites can be analyzed under identical conditions. The analytical properties of 1, 2, and eight metabolites were studied in plasma, serum, buffer solution, and urine. The detection limit in plasma was 4 X 10(-8) M for the glucuronides, 7 X 10(-9) M for the glycosides, and 1 X 10(-9) M for the aglycones. In plasma, 1, 2, doxorubicinol (3), 4'-epidoxorubicinol (4), doxorubicinone (5), and doxorubicinol aglycone (6) showed a linear concentration-response relationship from their detection limit up to 5 X 10(-6) M. A linear calibration graph for plasma samples was also obtained for 7-deoxydoxorubicinone (7) and 7-deoxydoxorubicinol (8); however, these compounds had a significantly lower upper limit (5 X 10(-7) M). Urine samples were acidified to pH 2.5 and analyzed by HPLC without further purification. A linear calibration curve was obtained in the clinically relevant range. The detection limit in urine was approximately 5 X 10(-8) M. Plasma and urine of two patients who had received 4'-epidoxorubicin by iv bolus injection were analyzed.     

Biotransformation of styrene in mice. Stereochemical aspects

Biotransformation of styrene and its toxic metabolite, phenyloxirane (1), in mice in vivo was studied. Mice were treated with single intraperitoneal doses of styrene (400 mg/kg of body weight), and with (R)-, (S)-, or racemic styrene oxide (150 mg/kg of body weight). Profiles of neutral and acidic metabolites were determined by GC/MS. Mandelic acid (3) and two mercapturic acids, N-acetyl-S-(2-hydroxy-2-phenylethyl)cysteine (5) and N-acetyl-S-(2-hydroxy-1-phenylethyl)cysteine (6), were found to be major urinary metabolites of both styrene and phenyloxirane. 1-Phenylethane-1,2-diol (2) was the main neutral metabolite. The rate of excretion of this metabolite, as determined by GC, was 5-10 times lower than that of mandelic acid. Several minor acidic metabolites were also identified. Among them, novel phenolic metabolites, namely, 2-(4-hydroxyphenyl)ethanol (7), (4-hydroxyphenyl)acetic acid (11), and two isomeric hydroxymandelic acids (12), are of toxicological significance. Main stereogenic metabolites were isolated as methyl esters from extracts of pooled acidified urine treated with diazomethane. The mandelic acid that was obtained was converted to diastereomeric Mosher's derivatives prior to analysis by NMR. Mercapturic acids were analyzed directly by (13)C NMR. Pure enantiomers of 1 were metabolized predominantly but not exclusively to corresponding enantiomers of 3. Styrene yielded predominantly (S)-mandelic acid. Fractions of mercapturic acids 5 and 6 isolated from urine amounted to 12-15% of the dose for all compounds that were administered. Conversion to mercapturic acids was highly regio- and stereoselective, yielding predominantly regioisomer 5. Styrene, as compared to racemic phenyloxirane, yielded slightly more diastereomers arising from (S)-1 than from (R)-1. These data can be explained by formation of a moderate excess of the less mutagenic (S)-1 in the metabolic activation of styrene in mice in vivo.     

Absorption, metabolism, and excretion of [(14)C]MK-0524, a prostaglandin D(2) receptor antagonist, in humans.

[(3R)-4-(4-Chlorobenzyl)-7-fluoro-5-(methylsulfonyl)-1,2,3,4-tetrahydrocyclopentaindol-3-yl]acetic acid (MK-0524) is a potent orally active human prostaglandin D(2) receptor 1 antagonist that is currently under development for the prevention of niacin-induced flushing. The metabolism and excretion of [(14)C]MK-0524 in humans were investigated in six healthy human volunteers following a single p.o. dose of 40 mg (202 microCi). [(14)C]MK-0524 was absorbed rapidly, with plasma C(max) achieved 1 to 1.5 h postdose. The major route of excretion of radioactivity was via the feces, with 68% of the administered dose recovered in feces. Urinary excretion averaged 22% of the administered dose, for a total excretion recovery of approximately 90%. The majority of the dose was excreted within 96 h following dosing. Parent compound was the primary radioactive component circulating in plasma, comprising 42 to 72% of the total radioactivity in plasma for up to 12 h. The only other radioactive component detected in plasma was M2, the acyl glucuronic acid conjugate of the parent compound. The major radioactive component in urine was M2, representing 64% of the total radioactivity. Minor metabolites included hydroxylated epimers (M1/M4) and their glucuronic acid conjugates, which occurred in the urine as urea adducts, formed presumably during storage of samples. Fecal radioactivity profiles mainly comprised the parent compound, originating from unabsorbed parent and/or hydrolyzed glucuronic acid conjugate of the parent compound. Therefore, in humans, MK-0524 was eliminated primarily via metabolism to the acyl glucuronic acid conjugate, followed by excretion of the conjugate into bile and eventually into feces.     

Disposition and metabolism of almotriptan in rats, dogs and monkeys

Almotriptan is a new highly potent selective 5-HT1B/1D receptor agonist developed for the treatment of migraine, and the disposition of almotriptan in different animal species is now addressed in the current study. Almotriptan was well absorbed in rats (69.1%) and dogs (100%) following oral treatment. The absolute bioavailability was variable reflecting different degrees of absorption and first-pass metabolism (18.7-79.6%). The elimination half-life was short and ranged between 0.7 and 3 h. The main route of elimination of almotriptan was urine with 75.6% and 80.4% of the dose recovered over a 168-h period in rats and dogs, respectively. The gamma-aminobutyric acid metabolite formed by oxidation of the pyrrolidine ring was the main metabolite found in urine, faeces, bile, and plasma of rats and in monkey urine. By contrast, the unchanged drug, the indole acetic acid metabolite formed by oxidative deamination of the dimethylaminoethyl group, and the N-oxide metabolite were the main metabolites in dog.     

Identification of new sulfur-containing metabolites of naphthalene in mouse urine

Seven acidic sulfur-containing metabolites were isolated from mouse urine following administration of naphthalene. The metabolites have been identified as (1-hydroxy-1,2-dihydro-2-naphthalenylthio)acetic acid (I), 2-hydroxy-3-(1-hydroxy-1,2-dihydro-2-naphthalenylthio)propanoic acid (II), (1,2,3-trihydroxy-1,2,3,4-tetrahydro-4-naphthalenylthio)acetic acid (III), and N-acetyl-S-(1-hydroxy-1,2-dihydro-2-naphthalenyl)-L-cysteine (IV). The dehydration products of I, II, and IV, namely 1-(naphthalenylthio)acetic acid (V), 2-hydroxy-3-(1-naphthalenylthio)propanoic acid (VI), and N-acetyl-S-(1-naphthalenyl)-L-cysteine (VII), respectively, were also present in several urinary extracts. Nine methylthio derivatives were identified in the neutral extract of urine. These metabolites were the following: 1-methylthionaphthalene, trans-1-hydroxy-2-methylthio-1,2-dihydronaphthalene, two stereoisomeric 1,2,3-trihydroxy-4-methylthio-1,2,3,4-tetrahydronaphthalenes, 1,3-di(methylthio)-2,4-dihydroxy-1,2,3,4-tetrahydronaphthalene, 1,4-di(methylthio)-2,3-dihydroxy-1,2,3,4-tetrahydronaphthalene, two methylthiohydroxy-naphthalenes, and a methylthiodihydroxydihydronaphthalene. Following intraperitoneal administration of N-acetyl-S-(1-hydroxy-1,2-dihydro-2-naphthalenyl)-L-cysteine to mice, the acidic metabolites I, II, and unchanged IV were found. The gas-chromatographic and gas chromatographic-mass spectral properties of the methyl ester-trimethylsilyl derivatives of the acidic sulfur metabolites of naphthalene are presented.     

In vivo and in vitro metabolism of 2-methylnaphthalene in the guinea pig

The metabolism of 2-methylnaphthalene (2-MN) in guinea pigs (in vivo and in vitro) was investigated. Excretion of 2-MN from guinea pigs took place rapidly. In the first 24 hr, nearly 80% of the orally administered 2-[3H]-MN was excreted in the urine in the form of several metabolites, and about 10% of it was recovered in the feces. The major metabolites in the urine were oxidative products of the methyl group of 2-MN (naphthoic acid and its glycine and glucuronic acid conjugates) and accounted for 76% of the total urinary radioactivity in the first 24 hr. S-(7-Methyl-1-naphthyl)cysteine and glucuronic acid and sulfate conjugates of 7-methyl-1-naphthol were also identified as minor metabolites (18% of the total urinary radioactivity). As an in vitro metabolite, the formation of S-(7-methyl-1-naphthyl)glutathione was indicated using the 9,000g supernatant of the homogenate of guinea pig liver. The oral administration of 2-MN (500 mg/kg) to guinea pigs significantly lowered the trichloroacetic acid-soluble sulfhydryl content in the liver.     

High-performance liquid chromatographic determination of aspirin and its metabolites in plasma and urine

A simple quantitative method for the rapid determination of aspirin and its metabolites, salicylic acid, salicyluric acid, and gentisic acid, in plasma and urine using o-toluic and o-anisic acids, respectively, as internal standards was developed. Plasma proteins were precipitated by the addition of acetonitrile and, after centrifugation, the supernatant fluid was injected directly onto a reverse-phase column. The mobile phase consisted of an isocratic mixture of water, methanol, and glacial acetic acid (64:25:1, v/v/v) and the separated components were detected at 238 nm using a UV detector. Concentrations greater than or equal to 0.5 microgram/ml could be quantitated for aspirin or its metabolites in plasma. The peak heights and peak height ratios to the internal standard, o-toluic acid, were linear for the concentration range of 0.5-200 micrograms/ml. The aspirin metabolites in urine were isolated by extracting the acidified urine with either and then reextracting the material into an aqueous buffer solution at pH 7.0. Twenty microliters of the buffer extract was directly injected onto the column. The separated components were detected and quantitated at 305 nm. Concentrations greater than or equal to 5 micrograms/ml of salicyluric acid, salicylic acid, and gentisic acid could be determined accurately. The peak heights and peak height ratios to the internal standard, o-anisic acid, were found to be linear for the concentration range of 5-200 micrograms/ml in urine.     

Metabolism of [2-14C]p-hydroxyphenyl acetic acid in rat, monkey and human hepatocytes and in bile-duct cannulated rats

We determined the metabolism of [2-(14)C]p-hydroxyphenyl acetic acid (p-HPA) in rat (male, Sprague-Dawley), monkey (male, Cynomolgus), and human (male, Caucasian) hepatocytes, and in bile-duct cannulated (BDC) rats (male, Sprague-Dawley). Unchanged p-HPA ranged from 87.0 to 92.6% of the total radioactivity (TRA) in the extracts of rat, monkey, and human hepatocytes. Metabolites M1 (a glucuronide conjugate of p-HPA) and M2 (a glycine conjugate of p-HPA) were detected, accounting for 1-4% of TRA. After an oral dose of [2-(14)C]p-HPA to BDC rats, p-HPA-related components was predominantly excreted in urine, accounting for 83% of the dose. Bile excretion was limited, accounting for only 1.5% of the dose. Unchanged p-HPA was the predominant radioactivity in plasma (84.6% of the TRA in 1-h pooled plasma) and urine (69.6% of the dose). Metabolites M1, M2, and M3 (a glucuronide of p-HPA) were all detected in plasma, urine, and bile as minor components. In summary, p-HPA was not metabolized extensively in rat, monkey, and human hepatocytes. In rats, absorption and elimination of p-HPA were nearly complete with urinary excretion of the unchanged p-HPA as the predominant route of elimination after oral dosing. No oxidative metabolites were detected, suggesting a minimal role for P450 enzymes in its overall metabolic clearance. Therefore, p-HPA has a low potential for drug-drug interactions mediated by the concomitant inhibitors and inducers of P450 enzymes.     

Metabolism and disposition of the oral absorption enhancer 14C-radiolabeled 8-(N-2-hydroxy-5-chlorobenzoyl)-amino-caprylic acid (5-CNAC) in healthy postmenopausal women and supplementary investigations in vitro

8-(N-2-hydroxy-5-chlorobenzoyl)-amino-caprylic acid (5-CNAC), a compound lacking pharmacological activity enhances the absorption of salmon calcitonin, when co-administered. Disposition and biotransformation of 5-CNAC was studied in six healthy postmenopausal women following a single oral dose of 200mg (14)C-radiolabeled 5-CNAC (as disodium monohydrate salt). Blood, plasma, urine and feces collected over 7 days were analyzed for radioactivity. Metabolite profiles were determined in plasma and excreta and metabolite structures were elucidated by LC-MS/MS, LC-(1)H NMR, enzymatic methods and by comparison with reference compounds. Oral 5-CNAC was safe and well tolerated in this study population. 5-CNAC absorption was rapid (t(max)=0.5h; C(max)=9.00 ± 2.74 μM (mean ± SD, n=6) and almost complete. The elimination half-life (t(?)) was 1.5 ± 1.1h. The radioactive dose was excreted mainly in urine (≥ 90%) in form of metabolites and 0.071% as intact 5-CNAC. Excretion of radioactivity in feces was minor and mostly as metabolites (<3%). Radioactivity in plasma reached C(max) (35.4 ± 7.9 μM) at 0.75 h and declined with a half-life of 13.9 ± 4.3h. 5-CNAC accounted for 5.8% of the plasma radioactivity AUC(0-24h). 5-CNAC was rapidly cleared from the systemic circulation, primarily by metabolism. Biotransformation of 5-CNAC involved: (a) stepwise degradation of the octanoic acid side chain and (b) conjugation of 5-CNAC and metabolites with glucuronic acid at the 2-phenolic hydroxyl group. The metabolism of 5-CNAC in vivo could be reproduced in vitro in human hepatocytes. No metabolism of 5-CNAC was observed in human liver microsomes.     

Metabolism and kinetics of amlodipine in man

1. The disposition of amlodipine, R,S,2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-3-ethoxycarbonyl- 5- methoxycarbonyl-6-methyl-1,4-dihydropyridine has been studied in two human volunteers using single oral and intravenous doses of 14C-amlodipine. The drug was well absorbed by the oral route while the mean oral bioavailability for unchanged drug was 62.5%. 2. Renal elimination was the major route of excretion with about 60% of the dosed radioactivity recovered in urine. Mean total recovered radioactivity in urine and faeces amounted to 84% for both the oral and intravenous routes. 3. Apart from a small amount of unchanged amlodipine (10% of urine 14C), only pyridine metabolites of amlodipine were excreted in urine. The majority (greater than 95%) of the metabolites excreted in the 0-72 h post-dose period were identified; the major metabolite was 2-([4-(2-chlorophenyl)-3-ethoxycarbonyl-5-methoxycarbonyl-6-methyl- 2-pyridyl]methoxy) acetic acid and this represented 33% of urinary radioactivity. The data indicate that oxidation of amlodipine to its pyridine analogue is the principal route of metabolism with subsequent metabolism by oxidative deamination, de-esterification and aliphatic hydroxylation. 4. For the two volunteers, amlodipine concentrations in plasma declined with a mean half-life of 33 h, while slower elimination of total drug-related material from plasma was observed, consistent with prolonged excretion (up to 12 days) of metabolites in urine and faeces. Only amlodipine and pyridine metabolites were found in the circulation. As these pyridine derivatives have minimal calcium antagonist activity the efficacy of amlodipine in man can most probably be attributed to the parent drug.     

Directly coupled HPLC-NMR and HPLC-MS approaches for the rapid characterisation of drug metabolites in urine: application to the human metabolism of naproxen

High resolution nuclear magnetic resonance (NMR) spectroscopy is a very powerful tool for the structural identification of xenobiotic metabolites in complex biological matrices such as plasma, urine and bile. However, these fluids are dominated by thousands of signals resulting from endogenous metabolites and it is advantageous when investigating drug metabolites in such matrices to simplify the spectra by including a separation step in the experiment by directly-coupling HPLC and NMR. Naproxen (6-methoxy-alpha-methyl-2-naphthyl acetic acid) is administered as the S-enantiomer and is metabolised in vivo to form its demethylated metabolite which is subsequently conjugated with beta-D-glucuronic acid as well as with sulfate. Naproxen is also metabolised by phase II metabolism directly to form a glycine conjugate as well as a glucuronic acid conjugate at the carboxyl group. In the present investigation, the metabolism of naproxen was investigated in urine samples with a very simple sample preparation using a combination of directly-coupled HPLC-1H NMR spectroscopy and HPLC-mass spectrometry (MS). A buffer system was developed which allows the same chromatographic method to be used for the HPLC-NMR as well as the HPLC-MS analysis. The combination of these methods is complementary in information content since the NMR spectra provide evidence to distinguish isomers such as the type of glucuronides formed, and the HPLC-MS data allow identification of molecules containing NMR-silent fragments such as occur in the sulfate ester.     

Pharmacokinetics and metabolism of the antiinflammatory agents S-H 766 in man]

Blood and plasma levels as well as urinary and fecal excretion were measured in humans after oral administration of radioactively labelled 4-[j-(2'-fluorobiphenylyl)]-4-hydroxycrotonic acid (S-H 766 MO). The radioactivity in the plasma reaches maximum values of about 10 mug eq./ml 1 to 2 h after application with either form. After repeated administration good agreement is found between the plasma levels measured and those simulated according to the pharmacokinetic parameters obtained after single application. The S-H 766 metabolites were investigated in blood and urine. The substance was found to undergo considerable metabolism, only approximately 2% being excreted in the urine unchanged. The conjugates, which constitute over 60% of the radioactivity of the urine, consist mainly of glucuronides and sulfates. The structure of the aglycones shows that the metabolism occurs along two pathways, by beta-oxidation of the aliphatic side chain into aryl acetic acids and by hydroxylation of the aromatic nucleus to phenolic compounds. It must be assumed that these biotransformations take place both simultaneously and successively.     

Metabolic fate of the new angiotensin-converting enzyme inhibitor imidapril in animals. 5th communication: isolation and identification of metabolites of imidapril in rats, dogs, and monkeys.

The metabolism of imidapril hydrochloride ((-)-(4S)-3-[(2S)-2-[[(1S)-1-ethoxycarbonyl-3-phenylpropyl]amino] propionyl]-1-methyl-2-oxoimidazolidine-4-carboxylic acid hydrochloride, imidapril, TA-6366, CAS 89396-94-1) was studied in rats and dogs after oral or intravenous administration of [N-methyl-14C]-imidapril or [alanine-3-14C]-imidapril, and in monkeys after oral administration of [alanine-3-14C]-imidapril. Radio-chromatographic analysis of the metabolites of imidapril from the plasma, urine, and bile of rats, dogs, or monkeys resulted in the detection of at least four metabolites. These four metabolites were isolated and characterized by high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry(GC-MS). Of these metabolites, M1 (6366 A, CAS 89371-44-8) was pharmacologically active; however, M2, M3, and M4 were inactive. There was no evidence of any glucuronides or sulfates of drug-related compounds, or of the piperazine-dione lactam type metabolites of imidapril or 6366 A in the urine of the animals used. Imidapril was metabolized by hydrolysis at the carboxylic ethyl ester side-chain to give M1, and by cleavage of the amide bond to form M2 and M3. M4 was formed by hydrolysis of M3 and/or cleavage of the amide bond of M1. Qualitatively, the same metabolites were found in all animal species tested; however, quantitatively, there were differences in the amounts of metabolites formed depending on the species.