Pharmacokinetics,metabolism and excretion of the glycine antagonist GV150526A in rat and dog

1. The pharmacokinetics, metabolic fate and excretion of 3-[-2(phenylcarbamoyl) ethenyl-4,6-dichloroindole-2-carboxylic acid (GV150526), a novel glycine antagonist for stroke, in rat and dog following intravenous administration of [C14]-GV150526A were investigated. 2. Studies were also performed in bile duct-cannulated animals to confirm the route of elimination and to obtain more information on metabolite identity. 3. Metabolites in plasma, urine and bile were identified by HPLC-MS/MS and NMR spectroscopy. 4. GV150526A was predominantly excreted in the faeces via the bile, with only trace metabolites of radioactivity in urine (< 5%). Radioactivity in rat bile was predominantly due to metabolites, whereas approximately 50% of the radioactivity in dog bile was due to parent GV150526. 5. The principal metabolites in bile were identified as glucuronide conjugates of the carboxylic acid, whereas in rat urine the main metabolite was a sulphate conjugate of an aromatic oxidation metabolite. Multiple glucuronide peaks were observed and identified as isomeric glucuronides and their anomers arising from acyl migration and muta-rotation.     

Disposition and metabolism of TAK-438 (vonoprazan fumarate), a novel potassium-competitive acid blocker,in rats and dogs

1.?Following oral administration of [¹⁴C]TAK-438, the radioactivity was rapidly absorbed in rats and dogs. The apparent absorption of the radioactivity was high in both species.

2.?After oral administration of [¹⁴C]TAK-438 to rats, the radioactivity in most tissues reached the maximum at 1-hour post-dose. By 168-hour post-dose, the concentrations of the radioactivity were at very low levels in nearly all the tissues. In addition, TAK-438F was the major component in the stomach, whereas TAK-438F was the minor component in the plasma and other tissues. High accumulation of TAK-438F in the stomach was observed after oral and intravenous administration.

3.?TAK-438F was a minor component in the plasma and excreta in both species. Its oxidative metabolite (M-I) and the glucuronide of a secondary metabolite formed by non-oxidative metabolism of M-I (M-II-G) were the major components in the rat and dog plasma, respectively. The glucuronide of M-I (M-I-G) and M-II-G were the major components in the rat bile and dog urine, respectively, and most components in feces were other unidentified metabolites.

4.?The administered radioactive dose was almost completely recovered. The major route of excretion of the drug-derived radioactivity was via the feces in rats and urine in dogs.     

Metabolism and excretion of atorvastatin in rats and dogs.

Atorvastatin (AT) is a second-generation potent inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase, clinically approved for lowering plasma cholesterol. Using a mixture of [D(5)/D(0)] AT and/or [(14)C]AT, the metabolic fate and excretion of AT were examined in rats and dogs following single and multiple oral doses. Limited biliary recycling was examined in one dog after a single dose of AT. AT-derived metabolites in bile samples were identified by metabolite screening of the [D(5)/D(0)] AT molecular clusters using tandem mass spectrometry. Bile was a major route of [(14)C] drug-derived excretion, accounting for 73 and 33% of the oral dose in the rat and dog, respectively. The remaining radioactivity was recovered in the feces; only trace amounts were excreted in urine. Radioactive components identified in rat and dog bile were the para- and ortho-hydroxy metabolites, a glucuronide conjugate of ortho-hydroxy AT, and unchanged AT. Two minor radioactive components were identified as beta-oxidation products of AT with one confirmed as a beta-oxidized AT derivative. The reappearance of AT and major metabolites in bile from a dog administered a sample of its previously excreted bile indicated biliary recycling is an important component in AT metabolism. Multiple dose administration in rats did not alter biliary metabolic profiles. Rat and dog plasma profiles after multiple dose administration were similar and showed no additional metabolites not found in bile. Examination of rat and dog bile and plasma indicates that AT primarily undergoes oxidative metabolism.     

Human hepatic metabolism of a novel 2-carboxyindole glycine antagonist for stroke: in vitro-in vivo correlations

1. The hepatic metabolism of 3-[-2(phenylcarbamoyl) ethenyl]-4,6-dichloroindole-2-carboxylic acid (GV150526), a novel glycine antagonist for stroke, was investigated. 2. After a single intravenous administration of 800 mg GV150526 to healthy volunteers, six metabolites were observed. The major metabolites detected in human plasma have been shown by mass spectrometry to be glucoronides and one sulphate conjugate. 3. After incubation of GV150526 for 6 and 24 h with human liver slices, three glucuronide metabolites were observed. After incubation of GV150526 with pooled human liver microsomes, only one metabolite was observed, with the same molecular weight and HPLC retention time as the synthetic standard GV217053 (GV150526 hydroxylated on the para-position of the phenyl ring). 4. GV150526 hydroxylase enzyme kinetics--a step before sulphation--was determined using pooled human microsomes and was shown to be catalysed by cytochrome P4502C9. Glucuronidation kinetics towards GV150526 using microsomal preparations were also determined. Glucuronidation of GV150526 was observed with UGT1A1 cDNA-expressed protein, but not with UGT1A6. 5. The above enzyme kinetic data were used to calculate intrinsic clearance after scaling-up and hepatic clearance were calculated. Since GV150526 has a high plasma protein binding capacity, the effect of GV150526 binding to microsomal protein was determined. Thus, enzyme kinetic data were corrected, plotting the free (unbound) concentration of GV150526 versus enzymatic velocities: apparent Vmax did not alter significantly but apparent Km was approximately 10-fold lower. Correlation of these corrected enzyme kinetic data to predict clearance with in vivo clearance of GV150526 was good when both fu(plasma) and fu(microsomes) were included in the clearance calculations.     

Isolation and identification of the polar metabolites of chlorpheniramine in the dog

The metabolites of chlorpheniramine were isolated from dog urine. After daily repeated dosing with chlorpheniramine, [methylene-14C]chlorpheniramine maleate was given as a tracer and urine was collected until less than 1% of the labeled dose was excreted daily. An average of 54% of the oral radioactive dose was recovered in the urine. In addition to the N-demethylated metabolites, one very polar metabolite accounting for about 18% and two less polar metabolites accounting for a total of about 30% of the total urine radioactivity were isolated. Hydrolysis studies of the most polar metabolite indicated that it was a conjugate, though not a glucuronide or sulfate. The metabolite identified after hydrolysis was 3-(p-chlorobenzyl)-3-(2-pyridyl)propionic acid. One of the two less polar metabolites was identified as the corresponding alcohol. The least abundant metabolite could not be identified.     

Preclinical characterization of 2-[3-[3-[(5-ethyl-4'-fluoro-2-hydroxy[1,1'-biphenyl]-4-yl)oxy]propoxy]-2-propylphenoxy]benzoic acid metabolism: in vitro species comparison and in vivo disposition in rats.

Assessment of the pharmacokinetics of [14C]2-[3-[3-[(5-ethyl-4'-fluoro-2-hydroxy[1,1'-biphenyl]-4-yl)oxy]propoxy]-2-propylphenoxy-]benzoic acid ([14C]LY293111), an experimental anti-cancer agent, suggested long-lived circulating metabolites in rats. In vivo metabolites of LY293111 were examined in plasma, bile, urine, and feces of Fischer 344 (F344) rats after oral administration of [14C]LY293111. Metabolites were profiled by high-performance liquid chromatography-radiochromatography, and identified by liquid chromatography (LC)/mass spectrometry and LC/NMR. The major in vivo metabolites of LY293111 identified in rats were phenolic (ether), acyl, and bisglucuronides of LY293111. Measurement of radioactivity in rat plasma confirmed that a fraction of LY293111-derived material was irreversibly bound to plasma protein and that this bound fraction increased over time. This was consistent with the observed disparity in half-lives between LY293111 and total radioactivity in rats and monkeys, and is likely due to covalent modification of proteins by the acyl glucuronide. In vitro metabolism of [14C]LY293111 in liver slices from CD-1 mice, F344 rats, rhesus and cynomolgus monkeys, and humans indicates that glucuronidation was the primary metabolic pathway in all species. The acyl glucuronide was the most prevalent radioactive peak (16% of total 14C) produced by F344 rat slices, whereas the ether glucuronide was the major metabolite in all other species (26-36% of total 14C). Several minor hydroxylated metabolites were detected in F344 rat slice extracts but were not observed in other species. The data presented suggest that covalent modification of proteins by LY293111 acyl glucuronide is possible in multiple species, although the relative reactivity of this metabolite appears to be low compared with those known to cause adverse drug reactions.     

Species differences in the elimination of a peroxisome proliferator-activated receptor agonist highlighted by oxidative metabolism of its acyl glucuronide.

A species difference was observed in the excretion pathway of 2-[[5,7-dipropyl-3-(trifluoromethyl)-1,2-benzisoxazol-6-yl]oxy]-2-methylpropanoic acid (MRL-C), an alpha-weighted dual peroxisome proliferator-activated receptor alpha/gamma agonist. After intravenous or oral administration of [14C]MRL-C to rats and dogs, radioactivity was excreted mainly into the bile as the acyl glucuronide metabolite of the parent compound. In contrast, when [14C]MRL-C was administered to monkeys, radioactivity was excreted into both the bile and the urine as the acyl glucuronide metabolite, together with several oxidative metabolites and their ether or acyl glucuronides. Incubations in hepatocytes from rats, dogs, monkeys, and humans showed the formation of the acyl glucuronide of the parent compound as the major metabolite in all species. The acyl glucuronide and several hydroxylated products, some which were glucuronidated at the carboxylic acid moiety, were observed in incubations of MRL-C with NADPH- and uridine 5'-diphosphoglucuronic acid-fortified liver microsomes. However, metabolism was more extensive in the monkey microsomes than in those from the other species. When the acyl glucuronide metabolite of MRL-C was incubated with NADPH-fortified liver microsomes, in the presence of saccharo-1,4-lactone, it underwent extensive oxidative metabolism in the monkey but considerably less in the rat, dog, and human liver microsomes. Collectively, these data suggested that the oxidative metabolism of the acyl glucuronide might have contributed to the observed in vivo species differences in the metabolism and excretion of MRL-C.     

The metabolic disposition of aprepitant, a substance P receptor antagonist, in rats and dogs.

The absorption, metabolism, and excretion of [14C]aprepitant, a potent and selective human substance P receptor antagonist for the treatment of chemotherapy-induced nausea and vomiting, was evaluated in rats and dogs. Aprepitant was metabolized extensively and no parent drug was detected in the urine of either species. The elimination of drug-related radioactivity, after i.v. or p.o. administration of [14C]aprepitant, was mainly via biliary excretion in rats and by way of both biliary and urinary excretion in dogs. Aprepitant was the major component in the plasma at the early time points (up to 8 h), and plasma metabolite profiles of aprepitant were qualitatively similar in rats and dogs. Several oxidative metabolites of aprepitant, derived from N-dealkylation, oxidation, and opening of the morpholine ring, were detected in the plasma. Glucuronidation represented an important pathway in the metabolism and excretion of aprepitant in rats and dogs. An acid-labile glucuronide of [14C]aprepitant accounted for approximately 18% of the oral dose in rat bile. The instability of this glucuronide, coupled with its presence in bile but absence in feces, suggested the potential for enterohepatic circulation of aprepitant via this conjugate. In dogs, the glucuronide of [14C]aprepitant, together with four glucuronides derived from phase I metabolites, were present as major metabolites in the bile, accounting collectively for approximately 14% of the radioactive dose over a 4- to 24-h period after i.v. dosing. Two very polar carboxylic acids, namely, 4-fluoro-alpha-hydroxybenzeneacetic acid and 4-fluoro-alpha-oxobenzeneacetic acid, were the predominant drug-related entities in rat and dog urine.     

Metabolism of 2-Benzylthio-5-Trifluoromethyl Benzoic Acid (BTBA) by the Rat,Dog and Man

Abstract

1. Benzylthio-5-trifluoromethylbenzoic acid (BTBA) is well absorbed by rat, dog and man. The time course of disappearance from plasma in all three species suggests enterohepatic circulation.

2. The principal excretion product in rat urine has been identified as 2-mercapto-5-trifluoromethylbenzoic acid arising via S-dealkylation.

3. 2-Mercapto-5-trifluoromethylbenzoic acid is also converted to the disulphide metabolite, [2,2′-dithiobis(5-trifluoromethyl)benzoic acid], which is secreted together with BTBA and their glucuronide conjugates in rat bile. Rapid hydrolysis in the intestine results in faecal elimination of largely free BTBA and disulphide metabolite (2,2′-dithiobis[5-trifluoromethyl]benzoic acid).

4. In man, approximately 50% of the dose is recovered from urine as amino-acid and glucuronide conjugates of the drug and its debenzyl metabolite (2-mercapto-5-trifluoromethylbenzoic acid).

5. The drug is not readily metabolized by the dog and is secreted with bile and eliminated unchanged with faeces.     

Comparative biotransformation of radiolabeled [(14)C]omapatrilat and stable-labeled [(13)C(2)]omapatrilat after oral administration to rats, dogs, and humans.

Omapatrilat, a novel vasopeptidase inhibitor, is under development for the treatment of hypertension and congestive heart failure. This study describes the comparative biotransformation of radiolabeled [(14)C]- and stable-labeled [(13)C(2)]omapatrilat after administration of single oral doses to rats, dogs, and humans. The metabolites were identified by a combination of methods including reduction, hydrolysis, and comparison of high performance liquid chromatography retention times with those of the synthetic standards. Urinary metabolites were further characterized by liquid chromatography tandem mass spectrometry analysis. Prominent metabolites identified in human plasma, which were also present in rat and dog plasma, were S-methyl omapatrilat and S-2-thiomethyl-3-phenylpropionic acid. Omapatrilat accounted for only a small portion of the extractable radioactivity in plasma in all three species. A portion of the plasma radioactivity was unextractable in all three species (27-53%). The majority of unextractable radioactivity in plasma was characterized after dithiothreitol reduction to be omapatrilat and (S)-2-thio-3-phenylpropionic acid, both apparently bound to plasma proteins by reversible disulfide bonds. The major human urinary metabolites were the amine hydrolysis product, diasteromeric sulfoxide of (S)-2-thiomethyl-3-phenylpropionic acid, acyl glucuronide of S-methyl omapatrilat, and S-methyl omapatrilat. The minor metabolites were acyl glucuronide of (S)-2-thiomethyl-3-phenylpropionic acid, L-cysteine mixed disulfide of omapatrilat, diastereomers of S-methyl sulfoxide of omapatrilat, and S-methyl omapatrilat ring sulfoxide. The metabolic profiles of dog and human urine were qualitatively similar whereas rat urine showed only metabolites arising from hydrolysis of omapatrilat. Unchanged omapatrilat was not found in rat, dog, or human urine samples indicating extensive metabolism in vivo.     

Human hepatic metabolism of a novel 2-carboxyindole glycine antagonist for stroke: in vitro-in vivo correlations

1. The hepatic metabolism of 3-[-2(phenylcarbamoyl) ethenyl]-4,6-dichloroindole-2-carboxylic acid (GV150526), a novel glycine antagonist for stroke, was investigated. 2. After a single intravenous administration of 800?mg GV150526 to healthy volunteers, six metabolites were observed. The major metabolites detected in human plasma have been shown by mass spectrometry to be glucoronides and one sulphate conjugate. 3. After incubation of GV150526 for 6 and 24 h with human liver slices, three glucuronide metabolites were observed. After incubation of GV150526 with pooled human liver microsomes, only one metabolite was observed, with the same molecular weight and HPLC retention time as the synthetic standard GV217053 (GV150526 hydroxylated on the para-position of the phenyl ring). 4. GV150526 hydroxylase enzyme kinetics-astep before sulphation-was determined using pooled human microsomes and was shown to be catalysed by cytochrome P4502C9. Glucuronidation kinetics towards GV150526 using microsomal preparations were also determined.Glucuronidation of GV150526 was observed with UGT1A1 cDNA-expressed protein, but not with UGT1A6. 5. The above enzyme kinetic data were used to calculate intrinsic clearance after scaling-up and hepatic clearance were calculated. Since GV150526 has a high plasma protein binding capacity, the effect of GV150526 binding to microsomal protein was determined. Thus, enzyme kinetic data were corrected, plotting the free (unbound) concentration of GV150526 versus enzymatic velocities: apparent V_max did not alter significantly but apparent K_m was ～ 10-fold lower. Correlation of these corrected enzyme kinetic data to predict clearance with in vivo clearance of GV150526 was good when both ?u_plasma and ?u_microsomes were included in the clearance calculations.     

Felbamate metabolism in the rat, rabbit, and dog.

Two major and one minor metabolite of felbamate (FBM) as well as unchanged drug were isolated and identified by electron impact and chemical ionization mass spectrometry from rat and dog urine after dosing with [14C]FBM. The metabolites were 2-(4-hydroxyphenyl)-1,3-propanediol dicarbamate (p-OHF), 2-hydroxy-2-phenyl-1,3-propanediol dicarbamate, and 2-phenyl-1,3-propanediol monocarbamate. The metabolites and FBM were excreted mainly in urine, where their sum accounted to 81-94% of the radioactivity in hydrolyzed rat urine samples, 71-82% in rabbit urine samples, and 69-83% in dog urine samples. The amount of metabolites in the conjugated form was estimated to be 20-35% in rat, 20-30% in rabbit, and 10-20% in dog urine. The major biliary metabolite in all three species was p-OHF, whereas the amount of FBM was small. Metabolites found in dog feces were the same as those in the urine.     

Metabolism and disposition of 14C-granisetron in rat,dog and man after intravenous and oral dosing

1. The disposition and metabolic fate of ¹⁴C-granisetron, a novel 5-HT₃ antagonist, was studied in rat, dog, and male human volunteers after intravenous and oral administration.

2. Complete absorption occurred from the gastrointestinal tract following oral dosing, but bioavailability was reduced by first-pass metabolism in all three species.

3. There were no sex-specific differences observed in radiometabolite patterns in rat or dog and there was no appreciable change in disposition with dose between 0·25 and 5 mg/kg in rat and 0·25 and 10mg/kg in dog. Additionally, there were no large differences in disposition associated with route of administration in rat, dog and man.

4. In rat and dog, 35–41% of the dose was excreted in urine and 52–62% in faeces, via the bile. Metabolites were largely present as glucuronide and sulphate conjugates, together with numerous minor polar metabolites. In man, about 60% of dosed radioactivity was excreted in urine and 36% in faeces after both intravenous and oral dosing. Unchanged granisetron was only excreted in urine (5–25% of dose).

5. The major metabolites were isolated and identified by MS spectroscopy and nmr. In rat, the dominant routes of biotransformation after both intravenous and oral dosing were 5-hydroxylation and N1-demethylation, followed by the formation of conjugates which were the major metabolites in urine, bile and plasma. In dog and man the major metabolite was 7-hydroxy-granisetron, with lesser quantities of the 6,7-dihydrodiol and/or their conjugates.     

Biotransformation of nevirapine, a non-nucleoside HIV-1 reverse transcriptase inhibitor, in mice, rats, rabbits, dogs, monkeys, and chimpanzees.

The study objectives were to characterize the metabolism of nevirapine (NVP) in mouse, rat, rabbit, dog, monkey, and chimpanzee after oral administration of carbon-14-labeled or -unlabeled NVP. Liquid scintillation counting quantitated radioactivity and bile, plasma, urine, and feces were profiled by HPLC/UV diode array and radioactivity detection. Metabolite structures were confirmed by UV spectral and chromatographic retention time comparisons with synthetic metabolite standards, by beta-glucuronidase incubations, and in one case, by direct probe electron impact ionization/mass spectroscopy, chemical ionization/mass spectroscopy, and NMR. NVP was completely absorbed in both sexes of all species except male and female dogs. Parent compound accounted for <6% of total urinary radioactivity and <5.1% of total fecal radioactivity, except in dogs where 41 to 46% of the radioactivity was excreted as parent compound. The drug was extensively metabolized in both sexes of all animal species studied. Oxidation to hydroxylated metabolites occurred before glucuronide conjugation and excretion in urine and feces. Hydroxylated metabolites were 2-, 3-, 8-, and 12-hydroxynevirapine (2-, 3-, 8-, and 12-OHNVP). 4-carboxynevirapine, formed by secondary oxidation of 12-OHNVP, was a major urinary metabolite in all species except the female rat. Glucuronides of the hydroxylated metabolites were major or minor metabolites, depending on the species. Rat plasma profiles differed from urinary profiles with NVP and 12-OHNVP accounting for the majority of the total radioactivity. Dog plasma profiles, however, were similar to the urinary profiles with 12-OHNVP, its glucuronide conjugate, 4-carboxynevirapine, and 3-OHNVP glucuronide being the major metabolites. Overall, the same metabolites are formed in animals as are formed in humans.     

Metabolism of loprazolam in rat, dog and man in vivo

The metabolism of 14C-loprazolam has been studied in rat, dog and man in vivo. In rat, the major metabolic pathways were hydroxylation on the benzodiazepine ring, and reduction and acetylation of the nitro group. Both metabolites were identified by co-chromatography with standards, and were present in urine and bile conjugated with glucuronic acid. In both dog and human urine and bile significant amounts of the piperazine-N-oxide were found. This N-oxide was identified by co-chromatography with authentic compound and by mass spectroscopy. Both loprazolam and the dog biliary metabolites were hydrolysed spontaneously to polar material. Neither treatment with beta-glucuronidase nor incubation with gut microflora had any further effect. Only polar metabolites were found in dog and human faeces. The principal non-polar material found in rat plasma was the diazepine-hydroxy compound, and little loprazolam was present. Significant levels of loprazolam and lower levels of an unidentified metabolite were found in ether extracts of dog and human plasma. Both the piperazine-N-oxide and loprazolam were found in similar quantities in chloroform extracts of human plasma, and at two hours after dosage, the N-oxide and loprazolam accounted for greater than 90% of the radioactivity present in the plasma.     

HPLC-NMR with severe column overloading: fast-track metabolite identification in urine and bile samples from rat and dog treated with [14C]-ZD6126

The subject of this study was the determination of the major urinary and biliary metabolites of [(14)C]-ZD6126 following i.v. administration to female and male bile duct cannulated rats at 10 mg/kg and 20 mg/kg, respectively, and male bile duct cannulated dogs at 6 mg/kg by HPLC-NMR spectroscopy. ZD6126 is a phosphorylated pro-drug, which is rapidly hydrolysed to the active metabolite, ZD6126 phenol. The results presented here demonstrate that [(14)C]-ZD6126 phenol is subsequently metabolised extensively by male dogs and both, male and female rats. Recovery of the dose in bile and urine was determined utilising the radiolabel, revealing biliary excretion as the major route of excretion (93%) in dog, with the majority of the radioactivity recovered in both biofluids in the first 6 h. In the rat, greater than 92% recovery was obtained within the first 24 h. The major route of excretion was via the bile 51-93% within the first 12 h. The administered phosphorylated pro-drug was not observed in any of the excreta samples. Metabolite profiles of bile and urine samples were determined by high performance liquid chromatography with radiochemical detection (HPLC-RAD), which revealed a number of radiolabelled components in each of the biofluids. The individual metabolites were subsequently identified by HPLC-NMR spectroscopy and HPLC-MS. In the male dog, the major component in urine and bile was the [(14)C]-ZD6126 phenol glucuronide, which accounted for 3% and 77% of the dose, respectively. [(14)C]-ZD6126 phenol was observed in urine at 1% of dose, but was not observed in bile. A sulphate conjugate of demethylated [(14)C]-ZD6126 phenol was identified in bile by HPLC-NMR and confirmed by HPLC-MS. In the rat, the bile contained two major radiolabelled components. One was identified as the [(14)C]-ZD6126 phenol glucuronide, the other as a glucuronide conjugate of demethylated [(14)C]-ZD6126 phenol. However, a marked difference in the proportions of these two components was observed between male and female rats, either due to a sex difference in metabolism or a difference in dose level. The glucuronide conjugate of the demethylated [(14)C]-ZD6126 phenol was present at higher concentration in the bile of male rats (4-34%), while the phenol glucuronide was present at higher concentration in the bile of female rats (8-70%) over a 0-6 h collection period. A third component was only observed in the bile samples (0-6 h and 6-12 h) of male rats. This was identified as being the same sulphate conjugate of demethylated [(14)C]-ZD6126 phenol as the one observed in dog bile. The rat urines contained two main metabolites in greatly varying concentrations, namely the demethylated [(14)C]-ZD6126 phenol glucuronide and the glucuronide of [(14)C]-ZD6126 phenol. Again, the differences in relative amounts between male and female rats were observed, the major metabolite in the urines from male rats being the demethylated [(14)C]-ZD6126 phenol (0-17% in 0-24 h), whilst the phenol glucuronide, accounting for 0.5-50% of the dose over 0-24 h, was the major metabolite in females. Methanolic extracts of the pooled biofluid samples were submitted for HPLC-NMR for the quick identification of the major metabolites. Following a single injection of the equivalent of 6-28 ml of the biofluids directly onto the HPLC-column with minimal sample preparation, the metabolites could be largely successfully isolated. Despite severe column overloading, the major metabolites of [(14)C]-ZD6126 could be positively identified, and the results are presented in this paper.     

Disposition of the opioid antagonist,nalmefene, in rat and dog

1. The disposition of nalmefene in rat and dog was studied using in vitro and in vivo methodology. In vitro metabolite profiles were obtained following incubation of nalmefene with liver microsomes and biological fluids were assayed to profile in vivo metabolites. Characterization of metabolites was accomplished using hplc, co-chromatography with synthetic standards, or LC/MS.

2. In rat, tissue distribution and metabolite plasma concentration-time data were obtained following intravenous bolus dosing of nalmefene.

3. The results indicate that the primary phase I metabolite of nalmefene from liver microsome incubations was the N-dealkylated metabolite, nornalmefene. Quantitative metabolite production was rat ? dog. In vivo, nornalmefene glucuronide was the major metabolite in rat urine, whereas nalmefene glucuronide(s) were predominant in dog urine.

4. More than 90% of the radioactive dose was recovered in the rat excreta and tissues 24?h after an intravenous bolus dose of ¹⁴C-nalmefene, with no apparent organ-specific retention of radioactivity.

5. Pharmacokinetic analysis of the rat plasma metabolite data indicated that terminal half-lives for nalmefene and nornalmefene were comparable (～ 1?h). However, C_max and AUC of nornalmefene were ≤ 7% that of corresponding nalmefene values.     

The Metabolic Fate of the Coronary Dilator 4-(3,4,5-Trimethoxycinnamoyl)-1-(N-Isopropylcarbamoylmethyl)- Piperazine in the Rat,Dog and Man

1. An oral dose of the coronary dilator 4-(3,4,5-trimethoxycinnamoyl)-1- (N-isopropylcarbamoylmethyl)-piperazine was readily absorbed and more than 75% of the dose was excreted within 24 h by the rat, dog and man. In 4 days, rat, dog and man excreted in the urine and faeces respectively 32.5 and 62.3%, 43.9 and 49.1%, and 57.8 and 43.3%. Faecal radioactivity was mainly excreted via the bile.

2. Plasma concentrations of radioactivity reached a maximum within 1 h in rats and dogs and within 2 h in man. For several h, more than 50% of the radioactivity circulating in the plasma of rats and more than 80% in man was due to unchanged drug.

3. Sequential whole-body autoradiography of the rat indicated that much of the radioactivity was distributed in the liver, kidneys and gastrointestinal tract and that there was significant uptake into the heart and lungs.

4. Although similar metabolites were excreted by the rat, dog and man, the relative proportions differed. 11.7, 2.3 and 28.8% respectively of the unchanged drug were excreted in the urine and 13.1, 19.5 and 10.4% respectively of the principal metabolite a glucuronide whose exact structure was not determined. Other metabolites included 4-(3,4,5-trimethoxycinnamoyl)-1-carbamoylmethyl piperazine and N-(3,4,5-trimethoxycinnamoyl)-piperazine.     

Lacidipine metabolism in rat and dog: identification and synthesis of main metabolites.

1. The main metabolites of lacidipine were isolated from bile and plasma of rats and dogs following an oral dose of the 14C-labelled drug (10 mg/kg for rats: 2 and 1 mg/kg for dogs). They were identified by comparison of chromatographic and spectral data with authentic reference compounds synthesized ad hoc. 2. Five metabolites (I-V) were isolated and identified in dog bile by gradient h.p.l.c. with u.v. detection and h.p.l.c.-thermospray mass spectrometry. In all metabolites the heterocyclic ring has been oxidized to pyridine. Further biotransformation reactions involved hydroxylation of the methyl substituents and hydrolysis of the ethyl and t-butyl ester groups to produce carboxylic acids and a lactone. Some of these metabolites also occurred as glucuronide conjugates. 3. A metabolite retaining the intact dihydropyridine ring, the des-ethyl analogue of lacidipine (VI), was isolated from rat plasma where it accounted for 60% of the total circulating radioactivity up to 24 h after administration. To characterize this metabolite, h.p.l.c. with photodiode array u.v. detection also was employed. This compound was detected in dog plasma, but there was no evidence of its presence in dog bile samples. 4. Profiles of circulating metabolites were qualitatively similar in rats and dogs. Identified metabolites accounted for the large majority of total radioactivity in all the analysed samples.     

Metabolism and disposition of GTS-21, a novel drug for Alzheimer's disease.

1. GTS-21, a novel drug for Alzheimer's disease, is currently under clinical development. In the current study, the metabolism and disposition of GTS-21 have been evaluated in rat and dog after single oral and intravenous administration. 2. Following oral administration of [14C]GTS-21 to rat, radioactivity was primarily excreted in the faeces (67%) via the bile with possible enterohepatic circulation. Urinary excretion of radioactivity in rat and dog was 20 and 19% respectively. 3. GTS-21 was rapidly and extensively absorbed after oral administration and rapidly cleared from plasma. The maximum concentration ratio of GTS-21 to total radioactivity in plasma was low, indicating first-pass or pre-systemic biotransformation. 4. In rat, GTS-21 showed linear pharmacokinetics over doses ranging from 1 to 10 mg/kg with an absolute bioavailability of 23%. In dog, the absolute bioavailability was 27% at an oral dose of 3 mg/kg. 5. GTS-21 was O-demethylated to yield compounds that were then subject to glucuronidation. Three of the metabolites in rat urine were isolated and characterized as 4-OH-GTS-21, 4-OH-GTS-21 glucuronide and 2-OH-GTS-21 glucuronide. The major urinary metabolites were 4-OH-GTS-21 glucuronide and 2-OH-GTS-21 glucuronide. 6. In vitro chemical inhibition of cytochrome P450 in human liver microsomes indicated that CYPIA2 and CYP2E1 were the isoforms primarily responsible for the O-demethylation of GTS-21, with some contribution from CYP3A.