Identification of novel metabolites of pioglitazone in rat and dog

1. Four new metabolites of pioglitazone were identified by liquid chromatography-mass spectrometry (LC-MS/MS) as being formed by hydroxylation (M-VII and M-VIII), opening of the thiazolidinedione ring (M-X) and by desaturation of the terminal ethyl side chain or tether ethoxy moiety (M-IX), respectively. The structure of one of the hydroxylated metabolites (M-VII) was confirmed by chemical modification using the Jones reaction. 2. Oxidative cleavage of the thiazolidinedione ring is a novel pathway not previously reported for pioglitazone. 3. The hydroxylated M-VII was detected in incubations with rat, dog and human liver and kidney microsomes, and in plasma from rats and dogs dosed orally with [3 H]pioglitazone. 4. The carboxylic acid derivative of M-VII (M-V) and its taurine conjugate were the major radioactive components in dog bile.     

Biotransformation of diclofenac sodium (Voltaren) in animals and in man. I. Isolation and identification of principal metabolites.

1. The anti-inflammatory agent diclofenac sodium (o-[(2,6-dichlorophenyl)amino]phenylacetic acid sodium salt) is extensively metabolized by rat, dog, baboon and man. The main metabolites were isolated from the urine of all species and from the bile of rat and dog and identified by spectroscopy. 2. Metabolism involves direct conjugation of the unchanged drug, or oxidation of the aromatic rings usually followed by conjugation. Sites of oxidation are either position 3' or 4' of the dichlorophenyl ring or, alternatively, position 5 of the phenyl ring attached to the acetic acid moiety. 3. In the urine of rat, baboon and man conjugates of the hydroxylated metabolites predominate, but the major metabolite in dog urine is the taurine conjugate of unchanged diclofenac. 4. In the bile of rat and dog, the main metabolite is the ester glucuroniade of unchanged diclofenac.     

Metabolism of 2-nitrofluorene, 2-aminofluorene and 2-acylaminofluorenes in rat and dog and the role of intestinal bacteria

1. The in vivo metabolism of 2-nitrofluorene (NF), an environmental pollutant, and 2-aminofluorene (AF) and its acylated derivatives, 2-formylaminofluorene (FAF) and 2-acetylaminofluorene (AAF), was examined in rat and dog. 2. 7-Hydroxy-2-nitrofluorene, 5-hydroxy-2-nitrofluorene, AF, AAF, FAF, 7-hydroxy-2-aminofluorene, 5-hydroxy-2-aminofluorene, 7-hydroxy-2-acetylaminofluorene, 5-hydroxy-2-acetylaminofluorene, 7-hydroxy-2-formylaminofluorene and 5-hydroxy-2-formylaminofluorene were identified as urinary and faecal metabolites of NF in rat and dog. 3. AAF and its hydroxylated derivatives were detected as major metabolites of NF in rat, but FAF and its hydroxylated metabolites were mainly excreted in dog. 4. AF, AAF, FAF and their hydroxylated metabolites were also identified as urinary and faecal metabolites of AF, AAF or FAF in rat, suggesting that AAF and FAF are interconverted via AF. 5. Treatment of rat and dog with antibiotics significantly decreased the urinary and faecal excretion of AF and its derivatives after oral administration of NF, and partly decreased the excretion of acylated metabolites after an oral dose of AF. 6. The caecal contents of untreated rats and some species of intestinal bacteria exhibited nitro-reductase activity toward NF, and acylating activity toward AF, affording AAF and FAF.     

Studies on antidiabetic agents. 11. Novel thiazolidinedione derivatives as potent hypoglycemic and hypolipidemic agents.

In the course of further chemical modification of the novel antidiabetic pioglitazone (AD-4833, U-72,107), a series of 5-[4-(2- or 4-azolylalkoxy)benzyl- or -benzylidene]-2,4-thiazolidinediones was prepared and evaluated for hypoglycemic and hypolipidemic activities in insulin-resistant, genetically obese, and diabetic KKA(y) mice. Replacement of the 2-pyridyl moiety of pioglitazone by a 2- or 4-oxazolyl or a 2- or 4-thiazolyl moiety greatly enhanced in vivo potency. The corresponding 5-benzylidene-type compounds, in which a methine was used as a linker between the benzene ring and the thiazolidinedione ring, also had potent biological activity. Among the compounds synthesized, 5-[4-[2-(5-methyl-2-phenyl-4-oxazolyl)ethoxy]benzyl]-2,4- thiazolidinedione (18) exhibited the most potent activity, more than 100 times that of pioglitazone. The synthesis and structure-activity relationships for this novel series of derivatives are detailed.     

Mass spectrometric and NMR characterization of metabolites of roxifiban, a potent and selective antagonist of the platelet glycoprotein IIb/IIIa receptor

1. The methyl ester prodrug roxifiban is an orally active, potent and selective antagonist of the platelet glycoprotein GPIIb/IIIa receptor and is being developed for the prevention and treatment of arterial thrombosis. 2. Roxifiban was rapidly hydrolyzed to the zwitterion XV459 in vivo and by liver slices from the rat, mouse and human and by intestinal cores from dog. XV459 was metabolized to only a small extent in vitro and in vivo. 3. Studies with rat and dog given radiolabelled roxifiban showed limited oral absorption with the majority of the radiolabel being excreted in faeces. After i.v. doses of 14C-roxifiban, most of the radioactivity was recovered in the urine of rat whereas the dog excreted significant amounts of radioactivity in bile and urine. 4. XV459 could be metabolized extrahepatically by dog gut flora to produce an isoxazoline ring-opened metabolite. In vitro hepatic metabolism of XV459 was mainly by hydroxylation at the prochiral and chiral centres of the isoxazoline ring. These hydroxylated metabolites were not detected in the urine and plasma of human volunteers administered roxifiban. 5. Initial LC/MS identification of metabolites was achieved by dosing the rat with an equimolar mixture of d0:d4 roxifiban and detecting isotopic clusters of pseudomolecular ions. Unequivocal characterization of these metabolites was achieved by LC/MS, LC/NMR and high-field NMR techniques using synthetic standards of the metabolites. 6. The synthesis of one hydroxylated metabolite enabled the assignment of the correct stereochemistry of the substituted hydroxyl group on the isoxazoline ring.     

Metabolism of 2-nitrofluorene, 2-aminofluorene and 2-acylaminofluorenes in rat and dog and the role of intestinal bacteria

1. The in vivo metabolism of 2-nitrofluorene (NF), an environmental pollutant, and 2-aminofluorene (AF) and its acylated derivatives, 2-formylaminofluorene (FAF) and 2-acetylaminofluorene (AAF), was examined in rat and dog. 2. 7-Hydroxy-2-nitrofluorene, 5-hydroxy-2-nitrofluorene, AF, AAF, FAF, 7-hydroxy-2-aminofluorene, 5-hydroxy-2-aminofluorene, 7-hydroxy-2-acetylaminofluorene, 5-hydroxy-2-acetylaminofluorene, 7-hydroxy-2-formylaminofluorene and 5-hydroxy-2-formylaminofluorene were identified as urinary and faecal metabolites of NF in rat and dog. 3. AAF and its hydroxylated derivatives were detected as major metabolites of NF in rat, but FAF and its hydroxylated metabolites were mainly excreted in dog. 4. AF, AAF, FAF and their hydroxylated metabolites were also identified as urinary and faecal metabolites of AF, AAF or FAF in rat, suggesting that AAF and FAF are interconverted via AF. 5. Treatment of rat and dog with antibiotics significantly decreased the urinary and faecal excretion of AF and its derivatives after oral administration of NF, and partly decreased the excretion of acylated metabolites after an oral dose of AF. 6. The caecal contents of untreated rats and some species of intestinal bacteria exhibited nitro-reductase activity toward NF, and acylating activity toward AF, affording AAF and FAF.     

Biotransformation of a new pyrrolidinone cognition-enhancing agent: isolation and identification of metabolites in human urine

1. The metabolites of N-(2,6-dimethylphenyl)-2-(2-oxo-1-pyrrolidinyl)acetamide (DMPPA; MH-1), in the urine of human volunteers have been investigated. 2. Ten metabolites together with the unchanged drug (MH-1) were isolated by h.p.l.c. and identified by n.m.r. and mass spectrometry as: three metabolites hydroxylated in the pyrrolidine ring of MH-1 (MH-2, MH-3 and MH-4), three metabolites hydroxylated in the dimethylphenyl ring of MH-1 (MH-6, MH-7 and MH-8), N-[(2,6-dimethylphenylcarbamoyl)methyl]-4-hydroxybutyrylamide++ + (MH-5), N-[(2,6-dimethylphenyl-carbamoyl)methyl]succinamic acid (MH-9), the 3-O-sulphate of MH-6 (MH-10) and the 3-O-sulphate of N-(2,6-dimethyl-3-hydroxyphenyl)-2-(5-hydroxy-2-oxo-1-pyrrolidinyl)aceta mide (MH-11). 3. DMPPA was extensively metabolized. The principal metabolic transformations were hydroxylation of the pyrrolidine ring at the C5 carbon followed by oxidative C-N cleavage, and hydroxylation of the phenyl ring followed by sulphate conjugation.     

Metabolic and non-metabolic factors determining troglitazone hepatotoxicity: a review

Troglitazone (TGZ), a thiazolidinedione class of antidiabetic agent, causes serious idiosyncratic hepatotoxicity. TGZ is metabolized into reactive metabolites that covalently bind to cellular macromolecules, one of which is oxidation at the chromane ring, a unique structure of TGZ, and another involves oxidative cleavage of the thiazolidinedione ring, a structure common to less hepatotoxic antidiabetics, rosiglitazone and pioglitazone. TGZ is cytotoxic to HepG2 cells and rat and human hepatocytes. However, the role of the reactive metabolite on the TGZ toxicity is controversial, because there was no correlation of the generation of the reactive metabolites with susceptibility to the TGZ cytotoxicity, and chemical inhibitors of drug metabolizing enzymes could not protect the cells against the toxicity. Mitochondrial dysfunction, especially mitochondrial permeability transition, may be a pathophysiological event, which is mediated by TGZ itself and is a major non-metabolic factor. Other events such as apoptosis and PPARgamma-dependent steatosis could be also mediated by TGZ, while inhibition of bile salt export pump, a cause of TGZ-induced cholestasis, may be caused by the TGZ sulfate. In conclusion, although the TGZ is biotransformed into chemically reactive metabolites, there is currently no potential evidence for involvement of the reactive metabolite in the TGZ-induced liver injury.     

Liquid chromatography-mass spectrometry and liquid chromatography-NMR characterization of in vitro metabolites of a potent and irreversible peptidomimetic inhibitor of rhinovirus 3C protease.

In vitro metabolism of AG7088 [trans-(4S,2'R,5'S,3"'S)-4-[2'-4-(4-fluorobenzyl)-6'-methyl-5'-[(5"-methylisoxazole-3"-carbonylamino]-4-oxoheptanoylamino]-5-(2"'-oxopyrrolidin-3-"'-yl)pent-2-enoic acid ethyl ester] was studied in liver microsomes isolated from mice, rats, rabbits, dogs, monkeys, and humans. The structures of the metabolites were characterized by liquid chromatography (LC)-tandem mass spectrometry and LC-NMR methods. Hydrolysis of the ethyl ester to produce metabolite M4 (AG7185) is the predominant pathway in all species, with the greatest activity observed in rodents and rabbits, followed by monkeys, dogs, and humans. Several hydroxylation products were identified as minor metabolites, including diastereomers M1 and M2, with a hydroxy group at the P1-lactam moiety, and M3, with a hydroxy group at the methyl position of the methylisoxazole ring. Rodent and rabbit liver microsomes formed almost exclusively the acid metabolite M4 (AG7185), with very little hydroxylated metabolites, whereas monkey liver microsomes formed more secondary metabolites (i.e., acid analogs of the hydroxylated metabolites). The overall metabolic profile of AG7088 formed in dog liver microsomes closely resembled that of human liver microsomes; therefore, this species may be the most appropriate animal model relative to humans for exposure to AG7088 and its metabolites.     

Metabolism of a new synthetic opioid tetrahydrofuranylfentanyl in fresh isolated human hepatocytes: Detection and confirmation of ring‐opened metabolites

The metabolism of a new synthetic opioid tetrahydrofuranylfentanyl (THF‐fentanyl) was investigated using fresh human hepatocytes. Fourteen metabolites of THF‐fentanyl, such as tetrahydrofuran ring‐opened metabolites, desphenethylated metabolites, hydroxylated metabolites, and hydroxylated and methoxylated metabolites and their glucuronides, were detected in the culture medium of hepatocytes incubated with THF‐fentanyl. Six metabolites, i.e. desphenethylated metabolite, 4′‐hydroxy‐THF‐fentanyl, β‐hydroxy‐THF‐fentanyl, 4′‐hydroxy‐3′‐methoxy‐THF‐fentanyl, ring‐opened alcohol metabolite, and ring‐opened carboxylic acid metabolite, were identified via chemically synthesized authentic standards. A ring‐opened alcohol metabolite and a ring‐opened carboxylic acid metabolite are thought to be formed by reduction or oxidation of the intermediate aldehyde, which was formed by ring‐opening of the metabolite hydroxylated at the carbon atom adjacent to the oxygen atom of the tetrahydrofuran ring. A ring‐opened carboxylic acid metabolite was the main metabolite of THF‐fentanyl based on the peak intensity.     

Biotransformation of a new pyrrolidinone cognition-enhancing agent: isolation and identification of metabolites in human urine

1. The metabolites of N-(2,6-dimethylphenyl)-2-(2-oxo-1-pyrrolidinyl)acetamide (DMPPA; MH-1), in the urine of human volunteers have been investigated.

2. Ten metabolites together with the unchanged drug (MH-1) were isolated by?h.p.l.c. and identified by n.m.r. and mass spectrometry as: three metabolites hydroxylated in the pyrrolidine ring of MH-1 (MH-2, MH-3 and MH-4), three metabolites hydroxylated in the dimethylphenyl ring of MH-1 (MH-6, MH-7 and MH-8), N-[(2,6-dimethyl-phenylcarbamoyl)methyl]-4-hydroxybutyrylamide (MH-5), N-[(2,6-dimethyl-phenyl-carbamoyl)methyl]succinamic acid (MH-9), the 3-O-sulphate of MH-6 (MH-10) and the 3-O-sulphate of N-(2,6-dimethyl-3-hydroxyphenyl)-2-(5-hydroxy-2-oxo-1-pyrrolidinyl)acetamide (MH-11).

3. DMPPA was extensively metabolized. The principal metabolic transformations were hydroxylation of the pyrrolidine ring at the C5 carbon followed by oxidative C-N cleavage, and hydroxylation of the phenyl ring followed by sulphate conjugation.     

Montelukast and zafirlukast do not affect the pharmacokinetics of the CYP2C8 substrate pioglitazone

Objective Pioglitazone, a thiazolidinedione antidiabetic drug, is metabolised mainly by the cytochrome P450 (CYP) 2C8 enzyme. The leukotriene receptor antagonists montelukast and zafirlukast have potently inhibited CYP2C8 activity and the metabolism of pioglitazone in vitro. Our objective was to determine whether montelukast and zafirlukast increase the plasma concentrations of pioglitazone in humans.Methods In a randomised, double-blind crossover study with three phases and a washout period of 3 weeks, 12 healthy volunteers took either 10 mg montelukast once daily and placebo once daily, or 20 mg zafirlukast twice daily, or placebo twice daily, for 6 days. On day 3, they received a single oral dose of 15 mg pioglitazone. The plasma concentrations of pioglitazone and its metabolites M-IV, M-III, M-V and M-XI were measured for 96 h.Results The total area under the plasma concentration-time curve of pioglitazone during the montelukast and zafirlukast phases was 101% (range 71–143%) and 103% (range 78–146%), respectively, of that during the placebo phase. Also, the peak plasma concentration and elimination half-life of pioglitazone remained unaffected by montelukast and zafirlukast. There were no statistically significant differences in the pharmacokinetics of any of the metabolites of pioglitazone between the phases.Conclusions Montelukast and zafirlukast do not increase the plasma concentrations of pioglitazone, indicating that their inhibitory effect on CYP2C8 is negligible in vivo, despite their strong inhibitory effect on CYP2C8 in vitro. The results highlight the importance of in vivo interaction studies and of the incorporation of relevant pharmacokinetic properties of drugs, including plasma protein binding data, to in vitro-in vivo interaction predictions.Supported by grants from the National Technology Agency (Tekes), the Helsinki University Central Hospital Research Fund and the Sigrid Jusélius Foundation, Finland.     

Mass spectrometric identification of urinary and plasma metabolites of 2-(6'-carboxyhexyl)-3-n-hexylcyclohexylamine, a new antiaggregating agent.

The compound IBI-P-05006, 2-(6'-carboxyhexyl)-3-n-hexylcyclohexylamine, is an antiaggregating agent under development. IBI-P-05006 is an in vitro inhibitor of platelet aggregation. The biotransformation of this compound has been studied in the dog and rat. We present here a study on the metabolites of IBI-P-05006 found in dog and rat urine, and in dog plasma. Analyses were done by gas chromatography-mass spectrometry. In dog urine 15 metabolites were identified. Some of them were also found in dog plasma and in rat urine. The unmetabolized drug was found only in plasma. 10 different hydroxylated metabolites were characterized. The hydroxyl groups were introduced in the hexyl chain in positions omega-4, omega-3, omega-2, omega-1 and omega.     

Mass spectrometric and NMR characterization of metabolites of roxifiban,a potent and selective antagonist of the platelet glycoprotein IIb/IIIa receptor

1. The methylester prodrug roxifiban is an orally active, potent and selective antagonist of the platelet glycoprotein GPIIb/IIIa receptor and is being developed for the prevention and treatment of arterial thrombosis. 2. Roxifiban was rapidly hydrolyzed to the zwitterion XV459 in vivo and by liver slices from the rat, mouse and human and by intestinal cores from dog. XV459 was metabolized to only a small extent in vitro and in vivo. 3. Studies with rat and dog givenradiolabelled roxifibanshowedlimited oralabsorption with the majority of the radiolabel being excreted in faeces. After i.v. doses of ¹⁴C-roxifiban, most of the radioactivity was recovered in the urine of rat whereas the dog excreted significant amounts of radioactivity in bile and urine. 4. XV459 could be metabolized extrahepatically by dog gut flora to produce an isoxazoline ring-opened metabolite. In vitro hepatic metabolism of XV459 was mainly by hydroxylation at the prochiraland chiralcentres of the isoxazolinering. These hydroxylated metabolites were not detected in the urine and plasma of human volunteers administered roxifiban. 5. Initial LC/MS identification of metabolites was achieved by dosing the rat with an equimolar mixture of d₀:d₄ roxifiban and detecting isotopic clusters of pseudomolecular ions. Unequivocal characterization of these metabolites was achieved by LC/MS, LC/NMR and high-field NMR techniques using synthetic standards of the metabolites. 6. The synthesis of one hydroxylated metabolite enabled the assignment of the correct stereochemistry of the substituted hydroxyl group on the isoxazoline ring.     

Nephrotoxic and hepatotoxic potential of imidazolidinedione-, oxazolidinedione- and thiazolidinedione-containing analogues of N-(3,5-dichlorophenyl)succinimide (NDPS) in Fischer 344 rats

Nephrotoxicity of the agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) in rats is believed to involve metabolism on the succinimide ring. To further investigate this hypothesis, we synthesized and tested the following NDPS analogues, which contain other cyclic imide rings and may therefore be metabolized differently than NDPS: 3-(3,5-dichlorophenyl)-2,4-oxazolidinedione (DCPO), 3-(3,5-dichlorophenyl)-2,4-imidazolidinedione (DCPI), 3-(3,5-dichlorophenyl)-1-methyl-2,4-imidazolidinedione (DCPM) and 3-(3,5-dichlorophenyl)-2,4-thiazolidinedione (DCPT). Male Fischer 344 rats were administered DCPO, DCPI, DCPM, DCPT (0.6 or 1.0 mmol/kg, i.p. in corn oil), NDPS (0.6 mmol/kg, i.p. in corn oil) or corn oil (4 ml/kg). As evidenced by diuresis, proteinuria, elevated blood urea nitrogen levels, increased kidney weights and proximal tubular damage, NDPS produced severe nephrotoxicity in the rats. In contrast, DCPO, DCPI, DCPM and DCPT were mild nephrotoxicants. None of the compounds elevated serum alanine transferase activity or liver weights in the rats, however DCPT produced centrilobular necrosis. These experiments confirm that NDPS-induced nephrotoxicity is critically dependent on the presence of the succinimide ring. Furthermore, replacement of the succinimide ring with a thiazolidinedione ring produced a more pronounced effect on the liver than on the kidney. Liver damage has been reported in type II diabetic patients taking troglitazone, rosiglitazone and pioglitazone. Since these compounds also contain a thiazolidinedione ring, DCPT may be useful for investigating the role of this structural feature in hepatotoxicity.     

In vitro metabolism of MK-0767 [(+/-)-5-[(2,4-dioxothiazolidin-5-yl)methyl]-2-methoxy-N-[[(4-trifluoromethyl)-phenyl] methyl]benzamide], a peroxisome proliferator-activated receptor alpha/gamma agonist. II. Identification of metabolites by liquid chromatography-tandem mass spectrometry.

The in vitro metabolism of MK-0767 [(+/-)-5-[(2,4-dioxothiazolidin-5-yl) methyl]-2-methoxy-N-[[(4-trifluoromethyl)-phenyl] methyl]benzamide], a novel 2,4-thiazolidinedione (TZD)-containing peroxisome proliferator-activated receptor alpha/gamma agonist, was studied in rat, dog, monkey, and human liver microsomes and hepatocytes, as well as in recombinant human CYP3A4-containing microsomes. Twenty-two metabolites (some at trace levels) were detected by liquid chromatography-tandem mass spectrometry analysis. All appeared to be phase I metabolites except for a glucuronide conjugate of a hydroxylated metabolite that was detected at trace levels. A constant neutral loss scan experiment performed on a triple quadrupole mass spectrometer proved to be very useful for resolving the metabolites from endogenous compounds. It was observed that the initial site of metabolism of MK-0767 was at the TZD ring leading to two major metabolites, namely the 5-hydroxy-TZD metabolite (M24) and the mercapto metabolite (M22). The latter was formed via the cleavage of the TZD ring with the elimination of the carbonyl adjacent to the sulfur atom. The structure of M24 was established by accurate mass measurements and NMR analysis. This hydroxy-TZD metabolite might represent an important precursor for a group of metabolites formed by TZD ring opening and subsequent loss of the sulfur moiety. The mercapto metabolite, on the other hand, is probably the key precursor for the TZD ring-opened metabolites with retention of the sulfur, even though the detailed mechanism of the ring scission remains to be characterized. From these studies, it was concluded that the TZD ring was the major site of metabolism of MK-0767. All the metabolites produced in vitro from human preparations were detected in the corresponding preparations from the nonclinical species.     

Flutamide metabolism in four different species in vitro and identification of flutamide metabolites in human patient urine by high performance liquid chromatography/tandem mass spectrometry.

A new metabolic scheme of flutamide is proposed in this article. Some patients treated with flutamide, a nonsteroidal antiandrogen, have developed severe hepatic dysfunction. Toxic metabolites have been proposed to be responsible for these negative effects. In this study, the qualitative aspects of the in vitro metabolism of flutamide in liver microsomes from human, dog, pig, and rat were evaluated. A direct comparison of the flutamide metabolism in liver and prostate microsomes from pig was made, and the in vivo metabolism of flutamide was investigated in urine from orally treated prostate cancer patients. Liquid chromatography/tandem mass spectrometry was used for analysis. The mass spectrometer was equipped with an electrospray interface and operated in the negative ion mode. In liver microsomes from pig, dog, and rat, extensive hydroxylation of flutamide occurred. One, two, or three hydroxy groups were attached, and isomeric forms were detected for both monohydroxylated and trihydroxylated drug. In pig liver microsomes, isomers of a third metabolite, hydroxylated 4-nitro-3-(trifluoromethyl)-aniline, were also found after incubation with either flutamide or 2-hydroxyflutamide. In human liver microsomes, the pharmacologically active 2-hydroxyflutamide was the only metabolite detected. Several phase I metabolites as well as four intact phase II metabolites could be recovered from the urine samples. For the first time in humans, glucuronic acid conjugates of hydroxylated 4-nitro-3-(trifluoromethyl)-aniline, and mono- and dihydroxylated flutamide were identified, together with hydroxylated 4-nitro-3-(trifluoromethyl)-aniline conjugated with sulfate. In addition, one mercapturic acid conjugate of hydroxylated flutamide, probably formed from flutamide via a reactive intermediate, was detected.     

Studies on the biotransformation of lonazolac, bromerguride, lisuride and terguride in laboratory animals and their hepatocytes

1. Metabolic patterns and the extents of metabolism of four drugs, namely [14C]lonazolac (LON), [14C]bromerguride)BRO), [14C]lisuride (LIS) and [3H]terguride (TER) have been studied in three experimental models, namely hepatocyte suspensions of rat, guinea pig, beagle dog and cynomolgus monkey, isolated perfused liver of rat and guinea pig and intact animals (rat, guinea pig, dog and monkey). 2. Selection of compounds was based on differences in phase I metabolic pathways. LON is exclusively hydroxylated in the N-substituting aromatic ring, BRO is mainly N-deethylated in the urea moiety, and LIS and TER are both degraded into numerous metabolites. 3. The decrease in unchanged drug levels in hepatocyte suspensions was characterized by half-lives, with LON as the most stable and LIS as the least stable compound. Marked interspecies differences were found. De-ethylation and aromatic hydroxylation were much slower in rat hepatocytes than in the liver cells of other species; BRO was slowly biodegraded in dog hepatocytes while LIS was broken down extremely quickly. 4. Liver perfusion experiments and studies in vivo were evaluated for the extents of metabolism of each drug. 5. Metabolism studies in hepatocytes did not show any quantitative correlation to those of metabolism in vivo. The suitability of evaluating parameters for in vitro studies is discussed.     

Identification of urinary metabolites of 2-methyl-3-(1,4,5,6-tetrahydronicotinoyl)pyrazolo[1,5-a]pyridine in rat, rabbit and dog.

The metabolism of KC-764 (2-methyl-3-(1,4,5,6-tetrahydronicotinoyl)pyrazolo[1,5-a]pyridine, CAS 94457-09-7) in rat, rabbit and dog was studied. The urine of animals dosed with 14C-KC-764 was extracted with ethyl acetate after treatment with beta-glucuronidase and arylsulfatase. The metabolites were purified by TLC and HPLC from the extract. Unchanged KC-764 and 16 metabolites were isolated and their structures were identified or proposed by NMR and MS spectrometry. The metabolism of KC-764 took place by the oxidation of the tetrahydropyridine ring, 6,7-position and 2-methyl group of the pyrazolopyridine ring, and their combinations. The oxidation of the tetrahydropyridine ring was predominant in dog, whereas the oxidation of the pyrazolopyridine ring was more important in rabbit. Rat produced the various metabolites by their combination. 6-Oxo and 6-ureido derivatives of the tetrahydropyridine ring were common major metabolites in all animal species studied.     

Urinary metabolites of a novel quinoxaline non-nucleoside reverse transcriptase inhibitor in rabbit, mouse and human: identification of fluorine NIH shift metabolites using NMR and tandem MS

1. The urinary metabolites of (S)-2-ethyl-7-fluoro-3-oxo-3,4-dihydro-2H-quinoxaline-carboxylic acid isopropylester (GW420867X) have been investigated in samples obtained following oral administration to rabbit, mouse and human. GW420867X underwent extensive biotransformation to form hydroxylated metabolites and glucuronide conjugates on the aromatic ring, and on the ethyl and isopropyl side-chains in all species. In rabbit urine, a minor metabolite was detected and characterized as a cysteine adduct that was not observed in mouse or man. 2. The hydroxylated metabolites and corresponding glucuronide conjugates were isolated by semi-preparative HPLC and characterized using NMR, LC-NMR and LC-MS/MS. The relative proportions of fluorine-containing metabolites were determined in animal species by 19F-NMR signal integration. 3. The fluorine atom of the aromatic ring underwent NIH shift rearrangement in the metabolites isolated and characterized in rabbit, mouse and human urine. 4. The characterization of the NIH shift metabolites in urine enabled the detection and confirmation of the presence of these metabolites in human plasma.