Metabolism of a novel hypnotic,N3-phenacyluridine,and hypnotic and sedative activities of its enantiomer metabolites in mouse

1. The metabolism of N³-phenacyluridine (3-phenacyl-1- beta-D-ribofuranosyluracil), a potent hypnotic nucleoside derivative, was studied in mouse. 2. Of the radioactivity, 65% was excreted in urine within 48 h after intraperitoneal (i.p.) administration of [³H] N³-phenacyluridine. The urinary metabolites N³-phenacyluracil and N³-alpha-hydroxy-beta-phenethyluridine were extracted, isolated and analyzed by mass spectrometry. 3. Racemates of N³-alpha-hydroxy-beta-phenethyluridine were synthesized and both isomers were separated as N³-(S)-(+)-alpha-hydroxy-beta-phenethyluridine and N³-(R)-(-)-alpha hydroxy-beta-phenethyluridine by hplc (CHIRALCEL-OJ column) with retentions of 13.8 and 17.9 min respectively. The reduction process took place with high stereo-selectivity, which gave an alcohol product in the urine with the same retention (17.9 min) as one of the synthetic isomers separated by hplc. 4. One of urinary metabolites was identified as N³-(S)-(+)-alpha-hydroxy-beta-phenethyluridine. N³-phenacyluridine was predominantly converted to an alcoholic metabolite of (S)-(+)-configuration. 5. N³-phenacyluracil and uridine were also identified as minor metabolites. 6. The pharmacological effects of the metabolites and related compounds were also evaluated in mouse. N³-(S)-(+)-alpha-hydroxy-beta-phenethyluridine, but not N ³-(R)-(-)-alpha hydroxy-beta-phenethyluridine, possessed hypnotic activity and potentiated pentobarbitalinduced sleeping time with a similar potency to the parent compound, N³-phenacyluridine. N³-alpha-hydroxy-beta-phenethyluridine (racemate) had almost two thirds of the hypnotic activity of N³-(S)-(+)- alpha-hydroxy-beta-phenethyluridine. No other metabolites exhibited hypnotic activities. 7. The present study indicates that N³-(S)-(+)-alpha-hydroxy-beta-phenethyluridine, a major metabolite of N³-phenacyluridine, is an active metabolite and contributes a significant CNS depressant effect.     

Possible existence of a novel receptor for uridine analogues in the central nervous system using two isomers, N3-(S)-(+)- and N3-(R)-(-)-alpha-hydroxy-beta-phenethyluridines.

Uridine analogue binding sites, the so-called uridine receptor, were observed in the experiments on specific [3H]N3-phenacyluridine binding to bovine synaptic membranes using two isomers, N3-(S)-(+)- and N3-(R)-(-)-alpha-hydroxy-beta-phenethyluridine, as ligands. The potent hypnotic, N3-(S)-(+)-alpha-hydroxy-beta-phenethyluridine, but not the (R)-isomer, strongly inhibited [3H]N3-phenacyluridine binding. The racemate had inhibitory activity intermediate between that of the two alpha-hydroxy-beta-phenethyluridines ((R)- or (S)-isomers). The inhibitory constants of these compounds were determined. The Ki values of N3-phenacyluridine, alpha-hydroxy-beta-phenethyluridine (racemate), N3-(R)-(-)-, and N3-(S)-(+)-alpha-hydroxy-beta-phenethyluridine were 0.65, 397.4, 1908, and 10.2 nM, respectively. The present results indicate the existence of uridine receptors in the central nervous system in relation to their hypnotic activities reported previously.     

A carbonyl reductase-catalyzing reduction of N3-phenacyluridine in rabbit liver

Carbonyl reductase activity for a novel hypnotic, N3-phenacyluridine, was mainly localized in the cytosol fraction of rabbit liver. The enzyme (N3-phenacyluridine reductase) which catalyzes the reduction of N3-phenacyluridine to N3-alpha-hydroxy-beta-phenethyluridine was purified from the cytosolic fraction of rabbit liver by various chromatographic techniques (DEAE-Sephacel, Red Sepharose CL-6B and hydroxylapatite). N3-Phenacyluridine reductase had a minimum molecular weight of 39kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. This enzyme required reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor and its optimal pH was 7.5. Flavonoids (quercetin and quercitrin) were potent inhibitors of the enzyme, but pyrazole or barbital had little effect. The apparent Km and Vmax values of the enzyme for the reduction of N3-phenacyluridine were 0.32 mM and 8.7 units/mg protein, respectively. A variety of carbonyl compounds, including N3-phenacyluridine, were effectively reduced by the enzyme. However, the enzyme purified from rabbit liver differs in several respects from known carbonyl reductases in rabbit liver.     

Species variation and stereoselectivity in the metabolism of nicotine enantiomers

1. High performance liquid radiochromatographic systems have been developed for the identification and quantification of 7 urinary metabolites of both S-(-)-[3H-N'-CH3]nicotine and R-(+)-[3H-N'-CH3] nicotine in guinea pig, hamster, rat and rabbit. 2. 3'-Hydroxycotinine was a major urinary metabolite of both S-(-)-nicotine and R-(+)-nicotine in guinea pig, hamster and rabbit. Cotinine was not generally a significant urinary metabolite of either nicotine enantiomer, except in rat, where it constituted 14.6 and 10.4%, respectively, of the total radiolabel in the urine after administration of [3H]-S-(-)-nicotine or [3H]-R-(+)-nicotine. Nicotine N'-oxide was an important urinary metabolite of both nicotine isomers in guinea pig and rat, but in both cases, was not observable in hamster and rabbit. No N-methylated urinary metabolite of S-(-)-nicotine could be detected in any of the species examined. In R-(+)-nicotine experiments, only guinea pig afforded N-methylated metabolites. Significant amounts of 2 unidentified polar, non-basic urinary metabolites of both S-(-)- and R-(+)-nicotine-treated animals were observed. 3. Analysis of the comparative metabolism of the nicotine enantiomers in the four animals species studied, showed that stereoselective differences in the formation of oxidative metabolites existed, particularly in the formation of 3'-hydroxycotinine and nicotine-N'-oxide. A clear stereospecificity was observed in the guinea pig, in that only the R-(+)-nicotine enantiomer was N-methylated in this species. 4. Sex differences appear to exist in the metabolism of nicotine enantiomers in the rat. Female rats excreted more of the unidentified polar metabolite B than male rats, whereas the converse was true for nicotine-N'-oxide. In experiments with R-(+)-nicotine, urinary levels of 3'-hydroxycotinine and R-(+)-nicotine in female rats were higher than in male rats. Conversely, higher amounts of nicotine-N'-oxide were observed in the urine of male rats compared to those in female rats.     

Enantioselective disposition of hydroxychloroquine after a single oral dose of the racemate to healthy subjects.

1. Stereoselectivity in the disposition of hydroxychloroquine was investigated in 23 healthy males following a single oral dose of 200 mg racemic HCQ (rac-HCQ) sulphate. Total concentrations (R+S) and R/S ratios of HCQ and its metabolites were measured by stereoselective h.p.l.c. 2. HCQ was detected in whole blood and urine, up to 91 and 85 days after dosing, respectively. Metabolites could not be detected in whole blood while in urine detectable concentrations were still present after 85 days. The blood concentrations of HCQ enantiomers were measurable until 168 h post-dose. 3. R(-)-HCQ accounted for 62 +/- 3% (mean +/- s.d.) of the AUC of rac-HCQ AUC. The elimination half-life of S(+)-HCQ (457 +/- 122 h) was significantly shorter than that of R(-)-HCQ (526 +/- 140 h), partly due to its faster urinary excretion and hepatic metabolism. Its renal clearance was twice that of R(-)-HCQ (4.61 +/- 4.01 vs 1.79 +/- 1.30 1 h-1), and metabolites derived from the S-isomer represented 80-90% of the urinary recovery of the dose. 4. Over 85 days, 4.4 +/- 2.9 and 3.3 +/- 1.8% of the dose was recovered in urine as unchanged S(+)-HCQ and R(-)-HCQ, respectively. For the first 2 weeks, S(+)-HCQ excretion rate clearly surpassed that of R(-)-HCQ whereas afterwards the inverse was observed. However, since the first 2 weeks account for 95% of rac-HCQ renal excretion, the total urinary excretion of S(+)-HCQ clearly surpassed that of R(-)-HCQ.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Forensic aspects of the metabolism and excretion of cannabinoids following oral ingestion of cannabis resin

Following oral ingestion of cannabis resin, delta 9-THC-11-oic acid and its O-ester glucuronide were detected using RIA and combined hplc/RIA and shown to be major plasma metabolites of delta 9-THC. delta 9-THC-11-oic acid was not excreted in the urine in significant concentrations, the glucuronide conjugate being the major urinary metabolite detected. delta 9-THC metabolites were detected in blood for up to 5 days and in urine for up to 12 days following a single oral dose of delta 9-THC (20 mg). Estimates for the half life of delta 9-THC-11-oic acid and its glucuronide in plasma, and total metabolites in urine have been obtained. Interpretation of blood or urine total cannabinoid levels is most difficult, however, drug/metabolite ratios and metabolite/metabolite ratios may have potential for indicating recent cannabis use.     

Absorption,metabolism and excretion after oral administration of a new Ca antagonist, 14C-benidipine hydrochloride to man

1. In healthy male volunteers, the absorption, metabolite profiles and excretion of Cbenidipine hydrochloride, a new Ca antagonist, were investigated after oral administration at a dose of 8?mg. 2. C-benidipine hydrochloride was rapidly absorbed, and the plasma concentration of radioactivity and unchanged drug reached a maximum of 71 2 ng eq. ml at 1 1?h and 2 56 ng ml at 0 6?h respectively, and then declined bi-exponentially. The half-life in the elimination phase was 14 7 and 5 3?h respectively. AUC of unchanged drug was low, about 1% of that of radioactivity. 3. Five days after administration,36 4% of the administered radioactivity was excreted in urine and 58 9% in faeces. 4. The metabolite profiles in plasma, urine and faeces were analysed by hplc. At 1?h after administration the predominant metabolites in plasma were M9 and M2, which accounted for 13 8 and 8 2% of the radioactivity respectively, whereas unchanged drug represented 1 2%. Predominant metabolites in urine 12?h after administration were M3 andM8,whichaccountedfor2 22and2 21%oftheadministeredradioactivityrespectively. Metabolites excreted in faeces 120?h after administration were very complex and poorly separated by hplc and could not be characterized: unchanged drug was not detected in the faeces.     

Species Variation and Stereoselectivity in the Metabolism of Nicotine Enantiomers

1. High performance liquid radiochromatographic systems have been developed for the identification and quantification of 7 urinary metabolites of both S-(-)-[³H-N'-CH₃]nicotine and R-(+)-[³H-N'-CH₃] nicotine in guinea pig, hamster, rat and rabbit.

2. 3′-Hydroxycotinine was a major urinary metabolite of both S-(-)-nicotine and R-(+)-nicotine in guinea pig, hamster and rabbit. Cotinine was not generally a significant urinary metabolite of either nicotine enantiomer, except in rat, where it constituted 14·6 and 10·4%, respectively, of the total radiolabel in the urine after administration of [³H]-S-(-)-nicotine or [³H]-R-(+)-nicotine. Nicotine N'-oxide was an important urinary metabolite of both nicotine isomers in guinea pig and rat, but in both cases, was not observable in hamster and rabbit. No N-methylated urinary metabolite of S-(-)-nicotine could be detected in any of the species examined. In R-(+)-nicotine experiments, only guinea pig afforded N-methylated metabolites. Significant amounts of 2 unidentified polar, non-basic urinary metabolites of both S-(-)- and R-(+)-nicotine-treated animals were observed.

3. Analysis of the comparative metabolism of the nicotine enantiomers in the four animals species studied, showed that stereoselective differences in the formation of oxidative metabolites existed, particularly in the formation of 3′-hydroxycotinine and nicotine-N'-oxide. A clear stereospecificity was observed in the guinea pig, in that only the R-(+)-nicotine enantiomer was N-methylated in this species.

4. Sex differences appear to exist in the metabolism of nicotine enantiomers in the rat. Female rats excreted more of the unidentified polar metabolite B than male rats, whereas the converse was true for nicotine-N'-oxide. In experiments with R-(+)-nicotine, urinary levels of 3′-hydroxycotinine and R-(+)-nicotine in female rats were higher than in male rats. Conversely, higher amounts of nicotine-N'-oxide were observed in the urine of male rats compared to those in female rats.     

Disposition of the aromatic amine, benzidine, in the rat: characterization of mutagenic urinary and biliary metabolites

The mutagenic and carcinogenic aromatic amine, benzidine (BZ), underwent extensive biotransformation in the rat. Three days after po (5.0 mg/kg) or iv (2.5 mg/kg administration of [14C]BZ, 90% of the radiolabel had been excreted in the urine (25%) and feces (65%); 7% was recovered in the animal. As the dose was increased from 0.5 to 50 mg/kg, the percentage of the dose excreted in urine increased twofold. In distribution studies, a major portion of the iv dose accumulated in the intestinal tract due to the excretion of 71% of the administered radiolabel in bile. The liver, which is a primary target organ of BZ carcinogenicity in rats, contained a higher concentration of radiolabel than other tissues studied. A minimum of 17 urinary and/or biliary metabolites were separated by HPLC. The major metabolites were N-acetyl-BZ(ABZ), N,N'-diacetyl-BZ(DABZ), BZ-N-glucuronide, ABZ-glucuronide, N-OH-DABZ glucuronide, 3-OH-DABZ glucuronide, and a glutathione conjugate of DABZ (3-GSH-DABZ). At low doses (0.5 to 5 mg/kg), 3-OH-DABZ glucuronide, 3-GSH-DABZ, and DABZ were the major urinary or biliary metabolites. However, at higher doses (50 mg/kg), N-OH-DABZ glucuronide, which was a minor metabolite at low doses, became a major urinary and biliary metabolite. Several urinary and biliary metabolites displayed significant mutagenicity in the Salmonella typhimurium (strain TA98)-liver S9-beta-glucuronidase assay. However, N-OH-DABZ glucuronide exhibited a mutagenic potency 10X greater than the other urinary metabolites. Results of these studies demonstrate that BZ is rapidly metabolized via N-acetylation, N-hydroxylation, and aromatic hydroxylation to a variety of mutagenic metabolites which are excreted in urine or bile primarily as glucuronide and/or glutathione conjugates. The most potent mutagen studied was also a major urinary and biliary metabolite.     

In vivo metabolism of isomeric naphthalene oxide glutathione conjugates

We have examined the fate of glutathione conjugates derived from naphthalene metabolism at various dose levels (5-80 mg/kg) in an effort to explore the potential use of urinary mercapturic acids as biomarkers of exposure to naphthalene and as indicators of the activity and stereoselectivity of cytochrome P-450-dependent naphthalene epoxidation. This approach extends previous studies which demonstrated a high degree of stereoselectivity in the formation of (+)-1R,2S-naphthalene oxide from naphthalene in target tissue microsomes (mouse lung), but not in microsomal preparations isolated from nontarget tissues such as mouse liver. To validate the use of mercapturic acids as indicators of epoxide formation in vivo, individual naphthalene oxide glutathione adduct isomers were administered iv to mice, and urinary metabolites were identified and quantified. Mercapturates accounted for 69-75% of the administered dose in the 8-hr urines of animals treated with trans-1-(S)-hydroxy-2-(S)-glutathionyl-1,2-dihydronaphthalene (adduct 1) and 76-84% for trans-1-(R)-hydroxy-2-(R)-glutathionyl-1,2-dihydronaphthalene (adduct 2). Only 39-57% of the dose of trans-1-(R)-glutathionyl-2-(R)-hydroxy-1,2-dihydronaphthalene (adduct 3) administered to mice was excreted as the mercapturic acid derivative; however, two additional metabolites were detected which were not present in the urine of animals treated with adducts 1 or 2. The first metabolite, accounting for 2-4% of the dose of adduct 3, was not identified. The second metabolite, isolated by HPLC and identified by mass spectrometry as (hydroxy-1,2-dihydronaphthalenylthio)pyruvic acid, accounted for 14-25% of the administered dose of adduct 3.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Identification of the metabolites of a new oxazolidinone MAO-A inhibitor in rat

1. Six metabolites present in rat urine after the oral administration of E2011 ((5R)3-[2-((1S)-3-cyano-1-hydroxypropyl)benzothiazol-6-yl]-5-methoxymethyl-2-oxazolidinone) were isolated with an Amberlite XAD-4 column and hplc, and termed HPM-1, HPM-2, HPM-31, HPM-32, HPM-33 and HPM-4. 2. To determine the correspondence of the findings of the metabolites between tlc (which was used in our previous study) and hplc, the six metabolites were isolated from rat urine after the administration of ¹⁴C-labelled E2011 with an Amberlite XAD-4 column and hplc, and then analysed by tlc. HPM-1, HPM-2, HPM-31, HPM-32, HPM-33 and HPM4 were identified as IM7, IM3, IM4, IM2, IM1 and E2011, respectively. 3. The structures of the metabolites were identified with nmr and mass spectrometry. One of the compounds identified, HPM-4, was the unchanged drug, E2011, and HPM-2 was O-desmethyl-E2011. Another metabolite (HPM-33), the main metabolite in the urine, was identified as (4S)-hydroxy-E2011, and the others were (4S)-hydroxy-O-desmethylE2011 (HPM-1), 2′′-hydroxy-E2011 (HPM-31) and (4R)-hydroxy-E2011 (HPM-32). 4. In conclusion, the main metabolic pathway of E2011 in the rat consisted of O-demethylation and hydroxylation.     

Isolation and identification of 3-hydroxy-3-methyloxindole, the major murine metabolite of 3-methylindole

3-Methylindole (3MI) is pneumotoxic to ruminants and rodents subsequent to metabolic oxidative activation by cytochrome P-450 monooxygenases. Goats are much more susceptible than mice and rats to 3MI-mediated lung damage, and these differences in species susceptibility may be reflected by differences in the metabolic products of 3MI. Radioactive 3MI was administered ip to Swiss-Webster mice, and the major nonpolar urinary metabolites were fractionated and separated by HPLC. Although 3-methyloxindole has been shown to be the major urinary metabolite of 3MI in goats, it was not detected in mouse urine. Instead, the major metabolite, 3-hydroxy-3-methyloxindole, was isolated and purified and its structure elucidated by 1H and 13C NMR, mass spectrometry, and IR spectroscopy. This is the first identification of this highly oxidized indole from mammalian sources. The production of this metabolite may be indicative of the formation of an electrophilic methyleneoxindole intermediate, which could be responsible for pneumotoxicity in this species.     

Disposition of the enantiomers of cromakalim in rat and cynomolgus monkey

1. Disposition of the 3R,4S(+) and 3S,4R(-) enantiomers of the racemic antihypertensive drug cromakalim has been studied in rats and cynomolgus monkeys using the ¹⁴C-drug labelled in either the 3R,4S(+) or the 3S,4R(-) enantiomer.

2. After oral administration to rat, blood concentrations of the 3R,4S(+) enantiomer were up to fourfold higher than those of the 3S,4R(-) enantiomer. Metabolism of the former was not as extensive as that of the latter and consequently plasma and urinary radio-metabolite patterns were quantitatively different.

3. In contrast to rat, there were much greater differences in the disposition of the two enantiomers following oral administration of cromakalim to the cynomolgus monkey. Plasma concentrations of the 3R,4S(+) enantiomer were approximately 100 x those of the 3S,4R(-) enantiomer and the rate of urinary ¹⁴C elimination for the 3R,4S(+) enantiomer was much faster than that for the 3S,4R(-) enantiomer. Plasma and urinary radio-metabolite patterns were very different for the two isomers. Metabolic end products of the 3R,4S(+) enantiomer were predominantly phase I metabolites whereas the 3S,4R(-) enantiomer was almost entirely metabolized by glucuronidation.

4. A study of the racemic drug alone would have led to a misunderstanding of the fate of the compound in these species.     

Species differences in beta-oxidative metabolism of a thromboxane A2-receptor antagonist [(+)-S-145] in rat, dog and monkey

1. The formation of beta-oxidized metabolites from (+)-S-145 [(+)-(Z)-7-[(1R, 2S, 3S, 4S)-3-(benzenesulphonamide)bicyclo-[2.2.]-hept-2-yl]-5-heptenoic acid] by liver homogenates were compared between rat, dog and monkey. Species differences were found in hepatic beta-oxidation capacities. The results agree with the qualitative and quantitative differences in beta-oxidized metabolite proportions among these species observed in vivo. 2. The activities of microsomal (+)-S-145-CoA synthesis, the initial step of the beta-oxidation, were determined. Species differences in their intrinsic clearances primarily agreed with those of the beta-oxidized metabolite formation. 3. (+)-S-145-CoA oxidation activities towards (+)-S-145-CoA by liver homogenates were much higher than the beta-oxidized metabolite formation in all species, indicating that formed (+)-S-145-CoA was immediately beta-oxidized in peroxisomes. The species differences were inconsistent with those of beta-oxidized metabolite formation in vitro. 4. Therefore, quantitative differences of hepatic (+)-S-145 beta-oxidation capacity in rat, dog and monkey were considered to be mainly due to the species difference in (+)-S-145-CoA formation.     

Identification of urinary catechol and methylated catechol metabolites of phenytoin in humans, monkeys, and dogs by GLC and GLC-mass spectrometry.

A catechol metabolite, 5-(3,4-dihydroxyphenyl)-5-phenylhydantoin, and a methylated catechol metabolite, 5-(3-methoxy-4-hydroxyphenyl)-5-phenylhydantoin, were identified as urinary metabolites in humans, monkeys, and dogs following the administration of phenytoin. These metabolites were separated from each other and from other known metabolites of phenytoin as n-butyl derivatives by GLC and positively identified by combined GLC-mass spectrometry.     

Comparative metabolic disposition of [1-Me14C]caffeine in rats, mice, and Chinese hamsters

The metabolic disposition of [1-Me14C]caffeine has been studied and compared in three male rodent species: the rat, the mouse, and the Chinese hamster. No interspecies differences appeared in urinary and fecal excretion of radioactivity. However, 1-methyldemethylation was significantly more important in the rat with 20.6 +/- 0.8% of the dose recovered as 14CO2 compared with the Chinese hamster, 16.1 +/- 2%, and the mouse, 13.9 +/- 0.9%. HPLC and TLC analysis of 1-methyl-labeled metabolites showed that the rat exhibits a significantly higher urinary excretion of the four trimethyl derivatives: caffeine, 1,3,7-trimethyluric acid, trimethylallantoin, and 6-amino-5-[N-formylmethylamino]-1,3-dimethyluracil [40.8% of total urine radioactivity) when compared with the Chinese hamster (21.1%) and the mouse (19.7%). Compared with man (6%), these rodents have a greater ability to excrete caffeine without any demethylation. The rat was also characterized by a higher excretion of theophylline while the Chinese hamster excreted more paraxanthine, 1-methylxanthine, and the uracil derivative of paraxanthine. In the mouse, in addition to 1-methylxanthine and 1-methyluric acid, higher amounts of 1,3- and 1,7-dimethyluric acid were found. The mouse was particularly characterized by the presence of an unknown polar metabolite amounting to 22 +/- 3% of urine radioactivity. This metabolite must be produced from paraxanthine because its quantitative formation was inversely related to the excretion of paraxanthine and its metabolites. The observations that this metabolite is neither 5-acetylamino-6-amino-3-methyluracil nor 5-acetylamino-6-formylamino-3-methyluracil reported in humans demonstrate that both quantitative and qualitative interspecies differences occur for caffeine metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Urinary screening for alprazolam, triazolam, and their metabolites with the EMIT d.a.u. benzodiazepine metabolite assay

Alprazolam (Xanax) and triazolam (Halcion) are relatively new triazolobenzodiazepines that are anxiolytic and hypnotic. This study assesses the reactivity of these drugs and their major metabolites in the EMIT d.a.u. benzodiazepine metabolite assay. Analytical standards of drugs and metabolites and urine specimens from patients receiving these drugs were analyzed by EMIT. Alprazolam and alpha-OH alprazolam gave an equivalent response to the EMIT low calibrator between 0.2 and 0.3 microgram/mL. Triazolam and alpha-OH triazolam were reactive between 0.3 and 0.5 microgram/mL. The assay was positive in 24 out of 27 random urine specimens from alprazolam-treated patients and in 8 out of 19 urine specimens from triazolam-treated patients. Positive urine results were confirmed by measuring the major urinary metabolites alpha-OH alprazolam and alpha-OH triazolam by HPLC. The study demonstrates that the EMIT assay can detect significant amounts of alprazolam and metabolites in the urine. The assay was negative in 58% of the specimens from individuals receiving triazolam, however.     

Biotransformation of cyclizine in greyhounds. 1: Identification and analysis of cyclizine and some basic metabolites in canine urine by gas chromatography-mass spectrometry

1. The partial in vivo biotransformation of Marezine [(cyclizine.HCl); 1-diphenylmethyl-4-methylpiperazine hydrochloride] in the racing greyhound and the excretion of the unconjugated and conjugated (Phase II) basic metabolites of cyclizine in canine urine are reported. 2. Using copolymeric bonded mixed-mode solid-phase extraction cartridges, the basic isolates from both unhydrolysed and enzyme hydrolysed urine samples were isolated, derivatized as trimethylsilyl ethers and analysed by positive-ion electron ionization gas chromatography-mass spectrometry (EI(+)-GC-MS). Selected samples were analysed by positive-ion methane chemical ionization (CI(+))-GC-MS to aid structure elucidation of the putative metabolites. 3. Cyclizine was the major component excreted in post-administration urine. Five substrate-related basic compounds (M1--> M5) were tentatively identified by EI(+)- and CI(+)-GC-MS. The major Phase I metabolite was identified as norcyclizine [1-diphenylmethylpiperazine] (M1), the other metabolites (M2 --> M5) were tentatively identified as monohydroxylated products based on MS data. 4. Cyclizine and the N(4)-desmethyl metabolite (M1) are excreted unconjugated; the other four hydroxylated metabolites are excreted as Phase II conjugates (glucuronides and/or sulphates). Structures of the putative basic metabolites are presented. At least four other basic metabolites were also detected in post-administration urine, but could not be characterized from GC-MS data. 5. All unhydrolysed post-administration urine samples were analysed by selected ion monitoring EI(+)-GC-MS to quantify cyclizine and norcyclizine (M1) using authentic cyclizine as the analyte and chlorcyclizine as the internal standard. The level of M1 is expressed as 'cyclizine equivalents'. The duration of urinary elimination of cyclizine and M1 was obtained from their excretion profiles. 6. From these studies, cyclizine and norcyclizine (M1) would be the target compounds of choice in the development of screening and confirmatory methods for the detection of cyclizine administration to racing greyhounds. Information on any of the other metabolites may also be of some value for confirmatory analysis.