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1. The metabolism of Zaleplon (CL-284,846; ZAL) has been studied in precision-cut human liver slices and liver cytosol preparations. 2. Human liver slices metabolized ZAL to a number of products including 5-oxo-ZAL (M2), N-desethyl-5-oxo-ZAL (M1) and N-desethyl-ZAL (DZAL), the latter metabolite being known to be formed by CYP3A forms. 3. Human liver cytosol preparations catalysed the metabolism of ZAL to M2. Kinetic analysis of three cytosol preparations revealed mean (+/- SEM) K(m) and V(max) of 93 +/- 18 mm and 317 +/- 241 pmol/min/mg protein, respectively. 4. Using 16 individual human liver cytosol preparations a 33-fold variability in the metabolism of 80 micro M ZAL to M2 was observed. Correlations were observed between M2 formation and the metabolism of the aldehyde oxidase substrates phenanthridine (r(2) = 0.774) and phthalazine (r(2) = 0.460). 5. The metabolism of 80 micro M ZAL to M2 in liver cytosol preparations was markedly inhibited by the aldehyde oxidase inhibitors chlorpromazine, promethazine, hydralazine and menadione. Additional kinetic analysis suggested that chlorpromazine and promethazine were non-competitive inhibitors of M2 formation with K(i) of 2.3 and 1.9 micro M, respectively. ZAL metabolism to M2 was also inhibited by cimetidine. 6. Incubations conducted with human liver cytosol and H(2)(18)O demonstrated that the oxygen atom incorporated into ZAL and DZAL to form M2 and M1, respectively, was derived from water and not from molecular oxygen. 7. In summary, by correlation analysis, chemical inhibition and H(2)(18)O incorporation studies, ZAL metabolism to M2 in human liver appears to be catalysed by aldehyde oxidase. With human liver slices, ZAL was metabolized to products dependent on both aldehyde oxidase and CYP3A forms.

1. The effect of cimetidine on the metabolism of zaleplon (ZAL) in human liver subcellular fractions and precision-cut liver slices was investigated. 2. ZAL was metabolized to a number of products including 5-oxo-ZAL (M2), which is known to be formed by aldehyde oxidase, N-desethyl-ZAL (DZAL), which is known to be formed by CYP3A forms, and N-desethyl-5-oxo-ZAL (M1). 3. Human liver microsomes catalysed the NADPH-dependent metabolism of ZAL to DZAL. Kinetic analysis of three microsomal preparations revealed mean (+/-SEM) S(50) and V(max) of 310 +/- 24 micro M and 920 +/- 274 pmol/min/mg protein, respectively. 4. Human liver cytosol preparations catalysed the metabolism of ZAL to M2. Kinetic analysis of three cytosol preparations revealed mean (+/-SEM), K(m) and V(max) of 124 +/- 14 micro M and 564 +/- 143 pmol/min/mg protein, respectively. 5. Cimetidine inhibited ZAL metabolism to DZAL in liver microsomes and to M2 in the liver cytosol. With a ZAL substrate concentration of 62 micro M, the calculated mean (+/-SEM, n = 3) IC50 were 596 +/- 103 and 231 +/- 23 micro M for DZAL and M2 formation, respectively. Kinetic analysis revealed that cimetidine was a competitive inhibitor of M2 formation in liver cytosol with a mean (+/-SEM, n = 3) K(i) of 155 +/- 16 micro M. 6. Freshly cut human liver slices metabolized ZAL to a number of products including 1, M2 and DZAL. 7. Cimetidine inhibited ZAL metabolism in liver slices to M1 and M2, but not to DZAL. Kinetic analysis revealed that cimetidine was a competitive inhibitor of M2 formation in liver slices with an average (n = 2 preparations) K(i) of 506 micro M. 8. The results demonstrate that cimetidine can inhibit both the CYP3A and aldehyde oxidase pathways of ZAL metabolism in the human liver. Cimetidine appears to be a more potent inhibitor of aldehyde oxidase than of CYP3A forms and hence in vivo is likely to have a more marked effect on ZAL metabolism to M2 than on DZAL formation. 9. The results also demonstrate that precision-cut liver slices may be a useful model system for in vitro drug-interaction studies.

1. The aim was to employ real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) technology (TaqMan to examine the induction of some selected cytochrome P450 (CYP) forms in precision-cut rat liver slices. 2. Taqman primers and probe sets were developed for rat CYP1A1, CYP1A2, CYP2B1 and CYP4A1 forms. 3. Rat liver slices were cultured in control medium or medium containing either 10 micro g ml(-1) Aroclor 1254 (ARO), 500 micro M sodium phenobarbitone (NaPB) or 50 micro M Wy-14643 (WY) for 3, 6 and 24 h. 4. Compared with control liver slices, treatment with ARO for 3 and 6 h produced 24- and 184-fold increases, respectively, in CYP1A1 mRNA levels, and after 24h produced an 85-fold increase in CYP1A2 mRNA levels. Levels of CYP1A1 and CYP1A2 mRNA were not markedly affected by NaPB and WY. 5. Treatment with ARO and PB for 24 h produced 10.6- and 23.8-fold increases, respectively, in CYP2B1 mRNA. Levels of CYP2B1 mRNA were not markedly affected by WY. 6. Treatment with WY, but not ARO and NaPB, for 24h produced a 20.4-fold increase in levels of CYP4A1 mRNA. 7. These results demonstrate that cultured liver slices may be used to evaluate the effect of xenobiotics on CYP form mRNA levels.

A LC-LC/MS/MS method has been developed that significantly increases the throughput in metabolism screening of drug candidates during lead optimization in discovery. This was accomplished by the reduction of sample preparation time through an on-line extraction of a drug and its metabolites from microsomal proteins using turbulent flow chromatography. Following its injection onto a column at turbulent flow, the drug and its metabolites are backwashed onto a reverse-phase column via on-line column switching and resolved chromatographically at a laminar flow of 2 mL/min. This tandem turbulent-laminar flow chromatographic system in a total cycle time of 8 min can achieve adequate separation of isomeric metabolites of venlafaxine, haloperidol, or adatanserin. Further improvement in throughput can be achieved by multiplexing both microsomal stability assessment and metabolite profiling into a single analysis. This is made possible by the ability of the ion-trap mass spectrometer to perform simultaneously multiple-reaction monitoring for microsomal stability and data-dependent multiple-stage mass spectrometric analysis for metabolite profiling within a single LC analysis. Such a LC-LC/MS/MS approach can dramatically shorten the time for providing metabolism feedback to the drug discovery process.

The metabolism of equilin sulfate was determined in female dogs receiving 2.5 mg/kg of [3H]equilin sulfate alone or in a preparation that contained all the components that are present in the conjugated equine estrogen product Premarin. The pharmacokinetic parameters of total radioactivity indicated that the drug is rapidly absorbed and it has a moderate half-life in plasma. The total radioactivity in plasma following administration of [3H]equilin sulfate as part of a mixture of conjugated equine estrogens had significantly lower peak concentration (Cmax), a lower area under the curve (AUC), a longer terminal half-life (t1/2) and a longer mean residence time (MRT) than when [3H]equilin sulfate was given alone, indicating that the other components in the conjugated equine estrogen preparation altered the pharmacokinetics of equilin sulfate. An average of 26.7 +/- 4.4% of the administered radioactive dose was excreted in urine of dogs receiving [3H]equilin sulfate. Again, a significantly lower percentage (21.4 +/- 6.3%, P = 0.023) was eliminated in urine of dogs receiving [3H]equilin sulfate in the conjugated equine estrogen preparation, indicating that the absorption of equilin sulfate was perhaps altered by the other components in the conjugated equine estrogen preparation. Metabolite profiles of plasma and urine were similar. Equilin, equilenin, 17 beta-dihydroequilenin, 17 beta-dihydroequilin, 17 alpha-dihydroequilenin and 17 alpha-dihydroequilin were present in both matrices. 17 beta-Dihydroequilin and equilin were the two major chromatographic peaks in plasma samples. 17 beta-Dihydroequilenin and 17 beta-dihydroequilin were the major metabolites in urine. In conclusion, following oral administration of [3H]equilin sulfate to dogs, the radioactivity is rapidly absorbed. The disposition of equilin sulfate is altered by the other components that are present in the conjugated equine estrogen preparation Premarin. The reduction of the 17-keto group and aromatization of ring-B are the major metabolic pathways of equilin in the dog.

In vitro models are being used increasingly during all phases of the drug development process in concert with the more traditional in vivo toxicological and pharmacokinetic evaluations. These in vitro models may be classified empirically as either validated in vitro screens, value-added screens or 'ad-hoc' mechanistic screens. The application of these screens is discussed with respect to their level of validation, standardization, uses of human tissue, level of iteration with in vivo studies, regulatory position and utility in the drug discovery and development process. The predictability and reproducibility of these screens is discussed, as well as future trends in regard to emerging technology and its application.

1. The pharmacokinetics of venlafaxine have been evaluated in mouse, rat, dog and rhesus monkey after i.v. and/or i.g. doses of venlafaxine from 2 to 120 mg/kg either as single or repeated doses. 2. In rat, dog and monkey, venlafaxine is a high clearance compound with a large volume of distribution after i.v. administration. 3. Absolute bioavailability was low in rat and rhesus monkey (12.6 and 6.5%, respectively) and moderate in dog (59.8%). Other species differences were seen, including an elimination half-life of venlafaxine that was longer in dog and rhesus monkey (2-4 h) than in rodent (around 1 h). 4. In mouse, rat and dog, exposure to venlafaxine increased more than proportionally with dose, suggesting saturation of elimination. Exposure of venlafaxine decreased with repeated dosing in mouse and rat, but was unchanged in dog. 5. Exposure of animals to the bioactive metabolite, O-desmethylvenlafaxine (ODV), was less than that of venlafaxine itself. ODV was not detected in dog and not measurable in rhesus monkey receiving venlafaxine.

1. The metabolic disposition of venlafaxine has been studied in mouse, rat, dog, rhesus monkey and man after oral doses (22, 22, 2, and 10 mg/kg, and 50 mg, respectively) of 14C-venlafaxine as the hydrochloride. 2. In all species, over 85% of the administered radioactivity was recovered in the urine within 72 h, indicating extensive absorption from the GI tract and renal excretion. 3. Venlafaxine was extensively metabolized, with only 13.0, 1.8, 7.9, 0.3 and 4.7% dose appearing as parent compound in urine of mouse, rat, dog, monkey and man, respectively. The metabolite profile varied significantly among species, but primary metabolic reactions were demethylations and the conjugation of phase I metabolites. Hydroxylation of the cyclohexyl ring also occurred in mouse, rat and monkey, and a cyclic product was formed in rat and monkey. Glucuronidation was the primary conjugation reaction, although sulphate conjugates were also detected in mouse urine. 4. While no metabolite constituted more than 20% dose in any species except man, the major urinary metabolites were: mouse, N,O-didesmethyl-venlafaxine glucuronide; rat, cis-1,4-dihydroxy-venlafaxine; dog, O-desmethyl-venlafaxine glucuronide; monkey, N,N,O-tridesmethyl-venlafaxine; and man, O-desmethyl-venlafaxine.

1. The excretion and metabolism of enciprazine, an anxiolytic drug, was examined in rat, dog and man. 2. In rats and dogs that received 14C-enciprazine dihydrochloride orally and by i.v. injection, the drug was well absorbed. Radioactivity was excreted predominantly in the faeces of rats, equally in urine and faeces of dogs, and to a major extent in human urine. 3. Metabolic profiles, which were evaluated in urine and in rat bile, were similar following oral and i.v. dosing to rats and dogs. 4. Unchanged drug was not detected in rat, dog or human excreta. Glucuronide conjugates of 4-hydroxyenciprazine, m-desmethylenciprazine, p-desmethylenciprazine and enciprazine were detected in the excreta of all three species. A glycol metabolite was present only in rat bile and human urine. A metabolite desmethylated in the phenyl ring of the phenylpiperazine moiety also appeared to be present only in human urine. 5. Structural confirmation of the major metabolites in human urine and rat bile was accomplished by h.p.l.c.-mass spectrometry.

The metabolic disposition of recainam, an antiarrhythmic drug, was compared in mice, rats, dogs, rhesus monkeys, and humans. Following oral administration of [14C]recainam-HCl, radioactivity was excreted predominantly in the urine of all species except the rat. Metabolite profiles were determined in excreta by HPLC comparisons with synthetic standards. In rodents and rhesus monkeys, urinary excretion of unchanged recainam accounted for 23-36% of the iv dose and 3-7% of the oral dose. Aside from quantitative differences attributable to presystemic biotransformation, metabolite profiles were qualitatively similar following oral or iv administration to rodents and rhesus monkeys. Recainam was extensively metabolized in all species except humans. In human subjects, 84% of the urinary radioactivity corresponded to parent drug. The major metabolites in mouse and rat urine and rat feces were m- and p-hydroxyrecainam. Desisopropylrecainam and dimethylphenylaminocarboxylamino propionic acid were the predominant metabolites in dog and rhesus monkey urine. Small amounts of desisopropylrecainam and p-hydroxyrecainam were excreted in human urine. Selective enzymatic hydrolysis revealed that the hydroxylated metabolites were conjugated to varying degrees among species. Conjugated metabolites were not present in rat urine or feces, while conjugates were detected in mouse, dog, and monkey urine. Structural confirmation of the dog urinary metabolites was accomplished by mass spectral analysis. The low extent of metabolism of recainam in humans suggests that there will not be wide variations between dose and plasma concentrations.

The pharmacokinetics of recainam, an anti-arrhythmic drug, were compared in mice, rats, rabbits, dogs, rhesus monkeys, and man. Bioavailability was virtually complete in monkeys and dogs, 67 per cent in man and 51 per cent in rats. Non-linear kinetics between the oral and i.v. dose in rabbits precluded estimation of bioavailability. Linear plasma dose proportionality occurred in dogs between 6 and 60 mg kg-1 oral doses and rhesus monkeys between 1 and 15 mg kg-1 i.v. doses. A greater than proportional increase in the plasma AUC of recainam occurred between oral doses ranging from 54-208 mg kg-1 in mice, 25-110 mg kg-1 in rats, and 50-100 mg kg-1 in rabbits. In human subjects, the AUC/unit dose was linear between 400 and 800 mg. The terminal elimination t1/2 of recainam ranged from 1-5h in laboratory animals and man. The plasma Cmax and AUC of recainam were virtually identical after single or multiple (21 day) oral doses in dogs. After an i.v. dose, plasma clearance of recainam (l kg-1 .h) was 4.9-5.2 in rats and rabbits and 0.4-1.9 in dogs, rhesus monkeys, and man. The steady state volume of distribution was 2-5 times larger than the total body water of laboratory animals and man. Recainam was very poorly bound (10-45 per cent) to the serum proteins of rodents, rabbits, dogs, rhesus monkeys and man. In rhesus monkeys and man, recainam accounted for 10 per cent and 70 per cent, respectively, of the plasma radioactivity at 6 h post-dose. The pharmacokinetic profile of recainam in dogs most closely resembled that of man.

The metabolic disposition of pelrinone, a cardiotonic drug, was studied in mouse, rat, rabbit, dog, monkey and man. Pelrinone was rapidly and extensively absorbed in rodents, dogs, monkeys and man. Except in rabbits, the major portion of the serum radioactivity was due to parent drug. Pelrinone was moderately bound to human serum proteins and weakly bound to serum proteins from animals. Radioactive compounds were rapidly eliminated from rat tissues with the highest concentrations found in organs associated with absorption and elimination. After a 1.0 mg/kg i.v. dose, the rapid elimination of pelrinone from mouse, rat and dog serum precluded estimation of an elimination half life (t1/2). However, after higher oral or i.v. doses, a more prolonged elimination phase was apparent and the t1/2 of pelrinone ranged from 8-10 h in rodents and dogs. In human subjects given escalating oral or i.v. doses of pelrinone, the elimination t1/2 was independent of dose and averaged 1-2 h. The serum AUC of pelrinone was linearly dose-related following oral doses up to 20 mg/kg in dogs and 100 mg in man. In mice, a greater proportional increase in AUC occurred between oral doses of 2-100 mg/kg while in rats, the serum AUC increased in less than proportional manner from 10-200 mg/kg p.o. In all species, radioactive compounds were excreted mainly in the urine. No metabolites were detected in dog and human urine while small amounts of unconjugated metabolites were excreted in mouse and rat urine.

1. The disposition of AY-30,068 (I), a new tetrahydrocarbazole analgesic drug, was studied in mice, rats, dogs, rhesus monkeys, and man. 2. Oral doses of the 14C-labelled drug in aqueous solution were well absorbed in rodents, but absorption of oral doses of the crystalline drug in dogs was poor. Due to the virtual absence of serum metabolites in rats and dogs, the bioavailability of I was nearly identical to the extent of absorption. Although a small first-pass effect was observed in mice, unchanged I represented a major portion of serum radioactivity. 3. A linear increase in the serum concentrations of I occurred at doses between 0.05 and 25 mg/kg in rats, 0.1 and 50 mg/kg in dogs, and 1-160 mg in man. In rhesus monkeys given a 0.5 mg/kg oral dose, the Cmax and AUC of I were similar to values obtained following a corresponding dose in dogs. 4. After i.v. administration of a 1.0 mg/kg dose the terminal elimination half-life (t1/2 beta) of I was 4 h in mice and 9-10 h in rats and dogs. In rodents, dogs, and several human subjects, the elimination of I was interrupted by secondary peaks. Enterohepatic circulation was confirmed in bile duct cannulated rats, where the t1/2 beta of I was decreased to 2.4 h. In rodents the serum clearance and apparent volume of distribution of I were 0.04-0.2 l/kg.h and 0.5-0.8 l/kg, respectively, and 0.6 l/kg.h and 9.8 l/kg in dogs. 5. In rodents and dogs dosed with 14C-labelled I, radioactivity was excreted almost entirely in the faeces. No unchanged I was detected in rat bile, while about 70% of the radioactivity corresponded to conjugates of parent drug.