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 effect of rifampicin on cytochrome P450 isoforms in the CYP1A and CYP3A subfamilies has been studied in 72-h cultured precision-cut human liver slices. 2. In cultured human liver slices 50 microM rifampicin induced testosterone 6 beta-hydroxylase activity, but had no effect on 7-ethoxyresorufin O-deethylase and 7-methoxyresorufin O-demethylase activities. 3. Western immunoblotting of liver slice microsomes was performed with antibodies to rat CYP1A2 and human CYP3A4. Compared with control (dimethyl sulphoxide only treated) liver slice microsomes, rifampicin increased levels of CYP3A4 but had no effect on CYP1A2. 4. These results demonstrate that rifampicin induces CYP3A isoforms, but not CYP1A2, in cultured human liver slices. Some variability in the magnitude of induction by rifampicin was observed in the six human liver samples examined. 5. These results demonstrate that cultured human liver slices may be used to evaluate the effects of xenobiotics on CYP3A isoforms.

1. The metabolism of Zaleplon (CL-284,846; ZAL) has been studied in human liver microsomal preparations and in cDNA-expressed human cytochrome P450 (CYP) isoforms. 2. Human liver microsomes catalysed the NADPH-dependent N-deethylation of ZAL to DZAL (CL-284,859), but not to two other known in vivo metabolites, namely M1 (CL-345,644) and M2 (CL-345,905). Sigmoidal kinetic plots were observed for ZAL deethylation indicating positive cooperativity. 3. The metabolism of ZAL to DZAL was determined in a characterized bank of 24 human liver microsomal preparations. Good correlations (r2 = 0.734-0.937) were observed with caffeine 8-hydroxylase, diazepam 3-hydroxylase, dextromethorphan N-demethylase and testosterone 2 beta-, 6 beta- and 15 beta-hydroxylase activities, which are all catalysed by CYP3A isoforms. In contrast, poor correlations (r2 = 0.152-0.428) were observed for enzymatic markers for CYP1A2, CYP2A6, CYP2C9/10, CYP2D6, CYP2E1 and CYP4A9/11. 4. The metabolism of ZAL to DZAL in human liver microsomes was inhibited to 6-15% of control by 5-50 microM of the mechanism-based CYP3A inhibitor troleandomycin. Whereas some inhibition of DZAL formation was observed in the presence of 200 microM diethyldithiocarbamate, 5-50 microM furafylline, 2-20 microM sulphaphenazole, 50-500 microM S-mephenytoin and 1-10 microM quinidine had little effect. 5. Using human B-lymphoblastoid cell microsomes containing cDNA-expressed CYP isoforms, ZAL was metabolized to DZAL by CYP3A4, hut not to any great extent by CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1. 6. In contrast with ZAL, the NADPH-dependent N-deethylation of M2 to M1 proceeded at only a very low rate with both human liver microsomes and cDNA-expressed CYP3A4. 7. In summary, by correlation analysis, chemical inhibition studies and the use of cDNA-expressed CYPs, ZAL N-deethylation to DZAL in human liver appears to be catalysed by CYP3A isoforms.

1. Following an oral dose of [14C]phenol (12.5 or 25 mg/kg) to sheep, pig and rat, urinary elimination of radioactivity was rapid, 80-90% dose being excreted in the first 8 h. 2. In anaesthetized, ureter-cannulated rats, 70-80% of an intraduodenal dose was eliminated in 2 h; 2% dose was excreted as phenol conjugates in the urine within 10 min. 3. The major urinary metabolites from phenol (25 mg/kg) were phenylglucuronide and phenylsulphate. In the sheep, pig and rat, the glucuronide accounted for 49%, 83% and 42% respectively, of the total urinary metabolites and sulphate accounted for 32%, 1% and 55%. Conjugates of quinol were minor urinary metabolites (less than 7%) in all three species. 4. In sheep some 12% of the urinary metabolites was conjugated with phosphate; this metabolite was not found in rat or pig.