Marker drugs for induced drug metabolism in man
Chlorpromazine and ibuprofen were considered as potential marker drugs for measuring induced, cytochrome P-450 mediated drug metabolism in man. The metabolism of a suitable marker drug must be altered in some way as a result of cytochrome P-450 induction. Accordingly the effects of cytochrome P-450 induction on the metabolism of chlorpromazine and ibuprofen was investigated in rats, both in vitro and in vivo. Various fluorescence derivatisation. procedures were compared for the assay of chlorpromazine (CPZ) and its metabolites. An improved assay, involving solvent extraction, thin layer chromatography (TLC) and fluorescence derivatisation, was developed for the study of CPZ metabolism in vitro. The compounds were extracted from microsomal protein suspensions most efficiently into 15% n-propanol in dichloromethane by double extraction at pH2 and 12 successively. CPZ, mono-desmethylCPZ (NOR1CPZ), didesmethy1CPZ (NOR2CPZ), 7-hydroxyCPZ (70HCPZ), CPZ N-oxide (CPZN0), CPZ sulphoxide (CPZS0) and CPZN0 sulphoxide were separated using TLC on silica-gel developed with methanol : acetone : ammonia (50:50:1). The compounds were eluted from the TLC plate with chloroform : methanol (2:1) and subjected to fluorescence derivatisation by oxidation with hydrogen peroxide. The highly fluorescent oxidised derivatives were identified as sulphoxides by comparison of fluorescence characteristics. The amounts of derivatised CPZ and metabolites were measured by quantitative fluorimetry relative to a derivatised internal standard, promazine. The effects of cytochrome P-450 induction on the hepatic microsomal metabolism of CPZ were investigated using male, Sprague-Dawley rats and the TLC-fluorescence assay. The major enzymatic product of microsomes from untreated rats was CPZN0. Both NOR1CPZ and 70HCPZ were also produced but in smaller amounts. No enzymatic production of either NOR2CPZ or CPZS0 was detected. Although pretreatment of rats with 3-methyl-cholanthrene (3MC) was without effect, phenobarbitone (PB) induction produced marked changes in CPZ metabolite profile, whereby the hepatic microsomal productions of both NOR1CPZ and 70HCPZ were increased (3.6- and 2.0-fold respectively) and formation of CPZNO decreased (6.1-fold). Ethanol-pretreatment in vivo slightly induced the hepatic microsomal production of NOR1CPZ, but significantly decreased those of both CPZNO and 7OHCPZ. Normal-phase liquid chromatography, with dichloromethane-alcohol mixtures containing ammonia as the mobile phase, was examined for its applicability to the separation of CPZ and several of its non-conjugated metabolites. The addition of ammonia to the mobile phase was found to be essential in order to achieve separations and to improve peak symmetry. A normal-phase system was developed and found to be suitable for separation of CPZ, NOR1CPZ, NOR2CPZ, 70HCPZ, CPZNO, NOR1CPZ-sulphoxide and NOR2CPZ sulphoxide. Chromatography was performed on a Spherisorb 5-silica column (25cm x 4-mm) with a mobile phase flowing at Iml/min. A linear gradient of changing solvent composition changing from ethanol : dichloromethane : ammonia (15:185:1) to methanol : dichloromethane : ammonia (25:175:1), was used. The normal-phase HPLC system was used to study the effect of PB-pretreatment on the disposition of radiolabelled CPZ in rats in vivo. PB induction enhanced the elimination of total radioactivity from whole blood. Furthermore, although there was no overall difference between control and PB-treated rats in the amounts of total radioactivity excreted in the urine, a significant alteration in the pattern of individual urinary metabolites was found. PB-treated rats excreted greater amounts of both NOR1CPZ and NOR2CPZ but relatively less CPZ and CPZS0, compared to control rats. In an initial study using a small number of animals and [14C]chlorpromazine, pre treatment with PB not only increased the overall excretion of total radioactivity in the faeces, but also specifically enhanced the faecal excretion of 70HCPZ and several unidentified polar metabolites. The effects of cytochrome P-450 induction on the hepatic microsomal metabolism of [14C] ibuprofen in rats were investigated. Two major metabolites and one other, minor metabolite were detected using TLC on silica-gel developed with toluene : acetic acid (9:1) and measured radiometrically. Purification and identification of the metabolites was not undertaken in the absence of reference samples of authentic metabolites. PB-treatment in vivo induced the overall microsomal metabolism of [14C] ibuprofen (2- to 3-fold) and altered the relative abundancies of the three metabolites. Reversed- and normal-phase liquid chromatographic assays were developed for ibuprofen and its microsomal metabolites. Reversed-phase chromatography was performed on a Spherisorb 5mu-ODS (octadecyl silane) column (25cm x 4mm) with a mobile phase flowing at 1.5ml/min. A linear gradient, changing the ratio of solvent (10% methanol in water : methanol) from 25:75 to 75:25 in 32min was used. Normal-phase chromatography was performed on a Partisil 10mu-silica column (25 x 4-mm) with a mobile phase flowing at 2ml/min. A linear gradient, changing the ratio of solvent (tetrahydrofuran, pH 2 with 10N HC1 : cyclohexane) from 1:9 to 1:1 in 20 min was used.