CORTISOL METABOLISM IN-VITRO .3. INHIBITION OF MICROSOMAL 6-BETA-HYDROXYLASE AND CYTOSOLIC 4-ENE-REDUCTASE

The in vitro metabolism of cortisol in human liver fractions is highly complex and variable. Cytosolic metabolism proceeds predominantly via A-ring reduction (to give 3 alpha,5 beta-tetrahydrocortisol; 3 alpha,5 beta-THF), while microsomal incubations generate upto 7 metabolites, including 6 beta-hydroxycortisol (6 beta-OHF), and 6 beta-hydroxycortisone (6 beta-OHE), products of the cytochrome P450 (CYP) 3A subfamily. The aim of the present study was, therefore, to examine two of the main enzymes involved in cortisol metabolism namely, microsomal 6 beta-hydroxylase and cytosolic 4-ene-reductase. In particular, we wished to assess the substrate specificity of these enzymes and identify compounds with inhibitory potential. Incubations for 30 min containing [H-3]cortisol, potential inhibitors, microsomal or cytosolic protein (3 mg), and co-factors were followed by radiometric HPLC analysis. The K-m value for 6 beta-OHF and 6 beta-OHE formation was 15.2+/-2.1 mu M (mean+/-SD; n=4) and the V-max value 6.43+/-0.45 pmol/min/mg microsomal protein. The most potent inhibitor of cortisol 6 beta-hydroxylase was ketoconazole (K-i=0.9+/-0.4 mu M; n=4), followed by gestodene (K-i=5.6+/-0.6 mu M) and cyclosporine (K-i=6.8+/-1.4 mu M). Both betamethasone and dexamethasone produced some inhibition (K-i=31.3 and 54.5 mu M, respectively). However, substrates for CYP2C (tolbutamide), CYP2D (quinidine), and CYP1A (theophylline) were essentially non-inhibitory. The K-m value for cortisol 4-ene-reductase was 26.5+/-11.2 mu M (n=4) and the V-max value 107.7+/-46.0 pmol/min/mg cytosolic protein. The most potent inhibitors were androstendione (K-i=17.8+/-3.3 mu M) and gestodene (K-i=23.8+/-3.8 mu M). Although both compounds have identical A-rings to cortisol, and undergo reduction, inhibition was non-competitive.