Cortisol metabolism in vitro—III. Inhibition of microsomal 6β—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 3,5β-tetrahydrocortisol; 3,5β-THF), while microsomal incubations generate upto 7 metabolites, including 6β-hydroxycortisol (6β-OHF), and 6β-hydroxycortisone (6β-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 6β-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 ³H]cortisol, potential inhibitors, microsomal or cytosolic protein (3 mg), and co-factors were followed by radiometric HPLC analysis. The K_m value for 6β-OHF and 6β-OHE formation was 15.2 ± 2.1 μM (mean ± SD; n = 4) and the V_max value 6.43 ± 0.45 pmol/min/mg microsomal protein. The most potent inhibitor of cortisol 6β-hydroxylase was ketoconazole (K_i = 0.9 ± 0.4 μM; N = 4), followed by gestodene (K_i = 5.6 ± 0.6 μM) and cyclosporine (K_i = 6.8 ± 1.4 μM). Both betamethasone and dexamethasone produced some inhibition (K_i = 31.3 and 54.5 μ, 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 μM (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 μM) and gestodene (K_i = 23.8 ± 3.8 μM). Although both compounds have identical A-rings to cortisol, and undergo reduction, inhibition was non-competitive.