Identification of the cytochrome P450 enzymes involved in the N-demethylation of sildenafil

Aims To characterize the cytochrome P450 (CYP) enzymes responsible for the N-demethylation of sildenafil to its main metabolite. UK-103 320, to invest igate the potential inhibitory effects of sildenafil on CYP enzymes and to evaluate the potential of selected drugs to affect sildenafil metabolism. Methods The metabolic pathways of sildenafil N-demethylation were studied u sing human liver microsomes, as well as microsomes expressing individual hu man CYP enzymes. Further studies to identify the individual enzymes were pe rformed at 2.5 and 250 muM sildenafil, and employed a combination of chemic al inhibition, correlation analysis, and metabolism by expressed recombinan t CYP enzymes. In addition, the effect of sildenafil oil the activity of th e six major drug metabolizing enzymes was investigated, Results Sildenafil conversion was found to be mediated by at least two CYP enzymes, for which the mean kinetic parameters were K-m1 =6(+/-3 muM), K-m2 =81(+/-45 muM), V-max1=22(+/-9) pmol) and V-max2= 138(+/-77 pmol) UK-103 32 0 formed min(-1) mg(-1). At 250 muM sildenafil, N-demethylation was primari ly mediated through the low-affinity, high-K-m enzyme (approximately 83%), whilst at 3.5 muM there was a greater role for the high-affinity, low-K-m e nzyme (approximately 61%). Ketoconazole strongly inhibited metabolism at bo th sildenafil concentrations and was the only significant inhibitor at 250 muM sildenafil. At the lower sildenafil concentration, sulphaphenazole and quinidine also inhibited formation of UK-103 320. Overall, 75% or more of t he N-demethylation of sildenafil at any concentration is probably attributa ble to CYP3A4. These results were supported by experiments using expressed human CYP enzymes, in which only CYP3A4 and CYP2C9 exhibited substantial si ldenafil N-demethylase activity (respective K-m values of 221 muM and 27 mu M). Sildenafil metabolism was inhibited by potent CYP3A4 inhibitors which a re used clinically, but was found to be only a weak inhibitor of drug metab olizing enzymes itself the strongest inhibition occurring against CYP2C9 (K -i = 80 muM). Conclusions Evidence is provided for CYP3A4 and to a lesser extent CYP2C9-m ediated metabolism of sildenafil. There is the possibility that elevated pl asma concentrations of sildenafil could occur with coadministration of know n inhibitors of CYP2C9 or CYP3A4. Since peak plasma concentrations of clini cal doses of sildenafil are only 200 ng ml(-1) (similar to0.4 muM) it is ve ry unlikely that sildenafil will significantly alter the plasma concentrati on of other compounds metabolized by cytochrome P450 enzymes.