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 investigate 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 using human liver microsomes, as well as microsomes expressing individual human CYP enzymes. Further studies to identify the individual enzymes were performed at 2.5 and 250 µ m sildenafil, and employed a combination of chemical inhibition, correlation analysis, and metabolism by expressed recombinant CYP enzymes. In addition, the effect of sildenafil on the activity of the 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 m 1  = 6(±3 µ m ), K m 2  = 81(±45 µ m ), V max1  = 22(±9 pmol) and V max2  = 138(±77 pmol) UK‐103 320 formed min −1 mg −1 . At 250 µ m sildenafil, N ‐demethylation was primarily mediated through the low‐affinity, high‐ K m enzyme (approximately 83%), whilst at 2.5 µ m there was a greater role for the high‐affinity, low‐ K m enzyme (approximately 61%). Ketoconazole strongly inhibited metabolism at both sildenafil concentrations and was the only significant inhibitor at 250 µ m sildenafil. At the lower sildenafil concentration, sulphaphenazole and quinidine also inhibited formation of UK‐103 320. Overall, 75% or more of the N ‐demethylation of sildenafil at any concentration is probably attributable to CYP3A4. These results were supported by experiments using expressed human CYP enzymes, in which only CYP3A4 and CYP2C9 exhibited substantial sildenafil N ‐demethylase activity (respective K m values of 221 µ m and 27 µ m ). Sildenafil metabolism was inhibited by potent CYP3A4 inhibitors which are used clinically, but was found to be only a weak inhibitor of drug metabolizing enzymes itself, the strongest inhibition occurring against CYP2C9 ( K i  = 80 µ m ). Conclusions  Evidence is provided for CYP3A4 and to a lesser extent CYP2C9‐mediated metabolism of sildenafil. There is the possibility that elevated plasma concentrations of sildenafil could occur with coadministration of known inhibitors of CYP2C9 or CYP3A4. Since peak plasma concentrations of clinical doses of sildenafil are only 200 ng ml −1 (∼0.4 µ m ) it is very unlikely that sildenafil will significantly alter the plasma concentration of other compounds metabolized by cytochrome P450 enzymes.