Dapsone activation of CYP2C9-mediated metabolism: Evidence for activation of multiple substrates and a two-site model

Dapsone activates CYP2C9-mediated metabolism in various expression systems and is itself metabolized by CYP2C9 to its hydroxylamine metabolite. Studie s were conducted with expressed CYP2C9 to characterize the kinetic effects of dapsone (0-100 muM) on (S)-flurbiprofen (2-300 muM), (S)-naproxen (10-18 00 muM), and piroxicam (5-900 muM) metabolism in 6 x 6 matrix design experi ments. The influence of (S)-flurbiprofen on dapsone hydroxylamine formation was also studied. Dapsone increased the Michaelis-Menten-derived V-max of flurbiprofen 4'-hydroxylation from 12.6 to 20.6 pmol/min/pmol P450, and low ered its K-m from 28.9 to 10.0 muM, suggesting that dapsone activates CYP2C 9-mediated flurbiprofen metabolism without displacing flurbiprofen from the active site, supporting a two-site model describing activation. Similar re sults were observed with piroxicam 5'-hydroxylation, as V-max was increased from 0.08 to 0.20 pmol/min/pmol P450 and K-m was decreased from 183 to 50 muM in the presence of dapsone. In addition, the kinetic profile for naprox en was converted from biphasic to hyperbolic in the presence of dapsone, wh ile exhibiting similar decreases in K-m and increases in V-max. Kinetic par ameters were also estimated using the two-site binding equation, with cu va lues <1 and <beta> values >1, indicative of activation. Additionally, dapso ne hydroxylamine formation was measured from incubations containing flurbip rofen, exhibiting a kinetic profile that was minimally affected by the pres ence of flurbiprofen. Overall, these results suggest that dapsone activates the metabolism of multiple substrates of CYP2C9 by binding within the acti ve site and causing positive cooperativity, thus lending further support to a two-site binding model of P450-mediated metabolism.