Activation of cytochrome P450 2C9-mediated metabolism: mechanistic evidence in support of kinetic observations

Abstract

Studies were designed to investigate the possible mechanisms associated with the kinetic observation of CYP2C9 activation by dapsone and its phase I metabolite, N-hydroxydapsone. Kinetic studies suggested that dapsone activated CYP2C9-mediated flurbiprofen 4(')-hydroxylation by decreasing the K(m) (alpha=0.2) and increasing the V(max) (beta=1.9). Interestingly, N-hydroxydapsone also activated flurbiprofen 4(')-hydroxylation by increasing V(max) (beta=1.5) but had no effect on K(m) (alpha=0.98). To study the effects of these modulators on the binding affinity of flurbiprofen, spectral binding studies were performed. In the presence of dapsone, the spectral binding constant (K(s)) for flurbiprofen was reduced from 14.1 to 2.1 microM, while in the presence of N-hydroxydapsone, the K(s) remained unchanged (14.0 microM), which suggests that dapsone causes an increase in the affinity of flurbiprofen for CYP2C9, whereas N-hydroxydapsone does not. Additionally, stoichiometry measurements under activation conditions in the presence of dapsone resulted in a doubling of both NADPH and oxygen consumption for flurbiprofen 4(')-hydroxylation, with an overall increase in metabolite formation and a decrease in formation of peroxide and excess water. Interestingly, the presence of N-hydroxydapsone generally caused the same effects on stoichiometry as those of flurbiprofen 4(')-hydroxylation but failed to reduce excess water formation, which suggests that, while N-hydroxydapsone activates CYP2C9, it does so less efficiently and possibly through a mechanism different from that of dapsone.