CHARACTERIZATION OF THE SELECTIVITY AND MECHANISM OF HUMAN CYTOCHROME-P450 INHIBITION BY THE HUMAN IMMUNODEFICIENCY VIRUS-PROTEASE INHIBITOR NELFINAVIR MESYLATE

In vitro studies with human liver microsomes and P450 probe substrates were performed to characterize selectivity and mechanism of cytochrom e P450 inhibition by nelfinavir mesylate. At therapeutic concentration s (steady-state plasma concentrations approximate to 4 mu M), nelfinav ir was found to be a competitive inhibitor of only testosterone 6 beta -hydroxylase (CYP3A4) with a K-i concentration of 4.8 mu M. At suprath erapeutic concentrations, nelfinavir competitively inhibited dextromet horphan O-demethylase (CYP2D6), S-mephenytoin 4-hydroxylase (CYP2C19), and phenacetin O-deethylase (CYP1A2) with K-i concentrations of 68, 1 26, and 190 mu M, respectively. Nelfinavir did not appreciably inhibit tolbutamide 4-hydroxylase (CYP2C9), paclitaxel 6 alpha-hydroxylase (C YP2C8), or chlorzoxaxone 6 beta-hydroxylase (CYP2E1) activities. The i nhibitory potency of nelfinavir toward CYP3A4 suggested the possibilit y of in vivo inhibition of this isoform, whereas in vivo inhibition of other P450s was considered unlikely. In a one-sequence crossover stud y in 12 healthy volunteers, nelfinavir inhibited the elimination of th e CYP3A substrate terfenadine and the carboxylate metabolite of terfen adine. The 24-hr urinary recoveries of 6 beta-hydroxycortisol were red uced by an average of 27% during nelfinavir treatment, consistent with CYP3A inhibition by nelfinavir. Inhibition of CYP3A4 by nelfinavir in vitro was NADPH-dependent requiring the catalytic formation of a meta bolite or a metabolic intermediate. The catechol metabolite of nelfina vir (M3) was considered unlikely to be responsible for inhibition as t he addition of catechol O-methyl transferase, S-adenosyl methionine, a nd ascorbic acid to the preincubation mixture did not protect against the loss of testosterone 6 beta-hydroxylase activity. Also, the additi on of M3 to human liver microsomes did not inhibit CYP3A4. Although in cubations with nelfinavir showed a time- and concentration-dependent l oss of CYP3A4 activity, the partial or complete recovery of enzyme act ivity upon dialysis indicated that inhibition was reversible. Microsom al incubations with nelfinavir and NADPH did not result in a loss of s pectral P450 content compared with the NADPH control. Glutathione, N-a cetylcysteine, and catalase did not attenuate CYP3A4 inhibition by nel finavir. Collectively, these results suggest that the probable mechani sm for CYP3A4 inhibition by nelfinavir is a transient metabolic interm ediate or stable metabolite that coordinates tightly but reversibly to the heme moiety of the P450.