title: Characterization of the selectivity and mechanism of human cytochrome P450 inhibition by the human immunodeficiency virus-protease inhibitor nelfinavir mesylate.
abstract: In vitro studies with human liver microsomes and P450 probe substrates were performed to characterize selectivity and mechanism of cytochrome P450 inhibition by nelfinavir mesylate. At therapeutic concentrations (steady-state plasma concentrations approximately 4 microM), nelfinavir was found to be a competitive inhibitor of only testosterone 6beta-hydroxylase (CYP3A4) with a Ki concentration of 4. 8 microM. At supratherapeutic concentrations, nelfinavir competitively inhibited dextromethorphan O-demethylase (CYP2D6), S-mephenytoin 4-hydroxylase (CYP2C19), and phenacetin O-deethylase (CYP1A2) with Ki concentrations of 68, 126, and 190 microM, respectively. Nelfinavir did not appreciably inhibit tolbutamide 4-hydroxylase (CYP2C9), paclitaxel 6alpha-hydroxylase (CYP2C8), or chlorzoxaxone 6beta-hydroxylase (CYP2E1) activities. The inhibitory potency of nelfinavir toward CYP3A4 suggested the possibility of in vivo inhibition of this isoform, whereas in vivo inhibition of other P450s was considered unlikely. In a one-sequence crossover study in 12 healthy volunteers, nelfinavir inhibited the elimination of the CYP3A substrate terfenadine and the carboxylate metabolite of terfenadine. The 24-hr urinary recoveries of 6beta-hydroxycortisol were reduced 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 metabolite or a metabolic intermediate. The catechol metabolite of nelfinavir (M3) was considered unlikely to be responsible for inhibition as the addition of catechol O-methyl transferase, S-adenosyl methionine, and ascorbic acid to the preincubation mixture did not protect against the loss of testosterone 6beta-hydroxylase activity. Also, the addition of M3 to human liver microsomes did not inhibit CYP3A4. Although incubations with nelfinavir showed a time- and concentration-dependent loss of CYP3A4 activity, the partial or complete recovery of enzyme activity upon dialysis indicated that inhibition was reversible. Microsomal incubations with nelfinavir and NADPH did not result in a loss of spectral P450 content compared with the NADPH control. Glutathione, N-acetylcysteine, and catalase did not attenuate CYP3A4 inhibition by nelfinavir. Collectively, these results suggest that the probable mechanism for CYP3A4 inhibition by nelfinavir is a transient metabolic intermediate or stable metabolite that coordinates tightly but reversibly to the heme moiety of the P450.
authors.affiliation: Agouron Pharmaceuticals Inc., San Diego, CA 92121, USA.
authors.forename: J H
authors.initials: JH
authors.lastname: Lillibridge
authors.forename: B H
authors.initials: BH
authors.lastname: Liang
authors.forename: B M
authors.initials: BM
authors.lastname: Kerr
authors.forename: S
authors.initials: S
authors.lastname: Webber
authors.forename: B
authors.initials: B
authors.lastname: Quart
authors.forename: B V
authors.initials: BV
authors.lastname: Shetty
authors.forename: C A
authors.initials: CA
authors.lastname: Lee
mesh_terms: D000293:Adolescent; D000328:Adult; D019380:Anti-HIV Agents / Q000494:pharmacology*; D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D017320:HIV Protease Inhibitors / Q000494:pharmacology*; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors*; D019888:Nelfinavir / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D019380:Anti-HIV Agents; D065607:Cytochrome P-450 Enzyme Inhibitors; D017320:HIV Protease Inhibitors; D007527:Isoenzymes; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D019888:Nelfinavir