OF THE HUMAN IMMUNODEFICIENCY VIRUS PROTEASE INHIBITORS INDINAVIR AND RITONAVIR BY HUMAN INTESTINAL MICROSOMES AND EXPRESSED CYTOCHROME P 4503 A 4 / 3 A 5 : MECHANISM-BASED INACTIVATION OF CYTOCHROME P 4503 A BY RITONAVIR

Both ritonavir and indinavir were readily metabolized by human intestinal microsomes. Comparison of the patterns of metabolites in incubations with enterocyte microsomes and expressed cytochrome P450 (CYP) isozymes and immunoinhibition and chemical inhibition studies showed the essential role of the CYP3A subfamily in the metabolism of both protease inhibitors by the small intestine. Ritonavir was similarly biotransformed by microsomes containing expressed CYP3A4 or CYP3A5 isozymes (KM 5 0.05– 0.07 mM, Vmax 5 1–1.4 nmol/min/nmol CYP). In contrast, both the patterns of metabolites and the enzyme kinetic parameters for the metabolism of indinavir by expressed CYP3A5 (KM 5 0.21 mM, Vmax 5 0.24 nmol/min/nmol CYP) and CYP3A4 (KM 5 0.04 mM, Vmax 5 0.68 nmol/min/nmol CYP) were different. The biotransformation of both indinavir and ritonavir in human enterocyte microsomes was characterized by very low KM values (0.2–0.4 mM for indinavir and <0.1 mM for ritonavir). The Vmax for indinavir metabolism was greater in enterocyte (163 6 35 pmol/min/mg protein) than in liver (68 6 44 pmol/min/mg protein) microsomes. The metabolism of ritonavir in liver and enterocyte microsomes was associated with inactivation of CYP3A. The initial Vmax for ritonavir metabolism by enterocyte microsomes was 89 6 59 pmol/min/mg protein. The apparent inactivation rate constants for intestinal CYP3A and expressed CYP3A4 were 0.078 and 0.135 min, respectively. Metabolic inactivation of CYP3A by ritonavir explains the improved bioavailability and pharmacokinetics of ritonavir and the sustained elevation of blood levels of other, concomitantly administered, substrates of CYP3A. Indinavir and ritonavir (fig. 1) represent a new class of anti-HIV 1 agents that selectively inhibit the HIV type 1 protease. The HIV protease cleaves viral precursor polyproteins (Darke et al., 1988). This process is essential for the maturation of infectious virions (Gottlinger et al., 1989). Numerous protease inhibitors have been discovered. Most of them display low oral bioavailability and rapid elimination. Indinavir is one of the first protease inhibitors with enhanced oral bioavailability, varying from 10 to 70% in different species (Vacca et al., 1994; Linet al., 1996). Its average half-life in humans is 1.8 hr. Improved pharmacokinetic properties (half-life, 3.1–5.7 hr) and high oral bioavailability in humans have been recently reported for ritonavir (Kempf et al., 1995; Hsuet al., 1997). It has been shown that the protease inhibitors indinavir, ritonavir, and saquinavir are metabolized primarily by isozymes of the CYP3A subfamily and to a lesser extent by CYP2D6 (Kumaret al., 1996; Chibaet al., 1996, 1997; Fitzsimmons and Collins, 1997). CYP3A is a major subfamily of oxidative enzymes in the small intestine and accounts for 70% of the total intestinal CYP content (Watkins et al., 1987). CYP3A4 and CYP3A5 are two CYP3A isozymes that are commonly expressed in the gastrointestinal tract. The small intestine has been shown to be an important site for presystemic metabolism of cyclosporine (Hebert et l., 1992), midazolam (Thummelt al., 1996), rifabutin (Iatsimirskaia et al., 1997), and possibly other CYP3A substrates, including saquinavir (Fitzsimmons and Collins, 1997). Biotransformation of protease inhibitors by CYP3A enzymes in the intestine may account for low and variable bioavailability. Recently, Chiba et al.(1997) showed that the small intestine is capable of metabolizing indinavir. However, the contribution of the gut to the first-pass metabolism of indinavir, estimated fromin vitro metabolic data ( Vmax/KM) and intestinal mucosal blood flow, was suggested to be minor. The metabolism of ritonavir by intestinal enzymes has not been examined. The coadministration of ritonavir significantly increased the plasma levels of other drugs metabolized by CYP3A, including saquinavir, for which a 63–83-fold increase in AUC was reported (Kempf et al., 1997). Ritonavir also potently inhibited the CYP-mediated metabolism of indinavir, saquinavir, and nelfinavir in human liver microsomes; however, none of these protease inhibitors altered the metabolism of ritonavir in vivo or in vitro. The objectives of the present study were to 1) determine and compare enzyme kinetic parameters 1 Abbreviations used are: HIV, human immunodeficiency virus; CYP, cytochrome P450; ACN, acetonitrile; TEA, triethylamine; TFA, trifluoroacetic acid; MAB3A4, monoclonal antibody to cytochrome P4503A4; kin, inactivation rate constant; ESI, electrospray ionization; CID, collision-induced dissociation. Send reprint requests to: Tatiana Koudriakova, Department of Pharmacology, The Ohio State University, 5084 Graves Hall, 333 W. 10th Ave., Columbus, OH 43210. 0090-9556/98/2606-0552–561$02.00/0 DRUG METABOLISM AND DISPOSITION Vol. 26, No. 6 Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. 552 at A PE T Jornals on N ovem er 6, 2017 dm d.aspurnals. org D ow nladed from