Introduction

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Recent studies demonstrated that a new class of protease inhibitors, associated with two nucleoside analogs during triple therapy, were very efficient against HIV¹ virus (Barry et al., 1997). These drugs, namely ritonavir, indinavir, and saquinavir, are potent and specific inhibitors of HIV-1 protease, one of the major enzymes encoded by the retrovirus. These protease inhibitors are extensively metabolized by the liver P450 3A4 enzyme (Chiba et al., 1996, 1997; Fitzsimmons and Collins, 1997; Kumar et al., 1996) that represent about 30% of total P450 in human liver (Shimada et al., 1994). Thus, these drugs have a high first-pass effect (Barry et al., 1997), resulting in the excretion of less than 5% of the unchanged form. As patients with HIV disease are likely to be taking a multiple prolonged drug regimen, this may lead to drug interactions as a result of competition for the same substrate binding site of enzyme. As P450 3A4 is involved in the metabolism of quite half of the drugs currently on the market (Guengerich, 1996; Li et al., 1995), numerous drug interactions can be expected. Among these drugs, methadone and buprenorphine, now widely used in the treatment of opioid addiction (Dole and Nyswander, 1965; Mello and Mendelson, 1980), are likely to be coadministered with protease inhibitors to acquired acquired immunodeficiency syndrome (AIDS) patients. Furthermore, as methadone and buprenorphine are specific substrates of P450 3A4 (Iribarne et al., 1996, 1997a), these two drugs are potential candidates to interact with protease inhibitors.

The aim of this study was to compare the inhibitory potential of the HIV protease inhibitors ritonavir, indinavir, and saquinavir against methadone and buprenorphine N-dealkylations catalyzed by P450 3A4 in a panel of human liver microsomes. As the prediction of in vivo metabolic drug interaction from in vitro data has made significant advances in the last decade (Bertz and Granneman, 1997), in vitro data allow us to estimate the likelihood of metabolic interactions between three protease inhibitors and two opioid substitutes.

	Materials and Methods

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Chemicals. Ritonavir (ABT-538) was a gift from Abbott Laboratories, indinavir (MK-639) was from Merck Research Laboratories, and saquinavir (RO-31-8959) was from Roche Research Center. Methadone was purchased from Sigma-Chimie (Saint-Quentin-Fallavier, France), and buprenorphine was a gift from Schering-Plough (Herouville-Saint-Clair, France). All solvents were from the highest quality available from Merck (Darmstadt, Germany). Stock solutions of protease inhibitors were 1 mM ritonavir in methanol, 1 mM saquinavir, and 1 mM indinavir both in H₂O/acetonitrile (1/1, v/v).

Human Liver Microsomal Preparations. Human liver samples were from multi-organ donors who died after traffic accidents. The medical history for each donor on drug taking before death was not known. Sampling was made in accordance to French law. Microsomal fractions were prepared by differential centrifugations as previously described (Berthou et al., 1991). Two banks of human liver microsomes were used. Samples termed Br0X were previously characterized by specific monooxygenase activities specific to P450 3A, including tamoxifen N-demethylation, testosterone 6-hydroxylation, estradiol 2-hydroxylation, toremifene N-demethylation, methadone N-demethylation, nifedipine oxidation, and buprenorphine N-dealkylation (Berthou et al., 1994; Iribarne, 1996, 1997a; Jacolot et al., 1991). Other activities specific to P450 1A, 2D6, 2E1, 2C9, and 2C19 have been also characterized for this bank of microsomes (Berthou et al., 1991; Iribarne et al., 1996). Samples termed BrX were characterized only by nifedipine oxidation, 4-nitrophenol hydroxylation, and ethoxyresorufin O-deethylation activities.

Methadone and Buprenorphine N-Dealkylations. Metabolisms of methadone and buprenorphine were performed as previously described in this laboratory (Iribarne et al., 1996, 1997a). Briefly, the 1-ml reaction mixture consisted of variable concentrations of methadone (100-2000 µM) or buprenorphine (20-500 µM) in 0.1 M potassium phosphate buffer, pH 7.4. It was incubated 20 min at 37°C with 0.5 mg of microsomal protein, 1 mM NADPH, and variable amounts of protease inhibitor. After organic extraction, metabolite analysis was performed by reversed phase high performance liquid chromatography-UV detection at 210 nm, as previously described (Iribarne et al., 1996, 1997a).

Kinetic Analysis and K_i Determinations. For each K_i determination, three kinetics were carried out. The first one did not contain protease inhibitor, but the same amount of solvent was needed to add inhibitor. The second and the third experiments contained inhibitor at two different final concentrations: saquinavir 5 and 10 µM, indinavir 1 and 5 µM, and ritonavir 50 and 100 nM. K_m and V_max were determined with Enzpack 3 Biosoft Software (Cambridge, UK).

Prediction of in Vivo Drug Interactions. The prediction of metabolic drug interactions was based upon the assumption of competition for the same substrate binding site of enzyme. The efficacy of the inhibition will depend on the K_i value of the inhibitor in relation to the K_m value of the drug, assuming that plasma concentrations of inhibitor and drug reflect liver concentrations (Bertz and Granneman, 1997; Iribarne et al., 1997b; Ring et al., 1995). The relation between these parameters is function of the mechanism of inhibition competitive, noncompetitive, or mixed-type (David, 1995; Ring et al., 1995). Therefore, percentage of inhibition can be semi-qualitatively predicted.

	Results and Discussion

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Metabolisms of ritonavir, indinavir, and saquinavir are characterized by very low K_m values, about 20 µM with human liver microsomes but only about 0.5 µM with pure recombinant P450 3A4 (Kumar et al., 1996), 1-2 µM (Chiba et al., 1997) and less than 0.5 µM (Fitzsimmons and Collins, 1997), respectively, whereas methadone and buprenorphine are characterized by high K_m values, 500 and 70 µM (Iribarne et al., 1996, 1997a), respectively. Therefore, it can be predicted that methadone or buprenorphine are not able to inhibit metabolism of protease inhibitors, even at high concentrations.

K_i Determinations. Ritonavir. Ritonavir was shown not to be a mechanism-based inhibitor of P450 3A4 as demonstrated by the following experiment. A preincubation of microsomal proteins in presence of 1 mM NADPH and 100 nM ritonavir in 0.1 M potassium phosphate buffer, pH 7.4, was carried out for 20 min at 37°C. This preincubation medium was then diluted 20-fold in a mixture containing 500 µM methadone, 1 mM NADPH in 0.1 M potassium phosphate buffer, pH 7.4. No significant inhibition of methadone N-demethylation was observed following such a preincubation step.

Indinavir. Buprenorphine incubations with variable amounts of indinavir showed a concentration-dependent inhibition. K_m increased, whereas V_max decreased in presence of 1 or 5 µM indinavir. Indinavir was a potent mixed-type inhibitor of buprenorphine N-dealkylation with an apparent K_i of 0.8 µM (fig. 1A).

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Secondary plot Km/Vmax (or slopes of the Lineweaver-Burk plot) against inhibitor concentration. (A) Mixed-type inhibition of buprenorphine N-dealkylation by indinavir. Human hepatic microsomes Br67 were incubated for 20 min with buprenorphine concentrations ranging from 20 to 500 µM in the absence or the presence of either 1 or 5 µM indinavir. Results were obtained from two different experiments. (B) Noncompetitive inhibition of methadone N-demethylation by indinavir. Human hepatic microsomes Br062 were incubated for 20 min with methadone concentrations ranging from 100 to 2000 µM in the absence or the presence of either 2 or 5 µM indinavir. Results were obtained from two different experiments. (C) Competitive inhibition of buprenorphine N-dealkylation by saquinavir. Human hepatic microsomes Br062 were incubated for 20 min with buprenorphine concentrations ranging from 20 to 500 µM in the absence or the presence of either 10 or 20 µM saquinavir. Results were obtained from two different experiments.

Saquinavir. Preliminary studies showed that saquinavir inhibited methadone N-demethylation and buprenorphine N-dealkylation in a concentration-dependent manner. Kinetics of methadone were carried out in absence or presence of 5 or 10 µM saquinavir. This inhibition was characterized by a concentration-dependent increase in K_m values and decrease in V_max values. This is consistent with a mixed-type inhibition with a K_i of 15 µM (table 1).

Co-Incubations of Protease Inhibitors and Methadone or Buprenorphine with Human Liver Microsomes. Methadone N-demethylation and buprenorphine N-dealkylation were highly significant correlated in the panel of 13 human liver microsomal preparations (r = 0.93; p<0.001) with a 5-fold interindividual variability.