ALF and MDZ have similar intestinal extraction but low and intermediate hepatic extraction, respectively. Systemic and oral clearances of ALF are excellent in vivo probes for hepatic and first-pass CYP3A activities and drug interactions. Miosis was an acceptable surrogate for plasma ALF. ALF miosis may be a suitable noninvasive in vivo probe for both hepatic and first-pass CYP3A.

Background Methadone clearance is highly variable and drug interactions are problematic. Both have been attributed to CYP3A, but actual mechanisms are unknown. Drug interactions can provide such mechanistic information. Ritonavir/indinavir, one of the earliest protease inhibitor combinations, may inhibit CYP3A. We assessed ritonavir/indinavir effects on methadone pharmacokinetics and pharmacodynamics, intestinal and hepatic CYP3A activity, and intestinal transporters (P-glycoprotein) activity. CYP3A and transporters were assessed with alfentanil and fexofenadine, respectively. Methods Twelve healthy human immunodeficiency virus-negative volunteers underwent a sequential 3-part crossover. On three consecutive days they received oral alfentanil/fexofenadine, intravenous alfentanil, and intravenous plus oral (deuterium-labeled) methadone, repeated after acute (3d) and steady-state (2 wk) ritonavir/indinavir. Plasma and urine analytes were measured by mass spectrometry. Opioid effects were assessed by miosis. Results Alfentanil apparent oral clearance was inhibited >97% by both acute and steady-state ritonavir/indinavir, and systemic clearance was inhibited >90%, due to diminished hepatic and intestinal extraction. Ritonavir/indinavir increased fexofenadine area under the plasma concentration-time curve 4-to 5-fold, suggesting significant inhibition of gastrointestinal P-glycoprotein. Ritonavir/indinavir slightly increased methadone N-demethylation, but had no significant effects on methadone plasma concentrations, or on systemic or apparent oral clearance, renal clearance, hepatic extraction or clearance, or bioavailability. Ritonavir/indinavir had no significant effects on methadone plasma concentration-effect relationships. Conclusions Inhibition of both hepatic and intestinal CYP3A activity is responsible for ritonavir/indinavir drug interactions. Methadone disposition was unchanged despite profound inhibition of CYP3A activity, suggesting little or no role for CYP3A in clinical methadone metabolism and clearance. Methadone bioavailability was unchanged despite inhibition of gastrointestinal P-glycoprotein activity, suggesting that this transporter does not limit methadone intestinal absorption.

Ritonavir diminishes methadone plasma concentrations, attributed to CYP3A induction, but actual mechanisms are unknown. We determined ritonavir effects on stereoselective methadone pharmacokinetics and clinical effects (pupillary miosis) in healthy HIV-negative volunteers. Subjects received intravenous plus oral (deuterium-labeled) racemic methadone after nothing, short-term (3 day) and steady-state ritonavir. Acute and steady-state ritonavir, respectively, caused 1.5- and 2-fold induction of systemic and apparent oral R- and S-methadone clearances. Ritonavir increased renal clearance 40-50%, and stereoselectively (S>R) increased methadone hepatic N-demethylation 50-80%, extraction 2-fold, and clearance 2-fold. Bioavailability was unchanged, despite significant inhibition of intestinal P-glycoprotein. Intestinal and hepatic CYP3A was inhibited >70%. Ritonavir shifted methadone plasma concentration-miosis curves leftward and upward. Rapid ritonavir induction of methadone clearance results from increased renal clearance and induced hepatic metabolism. Induction of methadone metabolism occurred despite profound CYP3A inhibition, suggesting no role for CYP3A in clinical methadone metabolism and clearance. Ritonavir may alter methadone pharmacodynamics.

The hepatic and first-pass cytochrome P4503A (CYP3A) probe alfentanil (ALF) is also metabolized in vitro by CYP3A5. Human hepatic microsomal ALF metabolism is higher in livers with at least one CYP3A5*1 allele and higher CYP3A5 protein content, compared with CYP3A5*3 homozygotes with little CYP3A5. The influence of CYP3A5 genotype on ALF pharmacokinetics and pharmacodynamics was studied, and compared to midazolam (MDZ), another CYP3A probe. Healthy volunteers (58 men, 41 women) were genotyped for CYP3A5 *1, *3, *6, and *7 alleles. They received intravenous MDZ then ALF, and oral MDZ and ALF the next day. Plasma MDZ and ALF concentrations were determined by mass spectrometry. Dark-adapted pupil diameters were determined coincident with blood sampling. In CYP3A5(*)3/(*)3 (n=62), (*)1/(*)3 (n=28), and (*)1/(*)1 (n=8) genotypes, systemic clearances of ALF were 4.6+/-1.8, 4.8+/-1.7, and 3.9+/-1.7 ml/kg/min and those of MDZ were 7.8+/-2.3, 7.7+/-2.3, and 6.0+/-1.4 ml/kg/min, respectively (not significant), and apparent oral clearances were 11.8+/-7.2, 13.3+/-6.1, and 12.6+/-8.2 ml/kg/min for ALF and 35.2+/-19.0, 36.4+/-15.7, and 29.4+/-9.3 ml/kg/min for MDZ (not significant). Clearances were not different between African Americans (n=25) and Whites (n=68), or between CYP3A5 genotypes within African Americans. ALF pharmacodynamics was not different between CYP3A5 genotypes. There was consistent concordance between ALF and MDZ, in clearances and extraction ratios. Thus, in a relatively large cohort of healthy subjects with constitutive CYP3A activity, CYP3A5 genotype had no effect on the systemic or apparent oral clearances, or pharmacodynamics, of the CYP3A probes ALF and MDZ, despite affecting their hepatic microsomal metabolism.

Ritonavir diminishes methadone plasma concentrations, attributed to CYP3A, but mechanisms are unknown. We determined short-term (2 day) and steady-state (2 week) ritonavir effects on intestinal/hepatic CYP3A (probed with IV and oral alfentanil, and miosis), P-glycoprotein (fexofenadine) and methadone pharmacokinetics and pharmacodynamics in healthy volunteers. Acute ritonavir increased the AUC0-∞/dose ratio (ritonavir/control) for oral alfentanil 25-fold. Steady-state ritonavir increased the AUC0-∞/dose ratio for intravenous and oral alfentanil 4- and 10-fold, respectively, reduced hepatic extraction (0.26 to 0.07), intestinal extraction (0.51 to zero), and increased bioavailability (37 to 95%). Acute ritonavir inhibits first-pass CYP3A >96%. Chronic ritonavir inhibits hepatic (>70%) and first-pass (>90%) CYP3A. Acute and steady-state ritonavir increased fexofenadine AUC0-∞ 2.8- and 1.4-fold, suggesting P-glycoprotein inhibition. Steady-state ritonavir caused mild apparent induction of P-glycoprotein and hepatic CYP3A, but net inhibition still predominated. Ritonavir inhibits both intestinal and hepatic CYP3A and drug transport. Alfentanil miosis noninvasively determined CYP3A inhibition by ritonavir.