Alysa Walker

Intravenous and oral alfentanil as in vivo probes for hepatic and first‐pass cytochrome P450 3A activity: Noninvasive assessment by use of pupillary miosis

Systemic clearance of intravenous (IV) alfentanil (ALF) is an in vivo probe for hepatic cytochrome P450 (CYP) 3A activity, miosis is a surrogate for plasma ALF concentrations, and IV ALF miosis is a noninvasive probe for hepatic CYP3A. This investigation characterized the bioavailability and first‐pass metabolism of oral ALF and tested the hypotheses that (1) first‐pass ALF clearance reflects first‐pass CYP3A activity, (2) miosis after oral ALF will reflect intestinal and hepatic CYP3A activity, and (3) miosis can approximate plasma concentration‐based pharmacokinetic measures for IV and oral ALF as a noninvasive in vivo probe for hepatic and first‐pass CYP3A activity and drug interactions. Results were compared with those for midazolam (MDZ), an alternative CYP3A probe.

Influence of CYP3A5 Genotype on the Pharmacokinetics and Pharmacodynamics of the Cytochrome P4503A Probes Alfentanil and Midazolam

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.

Mechanism of Ritonavir Changes in Methadone Pharmacokinetics and Pharmacodynamics: II. Ritonavir Effects on CYP3A and P‐Glycoprotein Activities

Ritonavir diminishes methadone plasma concentrations, an effect attributed to CYP3A induction, but the actual mechanisms are unknown. We determined short‐term (2‐day) and steady‐state (2‐week) ritonavir effects on intestinal and hepatic CYP3A4/5 (probed with intravenous (IV) and oral alfentanil (ALF) and with miosis) and P‐glycoprotein (P‐gp) (fexofenadine), and on methadone pharmacokinetics and pharmacodynamics in healthy volunteers. Acute ritonavir increased the area under the concentration‐time curve (AUC)0–∞/dose ratio (ritonavir/control) for oral ALF 25‐fold. Steady‐state ritonavir increased the AUC0–∞/dose ratio for IV and oral ALF 4‐ and 10‐fold, respectively; reduced hepatic extraction (from 0.26 to 0.07) and intestinal extraction (from 0.51 to 0); and increased bioavailability (from 37 to 95%). Acute ritonavir inhibits first‐pass CYP3A >96%. Chronic ritonavir inhibits hepatic CYP3A (>70%) and first‐pass CYP3A (>90%). Acute and steady‐state ritonavir increased the fexofenadine AUC0–∞ 2.8‐ and 1.4‐fold, respectively, suggesting P‐gp inhibition. Steady‐state compared with acute ritonavir caused mild apparent induction of P‐gp and hepatic CYP3A, but net inhibition still predominated. Ritonavir inhibited both intestinal and hepatic CYP3A and drug transport. ALF miosis noninvasively determined CYP3A inhibition by ritonavir.

Methadone Pharmacokinetics are Independent of Cytochrome P4503A (CYP3A) Activity and Gastrointestinal Drug Transport: Insights from Methadone Interactions with Ritonavir/Indinavir

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 three-part crossover. On three consecutive days, they received oral alfentanil/fexofenadine, intravenous alfentanil, and intravenous plus oral (deuterium-labeled) methadone, repeated after acute (3 days) and steady-state (2 weeks) ritonavir/indinavir. Plasma and urine analytes were measured by mass spectrometry. Opioid effects were assessed by miosis. Results:Alfentanil apparent oral clearance was inhibited more than 97% by both acute and steady-state ritonavir/indinavir, and systemic clearance was inhibited more than 90% due to diminished hepatic and intestinal extraction. Ritonavir/indinavir increased fexofenadine area under the plasma concentration-time curve four- to five-fold, suggesting significant inhibition of gastrointestinal P-glycoprotein. Ritonavir/indinavir slightly increased methadone N-demethylation, but it 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.

Mechanism of ritonavir changes in methadone pharmacokinetics and pharmacodynamics I. Evidence against CYP3A mediation of methadone clearance

Ritonavir diminishes methadone plasma concentrations, an effect attributed to CYP3A induction, but the actual mechanisms are unknown. We determined ritonavir effects on stereoselective methadone pharmacokinetics and clinical effects (pupillary miosis) in healthy human immunodeficiency virus–negative volunteers. Subjects received intravenous plus oral (deuterium‐labeled) racemic methadone after no ritonavir, short‐term (3‐day) ritonavir, 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 hepatic methadone N‐demethylation 50–80%, extraction twofold, and clearance twofold. 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.

Methadone metabolism and clearance are induced by nelfinavir despite inhibition of cytochrome P4503A (CYP3A) activity

BACKGROUND Methadone plasma concentrations are decreased by nelfinavir. Methadone clearance and the drug interactions have been attributed to CYP3A4, but actual mechanisms of methadone clearance and the nelfinavir interaction are unknown. We assessed nelfinavir effects on methadone pharmacokinetics and pharmacodynamics, intestinal and hepatic CYP3A4/5 activity, and intestinal P-glycoprotein transport activity. CYP3A4/5 and transporters were assessed using alfentanil and fexofenadine, respectively. METHODS Twelve healthy HIV-negative volunteers underwent a sequential crossover. On three consecutive days they received oral alfentanil plus fexofenadine, intravenous alfentanil, and intravenous plus oral methadone. This was repeated after nelfinavir. Plasma and urine analytes were measured by mass spectrometry. Opioid effects were measured by pupil diameter change (miosis). RESULTS Nelfinavir decreased intravenous and oral methadone plasma concentrations 40-50%. Systemic clearance, hepatic clearance, and hepatic extraction all increased 1.6- and 2-fold, respectively, for R- and S-methadone; apparent oral clearance increased 1.7- and 1.9-fold. Nelfinavir stereoselectively increased (S>R) methadone metabolism and metabolite formation clearance, and methadone renal clearance. Methadone bioavailability and P-glycoprotein activity were minimally affected. Nelfinavir decreased alfentanil systemic and apparent oral clearances 50 and 76%, respectively. Nelfinavir appeared to shift the methadone plasma concentration-effect (miosis) curve leftward and upward. CONCLUSIONS Nelfinavir induced methadone clearance by increasing renal clearance, and more so by stereoselectively increasing hepatic metabolism, extraction and clearance. Induction occurred despite 50% inhibition of hepatic CYP3A4/5 activity and more than 75% inhibition of first-pass CYP3A4/5 activity, suggesting little or no role for CYP3A in clinical methadone disposition. Nelfinavir may alter methadone pharmacodynamics, increasing clinical effects.

Lack of Indinavir Effects on Methadone Disposition Despite Inhibition of Hepatic and Intestinal Cytochrome P4503A (CYP3A)

Background: Methadone disposition and pharmacodynamics are highly susceptible to interactions with antiretroviral drugs. Methadone clearance and drug interactions have been attributed to cytochrome P4503A4 (CYP3A4), but actual mechanisms are unknown. Drug interactions can be clinically and mechanistically informative. This investigation assessed effects of the protease inhibitor indinavir on methadone pharmacokinetics and pharmacodynamics, hepatic and intestinal CYP3A4/5 activity (using alfentanil), and intestinal transporter activity (using fexofenadine). Methods: Twelve healthy volunteers underwent a sequential crossover. On three consecutive days they received oral alfentanil plus fexofenadine, intravenous alfentanil, and intravenous plus oral (deuterium-labeled) methadone. This was repeated after 2 weeks of indinavir. Plasma and urine analytes were measured by mass spectrometry. Opioid effects were measured by miosis. Results: Indinavir significantly inhibited hepatic and first-pass CYP3A activity. Intravenous alfentanil systemic clearance and hepatic extraction were reduced to 40–50% of control, apparent oral clearance to 30% of control, and intestinal extraction decreased by half, indicating 50% and 70% inhibition of hepatic and first-pass CYP3A activity. Indinavir increased fexofenadine area under the plasma concentration-time curve 3-fold, suggesting significant P-glycoprotein inhibition. Indinavir had no significant effects on methadone plasma concentrations, methadone N-demethylation, systemic or apparent oral clearance, renal clearance, hepatic extraction or clearance, or bioavailability. Methadone plasma concentration-effect relationships were unaffected by indinavir. Conclusions: Despite significant inhibition of hepatic and intestinal CYP3A activity, indinavir had no effect on methadone N-demethylation and clearance, suggesting little or no role for CYP3A in clinical disposition of single-dose methadone. Inhibition of gastrointestinal transporter activity had no influence of methadone bioavailability.

Evaluation of First‐Pass Cytochrome P4503A (CYP3A) and P‐Glycoprotein Activities Using Alfentanil and Fexofenadine in Combination

Cytochrome P4503A (CYP3A) and P‐glycoprotein (P‐gp) are major determinants of oral bioavailability. Development of in vivo probe(s), for both CYP3A and P‐gp, which could be administered in combination, is a current goal. Nevertheless, there is considerable overlap in CYP3A and P‐gp substrate selectivities; there are few discrete probes. Alfentanil is a selective CYP3A probe but not a P‐gp substrate. Fexofenadine is a P‐gp probe but not a CYP3A substrate. This investigation tested the hypothesis that alfentanil and fexofenadine could be administered in combination to probe first‐pass CYP3A and P‐gp activities in humans. Two 3‐way crossover studies were conducted in healthy volunteers. In the first protocol, subjects received oral alfentanil alone, fexofenadine alone, or fexofenadine 1 hour after alfentanil. In the second protocol, subjects abstained from citrus and apple products for 5 days and received fexofenadine alone, fexofenadine 1 hour after alfentanil, or alfentanil 4 hours after fexofenadine. An assay using solid‐phase extraction and electrospray liquid chromatography/mass spectrometry was developed for the simultaneous quantification of plasma alfentanil and fexofenadine. In both protocols, alfentanil plasma concentrations and area under the concentration versus time curve (AUC) were unaffected by fexofenadine or meal composition. Fexofenadine given 1 hour after alfentanil and followed 1 hour later by a meal containing orange or apple juice had a somewhat lower AUC compared with fexofenadine alone (geometric mean ratio with and without the interacting drug = 0.73, 90% confidence interval [CI] = 0.59–1.04). Fexofenadine given 1 hour after alfentanil and followed 2 hours later by a meal not containing citrus or apple products had an AUC that was unchanged compared with fexofenadine alone (ratio = 0.91, 90% CI = 0.70–1.35). These results show that alfentanil disposition was not affected by fexofenadine. A dosing regimen was identified in which fexofenadine disposition was not affected by alfentanil. The timing and content of meals after fexofenadine had a significant effect on fexofenadine disposition. Alfentanil and fexofenadine in combination appear to be a useful probe for evaluating both first‐pass CYP3A and P‐gp activities in humans.

Disposition and miotic effects of oral alfentanil: A potential noninvasive probe for first‐pass cytochrome P4503A activity

Systemic clearance of the opioid alfentanil after intravenous administration is an excellent in vivo probe for hepatic cytochrome P4503A (CYP3A) activity and drug interactions. Alfentanil effect (miosis) is a surrogate for plasma alfentanil concentrations, and alfentanil effect kinetics may be a suitable noninvasive probe for hepatic CYP3A. Oral alfentanil might be a probe for first‐pass CYP3A activity; however, it is not used clinically, and oral alfentanil disposition is unknown. This investigation evaluated the disposition and miotic effects of oral alfentanil.

Sensitivity of intravenous and oral alfentanil and pupillary miosis as minimally invasive and noninvasive probes for hepatic and first-pass CYP3A activity.

This investigation determined the ability of alfentanil miosis and single‐point concentrations to detect various degrees of CYP3A inhibition. Results were compared with those for midazolam, an alternative CYP3A probe. Twelve volunteers were studied in a randomized 4‐way crossover, targeting 12%, 25%, and 50% inhibition of hepatic CYP3A. They received 0, 100, 200, or 400 mg oral fluconazole, followed 1 hour later by 1 mg intravenous midazolam and then 15 μg/kg intravenous alfentanil 1 hour later. The next day, they received fluconazole, followed by3mg oral midazolam and 40 μg/kg oral alfentanil. Dark‐adapted pupil diameters were measured coincident with blood sampling. Area under the plasma concentration‐time curve (AUC) ratios (fluconazole/control) after 100,200, and 400 mg fluconazole were (geometric mean) 1.3 * , 1.4 * , and 2.0 * for intravenous midazolam and 1.2 * , 1.6 * , and 2.2 * for intravenous alfentanil ( * significantly different from control), indicating 16% to 21%, 31% to 36%, and 43% to 53% inhibition of hepatic CYP3A. Single‐point concentration ratios were 1.5 * , 1.8 * , and 2.4 * for intravenous midazolam (at 5 hours) and 1.2 * , 1.6 * , and 2.2 * for intravenous alfentanil (at 4 hours). Pupil miosis AUC ratios were 0.9, 1.0, and 1.2 * . After oral dosing, plasma AUC ratios were 2.3 * , 3.6 * , and 5.3 * for midazolam and 1.8 * , 2.9 * , and 4.9 * for alfentanil; plasma single‐point ratios were 2.4 * , 4.5 * , and 6.9 * for midazolam and 1.8 * , 2.9 * , and 4.9 * for alfentanil, and alfentanil miosis ratios were 1.1, 1.9 * , and 2.7 * . Plasma concentration AUC ratios of alfentanil and midazolam were equivalent for detecting hepatic and first‐pass CYP3A inhibition. Single‐point concentrations were an acceptable surrogate for formal AUC determinations and as sensitive as AUCs for detecting CYP3A inhibition. Alfentanil miosis could detect 50% to 70% inhibition of CYP3A activity, but was less sensitive than plasma AUCs. Further refinements are needed to increase the sensitivity of alfentanil miosis for detecting small CYP3A changes.