Influence of CYP2B6 Pharmacogenetics on Stereoselective Inhibition and Induction of Bupropion Metabolism by Efavirenz in Healthy Volunteers.

We investigated the acute and chronic effects of efavirenz, a widely used antiretroviral drug, and CYP2B6 genotypes on the disposition of racemic and stereoisomers of bupropion (BUP) and its active metabolites, 4-hydroxyBUP, threohydroBUP and erythrohydroBUP. The primary objective of this study was to test how multiple processes unique to the efavirenz-CYP2B6 genotype interaction influence the extent of efavirenz-mediated drug-drug interaction (DDI) with the CYP2B6 probe substrate BUP. In a three-phase, sequential, open-label study, healthy volunteers (N=53) were administered a single 100 mg oral dose of BUP alone (control phase), with a single 600 mg oral efavirenz dose (inhibition phase), and after 17-days pretreatment with efavirenz (600 mg/day) (induction phase). Compared to the control phase, we show for the first time that efavirenz significantly decreases and chronically increases the exposure of hydroxyBUP and its diastereomers, respectively, and these interactions were CYP2B6 genotype dependent. Chronic efavirenz enhances the elimination of racemic BUP and its enantiomers as well as of threo- and erythro-hydroBUP and their diastereomers, suggesting additional novel mechanisms underlying efavirenz interaction with BUP. The effects of efavirenz and genotypes were nonstereospecific. In conclusion, acute and chronic administration of efavirenz inhibits and induces CYP2B6 activity. Efavirenz-BUP interaction is complex involving time- and CYP2B6 genotype-dependent inhibition and induction of primary and secondary metabolic pathways. Our findings highlight important implications to the safety and efficacy of BUP, study design considerations for future efavirenz interactions, and individualized drug therapy based on CYP2B6 genotypes. Significance Statement The effects of acute and chronic doses of efavirenz on the disposition of racemic and stereoisomers of BUP and its active metabolites were investigated in healthy volunteers. Efavirenz causes an acute inhibition, but chronic induction of CYP2B6 in a genotype dependent manner. Chronic efavirenz induces BUP reduction and the elimination of BUP active metabolites. Efavirenz's effects were non-stereospecific. These data reveal novel mechanisms underlying efavirenz DDI with BUP and provide important insights into time- and CYP2B6 genotype dependent DDIs.

Inhibitory Effects of Probenecid on Pharmacokinetics of Tenofovir Disoproxil Fumarate and Emtricitabine for On-Demand HIV Preexposure Prophylaxis.

In a randomized, crossover pharmacokinetic study in healthy volunteers (N = 14), a single dose of 2 g probenecid (PRO)-boosted 600 mg tenofovir disoproxil fumarate (TDF)/400 mg emtricitabine (FTC) (test (T) +PRO) was compared with the current on-demand HIV preexposure prophylaxis from the IPERGAY study (a 600 mg TDF/400 mg FTC on day 1 and 300 mg TDF/200 mg FTC on days 2 and 3) (control, C IPERGAY). PRO increased mean single-dose area under the plasma concentration-time curve extrapolated to infinity (AUC0-∞,SD ) of tenofovir (TFV) and FTC by 61% and 68%, respectively. The TFV-diphosphate (TFV-DP) concentrations in peripheral blood mononuclear cells were higher (~30%) at 24 hours in T +PRO but then fell significantly lower (~40%) at 72 hours compared with C IPERGAY. The interaction between FTC and PRO was unexpected and novel. Further study is needed to determine if this PRO-boosted TDF/FTC regimen would be clinically effective.

Mechanistic Assessment of Extrahepatic Contributions to Glucuronidation of Integrase Strand Transfer Inhibitors.

Integrase strand transfer inhibitor (INSTI)-based regimens dominate initial human immunodeficiency virus treatment. Most INSTIs are metabolized predominantly via UDP-glucuronosyltransferases (UGTs). For drugs predominantly metabolized by UGTs, including INSTIs, in vitro data recovered from human liver microsomes (HLMs) alone often underpredict human oral clearance. While several factors may contribute, extrahepatic glucuronidation may contribute to this underprediction. Thus, we comprehensively characterized the kinetics for the glucuronidation of INSTIs (cabotegravir, dolutegravir, and raltegravir) using pooled human microsomal preparations from liver (HLMs), intestine (HIMs), and kidney (HKMs) tissues; human embryonic kidney 293 cells expressing individual UGTs; and recombinant UGTs. In vitro glucuronidation of cabotegravir (HLMs≈HKMs>>>HIMs), dolutegravir (HLMs>HIMs>>HKMs), and raltegravir (HLMs>HKMs>> HIMs) occurred in hepatic and extrahepatic tissues. The kinetic data from expression systems suggested the major enzymes in each tissue: hepatic UGT1A9 > UGT1A1 (dolutegravir and raltegravir) and UGT1A1 (cabotegravir), intestinal UGT1A3 > UGT1A8 > UGT1A1 (dolutegravir) and UGT1A8 > UGT1A1 (raltegravir), and renal UGT1A9 (dolutegravir and raltegravir). Enzymes catalyzing cabotegravir glucuronidation in the kidney and intestine could not be identified unequivocally. Using data from dolutegravir glucuronidation as a prototype, a "bottom-up" physiologically based pharmacokinetic model was developed in a stepwise approach and predicted dolutegravir oral clearance within 4.5-fold (hepatic data only), 2-fold (hepatic and intestinal data), and 32% (hepatic, intestinal, and renal data). These results suggest clinically meaningful glucuronidation of dolutegravir in tissues other than the liver. Incorporation of additional novel mechanistic and physiologic underpinnings of dolutegravir metabolism along with in silico approaches appears to be a powerful tool to accurately predict the clearance of dolutegravir from in vitro data.

The Cytochrome P450 Enzyme Responsible for the Production of (Z)-Norendoxifen in vitro.

Norendoxifen, an active metabolite of tamoxifen, is a potent aromatase inhibitor. Little information is available regarding production of norendoxifen in vitro. Here, we conducted a series of kinetic and inhibition studies in human liver microsomes (HLMs) and expressed P450s to study the metabolic disposition of norendoxifen. To validate that norendoxifen was the metabolite of endoxifen, metabolites in HLMs incubates of endoxifen were measured using a HPLC/MS/MS method. To further probe the specific isoforms involved in the metabolic route, endoxifen was incubated with recombinant P450s (CYP 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4, 3A5 and CYP4A11). Formation rates of norendoxifen were evaluated in the absence and presence of P450 isoform specific inhibitors using HLMs. The peak of norendoxifen was found in the incubations consisting of endoxifen, HLMs, and cofactors. The retention times of norendoxifen, endoxifen, and the internal standard (diphenhydramine) were 7.81, 7.97, and 5.86 min, respectively. The Km (app) and Vmax (app) values of norendoxifen formation from endoxifen in HLM was 47.8 µm and 35.39 pmol min-1 mg-1 . The apparent hepatic intrinsic clearances of norendoxifen formation were 0.74 µl mg-1 min. CYP3A5 and CYP2D6 were the major enzymes capable of norendoxifen formation from endoxifen with the rates of 0.26 and 0.86 pmol pmol-1 P450 × min. CYP1A2, 3A2, 2C9, and 2C19 also contributed to norendoxifen formation, but the contributions were at least 6-fold lower. One micromolar ketoconazole (CYP3A inhibitor) showed an inhibitory effect on the rates of norendoxifen formation by 45%, but 1 µm quinidine (CYP2D6 inhibitor) does not show any inhibitory effect. Norendoxifen, metabolism from endoxifen by multiple P450s that including CYP3A5.

Chiral Plasma Pharmacokinetics and Urinary Excretion of Bupropion and Metabolites in Healthy Volunteers.

Bupropion, widely used as an antidepressant and smoking cessation aid, undergoes complex metabolism to yield numerous metabolites with unique disposition, effect, and drug-drug interactions (DDIs) in humans. The stereoselective plasma and urinary pharmacokinetics of bupropion and its metabolites were evaluated to understand their potential contributions to bupropion effects. Healthy human volunteers (n = 15) were administered a single oral dose of racemic bupropion (100 mg), which was followed by collection of plasma and urine samples and determination of bupropion and metabolite concentrations using novel liquid chromatography-tandem mass spectrometry assays. Time-dependent, elimination rate-limited, stereoselective pharmacokinetics were observed for all bupropion metabolites. Area under the plasma concentration-time curve from zero to infinity ratios were on average approximately 65, 6, 6, and 4 and Cmax ratios were approximately 35, 6, 3, and 0.5 for (2R,3R)-/(2S,3S)-hydroxybupropion, R-/S-bupropion, (1S,2R)-/(1R,2S)-erythrohydrobupropion, and (1R,2R)-/(1S,2S)-threohydrobupropion, respectively. The R-/S-bupropion and (1R,2R)-/(1S,2S)-threohydrobupropion ratios are likely indicative of higher presystemic metabolism of S- versus R-bupropion by carbonyl reductases. Interestingly, the apparent renal clearance of (2S,3S)-hydroxybupropion was almost 10-fold higher than that of (2R,3R)-hydroxybupropion. The prediction of steady-state pharmacokinetics demonstrated differential stereospecific accumulation [partial area under the plasma concentration-time curve after the final simulated bupropion dose (300-312 hours) from 185 to 37,447 nM⋅h] and elimination [terminal half-life of approximately 7-46 hours] of bupropion metabolites, which may explain observed stereoselective differences in bupropion effect and DDI risk with CYP2D6 at steady state. Further elucidation of bupropion and metabolite disposition suggests that bupropion is not a reliable in vivo marker of CYP2B6 activity. In summary, to our knowledge, this is the first comprehensive report to provide novel insight into mechanisms underlying bupropion disposition by detailing the stereoselective pharmacokinetics of individual bupropion metabolites, which will enhance clinical understanding of bupropion's effects and DDIs with CYP2D6.

Efavirenz Inhibits the Human Ether-A-Go-Go Related Current (hERG) and Induces QT Interval Prolongation in CYP2B6*6*6 Allele Carriers.

BACKGROUND: Efavirenz (EFV) has been associated with torsade de pointes despite marginal QT interval lengthening. Since EFV is metabolized by the cytochrome P450 (CYP) 2B6 enzyme, we hypothesized that EFV would lengthen the rate-corrected QT (QTcF) interval in carriers of the CYP2B6*6 decreased functional allele. OBJECTIVE: The primary objective of this study was to evaluate EFV-associated QT interval changes with regard to CYP2B6 genotype and to explore mechanisms of QT interval lengthening. METHODS: EFV was administered to healthy volunteers (n = 57) as a single 600 mg dose followed by multiple doses to steady-state. Subjects were genotyped for known CYP2B6 alleles and ECGs and EFV plasma concentrations were obtained serially. Whole-cell, voltage-clamp experiments were performed on cells stably expressing hERG and exposed to EFV in the presence and absence of CYP2B6 expression. RESULTS: EFV demonstrated a gene-dose effect and exceeded the FDA criteria for QTcF interval prolongation in CYP2B6*6/*6 carriers. The largest mean time-matched differences ∆∆QTcF were observed at 6 hours (14 milliseconds; 95% CI [1; 27]), 12 hours (18 milliseconds; 95% CI [-4; 40]), and 18 hours (6 milliseconds; 95% CI [-1; 14]) in the CYP2B6*6/*6 genotype. EFV concentrations exceeding 0.4 µg/mL significantly inhibited outward hERG tail currents (P < 0.05). CONCLUSIONS: This study demonstrates that homozygous carriers of CYP2B6*6 allele may be at increased risk for EFV-induced QTcF interval prolongation via inhibition of hERG.

Stereoselective Glucuronidation of Bupropion Metabolites In Vitro and In Vivo.

Bupropion is a widely used antidepressant and smoking cessation aid in addition to being one of two US Food and Drug Administration-recommended probe substrates for evaluation of cytochrome P450 2B6 activity. Racemic bupropion undergoes oxidative and reductive metabolism, producing a complex profile of pharmacologically active metabolites with relatively little known about the mechanisms underlying their elimination. A liquid chromatography-tandem mass spectrometry assay was developed to simultaneously separate and detect glucuronide metabolites of (R,R)- and (S,S)-hydroxybupropion, (R,R)- and (S,S)-hydrobupropion (threo) and (S,R)- and (R,S)-hydrobupropion (erythro), in human urine and liver subcellular fractions to begin exploring mechanisms underlying enantioselective metabolism and elimination of bupropion metabolites. Human liver microsomal data revealed marked glucuronidation stereoselectivity [Cl(int), 11.4 versus 4.3 µl/min per milligram for the formation of (R,R)- and (S,S)-hydroxybupropion glucuronide; and Cl(max), 7.7 versus 1.1 µl/min per milligram for the formation of (R,R)- and (S,S)-hydrobupropion glucuronide], in concurrence with observed enantioselective urinary elimination of bupropion glucuronide conjugates. Approximately 10% of the administered bupropion dose was recovered in the urine as metabolites with glucuronide metabolites, accounting for approximately 40%, 15%, and 7% of the total excreted hydroxybupropion, erythro-hydrobupropion, and threo-hydrobupropion, respectively. Elimination pathways were further characterized using an expressed UDP-glucuronosyl transferase (UGT) panel with bupropion enantiomers (both individual and racemic) as substrates. UGT2B7 catalyzed the stereoselective formation of glucuronides of hydroxybupropion, (S,S)-hydrobupropion, (S,R)- and (R,S)-hydrobupropion; UGT1A9 catalyzed the formation of (R,R)-hydrobupropion glucuronide. These data systematically describe the metabolic pathways underlying bupropion metabolite disposition and significantly expand our knowledge of potential contributors to the interindividual and intraindividual variability in therapeutic and toxic effects of bupropion in humans.

In Vitro Metabolism of Montelukast by Cytochrome P450s and UDP-Glucuronosyltransferases.

Montelukast has been recommended as a selective in vitro and in vivo probe of cytochrome P450 (P450) CYP2C8 activity, but its selectivity toward this enzyme remains unclear. We performed detailed characterization of montelukast metabolism in vitro using human liver microsomes (HLMs), expressed P450s, and uridine 5'-diphospho-glucuronosyltransferases (UGTs). Kinetic and inhibition experiments performed at therapeutically relevant concentrations reveal that CYP2C8 and CYP2C9 are the principal enzymes responsible for montelukast 36-hydroxylation to 1,2-diol. CYP3A4 was the main catalyst of montelukast sulfoxidation and stereoselective 21-hydroxylation, and multiple P450s participated in montelukast 25-hydroxylation. We confirmed direct glucuronidation of montelukast to an acyl-glucuronide. We also identified a novel peak that appears consistent with an ether-glucuronide. Kinetic analysis in HLMs and experiments in expressed UGTs indicate that both metabolites were exclusively formed by UGT1A3. Comparison of in vitro intrinsic clearance in HLMs suggest that direct glucuronidation may play a greater role in the overall metabolism of montelukast than does P450-mediated oxidation, but the in vivo contribution of UGT1A3 needs further testing. In conclusion, our in vitro findings provide new insight toward montelukast metabolism. The utility of montelukast as a probe of CYP2C8 activity may be compromised owing to involvement of multiple P450s and UGT1A3 in its metabolism.