Ser100-Phosphorylated RORα Orchestrates CAR and HNF4α to Form Active Chromatin Complex in Response to Phenobarbital to Regulate Induction of CYP2B6.

We have previously shown that the retinoid-related orphan receptor alpha (RORα) phosphorylation plays a pivotal role in sulfotransferase 1E1 gene regulation within mouse liver. Here, we found serine 100-phosphorylated RORα orchestrates constitutive androstane receptor (CAR) and hepatocyte nuclear factor 4 alpha (HNF4α) to induce CYP2B6 by phenobarbital (PB) in human primary hepatocytes (HPHs). RORα knockdown using small interfering RNAs suppressed CYP2B6 mRNAs in HPH, whereas transient expression of RORα in COS-1 cells activated CYP2B6 promoter activity in reporter assays. Through chromatin immunoprecipitation (IP) and gel shift assays, we found that RORα in the form of phosphorylated (p-) S100 directly bound to a newly identified RORα response element (RORα response element on CYP2B6 promoter, -660/-649) within the CYP2B6 promoter in untreated or treated HPH. In PB-treated HPH, p-Ser100 RORα was both enriched in the distal phenobarbital response element module (PBREM) and the proximal okadaic acid response element (OARE), a known HNF4α binding site. Chromatin conformation capture assay revealed direct contact between the PBREM and OARE only in PB-treated HPH. Moreover, CAR preferably interacted with phosphomimetically mutated RORα at Ser100 residue in co-IP assay. A gel shift assay with a radiolabeled OARE module and nuclear extracts prepared from PB-treated mouse liver confirmed that HNF4α formed a complex with Ser 100-phosphorylated RORα, as shown by supershifted complexes with anti-p-Ser100 RORα and anti-HNF4α antibodies. Altogether, the results established that p-Ser100 RORα bridging the PBREM and OARE orchestrates CAR and HNF4α to form active chromatin complex during PB-induced CYP2B6 expression in human primary hepatocytes. SIGNIFICANCE STATEMENT: CYP2B6 is a vital enzyme for the metabolic elimination of xenobiotics, and it is prone to induction by xenobiotics, including phenobarbital via constitutive androstane receptor (CAR) and hepatocyte nuclear factor 4 alpha (HNF4α). Here, we show that retinoid-related orphan receptor alpha (RORα), through phosphorylated S100 residue, orchestrated CAR-HNF4α interaction on the CYP2B6 promoter in human primary hepatocyte cultures. These results signify not only the role of RORα in the molecular process of CYP2B6 induction, but it also reveals the importance of conserved phosphorylation sites within the DNA-binding domain of the receptor.

A New Approach of Mitigating CYP3A4 Induction Led to the Discovery of Potent Hepatitis B Virus (HBV) Capsid Inhibitor with Optimal ADMET Profiles.

Described herein is a new approach to mitigate CYP3A4 induction. In this unconventional approach, a fine-tuning of the dihedral angle between the C4 phenyl and the dihydropyrimidine core of the heteroaryldihydropyrimidine (HAP) class of capsid inhibitors successfully altered the structure-activity-relationships (SARs) of the unwanted CYP3A4 induction and the desired HBV capsid inhibition to more favorable values. This eventually led to the discovery of a new capsid inhibitor with significantly reduced CYP3A4 induction, excellent anti-HBV activity, favorable preclinical PK/PD profiles, and no early safety flags.

CYP2B6 Genotype-Dependent Inhibition of CYP1A2 and Induction of CYP2A6 by the Antiretroviral Drug Efavirenz in Healthy Volunteers.

We investigated the effect of efavirenz on the activities of cytochrome P450 (CYP)1A2, CYP2A6, xanthine oxidase (XO), and N-acetyltransferase 2 (NAT2), using caffeine as a probe. A single 150 mg oral dose of caffeine was administered to healthy volunteers (n = 58) on two separate occasions; with a single 600 mg oral dose of efavirenz and after treatment with 600 mg/day efavirenz for 17 days. Caffeine and its metabolites in plasma and urine were quantified using liquid chromatography/tandem-mass spectrometry. DNA was genotyped for CYP2B6*4 (785A>G), CYP2B6*9 (516G>T), and CYP2B6*18 (983T>C) alleles using TaqMan assays. Relative to single-dose efavirenz treatment, multiple doses of efavirenz decreased CYP1A2 (by 38%) and increased CYP2A6 (by 85%) activities (P < 0.05); XO and NAT2 activities were unaffected. CYP2B6*6*6 genotype was associated with lower CYP1A2 activity following both single and multiple doses of efavirenz. No similar association was noted for CYP2A6 activity. This is the first report showing that efavirenz reduces hepatic CYP1A2 and suggesting chronic efavirenz exposure likely enhances the elimination of CYP2A6 substrates. This is also the first to report the extent of efavirenz-CYP1A2 interaction may be efavirenz exposure-dependent and CYP2B6 genotype-dependent.

CYP2B6 Genetic Polymorphisms, Depression, and Viral Suppression in Adults Living with HIV Initiating Efavirenz-Containing Antiretroviral Therapy Regimens in Uganda: Pooled Analysis of Two Prospective Studies.

Single-nucleotide polymorphisms (SNPs) in CYP2B6 have been shown to predict variation in plasma efavirenz concentrations, but associations between these SNPs and efavirenz-mediated depression and viral suppression are less well described. We evaluated three SNPs in CYP2B6 (rs3745274, rs28399499, and rs4803419) in Ugandan persons living with HIV. To define exposure, we used previously published pharmacokinetic modeling data to categorize participants as normal, intermediate, and poor efavirenz metabolizers. Our outcomes were probable depression in the first 2 years after antiretroviral therapy (ART) initiation (mean score of >1.75 on the Hopkins Symptom Depression Checklist) and viral suppression 6 months after ART initiation. We fit generalized estimating equation and modified Poisson regression models adjusted for demographic, clinical, and psychosocial characteristics with or without individuals with depression at the time of ART initiation. Among 242 participants, there were no differences in the pre-ART depression or viral load by efavirenz metabolism strata (p > .05). Participants were classified as normal (32%), intermediate (50%), and poor (18%) metabolizers. Seven percent (56/242) of follow-up visits met criteria for depression. Eighty-five percent (167/202) of participants who completed a 6-month visit achieved viral suppression. CYP2B6 metabolizer strata did not have a statistically significant association with either depression [adjusted risk ratio (aRR) comparing intermediate or poor vs. normal, 1.46; 95% confidence interval (CI), 0.72-2.95] or 6-month viral suppression (aRR, 1.01; 95% CI, 0.88-1.15). However, in analyses restricted to participants without pre-ART depression, poorer CYP2B6 metabolism was associated with increased odds of depression (adjusted odds ratio, 4.11; 95% CI, 1.04-16.20). Efavirenz-metabolizing allele patterns are strongly associated with risk of incident depression. Future work should elucidate further region-specific gene-environment interactions and whether alternate polymorphisms may be associated with efavirenz metabolism.

Amenamevir: Studies of Potential CYP2C8- and CYP2B6-Mediated Pharmacokinetic Interactions With Montelukast and Bupropion in Healthy Volunteers.

Amenamevir (formerly ASP2151) induces cytochrome P450 (CYP)2B6 and CYP3A4 and inhibits CYP2C8.  We conducted 2 studies, 1 using montelukast as a probe to assess CYP2C8 and the other bupropion to assess CYP2B6.  The montelukast study examined the effect of amenamevir on the pharmacokinetics of montelukast in 24 healthy men: each subject received montelukast 10 mg alone, followed by montelukast 10 mg with amenamevir 400 mg, or vice versa after a washout period.  In the bupropion study, 24 subjects received a single dose of 150 mg bupropion on days 1, 15, 22, and 29, and repeated once-daily doses of 400 mg amenamevir on days 6-15.  Amenamevir increased peak concentration and area under the concentration-time curve of montelukast by about 22% (ratio 121.7%, 90%CI [114.8, 129.1]; 121% [116.2, 128.4], respectively) with a similar increase in hydroxymontelukast (ratio 121.4%, 90%CI [106.4, 138.5]; 125.6 % [111.3, 141.7]).  Amenamevir reduced peak concentration and area under the concentration-time curve of bupropion by 16% (84.29%, 90%CI [78.00, 91.10]; 84.07%, 90%CI [78.85, 89.63]), with recovery after 1 week; the pharmacokinetics of the primary metabolite hydroxybupropion was unaffected.  Thus, amenamevir increased plasma concentrations of montelukast and decreased those of bupropion, but it did not do so enough to require dose adjustment of coadministered substrates of either CYP2C8 or CYP2B6.

Population Pharmacokinetic Model Linking Plasma and Peripheral Blood Mononuclear Cell Concentrations of Efavirenz and Its Metabolite, 8-Hydroxy-Efavirenz, in HIV Patients.

The objectives of this study were to characterize the population pharmacokinetics (PK) of efavirenz (EFV) and 8-hydroxy-efavirenz (8OHEFV) in plasma and peripheral blood mononuclear cells (PBMCs) and to explore covariates affecting the PK parameters. Fifty-one patients had steady-state 0-to-24-h concentrations of EFV and 8OHEFV in plasma with corresponding concentrations in PBMCs, while 261 patients had one or two sparse concentrations at 16 ± 1 h postdose at weeks 4 and/or 16. The pharmacogenetic markers CYP2B6*6, CYP3A5*3, CYP3A5*6, UGT2B7*2, ABCB1 (3435C→T, 3842A→G), OATP1B1*1B, and OATP1B1*5, the presence of a rifampin-based antituberculosis (anti-TB) regimen, baseline body weight and organ function values, and demographic factors were explored as covariates. EFV concentration data were well described by a two-compartment model with first-order absorption (Ka ) and absorption lag time (Alag) (Ka = 0.2 h-1; Alag = 0.83 h; central compartment clearance [CLc/F] for CYP2B6*1/*1 = 18 liters/h, for CYP2B6*1/*6 = 14 liters/h, and for CYP2B6*6/*6 = 8.6 liters/h) and PBMCs as a peripheral compartment. EFV transfer from plasma to PBMCs was first order (CLp/F = 32 liters/h), followed by capacity-limited return (Vmax = 4,400 ng/ml/h; Km = 710 ng/ml). Similarly, 8OHEFV displayed a first-order elimination and distribution to PBMCs, with a capacity-limited return to plasma. No covariate relationships resulted in a significant explanation of interindividual variability (IIV) on the estimated PK parameters of EFV and 8OHEFV, except for CYP2B6*6 genotypes, which were consistent with prior evidence. Both EFV and 8OHEFV accumulated to higher concentrations in PBMCs than in plasma and were well described by first-order input and Michaelis-Menten kinetics removal from PBMCs. CYP2B6*6 genotype polymorphisms were associated with decreased EFV and 8OHEFV clearance.

Evaluation of CYP2B6 Induction and Prediction of Clinical Drug-Drug Interactions: Considerations from the IQ Consortium Induction Working Group-An Industry Perspective.

Drug-drug interactions (DDIs) due to CYP2B6 induction have recently gained prominence and clinical induction risk assessment is recommended by regulatory agencies. This work aimed to evaluate the potency of CYP2B6 versus CYP3A4 induction in vitro and from clinical studies and to assess the predictability of efavirenz versus bupropion as clinical probe substrates of CYP2B6 induction. The analysis indicates that the magnitude of CYP3A4 induction was higher than CYP2B6 both in vitro and in vivo. The magnitude of DDIs caused by induction could not be predicted for bupropion with static or dynamic models. On the other hand, the relative induction score, net effect, and physiologically based pharmacokinetics SimCYP models using efavirenz resulted in improved DDI predictions. Although bupropion and efavirenz have been used and are recommended by regulatory agencies as clinical CYP2B6 probe substrates for DDI studies, CYP3A4 contributes to the metabolism of both probes and is induced by all reference CYP2B6 inducers. Therefore, caution must be taken when interpreting clinical induction results because of the lack of selectivity of these probes. Although in vitro-in vivo extrapolation for efavirenz performed better than bupropion, interpretation of the clinical change in exposure is confounded by the coinduction of CYP2B6 and CYP3A4, as well as the increased contribution of CYP3A4 to efavirenz metabolism under induced conditions. Current methods and probe substrates preclude accurate prediction of CYP2B6 induction. Identification of a sensitive and selective clinical substrate for CYP2B6 (fraction metabolized > 0.9) is needed to improve in vitro-in vivo extrapolation for characterizing the potential for CYP2B6-mediated DDIs. Alternative strategies and a framework for evaluating the CYP2B6 induction risk are proposed.

Establishment of In Silico Prediction Models for CYP3A4 and CYP2B6 Induction in Human Hepatocytes by Multiple Regression Analysis Using Azole Compounds.

Drug-drug interactions (DDIs) via cytochrome P450 (P450) induction are one clinical problem leading to increased risk of adverse effects and the need for dosage adjustments and additional therapeutic monitoring. In silico models for predicting P450 induction are useful for avoiding DDI risk. In this study, we have established regression models for CYP3A4 and CYP2B6 induction in human hepatocytes using several physicochemical parameters for a set of azole compounds with different P450 induction as characteristics as model compounds. To obtain a well-correlated regression model, the compounds for CYP3A4 or CYP2B6 induction were independently selected from the tested azole compounds using principal component analysis with fold-induction data. Both of the multiple linear regression models obtained for CYP3A4 and CYP2B6 induction are represented by different sets of physicochemical parameters. The adjusted coefficients of determination for these models were of 0.8 and 0.9, respectively. The fold-induction of the validation compounds, another set of 12 azole-containing compounds, were predicted within twofold limits for both CYP3A4 and CYP2B6. The concordance for the prediction of CYP3A4 induction was 87% with another validation set, 23 marketed drugs. However, the prediction of CYP2B6 induction tended to be overestimated for these marketed drugs. The regression models show that lipophilicity mostly contributes to CYP3A4 induction, whereas not only the lipophilicity but also the molecular polarity is important for CYP2B6 induction. Our regression models, especially that for CYP3A4 induction, might provide useful methods to avoid potent CYP3A4 or CYP2B6 inducers during the lead optimization stage without performing induction assays in human hepatocytes.

Rifampin enhances cytochrome P450 (CYP) 2B6-mediated efavirenz 8-hydroxylation in healthy volunteers.

The effect of rifampin on the in vivo metabolism of the antiretroviral drug efavirenz was evaluated in healthy volunteers. In a cross-over placebo control trial, healthy subjects (n = 20) were administered a single 600 mg oral dose of efavirenz after pretreatment with placebo or rifampin (600 mg/day for 10 days). Plasma and urine concentrations of efavirenz, 8-hydroxyefavirenz and 8,14-dihydroxyefavirenz were measured by LC-MS/MS. Compared to placebo treatment, rifampin increased the oral clearance (by ∼2.5-fold) and decreased maximum plasma concentration (Cmax) and area under the plasma concentration-time curve (AUC0-∞) of efavirenz (by ∼1.6- and ∼2.5-fold respectively) (p < 0.001). Rifampin treatment substantially increased the Cmax and AUC0-12h of 8-hydroxyefavirenz and 8,14-dihydroxyefavirenz, metabolic ratio (AUC0-72h of metabolites to AUC0-72h efavirenz) and the amount of metabolites excreted in urine (Ae0-12hr) (all, p < 0.01). Female subjects had longer elimination half-life (1.6-2.2-fold) and larger weight-adjusted distribution volume (1.6-1.9-fold) of efavirenz than male subjects (p < 0.05) in placebo and rifampin treated groups respectively. In conclusion, rifampin enhances CYP2B6-mediated efavirenz 8-hydroxylation in vivo. The metabolism of a single oral dose of efavirenz may be a suitable in vivo marker of CYP2B6 activity to evaluate induction drug interactions involving this enzyme.

Optical Isomers of Atorvastatin, Rosuvastatin and Fluvastatin Enantiospecifically Activate Pregnane X Receptor PXR and Induce CYP2A6, CYP2B6 and CYP3A4 in Human Hepatocytes.

Atorvastatin, fluvastatin and rosuvastatin are drugs used for treatment of hypercholesterolemia. They cause numerous drug-drug interactions by inhibiting and inducing drug-metabolizing cytochromes P450. These three statins exist in four optical forms, but they are currently used as enantiopure drugs, i.e., only one single enantiomer. There are numerous evidences that efficacy, adverse effects and toxicity of drugs may be enantiospecific. Therefore, we investigated the effects of optical isomers of atorvastatin, fluvastatin and rosuvastatin on the expression of drug-metabolizing P450s in primary human hepatocytes, using western blots and RT-PCR for measurement of proteins and mRNAs, respectively. The activity of P450 transcriptional regulators, including pregnane X receptor (PXR), aryl hydrocarbon receptor (AhR) and glucocorticoid receptor (GR), was assessed by gene reporter assays and EMSA. Transcriptional activity of AhR was not influenced by any statin tested. Basal transcriptional activity of GR was not affected by tested statins, but dexamethasone-inducible activity of GR was dose-dependently and enantioselectively inhibited by fluvastatin. Basal and ligand-inducible transcriptional activity of PXR was dose-dependently influenced by all tested statins, and the potency and efficacy between individual optical isomers varied depending on statin and optical isomer. The expression of CYP1A1 and CYP1A2 in human hepatocytes was not influenced by tested statins. All statins induced CYP2A6, CYP2B6 and CYP3A4, and the effects on CYP2C9 were rather modulatory. The effects varied between statins and enantiomers and induction potency decreased in order: atorvastatin (RR>RS = SR>SS) > fluvastatin (SR>RS = SS>RR) >> rosuvastatin (only RS active). The data presented here might be of toxicological and clinical importance.

Characterization of CYP2B6 in a CYP2B6-humanized mouse model: inducibility in the liver by phenobarbital and dexamethasone and role in nicotine metabolism in vivo.

The aim of this study was to further characterize the expression and function of human CYP2B6 in a recently generated CYP2A13/2B6/2F1-transgenic (TG) mouse model, in which CYP2B6 is expressed selectively in the liver. The inducibility of CYP2B6 by phenobarbital (PB) and dexamethasone (DEX), known inducers of CYP2B6 in human liver, was examined in the TG mice, as well as in TG/Cyp2abfgs-null (or "CYP2B6-humanized") mice. Hepatic expression of CYP2B6 mRNA and protein was greatly induced by PB or DEX treatment in both TG and TG/Cyp2abfgs-null mice. Function of the transgenic CYP2B6 was first studied using bupropion as a probe substrate. In PB-treated mice, the rates of hepatic microsomal hydroxybupropion formation (at 50 µM bupropion) were >4-fold higher in TG/Cyp2abfgs-null than in Cyp2abfgs-null mice (for both male and female mice); the rate difference was accompanied by a 5-fold higher catalytic efficiency in the TG/Cyp2abfgs-null mice and was abolished by an antibody to CYP2B6. The ability of CYP2B6 to metabolize nicotine was then examined, both in vitro and in vivo. The rates of hepatic microsomal cotinine formation from nicotine were significantly higher in TG/Cyp2abfgs-null than in Cyp2abfgs-null mice, pretreated with PB or DEX. Furthermore, systemic nicotine metabolism was faster in TG/Cyp2abfgs-null than in Cyp2abfgs-null mice. Thus, the transgenic CYP2B6 was inducible and functional, and, in the absence of mouse CYP2A and CYP2B enzymes, it contributed to nicotine metabolism in vivo. The CYP2B6-humanized mouse will be valuable for studies on in vivo roles of hepatic CYP2B6 in xenobiotic metabolism and toxicity.

Evaluation of 309 molecules as inducers of CYP3A4, CYP2B6, CYP1A2, OATP1B1, OCT1, MDR1, MRP2, MRP3 and BCRP in cryopreserved human hepatocytes in sandwich culture.

1. Regulation of hepatic metabolism or transport may lead to increase in drug clearance and compromise efficacy or safety. In this study, cryopreserved human hepatocytes were used to assess the effect of 309 compounds on the activity and mRNA expression (using qPCR techniques) of CYP1A2, CYP2B6 and CYP3A4, as well as mRNA expression of six hepatic transport proteins: OATP1B1 (SCLO1B1), OCT1 (SLC22A1), MDR1 (ABCB1), MRP2 (ABCC2), MRP3 (ABCC3) and BCRP (ABCG2). 2. The results showed that 6% of compounds induced CYP1A2 activity (1.5-fold increase); 30% induced CYP2B6 while 23% induced CYP3A4. qPCR data identified 16, 33 or 32% inducers of CYP1A2, CYP2B6 or CYP3A4, respectively. MRP2 was induced by 27 compounds followed by MDR1 (16)>BCRP (9)>OCT1 (8)>OATP1B1 (5)>MRP3 (2). 3. CYP3A4 appeared to be down-regulated (≥2-fold decrease in mRNA expression) by 53 compounds, 10 for CYP2B6, 6 for OCT1, 4 for BCRP, 2 for CYP1A2 and OATP1B1 and 1 for MDR1 and MRP2. 4. Structure-activity relationship analysis showed that CYP2B6 and CYP3A4 inducers are bulky lipophilic molecules with a higher number of heavy atoms and a lower number of hydrogen bond donors. Finally, a strategy for testing CYP inducers in drug discovery is proposed.

Carboxymefloquine, the major metabolite of the antimalarial drug mefloquine, induces drug-metabolizing enzyme and transporter expression by activation of pregnane X receptor.

Malaria patients are frequently coinfected with HIV and mycobacteria causing tuberculosis, which increases the use of coadministered drugs and thereby enhances the risk of pharmacokinetic drug-drug interactions. Activation of the pregnane X receptor (PXR) by xenobiotics, which include many drugs, induces drug metabolism and transport, thereby resulting in possible attenuation or loss of the therapeutic responses to the drugs being coadministered. While several artemisinin-type antimalarial drugs have been shown to activate PXR, data on nonartemisinin-type antimalarials are still missing. Therefore, this study aimed to elucidate the potential of nonartemisinin antimalarial drugs and drug metabolites to activate PXR. We screened 16 clinically used antimalarial drugs and six major drug metabolites for binding to PXR using the two-hybrid PXR ligand binding domain assembly assay; this identified carboxymefloquine, the major and pharmacologically inactive metabolite of the antimalarial drug mefloquine, as a potential PXR ligand. Two-hybrid PXR-coactivator and -corepressor interaction assays and PXR-dependent promoter reporter gene assays confirmed carboxymefloquine to be a novel PXR agonist which specifically activated the human receptor. In the PXR-expressing intestinal LS174T cells and in primary human hepatocytes, carboxymefloquine induced the expression of drug-metabolizing enzymes and transporters on the mRNA and protein levels. The crucial role of PXR for the carboxymefloquine-dependent induction of gene expression was confirmed by small interfering RNA (siRNA)-mediated knockdown of the receptor. Thus, the clinical use of mefloquine may result in pharmacokinetic drug-drug interactions by means of its metabolite carboxymefloquine. Whether these in vitro findings are of in vivo relevance has to be addressed in future clinical drug-drug interaction studies.

Enhanced oral bioavailability of efavirenz by solid lipid nanoparticles: in vitro drug release and pharmacokinetics studies.

Solid lipid nanoparticle is an efficient lipid based drug delivery system which can enhance the bioavailability of poorly water soluble drugs. Efavirenz is a highly lipophilic drug from nonnucleoside inhibitor category for treatment of HIV. Present work illustrates development of an SLN formulation for Efavirenz with increased bioavailability. At first, suitable lipid component and surfactant were chosen. SLNs were prepared and analyzed for physical parameters, stability, and pharmacokinetic profile. Efavirenz loaded SLNs were formulated using Glyceryl monostearate as main lipid and Tween 80 as surfactant. ESLN-3 has shown mean particle size of 124.5 ± 3.2 nm with a PDI value of 0.234, negative zeta potential, and 86% drug entrapment. In vitro drug release study has shown 60.6-98.22% drug release in 24 h by various SLN formulations. Optimized SLNs have shown good stability at 40°C ± 2°C and 75 ± 5% relative humidity (RH) for 180 days. ESLN-3 exhibited 5.32-fold increase in peak plasma concentration (C max) and 10.98-fold increase in AUC in comparison to Efavirenz suspension (ES).