8-hydroxyefavirenz and efavirenz

The purpose of this study was to determine the demographic and pharmacogenetic covariates that influence the disposition of efavirenz (EFV) and its major metabolites. A population pharmacokinetic (PK) model was developed from a randomized, cross-over, drug-interaction study in healthy male Korean subjects (n = 17). Plasma concentrations of EFV and its hydroxy-metabolites (0-120 hours) were measured by LC/MS/MS. Genomic DNA was genotyped for variants in the cytochrome P450 (CYP) 2A6, 2B6, 3A5, and MDR1 genes. A PK model was built in a stepwise procedure using nonlinear mixed effect modeling in NONMEM 7. The covariate model was built using the generalized additive modeling and forward selection-backward elimination. Model-based simulations were performed to predict EFV steady-state concentrations following 200, 400, and 600 mg daily oral dose among different CYP2B6 genotypes. The final model included only CYP2B6 genotype as a covariate that predicts EFV clearance through the formation of 8-OH EFV that represented 65% to 80% of EFV clearance. The total clearance of EFV in CYP2B6*6/*6 genotype was ∼30% lower than CYP2B6*1/*1 or CYP2B6*1/*6 alleles (P < .001). Clopidogrel reduced both formation and elimination clearances of 8-OH EFV by 22% and 19%, respectively (P = .033 and .041). Other demographics and genotype of accessory CYP pathways did not predict EFV or metabolites PK. CYP2B6 genotype was the only significant predictor of EFV disposition. The developed model may serve as the foundation for further exploration of pharmacogenetic-based dosing of EFV.

Topics: Adult; Algorithms; Alkynes; Area Under Curve; ATP Binding Cassette Transporter, Subfamily B, Member 1; Benzoxazines; Chromatography, High Pressure Liquid; Clopidogrel; Computer Simulation; Cross-Over Studies; Cyclopropanes; Cytochrome P-450 Enzyme System; DNA; Drug Interactions; Humans; Male; Models, Biological; Pharmacogenetics; Population; Reverse Transcriptase Inhibitors; Tandem Mass Spectrometry; Ticlopidine; Treatment Outcome; Young Adult

Cytochrome P450 46A1 (CYP46A1) is a central nervous system-specific enzyme, which catalyzes cholesterol 24-hydroxylation. Currently CYP46A1 is being evaluated in a clinical trial for activation by small doses of the anti-HIV drug efavirenz. Eight efavirenz-related compounds were investigated for CYP46A1 activation in vitro, induction of a CYP46A1 spectral response, spectral

Topics: Alkynes; Allosteric Site; Benzoxazines; Catalytic Domain; Cholesterol; Cholesterol 24-Hydroxylase; Cyclopropanes; Cytochrome P-450 CYP3A Inducers; Humans; Hydroxylation; In Vitro Techniques; Pharmaceutical Preparations; Protein Conformation

Efavirenz (EFV), a widely used antiretroviral drug, is associated with idiosyncratic hepatotoxicity and dyslipidemia. Here we demonstrate that EFV stimulates the activation in primary hepatocytes of key cell stress regulators: inositol-requiring 1

Topics: Alkynes; Animals; Benzoxazines; Cells, Cultured; Cyclopropanes; Endoribonucleases; Female; Hepatocytes; Humans; Liver; Macaca; Male; Mice; Mice, Inbred C57BL; Phosphorylation; RNA Splicing; Signal Transduction; Transcription Factors; X-Box Binding Protein 1

Efavirenz (EFV), an antiretroviral that interacts clinically with co-administered drugs via activation of the pregnane X receptor (PXR), is extensively metabolized by the cytochromes P450. We tested whether its primary metabolite, 8-hydroxyEFV (8-OHEFV) can activate PXR and potentially contribute to PXR-mediated drug-drug interactions attributed to EFV. Luciferase reporter assays revealed that despite only differing from EFV by an oxygen atom, 8-OHEFV does not activate PXR. Corroborating this, treatment with EFV for 72 h elevated the mRNA abundance of the PXR target gene, Cyp3a11, by approximately 28-fold in primary hepatocytes isolated from PXR-humanized mice, whereas treatment with 8-OHEFV did not result in a change in Cyp3A11 mRNA levels. FRET-based competitive binding assays and isothermal calorimetry demonstrated that even with the lack of ability to activate PXR, 8-OHEFV displays an affinity for PXR (IC

Topics: Alkynes; Animals; Benzoxazines; Binding Sites; Cyclopropanes; Hepatocytes; Humans; Ligands; Mice; Molecular Docking Simulation; Molecular Structure; Pregnane X Receptor; Protein Binding; Structure-Activity Relationship

Efavirenz is an anti-HIV drug that presents relevant short- and long-term central nervous system adverse reactions. Its main metabolite (8-hydroxy-efavirenz) was demonstrated to be a more potent neurotoxin than efavirenz itself. This work was aimed to understand how efavirenz biotransformation to 8-hydroxy-efavirenz is related to its short- and long-term neuro-adverse reactions. To access those mechanisms, the expression and activity of Cyp2b enzymes as well as the thiolomic signature (low molecular weight thiols plus S-thiolated proteins) were longitudinally evaluated in the hepatic and brain tissues of rats exposed to efavirenz during 10 and 36days. Efavirenz and 8-hydroxy-efavirenz plasma concentrations were monitored at the same time points. Cyp2b induction had a delayed onset in liver (p<0.001), translating into increases in Cyp2b activity in liver and 8-hydroxy-efavirenz plasma concentration (p<0.001). Moreover, an increase in S-cysteinyl-glycinylated proteins (p<0.001) and in free low molecular weight thiols was also observed in liver. A distinct scenario was observed in hippocampus, which showed an underexpression of Cyp2b as well as a decrease in S-cysteinylated and S-glutathionylated proteins. Additionally, the observed changes in tissues were associated with a marked increase of S-glutathionylation in plasma. Our data suggest that the time course of efavirenz biotransformation results from different mechanisms for its short- and long-term neurotoxicity. The difference in the redox profile between liver and hippocampus might explain why, despite being mostly metabolized by the liver, this drug is neurotoxic. If translated to clinical practice, this evidence will have important implications in efavirenz short- and long-term neurotoxicity prevention and management.

Topics: Alkynes; Animals; Anti-HIV Agents; Aryl Hydrocarbon Hydroxylases; Benzoxazines; Biotransformation; Cyclopropanes; Cytochrome P-450 CYP2B1; Hippocampus; Liver; Male; Neurotoxicity Syndromes; Prefrontal Cortex; Rats, Wistar; Steroid Hydroxylases; Sulfhydryl Compounds

Cynomolgus monkeys are used frequently in preclinical studies for new drug development due to their evolutionary closeness to humans. An antiretroviral drug, efavirenz, is a typical probe substrate for human cytochrome P450 (P450) 2B6, but is mainly metabolized by cynomolgus monkey P450 2C9. In this study, plasma concentrations of efavirenz were assessed in six cynomolgus monkeys genotyped for P450 2C9 c.334 A > C (I112L) (three wild-type, one heterozygote and two homozygotes) by high performance liquid chromatography with tandem mass spectrometry. After intravenous administration at a dose of 1.0 mg/kg, biphasic plasma elimination curves of efavirenz were seen in these cynomolgus monkeys. The mean plasma concentration of the primary metabolite 8-hydroxyefavirenz (1 h after treatment, with hydrolysis by β-glucuronidase) in the wild-type group was significantly higher (4.0-fold) than the combined heterozygous and homozygous group mean. The area under the plasma concentration-time curve value of efavirenz in the homozygous group after oral administration at a dose of 2.0 mg/kg was significantly higher (2.0-fold) than the combined wild-type and heterozygous group. These results collectively indicated that P450 2C9 c.334 A > C (I112L) variation was associated with efavirenz metabolic clearance in vivo. Cynomolgus P450 2C9 polymorphism might account for interindividual variations of efavirenz metabolism in cynomolgus monkeys. Copyright © 2016 John Wiley & Sons, Ltd.

Topics: Alkynes; Animals; Anti-HIV Agents; Benzoxazines; Cyclopropanes; Cytochrome P-450 CYP2C9; Genotype; Macaca fascicularis; Reverse Transcriptase Inhibitors

Efavirenz is a drug of choice for adults and children infected with the human immunodeficiency virus. Notably, up to 35% of patients on efavirenz suffer from mood changes. This work aimed to investigate efavirenz biotransformation into 8-hydroxy-efavirenz as an up-stream event of mood changes and to evaluate the suitability of 8-hydroxy-efavirenz biomonitoring for the minimization of these manifestations. A case-control study with two age-matched groups of HIV-infected male patients was performed in a group without adverse central nervous system complaints (28 patients) and a group presenting mood changes (14 patients). The plasma concentration of non-conjugated 8-hydroxy-efavirenz was higher in patients with mood changes (p=0.020). An association between efavirenz and 8-hydroxy-efavirenz-glucuronide was found (Spearman r=0.414, p<0.010), only within therapeutic efavirenz concentrations. This correlation was not observed in patients with toxic (>4mg/L) plasma concentrations of the parent drug. We conclude that metabolism to 8-hydroxy-efavirenz is associated with efavirenz-related mood changes, which suggests that the concentration of this metabolite is a suitable parameter for therapeutic drug monitoring aimed at controlling these manifestations. Moreover, our data suggest that 8-hydroxy-efavirenz is able to cross the blood-brain barrier and that the peripheral detoxification of 8-hydroxy-efavirenz by glucuronidation may be inhibited by toxic efavirenz concentrations.

Topics: Adult; Alkynes; Anti-HIV Agents; Anxiety; Benzoxazines; Biomarkers, Pharmacological; Biotransformation; Blood-Brain Barrier; Case-Control Studies; CD4 Lymphocyte Count; Cyclopropanes; Drug Monitoring; Drug Therapy, Combination; Feasibility Studies; Glucuronides; HIV Infections; Humans; Male; Metabolic Detoxication, Phase II; Middle Aged; Mood Disorders; Neurotoxicity Syndromes; Prevalence; Reverse Transcriptase Inhibitors

Efavirenz is a widely prescribed non-nucleoside reverse transcriptase inhibitor for the treatment of HIV infections. To test for potential long-term consequences of efavirenz on brain cells, cultured primary astrocytes were incubated with this substance or with its primary metabolite 8-hydroxy efavirenz for up to 7 days. Both, efavirenz and 8-hydroxy efavirenz caused time- and concentration-dependent cell toxicity and stimulated in subtoxic concentrations the glycolytic flux (glucose consumption and lactate release) in astrocytes. As 8-hydroxy efavirenz was less toxic than efavirenz and stimulated glycolysis in lower concentrations we tested for a potential hydroxylation of efavirenz to 8-hydroxy efavirenz in astrocytes. Analysis of media and cell lysates by HPLC-UV and mass spectrometry revealed that after 3 days of incubation viable astrocytes had accumulated about 17 and 7 % of the applied efavirenz and 8-hydroxy efavirenz, respectively. However, in cultures treated with efavirenz neither 8-hydroxy efavirenz nor any other known metabolite of efavirenz was detectable. These data demonstrate that cultured rat astrocytes efficiently accumulate, but not metabolize, efavirenz and 8-hydroxy efavirenz and that the observed chronic stimulation of glycolysis is mediated by both efavirenz and 8-hydroxy efavirenz.

Topics: Alkynes; Animals; Animals, Newborn; Anti-HIV Agents; Astrocytes; Benzoxazines; Brain; Cell Survival; Cells, Cultured; Cyclopropanes; Glucose; Lactic Acid; Rats, Wistar; Reverse Transcriptase Inhibitors; Stereoisomerism

Efavirenz (EFV) is one of the most commonly prescribed antiretrovirals for use in the treatment of human immunodeficiency virus (HIV) infection. EFV is extensively metabolized by cytochrome P450 to a number of oxygenated products; however, the pharmacologic activity and distribution of these metabolites in anatomic compartments have yet to be explored. The systemic distribution of EFV oxidative metabolites was examined in blood plasma, seminal plasma, and cerebrospinal fluid from subjects on an EFV-based regimen. The 8-hydroxy EFV metabolite was detected in blood plasma, seminal plasma, and cerebrospinal fluid, with median concentrations of 314.5 ng/ml, 358.5 ng/ml, and 3.37 ng/ml, respectively. In contrast, 7-hydroxy and 8,14-hydroxy EFV were only detected in blood plasma and seminal plasma with median concentrations of 8.84 ng/ml and 10.23 ng/ml, and 5.63 ng/ml and 5.43 ng/ml, respectively. Interestingly, protein-free concentrations of metabolites were only detectable in seminal plasma, where a novel dihdyroxylated metabolite of EFV was also detected. This accumulation of protein-free EFV metabolites was demonstrated to be the result of differential protein binding in seminal plasma compared with that of blood plasma. In addition, the oxidative metabolites of EFV did not present with any significant pharmacologic activity toward HIV-1 as measured using an HIV green fluorescent protein single-round infectivity assay. This study is the first to report the physiologic distribution of metabolites of an antiretroviral into biologic compartments that the virus is known to distribute and to examine their anti-HIV activity. These data suggest that the male genital tract may be a novel compartment that should be considered in the evaluation of drug metabolite exposure.

Topics: Adult; Aged; Alkynes; Anti-HIV Agents; Benzoxazines; Biotransformation; CD4-Positive T-Lymphocytes; Cells, Cultured; Cyclopropanes; Cytochrome P-450 Enzyme System; Female; HIV Reverse Transcriptase; HIV-1; Humans; Hydroxylation; Isoenzymes; Male; Microsomes, Liver; Middle Aged; Oxidation-Reduction; Protein Binding; Recombinant Proteins; Reverse Transcriptase Inhibitors; Semen; Tissue Distribution

The objective of this study was to assess the incidence, timing and identify pharmacogenetic, efavirenz (EFV) pharmacokinetic and biochemical predictors of EFV-based antiretroviral therapy (ART) drug-induced liver injury (DILI). ART-naïve HIV patients (n = 285) were prospectively enrolled. Pretreatment laboratory evaluations included hepatitis B surface antigen and C antibody, CD4 count and viral load. Liver tests were done at baseline, 1st, 2nd, 4th, 8th, 12th, 24th and 48th weeks during ART. Plasma EFV and 8-hydroxyefvairenz concentration was determined at week 4 using liquid chromatography-mass spectrometry. CYP2B6, CYP3A5, ABCB1 3435C/T and UGT2B7*2 genotyping was done using Taqman genotyping assay. Data were analyzed using survival analysis and Cox proportional hazards model. The incidence of DILI was 15.7% or 27.9 per 100 person-years and that of severe injury was 3.4% or 6.13 per 100 person-years. The median time for the development of DILI and severe injury was 2 and 4 weeks after initiation of ART, respectively. There was significant association of DILI with lower baseline platelet, albumin, log plasma viral load and CD4 count (P = 0.031, 0.037, 0.06 and 0.019, respectively). Elevated baseline alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, plasma EFV level and CYP2B6*6 were good predictors for the development of DILI (P = 0.03, 0.01, 0.016, 0.017 and 0.04, respectively). We report for the first time CYP2B6*6 as a putative genetic marker and high plasma EFV concentration as intermediate biomarker for vulnerability to EFV-induced liver injury in HIV patients. CYP2B6 genotyping and/or regular monitoring of EFV and lever enzymes level during early therapy is advised for early diagnosis and management of DILI.

Topics: Adult; Alkynes; Antiretroviral Therapy, Highly Active; Aryl Hydrocarbon Hydroxylases; ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily B, Member 1; Benzoxazines; Chemical and Drug Induced Liver Injury; Cohort Studies; Cyclopropanes; Cytochrome P-450 CYP2B6; Cytochrome P-450 CYP3A; Female; Genotype; HIV Infections; Humans; Male; Oxidoreductases, N-Demethylating; Proportional Hazards Models; Prospective Studies

Despite combination antiretroviral therapies (cARTs), a significant proportion of HIV-infected patients develop HIV-associated neurocognitive disorders (HAND). Ongoing viral replication in the central nervous system (CNS) caused by poor brain penetration of cART may contribute to HAND. However, it has also been proposed that the toxic effects of long-term cART may contribute to HAND. A better understanding of the neurotoxic potential of cART is critically needed in light of the use of CNS-penetrating cARTs to contend with the virus reservoir in the brain. The efavirenz (EFV) metabolites 7-hydroxyefavirenz (7-OH-EFV) and 8-hydroxyefavirenz (8-OH-EFV) were synthesized and purified, and their chemical structures were confirmed by mass spectrometry and NMR. The effects of EFV, 7-OH-EFV, and 8-OH-EFV on calcium, dendritic spine morphology, and survival were determined in primary neurons. EFV, 7-OH-EFV, and 8-OH-EFV each induced neuronal damage in a dose-dependent manner. However, 8-OH-EFV was at least an order of magnitude more toxic than EFV or 7-OH-EFV, inducing considerable damage to dendritic spines at a 10 nM concentration. The 8-OH-EFV metabolite evoked calcium flux in neurons, which was mediated primarily by L-type voltage-operated calcium channels (VOCCs). Blockade of L-type VOCCs protected dendritic spines from 8-OH-EFV-induced damage. Concentrations of EFV and 8-OH-EFV in the cerebral spinal fluid of HIV-infected subjects taking EFV were within the range that damaged neurons in culture. These findings demonstrate that the 8-OH metabolite of EFV is a potent neurotoxin and highlight the importance of directly determining the effects of antiretroviral drugs and drug metabolites on neurons and other brain cells.

Topics: Alkynes; Animals; Anti-HIV Agents; Apoptosis; Benzoxazines; Calcium; Cell Survival; Cells, Cultured; Cyclopropanes; Cytosol; Dendritic Spines; Dose-Response Relationship, Drug; Female; Humans; Male; Middle Aged; Rats; Tandem Mass Spectrometry

We investigated the long-term effect of efavirenz autoinduction on its plasma/peripheral blood mononuclear cell (PBMC) exposure and the CD4 count, and the importance of sex and pharmacogenetic variations.. Treatment-naive HIV patients (n = 163) received efavirenz-based antiretroviral therapy. Plasma and intracellular (PBMC) concentrations of efavirenz and 8-hydroxyefavirenz were determined at weeks 4 and 16 of antiretroviral therapy. CD4 count was determined at baseline, and at weeks 12, 24 and 48. Genotyping for CYP2B6*6, CYP3A5*3, CYP3A5*6, CYP3A5*7, ABCB1 3435C/T and UGT2B7 (-327G→A, *2) was done.. There was a significant increase in the median plasma (32%) and intracellular (53%) 8-hydroxyefavirenz concentrations with a decrease in the efavirenz metabolic ratio (MR) (calculated by dividing the concentration of efavirenz by that of 8-hydroxyefavirenz) (20% and 5%, respectively) by week 16 compared with at week 4. While the CYP2B6 genotype significantly influenced efavirenz pharmacokinetics at weeks 4 and 16, the effect of the UGT2B7 genotype and sex was significant only at week 16. The Wilcoxon matched pairs test indicated that the change in 8-hydroxyefavirenz concentration and efavirenz MR over time was significant in females and in CYP2B6*1 and UGT2B7*1 carriers. The intracellular 8-hydroxyefavirenz level at week 16 was a negative predictor of the CD4 count at week 24 (P = 0.03) and at week 48 (P = 0.007). CYP2B6 (P = 0.02) and UGT2B7 (P = 0.05) genotypes predicted the CD4 count at week 48. Among CYP2B6*1/*1 and UGT2B7*1/*1 carriers there was no significant change in the mean CD4 count after week 24, while it continuously increased until week 48 in CYP2B6*6 and UGT2B7*2 carriers.. The effects of long-term efavirenz autoinduction on its plasma/PBMC exposure and the CD4 count over time display wide interpatient variability, partly due to sex and CYP2B6 and UGT2B7 genetic variation. Patients with the CYP2B6*1/*1 and UGT2B7*1/*1 genotypes are at risk of suboptimal immune recovery due to pronounced long-term autoinduction.

Topics: Adult; Alkynes; Anti-HIV Agents; Antiretroviral Therapy, Highly Active; Aryl Hydrocarbon Hydroxylases; Benzoxazines; CD4 Lymphocyte Count; Cyclopropanes; Cytochrome P-450 CYP2B6; Enzyme Induction; Female; Genotype; Glucuronosyltransferase; HIV Infections; HIV-1; Humans; Leukocytes, Mononuclear; Male; Oxidoreductases, N-Demethylating; Sex Factors

Studies on the biotransformation of the clinically important non-nucleoside reverse transcriptase inhibitor efavirenz have shown that oxidation and secondary conjugation are important components of the processing of this molecule in vivo. We have synthesized metabolites of efavirenz to confirm their structure and to evaluate their activity as antivirals.

Topics: Alkynes; Animals; Antiviral Agents; Benzoxazines; Biotransformation; Cyclopropanes; Humans; Molecular Structure; Oxazines; Reverse Transcriptase Inhibitors