Drug Efavirenz Research Articles

Development and validation of sensitive methods for simultaneous determination of 9 antiviral drugs in different various environmental matrices by UPLC-MS/MS

Trace antiviral drug contamination in aquatic ecosystems is becoming a significant environmental concern that requires an urgent efficient determination method. Here we developed sensitive and robust multi-residue determination methods to simultaneously extract and analyze 9 commonly used antiviral drugs (abacavir, zidovudine, efavirenz, nevirapine, ritonavir, lopinavir, lamivudine, telbivudine and entecavir) in surface water, wastewater, sediment, and sludge. Water samples were extracted with solid-phase extraction (SPE) technique using tandem hydrophilic–lipophilic balance and graphitized carbon black cartridges, while sediment and sludge samples were extracted using QuEChERS (quick, easy, cheap, effective, rugged, and safe) method. The extraction conditions of SPE (pH and cartridge type) and QuEChERS (acetic acid content, salts reagent, and purification sorbent) methods were carefully optimized. We observed that under optimum conditions, the method quantification limits of the 9 antiviral drugs in water and solid samples ranged from 0.05 to 19.23 ng L−1 and from 0.02 to 7.38 ng g−1, respectively. For environmental samples spiking 3 different concentrations, the recovery values for the most targeted antiviral drugs ranged from 70 to 130%, except for efavirenz. All targeted antiviral drugs were detected in wastewater samples except for entecavir. We also found abacavir, efavirenz, ritonavir, lopinavir, and telbivudine in sediment and sludge samples. Notably, telbivudine was identified in all environmental matrices, with a high concentration of 127 ng L−1 and 222 ng g−1 in water and sediment samples, respectively.

Effect of High-Energy Milling on the Dissolution of Anti-HIV Drug Efavirenz in Different Solvents

The anti-HIV drug efavirenz (EFV) displays low and variable bioavailability because of its poor aqueous solubility. Ball milling is a simple and cost-effective alternative to traditional micronization to improve the solubility and dissolution rate of EFV. A multibody dynamics model was employed to optimize the milling process parameters, while the motion of the balls in the mill jar was monitored in operando. This led to a better understanding of the milling dynamics for efficient comminution and enhancement of EFV dissolution. The variability of results for different EFV batches was also considered. Depending on the EFV batch, there were intrinsic differences in how the milling affected the dissolution behavior and inhibition of HIV-1 infection. High-energy grinding is more effective on EFV materials containing an amorphous fraction; it helps to remove agglomeration and enhances dissolution. Polyvinylpyrrolidone (PVP) addition improves the dissolution by forming a hydrophilic layer on the EFV surface, thereby increasing the drug wettability. Polymorphism also affects the quality, dosage, and effectiveness of the drug. The mechanical stress effect and PVP addition on the EFV polymorphic transformation were monitored by X-ray powder diffraction, while the residual of ground EFV was collected after dissolution, analyzed by scanning electron microscopy, and provided insights into the morphological changes.

Low‐level determination of 4‐chlorobutyl‐(S)‐[4‐chloro‐2‐(4‐cyclopropyl‐1,1,1‐trifluoro‐2‐hydroxy‐but‐3yn‐2‐yl)phenyl] carbamate (4‐CTHC) in efavirenz drug substance by LC–MS

A rapid, simple, sensitive, and selective liquid chromatography-tandem mass spectrometry (LC-MS) test method was developed and validated for the trace level determination of 4-chlorobutyl-(S)-[4-chloro-2-(4-cyclopropyl-1,1,1-trifluoro-2-hydroxy-but-3yn-2-yl)phenyl] carbamate (4-CTHC). 4-CTHC is a potential genotoxic impurity in efavirenz drug substance and the acceptable level is 2.5 μg ml-1 with respect to analyte concentration according to ICH M7(R1) Multidisciplinary Guidelines M7(R1). The LC-MS/MS analysis of 4-CTHC impurity was carried out on a Kinetex C18 (150 × 4.6 mm, 5.0 μm) column. In this test procedure, the mobile phase was prepared with buffer (0.1% formic acid in water) and acetonitrile in the ratio of 1:3 (v/v). The method set flow rate was 0.4 ml min-1 . The method was developed with a short run time of <10 min. Selective ion monitoring acquisition mode and negative polarity electrospray ionization mode were used as an MS method to quantify genotoxic impurities at 422.25 Da. The method showed linearity in a range of 0.64-3.71 μg ml-1 with a correlation coefficient of 0.9992. The RSD for intra-day and inter-day precision was found to be <5%. The method's accuracy was in the range of 106.5-112.4% for the genotoxic impurity of 4-CTHC. The procedure was validated as per the current ICH Q2 (R1) guidelines and proved suitable for stability testing in the quality control laboratory for pharmaceutical preparations.

Untimed Efavirenz Drug Levels After Switching From Brand to Generic Formulations: A Short Communication.

In British Columbia, antiretrovirals are distributed at no cost to patients via a publicly funded program, using generic formulations if available. A generic efavirenz-emtricitabine-tenofovir DF (EFV-FTC-TDF) combination pill became available in April 2018. The authors compared EFV untimed drug levels in subjects switching from brand to generic EFV-FTC-TDF. Archived plasma HIV viral load samples were identified for consenting participants who switched from brand to generic EFV-FTC-TDF; 3 preswitch and 2-3 postswitch samples, collected ≥1 month apart were assessed for each subject. "Untimed" EFV concentrations with unknown dosing and collection time were assessed using a validated liquid chromatography-tandem mass spectrometry method. Participants' mean, minimum, and maximum EFV levels were compared using the Wilcoxon signed rank test. Participants with EFV levels in the range associated with lower risks of virologic failure and central nervous system toxicity (1000-4000 ng/mL), preswitch and postswitch, were enumerated. EFV levels were assessed in 297 preswitch and 249 postswitch samples from 99 participants, having exposure to brand and generic EFV for a median of 103 (Q1-Q3: 87-116) and 10.3 (Q1-Q3: 8.9-11.7) months, respectively. The final brand sample was collected at a median of 98 days preswitch; the first generic sample was collected at a median of 133 days postswitch. No significant differences were observed in participant mean EFV levels before (median 1968 ng/mL; Q1-Q3: 1534-2878 ng/mL) and after (median 1987 ng/mL; Q1-Q3: 1521-2834 ng/mL) switch (P = 0.85). Eighty participants had mean EFV levels within the 1000-4000 ng/mL range on the brand drug, of which 74 remained within this range postswitch. There were no statistically significant differences between untimed EFV levels in patients switching from the brand to generic EFV combination pill. Given the long elimination half-life of EFV, untimed drug levels may be a convenient way to estimate product bioequivalence.

Efavirenz, atazanavir, and ritonavir disrupt sarcoplasmic reticulum Ca2+ homeostasis in skeletal muscles

While muscle fatigue, pain and weakness are common co-morbidities in HIV-1 infected people, their underlying cause remain poorly defined. To this end, we evaluated whether the common antiretroviral drugs efavirenz (EFV), atazanavir (ATV) and ritonavir (RTV) could be a contributing factor by pertubating sarcoplasmic reticulum (SR) Ca2+ cycling. In live-cell imaging, EFV (6.0 μM), ATV (6.0 μM), and RTV (3.0 μM) elicited Ca2+ transients and blebbing of the plasma membranes of C2C12 skeletal muscle myotubes. Pretreating C2C12 skeletal muscle myotubes with the SR Ca2+ release channel blocker ryanodine (50 μM), slowed the rate and amplitude of Ca2+ release from and reuptake of Ca2+ into the SR. EFV, ATV and RTV (1 nM - 20 μM) potentiated and then displaced [3H] ryanodine binding to rabbit skeletal muscle ryanodine receptor Ca2+ release channel (RyR1). These drugs at concentrations 0.25–31.2 μM also increased and or decreased the open probability of RyR1 by altering its gating and conductance. ATV (≤5 μM) potentiated and >5μM inhibited the ability of sarco (endo)plasmic reticulum Ca2+-ATPase (SERCA1) to hydrolyze ATP and transport Ca2+. RTV (2.5–31.5 μM) dose-dependently inhibited SERCA1-mediated, ATP-dependent Ca2+ transport. EFV (0.25–31.5 μM) had no measurable effect on SERCA1's ability to hydrolyze ATP and transport Ca2+. These data support the notion that EFV, ATV and RTV could be contributing to skeletal muscle co-morbidities in PLWH by modulating SR Ca2+ homeostasis.

Development of reverse phase high-performance liquid chromatographic method for the estimation of HIV non-nucleoside reverse transcriptase inhibitor drug efavirenz in the rat brain

BackgroundThe brain is the potential viral reservoir, and estimating the antiviral drug concentration in the brain is a hurdle to the researchers as very few animal models are available for this study. The objective of the study was to develop and validate the RP-HPLC method for the estimation of antiviral drug efavirenz (EFV) in the brain of healthy Wistar rats. EFV was the first-line antiretroviral medication. The optimized HPLC condition used for the analysis had the mobile phase methanol to water (9:1) ratio. The flow rate was set at 0.8 mL/min, while the detection wavelength was 248 nm.ResultsThe retention time was found to be 5.7 min, and the % RSD was found within the limit. Recovery was found to be nearly 78%. The validation results were found to be within the limit range; hence, the obtained method was accurate, specific, rapid, and repeatable for estimation of EFV in the brain.ConclusionThis method for estimation of EFV in the rat brain can be applicable for pharmacokinetic and toxicology study of EFV in the brain after administration of EFV to rats.Graphical abstract.

Safety and Efficacy of Fixed Drug Combination Tenofovir, Lamivudine and Efavirenz to Prevent Transmission of HIV

Introduction: Mother-to-child transmission of HIV can occur during pregnancy, labour, or breastfeeding. The first-line regimen for prophylaxis in HIV infected pregnant women is combination of Tenofovir, Lamivudine and Efavirenz (TLE). Aim: To evaluate the safety and efficacy of the TLE regimen in the Prevention of Mother-To-Child Transmission (PMTCT) of HIV. Materials and Methods: The present hospital-based, retrospective cohort study was conducted at ART centre, Prevention of Parent to Child Transmission (PPTCT) centre, and Department of Medicine, SP Medical College, Bikaner from July 2016 to June 2019. HIV positive gravidas, on triple-drug regimen TLE (Tenofovir 300 mg, Lamivudine 300 mg, Efavirenz 600 mg) before conception and, those detected HIV reactive antenatally during study period were included in this study and started on TLE regimen. After delivery, these newborns were given syrup Nevirapine as per the PPTCT guidelines. Infants were tested with Rapid test and PCR for HIV, at six weeks, six months, 12 months, and 18 months of life. Results: Out of 87 pregnant women, enrolled and delivered at the study institute, 85 were live births and two were stillbirths. Out of 85 live-born babies, four have died during infancy and two were lost to follow-up despite repeated counselling. Five babies were referred to nearby Anti-Retroviral Therapy (ART) centers. So, the study followed 74 babies out of which one girl child was found to be positive for HIV-1 at 18 months of age (transmission rate of HIV was 1.35 and efficacy of TLE 98.65%). No major adverse effects of TLE were noted and all women continued TLE. Conclusion: The use of a triple-drug regimen (TLE) declined the risk of transmission of HIV from mother-to-child at negligible level, without drug resistance and with safety and tolerability as compared to single drug.

Parametric Approaches in Population Pharmacokinetics

Population pharmacokinetic (popPK) approaches have spread widely throughout clinical pharmacology research, and every clinician should have some understanding of them. After a general introduction on the fundamentals and fields of application of these approaches, this review focuses on parametric popPK methods to provide the clinicians with the conceptual tools to interpret appropriately the results of parametric popPK analyses and to understand their clinical utility. The emphasis is put on the clinical questions that popPK methods are best suited to address. The basic principles of the methodology are introduced first, and then the main algorithms and reference software programs used in such analyses are presented. The description of data analysis and clinical applications of the parametric popPK approach (ie, use in simulations and therapeutic drug monitoring) are illustrated with the example of the antiretroviral drug efavirenz.

Efavirenz induced gynecomastia, hidden reality: a case series

The prognosis of HIV infection has considerably improved following the introduction of highly active anti-retroviral therapy by reducing AIDS related morbidity and mortality. At the same time, ART drugs are well known for their side effects. Gynaecomastia is a lesser known side effect of a commonly used anti-retroviral drug efavirenz. There are very few reports of HAART-induced gynaecomastia in resource-limited settings. The current study presents a series of three cases that developed ultrasound confirmed gynaecomastia following efavirenz containing HAART. Initial reports of gynaecomastia related to HAART were in HIV patients with lipodystrophy, they were termed as pseudogynaecomastia. Gradually, few reports of efavirenz related gynaecomastia were published wherein other causes of gynaecomastia were ruled out. Several hypothesis have been suggested for the pathophysiology of development of gynaecomastia related to efavirenz consumption. All other causes were ruled out in our patients too. The incidence of gynaecomastia is increasing in men with HIV on HAART therapy, proper identification and management will promote better drug adherence.

Efavirenz induces DNA damage response pathway in lung cancer.

The cell-cycle related genes are potential gene targets in understanding the effects of efavirenz (EFV) in lung cancer. The present study aimed at investigating the expression changes of cell-cycle related genes in response to EFV drug treatment in human non-small cell lung carcinoma (A549) and normal lung fibroblast (MRC-5) cells. The loss in nuclear integrity in response to EFV was detected by 4′, 6-diamidino-2-phenylindole (DAPI) staining. Gene expression profiling was performed using human cell cycle PathwayFinder RT2 Profiler™ PCR Array. The expression changes of 84 genes key to the cell cycle pathway in humans following EFV treatment was examined. The R2 PCR Array analysis revealed a change in expression of selected gene targets (including MAD2L2, CASP3, AURKB). This change in gene expression was at least a two-fold between test (EFV treated) and the control. RT-qPCR confirmed the PCR array data. In addition to this, the ATM signaling pathway was shown to be upregulated following EFV treatment in MRC-5 cells. In particular, ATM’s upstream activation resulted in p53 upregulation in normal lung fibroblasts. Interestingly, the p53 signaling pathway was activated irrespective of the repressed ATM pathway in A549 cells as revealed by the Ingenuity Pathway Analysis (IPA). These EFV effects are similar to those of ionizing radiation and this suggests that EFV has anti-tumour properties.

Polymeric Precipitation Inhibitor Assisted Supersaturable SMEDDS of Efavirenz Based on Experimental Observations and Molecular Mechanics.

Supersaturable SMEDDS, a versatile dosage form, was investigated for improving the biopharmaceutical attributes and eradicating the food effect of poorly water soluble drug efavirenz. The present research pursues the development of efavirenz loaded Supersaturable Self- Microemulsifying Drug Delivery System (SS SMEDDS) for improving biopharmaceutical performance. Preformulation studies were carried out to determine the optimized range of lipid excipients to develop stable supersaturated SMEDDS (ST SMEDDS). The SS SMEDD formulation was prepared by adding hydroxypropyl methylcellulose as a polymeric precipitation inhibitor. The developed SS SMEDDS were evaluated for supersaturation behavior by performing in vitro supersaturation studies and molecular simulations by in silico docking. Dissolution was performed in biorelevant media to simulate fed/fasted conditions in gastrointestinal regions. Absorption behavior was determined through in vivo pharmacokinetics approach. The optimized ST SMEDDS formulation containing Maisine® CC, Tween 80 and Transcutol-P exhibited thermodynamic stability with quick rate of emulsification. The optimized SS SMEDDS containing suitable polymeric precipitation inhibitor exhibited enhanced efavirenz concentration in in vitro supersaturation test. The theoretical simulations by molecular docking revealed strong intermolecular interactions with a docking score of -3.004 KJ/mol. The dissolution performance of marketed product in biorelevant dissolution media inferred the existence of food effect in the dissolution of efavirenz. However, in SS SMEDDS, no significant differences in drug release behavior under different fasted/fed conditions signify that the food effect was neutralized. In vivo pharmacokinetics revealed a significant increase in the absorption profile of efavirenz from SS SMEDDS than that of ST SMEDDS and marketed product. The designed delivery system indicated promising results in developing an effectual EFV formulation for HIV treatment.

CYP46A1-dependent and independent effects of efavirenz treatment.

Cholesterol excess in the brain is mainly disposed via cholesterol 24-hydroxylation catalysed by cytochrome P450 46A1, a CNS-specific enzyme. Cytochrome P450 46A1 is emerging as a promising therapeutic target for various brain diseases with both enzyme activation and inhibition having therapeutic potential. The rate of cholesterol 24-hydroxylation determines the rate of brain cholesterol turnover and the rate of sterol flux through the plasma membranes. The latter was shown to affect membrane properties and thereby membrane proteins and membrane-dependent processes. Previously we found that treatment of 5XFAD mice, an Alzheimer’s disease model, with a small dose of anti-HIV drug efavirenz allosterically activated cytochrome P450 46A1 in the brain and mitigated several disease manifestations. Herein, we generated Cyp46a1−/−5XFAD mice and treated them, along with 5XFAD animals, with efavirenz to ascertain cytochrome P450 46A1-dependent and independent drug effects. Efavirenz-treated versus control Cyp46a1−/−5XFAD and 5XFAD mice were compared for the brain sterol and steroid hormone content, amyloid β burden, protein and mRNA expression as well as synaptic ultrastructure. We found that the cytochrome P450 46A1-dependent efavirenz effects included changes in the levels of brain sterols, steroid hormones, and such proteins as glial fibrillary acidic protein, Iba1, Munc13-1, post-synaptic density-95, gephyrin, synaptophysin and synapsin-1. Changes in the expression of genes involved in neuroprotection, neurogenesis, synaptic function, inflammation, oxidative stress and apoptosis were also cytochrome P450 46A1-dependent. The total amyloid β load was the same in all groups of animals, except lack of cytochrome P450 46A1 decreased the production of the amyloid β40 species independent of treatment. In contrast, altered transcription of genes from cholinergic, monoaminergic, and peptidergic neurotransmission, steroid sulfation and production as well as vitamin D3 activation was the main CYP46A1-independent efavirenz effect. Collectively, the data obtained reveal that CYP46A1 controls cholesterol availability for the production of steroid hormones in the brain and the levels of biologically active neurosteroids. In addition, cytochrome P450 46A1 activity also seems to affect the levels of post-synaptic density-95, the main postsynaptic density protein, possibly by altering the calcium/calmodulin-dependent protein kinase II inhibitor 1 expression and activity of glycogen synthase kinase 3β. Even at a small dose, efavirenz likely acts as a transcriptional regulator, yet this regulation may not necessarily lead to functional effects. This study further confirmed that cytochrome P450 46A1 is a key enzyme for cholesterol homeostasis in the brain and that the therapeutic efavirenz effects on 5XFAD mice are likely realized via cytochrome P450 46A1 activation.

Privileged scaffold inspired design of novel oxime-biphenyl-DAPYs in treatment of HIV-1.

Oxime is a key pharmacophore in drug development. The biphenyl diarylpyrimidines (DAPYs) have been developed by our group as novel non-nucleoside reverse transcriptase inhibitors (NNRTIs). In this study, fourteen oxime-biphenyl-DAPYs were designed and synthesized through a privileged scaffold inspired design strategy. They exhibited promising activity toward wild type HIV-1 and single mutant strains. Compound 7d was found to be the most potent one against both wild type (EC50=12.1nM) and E138K mutant strains (EC50=0.0270µM). It also had a much lower cytotoxicity (CC50>292µM) and higher selective index (SI>24105) than those of the FDA-approved drugs efavirenz and etravirine. Molecular docking and dynamics simulation predicted and disclosed the binding mode of compound 7d with the RT, providing the explanation on the antiviral activity. These results were helpful for subsequent structural optimizations in anti-HIV-1 drug discovery.

A Bifunctional Nucleoside Probe for the Inhibition of the Human Immunodeficiency Virus-Type 1 Reverse Transcriptase.

Nucleoside analogs have proven effective for the inhibition of viral polymerases and are the foundation of many antiviral therapies. In this work, the antiretroviral potential of 6-azauracil analogs was assessed using activity-based protein profiling techniques and functional assays. Probes based on the 6-azauracil scaffold were examined and found to bind to HCV polymerase and HIV-1 reverse transcriptase through covalent modification of residues near the active site. The modified sites on the HIV-1 RT were examined using a mass spectrometry approach, and it was discovered that the azauracil moieties modified the enzyme in proximity to its active site. However, these scaffolds gave little or no inhibition of enzyme activity. Instead, a bifunctional inhibitor was prepared using click chemistry to link the 6-azauracil moiety to azidothymidine (AzT) and the corresponding triphosphate (AzTTP). These bifunctional inhibitors were found to have potent inhibitory function through a mode of action that includes both alkylation and chain termination. An in vitro assay demonstrated that the bifunctional inhibitor was 23-fold more effective in inhibiting HIV-1 RT activity than the parent AzTTP. The bifunctional inhibitor was also tested in HIV-1 permissive T cells where it decreased Gag expression similarly to the front-line drug Efavirenz with no evidence of cytotoxicity. This new bifunctional scaffold represents an interesting tool for inhibiting HIV-1 by covalently anchoring a chain-terminating nucleoside analog in the active site of the reverse transcriptase, preventing its removal and abolishing enzymatic activity, and represents a novel mode of action for inhibiting polymerases including reverse transcriptases.

Development and validation of a UV-Visible method for the determination of the active principle Efavirenz in tablets

Methods proposed by pharmacopeias to control the quality of Efavirenz, an antiretroviral drug used in the treatment of AIDS are based on expensive equipment which are most of the time not available in African countries. To solve this issue, using UV-visible spectrophotometry, a cheap and easy-to-use device available in many laboratories on the African continent; a sensitive, reliable, simple and rapid method has been validated for the determination of the active substance Evafirenz in Efavirenz tablets, to control their quality by checking its dosage. The accuracy profile approach was adopted. The response function gave a correlation coefficient R2 = 0.9987. The detection and quantification limits were 0.15625 μg / mL and 0.515625 μg / mL, respectively. The acceptability limit has been set at 15%. The tolerance limits for the different concentration levels (80, 100 and 120%) are represented respectively as follows: lower limits 86.2; 90.3 and 96.1%; upper limits 100.2; 106.3 and 113.7%. The tolerance limits are within the acceptability limits for values around 100% concentration (7 μg / mL). Therefore, the method was declared valid and reliable for the analysis of Efavirenz in Efavirenz tablets and could routinely be used in laboratories for the quality control of EFAVIRENZ drug.&#x0D; © 2020 International Formulae Group. All rights reserved.&#x0D; Keywords: Efavirenz, accuracy profile, spectrophotometry, pharmaceutical formulation

Exploring the Effects of a V104F/V477F Double Mutation on CYP2B6 Activity Towards Efavirenz

BackgroundThe Cytochromes P450 2B6 (CYP2B6) and 2A6 (CYP2A6) participate in the metabolism of the anti‐HIV drug Efavirenz (EFV). CYP2B6 monohydroxylation of EFV results in the formation of 8‐hydroxyefavirenz (8‐OHEFV) whereas CYP2A6 monohydroxylation of EFV forms 7‐hydroxyefavirenz (7‐OHEFV). To date, little is known about the structural characteristics of these enzymes that lead to differential EFV metabolite formation. In order to better understand such characteristics, we sought to mutate CYP2B6 towards CYP2A6 in hopes of uncovering amino acids critical for the formation of 8‐OHEFV. Specifically, we decided to focus on two phenylalanine residues in the CYP2A6 active site that are valines at the analogous positions in CYP2B6.MethodsThe valine residues at positions 104 and 477 of CYP2B6 were mutated to phenylalanines using site directed mutagenesis, creating a V104F/V477F double mutant. Wild type (WT) and mutant CYP2B6 expression was accomplished in COS7 cells and, following treatment with EFV, metabolites were extracted and detected using a novel gas chromatography‐mass spectrometry (GC‐MS) method after chemical derivatization using BSTFA + 10% TMCS. Protein expression was assessed via western blotting.ResultsWe observed little to no difference in the protein expression of WT and mutant CYP2B6 proteins. 8‐OHEFV production in the COS7 system was detected for wild type and mutant CYP2B6, but no 7‐OHEFV production was observed. Interestingly, GC‐MS analysis of 8‐OHEFV formation by the V104F/V477F mutant indicates a marked increase in 8‐OHEFV production, suggesting the combination of these mutations might enhance 8‐OHEFV formation by CYP2B6.Future GoalsFuture experiments will seek to characterize each single mutant and determine kinetic parameters for 8‐OHEFV formation by all three mutant CYP2B6 proteins.Support or Funding InformationFunding for this work was generously provided by the Department of Biology and Chemistry at Azusa Pacific University and the Faculty Research Council of Azusa Pacific University

Brain sterol flux mediated by cytochrome P450 46A1 affects membrane properties and membrane-dependent processes.

Cytochrome P450 46A1 encoded by CYP46A1 catalyzes cholesterol 24-hydroxylation and is a CNS-specific enzyme that controls cholesterol removal and turnover in the brain. Accumulating data suggest that increases in cytochrome P450 46A1 activity in mouse models of common neurodegenerative diseases affect various, apparently unlinked biological processes and pathways. Yet, the underlying reason for these multiple enzyme activity effects is currently unknown. Herein, we tested the hypothesis that cytochrome P450 46A1-mediated sterol flux alters physico-chemical properties of the plasma membranes and thereby membrane-dependent events. We used 9-month-old 5XFAD mice (an Alzheimer’s disease model) treated for 6 months with the anti-HIV drug efavirenz. These animals have previously been shown to have improved behavioural performance, increased cytochrome P450 46A1 activity in the brain, and increased sterol flux through the plasma membranes. We further examined 9-month-old Cyp46a1−/− mice, which have previously been observed to have cognitive deficits and decreased sterol flux through brain membranes. Synaptosomal fractions from the brain of efavirenz-treated 5XFAD mice had essentially unchanged cholesterol levels as compared to control 5XFAD mice. However with efavirenz treatment in these mice, there were changes in the membrane properties (increased cholesterol accessibility, ordering, osmotic resistance and thickness) as well as total glutamate content and ability to release glutamate in response to mild stimulation. Similarly, the cholesterol content in synaptosomal fractions from the brain of Cyp46a1−/− mice was essentially the same as in wild-type mice but knockout of Cyp46a1 was associated with changes in membrane properties and glutamate content and its exocytotic release. Changes in Cyp46a1−/− mice were in the opposite direction to those observed in efavirenz-treated versus control 5XFAD mice. Incubation of synaptosomal fractions with the inhibitors of glycogen synthase kinase 3, cyclin-dependent kinase 5, protein phosphatase 1/2 A, and protein phosphatase 2B revealed that increased sterol flux in efavirenz-treated versus control 5XFAD mice affected the ability of all four enzymes to modulate glutamate release. In contrast, in Cyp46a1−/− versus wild-type mice, decreased sterol flux altered the ability of only cyclin-dependent kinase 5 and protein phosphatase 2B to regulate the glutamate release. Collectively, our results support cytochrome P450 46A1-mediated sterol flux as an important contributor to the fundamental properties of the membranes, protein phosphorylation and synaptic transmission. Also, our data provide an explanation of how one enzyme, cytochrome P450 46A1, can affect multiple pathways and processes and serve as a common potential target for several neurodegenerative disorders.

Development and Validation of RP-Chiral HPLC Method for Determination of (R)-Enantiomer Excess Content in Efavirenz

simple, specific, linear, accurate and precise reverse phase chiral HPLC method was developed for the separation of efavirenz enantiomers by using the Lux Amylose-2 column containing amylose tris(5-chloro-2-methyl phenyl carbamate) as a stationary phase. The mobile phase consists of 0.1 % formic acid in water and acetonitrile (55:45, v/v). The flow rate was kept at 1.0 mL/min and the detection wavelength used 252 nm and the column temperature was set at 25 ºC. The limit of detection was 0.01 mg/mL and the limit of quantification was 0.04 mg/mL. The linearity calibration curve of (R)-enantiomer was shown well from the range of 0.04 mg/mL to 0.4 mg/mL. The values of the correlation coefficient were 0.999 and 0.999 for (R)-enantiomer and (S)-efavirenz, respectively. The percentage recoveries of (R)-enantiomer from efavirenz drug substance were ranged from 93.5% to 107.5%. The results demonstrated that developed RP-chiral HPLC method was simple, precise, robust and applicable for the estimation of (R)-enantiomer in efavirenz API. This method was validated in as per ICH Q2 (R1) and USP validation of compendial methods &lt;1225&gt;.

Modelling the influx and efflux waves in drug movement: a basis for Pharmacokinetic-Pharmacodynamic link of efavirenz

A novel compartmental modelling approach to investigate influx and efflux distribution of the drug efavirenz is suggested. The models are developed for a patient who had been on 600 mg daily dose of efavirenz. The work makes use of suggested wave motion to model drug distribution. It offers new insights into PK-PD(Pharmacokinetic-Pharmacodynamic) modelling. The distributional systems (immission and emission) of efavirenz are investigated and models are suggested. Each of the two distributional systems consists of four descriptors a source (desorbate), an underlay(a substrate/stimulus), a facilitator (propeller) and a product (adsorbate). The immission distribution is responsible for adsorbate formation. The immission distributional kinetics model is applied in projecting bioavailability (F = 0.194 5), extrinsic pass effects availability , immission distributional volumes , elimination rate constants and clearance parameters . Furthermore, the emission system model introduces a two-phased uptake for distribution of the efavirenz solution particle. The emission distribution is responsible for adsorbate degradation. It establishes a concentration-dependent peripheral uptake and a central one which is independent of concentration. The two distributional systems (waves) proposes a basis and subsequent space for a linear response. An elasticity measure is suggested for resistance to distributional flow. The developed models show how concentration can be used to characterise and predict distributional kinetics. Several relationships can be inferred from models suggested herein such as links to assertations from the Navier–Stokes and Noyes-Whitney equations.

Molecular Hybridization-Inspired Optimization of Diarylbenzopyrimidines as HIV-1 Nonnucleoside Reverse Transcriptase Inhibitors with Improved Activity against K103N and E138K Mutants and Pharmacokinetic Profiles.

Molecular hybridization is a powerful strategy in drug discovery. A series of novel diarylbenzopyrimidine (DABP) analogues were developed by the hybridization of FDA-approved drugs etravirine (ETR) and efavirenz (EFV) as potential HIV-1 nonnucleoside reverse transcriptase inhibitors (NNRTIs). Substituent modifications resulted in the identification of new DABPs with the combination of the strengths of the two drugs, especially compound 12d, which showed promising activity toward the EFV-resistant K103N mutant. 12d also had a favorable pharmacokinetic (PK) profile with liver microsome clearances of 14.4 μL/min/mg (human) and 33.2 μL/min/mg (rat) and an oral bioavailability of 15.5% in rat. However, its activity against the E138K mutant was still unsatisfactory; E138K is the most prevalent NNRTI resistance-associated mutant in ETR treatment. Further optimizations resulted in a highly potent compound (12z) with no substituents on the phenyl ring and a 2-methyl-6-nitro substitution pattern on the 4-cyanovinyl-2,6-disubstitued phenyl motif. The antiviral activity of this compound was much higher than those of ETR and EFV against the WT, E138K, and K103N variants (EC50 = 3.4, 4.3, and 3.6 nM, respectively), and the cytotoxicity was decreased while the selectivity index (SI) was increased. In particular, this compound exhibited acceptable intrinsic liver microsome stability (human, 34.5 μL/min/mg; rat, 33.2 μL/min/mg) and maintained the good PK profile of its parent compound EFV and showed an oral bioavailability of 16.5% in rat. Molecular docking and structure-activity relationship (SAR) analysis provided further insights into the binding of the DABPs with HIV-1 reverse transcriptase and provided a deeper understanding of the key structural features responsible for their interactions.