Analysis of ART effects and drug resistance in adult HIV/AIDS patients in Meigu County, Liangshan Prefecture, China.

BACKGROUND OBJECTIVE: This study aimed to understand the basic situation of adults with human immunodeficiency virus (HIV) receiving antiretroviral therapy (ART) in Meigu County, Liangshan Yi Autonomous Prefecture. The information of patients who had been on ART for more than 6 months, the effect of ART, the possible reasons for ART failure, knowledge of drug resistance among patients with ART failure and the possible reasons for the emergence of drug resistance were analyzed. METHODS: A total of 2753 people living with HIV (PLWH) were collected for HIV-1 RNA virus nucleic acid testing. Plasma specimens with HIV-1 RNA ≥ 1000 copies/mL were sent to the laboratory for nucleic acid extraction, PCR, electrophoresis and sequencing, and the sequencing results were submitted to the HIV drug resistance database of Stanford University for subtyping to determine the drug resistance mutation sites and drug sensitivity levels. RESULTS: A total of 2753 patients were enrolled in this study. Antiviral therapy failed in 288 patients and was successfully amplified in 245, of which 111 had resistance genes. The resistance rate to failure of viral suppression was 45.3% (111/245). The highest rates of resistance to NNRTIs were found for efavirenz (EFV) and nevirapine (NVP) (42.9%), and the highest rates of resistance to NRTIs were found for 3TC and emtricitabine (FTC) (15.9%). The most common NNRTI resistance mutation site was K103N (20.8%), followed by V179D (9.4%) and V106M (7.8%); the most common NRTI resistance mutation site was M184V/I/MV (14.3%), followed by K65R (6.9%); three PI-associated resistance mutation sites were identified. The subtype of the resistant strain was CRF07-BC in almost all patients (98.9%). CONCLUSIONS: Compared with the previous low ART efficacy in the county, this study showed that the overall virological failure (VF) resistance rate in the county is still low, dominated by resistance to EFV, NVP, 3TC, FTC, and didanosine (DDI). Due to economic constraints, the core regimen is still 3TC + TDF, but before initiating ART, testing for HIV-1 subtypes and resistance should be conducted to avoid resistance that can lead to VF, especially for patients with high risk factors for resistance as shown by epidemiologic investigations.

Predicting Drug Interactions with Human Equilibrative Nucleoside Transporters 1 and 2 Using Functional Knockout Cell Lines and Bayesian Modeling.

Equilibrative nucleoside transporters (ENTs) 1 and 2 facilitate nucleoside transport across the blood-testis barrier (BTB). Improving drug entry into the testes with drugs that use endogenous transport pathways may lead to more effective treatments for diseases within the reproductive tract. In this study, CRISPR/CRISPR-associated protein 9 was used to generate HeLa cell lines in which ENT expression was limited to ENT1 or ENT2. We characterized uridine transport in these cell lines and generated Bayesian models to predict interactions with the ENTs. Quantification of [3H]uridine uptake in the presence of the ENT-specific inhibitor S-(4-nitrobenzyl)-6-thioinosine (NBMPR) demonstrated functional loss of each transporter. Nine nucleoside reverse-transcriptase inhibitors and 37 nucleoside/heterocycle analogs were evaluated to identify ENT interactions. Twenty-one compounds inhibited uridine uptake and abacavir, nevirapine, ticagrelor, and uridine triacetate had different IC50 values for ENT1 and ENT2. Total accumulation of four identified inhibitors was measured with and without NBMPR to determine whether there was ENT-mediated transport. Clofarabine and cladribine were ENT1 and ENT2 substrates, whereas nevirapine and lexibulin were ENT1 and ENT2 nontransported inhibitors. Bayesian models generated using Assay Central machine learning software yielded reasonably high internal validation performance (receiver operator characteristic > 0.7). ENT1 IC50-based models were generated from ChEMBL; subvalidations using this training data set correctly predicted 58% of inhibitors when analyzing activity by percent uptake and 63% when using estimated-IC50 values. Determining drug interactions with these transporters can be useful in identifying and predicting compounds that are ENT1 and ENT2 substrates and can thereby circumvent the BTB through this transepithelial transport pathway in Sertoli cells. SIGNIFICANCE STATEMENT: This study is the first to predict drug interactions with equilibrative nucleoside transporter (ENT) 1 and ENT2 using Bayesian modeling. Novel CRISPR/CRISPR-associated protein 9 functional knockouts of ENT1 and ENT2 in HeLa S3 cells were generated and characterized. Determining drug interactions with these transporters can be useful in identifying and predicting compounds that are ENT1 and ENT2 substrates and can circumvent the blood-testis barrier through this transepithelial transport pathway in Sertoli cells.

The 2-hydroxy-nevirapine metabolite as a candidate for boosting apolipoprotein A1 and for modulating anti-HDL antibodies.

The antiretroviral nevirapine (NVP) is associated to a reduction of atherosclerotic lesions and increases in high-density lipoprotein (HDL)-cholesterol. Despite being a hepatotoxic drug, which forbids its re-purposing to other therapeutic areas, not all NVP metabolites have the same potential to induce toxicity. Our aim was to investigate the effects of NVP and its metabolites in an exploratory study, towards the identification of a candidate to boost HDL. A pilot prospective (n = 11) and a cross-sectional (n = 332) clinical study were performed with the following endpoints: HDL-cholesterol and apolipoprotein A1 (ApoA1) levels, anti-HDL and anti-ApoA1 antibodies titers, paraoxonase, arylesterase and lactonase activities of paraoxonase-1, and NVP's metabolite profile. NVP treatment increased HDL-cholesterol, ApoA1 and paraoxonase-1 activities, and lowered anti-HDL and anti-ApoA1 titers. In the prospective study, the temporal modulation induced by NVP was different for each HDL-related endpoint. The first observation was a decrease in the anti-HDL antibodies titers. In the cross-sectional study, the lower titers of anti-HDL antibodies were associated to the proportion of 2-hydroxy-NVP (p = 0.03). In vitro models of hepatocytes were employed to clarify the individual contribution of NVP's metabolites for ApoA1 modulation. Long-term incubations of NVP and 2-hydroxy-NVP in the metabolically competent 3D model caused an increase in ApoA1 reaching 43 % (p < 0.05) and 86 % (p < 0.001), respectively. These results support the contribution of drug biotransformation for NVP-induced HDL modulation, highlighting the role of 2-hydroxy-NVP as ApoA1 booster and its association to lower anti-HDL titers. This biotransformation-guided approach allowed us to identify a non-toxic NVP metabolite as a candidate for targeting HDL.

Differential Impact of Nevirapine on Artemether-Lumefantrine Pharmacokinetics in Individuals Stratified by CYP2B6 c.516G>T Genotypes.

There is an increased recognition of the need to identify and quantify the impact of genetic polymorphisms on drug-drug interactions. This study investigated the pharmacogenetics of the pharmacokinetic drug-drug interaction between nevirapine and artemether-lumefantrine in HIV-positive and HIV-negative adult Nigerian subjects. Thirty each of HIV-infected patients on nevirapine-based antiretroviral therapy and HIV-negative volunteers without clinical malaria, but with predetermined CYP2B6 c.516GG and TT genotypes, were administered a complete treatment dose of 3 days of artemether-lumefantrine. Rich pharmacokinetic sampling prior to and following the last dose was conducted, and the plasma concentrations of artemether/dihydroartemisinin and lumefantrine/desbutyl-lumefantrine were quantified using tandem mass spectrometry. Pharmacokinetic parameters of artemether-lumefantrine and its metabolites in HIV-infected patients on nevirapine were compared to those in the absence of nevirapine in HIV-negative volunteers. Overall, nevirapine reduced exposure to artemether and desbutyl-lumefantrine by 39 and 34%, respectively. These reductions were significantly greater in GG versus TT subjects for artemether (ratio of geometric mean [90% confidence interval]: 0.42 [0.29 to 0.61] versus 0.81 [0.51 to 1.28]) and for desbutyl-lumefantrine (0.56 [0.43 to 0.74] versus 0.75 [0.56 to 1.00]). On the contrary, it increased exposure to dihydroartemisinin and lumefantrine by 47 and 30%, respectively. These increases were significantly higher in TT versus GG subjects for dihydroartemisinin (1.67 [1.20 to 2.34] versus 1.25 [0.88 to 1.78]) and for lumefantrine (1.51 [1.20 to 1.90] versus 1.08 [0.82 to 1.42]). This study underscores the importance of incorporating pharmacogenetics into all drug-drug interaction studies with potential for genetic polymorphisms to influence drug disposition.

Anti-HIV drugs promote ß-amyloid deposition and impair learning and memory in BALB/c mice.

OBJECTIVES: Growing evidence suggested that antiretroviral (ARV) drugs may promote amyloid beta (Aß) accumulation in HIV-1-infected brain and the persistence of HIV-associated neurocognitive disorders (HANDs). It has also been shown that lipid peroxidation upregulates ß-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) expression and subsequently promotes Aß peptide production. In the present study, we examined whether chronic exposure to the anti-HIV drugs tenofovir disoproxil fumarate (TDF) and nevirapine induces lipid peroxidation thereby promoting BACE1 and Aß generation and consequently impair cognitive function in mice. METHODS: TDF or nevirapine was orally administered to female BALB/c mice once a day for 8 weeks. On the 7th week of treatment, spatial learning and memory were assessed using the Morris water maze test. The levels of lipid peroxidation, BACE1, amyloid ß 1-42 (Aß1-42) and Aß deposits were measured in the hippocampal tissue upon completion of treatment. RESULTS: Chronic administration of nevirapine induced spatial learning and memory impairment in the Morris water maze test, whereas TDF did not have an effect. TDF and nevirapine administration increased hippocampal lipid peroxidation and Aß1-42 concentration. Nevirapine further upregulated BACE1 expression and Aß deposits. CONCLUSION: Our results suggest that chronic exposure to TDF and nevirapine contributes to hippocampal lipid peroxidation and Aß accumulation, respectively, as well as spatial learning and memory deficits in mice even in the absence of HIV infection. These findings further support a possible link between ARV drug toxicity, Aß accumulation and the persistence of HANDs.

Nonnucleoside Reverse Transcriptase Inhibitor Hypersusceptibility and Resistance by Mutation of Residue 181 in HIV-1 Reverse Transcriptase.

Substitutions at residue Y181 in HIV-1 reverse transcriptase (RT), in particular, Y181C, Y181I, and Y181V, are associated with nonnucleoside RT inhibitor (NNRTI) cross-resistance. In this study, we used kinetic and thermodynamic approaches, in addition to molecular modeling, to gain insight into the mechanisms by which these substitutions confer resistance to nevirapine (NVP), efavirenz (EFV), and rilpivirine (RPV). Using pre-steady-state kinetics, we found that the dissociation constant (Kd ) values for inhibitor binding to the Y181C and Y181I RT-template/primer (T/P) complexes were significantly reduced. In the presence of saturating concentrations of inhibitor, the Y181C RT-T/P complex incorporated the next correct deoxynucleoside triphosphate (dNTP) more efficiently than the wild-type (WT) complex, and this phenotype correlated with decreased mobility of the RT on the T/P substrate. Interestingly, we found that the Y181F substitution in RT-which represents a transitional mutation between Y181 and Y181I/V, or a partial revertant-conferred hypersusceptibility to EFV and RPV at both the virus and enzyme levels. EFV and RPV bound more tightly to Y181F RT-T/P. Furthermore, inhibitor-bound Y181F RT-T/P was less efficient than the WT complex in incorporating the next correct dNTP, and this could be attributed to increased mobility of Y181F RT on the T/P substrate. Collectively, our data highlight the key role that Y181 in RT plays in NNRTI binding.

Incidence and types of HIV-1 drug resistance mutation among patients failing first-line antiretroviral therapy.

OBJECTIVE: This study aims to investigate the prevalence and types of drug resistance mutations among patients failing first-line antiretroviral therapy (ART). METHODS: Plasma samples from 112 patients with human immunodeficiency virus-1 (HIV-1) were collected for virus RNA extract and gene amplification. The mutations related to drug resistance were detected and the incidence was statistically analyzed, and the drug resistance rate against common drugs was also evaluated. RESULTS: 103 cases were successfully amplified, and the main drug resistance mutations in the reverse transcriptase (RT) region were M184V (50.49%), K103N (28.16%), Y181C (25.24%), and K65R (27.18%), while no drug main resistance mutation was found in the protease (PR) region. The incidence of drug resistance mutations was significantly different among patients with different ages, routes of infection, duration of treatment, initial ART regimens and viral load. The drug resistance rate to the common drugs was assessed, including Efavirenz (EFV, 71.84%), Nevirapine (NVP, 74.76%), Lamivudine (3TC, 66.02%), Zidovudine (AZT, 4.85%), Stavudine (D4T, 16.51%), and Tenofovir (TDF, 21.36%). CONCLUSION: The drug resistance mutations to NRTIs and NNRTIs are complex and highly prevalent, which was the leading cause of first-line ART failure. This study provides significant theoretical support for developing the second-line and third-line therapeutic schemes.

First discovery of a potential carbonate prodrug of NNRTI drug candidate RDEA427 with submicromolar inhibitory activity against HIV-1 K103N/Y181C double mutant strain.

In the present work, we described the synthesis, antiviral profiles and metabolic stability in human plasma of compound 6, a potential carbonate prodrug of HIV-1 NNRTI drug candidate RDEA427. Compound 6 was found to inhibit the wild-type (WT) and K103N/Y181C double mutant HIV-1 strains at nano- and submicromolar concentrations, respectively. Moreover, it displayed potent HIV-1 reverse transcriptase inhibitory activity (IC50 = 0.264 µM). Further stability test in human plasma showed that 6 could release its active form RDEA427 in a linearly time-independent manner, possibly acting as a potential prodrug.

FRET-based assay to screen inhibitors of HIV-1 reverse transcriptase and nucleocapsid protein.

During HIV-1 reverse transcription, the single-stranded RNA genome is converted into proviral double stranded DNA by Reverse Transcriptase (RT) within a reverse transcription complex composed of the genomic RNA and a number of HIV-1 encoded proteins, including the nucleocapsid protein NCp7. Here, we developed a one-step and one-pot RT polymerization assay. In this in vitro assay, RT polymerization is monitored in real-time by Förster resonance energy transfer (FRET) using a commercially available doubly-labeled primer/template DNA. The assay can monitor and quantify RT polymerization activity as well as its promotion by NCp7. Z-factor values as high as 0.89 were obtained, indicating that the assay is suitable for high-throughput drug screening. Using Nevirapine and AZT as prototypical RT inhibitors, reliable IC50 values were obtained from the changes in the RT polymerization kinetics. Interestingly, the assay can also detect NCp7 inhibitors, making it suitable for high-throughput screening of drugs targeting RT, NCp7 or simultaneously, both proteins.

Interaction of Nevirapine with the Peptide Binding Groove of HLA-DRB1*01:01 and Its Effect on the Conformation of HLA-Peptide Complex.

Human leukocyte antigen (HLA)-DRB1*01:01 has been shown to be involved in nevirapine-induced hepatic hypersensitivity reactions. In the present study, in silico docking simulations and molecular dynamics simulations were performed to predict the interaction mode of nevirapine with the peptide binding groove of HLA-DRB1*01:01 and its possible effect on the position and orientation of the ligand peptide derived from hemagglutinin (HA). In silico analyses suggested that nevirapine interacts with HLA-DRB1*01:01 around the P4 pocket within the peptide binding groove and the HA peptide stably binds on top of nevirapine at the groove. The analyses also showed that binding of nevirapine at the groove will significantly change the inter-helical distances of the groove. An in vitro competitive assay showed that nevirapine (1000 µM) increases the binding of the HA peptide to HLA-DRB1*01:01 in an allele-specific manner. These results indicate that nevirapine might interact directly with the P4 pocket and modifies its structure, which could change the orientation of loaded peptides and the conformation of HLA-DRB1*01:01; these changes could be distinctively recognized by T-cell receptors. Through this molecular mechanism, nevirapine might stimulate the immune system, resulting in hepatic hypersensitivity reactions.

Effect of co-administration of Hypoxis hemerocallidea extract and antiretroviral therapy (HAART) on the histomorphology and seminal parameters in Sprague Dawley rats.

Although the successful introduction and rollout of antiretroviral therapy has impacted positively on morbidity and mortality of HIV-positive patients, its interaction with plant-based adjuvants remain sparsely investigated. We report the interaction and effects of adjuvant treatment with highly active antiretroviral therapy (HAART) and Hypoxis hemeocallidea (HH) extracts on testicular structure of rats. A total of 63 pathogen-free adult male Sprague Dawley rats were divided into nine groups and treated according to protocols. HAART cocktail predisposed to significant negative testicular parameters of sperm count, motility and seminiferous tubular epithelial height (quantitatively) (p < .03) and also altered the histomorphology of tubules with diffuse hypoplasia in seminiferous tubules. The higher dose of HH showed a better ability to mitigate the altered parameters and compares favourably with vitamin C in this protocol. While HH did not show any deleterious impact on morphometric data, its role as adjuvant did not significantly reduce the negative impact of HAART on morphometric indices especially with the lower dosage. Further investigations are warranted on the interactions between HAART and Hypoxis.

Antiretrovirals, Methamphetamine, and HIV-1 Envelope Protein gp120 Compromise Neuronal Energy Homeostasis in Association with Various Degrees of Synaptic and Neuritic Damage.

HIV-1 infection frequently causes HIV-associated neurocognitive disorders (HAND) despite combination antiretroviral therapy (cART). Evidence is accumulating that components of cART can themselves be neurotoxic upon long-term exposure. In addition, abuse of psychostimulants, such as methamphetamine, seems to aggravate HAND and compromise antiretroviral therapy. However, the combined effect of virus and recreational and therapeutic drugs on the brain is poorly understood. Therefore, we exposed mixed neuronal-glial cerebrocortical cells to antiretrovirals (ARVs) (zidovudine [AZT], nevirapine [NVP], saquinavir [SQV], and 118-D-24) of four different pharmacological categories and to methamphetamine and, in some experiments, the HIV-1 gp120 protein for 24 h and 7 days. Subsequently, we assessed neuronal injury by fluorescence microscopy, using specific markers for neuronal dendrites and presynaptic terminals. We also analyzed the disturbance of neuronal ATP levels and assessed the involvement of autophagy by using immunofluorescence and Western blotting. ARVs caused alterations of neurites and presynaptic terminals primarily during the 7-day incubation and depending on the specific compounds and their combinations with and without methamphetamine. Similarly, the loss of neuronal ATP was context specific for each of the drugs or combinations thereof, with and without methamphetamine or viral gp120. Loss of ATP was associated with activation of AMP-activated protein kinase (AMPK) and autophagy, which, however, failed to restore normal levels of neuronal ATP. In contrast, boosting autophagy with rapamycin prevented the long-term drop of ATP during exposure to cART in combination with methamphetamine or gp120. Our findings indicate that the overall positive effect of cART on HIV infection is accompanied by detectable neurotoxicity, which in turn may be aggravated by methamphetamine.

Drug-drug interactions between bedaquiline and the antiretrovirals lopinavir/ritonavir and nevirapine in HIV-infected patients with drug-resistant TB.

OBJECTIVES: Bedaquiline is a new anti-TB drug, which is metabolized by cytochrome P450 (CYP) 3A4. Concomitant ART is important for all HIV-infected patients treated for TB, but several antiretrovirals inhibit or induce CYP3A4. Single-dose drug-drug interaction studies found no significant interactions with nevirapine or lopinavir/ritonavir, but these findings could be misleading, especially because of bedaquiline's long terminal t1/2. We evaluated the effect of nevirapine and lopinavir/ritonavir on bedaquiline exposure. METHODS: We conducted a parallel-group pharmacokinetic study of three groups of participants who were on bedaquiline as part of therapy for drug-resistant TB: no ART (HIV seronegative); nevirapine-based ART; and lopinavir/ritonavir-based ART. Non-compartmental analyses were done and exposure of bedaquiline and its M2 metabolite compared between the no-ART group and the two ART groups. RESULTS: We enrolled 48 participants: 17 in the no-ART group, 17 in the nevirapine group and 14 in the lopinavir/ritonavir group. The following median bedaquiline pharmacokinetic parameters were significantly higher in the lopinavir/ritonavir group than in the no-ART group: AUC(0-48) (67 002 versus 34 730 ng ·â€Šh/mL; P = 0.003); Tmax (6 versus 4 h; P = 0.003); and t1/2 (55 versus 31 h; P = 0.004). On multivariate analysis, bedaquiline exposure was increased by lopinavir/ritonavir, male sex and time on bedaquiline. Bedaquiline exposure was not significantly different between the nevirapine group and the no-ART group. M2 metabolite exposure was not significantly different in either of the antiretroviral groups compared with the no-ART group. CONCLUSIONS: Lopinavir/ritonavir significantly increased bedaquiline exposure. The clinical significance of this interaction remains to be determined.

Testing anti-HIV activity of antiretroviral agents in vitro using flow cytometry analysis of CEM-GFP cells infected with transfection-derived HIV-1 NL4-3.

An assay, specifically optimized to evaluate the anti-HIV activity of antiretrovirals by flow cytometry analysis, is described. As widely used anti-HIV agents, zidovudine (AZT), abacavir (ABC), 2',3'-dideoxyinosine (DDI), lamivudine (3TC), nevirapine (NVP), and efavirenz (EFV), and as drugs of recent approval raltegravir (RAL), etravirine (ETR), and rilpivirine (RPV), were utilized as reference drugs. HIV-1 NL4-3 virus was prepared by transfection of HEK293T cells with purified plasmid DNA and quantified by p24 antigen-capture assay. For infection, CEM-GFP cells were exposed to vehicle or to several concentrations of the drugs for 2 hr at 37 °C before HIV-1 NL4-3 was added to each sample. The adsorption was prolonged for 3 hr at 37 °C. After 72 hr of incubation, HIV-induced GFP expression in infected CEM-GFP cells was assessed by flow cytometry analysis and expressed as % positive cells. For comparison, p24 production in supernatants was assessed by a commercial ELISA kit. On the basis of IC50 values, the anti-HIV activity, as assayed by this method, was EFV > 3TC > AZT > NVP > DDI > ABC and ETR > RPV > RAL. The comparison between the IC50 values calculated through flow cytometry and p24 production revealed overlapping results, showing that the optimized protocol of CEM-GFP infection with HIV NL4-3 is a suitable method to perform quantitative, rapid and low-expensive screening tests to evaluate the in vitro effect of new candidate anti-HIV drugs.

Antileishmanial activity of antiretroviral drugs combined with miltefosine.

Co-infection of Leishmaniasis, a neglected tropical disease, with human immunodeficiency virus (HIV) has hindered treatment efficacy. In this study, we aim to evaluate the antileishmanial activity of two protease inhibitors (darunavir and atazanavir) and four reverse transcriptase inhibitors (tenofovir, efavirenz, neviraprine, and delavirdine mesylate) on Leishmania infantum. The activity of different antiretrovirals combinations and of antiretroviral with miltefosine, a drug used on leishmaniasis treatment, was also evaluated. Only two non-nucleoside reverse transcriptase inhibitors (NNRTIs) were active on L. infantum. Efavirenz showed the best antileishmanial activity on promastigotes cells with IC50 value of 26.1 µM followed by delavirdine mesylate with an IC50 value of 136.2 µM. Neviraprine, tenofovir, atazanavir, and darunavir were not active at the concentrations tested (IC50 > 200 µM). The efavirenz also showed high antileishmanial activity on intramacrophage amastigotes with IC50 of 12.59 µM. The interaction of efavirenz with miltefosine improved antileishmanial activity on promastigotes and intracellular amastigotes (IC50 values of 11. 8 µM and 8.89 µM, respectively). These results suggest that combined-therapy including efavirenz and miltefosine could be alternative options for treating Leishmaniasis and Leishmania/HIV co-infections.

Inhibition of CYP2B6 by Medicinal Plant Extracts: Implication for Use of Efavirenz and Nevirapine-Based Highly Active Anti-Retroviral Therapy (HAART) in Resource-Limited Settings.

Highly active antiretroviral therapy (HAART) has greatly improved health parameters of HIV infected individuals. However, there are several challenges associated with the chronic nature of HAART administration. For populations in health transition, dual use of medicinal plant extracts and conventional medicine poses a significant challenge. There is need to evaluate interactions between commonly used medicinal plant extracts and antiretroviral drugs used against HIV/AIDS. Efavirenz (EFV) and nevirapine (NVP) are the major components of HAART both metabolized by CYP2B6, an enzyme that can potentially be inhibited or induced by compounds found in medicinal plant extracts. The purpose of this study was to evaluate the effects of extracts of selected commonly used medicinal plants on CYP2B6 enzyme activity. Recombinant human CYP2B6 was used to evaluate inhibition, allowing the assessment of herb-drug interactions (HDI) of medicinal plants Hyptis suaveolens, Myrothamnus flabellifolius, Launaea taraxacifolia, Boerhavia diffusa and Newbouldia laevis. The potential of these medicinal extracts to cause HDI was ranked accordingly for reversible inhibition and also classified as potential time-dependent inhibitor (TDI) candidates. The most potent inhibitor for CYP2B6 was Hyptis suaveolens extract (IC50 = 19.09 ± 1.16 µg/mL), followed by Myrothamnus flabellifolius extract (IC50 = 23.66 ± 4.86 µg/mL), Launaea taraxacifolia extract (IC50 = 33.87 ± 1.54 µg/mL), and Boerhavia diffusa extract (IC50 = 34.93 ± 1.06 µg/mL). Newbouldia laevis extract, however, exhibited weak inhibitory effects (IC50 = 100 ± 8.71 µg/mL) on CYP2B6. Launaea taraxacifolia exhibited a TDI (3.17) effect on CYP2B6 and showed a high concentration of known CYP450 inhibitory phenolic compounds, chlorogenic acid and caffeic acid. The implication for these observations is that drugs that are metabolized by CYP2B6 when co-administered with these herbal medicines and when adequate amounts of the extracts reach the liver, there is a high likelihood of standard doses affecting drug plasma concentrations which could lead to toxicity.

The HEPT Analogue WPR-6 Is Active against a Broad Spectrum of Nonnucleoside Reverse Transcriptase Drug-Resistant HIV-1 Strains of Different Serotypes.

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are important components of the highly active antiretroviral therapy (HAART) used to treat human immunodeficiency type 1 virus (HIV-1). However, because of the emergence of drug resistance and the adverse effects of current anti-HIV drugs, it is essential to develop novel NNRTIs with an excellent safety profile, improved activity against NNRTI-resistant viruses, and enhanced activity against clinical isolates of different subtypes. Here, we have identified 1-[(benzyloxy)methyl]-6-(3,5-dimethylbenzyl)-5-iodopyrimidine-2,4(1H,3H)-dione (WPR-6), a novel NNRTI with a 50% effective concentration (EC50) of 2 to 4 nM against laboratory-adapted HIV-1 strain SF33 and an EC50 of 7 to 14 nM against nucleoside reverse transcriptase inhibitor-resistant HIV-1 strain 7391 with a therapeutic index of >1 × 10(4). A panel of five representative clinical virus isolates of different subtypes circulating predominantly in China was highly sensitive to WPR-6, with EC50s ranging from 1 to 6 nM. In addition, WPR-6 showed excellent antiviral potency against the most prevalent NNRTI-resistant viruses containing the K103N and Y181C mutations. To determine whether WPR-6 selects for novel resistant mutants, in vitro resistance selection was conducted with laboratory-adapted HIV-1 strain SF33 on MT-4 cells. The results demonstrated that V106I and Y188L were the two dominant NNRTI-associated resistance mutations detected in the breakthrough viruses. Taken together, these in vitro data indicate that WPR-6 has greater efficacy than the reference HEPT analogue TNK651 and the marketed drug nevirapine against HIV-1. However, to develop it as a new NNRTI, further improvement of its pharmacological properties is warranted.

Drug resistance mutations 18 months after discontinuation of nevirapine-based ART for prevention of mother-to-child transmission of HIV in Malawi.

OBJECTIVES: The objective of this study was to determine the prevalence of drug resistance mutations among HIV-positive women in Malawi 18 months after discontinuing nevirapine-based ART for the prevention of mother-to-child transmission. PATIENTS AND METHODS: HIV-infected antiretroviral-naive (except for single-dose nevirapine) pregnant Malawian women receiving a nevirapine-based triple antiretroviral regimen from Week 25 of gestation until 6 months of breastfeeding were included in this analysis. Drug resistance was assessed in HIV-DNA 24 months post-partum and at baseline (before the initiation of treatment). In patients with resistance, the presence of mutations was also evaluated in the corresponding plasma samples. RESULTS: Seven out of 42 (16.7%) women studied had archived drug resistance at Month 24 [six cases had NNRTI-associated mutations and two cases the M184I mutation]. In four cases, resistance mutations were already present at baseline (all NNRTI mutations). In three cases, there was an emergence of 'new' resistance (also present in the plasma in one case). Of the 35 women without resistance mutations at Month 24, only one subject had resistance mutations at baseline. Baseline resistance was significantly more common among women with mutations at 24 months compared with those harbouring a WT virus (4/7 versus 1/35, P < 0.001). CONCLUSIONS: Among women who had discontinued drugs 6 months post-partum, only 3/42 (7.1%) had accumulated new resistance mutations in HIV-DNA 2 years after delivery. These findings are reassuring in terms of the safety of the Option B strategy for the prevention of HIV mother-to-child transmission.

Evaluation of anti-ulcerogenic and ulcer-healing activities of nevirapine in rats.

BACKGROUND: Nevirapine is a very potent antiretroviral drug frequently used in the management of Human Immunodeficiency Virus (HIV). Opportunistic pathologies in HIV/AIDS patients include gastric ulcer and gastrointestinal haemorrhage. Hence, the impact of nevirapine on induced gastric ulcer was studied using Wistar rats. METHODS: Anti-ulcer activity of nevirapine was evaluated using cold restraint stress-induced, ethanol-induced and pylorus ligation-induced gastric ulcer models for acute ulceration; and acetic acid-induced ulcer model for the chronic ulceration in Wistar rats. RESULTS: Nevirapine (9 mg/kg, 18 mg/kg and 36 mg/kg) showed significant (P < 0.05) reduction in ulcer severity score and ulcer index as compared to the control in the models,.with corresponding increase in percentage inhibition. Histopathological studies showed that nevirapine has a positive effect on the healing of gastric ulcer in the groups treated with the nevirapine compared with the control. The induced ulcers healed up in all the groups administered with nevirapine compared to what was found in the omeprazole group where manifestations of ulcer like inflammatory cells infiltration is still present. CONCLUSION: Nevirapine may possess highly therapeutic effect in the treatment and prevention of gastrointestinal complications that might come with the presence of HIV virus in patients.

Anti-HIV host factor SAMHD1 regulates viral sensitivity to nucleoside reverse transcriptase inhibitors via modulation of cellular deoxyribonucleoside triphosphate (dNTP) levels.

Newly identified anti-HIV host factor, SAMHD1, restricts replication of lentiviruses such as HIV-1, HIV-2, and simian immunodeficiency virus in macrophages by enzymatically hydrolyzing and depleting cellular dNTPs, which are the substrates of viral DNA polymerases. HIV-2 and some simian immunodeficiency viruses express viral protein X (VPX), which counteracts SAMHD1 and elevates cellular dNTPs, enhancing viral replication in macrophages. Because nucleoside reverse transcriptase inhibitors (NRTIs), the most commonly used anti-HIV drugs, compete against cellular dNTPs for incorporation into proviral DNA, we tested whether SAMHD1 directly affects the efficacy of NRTIs in inhibiting HIV-1. We found that reduction of SAMHD1 levels with the use of virus-like particles expressing Vpx- and SAMHD1-specific shRNA subsequently elevates cellular dNTPs and significantly decreases HIV-1 sensitivity to various NRTIs in macrophages. However, virus-like particles +Vpx treatment of activated CD4(+) T cells only minimally reduced NRTI efficacy. Furthermore, with the use of HPLC, we could not detect SAMHD1-mediated hydrolysis of NRTI-triphosphates, verifying that the reduced sensitivity of HIV-1 to NRTIs upon SAMHD1 degradation is most likely caused by the elevation in cellular dNTPs.