Non-nucleoside Reverse Transcriptase Inhibitors Nevirapine Research Articles

Structural Studies and Structure Activity Relationships for Novel Computationally Designed Non-nucleoside Inhibitors and Their Interactions With HIV-1 Reverse Transcriptase.

Reverse transcriptase (RT) from the human immunodeficiency virus continues to be an attractive drug target for antiretroviral therapy. June 2022 will commemorate the 30th anniversary of the first Human Immunodeficiency Virus (HIV) RT crystal structure complex that was solved with non-nucleoside reverse transcriptase inhibitor nevirapine. The release of this structure opened opportunities for designing many families of non-nucleoside reverse transcriptase inhibitors (NNRTIs). In paying tribute to the first RT-nevirapine structure, we have developed several compound classes targeting the non-nucleoside inhibitor binding pocket of HIV RT. Extensive analysis of crystal structures of RT in complex with the compounds informed iterations of structure-based drug design. Structures of seven additional complexes were determined and analyzed to summarize key interactions with residues in the non-nucleoside inhibitor binding pocket (NNIBP) of RT. Additional insights comparing structures with antiviral data and results from molecular dynamics simulations elucidate key interactions and dynamics between the nucleotide and non-nucleoside binding sites.

Immunological and Virological Outcomes among Treatment Experienced HIV-Infected Patients on Dolutegravir Regimen in Tanzania

Introduction The integrase inhibitor dolutegravir (DTG) has recently replaced the non-nucleoside reverse transcriptase inhibitors (efavirenz and nevirapine) in several Sub-Saharan African countries, including Tanzania. Since this change, there is scarce data on the current treatment outcomes focusing on treatment-experienced HIV-infected patients switched to DTG based regimens in Tanzania. Objectives This study aimed to investigate immunological and virological outcomes among treatment-experienced HIV-infected patients who were switched to DTG based regimen in Tanzania. Results We enrolled 397 patients, majority (65%) were female patients with mean age of 42.7 (95% CI; 40.7 – 44.7) years. The mean baseline CD4+ cell count was 457.1 (95% CI; 426.6 – 489.8) cells/mm3 with 8.3% of patients with CD4+ cell count <200 cells/mm3. The mean baseline viral load (VL) was 169.8 (95% CI; 128.8 – 223.9) copies/ml, whereby 20.6% had VL ≥ 1000 copies/ml. After the use of a new fixed dose combination of Tenofovir (TDF) 300 mg + Lamivudine (3TC) 300 mg + DTG 50 mg (TLD) for at least 24 weeks, the overall rate of virological suppression (<50 copies/ml) was 89.9% (95%CI 86.7% - 92.7%). The significant predictors of virological failure were overall duration on ART use (p = 0.004), duration on TDF + 3TC, and Efavirenz (EFV) (TLE) (p = 0.007), and baseline VL (p < 0.001). Conclusion TLD regimen has provided favorable preliminary results among patients who previously used TLE in HIV/AIDS treatment by showing good virological and immunological outcomes. The long-term treatment outcomes require further investigation.

Impact of HIV-1 Resistance-Associated Mutations on Susceptibility to Doravirine: Analysis of Real-World Clinical Isolates

ABSTRACTClinical management of human immunodeficiency virus type-1 (HIV-1) infection may be negatively impacted by either acquired or transmitted drug resistance. Here, we aim to extend our understanding of the impact of resistance-associated mutations (RAMs) on the susceptibility of clinical isolates to the nonnucleoside reverse transcriptase inhibitor (NNRTI) doravirine. Clinical isolates from people living with HIV-1 undergoing routine testing for susceptibility to doravirine and other approved NNRTIs (etravirine, rilpivirine, efavirenz, and nevirapine) were collected from August 2018 to August 2019. Susceptibility in the presence/absence of NNRTI and nucleos(t)ide reverse transcriptase inhibitor (NRTI) mutations was determined using cutoffs for relative fold change in inhibition (ratio of the 50% inhibitory concentration [IC50] of patient virus compared with the IC50 of a wild-type reference strain). Biological cutoffs of 3- to 15-fold change were investigated for doravirine, with preestablished cutoffs used for the other NNRTIs. Of 4,070 clinical isolates, 42.9% had ≥1 NNRTI RAM. More isolates were susceptible to doravirine (92.5–96.7%) than to etravirine (91.5%), rilpivirine (89.5%), efavirenz (81.5%), or nevirapine (77.5%). Based on a 3-fold cutoff, doravirine susceptibility was retained in 44.7–65.8% of isolates resistant to another NNRTI and 28.5% of isolates resistant to all other tested NNRTIs. The presence of NRTI RAMs, including thymidine analog mutations, was associated with doravirine hypersusceptibility in some isolates, particularly in the absence of NNRTI RAMs. These results support the favorable resistance profile of doravirine and are of particular importance given the challenge posed by both acquired and transmitted resistance.

NNRTI and Liver Damage: Evidence of Their Association and the Mechanisms Involved.

Due to the improved effectiveness and safety of combined antiretroviral therapy, human immunodeficiency virus (HIV) infection has become a manageable, chronic condition rather than a mortal disease. However, HIV patients are at increased risk of experiencing non-AIDS-defining illnesses, with liver-related injury standing out as one of the leading causes of death among these patients. In addition to more HIV-specific processes, such as antiretroviral drug-related toxicity and direct injury to the liver by the virus itself, its pathogenesis is related to conditions that are also common in the general population, such as alcoholic and non-alcoholic fatty liver disease, viral hepatitis, and ageing. Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are essential components of combined anti-HIV treatment due to their unique antiviral activity, high specificity, and acceptable toxicity. While first-generation NNRTIs (nevirapine and efavirenz) have been related largely to liver toxicity, those belonging to the second generation (etravirine, rilpivirine and doravirine) seem to be generally safe for the liver. Indeed, there is preclinical evidence of rilpivirine being hepatoprotective in different models of liver injury, independently of the presence of HIV. The present study aims to review the mechanisms by which currently available anti-HIV drugs belonging to the NNRTI family may participate in the development of liver disease.

Association of CYP2B6 Genetic Variation with Efavirenz and Nevirapine Drug Resistance in HIV-1 Patients from Botswana.

PurposeCYP2B6 liver enzyme metabolizes the two non-nucleoside reverse transcriptase inhibitors Efavirenz (EFV) and Nevirapine (NVP) used in the antiretroviral therapy (ART) regimens for HIV-infected individuals. Polymorphisms of the CYP2B6 gene influence drug levels in plasma and possibly virological outcomes. The aim of this study was to explore the potential impact of CYP2B6 genotype and haplotype variation on the risk of developing EFV/NVP drug resistance mutations (DRMs) in HIV-1 patients receiving EFV-/NVP-containing regimens in Botswana.Patients and MethodsParticipants were a sub-sample of a larger study (Tshepo study) conducted in Gaborone, Botswana, among HIV-infected individuals taking EFV/NVP containing ART. Study samples were retrieved and assigned to cases (with DRMs) and controls (without DRMs). Four single-nucleotide polymorphisms (SNPs) in the CYP2B6 gene (−82T>C; 516G>T; 785A>G; 983T>C) were genotyped, the haplotypes reconstructed, and the metabolic score assigned. The possible association between drug resistance and several independent factors (baseline characteristics and CYP2B6 genotypes) was assessed by Binary Logistic Regression (BLR) analysis. EFV/NVP resistance status and CYP2B6 haplotypes were also analyzed using Z-test, chi-square and Fisher’s exact test statistics.ResultsTwo hundred and twenty-seven samples were analysed (40 with DRMs, 187 without DRMs). BLR analysis showed an association between EFV/NVP resistance and CYP2B6 516G allele (OR: 2.26; 95% CI: 1.27–4.01; P=0.005). Moreover, haplotype analysis revealed that the proportion of EFV/NVP-resistant infections was higher among CYP2B6 fast than extensive/slow metabolizers (30.8% vs 16.8%; P=0.035), with the 516G allele more represented in the haplotypes of fast than extensive/slow metabolizers (100.0% vs 53.8%; P<0.001).ConclusionWe demonstrated that the CYP2B6 516G allele, and even more when combined in fast metabolic haplotypes, is associated with the presence of EFV/NVP resistance, strengthening the need to assess the CYP2B6 genetic profiles in HIV-infected patients in order to improve the virologic outcomes of NNRTI containing ART.

Analysis of Pharmacovigilance Databases for Dolutegravir Safety in Pregnancy.

The Botswana Tsepamo study reported neural tube defects (NTDs) in 4 of 426 (0.94%) infants of women receiving preconception dolutegravir (DTG) antiretroviral therapy (ART) vs 14 of 11 300 (0.12%) receiving preconception non-DTG ART. Data are needed to investigate this potential safety signal. Clinicians, patients, and pharmaceutical companies can report adverse drug reactions (ADRs) to pharmacovigilance databases. Data from ADRs reported to various pharmacovigilance databases were searched for NTDs. Four pharmacovigilance databases (World Health Organization [WHO] VigiAccess; United Kingdom Medicines Health Regulatory Authority [UK MHRA]; European Medicines Agency [EMA] EudraVigilance; US Food and Drug Administration Adverse Event Reporting System [FAERS]) with online data availability were analyzed for NTD reports for 4 integrase inhibitors (DTG, raltegravir, elvitegravir, bictegravir), 2 protease inhibitors (darunavir, atazanavir), and 2 nonnucleoside reverse transcriptase inhibitors (nevirapine, efavirenz). Reports in the system organ class "congenital or familial disorders" were searched for NTDs. NTDs have been reported among infants born from women taking a wide range of antiretrovirals in 4 pharmacovigilance databases (WHO VigiAccess, 116 reactions; UK MHRA, 8 cases; EMA EudraVigilance, 20 cases; FAERS, 44 cases). Six NTDs were identified for DTG across the pharmacovigilance databases. Cases were very hard to interpret, given the lack of clear denominators. Pharmacovigilance databases have many limitations, most importantly lack of a clear denominator for patients exposed to the drug of interest and duplicate cases that are difficult to identify. Given widespread use of new antiretroviral drugs worldwide and anticipated use of new drugs, prospective follow-up of pregnant women and birth surveillance studies such as Tsepamo are critically needed.

Site‐Specific Dueteration Decreases Cytrochrome P450 Production of 12‐Hydroxy‐Nevirapine through the Kinetic Isotope Effect

The non‐nucleoside reverse transcriptase inhibitor nevirapine (NVP), classified as an essential medication by the World Health Organization, has been shown to cause life‐threatening hepatotoxicity and skin rash in patients. The 12‐hydroxyNVP (12‐OHNVP) metabolite of NVP has been implicated in these toxicities and here we aimed to gain a detailed understanding of utility of introducing deuteriums at the 12‐position of NVP as an approach to decreasing the formation of this metabolite. To do so, an ultra‐high performance liquid chromatograph tandem high‐resolution mass spectrometry assay was developed and employed to detect 12‐OHNVP formation following incubation of NVP with human liver microsomes and individual cDNA‐expressed enzymes. Through this work, we found that formation of 12‐OHNVP was most abundant following incubation with cytochrome P450 (P450; CYP) 2C19 although each of the P450s tested, with the exception of CYP3A5, catalyzed the formation of 12‐OHNVP to some extent. In line with this, formation of 12‐OHNVP by human liver microsomes was not abrogated in the presence of any established small molecule inhibitors of P450 activity including ketoconazole (CYP3A inhibitor). In order to test whether the deuterium kinetic isotope effect could be employed to control 12‐OHNVP production by all P450s we investigated the impact of 12th position deuteration of NVP on the formation of this metabolite. Human liver microsomes were incubated with 10 μM NVP or 10 μM 12‐d3NVP for 30 minute and the production of the 12‐OHNVP was measured. The abundance of 12‐OHNVP was 6.8‐fold lower when human liver microsomes were incubated with 12‐d3NVP, as compared to undeuterated NVP incubations. Of note, no difference in the formation of other P450‐dependent metabolites of NVP was observed. In order to test the impact of deuterium kinetic isotope effects on the metabolism of NVP in a whole cell system, we measured metabolite levels in the medium of primary mouse hepatocytes treated with 10 μM NVP and 12‐d3NVP for 24 hours. A 4.7‐fold decrease was observed in 12‐OHNVP production in the medium of cells treated with deuterated NVP as compared to undeuterated NVP. As with the human liver microsome experiments, no significant difference was observed in the production of other P450‐dependent metabolites between treatments. These results demonstrate that dueteration at the 12th position can markedly decrease cytochrome P450 production of 12‐OHNVP through the kinetic isotope effect. The impact of this controlled metabolism on NVP toxicity will be a subject of further research. This work was funded by NSF GRFP DGE‐1232825 awarded to CJSH and NIH R01 GM103853.Support or Funding InformationThis work was funded by NSF GRFP DGE‐1232825 awarded to CJSH and NIH R01 GM103853.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Fulminant Hepatitis Following Ritonavir-Nevirapine-Efavirenz Regimen in a Female HIV Patient in Pointe-Noire in 2016

Current antiretroviral therapy has reduced morbidity and mortality of HIV patients. However, their induced hepatotoxicity constitutes a risk. In this issue, we report a clinical case of fulminant hepatitis, observed in the HIV unit of the hepatogastroenterology department of the General hospital of Loandjili in Pointe-Noire. The patient is a 36-year-old female HIV treated with triple-dug combination antiretroviral therapies (ART) including one antiprotease (ritonavir) and two non-nucleoside reverse transcriptase inhibitors (nevirapine and efavirenz). He developed fulminant hepatitis five years after treatment initiation. He succumbed to the side effects. Although antiretroviral combination therapies are the standard of care for HIV infection, increased vigilance is warranted to early identify this side effect and adjust treatment in order to prevent fatal consequences.

Prevalence of Asymptomatic Parasitemia and Gametocytemia in HIV-Infected Children on Differing Antiretroviral Therapy.

.Laboratory data and prior pediatric reports indicate that HIV protease inhibitor (PI)–based antiretroviral therapy (ARV) kills gametocytes and reduces rates of gametocytemia, but not asymptomatic parasitemia, in a high malaria-transmission area. To determine whether ARV regimen impacts these rates in areas with less-intense malaria transmission, we compared asymptomatic parasitemia and gametocytemia rates in HIV-infected children by ARV regimen in Lilongwe, Malawi, an area of low-to-moderate transmission intensity. HIV PI lopinavir–ritonavir (LPV–rtv) ARV– or non-nucleoside reverse transcriptase inhibitor nevirapine ARV–treated children did not differ in the rates of polymerase chain reaction-detected asymptomatic parasitemia (relative risk [RR] 0.43 95% confidence interval [CI] [0.16, 1.18], P value 0.10) or microscopically detected gametocytemia with LPV–rtv ARV during symptomatic malaria (RR 0.48 95% CI [0.22,1.04] P value 0.06). LPV–rtv ARV was not associated with reduced rates of asymptomatic parasitemia, or gametocytemia on days of symptomatic malaria episodes, in HIV-infected children. Larger studies should evaluate whether ARV impacts transmission.

Ultra-Deep Sequencing Analysis on HIV Drug-Resistance-Associated Mutations Among HIV-Infected Individuals: First Report from the Philippines.

A sharp increase in the number of people living with HIV has been documented in the Philippines. In response, the government has instituted antiretroviral therapy (ART) nationwide through HIV treatment hubs. However, no data presently exist on the status of ART drug-resistance-associated mutations (DRMs). In this study, we aim at analyzing DRM profiles in the Philippines and at providing comprehensive data on DRMs to guide treatment decisions and prevent viral failures. We conducted a cross-sectional study in 119 volunteers who tested positive for HIV from more than 8,000 participants screened for HIV across the nation through the 2013 Integrated HIV Behavioral and Serologic Surveillance (IHBSS) program. Amplicons were generated from plasma RNA by using primers designed to analyze diverse HIV-1 isolates targeting the reverse transcriptase region and sequenced on a 454 ultra-deep sequencing (UDS) platform to assess DRMs. DRMs were defined by using the Stanford HIV drug resistance database, and we found only 2 from 110 evaluable individuals with major HIV variants (>20% prevalence) that were highly resistant to the non-nucleoside reverse transcriptase inhibitor (NNRTI: efavirenz and nevirapine). However, a larger fraction of individuals harbored minority drug-resistant HIV variants (0.5%-20% prevalence) and they were highly resistant to NNRTI nevirapine (89/110), rilpivirine (5/110), and efavirenz (49/110). This study is the first report on the presence of HIV drug resistance in the Philippines and demonstrates the utility of UDS in assisting the detection of HIV minor variants. Monitoring for ART-DRMs will assist in improving HIV management strategies in curtailing the evolving epidemic in the Philippines.

High rates of virological failure and drug resistance in perinatally HIV-1-infected children and adolescents receiving lifelong antiretroviral therapy in routine clinics in Togo.

IntroductionAntiretroviral treatment (ART) has been scaled up over the last decade but compared to adults, children living with HIV are less likely to receive ART. Moreover, children and adolescents are more vulnerable than adults to virological failure (VF) and emergence of drug resistance. In this study we determined virological outcome in perinatally HIV-1-infected children and adolescents receiving ART in Togo.MethodsHIV viral load (VL) testing was consecutively proposed to all children and adolescents who were on ART for at least 12 months when attending HIV healthcare services for their routine follow-up visit (June to September 2014). Plasma HIV-1 VL was measured using the m2000 RealTime HIV-1 assay (Abbott Molecular, Des Plaines, IL, USA). Genotypic drug resistance was done for all samples with VL>1000 copies/ml.Results and discussionAmong 283 perinatally HIV-1-infected children and adolescents included, 167 (59%) were adolescents and 116 (41%) were children. The median duration on ART was 48 months (interquartile range: 28 to 68 months). For 228 (80.6%), the current ART combination consisted of two nucleoside reverse transcriptase inhibitors (NRTIs) (zidovudine and lamivudine) and one non-nucleoside reverse transcriptase inhibitor (NNRTI) (nevirapine or efavirenz). Only 28 (9.9%) were on a protease inhibitor (PI)-based regimen. VL was below the detection limit (i.e. 40 copies/ml) for 102 (36%), between 40 and 1000 copies/ml for 35 (12.4%) and above 1000 copies/ml for 146 (51.6%). Genotypic drug-resistance testing was successful for 125/146 (85.6%); 110/125 (88.0%) were resistant to both NRTIs and NNRTIs, 1/125 (0.8%) to NRTIs only, 4/125 (3.2%) to NNRTIs only and three harboured viruses resistant to reverse transcriptase and PIs. Overall, 86% (108/125) of children and adolescents experiencing VF and successfully genotyped, corresponding thus to at least 38% of the study population, had either no effective ART or had only a single effective drug in their current ART regimen.ConclusionsOur study provided important information on virological outcome on lifelong ART in perinatally HIV-1-infected children and adolescents who were still on ART and continued to attend antiretroviral (ARV) clinics for follow-up visits. Actual conditions for scaling up and monitoring lifelong ART in children in resource-limited countries can have dramatic long-term outcomes and illustrate that paediatric ART receives inadequate attention.

Resistance to reverse transcriptase inhibitors used in the treatment and prevention of HIV-1 infection.

Inhibitors that target the retroviral enzyme reverse transcriptase (RT) have played an indispensable role in the treatment and prevention of HIV-1 infection. They can be grouped into two distinct therapeutic groups, namely the nucleoside and nucleotide RT inhibitors (NRTIs), and the non-nucleoside RT inhibitors (NNRTIs). NRTIs form the backbones of most first- and second-line antiretroviral therapy (ART) regimens formulated for the treatment of HIV-1 infection. They are also used to prevent mother-to-child transmission, and as pre-exposure prophylaxis in individuals at risk of HIV-1 infection. The NNRTIs nevirapine (NVP), efavirenz and rilpivirine also used to form part of first-line ART regimens, although this is no longer recommended, while etravirine can be used in salvage ART regimens. A single-dose of NVP administered to both mother and child has routinely been used in resource-limited settings to reduce the rate of HIV-1 transmission. Unfortunately, the development of HIV-1 resistance to RT inhibitors can compromise the efficacy of these antiviral drugs in both the treatment and prevention arenas. Here, we provide an up-to-date review on drug-resistance mutations in HIV-1 RT, and discuss their cross-resistance profiles, molecular mechanisms and clinical significance.

Efavirenz Has the Highest Anti-Proliferative Effect of Non-Nucleoside Reverse Transcriptase Inhibitors against Pancreatic Cancer Cells.

BackgroundCancer prevention and therapy in HIV-1-infected patients will play an important role in future. The non-nucleoside reverse transcriptase inhibitors (NNRTI) Efavirenz and Nevirapine are cytotoxic against cancer cells in vitro. As other NNRTIs have not been studied so far, all clinically used NNRTIs were tested and the in vitro toxic concentrations were compared to drug levels in patients to predict possible anti-cancer effects in vivo.MethodsCytotoxicity was studied by Annexin-V-APC/7AAD staining and flow cytometry in the pancreatic cancer cell lines BxPC-3 and Panc-1 and confirmed by colony formation assays. The 50% effective cytotoxic concentrations (EC50) were calculated and compared to the blood levels in our patients and published data.ResultsThe in vitro EC50 of the different drugs in the BxPC-3 pancreatic cancer cells were: Efavirenz 31.5μmol/l (= 9944ng/ml), Nevirapine 239μmol/l (= 63786ng/ml), Etravirine 89.0μmol/l (= 38740ng/ml), Lersivirine 543μmol/l (= 168523ng/ml), Delavirdine 171μmol/l (= 78072ng/ml), Rilpivirine 24.4μmol/l (= 8941ng/ml). As Efavirenz and Rilpivirine had the highest cytotoxic potential and Nevirapine is frequently used in HIV-1 positive patients, the results of these three drugs were further studied in Panc-1 pancreatic cancer cells and confirmed with colony formation assays. 205 patient blood levels of Efavirenz, 127 of Rilpivirine and 31 of Nevirapine were analyzed. The mean blood level of Efavirenz was 3587ng/ml (range 162–15363ng/ml), of Rilpivirine 144ng/ml (range 0-572ng/ml) and of Nevirapine 4955ng/ml (range 1856–8697ng/ml). Blood levels from our patients and from published data had comparable Efavirenz levels to the in vitro toxic EC50 in about 1 to 5% of all patients.ConclusionAll studied NNRTIs were toxic against cancer cells. A low percentage of patients taking Efavirenz reached in vitro cytotoxic blood levels. It can be speculated that in HIV-1 positive patients having high Efavirenz blood levels pancreatic cancer incidence might be reduced. Efavirenz might be a new option in the treatment of cancer.

Crystal and Molecular Structures of Three Organic Salts from Nevirapine

The preparation, X-ray crystal structure, Fourier Transform infrared (FTIR) spectroscopy, and elemental analysis of three compounds of the non-nucleoside reverse transcriptase inhibitor nevirapine are reported. XRD and FTIR analysis indicated that all these complexes are organic salt. The compound 1 crystallizes in the triclinic, space group P−1, with a = 9.147(1) A, b = 9.494(2) A, c = 11.948(2) A, α = 111.079(2)°, β = 90.0890(10)°, γ = 95.0780(10)°,V = 963.7(3) A3, Z = 2. The compound 2 crystallizes in the monoclinic, space group C2/c, with a = 21.608(2) A, b = 12.860(1) A, c = 15.653(1) A, β = 92.2940(10)°, V = 4346.1(6) A3, Z = 8. The compound 3 crystallizes in the monoclinic, space group P2(1)/c, with a = 9.013(1) A, b = 26.580(2) A, c = 8.360(1) A, β = 114.789(2)°, V = 1818.3(3) A3, Z = 4. Nevirapine molecules are linked essentially by the N–H···O hydrogen bonds, forming centrosymmetric dimers. In the dimers every nevirapine displays a butterfly-like conformation with the cyclopropyl group directed towards the concave face of the molecule. In this work, the hydrogen bonding interactions in the salts have been investigated. The role of weak and strong hydrogen bonding in the crystal packing is ascertained. In the three prepared salts there are plenty of weak nonbonding interactions such as directional hydrogen bonds of N–H···O (neutral and ionic), intra- and interchain CH–N, CHn···O (n = 1, 2, 3), CH-S, O-C contact, O-π, CHn-π (n = 1 and 2), and π-π interactions. Due to these collective weak interactions, all the compounds displayed 3D framework structures.

Predicted residual activity of rilpivirine in HIV-1 infected patients failing therapy including NNRTIs efavirenz or nevirapine

Rilpivirine is a second-generation nonnucleoside reverse-transcriptase inhibitor (NNRTI) currently indicated for first-line therapy, but its clinical benefit for HIV-1 infected patients failing first-generation NNRTIs is largely undefined. This study quantified the extent of genotypic rilpivirine resistance in viral isolates from 1212 patients upon failure of efavirenz- or nevirapine-containing antiretroviral treatment, of whom more than respectively 80% and 90% showed high-level genotypic resistance to the failing NNRTI. Of all study patients, 47% showed a rilpivirine resistance-associated mutation (RPV-RAM), whereas preserved residual rilpivirine activity was predicted in half of the patients by three genotypic drug resistance interpretation algorithms. An NNRTI-dependent impact on rilpivirine resistance was detected. Compared with the use of nevirapine, the use of efavirenz was associated with a 32% lower risk of having a RPV-RAM and a 50% lower risk of predicted reduced rilpivirine susceptibility. Most prevalent RPV-RAMs after nevirapine experience were Y181C and H221Y, whereas L100I+K103N, Y188L and K101E occurred most in efavirenz-experienced patients. Predicted rilpivirine activity was not affected by HIV-1 subtype, although frequency of individual mutations differed across subtypes. In conclusion, this genotypic resistance analysis strongly suggests that the latest NNRTI, rilpivirine, may retain activity in a large proportion of HIV-1 patients in whom resistance failed while they were on an efavirenz- or nevirapine-containing regimen, and may present an attractive option for second-line treatment given its good safety profile and dosing convenience. However, prospective clinical studies assessing the effectiveness of rilpivirine for NNRTI-experienced patients are warranted to validate knowledge derived from genotypic and phenotypic drug resistance studies.

Differential activation of human constitutive androstane receptor and its SV23 and SV24 splice variants by rilpivirine and etravirine.

Rilpivirine and etravirine are second-generation non-nucleoside reverse transcriptase inhibitors (NNRTIs) indicated for the treatment of HIV/AIDS. The constitutive androstane receptor (CAR) regulates the expression of genes involved in various biological processes, including the transport and biotransformation of drugs. We investigated the effect of rilpivirine and etravirine on the activity of the wild-type human CAR (hCAR-WT) and its hCAR-SV23 and hCAR-SV24 splice variants, and compared it with first-generation NNRTIs (efavirenz, nevirapine, and delavirdine). Receptor activation, ligand-binding domain (LBD) transactivation, and co-activator recruitment were investigated in transiently transfected, NNRTI-treated HepG2 cells. Nuclear translocation of green fluorescent protein-tagged hCAR-WT and CYP2B6 gene expression were assessed in NNRTI-treated human hepatocytes. Rilpivirine and etravirine activated hCAR-WT, but not hCAR-SV23 or hCAR-SV24, and without transactivating the LBD or recruiting steroid receptor coactivators SRC-1, SRC-2, or SRC-3. Among the first-generation NNRTIs investigated, only efavirenz activated hCAR-WT, hCAR-SV23, and hCAR-SV24, but none of them transactivated the LBD of these receptors or substantively recruited SRC-1, SRC-2, or SRC-3. Rilpivirine, etravirine, and efavirenz triggered nuclear translocation of hCAR-WT and increased hCAR target gene (CYP2B6) expression. NNRTIs activate hCAR-WT, hCAR-SV23, and hCAR-SV24 in a drug-specific and isoform-selective manner. The activation occurs by a mechanism that does not appear to involve binding to the LBD or recruitment of SRC-1, SRC-2, or SRC-3.

Pharmacokinetics of injectable, long-acting nevirapine for HIV prophylaxis in breastfeeding infants.

Mother-to-child transmission (MTCT) of HIV-1 remains a global health problem. The World Health Organization (WHO) recommendations advise the administration of a once-daily, oral, prophylactic regimen of the nonnucleoside reverse transcriptase inhibitor nevirapine (NVP) from birth until 4 to 6 weeks of age for infants born to HIV-infected mothers in regions without access to safe and nutritionally adequate alternatives to breast milk. A critical factor driving the successful implementation of the WHO guidelines involves sustaining high adherence to the frequent dosing. With these challenges in mind, we have developed the first injectable, sustained-release NVP formulations with the goal of providing, for 6 weeks or longer, preventative plasma drug levels from a single subcutaneous administration at birth. The long-acting NVP consists of large (>50 μm), monodisperse NVP particles coated with biocompatible polymers that control the drug release kinetics. Two lead formulations exhibiting burst-free, sustained-release kinetics for up to 75 days in vitro were developed. Subsequent in vivo studies in rats demonstrated no toxicity related to the formulations. Rat plasma NVP concentrations were above the analytical assay's limit of quantification for up to 28 days. Pharmacokinetic analysis of the rat plasma NVP concentration-time data allowed absorption rate constants to be calculated. These data then were used to simulate infant NVP exposure from a single injected dose (<200 mg) of our long-acting formulations, demonstrating preliminary feasibility of the technology to maintain safe, preventative NVP plasma levels (0.2 to 3.0 μg ml(-1)) for 6 weeks or longer.

Pharmacokinetic interaction between nevirapine and nortriptyline in rats: inhibition of nevirapine metabolism by nortriptyline.

One of the most frequent comorbidities of HIV infection is depression, with a lifetime prevalence of 22 to 45%. Therefore, it was decided to study a potential pharmacokinetic interaction between the nonnucleoside reverse transcriptase inhibitor nevirapine (NVP) and the tricyclic antidepressant nortriptyline (NT). NVP and NT were administered to rats either orally, intraduodenally, or intravenously, and the changes in plasma levels and pharmacokinetic parameters were analyzed. Experiments with rat and human hepatic microsomes were carried out to evaluate the inhibitory effects of NT on NVP metabolism. NVP plasma concentrations were significantly higher when this drug was coadministered with NT. The maximum plasma concentrations of NVP were increased 2 to 5 times and the total plasma clearance was decreased 7-fold in the presence of NT. However, statistically significant differences in the pharmacokinetic parameters of NT in the absence and presence of NVP were not found. In vitro studies with rat and human hepatic microsomes confirmed the inhibition of NVP hepatic metabolism by NT in a concentration-dependent way, with the inhibition being more intense in the case of rat microsomes. In conclusion, a pharmacokinetic interaction between NVP and NT was detected. This interaction was a consequence of the inhibition of hepatic metabolism of NVP by NT. In vivo human studies are required to evaluate the effects of this interaction on the pharmacokinetics of NVP before it can be taken into account for patients receiving NVP.

Genome‐wide association study of nevirapine hypersensitivity in a malawian HIV‐infected population

The non-nucleoside reverse transcriptase inhibitor nevirapine is used in the treatment of HIV in many developing countries. Its use is associated with occurrence of hypersensitivity in 6-10% of patients. This hypersensitivity can manifest as a number of phenotypes which include the severe skin blistering reactions Stevens Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN). The aim was to undertake a genome wide association study (GWAS) in order to identify genetic variants associated with predisposition to nevirapine-induced hypersensitivity. A total of 333 nevirapine-exposed (151 hypersensitive and 182 tolerant) , HIV-infected Malawian adults were genotyped for 826,551 genotyped SNPs using the Illumina HumanOmni1-Quad_v1 chip. A replication cohort of 62 hypersensitive and 59 tolerant patients from Malawi and Uganda was genotyped for 40 SNPs statistically significantly associated with a hypersensitive phenotype in the main cohort (p<5x10-5) using the Sequenom iPLEX platform or TaqMan allelic discrimination. Logistic regression analysis identified 40 statistically significant SNP signals associated with a nevirapine hypersensitivity phenotype. Only 1 SNP association signal (in the HLA-C locus, associated with SJS/TEN) was statistically significant in both our main discovery (=1.48x10-6) and enriched replication cohort (38 cases and 59 controls) (p=9.6x10-5). Meta-analysis determined the odds ratio as 5.17 (p=2.61x10-10). Data suggest this SNP to be a strong proxy for HLA-C*04:01 carriage (96% co-occurrence). We have confirmed that, in a sub-Saharan African population, HLA-C*04:01 carriage confers a significant risk for nevirapine-induced SJS_TEN though not to less severe hypersensitivity phenotypes. No other significant high penetrance genetic risk factors were identified.

Unraveling the Gymnastics of Reverse Transcription Through Single Molecule Spectroscopy

AIDS Research and Human RetrovirusesVol. 30, No. 3 Images in HIV ResearchFree AccessUnraveling the Gymnastics of Reverse Transcription Through Single Molecule SpectroscopyElio A. Abbondanzieri and Stuart F.J. Le GriceElio A. AbbondanzieriDepartment of Bionanoscience, Delft University of Technology, Delft, The Netherlands.Search for more papers by this author and Stuart F.J. Le GriceRT Biochemistry Section, HIV Drug Resistance Program, National Cancer Institute-Frederick, Frederick, Maryland.Search for more papers by this authorPublished Online:3 Mar 2014https://doi.org/10.1089/aid.2014.0023AboutSectionsPDF/EPUB ToolsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail Synthesizing integration-competent, double-stranded DNA from the single-stranded human immunodeficiency virus (HIV) RNA genome might more aptly be described as molecular gymnastics (Fig. 1). Key elements of this routine include intramolecular and intermolecular transfer of the replication complex between genome termini, strand displacement synthesis, selecting plus-strand RNA primers from within the RNA/DNA replication intermediate, their excision from nascent DNA, and phased A-tract-induced cessation of DNA synthesis near the center of the genome. While site-directed mutagenesis identifies residues of HIV-1 reverse transcriptase (RT) that mediate these functions, and X-ray crystallography gives elegant, but static snapshots of nucleoprotein complexes, they cannot assess conformational dynamics of this remarkably versatile enzyme. To better understand how HIV-1 RT interacts with nucleic acid required designing a single molecule strategy based on Foerster resonance energy transfer (FRET), placing fluorophores into the enzyme and its nucleic acid substrates.FIG. 1. Multistep acrobatics and reverse transcription.Studies with duplex DNA predictably recapitulated an enzyme with its DNA polymerase catalytic center straddled over the primer terminus. However, an unexpected observation of Abbondanzieri et al.1 was that this orientation reversed when the DNA primer was replaced with RNA. Later studies examining polypurine tract recognition not only confirmed that HIV-1 RT could assume different orientations, but also was sufficiently agile to “flip” between these during a single binding event. Such reversal in RT orientation could help explain how its N-terminal DNA polymerase and C-terminal ribonuclease H (RNase H) domains can recognize the PPT primer terminus at different steps of plus-strand synthesis.Another concept we accept when studying HIV-1 RT is that it is a distributive polymerase, i.e., it frequently dissociates from its nucleic acid substrate. How then, upon rebinding, does the enzyme locate the primer terminus to reengage in DNA synthesis? Subsequent studies of Liu et al. illustrated the capacity of RT to slide on the nucleic acid duplex, a property that could be enhanced in the presence of nonnucleoside RT inhibitors or restricted by forming a ternary complex. However, the story does not end here. Since there appears to be no orientation (or directional) bias during sliding, the polymerization target site could be accessed by the retroviral polymerase in two different orientations. Single molecule spectroscopy not only confirmed this, but elegantly demonstrated that an enzyme accessing the primer terminus in an “RNase H mode” could spontaneously flip into the polymerization mode, thereby linking the properties of flipping and sliding.2A state-of-the-art technology, high-profile publications, but what is the significance? For many years, we have presumed that nonnucleoside inhibition of HIV-1 RT occurred through a “grinding of the gears” and an inability to support DNA synthesis. However, examining the interaction of HIV-1 RT with its PPT by single molecule spectroscopy revealed that the nonnucleoside reverse transcriptase inhibitor (NNRTI) nevirapine promotes a reversal of enzyme orientation, positioning the RNase H domain over the primer terminus, thereby inhibiting synthesis of plus-strand DNA. These and related studies on tRNA-primed initiation of minus-strand DNA synthesis3 show that, in a different sense, HIV fitness requires nimble and flexible maneuvering of its DNA polymerase!References1 Abbondanzieri EA, Bokinsky G, Rausch JW, Zhang J, Le Grice SFJ, and Zhuang X: Dynamic binding orientations determine the enzymatic activity of HIV reverse transcriptase. Nature 2008;453:184–189. Crossref, Medline, Google Scholar2 Liu S, Abbondanzieri EA, Rausch JW, Le Grice SFJ, and Zhuang X: Slide into action dynamic shuttling of HIV reverse transcriptase on nucleic acid substrates. Science 2008;322:1092–1097. Crossref, Medline, Google Scholar3 Liu S, Harada BT, Miller JT, Le Grice SFJ, and Zhuang X: Initiation complex dynamics direct the transitions between distinct phases of early HIV reverse transcription. Nat Struct Mol Biol 2010;17:1453–1460. Crossref, Medline, Google ScholarFiguresReferencesRelatedDetails Volume 30Issue 3Mar 2014 InformationCopyright 2014, Mary Ann Liebert, Inc.To cite this article:Elio A. Abbondanzieri and Stuart F.J. Le Grice.Unraveling the Gymnastics of Reverse Transcription Through Single Molecule Spectroscopy.AIDS Research and Human Retroviruses.Mar 2014.209-210.http://doi.org/10.1089/aid.2014.0023Published in Volume: 30 Issue 3: March 3, 2014PDF download