Analysis of human immunodeficiency virus type 1 drug resistance in children receiving nucleoside analogue reverse-transcriptase inhibitors plus nevirapine, nelfinavir, or ritonavir (Pediatric AIDS Clinical Trials Group 377)

In Pediatric AIDS Clinical Trials Group 377, antiretroviral therapy-experienced children were randomized to 4 treatment arms that included different combinations of stavudine, lamivudine (3TC), nevirapine (Nvp), nelfinavir (Nfv), and ritonavir (Rtv). Previous treatment with zidovudine (Zdv), didanosine (ddI), or zalcitabine (ddC) was acceptable. Drug resistance ((R)) mutations were assessed before study treatment (baseline) and at virologic failure. Zdv(R), ddI(R), and ddC(R) mutations were detected frequently at baseline but were not associated with virologic failure. Children with drug resistance mutations at baseline had greater reductions in virus load over time than did children who did not. Nvp(R) and 3TC(R) mutations were detected frequently at virologic failure, and Nvp(R) mutations were more common among children receiving 3-drug versus 4-drug Nvp-containing regimens. Children who were maintained on their study regimen after virologic failure accumulated additional Nvp(R) and 3TC(R) mutations plus Rtv(R) and Nfv(R) mutations. However, Rtv(R) and Nfv(R) mutations were detected at unexpectedly low rates.

Estimating Frequencies of Minority Nevirapine-Resistant Strains in Chronically HIV-1-Infected Individuals Naive to Nevirapine by Using Stochastic Simulations and a Mathematical Model

Nevirapine forms the mainstay of our efforts to curtail the pediatric AIDS epidemic through prevention of mother-to-child transmission of HIV-1. A key limitation, however, is the rapid selection of HIV-1 strains resistant to nevirapine following the administration of a single dose. This rapid selection of resistance suggests that nevirapine-resistant strains preexist in HIV-1 patients and may adversely affect outcomes of treatment. The frequencies of nevirapine-resistant strains in vivo, however, remain poorly estimated, possibly because they exist as a minority below current assay detection limits. Here, we employ stochastic simulations and a mathematical model to estimate the frequencies of strains carrying different combinations of the common nevirapine resistance mutations K103N, V106A, Y181C, Y188C, and G190A in chronically infected HIV-1 patients naive to nevirapine. We estimate the relative fitness of mutant strains from an independent analysis of previous competitive growth assays. We predict that single mutants are likely to preexist in patients at frequencies (similar to 0.01% to 0.001%) near or below current assay detection limits (>0.01%), emphasizing the need for more-sensitive assays. The existence of double mutants is subject to large stochastic variations. Triple and higher mutants are predicted not to exist. Our estimates are robust to variations in the recombination rate, cellular superinfection frequency, and the effective population size. Thus, with 10(7) to 10(8) infected cells in HIV-1 patients, even when undetected, nevirapine-resistant genomes may exist in substantial numbers and compromise efforts to prevent mother-to-child transmission of HIV-1, accelerate the failure of subsequent antiretroviral treatments, and facilitate the transmission of drug resistance.

New insights on nevirapine use: A mechanistic perspective of its toxic events

Introdução: A nevirapina (NVP) é um fármaco amplamente utilizado para o tratamento da infecção pelo vírus da imunodeficiência humana de tipo 1 (VIH-1), no entanto, a sua utilização na terapêutica crónica tem sido associada à toxicidade hepática e cutânea. O sexo feminino é um factor de risco para o desenvolvimento destes eventos tóxicos, mas as razões para essa diferença entre o sexo feminino e masculino não estão completamente esclarecidas. Diferenças na biotransformação da NVP e na formação de metabolitos tóxicos podem ser as causas subjacentes. O presente trabalho teve como objectivo explorar as diferenças entre homens e mulheres na biotransformação da NVP, como um potencial factor de toxicidade induzida por este fármaco anti-retroviral. Materiais e Métodos: Todos os indivíduos incluídos no presente estudo eram adultos com infecção por VIH-1 confirmada, tratados com 400 mg de NVP uma vez ao dia, durante pelo menos 1 mês. Foram colhidas amostras de sangue e os níveis de NVP e dos metabolitos de fase I foram determinados por cromatografia líquida de alta performance. Os dados antropométricos e clínicos e os perfis de metabolitos foram avaliados de forma a averiguar possíveis diferenças relacionadas com o sexo dos indivíduos. Resultados: Foram incluídos 52 doentes (63% do sexo masculino). O peso corporal foi inferior nas mulheres (p = 0.028) e o sexo feminino foi associado a maiores níveis de fosfatase alcalina (p = 0.036) e lactato desidrogenase (p = 0.037). Os níveis plasmáticos de NVP (p = 0.030) e 3-hidroxi-NVP (p = 0.035), assim como as proporções de 12-hidroxi-NVP (p = 0.037) e 3-hidroxi-NVP (p = 0.001) foram maiores nas mulheres, quando ajustados pelo peso corporal dos indivíduos. Discussão: Existem diferenças na biotransformação da NVP entre homens e mulheres, particularmente na formação de 12-hidroxi-NVP e 3-hidroxi-NVP. Estes resultados apontam para uma formação de metabolitos reactivos, que é dependente do sexo e que pode contribuir para o perfil de dimorfismo sexual associado às reacções tóxicas induzidas pela NVP.

Analysis of phase transition and dehydration processes of nevirapine

Solid-state characterization of crystalline drugs is an important pre-formulation step for the development and design of solid dosage forms, such as pellets and tablets. In this study, phase transition and dehydration processes of nevirapine have been studied by differential scanning calorimetry and thermogravimetry differential thermal analysis to overcome the problems of drug formulation, namely poor solubility and poor content uniformity. Phase solubility studies elucidated the mechanism of enhanced nevirapine solubility.

The effect of efavirenz versus nevirapine-containing regimens on immunologic, virologic and clinical outcomes in a prospective observational study

OBJECTIVE: To compare regimens consisting of either efavirenz or nevirapine and two or more nucleoside reverse transcriptase inhibitors (NRTIs) among HIV-infected, antiretroviral-naive, and AIDS-free individuals with respect to clinical, immunologic, and virologic outcomes. DESIGN: Prospective studies of HIV-infected individuals in Europe and the US included in the HIV-CAUSAL Collaboration. METHODS: Antiretroviral therapy-naive and AIDS-free individuals were followed from the time they started an NRTI, efavirenz or nevirapine, classified as following one or both types of regimens at baseline, and censored when they started an ineligible drug or at 6 months if their regimen was not yet complete. We estimated the 'intention-to-treat' effect for nevirapine versus efavirenz regimens on clinical, immunologic, and virologic outcomes. Our models included baseline covariates and adjusted for potential bias introduced by censoring via inverse probability weighting. RESULTS: A total of 15 336 individuals initiated an efavirenz regimen (274 deaths, 774 AIDS-defining illnesses) and 8129 individuals initiated a nevirapine regimen (203 deaths, 441 AIDS-defining illnesses). The intention-to-treat hazard ratios [95% confidence interval (CI)] for nevirapine versus efavirenz regimens were 1.59 (1.27, 1.98) for death and 1.28 (1.09, 1.50) for AIDS-defining illness. Individuals on nevirapine regimens experienced a smaller 12-month increase in CD4 cell count by 11.49 cells/mul and were 52% more likely to have virologic failure at 12 months as those on efavirenz regimens. CONCLUSIONS: Our intention-to-treat estimates are consistent with a lower mortality, a lower incidence of AIDS-defining illness, a larger 12-month increase in CD4 cell count, and a smaller risk of virologic failure at 12 months for efavirenz compared with nevirapine.

Predicting and diagnosing abacavir and nevirapine drug hypersensitivity: from bedside to bench and back again

There is a growing discussion surrounding the issue of personalized approaches to drug prescription based on an individual's genetic makeup. This field of investigation has focused primarily on identifying genetic factors that influence drug metabolism and cellular disposition, thereby contributing to dose-dependent toxicities and/or variable drug efficacy. However, pharmacogenetic approaches have also proved valuable in predicting drug hypersensitivity reactions in selected patient populations, including HIV-infected patients receiving long-term antiretroviral therapy. In this instance, susceptibility has been strongly linked to genetic loci involved in antigen recognition and presentation to the immune system--most notably within the major histocompatibility complex (MHC) region--consistent with the notion that hypersensitivity reactions represent drug-specific immune responses that are largely dose independent. Here the authors describe their experiences with the development of pharmacogenetic approaches to hypersensitivity reactions associated with abacavir and nevirapine, two commonly prescribed antiretroviral drugs. It is demonstrated that prospective screening tests to identify and exclude individuals with a certain genetic makeup may be largely successful in decreasing or eliminating incidence of these adverse drug reactions in certain populations. This review also explores the broader implications of these findings.

Influence of CYP2B6 polymorphism on plasma and intracellular concentrations and toxicity of efavirenz and nevirapine in HIV-infected patients

BACKGROUND: Efavirenz (EFV) and nevirapine (NVP) are metabolized by cytochrome P450 2B6 (CYP2B6). Allele 516 G>T (Gln172His) is associated with diminished activity of this isoenzyme, and may lead to differences in drug exposure. METHODS: We evaluated this allele as a pharmacogenetic marker of EFV and NVP pharmacokinetics and EFV toxicity in 167 participants receiving EFV and 59 receiving NVP recruited within the genetics project of the Swiss HIV Cohort Study. Drug concentrations were measured in plasma and in peripheral blood mononuclear cells (PBMCs) from the same sample. Neuropsychological toxicity of EFV (sleep disorders, mood disorders, fatigue) was assessed using a standardized questionnaire. RESULTS AND CONCLUSIONS: CYP2B6 516TT was associated with greater plasma and intracellular exposure to EFV, and greater plasma exposure to NVP. Intracellular drug concentration, and CYP2B6 genotype were predictors of EFV neuropsychological toxicity. CYP2B6 genotyping may be useful to complement an individualization strategy based on plasma drug determinations to increase the safety and tolerability of EFV.

Risk factors for treatment-limiting toxicities in patients starting nevirapine-containing antiretroviral therapy

BACKGROUND: This collaboration of seven observational clinical cohorts investigated risk factors for treatment-limiting toxicities in both antiretroviral-naive and experienced patients starting nevirapine-based combination antiretroviral therapy (NVPc). METHODS: Patients starting NVPc after 1 January 1998 were included. CD4 cell count at starting NVPc was classified as high (>400/microl/>250/microl for men/women, respectively) or low. Cox models were used to investigate risk factors for discontinuations due to hypersensitivity reactions (HSR, n = 6547) and discontinuation of NVPc due to treatment-limiting toxicities and/or patient/physician choice (TOXPC, n = 10,186). Patients were classified according to prior antiretroviral treatment experience and CD4 cell count/viral load at start NVPc. Models were stratified by cohort and adjusted for age, sex, nadir CD4 cell count, calendar year of starting NVPc and mode of transmission. RESULTS: Median time from starting NVPc to TOXPC and HSR were 162 days [interquartile range (IQR) 31-737] and 30 days (IQR 17-60), respectively. In adjusted Cox analyses, compared to naive patients with a low CD4 cell count, treatment-experienced patients with high CD4 cell count and viral load more than 400 had a significantly increased risk for HSR [hazard ratio 1.45, confidence interval (CI) 1.03-2.03] and TOXPC within 18 weeks (hazard ratio 1.34, CI 1.08-1.67). In contrast, treatment-experienced patients with high CD4 cell count and viral load less than 400 had no increased risk for HSR 1.10 (0.82-1.46) or TOXPC within 18 weeks (hazard ratio 0.94, CI 0.78-1.13). CONCLUSION: Our results suggest it may be relatively well tolerated to initiate NVPc in antiretroviral-experienced patients with high CD4 cell counts provided there is no detectable viremia.

Identification of protein targets of nevirapine reactive metabolites using click chemistry and mass spectrometry-based differential proteomics

Abstract : Adverse drug reactions (ADRs) are undesirable effects caused after administration of a single dose or prolonged administration of drug or result from the combination of two or more drugs. Idiosyncratic drug reaction (IDR) is an adverse reaction that does not occur in most patients treated with a drug and does not involve the therapeutic effect of the drug. IDRs are unpredictable and often life-threatening. Idiosyncratic reaction is dependent on drug chemical characteristics or individual immunological response. IDRs are a major problem for drug development because they are usually not detected during clinical trials. In this study we focused on IDRs of Nevirapine (NVP), which is a non-nucleoside reverse transcriptase inhibitor used for the treatment of Human Immunodeficiency Virus (HIV) infections. The use of NVP is limited by a relatively high incidence of skin rash. NVP also causes a rash in female Brown Norway (BN) rats, which we use as animal model for this study. Our hypothesis is that idiosyncratic skin reactions associated with NVP treatment are due to post-translational modifications of proteins (e.g., glutathionylation) detectable by MS. The main objective of this study was to identify the proteins that are targeted by a reactive metabolite of Nevirapine in the skin. The specific objectives derived from the general objective were as follow: 1) To implement the click chemistry approach to detect proteins modified by a reactive NVP-Alkyne (NVP-ALK) metabolite. The purpose of using NVP-ALK was to couple it with Biotin using cycloaddition Click Chemistry reaction. 2) To detect protein modification using Western blotting and Mass Spectrometry techniques, which is important to understand the mechanism of NVP induced toxicity. 3) To identify the proteins using MASCOT search engine for protein identification, by comparing obtained spectrum from Mass Spectrometry with theoretical spectrum to find a matching peptide sequence. 4) To test if the drug or drug metabolites can cause harmful effects, as the induction of oxidative stress in cells (via protein glutathionylation). Oxidative stress causes cell damage that mediates signals, which likely induces the immune response. The results showed that Nevirapine is metabolized to a reactive metabolite, which causes protein modification. The extracted protein from the treated BN rats matched 10% of keratin, which implies that keratin was the protein targeted by the NVP-ALK.

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

This is an open access article under the CC BY-NC-ND license - http://creativecommons.org/licenses/by-nc-nd/4.0/

Development of Nevirapine resistance in children exposed to the prevention of mother-to-child HIV-1 transmission programme in Maputo, Mozambique

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Inhibitors of human immunodeficiency virus type 1 reverse transcriptase target distinct phases of early reverse transcription

Early HIV-1 reverse transcription can be separated into initiation and elongation phases. Here we show, using PCR analysis of negative-strand strong-stop DNA [(−)ssDNA] synthesis in intact virus, that different reverse transcriptase (RT) inhibitors affect distinct phases of early natural endogenous reverse transcription (NERT). The effects of nevirapine on NERT were consistent with a mechanism of action including both specific and nonspecific binding events. The nonspecific component of this inhibition targeted the elongation reaction, whereas the specific effect seemed principally to be directed at very early events (initiation or the initiation-elongation switch). In contrast, foscarnet and the nucleoside analog ddATP inhibited both early and late (−)ssDNA synthesis in a similar manner. We also examined compounds that targeted other viral proteins and found that Ro24-7429 (a Tat antagonist) and rosmarinic acid (an integrase inhibitor) also directly inhibited RT. Our results indicate that NERT can be used to identify and evaluate compounds that directly target the reverse transcription complex.

S-DABO 类衍生物及AZT-氟喹喏酮类偶联物体外抗HIV 活性及机制研究

本论文由2 个相对独立的部分组成: S-DABO 类衍生物体外抗HIV 活性及 其机制研究和AZT－氟喹喏酮类偶联物体外抗HIV-1 活性及其机制研究。 HIV 逆转录酶抑制剂一直是抗HIV 药物研发的热点。该类抑制剂靶定在病 毒复制周期早期，为HAART 疗法提供了很多新的药物组合。目前FDA 批准上 市的逆转录酶抑制剂虽然有很多，但由于较严重的毒副作用、HIV 病毒易变异、 耐药性的出现等问题还需要开发更多的新的逆转录酶抑制剂。本论文对23 个 S-DABO 类化合物和8 个AZT－氟喹喏酮类偶联物的体外抗HIV 活性进行检 测，并对其中活性较高的化合物进行靶点和机制研究。 23 个S-DABO 类化合物采用对C8166 细胞的毒性试验，对HIV-1ⅢB 诱导的 合胞体形成的抑制试验和对HIV-1ⅢB 急性感染的MT-4 细胞的保护试验进行抗 HIV-1 活性初步筛选。试验结果发现所有化合物均对多种HIV 宿主细胞毒性小， 其中22 个化合物具有抗HIV-1 活性，特别是化合物RZK-4 和RZK-5，其对 HIV-1ⅢB 诱导的合胞体形成的SI 值（Selective index）分别为>16666 和>38462； RZK-4 和RZK-5 对HIV-1ⅢB 急性感染的MT-4 细胞的保护的SI 值分别为2666.67 和2150.54，与相应的阳性对照药品NVP（Nevirapine）的SI 值相接近。以p24 抗原水平为指标，对其中20 个化合物的抗HIV-1 活性进行确证，发现这20 个 化合物均能抑制 HIV-1ⅢB p24 抗原的产生，其中RZK-4 和RZK-5 的EC50 值分 别为5.93 和5.74ng/ml，比相应的阳性对照药品NVP（Nevirapine）的EC50 值 要低（27.3ng/ml）。这些化合物对试验株HIV-1MN、临床分离株HIV-1KM018、非 核苷类抑制剂耐药株HIV-1ⅢB A17 也有较好的抑制效果。但这23 个S-DABO 类化合物对HIV-2 病毒株均无抑制作用。通过检测化合物对感染与未感染细胞的 融合的抑制、对HIV-1 逆转录酶和蛋白酶活性的抑制、对慢性感染H9 细胞 （H9/HIV-1ⅢB）中病毒复制的抑制等试验来探讨化合物的抗HIV-1 机制。结果 显示：有20 个化合物对HIV-1 蛋白酶（PR）有抑制作用，其中有17 个化合物 对HIV-1 逆转录酶（RT）有抑制作用；但所有化合物均不能抑制感染与未感染 细胞的融合，也不能抑制慢性感染H9 细胞中病毒的复制。试验结果表明，这 23 个S-DABO 类化合物主要通过抑制HIV-1 逆转录酶来发挥作用，它们是典型 的非核苷类RT 抑制剂。 8 个AZT－氟喹喏酮类偶联物采用对C8166 细胞的毒性试验，对HIV-1ⅢB 诱导的合胞体形成的抑制试验和对HIV-1ⅢB急性感染的MT-4 细胞的保护试验 进行抗HIV-1 活性初步筛选。试验结果发现其中2 个化合物SRLZ 和SROZ 有 较显著的抗HIV-1 活性，其对HIV-1ⅢB诱导的合胞体形成抑制的SI 值分别为 >41667 和>105263；对HIV-1ⅢB 急性感染的MT-4 细胞的保护的SI 值分别为 30162 和 6368，与AZT（Zidothymidine）的SI 值相近似。以p24 抗原水平为 指标，对其抗HIV-1活性进行确证，发现化合物SRLZ和SROZ均能抑制HIV-1ⅢB p24 抗原的产生，其EC50 值分别为 0.71 和2.1ng/ml,比相应的阳性对照药品AZT 的EC50 值要低（3.5ng/ml）。化合物SRLZ 和SROZ 对临床分离株HIV-1KM018 也有较好的抑制活性，其EC50 值分别为1.4 和22ng/ml。通过检测化合物对慢 性感染H9 细胞（H9/HIV-1ⅢB）中病毒复制的抑制试验来探讨化合物的抗HIV-1 机制，结果表明化合物SRLZ 和SROZ 均不能抑制慢性感染H9 细胞中病毒的 复制。通过检测化合物对金黄色葡萄球菌的抑制作用来检测其抗菌活性，化合 物SRLZ 和SROZ 对金黄色葡萄球菌有较好的抑制作用，其MIC（Minimum inhibitory concentration）值分别为14.65 和7.32μg/ml，与其相应的阳性对照药 物的MIC 值相类似。试验结果表明：药物—药物偶连这种化学修饰方法并没有 改变AZT－氟喹喏酮类偶联物的抗HIV 作用靶点，但也没有较大地影响到其体 外抗病毒活性和抗微生物活性。

Validation of Thin Layer Chromatographic Methods

Thin-layer and high-performance thin-layer chromatography (TLC/HPTLC) methods for assaying compound(s) in a sample must be validated to ensure that they are fit for their intended purpose and, where applicable, meet the strict regulatory requirements for controlled products. Two validation approaches are identified in the literature, i.e. the classic and the alternative, which is using accuracy profiles.Detailed procedures of the two approaches are discussed based on the validation of methods for pharmaceutical analysis, which is an area considered having more strict requirements. Estimation of the measurement uncertainty from the validation approach using accuracy profiles is also described.Examples of HPTLC methods, developed and validated to assay sulfamethoxazole and trimethoprim on the one hand and lamivudine, stavudine, and nevirapine on the other, in their fixed-dose combination tablets, are further elaborated.

Supra-treatment threshold neonatal jaundice: Incidence in HIV-exposed compared to non-exposed neonates at Queen Elizabeth Central Hospital in Blantyre, Malawi

INTRODUCTION: Jaundice is the yellowish pigmentation of the skin, sclera, and mucous membranes resulting from bilirubin deposition. Children born to mothers with HIV are more likely to be born premature, with low birth weight, and to become septic-all risk factors for neonatal jaundice. Further, there has been a change in the prevention of mother-to-child transmission (PMTCT) of HIV guidelines from single-dose nevirapine to a six-week course, all of which theoretically put HIV-exposed newborns at greater risk of developing neonatal jaundice.

AIM: We carried out a study to determine the incidence of severe and clinical neonatal jaundice in HIV-exposed neonates admitted to the Chatinkha Nursery (CN) neonatal unit at Queen Elizabeth Central Hospital (QECH) in Blantyre.

METHODS: Over a period of four weeks, the incidence among non-exposed neonates was also determined for comparison between the two groups of infants. Clinical jaundice was defined as transcutaneous bilirubin levels greater than 5 mg/dL and severe jaundice as bilirubin levels above the age-specific treatment threshold according the QECH guidelines. Case notes of babies admitted were retrieved and information on birth date, gestational age, birth weight, HIV status of mother, type of feeding, mode of delivery, VDRL status of mother, serum bilirubin, duration of stay in CN, and outcome were extracted.

RESULTS: Of the 149 neonates who were recruited, 17 (11.4%) were HIV-exposed. One (5.88%) of the 17 HIV-exposed and 19 (14.4%) of 132 HIV-non-exposed infants developed severe jaundice requiring therapeutic intervention (p = 0.378). Eight (47%) of the HIV-exposed and 107 (81%) of the non-exposed neonates had clinical jaundice of bilirubin levels greater than 5 mg/dL (p < 0.001).

CONCLUSIONS: The study showed a significant difference in the incidence of clinical jaundice between the HIV-exposed and HIV-non-exposed neonates. Contrary to our hypothesis, however, the incidence was greater in HIV-non-exposed than in HIV-exposed infants.