Development of a pseudovirus assay and evaluation to screen natural products for inhibition of HIV-1 subtype C reverse transcriptase.

ETHNOPHARMACOLOGICAL RELEVANCE: Medicinal plants are used in the management of Human Immunodeficiency Virus and Acquired Immunodeficiency Syndrome (HIV/AIDS) in many developing country settings where HIV-1 subtype C drives the epidemic. Efforts to identify plant derived molecules with anti-HIV properties require reproducible assay systems for routine screening of selected plant compounds. Although a number of standardized HIV-1 pseudoviruses have been generated to assess infectivity, replicability or reproducibility, HIV-1 subtype C (HIV-1-C) pseudoviruses have not been comprehensively characterized to identify inhibitory plant substances. AIM OF THE STUDY: The current study aimed at developing an HIV-1-C pseudovirus assay, and evaluate plant substances targeting reverse transcriptase (RT) activity. MATERIALS AND METHODS: HIV-1 subtype C pseudoviruses containing a luciferase reporter gene were generated by transfection of human 293T cells. HIV-1 subtype B (HIV-1-B) wild type pseudoviruses and mutants resistant to nucleoside and non-nucleoside RT inhibitors were also generated and used as controls. Selected plant substances and the RT inhibitors Zidovudine (AZT) and Nevirapine (NVP), were used to evaluate inhibition. Pseudovirus infectivity was determined by luciferase measurement in CF2/CD4+/CCR5 cells, and cytotoxicity was determined using the MTT assay. AZT and NVP inhibited wild type pseudoviruses in a dose dependent manner, with IC50 values in the nanomolar range. RESULTS: Pseudoviruses harbouring RT drug resistance mutations were poorly suppressed by AZT and NVP. Catechin, obtained from Peltophorum africanum inhibited HIV-1-C and HIV-1-B pseudoviruses with selective indices of 6304 µM (IC50: 0.49 µM, CC50: 3089 µM) and 1343 µM (IC50: 2.3 µM, CC50: 3089 µM), respectively; while the methanol root crude extract of Elaeodendron transvaalense gave IC50 values of 11.11 µg/ml and 16.86 µg/ml, respectively. CONCLUSION: The developed HIV-1-C pseudovirus assay can be used to screen plant substances for RT inhibition, and may have utility in settings with limited access to high level biosafety facilities.

Islatravir for the treatment and prevention of infection with the human immunodeficiency virus type 1.

PURPOSE OF REVIEW: To discuss the potential role of islatravir (ISL), a novel reverse transcriptase translocation inhibitor, in the treatment and prevention of human immunodeficiency virus type 1 (HIV-1) infection. RECENT FINDINGS: Islatravir (4'-ethynyl-2-fluoro-2'-deoxyadenosine, MK-8591) is a long-acting first-in-class nucleoside reverse transcriptase translocation inhibitor with the potential for versatile dosing routes and dosing intervals. It demonstrated robust antiviral activity when dosed once daily and once weekly in HIV-1-infected individuals and SIV-infected rhesus macaques. In clinical trials of ISL in combination with doravirine and lamivudine, daily oral administration resulted in high levels of virologic suppression in HIV-infected individuals. In preclinical studies, ISL dosed orally once-weekly as preexposure prophylaxis (PrEP), protected rhesus macaques against SHIV infection via the mucosal route in the low-dose rectal challenge model. Most recently, data in healthy HIV-1-uninfected individuals demonstrated the feasibility of formulating of ISL as an implant. In these studies, levels of intracellular ISL-triphosphate were consistent with the potential for a once-yearly implantable administration of ISL as PrEP. SUMMARY: Islatravir is a promising new agent for both the treatment and prevention of HIV-1 infection.

Clinical features of hypophosphatemic osteomalacia induced by long-term low-dose adefovir dipivoxil.

OBJECTIVE: To investigate the predictors of hypophosphatemic osteomalacia induced by adefovir dipivoxil (ADV) and to monitor for early detection. PATIENTS AND METHODS: Hospitalized patients who were diagnosed with ADV-related hypo-phosphatemic osteomalacia were recruited and retrospectively analyzed in our hospital from January 2012 to December 2016. A telephone interview was conducted at 1, 3, 6, 9, 12, and 24 months after cessation of ADV. RESULTS: In the 8 patients enrolled in the study, the hypophosphatemic osteomalacia symptoms developed at an average of 5.14 (4-7) years since ADV treatment (10 mg/d). The average alkaline phosphatase (ALP) level was 279.50 (137-548) U/L, which was significantly higher than the normal level (45-125 U/L). The serum phosphorus level was an average of 0.59 (0.43-0.69) mmol/L, which was lower than the normal range (2.06-2.60 mmol/L). Serum calcium levels of the enrolled patients remained within normal limits. Reduced estimated glomerular filtration rate (eGFR <29 mL/min/1.73 m2) was seen in 4 cases. The clinical manifestations were mainly progressive systemic bone and joint pain, frequent fractures, trouble in walking, height reduction (4-6 cm), and so on. After cessation of ADV, symptoms like bone pain resolved gradually. Serum phosphorus level restored to normal in 4.5 months after the withdrawal of ADV. However, in 4 patients, renal function failed to return to normal in 24 months. CONCLUSION: More attention should be paid to the duration of ADV treatment. The level of serum phosphorus and ALP, as well as renal function, should be monitored for early detection of potential adverse drug reactions.

Renal histoarchitectural changes in nevirapine therapy: possible role of kolaviron and vitamin C in an experimental animal model.

BACKGROUND: There is paucity of literature regarding the nephrotoxicity of antiretroviral drugs and its interaction with plant-based adjuvants. This study investigates the attenuating effect of kolaviron in nevirapine-therapy on the histological structure of the kidneys of adult male Sprague-Dawley rats. OBJECTIVE: To determine the attenuating influence of anti-oxidant status of kolaviron on the kidneys of experimental animals following nevirapine administration. METHODS: Forty eight pathogen-free adult male Sprague-Dawley rats were used for this study. The animals were divided into 8 groups (A-H) with 6 animals in each group. Group A was given normal saline as the control; group B was given nevirapine; group C was given kolaviron; group D was given vitamin C; group E was given nevirapine and kolaviron; group F was given nevirapine and vitamin C; Group G was given nevirapine and kolaviron (kolaviron withdrawn after day 28) and group H was given corn oil. The experiment lasted 56 days after which the animals were sacrificed, blood samples were collected through cardiac puncture for serum analysis and the kidneys were harvested and prepared for H& E histological examination. RESULTS: Nevirapine caused histoarchitectural damage in the glomerular apparatus with resultant increase in kidney/body weight ratio (p<0.001). Adjuvant treatment with kolaviron attenuated these nephrotoxic effects. Serum anti-oxidant enzyme (SOD and CAT) activities were significantly reduced in kolaviron and vitamin C treated animals, whereas in the nevirapine group these parameters were significantly elevated (P<0.05). However, co-administration of nevirapine and vitamin C did not improve the histoarchitecture of the kidney. CONCLUSION: Adjuvant treatment with kolaviron (an anti-oxidant) for 56 days appears to attenuate the nephrotoxicity of nevirapine in this model.

Effect of rifampicin and efavirenz on moxifloxacin concentrations when co-administered in patients with drug-susceptible TB.

Objectives: We compared the pharmacokinetics of moxifloxacin during rifampicin co-treatment or when dosed alone in African patients with drug-susceptible recurrent TB. Methods: Patients in the intervention arm of the Improving Retreatment Success (IMPRESS) randomized controlled TB trial received 400 mg of moxifloxacin, with rifampicin, isoniazid and pyrazinamide in the treatment regimen. Moxifloxacin concentrations were measured in plasma during rifampicin-based TB treatment and again 4 weeks after treatment completion, when given alone as a single dose. Moxifloxacin concentration-time data were analysed using non-linear mixed-effects models. Results: We included 58 patients; 42 (72.4%) were HIV co-infected and 40 (95%) of these were on efavirenz-based ART. Moxifloxacin pharmacokinetics was best described using a two-compartment disposition model with first-order lagged absorption and elimination using a semi-mechanistic model describing hepatic extraction. Oral clearance (CL/F) of moxifloxacin during rifampicin-based TB treatment was 24.3 L/h for a typical patient (fat-free mass of 47 kg), resulting in an AUC of 16.5 mg·h/L. This exposure was 7.8% lower than the AUC following the single dose of moxifloxacin given alone after TB treatment completion. In HIV-co-infected patients taking efavirenz-based ART, CL/F of moxifloxacin was increased by 42.4%, resulting in a further 30% reduction in moxifloxacin AUC. Conclusions: Moxifloxacin clearance was high and plasma concentrations low in our patients overall. Moxifloxacin AUC was further decreased by co-administration of efavirenz-based ART and, to a lesser extent, rifampicin. The clinical relevance of the low moxifloxacin concentrations for TB treatment outcomes and the need for moxifloxacin dose adjustment in the presence of rifampicin and efavirenz co-treatment need further investigation.

Multicompartmental Pharmacokinetic Model of Tenofovir Delivery to the Rectal Mucosa by an Enema.

Rectal enemas that contain prophylactic levels of anti-HIV microbicides such as tenofovir have emerged as a promising dosage form to prevent sexually transmitted HIV infections. The enema vehicle is promising due to its likely ability to deliver a large amount of drug along the length of the rectal canal. Computational models of microbicide drug delivery by enemas can help their design process by determining key factors governing drug transport and, more specifically, the time history and degree of protection. They can also inform interpretations of experimental pharmacokinetic measures such as drug concentrations in biopsies. The present work begins rectal microbicide PK modeling, for enema vehicles. Results here show that a paramount factor in drug transport is the time of enema retention; direct connectivity between enema fluid and the fluid within rectal crypts is also important. Computations of the percentage of stromal volume protected by a single enema dose indicate that even with only a minute of enema retention, protection of 100% can be achieved after around 14 minutes post dose. Concentrations in biopsies are dependent on biopsy thickness; and control and/or knowledge of thickness could improve accuracy and decrease variability in biopsy measurements. Results here provide evidence that enemas are a promising dosage form for rectal microbicide delivery, and offer insights into their rational design.

Maraviroc and reverse transcriptase inhibitors combinations as potential preexposure prophylaxis candidates.

OBJECTIVE: Receptive anal intercourse in both men and women is associated with the highest probability for sexual acquisition of HIV infection. As part of a program to develop an effective prevention strategy, we performed an ex-vivo preclinical evaluation to determine the efficacy of multiple double combinations of maraviroc (MVC) and reverse transcriptase inhibitors (RTIs). DESIGN: The entry inhibitor, MVC, a nucleotide RTI, tenofovir and two non-nucleoside RTIs, UC781 and TMC120 (dapivirine, DPV), were used in double, combinations against a panel of CCR5-using clade B and clade C HIV-1 isolates and against MVC-escape variants. A gel-formulated version of MVC-DPV combination was also tested. METHODS: Indicator cells, cocultures of immature dendritic cells with CD4T cells, and colorectal tissue explants were used to assess antiviral activity of drug combinations. RESULTS: All dual MVC-RTI combinations tested inhibited MVC-sensitive and resistant isolates in cellular and colorectal explants models. All the combinations were positive with no reduction in the activity of MVC. In tissue explants, the combinations against all viral isolates tested produced an increase in the activity of MVC. An initial gel-formulation of MVC-DPV combination showed greater and prolonged antiviral activity of MVC in mucosal tissue explants. CONCLUSION: This study demonstrates that combinations based on antiretroviral drugs inhibiting HIV transmission at viral entry and reverse transcription have potential as prevention strategies against colorectal transmission of HIV-1 including MVC-resistant isolates. Preclinical evaluation with colorectal tissue explants indicates that a gel-formulation of MVC-DPV is an effective candidate colorectal microbicide.

Naringin Reverses Hepatocyte Apoptosis and Oxidative Stress Associated with HIV-1 Nucleotide Reverse Transcriptase Inhibitors-Induced Metabolic Complications.

Nucleoside Reverse Transcriptase Inhibitors (NRTIs) have not only improved therapeutic outcomes in the treatment of HIV infection but have also led to an increase in associated metabolic complications of NRTIs. Naringin's effects in mitigating NRTI-induced complications were investigated in this study. Wistar rats, randomly allotted into seven groups (n = 7) were orally treated daily for 56 days with 100 mg/kg zidovudine (AZT) (groups I, II III), 50 mg/kg stavudine (d4T) (groups IV, V, VI) and 3 mL/kg of distilled water (group VII). Additionally, rats in groups II and V were similarly treated with 50 mg/kg naringin, while groups III and VI were treated with 45 mg/kg vitamin E. AZT or d4T treatment significantly reduced body weight and plasma high density lipoprotein concentrations but increased liver weights, plasma triglycerides and total cholesterol compared to controls, respectively. Furthermore, AZT or d4T treatment significantly increased oxidative stress, adiposity index and expression of Bax protein, but reduced Bcl-2 protein expression compared to controls, respectively. However, either naringin or vitamin E significantly mitigated AZT- or d4T-induced weight loss, dyslipidemia, oxidative stress and hepatocyte apoptosis compared to AZT- or d4T-only treated rats. Our results suggest that naringin reverses metabolic complications associated with NRTIs by ameliorating oxidative stress and apoptosis. This implies that naringin supplements could mitigate lipodystrophy and dyslipidemia associated with NRTI therapy.

Natural Plant Alkaloid (Emetine) Inhibits HIV-1 Replication by Interfering with Reverse Transcriptase Activity.

Ipecac alkaloids are secondary metabolites produced in the medicinal plant Psychotria ipecacuanha. Emetine is the main alkaloid of ipecac and one of the active compounds in syrup of Ipecac with emetic property. Here we evaluated emetine's potential as an antiviral agent against Human Immunodeficiency Virus. We performed in vitro Reverse Transcriptase (RT) Assay and Natural Endogenous Reverse Transcriptase Activity Assay (NERT) to evaluate HIV RT inhibition. Emetine molecular docking on HIV-1 RT was also analyzed. Phenotypic assays were performed in non-lymphocytic and in Peripheral Blood Mononuclear Cells (PBMC) with HIV-1 wild-type and HIV-harboring RT-resistant mutation to Nucleoside Reverse Transcriptase Inhibitors (M184V). Our results showed that HIV-1 RT was blocked in the presence of emetine in both models: in vitro reactions with isolated HIV-1 RT and intravirion, measured by NERT. Emetine revealed a strong potential of inhibiting HIV-1 replication in both cellular models, reaching 80% of reduction in HIV-1 infection, with low cytotoxic effect. Emetine also blocked HIV-1 infection of RT M184V mutant. These results suggest that emetine is able to penetrate in intact HIV particles, and bind and block reverse transcription reaction, suggesting that it can be used as anti-HIV microbicide. Taken together, our findings provide additional pharmacological information on the potential therapeutic effects of emetine.

Nonclinical Safety Profile of BMS-986001, a Nucleoside Transcriptase Inhibitor for Combination Retroviral Therapy.

Nucleoside reverse transcriptase inhibitors (NRTIs)/nucleotide reverse transcriptase inhibitors are key components of combination antiretroviral therapy for HIV infection. First-generation NRTIs are associated with mitochondrial toxicity in patients, mainly due to inhibition of human DNA polymerase γ (hDNA polγ) that manifests as adverse events such as lipodystrophy, lactic acidosis, myopathy, cardiomyopathy, or nephropathy in patients. In chronic nonclinical studies in rodents and nonrodents, eukaryotic (host) mitochondrial toxicity manifests as some drug-specific toxicities similar to human toxicity. BMS-986001, a novel thymidine analog with minimal hDNA polγ inhibition, has demonstrated antiretroviral activity in early clinical studies. The primary toxicity of BMS-986001 in rats and monkeys is bone marrow dyserythropoiesis with associated decreases in red blood cell mass. Additionally, at high doses, severe platelet reductions accompanied by cutaneous petechiae began during weeks 8 and 11 in 3 of 60 monkeys in chronic toxicity studies. In a 6-month study, platelet reductions required euthanasia of the 2 affected monkeys (300 mg/kg/d) at week 14, but with dose reduction (200 mg/kg/d) remaining monkeys had no platelet changes. One affected monkey (200 mg/kg/d) in a 9-month study completed dosing and its platelet counts recovered during a 1-month recovery. Formation of platelet-bound immunoglobulin in the presence of BMS-986001, together with rapid and complete platelet recovery in the absence of BMS-986001, suggested that platelet decreases in monkeys may be immune mediated. No findings indicative of mitochondrial toxicity were observed in rats or monkeys given BMS-986001, suggesting an improved safety profile compared to marketed NRTI or tenofovir disoproxil fumarate.

Switching to darunavir/ritonavir monotherapy vs. triple-therapy on body fat redistribution and bone mass in HIV-infected adults: the Monarch randomized controlled trial.

Changes in body fat distribution and bone mass in HIV-infected patients may be associated with long-term use of nucleoside reverse transcriptase inhibitors (NRTIs). The Monarch trial recruited 30 patients receiving non-nucleoside reverse transcriptase inhibitor or protease inhibitor-based highly active antiretroviral therapy, with HIV RNA <40 copies/mL. Patients were randomized to either darunavir/ritonavir 800/100 mg once daily monotherapy or darunavir/ritonavir 800/100 mg once daily + two NRTIs. Bone mass, peripheral lipoatrophy and central fat accumulation were assessed using dual-energy X-ray absorptiometry scanning, supplemented by computed tomography scans. Median age was 43 years, 77% were men. Visceral adipose tissue remained stable from baseline to Week 48 in the whole group (p = 0.261) with no significant difference between arms (p = 0.56). There was a significant reduction in insulin resistance (HOMA-IR, p = 0.013) over 48 weeks in the whole group, but not of body mass index (p = 0.24). In the darunavir/ritonavir monotherapy arm, there was a small but significant increase in both lumbar and femur bone mineral density at 48 weeks and was observed after correction for baseline values. The absolute change in lumbar bone mineral density at 48 weeks was more pronounced in the darunavir/ritonavir arm compared with the darunavir/ritonavir + 2NRTIs arm. In this study, discontinuing nucleoside analogues and switching to darunavir/ritonavir monotherapy was associated with a small but statistically significant increase in bone mineral density, but stable levels of limb fat and visceral adipose tissue.

[Resistance profile of rilpivirine]. / Perfil de resistencia de rilpivirina.

Rilpivirine (RPV) is a new second-generation nonnucleoside reverse transcriptase inhibitor (NNRTI) approved for use in combination with two nucleoside/nucleotide reverse transcriptase inhibitors (NRTI) as initial therapy in treatment-naïve HIV-1-infected patients with a baseline viral load ≤100,000 copies/mL. RPV is a diarylpyrimidine derivative with potent in vitro activity against multiple HIV-1 variants with resistance mutations to first-generation NNRTI such as K103N. In vitro studies and phase III clinical trials have allowed the identification of 16 mutations associated with resistance to RPV K101E/P, E138A/G/K/Q/R, V179L, Y181C/I/V, Y188L, H221Y, F227C and M230I/L. The risk of virologic failure in patients receiving RPV plus 2 NRTI with plasma viral load ≤ 100,000 copies/mL is low, but a high percentage of patients failing RPV develop resistance mutations to both RPV and NRTI. The most common resistance mutation that emerges in this setting is E138K. This mutation is usually associated with M184I due to a double compensatory effect of this combination, which confers resistance to RPV, as well as to lamivudine and emtricitabine. The emergence of RPV resistance confers cross-resistance to all NNRTI and, importantly, high percentages of cross-resistance to etravirine.

Tenofovir monotherapy and tenofovir plus entecavir combination as rescue therapy for entecavir partial responders.

BACKGROUND AND AIMS: Despite high potency, a significant proportion of patients treated with entecavir achieve only partial viral suppression. Our goal was to examine the complete viral suppression rate (undetectable HBV DNA PCR levels) with alternative therapies in such patients. METHODS: We retrospectively studied 42 consecutive patients with partial response to entecavir (detectable HBV DNA at ≥12 months of therapy) who were treated at three clinics with rescue therapies: entecavir + adefovir (n = 5), tenofovir (n = 6), and entecavir + tenofovir (n = 31). Antiviral resistance was excluded by negative mutation analysis and/or absence of virologic breakthrough (increase >1 log(10)IU/mL from nadir). RESULTS: All patients were Asian and 57 % were male with a median age of 36 (22-64) years. Only a few patients had prior exposure to lamivudine (7 %) or adefovir (7 %). Almost all patients (95 %) had positive HBeAg. Overall, the complete viral suppression rate was 79 %, and the alanine aminotransferase normalization rate was 83 % in entecavir partial responders after 6 months on rescue therapies. Cumulative complete viral suppression rates were significantly different (P = 0.0164) among the entecavir + adefovir, tenofovir, and entecavir + tenofovir treatment groups at 6 months (20 vs. 83 vs. 83 %, respectively) and 12 months (20 vs. 100 vs. 97 %). All three patients without complete viral suppression on entecavir + adefovir became aviremic 6 months after switching to entecavir + tenofovir. CONCLUSIONS: Virologic response to entecavir + tenofovir combination therapy and tenofovir monotherapy appeared to be similar in most patients, but not with the entecavir + adefovir combination.

High dose lamivudine in HBV-related cirrhotic patients with unsatisfactory response after adefovir add-on.

BACKGROUND: Before tenofovir approval for chronic hepatitis B therapy, the clinical management of patients with suboptimal response or virological breakthrough during combination treatment with lamivudine and adefovir dipivoxil was a difficult clinical challenge. AIMS: In order to improve virologic response and reduce the risk of decompensation, we evaluate the efficacy of a high dose of lamivudine on chronic HBV patients who have previously presented an unsatisfactory response during treatment with lamivudine 100mg/day and adefovir 10mg/day. METHODS: Six patients with HBV-related liver cirrhosis were prospectively enrolled. All were HBeAg-negative and presented a suboptimal response or virological breakthrough after "adefovir add-on" because of development of clinical breakthrough during Lamivudine treatment. Lamivudine dose was increased to 200 or 300 mg, depending on viral load. After 12 months of follow-up, virological and biochemical response were evaluated. RESULTS: After 12 months of high-dose lamivudine, all patients (6/6, 100%) achieved a significant decrease of serum HBV DNA (mean reduction 2,62 ± 1,15 Log10 UI/ml, P = 0.03) and normalized ALT. In three patients (3/6, 50%), HBV DNA became undetectable within 6 months. No patient developed liver decompensation and no significant changes occurred in serum creatinine, serum and urinary electrolytes. No adverse events were registered. CONCLUSIONS: In our experience, rescue strategy with high-dose lamivudine inhibited viral replication leading to undetectability of serum HBVDNA. This rescue treatment presented a good safety profile, without adverse events during the study period. Customized increase of nucleos(t)ide analogues dose in difficult-to-treat patients may be a proficient approach in challenging clinical setting.

Effect of short term and chronic administration of Sutherlandia frutescens on pharmacokinetics of nevirapine in rats.

Sutherlandia frutescens (sutherlandia), an African herbal supplement was recommended by the South African Ministry of Health for the treatment of AIDS patients. However, no reports yet exist delineating the effect of sutherlandia on pharmacokinetics of antiretroviral agents. Therefore, this investigation aimed at screening the effects of short term and chronic exposure of sutherlandia on oral bioavailability and pharmacokinetics of nevirapine (NVP), a non-nucleoside reverse transcriptase inhibitor, in Sprague Dawley rats. NVP (6 mg/kg) was administered orally alone (control) and with co-administration of sutherlandia; short term (12 mg/kg single dose) and long term (12 mg/kg, once a day for 5 days). No significant difference in the pharmacokinetic parameters of NVP was found upon short-term co-administration of Sutherlandia. However, there was a 50% decrease (p<0.05) in the AUC and C(max) values of NVP after 5 days of chronic exposure with Sutherlandia. In addition, quantitative RT-PCR studies demonstrated a 2-3-fold increase in the hepatic and intestinal mRNA expression of CYP3A2, relative to vehicle control. To further confirm, if this could translate into a clinically relevant pharmacokinetic interaction in patients, we tested this hypothesis employing LS-180 cells as an in vitro induction model for human CYP3A4. Ninety-six hours post treatment, similar to positive control rifampicin (25 µM), sutherlandia extract (300 µg/mL) resulted in elevated m-RNA expression levels and functional activity of CYP3A4 (human homologue of rodent CYP3A2) in LS-180 cells. Taken together, these results suggest that a potential drug-herb interaction is possible when NVP is co-administered with S. frutescens, although this hypothesis still remains to be investigated in a clinical setting.

One percent tenofovir applied topically to humanized BLT mice and used according to the CAPRISA 004 experimental design demonstrates partial protection from vaginal HIV infection, validating the BLT model for evaluation of new microbicide candidates.

Recent iPrEx clinical trial results provided evidence that systemic preexposure prophylaxis (PrEP) with emtricitabine (FTC) and tenofovir disoproxil fumarate (TDF) can partially prevent rectal HIV transmission in humans. Similarly, we have previously demonstrated that systemic administration of the same FTC-TDF combination efficiently prevented rectal transmission in humanized bone marrow/liver/thymus (BLT) mice. The CAPRISA 004 trial recently demonstrated that topical application of the tenofovir could partially prevent vaginal HIV-1 transmission in humans. To further validate the usefulness of the BLT mouse model for testing HIV prevention strategies, we evaluated the topical administration of tenofovir as used in CAPRISA 004 to prevent vaginal HIV transmission in BLT mice. Our results demonstrate that vaginally administered 1% tenofovir significantly reduced HIV transmission in BLT mice (P = 0.002). Together with the results obtained after systemic antiretroviral PrEP, these topical inhibitor data serve to validate the use of humanized BLT mice to evaluate both systemic and topical inhibitors of HIV transmission. Based on these observations, we tested six additional microbicide candidates for their ability to prevent vaginal HIV transmission: a C-peptide fusion inhibitor (C52L), a membrane-disrupting amphipathic peptide inhibitor (C5A), a trimeric d-peptide fusion inhibitor (PIE12-Trimer), a combination of reverse transcriptase inhibitors (FTC-TDF), a thioester zinc finger inhibitor (TC247), and a small-molecule Rac inhibitor (NSC23766). No protection was seen with the Rac inhibitor NSC23766. The thioester compound TC247 offered partial protection. Significant protection was afforded by FTC-TDF, and complete protection was offered by three different peptide inhibitors tested. Our results demonstrate that these effective topical inhibitors have excellent potential to prevent vaginal HIV transmission in humans.

Role of pyrimidine depletion in the mitochondrial cardiotoxicity of nucleoside analogue reverse transcriptase inhibitors.

OBJECTIVE: Long-term antiretroviral treatment with nucleoside analogue reverse transcriptase inhibitors (NRTI) may result in a cardiomyopathy due to mitochondrial DNA (mtDNA) depletion. An intact mitochondrial function is required for the synthesis of intramyocardial pyrimidine nucleotides, which in turn are building blocks of mtDNA. We investigated if NRTI-related cardiomyopathy can be prevented with pyrimidine precursors. METHODS: Mice were fed with zidovudine or zalcitabine with or without simultaneous Mitocnol, a dietary supplement with high uridine bioavailability. Myocardia were examined after 9 weeks. RESULTS: Both NRTI induced a cardiomyopathy with mitochondrial enlargement, a disrupted cristal architecture on electron microscopy and diminished myocardial mtDNA copy numbers. The myocardial mtDNA-encoded cytochrome c-oxidase I subunit was impaired more profoundly than the nucleus-encoded cytochrome c-oxidase IV subunit. The myocardial formation of reactive oxygen species and mtDNA mutations was enhanced in zidovudine and zalcitabine treated animals. Mitocnol attenuated or normalized all myocardial pathology when given with both NRTI, but by itself had no intrinsic effects and no apparent adverse effects. CONCLUSIONS: Zidovudine and zalcitabine induce a mitochondrial cardiomyopathy, which is antagonized with uridine supplementation, implicating pyrimidine pool depletion in its pathogenesis. Pyrimidine pool replenishment may be exploited clinically because uridine is well tolerated.

The genetic toxicity effects of lamivudine and stavudine antiretroviral agents.

IMPORTANCE OF THE FIELD: The nucleoside reverse transcriptase inhibitors (NRTIs) are used in antiretroviral therapy worldwide for the treatment of HIV infections. These drugs act by blocking reverse transcriptase enzyme activity, causing pro-viral DNA chain termination. As a consequence, NRTIs could cause genomic instability and loss of heterozygosity. AREAS COVERED IN THIS REVIEW: This review highlights the toxic and genotoxic effects of NRTIs, particularly lamivudine (3TC) and stavudine (d4T) analogues. In addition, a battery of short-term in vitro and in vivo systems are described to explain the potential genotoxic effects of these NRTIs as a single drug or a complexity of highly active antiretroviral therapy. WHAT THE READER WILL GAIN: The readers will gain an understanding of a secondary effect that could be induced by 3TC and d4T treatments. TAKE HOME MESSAGE: Considering that AIDS has become a chronic disease, more comprehensive toxic genetic studies are needed, with particular attention to the genetic alterations induced by NRTIs. These alterations play a primary role in carcinogenesis and are also involved in secondary and subsequent steps of carcinogenesis.

Efficacy of Carraguard-based microbicides in vivo despite variable in vitro activity.

Anti-HIV microbicides are being investigated in clinical trials and understanding how promising strategies work, coincident with demonstrating efficacy in vivo, is central to advancing new generation microbicides. We evaluated Carraguard and a new generation Carraguard-based formulation containing the non-nucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 (PC-817). Since dendritic cells (DCs) are believed to be important in HIV transmission, the formulations were tested for the ability to limit DC-driven infection in vitro versus vaginal infection of macaques with RT-SHIV (SIVmac239 bearing HIV reverse transcriptase). Carraguard showed limited activity against cell-free and mature DC-driven RT-SHIV infections and, surprisingly, low doses of Carraguard enhanced infection. However, nanomolar amounts of MIV-150 overcame enhancement and blocked DC-transmitted infection. In contrast, Carraguard impeded infection of immature DCs coincident with DC maturation. Despite this variable activity in vitro, Carraguard and PC-817 prevented vaginal transmission of RT-SHIV when applied 30 min prior to challenge. PC-817 appeared no more effective than Carraguard in vivo, due to the limited activity of a single dose of MIV-150 and the dominant barrier effect of Carraguard. However, 3 doses of MIV-150 in placebo gel at and around challenge limited vaginal infection, demonstrating the potential activity of a topically applied NNRTI. These data demonstrate discordant observations when comparing in vitro and in vivo efficacy of Carraguard-based microbicides, highlighting the difficulties in testing putative anti-viral strategies in vitro to predict in vivo activity. This work also underscores the potential of Carraguard-based formulations for the delivery of anti-viral drugs to prevent vaginal HIV infection.

Effectiveness of antiretroviral therapy among HIV-infected children in sub-Saharan Africa.

Assessment of antiretroviral treatment programmes for HIV-infected children in sub-Saharan Africa is important to enable the development of effective care and improve treatment outcomes. We review the effectiveness of paediatric antiretroviral treatment programmes in sub-Saharan Africa and discuss the implications of these findings for the care and treatment of HIV-infected children in this region. Available reports indicate that programmes in sub-Saharan Africa achieve treatment outcomes similar to those in North America and Europe. However, progress in several areas is required to improve the care of HIV-infected children in sub-Saharan Africa. The findings emphasise the need for low-cost diagnostic tests that allow for earlier identification of HIV infection in infants living in sub-Saharan Africa, improved access to antiretroviral treatment programmes, including expansion of care into rural areas, and the integration of antiretroviral treatment programmes with other health-care services, such as nutritional support.