Antileishmanial activity of antiretroviral drugs combined with miltefosine.

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

Role of the K101E substitution in HIV-1 reverse transcriptase in resistance to rilpivirine and other nonnucleoside reverse transcriptase inhibitors.

Resistance to the recently approved nonnucleoside reverse transcriptase inhibitor (NNRTI) rilpivirine (RPV) commonly involves substitutions at positions E138K and K101E in HIV-1 reverse transcriptase (RT), together with an M184I substitution that is associated with resistance to coutilized emtricitabine (FTC). Previous biochemical and virological studies have shown that compensatory interactions between substitutions E138K and M184I can restore enzyme processivity and the viral replication capacity. Structural modeling studies have also shown that disruption of the salt bridge between K101 and E138 can affect RPV binding. The current study was designed to investigate the impact of K101E, alone or in combination with E138K and/or M184I, on drug susceptibility, viral replication capacity, and enzyme function. We show here that K101E can be selected in cell culture by the NNRTIs etravirine (ETR), efavirenz (EFV), and dapivirine (DPV) as well as by RPV. Recombinant RT enzymes and viruses containing K101E, but not E138K, were highly resistant to nevirapine (NVP) and delavirdine (DLV) as well as ETR and RPV, but not EFV. The addition of K101E to E138K slightly enhanced ETR and RPV resistance compared to that obtained with E138K alone but restored susceptibility to NVP and DLV. The K101E substitution can compensate for deficits in viral replication capacity and enzyme processivity associated with M184I, while M184I can compensate for the diminished efficiency of DNA polymerization associated with K101E. The coexistence of K101E and E138K does not impair either viral replication or enzyme fitness. We conclude that K101E can play a significant role in resistance to RPV.

Synthesis and biological evaluation of pyridazine derivatives as novel HIV-1 NNRTIs.

In continuation of our efforts toward identification and optimization of novel non-nucleoside reverse transcriptase inhibitors (NNRTIs), we have employed a structure-based bioisosterism strategy, with which a new series of diarylpyridazine (DAPD) derivatives were synthesized and evaluated for their anti-HIV-1 (human immunodeficiency virus type 1) activity. Most of the title compounds displayed excellent anti-HIV-1 activity at submicromolar concentrations ranging from 34 nM to 5.08 µM. The most promising compound 8g inhibited HIV-1 IIIB in MT-4 cells at a low EC50 value (0.034 µM), which was lower than the reference drug nevirapine and delavirdine. The structure activity relationships (SARs) were discussed and rationalized by docking simulations.

Methylmethacrylate-sulfopropylmethacrylate nanoparticles with surface RMP-7 for targeting delivery of antiretroviral drugs across the blood-brain barrier.

This study investigates the capability of methylmethacrylate-sulfopropylmethacrylate (MMA-SPM) nanoparticles (NPs) with grafted RMP-7 (RMP-7/MMA-SPM NPs) to deliver stavudine (D4T), delavirdine (DLV), and saquinavir (SQV) across the blood-brain barrier (BBB). The permeability coefficients of the three drugs across the BBB were evaluated by a co-culture model containing human brain-microvascular endothelial cells and human astrocytes. An increase in the concentration of ammonium persulfate (APS), the polymerization initiator, enhanced the particle size of drug-loaded RMP-7/MMA-SPM NPs. When the concentration of APS was 0.6%, the average particle diameter was smaller than 50 nm. These spherical drug carriers were uniform in size and displayed a dominant topography of discrete hillocks and deep pits in deposited film. Smaller RMP-7/MMA-SPM NPs yielded a larger drug loading efficiency. The order of drug in the loading efficiency and in the particle uptake was, respectively, D4T>DLV>SQV and D4T>SQV>DLV. Endocytosis of RMP-7/MMA-SPM NPs and tight junction mediation can improve the permeability of D4T, DLV, and SQV across the BBB.

Human immunodeficiency virus type 1 resistance or cross-resistance to nonnucleoside reverse transcriptase inhibitors currently under development as microbicides.

Microbicides based on nonnucleoside reverse transcriptase inhibitors (NNRTIs) are currently being developed to protect women from HIV acquisition through sexual contact. However, the large-scale introduction of these products raises two major concerns. First, when these microbicides are used by undiagnosed HIV-positive women, they could potentially select for viral resistance, which may compromise subsequent therapeutic options. Second, NNRTI-based microbicides that are inactive against NNRTI-resistant strains might promote the selective transmission of these viruses. In order to address these concerns, drug resistance was selected in vitro by the serial passage of three viral isolates from subtypes B and C and CRF02_AG (a circulating recombinant form) in activated peripheral blood mononuclear cells (PBMCs) under conditions of increasing concentrations of three NNRTIs (i.e., TMC120, UC781, and MIV-160) that are currently being developed as candidate microbicides. TMC120 and MIV-160 displayed a high genetic barrier to resistance development, whereas resistance to UC781 emerged rapidly, similarly to efavirenz and nevirapine. Phenotypically, the selected viruses appeared to be highly cross-resistant to current first-line therapeutic NNRTIs (i.e., delavirdine, nevirapine, and efavirenz), although they retained some susceptibility to the more recently developed NNRTIs lersivirine and etravirine. The ability of UC781, TMC120, and MIV-160 to inhibit the in vitro-selected NNRTI-resistant viruses was also limited, although residual activity could be observed for the candidate microbicide NNRTI MIV-170. Interestingly, only four p2/p7/p1/p6/PR/RT/INT recombinant NNRTI-resistant viruses (i.e., TMC120-resistant VI829, EFV-resistant VI829, MIV-160-resistant VI829, and EFV-resistant MP568) showed impairments in replicative fitness. Overall, these in vitro analyses demonstrate that due to potential cross-resistance, the large-scale introduction of single-NNRTI-based microbicides should be considered with caution.

Progress of bis(heteroaryl)piperazines (BHAPs) as non-nucleoside reverse transcriptase inhibitors (NNRTIs) against human immunodeficiency virus type 1 (HIV-1).

Since the first case of acquired immunodeficiency syndrome (AIDS) was reported in 1981, AIDS, as the global disease affecting 33.2 million people in 2007, has always been an unsolved problem worldwide. Reverse transcriptase (RT) is a crucial enzyme in the life cycle of human immunodeficiency virus type 1 (HIV-1), and thereby has been the prime drugs target for antiretroviral (ARV) therapy against AIDS. To date, two classes of RT inhibitors (RTIs), e.g., nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs), and a lot of compounds tested as RTIs have been described. To our knowledge, bis(heteroaryl)piperazines (BHAPs) have been considered as one class of promising NNRTIs, such as structurally and chemically related NNRTI delavirdine, which was approved by the U. S. Food and Drug Administration (FDA) for the treatment of HIV-1 infection in 1997. In this mini-review, we make attempts to report the progress of synthesis and structure-activity relationship (SAR) of BHAPs, in the meantime, the synergistic inhibition of HIV-1 replication by combining delavirdine with other HIV-1 inhibitors is also discussed. It will pave the way for the design and development of BHAPs as anti-HIV-1 agents in AIDS chemotherapy in the future.

[Establishment of pharmacological evaluation system for non-nucleoside reverse-transcriptase inhibitors resistant HIV-1].

Consistent non-nucleoside reverse-transcriptase inhibitors (NNRTIs) resistant HIV-1 strains occurred due to the clinical use for more than ten years of efavirenz (EFV), nevirapine (NVP), and delavirdine (DLV). In this study, we established nine cell-based pharmacological models according to most NNRTIs-resistant clinical tested strains, Resistant mutations were introduced into vector, pNL4-3.Luc.R-E-, by overlapping PCR. Then, pseudovirions were produced by co-transfection of VSV-G plasmid and pNL4-3.Luc.R-E- -mut. All nine recombinant VSVG/HIV-mut pseudovirions (VSVG/HIV-wt, VSVG/HIV(-K103N), VSVG/HIV(-Y181C), VSVG/HIV(-L100I,K103N), VSVG/HIV(-Y188L), VSVG/HIV(-K103N,Y181C), VSVG/HIV(-K103N,P225H), VSVG/HIV(-K103N,Y188L), VSVG/HIV(-K103N,G109A) and VSVG/HIV(-K103N,V108I)) had high efficient infectivity. Furthermore, they all showed resistant characteristics to EFV and NVP with IC50 changes consisting with clinical reports, not to nucleoside reverse-transcriptase inhibitors (AZT and d4T). This series safe cell-based model, which could be carried out in BSL-2 laboratory, can be used for evaluating NNRTIs candidates.

A study of 3-substituted benzylidene-1,3-dihydro-indoline derivatives as antimicrobial and antiviral agents.

3-Substituted benzylidene-1,3-dihydro-indoline derivatives were tested for their in vitro antibacterial activity against the Gram-negative bacteria Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, and the Gram-positive bacteria Bacillus subtilis, Staphylococcus aureus, and for their their in vitro antifungal activity against Candida krusei and Candida albicans. The minimum inhibitory concentration (MIC) values were determined by the 2-fold serial dilution technique in Mueller Hinton broth and Sabouraud dextrose agar using antibacterial and antifungal assays, respectively. For comparison of the antimicrobial activity, rifampicin, ampicillin trihydrate, gentamicin sulfate, and ofloxacin were used as reference antibacterial agents, and fluconazole and amphotericin B were employed as reference antifungal agents. The most active compound 10 showed notable inhibition against Bacillus subtilis, Staphylococcus aureus, and Candida krusei. Compounds 1 and 6 were found slightly effective against Klebsiella pneumoniae and Escherichia coli. In addition, compounds 13 and 14 showed inhibition against Bacillus subtilis and Staphylococcus aureus. Indole derivatives were also tested in vitro for replication of the HepAD38 cell line and compared with lamivudine (3TC, L-2',3'-dideoxy-3'-thiacytidine). The IC50 values of the compounds were found to be >1000 microM against HBV except for compound 13 which exhibited activity with an IC50 value of 500 microM.

The human immunodeficiency virus type 1 nonnucleoside reverse transcriptase inhibitor resistance mutation I132M confers hypersensitivity to nucleoside analogs.

We previously identified a rare mutation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), I132M, which confers high-level resistance to the nonnucleoside RT inhibitors (NNRTIs) nevirapine and delavirdine. In this study, we have further characterized the role of this mutation in viral replication capacity and in resistance to other RT inhibitors. Surprisingly, our data show that I132M confers marked hypersusceptibility to the nucleoside analogs lamivudine (3TC) and tenofovir at both the virus and enzyme levels. Subunit-selective mutagenesis studies revealed that the mutation in the p51 subunit of RT was responsible for the increased sensitivity to the drugs, and transient kinetic analyses showed that this hypersusceptibility was due to I132M decreasing the enzyme's affinity for the natural dCTP substrate but increasing its affinity for 3TC-triphosphate. Furthermore, the replication capacity of HIV-1 containing I132M is severely impaired. This decrease in viral replication capacity could be partially or completely compensated for by the A62V or L214I mutation, respectively. Taken together, these results help to explain the infrequent selection of I132M in patients for whom NNRTI regimens are failing and furthermore demonstrate that a single mutation outside of the polymerase active site and inside of the p51 subunit of RT can significantly influence nucleotide selectivity.

Monitoring the development of non-nucleoside reverse transcriptase inhibitor-associated resistant HIV-1 using an electrochemiluminescence-based reverse transcriptase polymerase assay.

Reverse transcriptase (RT) plays an essential role in the HIV-1 replication process, which converts a single-strand RNA into a double-strand DNA via polymerase and RNase H activities. Therefore, inhibition of RT has been one of the primary therapeutic strategies for suppressing the replication of HIV-1. To facilitate the process of discovering the next generation of antiretroviral agents, this study presents a highly sensitive and nonradioactive RT polymerase assay that is based on electrochemiluminescence (ECL) technology, where a ruthenylated dUTP (Ru-dUTP) is employed as one of the dNTPs. The concentration of the RT enzymes required for the assay can be as low as 1 pM, enabling us to evaluate inhibitors with low picomolar potency. More importantly, the assay is capable of detecting endogenous RT activity in cell-free viruses. Therefore, the assay was applied to monitor the development of resistance mutation(s) by viruses under the treatment with a non-nucleoside reverse transcriptase inhibitor (NNRTI) in cell culture. The magnitude of resistance of the resulting mutant viruses was assessed directly by the assay, eliminating the need for cloning, expressing, and purifying the RT mutants.

Emergence of NNRTI drug resistance mutations after single-dose nevirapine exposure in HIV type 1 subtype C-infected infants in India.

A feasibility study for providing single-dose nevirapine (SD-NVP) prophylaxis for prevention of mother-to-child transmission (PMTCT) of HIV infection provided an opportunity to study the emergence of nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance mutations as a result of single-dose administration. The study aimed at the detection of NNRTI drug resistance mutations arising as a result of SD-NVP. A total of 19 and 13 samples collected at 48 h and 2 months postpartum, respectively, from infants that were given SD-NVP were studied for the presence of NNRTI drug resistance mutations by PCR amplification and sequencing of the HIV-1 pol gene using HIV proviral DNA. The drug resistance mutational analysis of final sequences was carried out using the Stanford University HIV Drug Resistance database (http://hivdb.stanford.edu/hiv). Mutations associated with NNRTI drug resistance were observed in two (10.5%) and six (46.15%) samples at 48 h and at 2 months, respectively. K103N, one of the most common mutations, was not observed in any of the samples. The emergence of NVP resistance must be weighed against the simplicity, efficacy, and cost effectiveness of SD-NVP prophylaxis in PMTCT settings in developing countries.

[HIV-1 genotypic resistance profiles in children failing highly active antiretroviral therapy].

OBJECTIVE: To study the genotypic resistance profiles of HIV-1 children failing highly active antiretroviral therapy (HAART) so as to provide helpful information for the treatment regime of Chinese children infected with HIV-1. METHODS: Peripheral venous blood samples were collected from 20 HIV-1 infected children of Henan province, aged 9 (3 - 14). Nested RT-PCR was used to amplify part of the RT (40 -250 aa) gene. The PCR products of RT gene underwent nucleotide sequencing, the resulting nucleotide sequences were analyzed by the HIVdb data offered by the Stanford University web site to find the drug resistance mutations. RESULTS: (1) Phylogenetic analysis revealed that 20 of the RT sequences were classified as subtype B. (2) According to the genotypic analysis, 20 , 15, and 13 children showed high level resistance to the nevirapine. (NVP), delavirdine (DLV), and efavirenz (EFV) respectively; 7 and 5 children showed high and intermediate level resistance to azidothymidine (AZT) respectively. Five children showed potential low-level and intermediate level resistance to lamivudine (3TC), and 11 showed high level resistance to 3TC; 11 showed intermediate and high level resistance to stavudine (d4T) and didanoside (ddI) respectively; and 19 and 12 children showed resistance to abacavir (ABC) and tenofovir (TDF) which had never been taken by these children. CONCLUSION: The emergence of HIV resistant strains during antiretroviral therapy is one of the main reasons for treatment failure in HIV-infected children.

Biosensor-based kinetic characterization of the interaction between HIV-1 reverse transcriptase and non-nucleoside inhibitors.

Details of the interaction between HIV-1 reverse transcriptase and non-nucleoside inhibitors (NNRTIs) have been elucidated using a biosensor-based approach. This initial study was performed with HIV-1 reverse transcriptase mutant K103N, the phenethylthioazolylthiourea compound (PETT) MIV-150, and the three NNRTIs licensed for clinical use: nevirapine, delavirdine, and efavirenz. Mathematical evaluation of the experimental data with several interaction models revealed that the four inhibitors interacted with HIV-1 RT with varying degrees of complexity. The simplest adequate model accounted for two different conformations of the free enzyme, of which only one can bind the inhibitor, consistent with a previously hypothesized population-shift model including a preformation of the NNRTI binding site. In addition, a heterogeneous binding was observed for delavirdine, efavirenz, and MIV-150, indicating that two noncompetitive and kinetically distinct enzyme-inhibitor complexes could be formed. Furthermore, for these compounds, there were indications for ligand-induced conformational changes.

Reverse transcriptase mutations 118I, 208Y, and 215Y cause HIV-1 hypersusceptibility to non-nucleoside reverse transcriptase inhibitors.

BACKGROUND: HIV-1 hypersusceptibility to non-nucleoside reverse transcriptase inhibitors (NNRTI) improves the response to NNRTI-containing regimens. The genetic basis for NNRTI hypersusceptibility was partly defined in our earlier analyses of a paired genotype-phenotype dataset of viral isolates from treatment-experienced patients, in which we identified reverse transcriptase mutations V118I, H208Y, and T215Y as being strongly associated with NNRTI hypersusceptibility. OBJECTIVES: We evaluated the role of these mutations in NNRTI hypersusceptibility by site-directed mutagenesis and phenotypic analysis of HIV-1 recombinants. METHODS: Drug susceptibility and replication capacity were determined in single cycle assays. Hypersusceptibility was defined by a statistically significant (P < 0.01; Student's t-test) mean fold-change in 50% inhibitory concentration (IC50) of less than 0.4. RESULTS: The single mutations V118I, H208Y, and T215Y did not show hypersusceptibility to efavirenz with mean fold-change of 0.58, 0.55, and 0.70, respectively (P < 0.01 and P = 0.12). The H208Y/T215Y and V118I/H208Y/T215Y mutants showed marked hypersusceptibility to efavirenz, having mean fold-change values of 0.27 and 0.20, respectively (P < 0.001). In addition, H208Y/T215Y, V118I/T215Y, and V118I/H208Y/T215Y were hypersusceptible to delavirdine and nevirapine. The V118I/T215Y mutant was not replication impaired; whereas H208Y/T215Y and V118I/H208Y/T215Y had significantly (P < 0.01) reduced replication capacities of 40 and 35% of wild-type, respectively. CONCLUSION: Different combinations of V118I, H208Y, and T215Y produce NNRTI hypersusceptibility. The V118I/T215Y mutant is hypersusceptible to delavirdine and nevirapine without reduced replication capacity, whereas the H208Y/T215Y and V118I/H208Y/T215Y mutants are hypersusceptible to all NNRTI and show impaired replication. These findings suggest that more than one mechanism is involved in NNRTI hypersusceptibility.

Phenotypic drug resistance patterns in subtype A HIV-1 clones with nonnucleoside reverse transcriptase resistance mutations.

We analyzed the nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI) susceptibility of 29 subtype A HIV-1 clones isolated from 10 Ugandan women after single-dose nevirapine (NVP) administration. Six clones had no NNRTI resistance-associated mutations ("wild type"), eight had K103N, nine had Y181C, five had G190A, and one had Y181S. Three clones displayed unexpected phenotypic drug susceptibility/resistance based on their RT genotypes. One wild-type clone had reduced susceptibility to NVP, delavirdine (DLV), and efavirenz (EFV), one clone with K103N was susceptible to all three NNRTIs, and one clone with G190A had extreme hypersusceptibility to DLV. Three unusual HIV-1 RT amino acid substitutions may have contributed to the unexpected phenotypes of the clones: I31T, N136S, and N265D. These polymorphisms were rarely detected among 47,900 HIV-1 genotypes from clinical samples of predominantly United States origin. Further studies are needed to define the genetic correlates of antiretroviral drug resistance in nonsubtype B HIV-1.

Fármacos antirretrovirales (2º parte) / Antiretroviral medicine (Part Two)

En esta segunda parte se presentan los fármacos antirretrovirales incluidos en el grupo de los inhibidores NO Nucleósidos de la Transcriptasa Inversa y los inhibidores de Proteasa (AU)

Effects of the G190A substitution of HIV reverse transcriptase on phenotypic susceptibility of patient isolates to delavirdine.

BACKGROUND: Cross resistance is common among the non-nucleoside reverse transcriptase inhibitors (NNRTIs). G190A appears in 5-15% of the patients treated with nevirapine or efavirenz who develop clinical resistance. OBJECTIVES: In this study we investigated the effect of G190A and other NNRTI substitutions on the phenotypic susceptibility to this class of drugs. STUDY DESIGN: We identified 15 individuals, who after treatment with NNRTIs (nevirapine or efavirenz; median exposure of 20 months), developed isolated G190A, G190A in combination with K103N, or K103N alone. Phenotypic and genotypic analyses of stored plasma specimens were performed before and after the mutations occurred to assess NNRTI susceptibility. RESULTS: All isolates that developed only G190A substitution became less susceptible to nevirapine (median: 125-fold) and efavirenz (median: 10-fold) but were 2.5-fold more sensitive to delavirdine (Wilcoxon P = 0.06). In the group with only K103N substitution, acquisition of resistance to all NNRTIs was observed. In the group with the double substitutions, G190A and K103N, delavirdine susceptibility decreased 13-fold, while resistance to nevirapine and efavirenz decreased by 239- and 154-folds, respectively (Kruskal-Wallis H P = 0.009). CONCLUSIONS: The data suggest that the presence of a G190A substitution attenuates the phenotypic resistance associated with a K103N substitution, although resistance is still present. The in vivo significance of the increased phenotypic susceptibility to delavirdine is not known but could be evaluated in a clinical trial.

Clinical utility of current NNRTIs and perspectives of new agents in this class under development.

Highly active antiretroviral therapy (HAART) has significantly reduced the number of deaths caused by AIDS. However, the antiviral efficacy of HAART comprising protease inhibitors (PIs) and nucleoside reverse transcriptase inhibitors (NRTIs) is frequently accompanied by a decrease in patients' quality of life. PI-based therapies often fail due to poor adherence caused by heavy pill burden, complex dosing schedules and undesirable side effects. The current trend is to switch from PI-based to PI-sparing regimens consisting of non-nucleoside reverse transcriptase inhibitors (NNRTIs) and NRTIs. Despite some encouraging results from NNRTI-containing therapies, two major concerns in using the currently available NNRTIs remain: 1) low genetic barrier to the emergence of resistance and 2) cross-resistance due to single mutations that often render the whole class of NNRTIs ineffective. Clearly, new and improved NNRTIs are needed to address these concerns.

Sex-based differences in saquinavir pharmacology and virologic response in AIDS Clinical Trials Group Study 359.

AIDS Clinical Trials Group study 359 was a controlled study of saquinavir with either ritonavir or nelfinavir, together with delavirdine, adefovir, or both, in indinavir-experienced persons. Saquinavir was common in all study arms, and the study investigated relationships among characteristics of patients, saquinavir area under the curve (AUC) and trough concentrations (C(min)), and virologic response. Concentrations of saquinavir were higher when it was combined with ritonavir than when it was combined with nelfinavir and were lower with adefovir-containing regimens. Females had higher AUC and C(min) values than did males. Higher saquinavir AUC and C(min) values were associated with a greater likelihood of human immunodeficiency virus (HIV) RNA levels