Vicriviroc Research Articles

V1 and V2 Domains of HIV Envelope Contribute to CCR5 Antagonist Resistance.

Vicriviroc (VCV) is a CCR5 antagonist that blocks the viral entry of CCR5-tropic (R5) virions by binding to and inducing a conformational change in the chemokine receptor. Clinical resistance to CCR5 antagonists occurs in two phases, competitive and noncompetitive stages. In this study, we analyzed two subjects, from a phase 2b VCV clinical trial, whose quasispecies contained R5 and dual-mixed virions at the earliest recorded time of virological failure (VF). Genotypic analysis of R5-tropic patient-derived envelope genes revealed significant changes in the V1/V2 coding domain and convergence toward a more homogenous sequence under VCV therapy. Additionally, a small population of baseline clones sharing similar V1/V2 and V3 domains with the predominant VF isolate was observed. These clones were denoted preresistant based on their genotype. Preresistant clones and chimeric clones containing V1/V2 regions isolated during VF displayed high 50% inhibitory concentration (IC50) values relative to those at baseline, consistent with early competitive resistance. Genotypic analysis of the dual-tropic clones also showed significant changes in the V1/V2 region, different from the resistant R5-tropic viruses. Our findings suggest that the V1/V2 domain plays a key role in the initial step of development of drug resistance.IMPORTANCE It is believed that each CCR5 antagonist-resistant isolate will develop its own unique set of mutations, making it difficult to identify a signature mutation that can effectively predict CCR5 antagonist resistance. This may explain why we do not observe shared mutations among clinical studies. The present study examined the earliest events in the development of drug resistance with viral quasispecies that continued the use of CCR5 for entry. Genotypic and phenotypic assays demonstrated a distinct role of the variable domain V1/V2 in competitive resistance to CCR5 antagonist therapy. Thus, future studies analyzing the development of clinical resistance should focus on the relationship between the V1/V2 and V3 domains.

Effects of sequence changes in the HIV-1 gp41 fusion peptide on CCR5 inhibitor resistance

A rare pathway of HIV-1 resistance to small molecule CCR5 inhibitors such as Vicriviroc (VCV) involves changes solely in the gp41 fusion peptide (FP). Here, we show that the G516V change is critical to VCV resistance in PBMC and TZM-bl cells, although it must be accompanied by either M518V or F519I to have a substantial impact. Modeling VCV inhibition data from the two cell types indicated that G516V allows both double mutants to use VCV-CCR5 complexes for entry. The model further identified F519I as an independent determinant of preference for the unoccupied, high-VCV affinity form of CCR5. From inhibitor-free reversion cultures, we also identified a substitution in the inner domain of gp120, T244A, which appears to counter the resistance phenotype created by the FP substitutions. Examining the interplay of these changes will enhance our understanding of Env complex interactions that influence both HIV-1 entry and resistance to CCR5 inhibitors.

Vicriviroc Resistance Decay and Relative Replicative Fitness in HIV-1 Clinical Isolates under Sequential Drug Selection Pressures

We previously described an HIV-1-infected individual who developed resistance to vicriviroc (VCV), an investigational CCR5 antagonist, during 28 weeks of therapy (Tsibris AM et al., J. Virol. 82:8210-8214, 2008). To investigate the decay of VCV resistance mutations, a standard clonal analysis of full-length env (gp160) was performed on plasma HIV-1 samples obtained at week 28 (the time of VCV discontinuation) and at three subsequent time points (weeks 30, 42, and 48). During 132 days, VCV-resistant HIV-1 was replaced by VCV-sensitive viruses whose V3 loop sequences differed from the dominant pretreatment forms. A deep-sequencing analysis showed that the week 48 VCV-sensitive V3 loop form emerged from a preexisting viral variant. Enfuvirtide was added to the antiretroviral regimen at week 30; by week 48, enfuvirtide treatment selected for either the G36D or N43D HR-1 mutation. Growth competition experiments demonstrated that viruses incorporating the dominant week 28 VCV-resistant env were less fit than week 0 viruses in the absence of VCV but more fit than week 48 viruses. This week 48 fitness deficit persisted when G36D was corrected by either site-directed mutagenesis or week 48 gp41 domain swapping. The correction of N43D, in contrast, restored fitness relative to that of week 28, but not week 0, viruses. Virus entry kinetics correlated with observed fitness differences; the slower entry of enfuvirtide-resistant viruses corrected to wild-type rates in the presence of enfuvirtide. These findings suggest that while VCV and enfuvirtide select for resistance mutations in only one env subunit, gp120 and gp41 coevolve to maximize viral fitness under sequential drug selection pressures.

Differential Use of CCR5 by HIV-1 Clinical Isolates Resistant to Small-Molecule CCR5 Antagonists

How HIV-1 resistant to small-molecule CCR5 antagonists uses the coreceptor for entry has been studied in a limited number of isolates. We characterized dependence on the N terminus (NT) and the second extracellular loop (ECL2) of CCR5 of three vicriviroc (VCV)-resistant clinical isolates broadly cross-resistant to other CCR5 antagonists. Pseudoviruses were constructed to assess CCR5 use by VCV-sensitive and -resistant envelopes of subtype B and C viruses. We determined the extent of entry inhibition by monoclonal antibodies (MAbs) directed against the NT and ECL2 in the presence and absence of VCV and the capacity of these pseudoviruses to use CCR5 mutants that contained scanning alanine substitutions in the CCR5 NT and ECL2 domains. Sensitive and resistant viruses were completely and competitively inhibited by the ECL2-specific MAb 2D7, whereas the NT-specific MAb CTC5 led to partial noncompetitive inhibition. VCV-resistant clones showed greater sensitivity to 2D7 than VCV-sensitive clones, but in the presence of saturating VCV concentrations, the 2D7 susceptibilities of two VCV-resistant viruses were similar to that of VCV-sensitive virus. The entry of VCV-sensitive and -resistant isolates was impaired to differing degrees by alanine mutations in CCR5; substitutions in NT had the greatest effect on viral entry. HIV-1 clinical isolates broadly resistant to CCR5 antagonists demonstrated significant heterogeneity in their use of CCR5. This heterogeneity makes it difficult to draw general conclusions about the relationship between patterns of CCR5 antagonist resistance and the use of specific CCR5 domains for entry.

HIV-1 Clinical Isolates Resistant to CCR5 Antagonists Exhibit Delayed Entry Kinetics That Are Corrected in the Presence of Drug

HIV CCR5 antagonists select for env gene mutations that enable virus entry via drug-bound coreceptor. To investigate the mechanisms responsible for viral adaptation to drug-bound coreceptor-mediated entry, we studied viral isolates from three participants who developed CCR5 antagonist resistance during treatment with vicriviroc (VCV), an investigational small-molecule CCR5 antagonist. VCV-sensitive and -resistant viruses were isolated from one HIV subtype C- and two subtype B-infected participants; VCV-resistant isolates had mutations in the V3 loop of gp120 and were cross-resistant to TAK-779, an investigational antagonist, and maraviroc (MVC). All three resistant isolates contained a 306P mutation but had variable mutations elsewhere in the V3 stem. We used a virus-cell β-lactamase (BlaM) fusion assay to determine the entry kinetics of recombinant viruses that incorporated full-length VCV-sensitive and -resistant envelopes. VCV-resistant isolates exhibited delayed entry rates in the absence of drug, relative to pretherapy VCV-sensitive isolates. The addition of drug corrected these delays. These findings were generalizable across target cell types with a range of CD4 and CCR5 surface densities and were observed when either population-derived or clonal envelopes were used to construct recombinant viruses. V3 loop mutations alone were sufficient to restore virus entry in the presence of drug, and the accumulation of V3 mutations during VCV therapy led to progressively higher rates of viral entry. We propose that the restoration of pre-CCR5 antagonist therapy HIV entry kinetics drives the selection of V3 loop mutations and may represent a common mechanism that underlies the emergence of CCR5 antagonist resistance.

128 Suppression of CCR5 Density as a Novel Way to Enhance the Anti-HIV Activity of Fusion Inhibitors and CCR5 Antagonists

The CCR5 chemokine receptor plays a crucial role in HIV-1 infection. We have established methods to quantify CCR5 density and to evaluate its impact on virus infectivity in spreading/single cycle infection and direct virus-cell fusion assays. In our preliminary studies, we use a stochiometry model (Kuhmann et al, 2006) to calculate a number of CCR5 molecules needed for HIV-1 pseudovirus infection. In addition, using low doses of the drug Rapamycin, a CCR5 suppressor, we demonstrate decreased R5 HIV-1 infectivity and enhanced potency of entry inhibitors. Our data show that Rapamycin reduction of CCR5 density restores sensitivity of drug-resistant R5 HIV-1 to fusion inhibitor Enfuvirtide (T-20) and to all of three new generation of CCR5 antagonists: Vicriviroc, Maraviroc and Aplaviroc. We found that Rapamycin induced reduction of CCR5 density in lymphocytes increased sensitivity to Vicriviroc (VCV) in VCV-resistant strains, inhibiting production by ∼ 90%. Novel Beta-lactamase (BlaM) entry assay revealed the differences in the activity between VCV sensitive and VCV resistant virus. In the case of VCV sensitive virus, we observed complete inhibition in cell lines with high and physiological CCR5 levels. In the case of the resistant virus, there is no inhibition in higher CCR5 expressive cells, but in cells with physiological expression of CCR5, we do observe susceptibility to VCV resistant virus. As an alternative anti-HIV therapy approach, we successfully employ the CCR5 antibodies in order to determine the affinity of resistant virus Envelope in cases when it is free versus occupied CCR5 site by the CCR5 antagonist, Maraviroc. Identifying the most effective drug combinations of Rapamycin or CCR5 antibodies with the CCR5 antagonists (with the lowest possible side effects) has relevant clinical implications in anti-viral therapy.

Mapping and Characterization of Vicriviroc Resistance Mutations From HIV-1 Isolated from Treatment-Experienced Subjects Enrolled in a Phase II Study (VICTOR-E1)

In the phase 2 VICTOR-E1 study, treatment-experienced subjects receiving 20 mg or 30 mg of the CCR5 antagonist vicriviroc (VCV), with a boosted protease containing optimized background regimen, experienced significantly greater reductions in HIV-1 viral load compared with control subjects. Among the 79 VCV-treated subjects, 15 experienced virologic failure, and of these 5 had VCV-resistant virus. This study investigated the molecular basis for the changes in susceptibility to VCV in these subjects. Sequence analysis and phenotypic susceptibility testing was performed on envelope clones from VCV-resistant virus. For select clones, an exchange of mutations in the V3 loop was performed between phenotypically resistant clones and the corresponding susceptible clones. Phenotypic resistance was manifest by reductions in the maximum percent inhibition. Clonal analysis of envelopes from the 5 subjects identified multiple amino acid changes in gp160 that were exclusive to the resistant clones, however, none of the changes were conserved between subjects. Introduction of V3 loop substitutions from the resistant clones into the matched susceptible clones was not sufficient to reproduce the resistant phenotype. Likewise, changing the substitutions in the V3 loops from resistant clones to match susceptible clones only restored susceptibility in 1 clone. There were no clearly conserved patterns of mutations in gp160 associated with phenotypic resistance to VCV and mutations both within and outside of the V3 loop contributed to the resistance phenotype. These data suggest that genotypic tests for VCV susceptibility may require larger training sets and additional information beyond V3 sequences.

Resistance of a human immunodeficiency virus type 1 isolate to a small molecule CCR5 inhibitor can involve sequence changes in both gp120 and gp41

Here, we describe the genetic pathways taken by a human immunodeficiency virus type 1 (HIV-1) isolate, D101.12, to become resistant to the small molecule CCR5 inhibitor, vicriviroc (VCV), in vitro. Resistant D101.12 variants contained at least one substitution in the gp120 V3 region (H308P), plus one of two patterns of gp41 sequence changes involving the fusion peptide (FP) and a downstream residue: G514V+V535M or M518V+F519L+V535M. Studies of Env-chimeric and point-substituted viruses in peripheral blood mononuclear cells (PBMC) and TZM-bl cells showed that resistance can arise from the cooperative action of gp120 and gp41 changes, while retaining CCR5 usage. Modeling the VCV inhibition data from the two cell types suggests that D101.12 discriminates between high- and low-VCV affinity forms of CCR5 less than D1/85.16, a resistant virus with three FP substitutions.

Evolution of CCR5 Antagonist Resistance in an HIV-1 Subtype C Clinical Isolate

We previously reported vicriviroc (VCV) resistance in an HIV-infected subject and used deep sequencing and clonal analyses to track the evolution of V3 sequence forms over 28 weeks of therapy. Here, we test the contribution of gp120 mutations to CCR5 antagonist resistance and investigate why certain minority V3 variants emerged as the dominant species under drug pressure. Nineteen site-directed HIV-1 mutants were generated that contained gp120 VCV resistance mutations. Viral sensitivities to VCV, maraviroc, TAK-779, and HGS004 were determined. Three patterns of susceptibilities were observed as follows: sigmoid inhibition curves with 50% inhibitory concentration similar to pretreatment virus [07J-week 0 (W0)], single mutants with decreased 50% inhibitory concentrations compared with 07J-W0, and mutants that contained ≥5 of 7 VCV resistance mutations with flattened inhibition curves and decreased or negative percent maximal inhibition. Substitutions such as S306P, which sensitized virus to CCR5 antagonists when present as single mutations, were not detected in the baseline virus population but were necessary for maximal resistance when incorporated into V3 backbones that included preexisting VCV resistance mutations. CCR5 antagonist resistance was reproduced only when a majority of V3 mutations were present. Minority V3 loop variants may serve as a scaffold upon which additional mutations lead to complete VCV resistance.

Clinical resistance to vicriviroc through adaptive V3 loop mutations in HIV-1 subtype D gp120 that alter interactions with the N-terminus and ECL2 of CCR5

The HIV-1 CCR5 co-receptor is a member of the chemokine receptor family of G-protein coupled receptors; for which a number of small molecule antagonists, such as vicriviroc (VCV), have been developed to inhibit HIV-1 R5-tropic replication. In this study, we analyzed an HIV-1 subtype D envelope gene from a clinical trial subject who developed complete resistance to VCV. The HIV-1 resistant envelope has six predominant amino acid changes in the V3 loop, together with one change in the C4 domain of gp120, which are fully responsible for the resistance phenotype. V3 loop mutations Q315E and R321G are essential for resistance to VCV, whereas E328K and G429R in C4 contribute significantly to the infectivity of the resistant variant. Collectively, these amino acid changes influenced the interaction of gp120 with both the N-terminus and ECL2 region of CCR5.

Vicriviroc and Peripheral Neuropathy: Results from AIDS Clinical Trials Group 5211

Purpose: To evaluate the effect of vicriviroc (VCV) on peripheral neuropathy (PN), the most prevalent neurological complication of HIV infection in HIV-1–infected treatment- experienced population.Method: A5211 is a randomized placebo- controlled trial evaluating VCV in treatment-experienced HIV participants failing current therapy. Participants were randomized to VCV (5, 10, or 15 mg) or placebo with optimized ritonavir-containing antiretroviral therapy and followed for 48 weeks. PN was defined as having at least mild loss of vibration bilaterally or ankle reflexes absent or hypoactive bilaterally. We estimated the association between VCV (pooled doses) with PN using a logistic generalized estimating equation. Additional outcomes included symptomatic neuropathy (SPN), painful neuropathy (PPN), and neuropathic signs and symptoms.Results: 118 participants (92% male, 65% white, median age of 46 years, median baseline CD4 139, median HIV-1 RNA 4.58 log) were randomized (90 on VCV and 28 on placebo). VCV therapy did not result in a statistically significant difference relative to placebo in PN (OR = 1.52;P = .39; 95% CI 0.59, 3.90) after controlling for baseline PN status and baseline neurotoxic nucleoside reverse transcriptase inhibitor(s) use.Conclusion: Treatment with VCV over 48 weeks failed to result in statistically significant effect on PN in treatment-experienced participants with HIV infection relative to placebo, however potentially important effects cannot be ruled out.

Reduction of CCR5 with low-dose rapamycin enhances the antiviral activity of vicriviroc against both sensitive and drug-resistant HIV-1

Vicriviroc (VCV) is a chemokine (C-C motif) receptor 5 (CCR5) antagonist with potent anti-HIV activity that currently is being evaluated in phase III clinical trials. In the present study, donor CCR5 density (CCR5 receptors/CD4 lymphocytes) inversely correlated with VCV antiviral activity (Spearman's correlation test; r = 0.746, P = 0.0034). Low doses of the transplant drug rapamycin (RAPA) reduced CCR5 density and enhanced VCV antiviral activity. In drug interaction studies, the RAPA/VCV combination had considerable antiviral synergy (combination indexes of 0.1-0.04) in both multicycle and single-cycle infection of lymphocytes. The synergy between RAPA and VCV translated into dose reduction indexes of 8- to 41-fold reductions for RAPA and 19- to 658-fold reductions for VCV. RAPA enhanced VCV antiviral activity against both B and non-B clade isolates, potently suppressing clade G viruses with reported reduced sensitivities to VCV and to the licensed CCR5 antagonist maraviroc. Importantly, RAPA reduction of CCR5 density in lymphocytes sensitized VCV-resistant strains to VCV, inhibiting virus production by approximately 90%. We further demonstrated the role of CCR5 density on VCV activity against resistant virus in donor lymphocytes and in cell lines expressing varying CCR5 densities. Together, these results suggest that low doses of RAPA may increase the durability of VCV-containing regimens in patients by enhancing VCV viral suppression, by allowing the use of lower doses of VCV with reduced potential for toxicity, and by controlling emerging VCV-resistant variants.

Phase II Study of Vicriviroc versus Efavirenz (both with Zidovudine/Lamivudine) in Treatment‐Naive Subjects with HIV‐1 Infection

Vicriviroc (VCV) is a CCR5 antagonist with nanomolar activity against human immunodeficiency virus (HIV) replication in vitro and in vivo. We report the results of a phase II dose-finding study of VCV plus dual nucleoside reverse-transcriptase inhibitors (NRTIs) in the treatment-naive HIV-1-infected subjects. This study was a randomized, double-blind, placebo-controlled trial that began with a 14-day comparison of 3 dosages of VCV with placebo in treatment-naive subjects infected with CCR5-using HIV-1. After 14 days of monotherapy, lamivudine/zidovudine was added to the VCV arms; subjects receiving placebo were treated with efavirenz and lamivudine/zidovudine; the planned treatment duration was 48 weeks. Ninety-two subjects enrolled. After 14 days of once-daily monotherapy, the mean viral loads decreased from baseline values by 0.07 log(10) copies/mL in the placebo arm, 0.93 log(10) copies/mL in the VCV 25 mg arm, 1.18 log(10) copies/mL in the VCV 50 mg arm, and 1.34 log(10) copies/mL in the VCV 75 mg arm (P < .001 for each VCV arm vs. the placebo arm). The combination-therapy portion of the study was stopped because of increased rates of virologic failure in the VCV 25 mg/day arm (relative hazard [RH], 21.6; 95% confidence interval [CI], 2.8-168.9) and the VCV 50 mg/day arm (RH, 11.7; 95% CI, 1.5-92.9), compared with that in the control arm. VCV administered with dual NRTIs in treatment-naive subjects with HIV-1 infection had increased rates of virologic failure, compared with efavirenz plus dual NRTIs. No treatment-limiting toxicity was observed. Study of higher doses of VCV as part of combination therapy is warranted.

In Vivo Emergence of Vicriviroc Resistance in a Human Immunodeficiency Virus Type 1 Subtype C-Infected Subject

Little is known about the in vivo development of resistance to human immunodeficiency virus type 1 (HIV-1) CCR5 antagonists. We studied 29 subjects with virologic failure from a phase IIb study of the CCR5 antagonist vicriviroc (VCV) and identified one individual with HIV-1 subtype C who developed VCV resistance. Studies with chimeric envelopes demonstrated that changes within the V3 loop were sufficient to confer VCV resistance. Resistant virus showed VCV-enhanced replication, cross-resistance to another CCR5 antagonist, TAK779, and increased sensitivity to aminooxypentane-RANTES and the CCR5 monoclonal antibody HGS004. Pretreatment V3 loop sequences reemerged following VCV discontinuation, implying that VCV resistance has associated fitness costs.

Vicriviroc in Treatment-Naive Patients

Vicriviroc is an investigational CCR5 inhibitor that seemed promising in industry-funded, phase I clinical trials involving treatment-naive HIV-infected patients. However, recent results from a phase II trial of this drug (also industry funded) are less encouraging. A total of 92 treatment-naive patients with CCR5-tropic virus only were randomized to receive double-blinded, once-daily vicriviroc (25 mg, 50 mg, or 75 mg) or placebo for 14 days, followed by combination therapy with either vicriviroc or open-label efavirenz, each with AZT/3TC, for …

Response Normalized Liquid Chromatography Nanospray Ionization Mass Spectrometry

The widely different LC-MS response observed for many structurally different compounds limits the use of LC-MS in full scan detection mode for quantitative determination of drugs and metabolites without using reference standard. The recently introduced nanospray ionization (NSI) technique shows comparable MS response for some compounds under non-LC-MS conditions. However, in the presence of numerous endogenous compounds commonly associated with biological samples such as urine, plasma, and bile, LC-MS is required to separate, detect, identify, and measure individual analytes. An LC-NSI-MS system was devised and the MS response obtained in this system for a variety of pharmaceutical drugs and their metabolites. The set-up involves two high-performance liquid chromatography (HPLC) systems, a chip-based NSI source and a quadrupole-time-of-flight (Q-TOF) mass spectrometer. Herein this is referred to as the response normalized-liquid chromatography NSI-MS (RNLC-NSI-MS) system. One HPLC unit performs the analytical separation, while the other unit adds solvent post-column with an exact reverse of the mobile phase composition such that the final composition entering the NSI source is isocratic throughout the entire HPLC run. The data obtained from four different structural classes of compounds [vicriviroc (VCV), desloratadine (DL), tolbutamide, and cocaine] and their metabolites indicate that by maintaining the solvent composition unchanged across the HPLC run, the influence of the solvent environment on the ionization efficiency is minimized. In comparison to responses obtained from radiochromatograms, responses from conventional LC-ESI-MS overestimated the VCV and DL responses, respectively, by 6- and 20-fold. Although VCV and DL responses obtained using LC-NSI-MS are within 2- to 6-fold from the respective radiochromatographic responses, the response normalization modification results in nearly uniform LC-NSI-MS response for all compounds evaluated.

Vicriviroc in Treatment-Experienced Patients: 24-Week Results

Vicriviroc is one of two CCR5 inhibitors being developed to treat HIV infection. In this phase II study (ACTG 5211), investigators tested the safety

Malignancies reported with vicriviroc

Malignancies reported with vicriviroc

Malignancies reported with vicriviroc

Malignancies reported with vicriviroc