Maraviroc for previously treated patients with R5 HIV-1 infection

BACKGROUND CC chemokine receptor 5 antagonists are a new class of antiretroviral agents. METHODS We conducted two double-blind, placebo-controlled, phase 3 studies--Maraviroc versus Optimized Therapy in Viremic Antiretroviral Treatment-Experienced Patients (MOTIVATE) 1 and MOTIVATE 2--with patients who had R5 human immunodeficiency virus type 1 (HIV-1) only. They had been treated with or had resistance to three antiretroviral-drug classes and had HIV-1 RNA levels of more than 5000 copies per milliliter. The patients were randomly assigned to one of three antiretroviral regimens consisting of maraviroc once daily, maraviroc twice daily, or placebo, each of which included optimized background therapy (OBT) based on treatment history and drug-resistance testing. Safety and efficacy were assessed after 48 weeks. RESULTS A total of 1049 patients received the randomly assigned study drug; the mean baseline HIV-1 RNA level was 72,400 copies per milliliter, and the median CD4 cell count was 169 per cubic millimeter. At 48 weeks, in both studies, the mean change in HIV-1 RNA from baseline was greater with maraviroc than with placebo: -1.66 and -1.82 log(10) copies per milliliter with the once-daily and twice-daily regimens, respectively, versus -0.80 with placebo in MOTIVATE 1, and -1.72 and -1.87 log(10) copies per milliliter, respectively, versus -0.76 with placebo in MOTIVATE 2. More patients receiving maraviroc once or twice daily had HIV-1 RNA levels of less than 50 copies per milliliter (42% and 47%, respectively, vs. 16% in the placebo group in MOTIVATE 1; 45% in both maraviroc groups vs. 18% in MOTIVATE 2; P<0.001 for both comparisons in each study). The change from baseline in CD4 counts was also greater with maraviroc once or twice daily than with placebo (increases of 113 and 122 per cubic millimeter, respectively, vs. 54 in MOTIVATE 1; increases of 122 and 128 per cubic millimeter, respectively, vs. 69 in MOTIVATE 2; P<0.001 for both comparisons in each study). Frequencies of adverse events were similar among the groups. CONCLUSIONS Maraviroc, as compared with placebo, resulted in significantly greater suppression of HIV-1 and greater increases in CD4 cell counts at 48 weeks in previously treated patients with R5 HIV-1 who were receiving OBT. (ClinicalTrials.gov numbers, NCT00098306 and NCT00098722.

Prevalence of HIV-positive test by ethnicity and region.

<p>Prevalence of HIV-positive test by ethnicity and region.</p

Patient characteristics by European region.

1<p>North:Denmark, Sweden, Netherlands, UK, West Central: Austria, Belgium, Germany, East Central: Belarus, Bosnia, Croatia, Poland, Ukraine and South: Italy and Spain.</p>2<p>IQR:Interquartile range.</p>3<p>STI: Sexually transmitted infection, LYM: Malignant lymphoma, CAN: Cervical or anal cancer/dysplasia, HZV: Herpes zoster, HEP: Hepatitis B or C, MON: Ongoing mononucleosis-like illness, CYT: Unexplained leukocytopenia/thrombocytopenia lasting >4 weeks, SEB: Seborrheic dermatitis/exanthema (SEB).</p><p>Missing data: 10 (0.3%) gender, 120 (3.3%) ethnicity, 63 (1.8%) age, 120 (3.3%) sexual orientation, 291 (8.1%) previous HIV test.</p

Prevalence of HIV by indicator condition.

*<p>Unpublished prevalence data from participating study sites.</p>**<p>includes MSM, IDU prevalence.</p>***<p>UNAIDS adults aged 15–49 country HIV prevalence rate <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052845#pone.0052845-UNAIDS2" target="_blank">[31]</a>.</p

Compounds releasing active P-TEFb and PKC agonists act synergistically to increase HIV-1 production.

<p>J-Lat 9.2 (panels <b>A</b> and <b>C</b>) and U1 (panels <b>B</b> and <b>D</b>) cell lines were mock-treated or treated with two doses of JQ1, I-BET, I-BET151, HMBA alone or in combination with three doses of either bryostatin-1 or prostratin as indicated. At 24 hours post-treatment, viral production was estimated by measuring CA-p24 antigen concentration in culture supernatants. The mock-treated value was arbitrarily set at a value of 1. Means and standard errors of the means from duplicate samples are indicated. One representative experiment from three is represented. For each combinatory treatment, the fold-synergy was calculated by dividing the effect observed after co-treatments by the sum of the effects obtained after the individual treatments.</p

PKC agonist+BETi/HMBA combined treatments increase HIV-1 expression in a higher proportion of cells than the drug alone and synergistically enhance viral transcription.

<p>The J-Lat 9.2 cells (panel <b>A</b>) or CHME-5/HIV microglial cells (panel <b>B</b>) harbor latent HIV-1 provirus containing <i>gfp</i> gene. The cells were mock-treated, treated with JQ1 (0.5μM), I-BET (0.5μM), I-BET151 (0.5μM), HMBA (5mM), bryostatin-1 (10nM) and prostratin (2.5μM) alone or in combination as indicated. At 24 hours post-treatment, cells were analyzed by flow cytometry to quantify the proportion of cells expressing GFP. Means and standard errors of the means from duplicate samples are indicated. One representative experiment from three is represented. For each combinatory treatment, the fold-synergy was calculated by dividing the effect observed after co-treatments by the sum of the effects obtained from individual treatments. Panels <b>C</b>-<b>F.</b> Measurement of initiated and elongated HIV-1 transcripts following drug treatment. Total RNA was extracted from J-Lat 9.2 (panels <b>C</b> and <b>D</b>), U1 (panel <b>E</b>), CHME-5/HIV (panel <b>F</b>) cells which were mock-treated or treated with BETi, HMBA, bryostatin-1 and prostratin for 24 hours at concentrations described in Fig 2A and 2B. Initiated (primers TAR) or elongated (primers <i>tat</i> or <i>env</i>) transcripts were quantified by quantitative real-time RT-PCR. Values were normalized using β-actin gene primers and were presented as fold inductions relative to the values measured in mock-treated cells, which were arbitrarily set at a value of 1. Means and standard errors of the means from duplicate samples are indicated. One representative experiment from two is represented. For each combinatory treatment, the fold-synergy was calculated by dividing the effect observed after co-treatments by the sum of the effects after the individual treatments.</p

PKC agonists and compounds releasing active P-TEFb induce HIV-1 recovery in CD8⁺-depleted PBMCs from cART-treated HIV⁺ aviremic patients.

<p><i>Ex vivo</i> cultures of CD8⁺-depleted PBMCs purified from blood of 24 patients were mock-treated or treated with anti-CD3+anti-CD28 antibodies, prostratin (0.5μM), bryostatin-1 (5nM), JQ1 (0.25μM), I-BET (0.25μM), I-BET151 (0.25μM) or HMBA (5mM) alone or in combination as indicated. Six days post-treatment, concentrations of genomic viral RNA in culture supernatants were determined and the values were expressed as HIV-1 RNA copies/ml. Total HIV-1 DNA was expressed as HIV-1 DNA copies/10⁶ CD8⁺-depleted PBMCs. Values representing higher viral production after the combined treatment than after the single drug treatments are shown in grey. Values representing reactivation of the virus observed exclusively after combined treatments are shown in black. ‘I’ indicates below the 150 HIV-1 RNA copies/ml limit of detection. ‘//’ indicates not tested conditions.</p

Expression of cell surface activation markers following PKC agonists and JQ1 treatments.

<p>Blood from 4 uninfected donors was split and one half was used to purify resting CD4⁺ T cells (panels <b>A</b>-<b>E</b>) and the other half was used to purify CD8⁺-depleted PBMCs (panels <b>F</b>-<b>I</b>). Cell cultures were mock-treated, treated with anti-CD3+anti-CD28 antibodies (C+), JQ1 [0.25μM], bryostatin-1 [5nM], prostratin [0.5μM] or ing-B [10nM] alone or in combination for 6 days. Cells were incubated with anti-CD38 (panels <b>A</b> and <b>F</b>), anti-CD69 (panels <b>B</b> and <b>G</b>), anti-HLA-DR (panels C and H), anti-CD25 (panels <b>D</b> and <b>I</b>) or anti-CD4 (panel <b>E</b>) antibodies prior to flow cytometry analysis. The results are presented as percentage of marker expression in the population of CD4⁺ cells (panels <b>A-D</b> and panels <b>F-I</b>) and as median fluorescence intensity (MFI) of CD4⁺ cells (panel <b>E</b>). Dashed line indicates the percentage of expression obtained in mock-treated cells. The means are represented. Statistical comparisons are indicated. Statistically relevant and not statistically relevant comparisons are indicated by asterisk and “ns”, respectively.</p

PKC agonists synergize with BETi in releasing active P-TEFb.

<p>Jurkat cells were mock-treated, treated with JQ1 (0.25μM), bryostatin-1 (5nM) alone or in combination for 1 hour (Panel <b>A</b>) or 24 hours (Panel <b>B</b>). Nuclear extracts were prepared from treated cells and subjected to immunoprecipitations (IP) with an anti-CDK9 antibody or the control IgG. The complexes were immunodetected for the presence of CycT1 and HEXIM-1 by Western blotting (right panels). Input controls for CDK9, CycT1 and HEXIM1 are presented (left panels). Levels of β-actin were measured to control protein loading. Panels <b>A</b> and <b>B</b>. Histograms represent quantification of band intensities normalized to CDK9 levels in the IP and then normalized to mock-treated condition. Panel <b>C.</b> HeLa cells expressing YC.P-TEFb and VN.CTD were left untreated or were treated as indicated for 1 hour. Venus-positive cells were detected by fluorescence microscopy (upper panels). Bright-field images were also taken (lower panels). Panel <b>D.</b> HeLa cells expressing YC.P-TEFb and VN.CTD were treated as outlined in C and Venus-positive cells were counted and averaged from three different areas. Error bars represent differences between counts of Venus-positive cells from the randomly chosen fields under the microscope. Panel <b>E.</b> Hela cells were transfected with the Hex1(-104)Luc reporter plasmid. At 24 hours post-transfection, cells were mock-treated or treated with the different compounds as indicated. Luciferase activities in cell extracts were measured 24 hours after drug treatments and reported as fold increases over the activity observed in mock-treated condition (transfection of the reporter plasmid without drug treatment) and arbitrarily set at a value of 1. An experiment performed in duplicate representative of two independent experiments is shown.</p

PKC agonists and compounds releasing active P-TEFb induce HIV-1 recovery in CD8⁺-depleted PBMCs and in resting CD4⁺ T cells from cART-treated HIV⁺ aviremic patients.

<p>Panel <b>A.</b><i>Ex vivo</i> cultures of CD8⁺-depleted PBMCs purified from blood of 24 patients were mock-treated, treated with anti-CD3+anti-CD28 antibodies (C+), prostratin (0.5μM), bryostatin-1 (5nM), JQ1 (0.25μM), I-BET (0.25μM), I-BET151 (0.25μM) or HMBA (5mM) alone or in combination as indicated. Six days post-treatment, concentrations of genomic viral RNA were measured in culture supernatants. Statistical comparisons to positive control are indicated if p<0.05 (superiority to positive control). Panel <b>B.</b><i>Ex vivo</i> cultures of resting CD4⁺ T cells purified from blood of 15 patients were mock-treated, treated with anti-CD3+anti-CD28 antibodies (C+), prostratin (0.5μM), bryostatin-1 (5nM), ing-B (10nM), JQ1 (0.25μM), alone or in combination as indicated. Six days post-treatment, concentrations of genomic viral RNA in culture supernatants were determined. Statistical comparisons to positive control are indicated if p>0.05 (non-inferiority). Panels <b>A</b> and <b>B</b>. Dashed line indicates the 150 HIV-1 RNA copies/ml limit of detection. Panel <b>C</b>. <i>Ex vivo</i> cultures of CD8⁺-depleted PBMCs purified from blood of 7 HIV⁺ patients were mock-treated, treated with anti-CD3+anti-CD28 antibodies (C+), bryostatin-1 (5nM), JQ1 (0.25μM) or ing-B (10nM) alone or in combination in the presence of cART. One day post-treatment, concentrations of genomic viral RNA in culture supernatants were determined. Statistical comparisons to positive control are indicated if p<0.05 (superiority to positive control). Panel <b>D.</b><i>Ex vivo</i> cultures of CD8⁺-depleted PBMCs purified from blood of 11 HIV⁺ patients were mock-treated, treated with anti-CD3+anti-CD28 antibodies (C+), bryostatin-1 (5nM), JQ1 (0.25μM) or ing-B (10nM) alone or in combination in the presence of cART. Three days post-treatment, concentrations of genomic viral RNA in culture supernatants were determined. Statistical comparisons to positive control are indicated if p>0.05 (non-inferiority to positive control). Panel <b>E.</b><i>Ex vivo</i> cultures of CD8⁺-depleted PBMCs purified from blood of 11 HIV⁺ patients were mock-treated, treated with anti-CD3+anti-CD28 antibodies (C+), bryostatin-1 (5nM), ing-B (10nM) or JQ1 (0.25μM) alone or in combination in the presence of cART. Six days post-treatment, concentrations of genomic viral RNA in culture supernatants were determined. Statistical comparisons to positive control are not indicated because p<0.05 for all the conditions (superiority of the positive control). Panels <b>C-E.</b> Dashed line indicates the 15 HIV-1 RNA copies/ml limit of detection. Panels <b>A-E.</b> Each symbol represents one cART-treated HIV⁺ patient. The means are represented.</p