ABSTRACT
BACKGROUND: Patients with chronic hepatitis C virus (HCV) infection who have not had a response to therapy with peginterferon and ribavirin may benefit from the addition of multiple direct-acting antiviral agents to their treatment regimen. METHODS: This open-label, phase 2a study included an exploratory cohort of 21 patients with chronic HCV genotype 1 infection who had not had a response to previous therapy (i.e., had not had ≥2 log(10) decline in HCV RNA after ≥12 weeks of treatment with peginterferon and ribavirin). We randomly assigned patients to receive the NS5A replication complex inhibitor daclatasvir (60 mg once daily) and the NS3 protease inhibitor asunaprevir (600 mg twice daily) alone (group A, 11 patients) or in combination with peginterferon alfa-2a and ribavirin (group B, 10 patients) for 24 weeks. The primary end point was the percentage of patients with a sustained virologic response 12 weeks after the end of the treatment period. RESULTS: A total of 4 patients in group A (36%; 2 of 9 with HCV genotype 1a and 2 of 2 with genotype 1b) had a sustained virologic response at 12 weeks after treatment and also at 24 weeks after treatment.. Six patients (all with HCV genotype 1a) had viral breakthrough while receiving therapy, and resistance mutations to both antiviral agents were found in all cases; 1 patient had a viral response at the end of treatment but had a relapse after the treatment period. All 10 patients in group B had a sustained virologic response at 12 weeks after treatment, and 9 had a sustained virologic response at 24 weeks after treatment. Diarrhea was the most common adverse event in both groups. Six patients had transient elevations of alanine aminotransferase levels to more than 3 times the upper limit of the normal range. CONCLUSIONS: This preliminary study involving patients with HCV genotype 1 infection who had not had a response to prior therapy showed that a sustained virologic response can be achieved with two direct-acting antiviral agents only. In addition, a high rate of sustained virologic response was achieved when the two direct-acting antiviral agents were combined with peginterferon alfa-2a and ribavirin. (Funded by Bristol-Myers Squibb; ClinicalTrials.gov number, NCT01012895.).
Subject(s)
Antiviral Agents/therapeutic use , Hepacivirus/genetics , Hepatitis C, Chronic/drug therapy , Imidazoles/therapeutic use , Adult , Antiviral Agents/adverse effects , Carbamates , Drug Resistance, Viral , Drug Therapy, Combination , Female , Genotype , Humans , Imidazoles/adverse effects , Interferon-alpha/adverse effects , Interferon-alpha/therapeutic use , Male , Middle Aged , Polyethylene Glycols/adverse effects , Polyethylene Glycols/therapeutic use , Pyrrolidines , RNA, Viral/blood , Recombinant Proteins/adverse effects , Recombinant Proteins/therapeutic use , Recurrence , Ribavirin/adverse effects , Ribavirin/therapeutic use , Valine/analogs & derivativesSubject(s)
Monitoring, Ambulatory/methods , Polysomnography/methods , Sleep Apnea Syndromes/diagnosis , Equipment Design , Heart Rate/physiology , Humans , Medical Laboratory Science/instrumentation , Microcomputers , Monitoring, Ambulatory/instrumentation , Oximetry/instrumentation , Oxygen/blood , Plethysmography/instrumentation , Polysomnography/instrumentation , Pulse , Respiration , Sleep Apnea Syndromes/classification , Sleep, REM/physiology , Vascular Resistance/physiologyABSTRACT
Two major interferon (IFN)-mediated antiviral defense enzymes are double-stranded (ds)RNA-dependent 2',5'-oligoadenylate (2-5A) synthetase (2-5OAS) and p68 kinase (PKR). When activated by dsRNA, 2-5OAS synthesizes 2-5A, which binds to and activates RNase L. Activated RNase L hydrolyzes single-stranded viral RNA, thereby inhibiting viral protein synthesis. HIV-1 inhibits the IFN-mediated intracellular antiviral pathways. We have reported the synthesis and characterization of a nuclease-resistant 2-5A agonist (2-5A(N6B)) that overcomes the HIV-1 induced blockades by restoring the 2-5OAS/RNase L antiviral pathway (Homan JW, et al., J Acquir Immune Defic Syndr 2002;30:9-20). The objective of this study was to test the effect of 2-5A(N6B) on chronically infected CD4(+) T lymphocytes and CD14(+) monocytes derived from HIV-1-seropositive individuals. Wild-type HIV-1 replication was effectively inhibited by 2-5A(N6B) in CD4(+) T lymphocytes and CD14(+) monocytes purified from HIV-1 seropositive individuals (n = 18) compared to untreated cells. We also assessed the cytotoxicity of 2-5A(N6B) and report that 2-5A(N6B) exerts its anti-HIV-1 activity with no evidence of cytotoxicity (IC(90) > 100,000 nM). Furthermore, 2-5A(N6B) did not alter the cellular RNA profile, affect CCR5 or CXCR4 coreceptor expression, or activate caspase-dependent apoptosis. Evidence is also provided to show that 2-5A(N6B), and naturally occurring 2-5A(4), act as ligands to activate human Toll-like receptor 4. These results indicate that the 2-5A agonist 2-5A(N6B) has the potential to enhance host cell innate and acquired immune defense mechanisms against HIV-1 infection.
Subject(s)
Adenine Nucleotides/pharmacology , Anti-HIV Agents/pharmacology , CD4-Positive T-Lymphocytes/virology , HIV Infections/metabolism , HIV-1/drug effects , Lipopolysaccharide Receptors , Oligoribonucleotides/pharmacology , Adenine Nucleotides/agonists , Adenine Nucleotides/chemical synthesis , Adult , Aged , CD4 Lymphocyte Count , CD4-Positive T-Lymphocytes/drug effects , Cells, Cultured , Dose-Response Relationship, Drug , Female , Flow Cytometry , HIV Infections/drug therapy , HIV Seropositivity , HIV-1/physiology , Humans , Immunologic Factors/pharmacology , Male , Middle Aged , Monocytes/cytology , Monocytes/immunology , Oligoribonucleotides/agonists , Oligoribonucleotides/chemical synthesis , Reverse Transcriptase Polymerase Chain Reaction , Toll-Like Receptor 4/metabolism , Virus Replication/drug effectsABSTRACT
Adenosine, released by cells in an injurious or hypoxic environment, possesses potent anti-inflammatory effects by inhibiting the production of proinflammatory cytokines and superoxide anions (O2-). We hypothesized that adenosine compounds also induced heterologous desensitization of chemokine receptors, which played a critical role in leukocyte trafficking. Our studies using adenosine receptor subtype-specific agonists revealed that pretreatment with adenosine compounds suppressed RANTES-induced chemotaxis and Ca2+ flux through activation of A2a adenosine receptor. Adenosine compounds also desensitized IL-8- and MCP-1-induced chemotaxis, but not that induced by fMLP. Activation of protein kinase A (PKA), a component of the signaling pathway induced by the A2a receptor, was sufficient to desensitize RANTES-induced chemotaxis. Inhibition of PKA reversed the desensitization effects of adenosine compounds, suggesting that PKA was necessary for A2a receptor-mediated heterologous desensitization. In a mouse model, prior activation of A2a receptors blocked RANTES-induced recruitment of leukocytes in an air pouch. Moreover, the A2a receptor-induced cross-desensitization also reduced the susceptibility of monocytes to infection by an R5 strain of HIV-1. Our results suggest that activation of A2a adenosine receptors suppresses chemokine receptor function, and such receptor cross-talk was based on the simple mechanism of PKA-mediated heterologous desensitization, thus contributing to the antiinflammatory activity of adenosine.
Subject(s)
Adenosine A2 Receptor Agonists , Adenosine/pharmacology , Receptors, Chemokine/drug effects , Adenosine/analogs & derivatives , Animals , Chemokine CCL5/administration & dosage , Chemokine CCL5/antagonists & inhibitors , Chemotaxis/drug effects , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic AMP-Dependent Protein Kinases/physiology , Gene Expression Profiling , HIV Infections/metabolism , Humans , Leukocytes/drug effects , Leukocytes/metabolism , Mice , Mice, Inbred BALB C , Phenethylamines/pharmacology , Receptor, Adenosine A2A/genetics , Receptor, Adenosine A2A/metabolism , Receptors, Chemokine/metabolism , Reverse Transcriptase Polymerase Chain Reaction/methods , Sensitivity and Specificity , Structure-Activity RelationshipABSTRACT
Previous studies from this laboratory evaluated the role of p68 kinase (PKR) in the control of HIV-1 replication via retrovirus-mediated gene transfer. PKR was studied because it is a key component of the interferon (IFN)-associated innate antiviral defense pathway in mammalian cells. In this study, CD34(+) hematopoietic stem cells (HSC) were transduced with an HIV-1-based lentiviral vector encoding the PKR transgene (pHIV-PIB) and cultured under conditions that support in vitro differentiation. With high-titer pseudotyped vector stocks, the histogram suggests 100% transduction of the HSC because the cells were blasticidin resistant. Analysis of transduced cells by hybridization revealed an average proviral vector copy number of 1.8 and 2.1 copies of vector sequence per cell. Increased PKR expression and activity (phosphorylation of eukaryotic initiation factor 2alpha [eIF2alpha]) were demonstrated in PKR-transduced, differentiated HSC. There was minimal reduction in cell viability and no induction of apoptosis after transduction of PKR. HSC transduced with the pHIV-PIB lentiviral vector demonstrated normal differentiation into CD34-derived T cell progeny. Two days after HIV-1 infection, lentivirus-mediated transduction of PKR inhibited HIV-1 replication by 72% in T cell progeny compared with cells transduced with the empty vector control (pHIV-IB). By days 5 and 7 post-HIV-1 infection, the surviving PKR-transduced cells were protected from HIV-1 infection, as evidenced by a decrease in p24 antigen expression of at least two orders of magnitude. Our results demonstrate that PKR can be effectively delivered to HSC by a lentiviral vector and can protect CD34-derived T cell progeny from HIV-1 infection. These results provide support for application of the innate antiviral defense pathway in a gene therapy setting to the treatment of HIV-1 infection.