RESUMEN
Capsid assembly is critical in the hepatitis B virus (HBV) life cycle, mediated by the viral core protein. Capsid assembly is the target for new anti-viral therapeutics known as capsid assembly modulators (CAMs) of which the CAM-aberrant (CAM-A) class induces aberrant shaped core protein structures and leads to hepatocyte cell death. This study aimed to identify the mechanism of action of CAM-A modulators leading to HBV-infected hepatocyte elimination where CAM-A-mediated hepatitis B surface antigen (HBsAg) reduction was evaluated in a stable HBV replicating cell line and in AAV-HBV-transduced C57BL/6, C57BL/6 SCID, and HBV-infected chimeric mice with humanized livers. Results showed that in vivo treatment with CAM-A modulators induced pronounced reductions in hepatitis B e antigen (HBeAg) and HBsAg, associated with a transient alanine amino transferase (ALT) increase. Both HBsAg and HBeAg reductions and ALT increase were delayed in C57BL/6 SCID and chimeric mice, suggesting that adaptive immune responses may indirectly contribute. However, CD8+ T cell depletion in transduced wild-type mice did not impact antigen reduction, indicating that CD8+ T cell responses are not essential. Transient ALT elevation in AAV-HBV-transduced mice coincided with a transient increase in endoplasmic reticulum stress and apoptosis markers, followed by detection of a proliferation marker. Microarray data revealed antigen presentation pathway (major histocompatibility complex class I molecules) upregulation, overlapping with the apoptosis. Combination treatment with HBV-specific siRNA demonstrated that CAM-A-mediated HBsAg reduction is dependent on de novo core protein translation. To conclude, CAM-A treatment eradicates HBV-infected hepatocytes with high core protein levels through the induction of apoptosis, which can be a promising approach as part of a regimen to achieve functional cure. IMPORTANCE: Treatment with hepatitis B virus (HBV) capsid assembly modulators that induce the formation of aberrant HBV core protein structures (CAM-A) leads to programmed cell death, apoptosis, of HBV-infected hepatocytes and subsequent reduction of HBV antigens, which differentiates CAM-A from other CAMs. The effect is dependent on the de novo synthesis and high levels of core protein.
Asunto(s)
Antivirales , Apoptosis , Regulación Viral de la Expresión Génica , Antígenos del Núcleo de la Hepatitis B , Virus de la Hepatitis B , Hepatocitos , Biosíntesis de Proteínas , Animales , Ratones , Antivirales/farmacología , Antivirales/uso terapéutico , Apoptosis/efectos de los fármacos , Cápside/química , Cápside/clasificación , Cápside/efectos de los fármacos , Cápside/metabolismo , Proteínas de la Cápside/metabolismo , Hepatitis B/tratamiento farmacológico , Hepatitis B/inmunología , Hepatitis B/metabolismo , Hepatitis B/virología , Antígenos del Núcleo de la Hepatitis B/biosíntesis , Antígenos del Núcleo de la Hepatitis B/metabolismo , Antígenos e de la Hepatitis B/metabolismo , Antígenos de Superficie de la Hepatitis B/metabolismo , Virus de la Hepatitis B/crecimiento & desarrollo , Virus de la Hepatitis B/inmunología , Virus de la Hepatitis B/metabolismo , Virus de la Hepatitis B/patogenicidad , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hepatocitos/patología , Hepatocitos/virología , Ratones Endogámicos C57BL , Ratones SCID , Replicación Viral , Línea Celular , Linfocitos T CD8-positivos/inmunología , Presentación de AntígenoRESUMEN
Chronic Hepatitis B Virus (HBV) infection remains a global burden. To identify small molecule RIG-I agonists as antivirals against HBV, we developed an HBV-pgRNA-based interferon-ß (IFN-ß) luciferase reporter assay with high level of assay sensitivity, specificity and robustness. Through HTS screening, lead compound (JJ#1) was identified to activate RIG-I signaling pathway by inducing TBK1 phosphorylation. Knockdown experiments demonstrated that JJ#1-induced retinoic acid-inducible gene 1 (RIG-I) signaling pathway activation was MAVS-dependent. Furthermore, JJ#1 exhibited HBV antiviral potency in HBV-infected cell models by reducing HBV DNA and antigens (HBsAg and HBeAg).
Asunto(s)
Hepatitis B Crónica , Hepatitis B , Humanos , Virus de la Hepatitis B , Tretinoina , Fosforilación , Antivirales/farmacologíaRESUMEN
HIV infection is controlled immunologically in a small subset of infected individuals without antiretroviral therapy (ART), though the mechanism of control is unclear. CD8+ T cells are a critical component of HIV control in many immunological controllers. NK cells are also believed to have a role in controlling HIV infection, though their role is less well characterized. We used mass cytometry to simultaneously measure the levels of expression of 24 surface markers on peripheral NK cells from HIV-infected subjects with various degrees of HIV natural control; we then used machine learning to identify NK cell subpopulations that differentiate HIV controllers from noncontrollers. Using CITRUS (cluster identification, characterization, and regression), we identified 3 NK cell subpopulations that differentiated subjects with chronic HIV viremia (viremic noncontrollers [VNC]) from individuals with undetectable HIV viremia without ART (elite controllers [EC]). In a parallel approach, we identified 11 NK cell subpopulations that differentiated HIV-infected subject groups using k-means clustering after dimensionality reduction by t-neighbor stochastic neighbor embedding (tSNE) or linear discriminant analysis (LDA). Among these additional 11 subpopulations, the frequencies of 5 correlated with HIV DNA levels; importantly, significance was retained in 2 subpopulations in analyses that included only cohorts without detectable viremia. By comparing the surface marker expression patterns of all identified subpopulations, we revealed that the CD11b+ CD57- CD161+ Siglec-7+ subpopulation of CD56dim CD16+ NK cells are more abundant in EC and HIV-negative controls than in VNC and that the frequency of these cells correlated with HIV DNA levels. We hypothesize that this population may have a role in immunological control of HIV infection.IMPORTANCE HIV infection results in the establishment of a stable reservoir of latently infected cells; ART is usually required to keep viral replication under control and disease progression at bay, though a small subset of HIV-infected subjects can control HIV infection without ART through immunological mechanisms. In this study, we sought to identify subpopulations of NK cells that may be involved in the natural immunological control of HIV infection. We used mass cytometry to measure surface marker expression on peripheral NK cells. Using two distinct semisupervised machine learning approaches, we identified a CD11b+ CD57- CD161+ Siglec-7+ subpopulation of CD56dim CD16+ NK cells that differentiates HIV controllers from noncontrollers. These cells can be sorted out for future functional studies to assess their potential role in the immunological control of HIV infection.
Asunto(s)
Infecciones por VIH/inmunología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/virología , Antígeno CD11b/inmunología , Antígeno CD56/inmunología , Antígenos CD57/inmunología , Linfocitos T CD8-positivos/virología , Línea Celular Tumoral , ADN Viral/inmunología , Infecciones por VIH/virología , VIH-1/inmunología , Humanos , Células K562 , Subfamilia B de Receptores Similares a Lectina de Células NK/inmunología , Receptores de IgG/inmunología , Viremia/inmunología , Viremia/virologíaRESUMEN
OBJECTIVE: Although bone marrow, liver, thymus (BLT)-humanized mice provide a robust model for HIV-1 infection and enable evaluation of cure strategies dependent on endogenous immune responses, most mice develop graft versus host disease (GVHD), limiting their utility for extended HIV cure studies. This study aimed to: evaluate the GVHD-resistant C57 black 6 (C57BL/6) recombination activating gene 2 (Rag2)γcCD47 triple knockout (TKO)-BLT mouse as a model to establish HIV-1 latency. Determine whether TKO-BLT mice could be maintained on antiretroviral therapy (ART) for extended periods of time. Assess the rapidity of viral rebound following therapy interruption. DESIGN: TKO-BLT mice were HIV-1 infected, treated with various ART regimens over extended periods of time and assayed for viral rebound following therapy interruption. METHODS: Daily subcutaneous injection and oral ART-mediated suppression of HIV-1 infection was tested at various doses in TKO-BLT mice. Mice were monitored for suppression of viremia and cellular HIV-1 RNA and DNA prior to and following therapy interruption. RESULTS: Mice remained healthy for 45 weeks posthumanization and could be treated with ART for up to 18 weeks. Viremia was suppressed to less than 200 copies/ml in the majority of mice with significant reductions in cellular HIV-1 RNA and DNA. Treatment interruption resulted in rapid viral recrudescence. CONCLUSION: HIV-1 latency can be maintained in TKO-BLT mice over extended periods on ART and rapid viral rebound occurs following therapy removal. The additional 15-18 weeks of healthy longevity compared with other BLT models provides sufficient time to examine the decay kinetics of the latent reservoir as well as observe delays in recrudescence in HIV-1 cure studies.
Asunto(s)
Modelos Animales de Enfermedad , Infecciones por VIH/tratamiento farmacológico , Ratones Transgénicos , Administración Oral , Animales , Antirretrovirales/administración & dosificación , VIH-1/aislamiento & purificación , VIH-1/fisiología , Humanos , Inyecciones Subcutáneas , Ratones Endogámicos C57BL , Ratones Noqueados , Resultado del Tratamiento , Carga Viral , Latencia del VirusRESUMEN
Antiretroviral therapy can suppress HIV replication to undetectable levels but does not eliminate latent HIV, thus necessitating lifelong therapy. Recent efforts to target this persistent reservoir have focused on inducing the expression of latent HIV so that infected cells may be recognized and eliminated by the immune system. Toll-like receptor (TLR) activation stimulates antiviral immunity and has been shown to induce HIV from latently infected cells. Activation of TLR7 leads to the production of several stimulatory cytokines, including type I interferons (IFNs). In this study, we show that the selective TLR7 agonist GS-9620 induced HIV in peripheral blood mononuclear cells (PBMCs) from HIV-infected individuals on suppressive antiretroviral therapy. GS-9620 increased extracellular HIV RNA 1.5- to 2-fold through a mechanism that required type I IFN signaling. GS-9620 also activated HIV-specific T cells and enhanced antibody-mediated clearance of HIV-infected cells. Activation by GS-9620 in combination with HIV peptide stimulation increased CD8 T cell degranulation, production of intracellular cytokines, and cytolytic activity. T cell activation was again dependent on type I IFNs produced by plasmacytoid dendritic cells. GS-9620 induced phagocytic cell maturation and improved effector-mediated killing of HIV-infected CD4 T cells by the HIV envelope-specific broadly neutralizing antibody PGT121. Collectively, these data show that GS-9620 can activate HIV production and improve the effector functions that target latently infected cells. GS-9620 may effectively complement orthogonal therapies designed to stimulate antiviral immunity, such as therapeutic vaccines or broadly neutralizing antibodies. Clinical studies are under way to determine if GS-9620 can target HIV reservoirs.IMPORTANCE Though antiretroviral therapies effectively suppress viral replication, they do not eliminate integrated proviral DNA. This stable intermediate of viral infection is persistently maintained in reservoirs of latently infected cells. Consequently, lifelong therapy is required to maintain viral suppression. Ultimately, new therapies that specifically target and eliminate the latent HIV reservoir are needed. Toll-like receptor agonists are potent enhancers of innate antiviral immunity that can also improve the adaptive immune response. Here, we show that a highly selective TLR7 agonist, GS-9620, activated HIV from peripheral blood mononuclear cells isolated from HIV-infected individuals with suppressed infection. GS-9620 also improved immune effector functions that specifically targeted HIV-infected cells. Previously published studies on the compound in other chronic viral infections show that it can effectively induce immune activation at safe and tolerable clinical doses. Together, the results of these studies suggest that GS-9620 may be useful for treating HIV-infected individuals on suppressive antiretroviral therapy.
Asunto(s)
Infecciones por VIH/virología , VIH/inmunología , VIH/fisiología , Factores Inmunológicos/farmacología , Pteridinas/farmacología , Receptor Toll-Like 7/agonistas , Activación Viral/efectos de los fármacos , Antirretrovirales/uso terapéutico , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/efectos de los fármacos , Humanos , Interferón Tipo I/metabolismo , Activación de Linfocitos/efectos de los fármacos , Fagocitos/efectos de los fármacos , Fagocitos/fisiología , ARN Viral/sangre , Carga ViralRESUMEN
The interferon-free combination of once-daily faldaprevir 120 mg, twice-daily deleobuvir 600 mg, and weight-based ribavirin was evaluated in two Phase III studies (HCVerso1, HCVerso2) in hepatitis C virus genotype-1b-infected, treatment-naïve patients, including those ineligible for peginterferon (HCVerso2). Patients without cirrhosis were randomized to 16 weeks (Arm 1; n=208 HCVerso1, n=213 HCVerso2) or 24 weeks (Arm 2; n=211 in both studies) of faldaprevir + deleobuvir + ribavirin. Patients with compensated cirrhosis received open-label faldaprevir + deleobuvir + ribavirin for 24 weeks (Arm 3; n=51, n=72). Primary endpoints were comparisons of adjusted sustained virologic response (SVR) rates with historical rates: 71% (HCVerso1) and 68% (HCVerso2). Adjusted SVR12 rates were significantly greater than historical controls for Arms 1 and 2 in HCVerso2 (76%, 95% confidence interval [CI] 71-81, P=0.002; 81%, 95% CI 76-86, P<0.0001) and Arm 2 in HCVerso1 (81%, 95% CI 77-86, P<0.0001), but not for Arm 1 of HCVerso1 (72%, 95% CI 66-77, P=0.3989). Unadjusted SVR12 rates in Arms 1, 2, and 3 were 71.6%, 82.5%, and 72.5%, respectively, in HCVerso1 and 75.6%, 82.0%, and 73.6%, respectively, in HCVerso2. Virologic breakthrough and relapse occurred in 24-week arms in 8%-9% and 1% of patients, respectively, and in 16-week arms in 7%-8% and 9%-11% of patients, respectively. The most common adverse events were nausea (46%-61%) and vomiting (29%-35%). Adverse events resulted in discontinuation of all medications in 6%-8% of patients. In treatment-naïve patients with hepatitis C virus genotype-1b infection, with or without cirrhosis, faldaprevir + deleobuvir + ribavirin treatment for 24 weeks resulted in adjusted SVR12 rates significantly higher than historical controls. Both studies were registered in ClinicalTrials.gov (NCT01732796, NCT01728324).
RESUMEN
Bictegravir (BIC; GS-9883), a novel, potent, once-daily, unboosted inhibitor of HIV-1 integrase (IN), specifically targets IN strand transfer activity (50% inhibitory concentration [IC50] of 7.5 ± 0.3 nM) and HIV-1 integration in cells. BIC exhibits potent and selective in vitro antiretroviral activity in both T-cell lines and primary human T lymphocytes, with 50% effective concentrations ranging from 1.5 to 2.4 nM and selectivity indices up to 8,700 relative to cytotoxicity. BIC exhibits synergistic in vitro antiviral effects in pairwise combinations with tenofovir alafenamide, emtricitabine, or darunavir and maintains potent antiviral activity against HIV-1 variants resistant to other classes of antiretrovirals. BIC displayed an in vitro resistance profile that was markedly improved compared to the integrase strand transfer inhibitors (INSTIs) raltegravir (RAL) and elvitegravir (EVG), and comparable to that of dolutegravir (DTG), against nine INSTI-resistant site-directed HIV-1 mutants. BIC displayed statistically improved antiviral activity relative to EVG, RAL, and DTG against a panel of 47 patient-derived HIV-1 isolates with high-level INSTI resistance; 13 of 47 tested isolates exhibited >2-fold lower resistance to BIC than DTG. In dose-escalation experiments conducted in vitro, BIC and DTG exhibited higher barriers to resistance than EVG, selecting for HIV-1 variants with reduced phenotypic susceptibility at days 71, 87, and 20, respectively. A recombinant virus with the BIC-selected M50I/R263K dual mutations in IN exhibited only 2.8-fold reduced susceptibility to BIC compared to wild-type virus. All BIC-selected variants exhibited low to intermediate levels of cross-resistance to RAL, DTG, and EVG (<8-fold) but remained susceptible to other classes of antiretrovirals. A high barrier to in vitro resistance emergence for both BIC and DTG was also observed in viral breakthrough studies in the presence of constant clinically relevant drug concentrations. The overall virologic profile of BIC supports its ongoing clinical investigation in combination with other antiretroviral agents for both treatment-naive and -experienced HIV-infected patients.
Asunto(s)
Farmacorresistencia Viral/efectos de los fármacos , Inhibidores de Integrasa VIH/farmacología , Integrasa de VIH/metabolismo , VIH-1/efectos de los fármacos , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Amidas , Fármacos Anti-VIH/farmacología , Línea Celular , Sinergismo Farmacológico , Integrasa de VIH/genética , VIH-1/genética , VIH-1/aislamiento & purificación , Compuestos Heterocíclicos con 3 Anillos/farmacología , Humanos , Mutación , Oxazinas , Piperazinas , Piridonas , Raltegravir Potásico/farmacologíaRESUMEN
BACKGROUND & AIM: The resistance profile of anti-hepatitis C virus (HCV) agents used in combination is important to guide optimal treatment regimens. We evaluated baseline and treatment-emergent NS3/4A and NS5B amino-acid variants among HCV genotype (GT)-1a and -1b-infected patients treated with faldaprevir (HCV protease inhibitor), deleobuvir (HCV polymerase non-nucleoside inhibitor), and ribavirin in multiple clinical studies. METHODS: HCV NS3/4A and NS5B population sequencing (Sanger method) was performed on all baseline plasma samples (n = 1425 NS3; n = 1556 NS5B) and on post-baseline plasma samples from patients with virologic failure (n = 113 GT-1a; n = 221 GT-1b). Persistence and time to loss of resistance-associated variants (RAVs) was estimated using Kaplan-Meier analysis. RESULTS: Faldaprevir RAVs (NS3 R155 and D168) and deleobuvir RAVs (NS5B 495 and 496) were rare (<1%) at baseline. Virologic response to faldaprevir/deleobuvir/ribavirin was not compromised by common baseline NS3 polymorphisms (e.g. Q80K in 17.5% of GT-1a) or by NS5B A421V, present in 20% of GT-1a. In GT-1b, alanine at NS5B codon 499 (present in 15% of baseline sequences) was associated with reduced response. Treatment-emergent RAVs consolidated previous findings: NS3 R155 and D168 were key faldaprevir RAVs; NS5B A421 and P495 were key deleobuvir RAVs. Among on-treatment virologic breakthroughs, RAVs emerged in both NS3 and NS5B (>90%). Virologic relapse was associated with RAVs in both NS3 and NS5B (53% GT-1b; 52% GT-1b); some virologic relapses had NS3 RAVs only (47% GT-1a; 17% GT-1b). Median time to loss of GT-1b NS5B P495 RAVs post-treatment (5 months) was less than that of GT-1b NS3 D168 (8.5 months) and GT-1a R155 RAVs (11.5 months). CONCLUSION: Faldaprevir and deleobuvir RAVs are more prevalent among virologic failures than at baseline. Treatment response was not compromised by common NS3 polymorphisms; however, alanine at NS5B amino acid 499 at baseline (wild-type in GT-1a, polymorphism in GT-1b) may reduce response to this deleobuvir-based regimen.
Asunto(s)
Acrilatos/uso terapéutico , Bencimidazoles/uso terapéutico , Proteínas Portadoras/antagonistas & inhibidores , Farmacorresistencia Viral/efectos de los fármacos , Hepatitis C Crónica/tratamiento farmacológico , Mutación/genética , Oligopéptidos/uso terapéutico , Tiazoles/uso terapéutico , Proteínas no Estructurales Virales/antagonistas & inhibidores , Sustitución de Aminoácidos , Ácidos Aminoisobutíricos , Antivirales/uso terapéutico , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Ensayos Clínicos Fase I como Asunto , Ensayos Clínicos Fase II como Asunto , Ensayos Clínicos Fase III como Asunto , Hepacivirus/efectos de los fármacos , Hepacivirus/genética , Hepacivirus/metabolismo , Hepatitis C Crónica/genética , Hepatitis C Crónica/virología , Humanos , Péptidos y Proteínas de Señalización Intracelular , Leucina/análogos & derivados , Polimorfismo Genético/efectos de los fármacos , Polimorfismo Genético/genética , Prolina/análogos & derivados , Inhibidores de Proteasas/uso terapéutico , Quinolinas , Resultado del Tratamiento , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismoRESUMEN
We describe our efforts to identify analogs of thumb pocket 1 HCV NS5B inhibitor 1 (aza-analog of BI 207524) with improved plasma to liver partitioning and a predicted human half-life consistent with achieving a strong antiviral effect at a reasonable dose in HCV-infected patients. Compounds 3 and 7 were identified that met these criteria but exhibited off-target promiscuity in an in vitro pharmacology screen and in vivo toxicity in rats. High lipophilicity in this class was found to correlate with increased probability for promiscuous behavior and toxicity. The synthesis of an 8×11 matrix of analogs allowed the identification of C3, an inhibitor that displayed comparable potency to 1, improved partitioning to the liver and reduced lipophilicity. Although C3 displayed reduced propensity for in vitro off-target inhibition and the toxicity profile in rats was improved, the predicted human half-life of this compound was short, resulting in unacceptable dosing requirements to maintain a strong antiviral effect in patients.
Asunto(s)
Acrilatos/química , Acrilatos/farmacología , Antivirales/química , Antivirales/farmacología , Hepacivirus/efectos de los fármacos , Indoles/química , Indoles/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Acrilatos/farmacocinética , Acrilatos/toxicidad , Animales , Antivirales/farmacocinética , Antivirales/toxicidad , Perros , Haplorrinos , Hepatitis C/tratamiento farmacológico , Hepatitis C/virología , Humanos , Indoles/farmacocinética , Indoles/toxicidad , Lípidos/química , Hígado/metabolismo , Hígado/virología , Ratas , Proteínas no Estructurales Virales/metabolismoRESUMEN
A series of heterocyclic aza-analogs of BI 207524 (2), a potent HCV NS5B polymerase thumb pocket 1 inhibitor, was investigated with the goal to reduce the liability associated with the release of a genotoxic aniline metabolite in vivo. Analog 4, containing a 2-aminopyridine aniline isostere that is negative in the Ames test was identified, and was found to provide comparable GT1a/1b potency to 2. Although the cross-species PK profile, poor predicted human liver distribution of analog 4 and allometry principles projected high doses to achieve a strong antiviral response in patients, this work has provided a path forward toward the design of novel thumb pocket 1 NS5B polymerase inhibitors with improved safety profiles.
Asunto(s)
Acrilatos/metabolismo , Acrilatos/farmacología , Compuestos de Anilina/metabolismo , Antivirales/metabolismo , Antivirales/farmacología , Hepacivirus/efectos de los fármacos , Indoles/metabolismo , Indoles/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Acrilatos/química , Acrilatos/farmacocinética , Animales , Antivirales/química , Antivirales/farmacocinética , Perros , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacocinética , Inhibidores Enzimáticos/farmacología , Haplorrinos , Hepacivirus/enzimología , Hepatitis C/tratamiento farmacológico , Hepatitis C/virología , Humanos , Indoles/química , Indoles/farmacocinética , Ratas , Proteínas no Estructurales Virales/metabolismoRESUMEN
BACKGROUND & AIMS: The efficacy and tolerability of faldaprevir, a potent hepatitis C virus (HCV) NS3/4A protease inhibitor, plus peginterferon (PegIFN) and ribavirin (RBV) was assessed in a double-blind, placebo-controlled phase 3 study of treatment-naïve patients with HCV genotype-1 infection. METHODS: Patients were randomly assigned (1:2:2) to PegIFN/RBV plus: placebo (arm 1, n = 132) for 24 weeks; faldaprevir (120 mg, once daily) for 12 or 24 weeks (arm 2, n = 259); or faldaprevir (240 mg, once daily) for 12 weeks (arm 3, n = 261). In arms 2 and 3, patients with early treatment success (HCV-RNA <25 IU/ml at week 4 and undetectable at week 8) stopped all treatment at week 24. Other patients received PegIFN/RBV until week 48 unless they met futility criteria. The primary endpoint was sustained virologic response 12 weeks post-treatment (SVR12). RESULTS: SVR12 was achieved by 52%, 79%, and 80% of patients in arms 1, 2, and 3, respectively (estimated difference for arms 2 and 3 vs. arm 1: 27%, 95% confidence interval 17%-36%; and 29%, 95% confidence interval, 19%-38%, respectively; p < 0.0001 for both). Early treatment success was achieved by 87% (arm 2) and 89% (arm 3) of patients, of whom 86% and 89% achieved SVR12. Adverse event rates were similar among groups; few adverse events led to discontinuation of all regimen components. CONCLUSIONS: Faldaprevir plus PegIFN/RBV significantly increased SVR12, compared with PegIFN/RBV, in treatment-naïve patients with HCV genotype-1 infection. No differences were seen in responses of patients given faldaprevir once daily at 120 or 240 mg.
Asunto(s)
Antivirales/administración & dosificación , Hepatitis C Crónica/tratamiento farmacológico , Hepatitis C Crónica/virología , Interferón-alfa/administración & dosificación , Oligopéptidos/administración & dosificación , Polietilenglicoles/administración & dosificación , Ribavirina/administración & dosificación , Tiazoles/administración & dosificación , Adulto , Ácidos Aminoisobutíricos , Antivirales/efectos adversos , Método Doble Ciego , Quimioterapia Combinada , Femenino , Genotipo , Hepacivirus/clasificación , Hepacivirus/efectos de los fármacos , Hepacivirus/genética , Humanos , Interferón-alfa/efectos adversos , Leucina/análogos & derivados , Masculino , Persona de Mediana Edad , Oligopéptidos/efectos adversos , Polietilenglicoles/efectos adversos , Prolina/análogos & derivados , Quinolinas , ARN Viral/sangre , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/efectos adversos , Ribavirina/efectos adversos , Tiazoles/efectos adversosRESUMEN
A prodrug approach was developed to address the low oral bioavailability of a poorly soluble (<0.1µg/mL in pH 6.8 buffer) but highly permeable thumb pocket 1 HCV NS5B polymerase inhibitor. Bioconversion rates of structurally diverse prodrug derivatives were evaluated in a panel of in vitro assays using microsomes, from either liver or intestinal tissues, simulated intestinal fluids, simulated gastric fluids or plasma. In vivo bioconversion of promising candidates was evaluated following oral administration to rats. The most successful strategy involved modification of the parent drug carboxylic acid moiety to glycolic amide esters which improved solubility in lipid-based self-emulsifying drug delivery systems (SEDDS). Crystalline prodrug analog 36 (mp 161°C) showed good solubility in individual SEDDS components (up to 80mg/mL) compared to parent 2 (<3mg/mL; mp 267°C) and cross-species bioconversions which correlated with in vitro stability in liver microsomes.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Emulsiones/administración & dosificación , Inhibidores de la Síntesis del Ácido Nucleico/administración & dosificación , Profármacos/administración & dosificación , Proteínas no Estructurales Virales/antagonistas & inhibidores , Administración Oral , Animales , Emulsiones/química , Emulsiones/metabolismo , Microsomas/efectos de los fármacos , Microsomas/metabolismo , Inhibidores de la Síntesis del Ácido Nucleico/química , Inhibidores de la Síntesis del Ácido Nucleico/metabolismo , Profármacos/química , Profármacos/metabolismo , Ratas , Solubilidad , Proteínas no Estructurales Virales/metabolismoRESUMEN
The development of interferon-free regimens for the treatment of chronic HCV infection constitutes a preferred option that is expected in the future to provide patients with improved efficacy, better tolerability, and reduced risk for emergence of drug-resistant virus. We have pursued non-nucleoside NS5B polymerase allosteric inhibitors as combination partners with other direct acting antivirals (DAAs) having a complementary mechanism of action. Herein, we describe the discovery of a potent follow-up compound (BI 207524, 27) to the first thumb pocket 1 NS5B inhibitor to demonstrate antiviral activity in genotype 1 HCV infected patients, BILB 1941 (1). Cell-based replicon potency was significantly improved through electronic modulation of the pKa of the carboxylic acid function of the lead molecule. Subsequent ADME-PK optimization lead to 27, a predicted low clearance compound in man. The preclinical profile of inhibitor 27 is discussed, as well as the identification of a genotoxic metabolite that led to the discontinuation of the development of this compound.
Asunto(s)
Acrilatos/síntesis química , Antivirales/síntesis química , Antivirales/metabolismo , Indoles/síntesis química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Acrilatos/metabolismo , Acrilatos/farmacocinética , Animales , Antivirales/farmacología , Cinamatos/síntesis química , Cinamatos/metabolismo , Cinamatos/farmacocinética , Perros , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacocinética , Hepatitis C Crónica , Humanos , Indoles/metabolismo , Indoles/farmacocinética , Macaca mulatta , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Ratas , Relación Estructura-ActividadRESUMEN
Previous investigations identified 2'-C-Me-branched ribo-C-nucleoside adenosine analogues, 1, which contains a pyrrolo[2,1-f][1,2,4]triazin-4-amine heterocyclic base, and 2, which contains an imidazo[2,1-f][1,2,4]triazin-4-amine heterocyclic base as two compounds with promising anti-HCV in vitro activity. This Letter describes the synthesis and evaluation of a series of novel analogues of these compounds substituted at the 2-, 7-, and 8-positions of the heterocyclic bases. A number of active new HCV inhibitors were identified but most compounds also demonstrated unacceptable cytotoxicity. However, the 7-fluoro analogue of 1 displayed good potency with a promising cytotherapeutic margin.
Asunto(s)
Antivirales/farmacología , Proliferación Celular/efectos de los fármacos , Hepacivirus/efectos de los fármacos , Imidazoles/química , Nucleósidos/farmacología , Pirroles/química , Triazinas/química , Replicación Viral/efectos de los fármacos , Antivirales/química , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/virología , Hepacivirus/genética , Hepatitis C/tratamiento farmacológico , Hepatitis C/virología , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/virología , Estructura Molecular , Nucleósidos/química , ARN Viral/genética , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
BACKGROUND: Coexpression of CD160 and PD-1 on HIV-specific CD8+ T-cells defines a highly exhausted T-cell subset. CD160 binds to Herpes Virus Entry Mediator (HVEM) and blocking this interaction with HVEM antibodies reverses T-cell exhaustion. As HVEM binds both inhibitory and activatory receptors, our aim in the current study was to assess the impact of CD160-specific antibodies on the enhancement of T-cell activation. METHODS: Expression of the two CD160 isoforms; glycosylphosphatidylinositol-anchored (CD160-GPI) and the transmembrane isoforms (CD160-TM) was assessed in CD4 and CD8 primary T-cells by quantitative RT-PCR and Flow-cytometry. Binding of these isoforms to HVEM ligand and the differential capacities of CD160 and HVEM specific antibodies to inhibit this binding were further evaluated using a Time-Resolved Fluorescence assay (TRF). The impact of both CD160 and HVEM specific antibodies on enhancing T-cell functionality upon antigenic stimulation was performed in comparative ex vivo studies using primary cells from HIV-infected subjects stimulated with HIV antigens in the presence or absence of blocking antibodies to the key inhibitory receptor PD-1. RESULTS: We first show that both CD160 isoforms, CD160-GPI and CD160-TM, were expressed in human primary CD4+ and CD8+ T-cells. The two isoforms were also recognized by the HVEM ligand, although this binding was less pronounced with the CD160-TM isoform. Mechanistic studies revealed that although HVEM specific antibodies blocked its binding to CD160-GPI, surprisingly, these antibodies enhanced HVEM binding to CD160-TM, suggesting that potential antibody-mediated HVEM multimerization and/or induced conformational changes may be required for optimal CD160-TM binding. Triggering of CD160-GPI over-expressed on Jurkat cells with either bead-bound HVEM-Fc or anti-CD160 monoclonal antibodies enhanced cell activation, consistent with a positive co-stimulatory role for CD160-GPI. However, CD160-TM did not respond to this stimulation, likely due to the lack of optimal HVEM binding. Finally, ex vivo assays using PBMCs from HIV viremic subjects showed that the use of CD160-GPI-specific antibodies combined with blockade of PD-1 synergistically enhanced the proliferation of HIV-1 specific CD8+ T-cells upon antigenic stimulation. CONCLUSIONS: Antibodies targeting CD160-GPI complement the blockade of PD-1 to enhance HIV-specific T-cell responses and warrant further investigation in the development of novel immunotherapeutic approaches.
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Antígenos CD/genética , Antígenos CD/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Activación de Linfocitos/genética , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Adulto , Animales , Antígenos CD/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Células CHO , Cricetinae , Cricetulus , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/inmunología , Proteínas Ligadas a GPI/metabolismo , Infecciones por VIH/genética , Infecciones por VIH/metabolismo , Humanos , Células Jurkat , Masculino , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Isoformas de Proteínas/metabolismo , Receptores Inmunológicos/inmunología , Miembro 14 de Receptores del Factor de Necrosis Tumoral/metabolismoRESUMEN
BACKGROUND & AIMS: Chronic hepatitis C virus (HCV) infection is characterised by a failure of virus-specific CD8+ T cells that is mainly caused by viral escape and T cell exhaustion. Constant antigen stimulation has been suggested to contribute to HCV-specific CD8+ T cell exhaustion. However, IFN-based therapies failed to recover HCV-specific CD8+ T cell function suggesting that the damage to CD8+ T cells may be permanent even after antigen removal. It was therefore the objective of this study to analyse the impact of inhibition of ongoing viral replication by IFN-free therapy with direct acting antivirals (DAA) on the phenotype and function of HCV-specific CD8+ T cells. METHODS: Virus-specific CD8+ T cells obtained from a patient cohort of 51 previously untreated chronically infected patients undergoing IFN-free therapy with a combination of faldaprevir (a protease inhibitor) and deleobuvir (a non-nucleoside polymerase inhibitor) with or without ribavirin were analysed ex vivo and after in vitro expansion at baseline, wk4, wk 12, and after treatment. RESULTS: Our results show the rapid restoration of proliferative HCV-specific CD8+ T cells in the majority of patients with SVR12 within 4 weeks of therapy suggesting that IFN-free therapy mediated antigen removal may restore CD8+ T cell function. CONCLUSIONS: This study indicates a specific restoration of proliferative HCV-specific CD8+ T cells under IFN-free therapy. This is in contrast to PegIFN-based therapies that have been shown not to restore T cell function during and after chronic infection.
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Antivirales/farmacología , Antivirales/uso terapéutico , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/fisiología , Hepacivirus , Hepatitis C Crónica/tratamiento farmacológico , Acrilatos/farmacología , Acrilatos/uso terapéutico , Adulto , Anciano , Ácidos Aminoisobutíricos , Bencimidazoles/farmacología , Bencimidazoles/uso terapéutico , Linfocitos T CD8-positivos/patología , Proliferación Celular/efectos de los fármacos , Proliferación Celular/fisiología , Células Cultivadas , Contraindicaciones , Quimioterapia Combinada , Femenino , Hepacivirus/efectos de los fármacos , Hepacivirus/fisiología , Hepatitis C Crónica/patología , Humanos , Técnicas In Vitro , Interferón alfa-2 , Interferón-alfa , Leucina/análogos & derivados , Masculino , Persona de Mediana Edad , Oligopéptidos/farmacología , Oligopéptidos/uso terapéutico , Polietilenglicoles , Prolina/análogos & derivados , Quinolinas , Proteínas Recombinantes , Ribavirina/farmacología , Ribavirina/uso terapéutico , Tiazoles/farmacología , Tiazoles/uso terapéutico , Resultado del Tratamiento , Replicación Viral/efectos de los fármacos , Replicación Viral/fisiologíaRESUMEN
Nucleoside analogues have long been recognized as prospects for the discovery of direct acting antivirals (DAAs) to treat hepatitis C virus because they have generally exhibited cross-genotype activity and a high barrier to resistance. C-Nucleosides have the potential for improved metabolism and pharmacokinetic properties over their N-nucleoside counterparts due to the presence of a strong carbon-carbon glycosidic bond and a non-natural heterocyclic base. Three 2'CMe-C-adenosine analogues and two 2'CMe-guanosine analogues were synthesized and evaluated for their anti-HCV efficacy. The nucleotide triphosphates of four of these analogues were found to inhibit the NS5B polymerase, and adenosine analogue 1 was discovered to have excellent pharmacokinetic properties demonstrating the potential of this drug class.
RESUMEN
The design and preliminary SAR of a new series of 1H-quinazolin-4-one (QAZ) allosteric HCV NS5B thumb pocket 2 (TP-2) inhibitors was recently reported. To support optimization efforts, a molecular dynamics (MD) based modeling workflow was implemented, providing information on QAZ binding interactions with NS5B. This approach predicted a small but critical ligand-binding induced movement of a protein backbone region which increases the pocket size and improves access to the backbone carbonyl groups of Val 494 and Pro 495. This localized backbone shift was consistent with key SAR results and was subsequently confirmed by X-ray crystallography. The MD protocol guided the design of inhibitors, exploiting novel H-bond interactions with the two backbone carbonyl groups, leading to the first thumb pocket 2 NS5B inhibitor with picomolar antiviral potency in genotype (gt) 1a and 1b replicons (EC50 = 120 and 110 pM, respectively) and with EC50 ≤ 80 nM against gt 2-6.
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Antivirales/farmacología , Inhibidores Enzimáticos/farmacología , Hepacivirus/efectos de los fármacos , Replicón/efectos de los fármacos , Proteínas no Estructurales Virales/antagonistas & inhibidores , Regulación Alostérica , Antivirales/química , Línea Celular , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Hepacivirus/enzimología , Hepacivirus/genética , Simulación de Dinámica Molecular , Relación Estructura-ActividadRESUMEN
Conformational restrictions of flexible torsion angles were used to guide the identification of new chemotypes of HCV NS5B inhibitors. Sites for rigidification were based on an acquired conformational understanding of compound binding requirements and the roles of substituents in the free and bound states. Chemical bioisosteres of amide bonds were explored to improve cell-based potency. Examples are shown, including the design concept that led to the discovery of the phase III clinical candidate deleobuvir (BI 207127). The structure-based strategies employed have general utility in drug design.
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Antivirales/farmacología , Bencimidazoles/farmacología , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Hepacivirus/efectos de los fármacos , Indoles/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Antivirales/química , Bencimidazoles/química , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Hepacivirus/enzimología , Indoles/química , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación MolecularRESUMEN
An anthranilic acid series of allosteric thumb pocket 2 HCV NS5B polymerase inhibitors exhibited hindered rotation along a covalent bond axis, and the existence of atropisomer chirality was confirmed by NMR, HPLC analysis on chiral supports, and computational studies. A thorough understanding of the concerted rotational properties and the influence exerted by substituents involved in this steric phenomenon was attained through biophysical studies on a series of truncated analogues. The racemization half-life of a compound within this series was determined to be 69 min, which was consistent with a class 2 atropisomer (intermediate conformational exchange). It was further found by X-ray crystallography that one enantiomer of a compound bound to the intended HCV NS5B polymerase target whereas the mirror image atropisomer was able to bind to an unrelated HIV matrix target. Analogues were then identified that selectively inhibited the former. These studies highlight that atropisomer chirality can lead to distinct entities with specific properties, and the phenomenon of atropisomerism in drug discovery should be evaluated and appropriately managed.