RESUMEN
Retroscreen (hVIVO) have developed an RSV human viral challenge model (hVCM) for testing the efficacy of novel antiviral therapies by monitoring changes in viral load and symptoms. The integrated cycler technology and Simplexa™ kits (Focus Diagnostics) currently provide fast, qualitative and sensitive diagnostic testing in hospitals and other healthcare facilities for patients with well-established respiratory illness. We have developed a novel use of qualitative integrated cycler PCR (qicPCR) technology to identify onset of RSV infection enabling an informed dosing clinical protocol in the RSV hVCM. We have validated qicPCR detection of RSV in spiked nasal wash aspirates and demonstrate that the qicPCR assay is 94% concordant with RSV plaque assay data in nasal wash samples from 53 RSV inoculated human volunteers in the hVCM. The use of qicPCR for informed dosing was successfully implemented in a recent clinical trial demonstrating efficacy of the RSV entry inhibitor GS-5806 in the hVCM (NCT01756482). Comparison of qicPCR positivity in relation to nasal wash viral load measured by both RT-qPCR and plaque assay shows that the therapeutic exposure was correctly initiated prior to onset and peak of RSV viral shedding and symptoms in the majority of volunteers.
Asunto(s)
Antivirales/administración & dosificación , Cavidad Nasal/virología , Reacción en Cadena de la Polimerasa/métodos , Infecciones por Virus Sincitial Respiratorio/diagnóstico , Virus Sincitiales Respiratorios/aislamiento & purificación , Carga Viral/métodos , Ensayos Clínicos como Asunto , Humanos , Infecciones por Virus Sincitial Respiratorio/tratamiento farmacológico , Infecciones por Virus Sincitial Respiratorio/patología , Infecciones por Virus Sincitial Respiratorio/virologíaRESUMEN
BACKGROUND AND OBJECTIVE: Synthetic TLR7 agonists have been proposed as oral replacements for interferonα (IFNα) therapy in the treatment of hepatitis C virus infection. However, adverse effects, such as lymphopenia and cardiovascular irregularities, have been observed in the clinical following treatment with TLR7 agonists. We wished to understand and characterise the relationship between TLR7 agonism and adverse effects. METHODS: We compared responses to two prototypic TLR7 agonists (Resiquimod: R-848; and PF-04878691) in a mouse model and compared the responses to treatment with IFNα. We measured clinically relevant adverse effects such as lymphopenia and cardiovascular irregularities and related them to plasma drug levels and clinically relevant efficacy biomarkers such as the pro-inflammatory cytokine IP-10, 2'5'OAS and TLR7 receptor expression. RESULTS: By 2 h post dose all agents had induced a dose-dependent transient lymphopenia. IFNα increased heart rate immediately following dosing, persisting for 5 h, whilst PF-04878691 induced significant reductions in blood pressure. Lymphopenia co-incided with maximum plasma drug levels, raised levels of IP-10 and the auto-induction of TLR7 expression in the blood and lymph nodes. Peak levels of 2'5'OAS occurred at 24 h post-dose and only at doses which also induced lymphopenia. CONCLUSIONS: We conclude that systemic delivery of TLR7 agonists or IFNα induces similar exaggerated pharmacology, consistent with there being a narrow therapeutic window between efficacy and safety. This clinically validated mouse model will help to investigate whether more potent agonists or optimised dosing schedules, will be successful strategies for targeting TLR7 in patients.
Asunto(s)
Aminoquinolinas/efectos adversos , Hipotensión/inducido químicamente , Imidazoles/efectos adversos , Linfopenia/inducido químicamente , Sulfonamidas/efectos adversos , Receptor Toll-Like 7/agonistas , 2',5'-Oligoadenilato Sintetasa/metabolismo , Aminoquinolinas/sangre , Aminoquinolinas/farmacocinética , Animales , Biomarcadores/metabolismo , Presión Sanguínea/efectos de los fármacos , Quimiocina CXCL10/metabolismo , Femenino , Frecuencia Cardíaca/efectos de los fármacos , Hipotensión/metabolismo , Imidazoles/sangre , Imidazoles/farmacocinética , Interferón-alfa/efectos adversos , Interferón-alfa/sangre , Interferón-alfa/farmacocinética , Hígado/efectos de los fármacos , Hígado/metabolismo , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/metabolismo , Recuento de Linfocitos , Linfopenia/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Quinolinas , Sulfonamidas/sangre , Sulfonamidas/farmacocinética , Receptor Toll-Like 7/genética , Receptor Toll-Like 7/metabolismoRESUMEN
The discovery of a series of highly potent and novel TLR7 agonist interferon inducers is described. Structure-activity relationships are presented, along with pharmacokinetic studies of a lead molecule from this series of N9-pyridylmethyl-8-oxo-3-deazapurine analogues. A rationale for the very high potency observed is offered. An investigation of the clearance mechanism of this class of compounds in rat was carried out, resulting in aldehyde oxidase mediated oxidation being identified as a key component of the high clearance observed. A possible solution to this problem is discussed.
Asunto(s)
Antivirales/síntesis química , Antivirales/farmacología , Hepacivirus/efectos de los fármacos , Hepatitis C/tratamiento farmacológico , Interferones/agonistas , Receptor Toll-Like 7/agonistas , Aldehído Oxidasa/metabolismo , Animales , Antivirales/química , Antivirales/farmacocinética , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos , Hepacivirus/fisiología , Hepatitis C/virología , Humanos , Inyecciones Intravenosas , Inductores de Interferón/síntesis química , Inductores de Interferón/química , Inductores de Interferón/farmacocinética , Inductores de Interferón/farmacología , Microsomas Hepáticos/metabolismo , Terapia Molecular Dirigida , Peso Molecular , Purinas/síntesis química , Purinas/metabolismo , Ratas , Solubilidad , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
We have screened 47 locked nucleic acid (LNA) antisense oligonucleotides (ASOs) targeting conserved (>95% homology) sequences in the hepatitis C virus (HCV) genome using the subgenomic HCV replicon assay and generated both antiviral (50% effective concentration [EC(50)]) and cytotoxic (50% cytotoxic concentration [CC(50)]) dose-response curves to allow measurement of the selectivity index (SI). This comprehensive approach has identified an LNA ASO with potent antiviral activity (EC(50) = 4 nM) and low cytotoxicity (CC(50) >880 nM) targeting the 25- to 40-nucleotide region (nt) of the HCV internal ribosome entry site (IRES) containing the distal and proximal miR-122 binding sites. LNA ASOs targeting previously known accessible regions of the IRES, namely, loop III and the initiation codon in loop IV, had poor SI values. We optimized the LNA ASO sequence by performing a 1-nucleotide walk through the 25- to 40-nt region and show that the boundaries for antiviral efficacy are extremely precise. Furthermore, we have optimized the format for the LNA ASO using different gapmer and mixomer patterns and show that RNase H is required for antiviral activity. We demonstrate that RNase H-refractory ASOs targeting the 25- to 40-nt region have no antiviral effect, revealing important regulatory features of the 25- to 40-nt region and suggesting that RNase H-refractory LNA ASOs can act as potential surrogates for proviral functions of miR-122. We confirm the antisense mechanism of action using mismatched LNA ASOs. Finally, we have performed pharmacokinetic experiments to demonstrate that the LNA ASOs have a very long half-life (>5 days) and attain hepatic maximum concentrations >100 times the concentration required for in vitro antiviral activity.
Asunto(s)
Antivirales/farmacología , Antivirales/farmacocinética , Hepacivirus/efectos de los fármacos , Oligonucleótidos Antisentido/farmacología , Oligonucleótidos Antisentido/farmacocinética , Ribosomas/virología , Animales , Línea Celular , Humanos , Riñón/metabolismo , Hígado/metabolismo , RatonesRESUMEN
The synthesis and structure-activity relationships of a series of novel interferon inducers are described. Pharmacokinetic studies and efficacy assessment of a series of 8-oxo-3-deazapurine analogues led to the identification of compound 33, a potent and selective agonist of the TLR7 receptor with an excellent in vivo efficacy profile in a mouse model.
Asunto(s)
Antivirales/química , Hepacivirus/efectos de los fármacos , Purinas/química , Infecciones por Virus ARN/tratamiento farmacológico , Receptor Toll-Like 7/agonistas , Administración Oral , Animales , Antivirales/síntesis química , Antivirales/uso terapéutico , Diseño de Fármacos , Ratones , Modelos Animales , Purinas/farmacocinética , Purinas/uso terapéutico , Ratas , Relación Estructura-Actividad , Receptor Toll-Like 7/metabolismoRESUMEN
BACKGROUND: The BALB/c mouse is commonly used to study RSV infection and disease. However, despite the many advantages of this well-characterised model, the inoculum is large, viral replication is restricted and only a very small amount of virus can be recovered from infected animals. A key question in this model is the fate of the administered virus. Is replication really being measured or is the model measuring the survival of the virus over time? To answer these questions we developed a highly sensitive strand-specific quantitative PCR (QPCR) able to accurately quantify the amount of RSV replication in the BALB/c mouse lung, allowing characterisation of RSV negative and positive strand RNA dynamics. RESULTS: In the mouse lung, no increase in RSV genome was seen above the background of the original inoculum whilst only a limited transient increase (< 1 log) in positive strand, replicative intermediate (RI) RNA occurred. This RNA did however persist at detectable levels for 59 days post infection. As expected, ribavirin therapy reduced levels of infectious virus and RI RNA in the mouse lung. However, whilst Palivizumab therapy was also able to reduce levels of infectious virus, it failed to prevent production of intracellular RI RNA. A comparison of RSV RNA kinetics in human (A549) and mouse (KLN205) cell lines demonstrated that RSV replication was also severely delayed and impaired in vitro in the mouse cells. CONCLUSIONS: This is the first time that such a sensitive strand-specific QPCR technique has been to the RSV mouse system. We have accurately quantified the restricted and abortive nature of RSV replication in the mouse. Further in vitro studies in human and mouse cells suggest this restricted replication is due at least in part to species-specific host cell-viral interactions.
Asunto(s)
Reacción en Cadena de la Polimerasa/métodos , ARN Viral/biosíntesis , ARN Viral/genética , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitial Respiratorio Humano/patogenicidad , Replicación Viral , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Pulmón/virología , Ratones , Ratones Endogámicos BALB C , Virus Sincitial Respiratorio Humano/genética , Virus Sincitial Respiratorio Humano/crecimiento & desarrolloRESUMEN
Toll-like receptors recognize invading microorganisms and activate innate immune responses. Their discovery has opened up a range of therapeutic possibilities, in particular for infectious diseases. Responses to TLR agonists have been largely studied in mice and little information exists in other species. Given that rats are commonly used for pharmacokinetic and toxicology studies in drug development, we compared TLR7 responses in rat and human. Stimulation of rat and human peripheral blood mononuclear cells with the TLR7 agonist SM360320 showed that in rat cells, the interferon-induced gene, 2', 5' oligoadenylate synthase and tumor necrosis factor alpha were induced at lower concentrations and to a greater degree compared with human cells. Both human and rat cells demonstrated tolerance and could not be restimulated following initial treatment with high concentrations of SM360320. Reducing the concentration of the initial treatment allowed cells to be restimulated following a period of recovery. The initial treatment concentration had to be reduced to a greater extent to enable restimulation of rat cells compared with human cells. Dosing whole rats repeatedly with different concentrations of SM360320 confirmed the in vitro results. Treatment of human cells with high concentrations of interferon alpha did not induce tolerance to subsequent treatment with SM360320 indicating that tolerance occurs in the TLR7 signaling pathway, rather than the interferon signaling pathway. We conclude that rat and human cells respond differently to TLR7 activation and that these differences should be considered when using rat as a model to study TLR7 agonists.
Asunto(s)
Leucocitos Mononucleares/inmunología , Receptor Toll-Like 7/agonistas , Factor de Necrosis Tumoral alfa/metabolismo , 2',5'-Oligoadenilato Sintetasa/efectos de los fármacos , 2',5'-Oligoadenilato Sintetasa/metabolismo , Adenina/análogos & derivados , Adenina/farmacología , Animales , Humanos , Interferón-alfa/farmacología , Leucocitos Mononucleares/efectos de los fármacos , Ratas , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Receptor Toll-Like 7/inmunología , Factor de Necrosis Tumoral alfa/agonistasRESUMEN
Toll-like receptors (TLRs) are key mediators of innate immunity, and their activation by microbial components leads to the production of cytokines and interferons. Recombinant alpha interferon has been used to treat several viral diseases and is the current standard of care for hepatitis C virus (HCV) infection. Recently, agonists of TLR7 and TLR9 have been shown to have clinical efficacy in HCV patients, and this is correlated with their ability to induce endogenous type I interferon production. We have carried out a comprehensive study of agonists of TLRs 1 to 9 to determine if any additional TLRs can induce antiviral molecules from human peripheral blood mononuclear cells (PBMCs). The agonists were incubated with PBMCs, and the supernatant was then removed and added to HCV replicon cells to assess antiviral activity. Agonists of TLRs 3, 4, 7, 8, and 9 were found to be potent inducers of antiviral activity in PBMC supernatants, and the activity correlated with the induction of alpha interferon and the interferon-induced antiviral biomarker 2',5'-oligoadenylate synthase. Antiviral activity of TLR7 and TLR8 agonists was blocked by an antibody that binds to the type I interferon receptor, confirming that the antiviral activity results from type I interferon induction. TLR4 and TLR8 agonists were found to strongly induce the proinflammatory cytokines interleukin 1beta and tumor necrosis factor alpha at concentrations similar to those inducing antiviral activity. This raises concerns about adverse side effects if these were to be used as antiviral agents. We therefore conclude that TLRs 3, 7, and 9 represent the most attractive targets for the development of new HCV therapies.
Asunto(s)
Antivirales/farmacología , Hepacivirus/efectos de los fármacos , Receptores Toll-Like/agonistas , Línea Celular Tumoral , Citocinas/metabolismo , Hepatitis C/tratamiento farmacológico , Hepatitis C/virología , Humanos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/virología , Receptor Toll-Like 3/agonistas , Receptor Toll-Like 7/agonistas , Receptor Toll-Like 9/agonistasRESUMEN
A series of 4'-substituted ribonucleoside derivatives has been prepared and evaluated for inhibition of hepatitis C virus (HCV) RNA replication in cell culture. The most potent and non-cytotoxic derivative was compound 28 (4'-azidocytidine, R1479) with an IC(50) of 1.28 microM in the HCV replicon system. The triphosphate of compound 28 was prepared and shown to be an inhibitor of RNA synthesis mediated by NS5B (IC(50)=320 nM), the RNA polymerase encoded by HCV. Data on related analogues have been used to generate some preliminary requirements for activity within this series of nucleosides.
Asunto(s)
Antivirales/química , Química Farmacéutica/métodos , Citidina/análogos & derivados , Hepacivirus/genética , Ribonucleósidos/química , Replicación Viral/efectos de los fármacos , Citidina/farmacología , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Concentración 50 Inhibidora , Modelos Químicos , Conformación Molecular , Nucleósidos/química , ARN/química , UridinaRESUMEN
Hepatitis C virus (HCV) polymerase activity is essential for HCV replication. Targeted screening of nucleoside analogs identified R1479 (4'-azidocytidine) as a specific inhibitor of HCV replication in the HCV subgenomic replicon system (IC(50) = 1.28 microM) with similar potency compared with 2'-C-methylcytidine (IC(50) = 1.13 microM). R1479 showed no effect on cell viability or proliferation of HCV replicon or Huh-7 cells at concentrations up to 2 mM. HCV replicon RNA could be fully cleared from replicon cells after prolonged incubation with R1479. The corresponding 5'-triphosphate derivative (R1479-TP) is a potent inhibitor of native HCV replicase isolated from replicon cells and of recombinant HCV polymerase (NS5B)-mediated RNA synthesis activity. R1479-TP inhibited RNA synthesis as a CTP-competitive inhibitor with a K(i) of 40 nM. On an HCV RNA-derived template substrate (complementary internal ribosome entry site), R1479-TP showed similar potency of NS5B inhibition compared with 3'-dCTP. R1479-TP was incorporated into nascent RNA by HCV polymerase and reduced further elongation with similar efficiency compared with 3'-dCTP under the reaction conditions. The S282T point mutation in the coding sequence of NS5B confers resistance to inhibition by 2'-C-MeATP and other 2'-methyl-nucleotides. In contrast, the S282T mutation did not confer cross-resistance to R1479.