RESUMEN
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
BACKGROUND: This study presents preclinical data of a novel interferon (IFN)-α8 fusion protein, PF-04849285, and compares it with IFN-α2 and pegylated IFN-α2; the latter being the current standard of care for HCV. METHODS: The antiviral properties were evaluated in vitro using the HCV replication assay (replicon) and the general encephalomyocarditis virus assay. The binding affinity to both IFNR-subunits was assessed using surface plasmon resonance. Ex vivo experiments using cynomolgus monkey and human blood were used for the evaluation of induction of IFN-inducible biomarkers (interferon inducible protein 10 [IP-10], 2'-5'-oligoadenylate synthetase [OAS2] and interleukin-6 [IL-6]). The molecule was tested intravenously and subcutaneously in cynomolgus monkey in a single dose study for two weeks at 0.01, 1, 5 and 20 mg/kg. Each route and dose combination was given to a single male animal, blood samples were collected for evaluation of biomarkers and pharmacokinetics. The compound was also tested in cynomolgus monkey in a multiple dose study for four weeks, with a twice-a-week dosing prior to a three-week wash-out period for toxicokinetics, pharmacokinetics, and biomarker evaluation at 20, 50 or 100 mg/kg subcutaneously and 20 mg/kg intravenously. RESULTS: The molecule is 10× more potent than the pegylated IFN-α2a, with potency similar to the unmodified IFN-α2a. No unanticipated findings were observed in cynomolgus monkey when dosed up to 20 mg/kg, >10,000-fold margin over the anticipated efficacious human dose. CONCLUSIONS: The biomarker and toxicological findings were consistent with a potent IFN molecule. The potency and pharmacokinetic properties of the molecule are consistent with dosing at least every two weeks with the potential for monthly dosing' and not 'at least twice daily' as presented in the original [corrected].
Asunto(s)
Antivirales/farmacología , Hepatitis C/tratamiento farmacológico , Interferón-alfa/farmacología , Proteínas Recombinantes de Fusión/farmacología , Animales , Antivirales/farmacocinética , Antivirales/toxicidad , Línea Celular , Evaluación Preclínica de Medicamentos , Virus de la Encefalomiocarditis/efectos de los fármacos , Femenino , Hepacivirus/efectos de los fármacos , Humanos , Interferón alfa-2 , Interferón-alfa/administración & dosificación , Interferón-alfa/farmacocinética , Interferón-alfa/toxicidad , Macaca fascicularis , Masculino , Receptores de Interferón/metabolismo , Proteínas Recombinantes de Fusión/farmacocinética , Proteínas Recombinantes de Fusión/toxicidad , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/farmacología , Resultado del Tratamiento , Replicación Viral/efectos de los fármacosRESUMEN
BACKGROUND: A significant challenge of oligonucleotide bioanalysis is the selective extraction from complex tissue samples, where the molecules that distribute into the intracellular space are extensively protein bound and sit amongst a high concentration of endogenous nucleic acid material. Published analytical methodology currently purports extensive sample preparation requirements that include cell lysis steps, homogenization and dual cleanup with liquid-liquid extraction and solid-phase extraction, prior to injection. RESULTS: We have developed a simple liquid-liquid extraction approach to rapidly isolate antisense oligonucleotides from biological tissues with high recovery and combined these preparative steps with a robust monolithic column LC-MS/MS setup. The platform showed improved chromatographic resolution and detection sensitivity over standard reversed-phase columns and required a low sample volume. CONCLUSION: The high-throughput method was sufficient to accurately quantify multiple antisense oligonucleotides in mouse tissue and plasma down to low ng/g and ng/ml levels, respectively, for pharmacokinetic determination, and exhibited a high degree of specificity.
Asunto(s)
Cromatografía Liquida/métodos , Oligonucleótidos Antisentido/análisis , Espectrometría de Masas en Tándem/métodos , Animales , Cromatografía Liquida/instrumentación , Riñón/química , Hígado/química , Ratones , Oligonucleótidos Antisentido/farmacocinética , Estándares de Referencia , Extracción en Fase Sólida/métodos , Espectrometría de Masas en Tándem/instrumentaciónRESUMEN
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
AIM: To assess the translation of pharmacokinetic-pharmacodynamic (PK-PD) relationships for heart rate effects of PF-00821385 in dog and man. METHODS: Cardiovascular telemetric parameters and concentration data were available for animals receiving active doses (0.5-120 mg kg(-1), n= 4) or vehicle. PF-00821385 was administered to 24 volunteers and pharmacokinetic and vital signs data were collected. PK-PD models were fitted using nonlinear mixed effects. RESULTS: Compartmental models with linear absorption and clearance were used to describe pharmacokinetic disposition in animal and man. Diurnal variation in heart and pulse rate was best described with a single cosine function in both dog and man. Canine and human heart rate change were described by a linear model with free drug slope 1.76 bpm microM(-1)[95% confidence interval (CI) 1.17, 2.35] in the dog and 0.76 bpm microM(-1) (95% CI 0.54, 1.14) in man. CONCLUSIONS: The preclinical translational of concentration-response has been described and the potential for further interspecies extrapolation and optimization of clinical trial design is addressed.
Asunto(s)
Fármacos Anti-VIH/farmacología , Fármacos Anti-VIH/farmacocinética , Frecuencia Cardíaca/efectos de los fármacos , Piperazinas/farmacología , Piridinas/farmacología , Administración Oral , Adulto , Animales , Fármacos Anti-VIH/administración & dosificación , Estudios de Cohortes , Perros , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Infecciones por VIH/tratamiento farmacológico , Humanos , Modelos Lineales , Masculino , Tasa de Depuración Metabólica , Persona de Mediana Edad , Adulto JovenRESUMEN
Recombinant alpha interferon (IFN-alpha) is used in the treatment of hepatitis C virus (HCV)-infected patients but is not optimal in terms of efficacy or tolerability. Toll-like 7 receptor (TLR-7) agonists stimulate the innate immune system to produce, among other cytokines, IFN-alpha and are being evaluated as alternative drugs to treat HCV infection. This paper describes the application of pharmacokinetic-pharmacodynamic (PK-PD) modeling to understanding the behavior of a TLR-7 agonist [9-benzyl-8-hydroxy-2-(2-methoxyethoxy) adenine (BHMA)] in mice, using IFN-alpha as a biomarker. This is the first report of such a PK-PD model, and the conclusions may be of utility in the clinical development of TLR-7 agonists for HCV infection.
Asunto(s)
Adenina/análogos & derivados , Antivirales , Interferón-alfa/metabolismo , Receptor Toll-Like 7/agonistas , Adenina/administración & dosificación , Adenina/inmunología , Adenina/farmacocinética , Animales , Animales no Consanguíneos , Antivirales/administración & dosificación , Antivirales/inmunología , Antivirales/farmacocinética , Línea Celular Tumoral , Hepacivirus/efectos de los fármacos , Hepatitis C/inmunología , Hepatitis C/virología , Humanos , Ratones , Modelos AnimalesRESUMEN
The synthesis and structure-activity relationship of a series of novel gp120-CD4 inhibitors are described. Pharmacokinetic studies and antiviral spectrum assessment of lead compounds led to the identification of compound 36, a potent gp120-CD4 inhibitor which exhibited antiviral potency across a spectrum of 25 clade B isolates.