RESUMEN
Benzodiazepine drugs, through interaction with GABA(Aα1), GABA(Aα2,3), and GABA(Aα5) subunits, modulate cortical network oscillations, as reflected by a complex signature in the EEG power spectrum. Recent drug discovery efforts have developed GABA(Aα2,3)-subunit-selective partial modulators in an effort to dissociate the side effect liabilities from the efficacy imparted by benzodiazepines. Here, we evaluated rat EEG and behavioral end points during dosing of nine chemically distinct compounds that we confirmed statistically to selectively to enhance GABA(Aα2,3)-mediated vs. GABA(Aα1) or GABA(Aα5) currents in voltage clamped oocytes transfected with those GABA(A) subunits. These compounds were shown with in vivo receptor occupancy techniques to competitively displace [(3)H]flumazenil in multiple brain regions following peripheral administration at increasing doses. Over the same dose range, the compounds all produced dose-dependent EEG spectral power increases in the ß- and and γ-bands. Finally, the dose range that increased γ-power coincided with that eliciting punished over unpunished responding in a behavioral conflict model of anxiety, indicative of anxiolysis without sedation. EEG γ-band power increases showed a significant positive correlation to in vitro GABA(Aα2,3) modulatory intrinsic activity across the compound set, further supporting a hypothesis that this EEG signature was linked specifically to pharmacological modulation of GABA(Aα2,3) signaling. These findings encourage further evaluation of this EEG signature as a noninvasive clinical translational biomarker that could ultimately facilitate development of GABA(Aα2,3)-subtype-selective drugs for anxiety and potentially other indications.
Asunto(s)
Ansiolíticos/farmacología , Ritmo beta/efectos de los fármacos , Encéfalo/efectos de los fármacos , GABAérgicos/farmacología , Ritmo Gamma/efectos de los fármacos , Animales , Ansiolíticos/farmacocinética , Ansiedad/tratamiento farmacológico , Ansiedad/fisiopatología , Percepción Auditiva/efectos de los fármacos , Percepción Auditiva/fisiología , Ritmo beta/fisiología , Encéfalo/fisiopatología , Condicionamiento Operante/efectos de los fármacos , Condicionamiento Operante/fisiología , Conflicto Psicológico , Relación Dosis-Respuesta a Droga , Electrodos Implantados , Electroencefalografía , GABAérgicos/farmacocinética , Ritmo Gamma/fisiología , Modelos Lineales , Masculino , Técnicas de Placa-Clamp , Ratas Long-Evans , Ratas Sprague-Dawley , Receptores de GABA-A/metabolismoRESUMEN
Positive modulators at the benzodiazepine site of α2- and α3-containing GABA(A) receptors are believed to be anxiolytic. Through oocyte voltage clamp studies, we have discovered two series of compounds that are positive modulators at α2-/α3-containing GABA(A) receptors and that show no functional activity at α1-containing GABA(A) receptors. We report studies to improve this functional selectivity and ultimately deliver clinical candidates. The functional SAR of cinnolines and quinolines that are positive allosteric modulators of the α2- and α3-containing GABA(A) receptors, while simultaneously neutral antagonists at α1-containing GABA(A) receptors, is described. Such functionally selective modulators of GABA(A) receptors are expected to be useful in the treatment of anxiety and other psychiatric illnesses.
Asunto(s)
Receptores de GABA-A/química , Regulación Alostérica , Ansiolíticos/síntesis química , Ansiolíticos/química , Ansiolíticos/farmacología , Benzodiazepinas/química , Antagonistas de Receptores de GABA-A/síntesis química , Antagonistas de Receptores de GABA-A/química , Antagonistas de Receptores de GABA-A/farmacología , Compuestos Heterocíclicos con 2 Anillos/química , Quinolinas/química , Receptores de GABA-A/metabolismo , Relación Estructura-ActividadRESUMEN
Positive modulators at benzodiazepine sites of α2- and α3-containing GABA(A) receptors are believed to be anxiolytic. Negative allosteric modulators of α5-containing GABA(A) receptors enhance cognition. By oocyte two-electrode voltage clamp and subsequent structure-activity relationship studies, we discovered cinnoline and quinoline derivatives that were both positive modulators at α2-/α3-containing GABA(A) receptors and negative modulators at α5-containing GABA(A) receptors. In addition, these compounds showed no functional activity at α1-containing GABA(A) receptors. Such dual functional modulators of GABA(A) receptors might be useful for treating comorbidity of anxiety and cognitive impairments in neurological and psychiatric illnesses.
Asunto(s)
Receptores de GABA-A/química , Regulación Alostérica , Benzodiazepinas/química , Compuestos Heterocíclicos con 2 Anillos/síntesis química , Compuestos Heterocíclicos con 2 Anillos/química , Compuestos Heterocíclicos con 2 Anillos/farmacología , Técnicas de Placa-Clamp , Teoría Cuántica , Quinolinas/síntesis química , Quinolinas/química , Quinolinas/farmacología , Receptores de GABA-A/metabolismo , Relación Estructura-ActividadRESUMEN
INTRODUCTION: Gyrolab™ technology presents a technology breakthrough for large molecule bioanalysis to support biologic drug development. The advantages of this innovative platform include fully automated nanoscale immunoassay capability, better assay reproducibility and data quality, small reagent and sample volumes, and rapid assay development and validation as a result of reduced run time. Although Gyrolab has been increasingly used in method development in discovery environment, few fully validated Gyrolab assays have been reported. Here we report a method validation of a Gyrolab assay to determine rituximab levels in human serum. METHODS: Rituximab is captured on a Bioaffy™ CD by a biotinylated rat anti-idiotypic monoclonal antibody against rituximab and detected by an Alexa Fluor®-labeled anti-human IgG antibody. Assay conditions were optimized to give required sensitivity and dynamic range. The assay validation was conducted according to the current industry standards for GLP-regulated immunoassays. RESULTS: The intrabatch precision and accuracy for the assay were determined using spiked human serum samples and shown to have a coefficient of variation (CV) of <11% with a mean bias <20%. The interbatch precision (CV) and absolute mean bias were both <12% with the total error <25%. Adequate spike recovery was demonstrated in serum samples of healthy individuals and solid tumor patients. The dilutional linearity test showed that the determined concentrations adjusted with various dilution factors had a linear relationship with the expected concentrations and that there was no hook effect. The method has been validated for the quantification of rituximab in human serum from 90 to 60,000 ng/mL with a minimum required dilution of 30. DISCUSSION: The Gyrolab assay was proved to be accurate, precise and selective, with a comparable sensitivity to the ELISA method, but provided an automated nanoscale assay with a significantly wider assay dynamic range for the determination of rituximab in human serum during pharmacokinetics/toxicokinetics studies.