RESUMEN
AIM: Ki67 is a prognostic and/or predictive biomarker in patients with malignancies. Flow cytometry is a powerful technology for single-cell multiparameter analysis. RESULTS: We developed and validated a multicolor quantitative flow cytometry assay for detection of intracellular Ki67 expression in various immune cell subsets from human blood. The assay was optimized and showed excellent precisions. Assessment of the sample stability indicated that percentage changes from the fresh sample for the reportable results of interest were within 20%, up to 72 h after blood collection in the Cyto-Chex® BCT tube. CONCLUSION: The validated assay is sufficiently robust to analyze clinical samples. Easy access to peripheral blood enables continuous monitoring of Ki67 expression in blood as a biomarker, for example, for immunotherapy studies.
Asunto(s)
Citometría de Flujo , Antígeno Ki-67/sangre , Adulto , Anticuerpos Monoclonales/inmunología , Biomarcadores/sangre , Humanos , Antígeno Ki-67/inmunología , Linfocitos/metabolismo , Neoplasias/diagnóstico , Estabilidad ProteicaRESUMEN
A series of pyrrolo-benzo-1,4-diazine analogs have been synthesized to improve the profile of the previous lead compound 1. The syntheses, structure-activity relationships, and selected pharmacokinetic data of these analogs are described. The optimization efforts allowed the identification of 33, a quinoline amide exhibiting potent Na(v)1.7 inhibitory activity and moderate selectivity over Na(v)1.5. Compound 33 displayed anti-nociceptive oral efficacy in a rat CFA inflammatory pain model at 100 mpk and in a rat spinal nerve ligation neuropathic pain model with an EC50 75 µM.
Asunto(s)
Analgésicos/farmacología , Ganglios Espinales/efectos de los fármacos , Canal de Sodio Activado por Voltaje NAV1.7/química , Neuralgia/tratamiento farmacológico , Bloqueadores de los Canales de Sodio/farmacología , Nervios Espinales/efectos de los fármacos , Compuestos de Espiro/farmacología , Analgésicos/química , Animales , Estructura Molecular , Técnicas de Placa-Clamp , Quinoxalinas/química , Ratas , Bloqueadores de los Canales de Sodio/química , Compuestos de Espiro/química , Relación Estructura-ActividadRESUMEN
A series of pyrrolo-benzo-1,4-diazine analogs have been synthesized and displayed potent Nav1.7 inhibitory activity and moderate selectivity over Nav1.5. The syntheses, structure-activity relationships, and selected pharmacokinetic data of these analogs are described. Compound 41 displayed anti-nociceptive efficacy in the rat CFA pain model at 100 mpk oral dosing.
Asunto(s)
Descubrimiento de Drogas , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Quinoxalinas/farmacología , Bloqueadores de los Canales de Sodio/farmacología , Compuestos de Espiro/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Quinoxalinas/síntesis química , Quinoxalinas/química , Bloqueadores de los Canales de Sodio/síntesis química , Bloqueadores de los Canales de Sodio/química , Compuestos de Espiro/síntesis química , Compuestos de Espiro/química , Relación Estructura-ActividadRESUMEN
In vitro screens using cellular preparations expressing human Ether-à-go-go related gene (hERG) potassium channels have become an intrinsic tool for evaluating cardiac liability of compounds during early preclinical stage development. Although hERG channel blocking effects are most reliably evaluated using the low-throughput, manual patch clamp technique, methods and technologies that yield hERG activity data in multiwell format are required to address increased throughput requirements. In most cases, multiwell approaches to measuring hERG activity involve achieving a reasonable balance between throughput and data fidelity. Here we compared two functional multiwell hERG assays: a fluorescence-based fluorometric imaging plate reader (FLIPR(®)) screen measuring thallium (Tl(+)) influx through hERG channels and an automated patch clamp assay using an IonWorks Quattro(®). Mean Z' values for FLIPR-Tl(+) and IonWorks Quattro assays were similar, 0.57 ± 0.09 (±SD; n = 10) versus 0.63 ± 0.11 (n = 12), respectively. IC50 determinations for a set of 17 reference compounds were used to evaluate potency shifts relative to conventional voltage clamp data. The reference compound set included members that are known to exert severe potency shifts in multiwell assays. Mean potency shift values for FLIPR-Tl(+) and IonWorks Quattro assays were 117- and 8-fold, respectively. On the basis of reduced potency shifts and low data variability, we conclude that the IonWorks Quattro screen was a better predictor of hERG activity in conventional whole-cell patch clamp than the Tl(+) influx assay.
Asunto(s)
Evaluación Preclínica de Medicamentos , Canales de Potasio Éter-A-Go-Go/antagonistas & inhibidores , Bloqueadores de los Canales de Potasio/farmacología , Talio/metabolismo , Células Cultivadas , Descubrimiento de Drogas , Canales de Potasio Éter-A-Go-Go/fisiología , Células HEK293 , Ensayos Analíticos de Alto Rendimiento , Humanos , Mediciones Luminiscentes , Potenciales de la Membrana/efectos de los fármacos , Técnicas de Placa-ClampRESUMEN
A series of spiro-azetidines and azetidinones has been evaluated as novel blockers of the T-type calcium channel (Ca(V)3.2) which is a new therapeutic target for the potential treatment of both inflammatory and neuropathic pain. Confirmation and optimization of the potency, selectivity and DMPK properties of leads will be described.
Asunto(s)
Azetidinas/química , Bloqueadores de los Canales de Calcio/síntesis química , Canales de Calcio Tipo T/química , Piperidinas/química , Compuestos de Espiro/química , Animales , Azetidinas/síntesis química , Azetidinas/uso terapéutico , Bloqueadores de los Canales de Calcio/química , Bloqueadores de los Canales de Calcio/uso terapéutico , Canales de Calcio Tipo T/metabolismo , Diseño de Fármacos , Humanos , Dolor/tratamiento farmacológico , Unión Proteica , Ratas , Relación Estructura-ActividadRESUMEN
T-type calcium channel antagonists were designed using a protocol involving the program SPROUT and constrained by a ComFA-based pharmacophore model. Scaffolds generated by SPROUT were evaluated based on their ability to be translated into structures that were synthetically tractable. From this exercise, a novel series of potent and selective T-type channel antagonists containing a biaryl sulfonamide core were discovered.
Asunto(s)
Bloqueadores de los Canales de Calcio/química , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo T/efectos de los fármacos , Sulfonamidas/química , Sulfonamidas/farmacología , Animales , Cristalografía por Rayos X , Diseño de Fármacos , Técnicas In Vitro , Técnicas de Placa-Clamp , Relación Estructura-ActividadRESUMEN
Contractility studies in isolated feline myocytes have demonstrated that sphingosine, a metabolite stimulated by tumor necrosis factor (TNF) binding, decreases intracellular calcium release and depresses inotropic activity. This study investigated the electrophysiologic effects of sphingosine in isolated cat myocytes as well as the cardiodynamic consequence of TNF, sphingosine, and its metabolic precursors in vivo. In cat myocytes, sphingosine markedly decreased action potential duration, lowered action potential plateau, and inhibited L-type calcium current (I(Ca-L)). After administration of TNF, sphingomyelin, C2-ceramide, or sphingosine, only C2-ceramide and sphingosine depressed cardiac function in normal rats. Negative inotropic effects of C2-ceramide were attenuated by N-oleoylethanolamine (NOE), a ceramidase inhibitor that blocks sphingosine formation. Rats pretreated with NOE before undergoing 30 min of acute regional myocardial ischemia followed by 150 min of reperfusion exhibited improved survival. Most deaths could be attributed to acute pump failure accompanied by bradycardia. Myocardial infarct size and peak serum TNF were not different between NOE- and vehicle-treated groups (3,908 +/- 1097 pg/ml and 3,027 +/- 846 pg/ml, respectively). These results indicate that sphingosine exerts direct inhibitory effects on the action potential and I(Ca-L) in isolated feline myocytes, consistent with previously reported sphingosine activity on I(Ca-L) in isolated rat myocytes. The in vivo study suggests that reducing sphingosine production with N-oleoylethanolamine attenuates cardiodepression and can improve overall survival after ischemic injury. Clearly, agents that modulate sphingosine production limit cardiodepression and may provide a therapeutic benefit in clinical conditions of myocardial inflammatory injury.