RESUMO
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.
Assuntos
Citometria de Fluxo , Antígeno Ki-67/sangue , Adulto , Anticorpos Monoclonais/imunologia , Biomarcadores/sangue , Humanos , Antígeno Ki-67/imunologia , Linfócitos/metabolismo , Neoplasias/diagnóstico , Estabilidade ProteicaRESUMO
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.
Assuntos
Analgésicos/farmacologia , Gânglios Espinais/efeitos dos fármacos , Canal de Sódio Disparado por Voltagem NAV1.7/química , Neuralgia/tratamento farmacológico , Bloqueadores dos Canais de Sódio/farmacologia , Nervos Espinhais/efeitos dos fármacos , Compostos de Espiro/farmacologia , Analgésicos/química , Animais , Estrutura Molecular , Técnicas de Patch-Clamp , Quinoxalinas/química , Ratos , Bloqueadores dos Canais de Sódio/química , Compostos de Espiro/química , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Descoberta de Drogas , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Quinoxalinas/farmacologia , Bloqueadores dos Canais de Sódio/farmacologia , Compostos de Espiro/farmacologia , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Quinoxalinas/síntese química , Quinoxalinas/química , Bloqueadores dos Canais de Sódio/síntese química , Bloqueadores dos Canais de Sódio/química , Compostos de Espiro/síntese química , Compostos de Espiro/química , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Avaliação Pré-Clínica de Medicamentos , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Bloqueadores dos Canais de Potássio/farmacologia , Tálio/metabolismo , Células Cultivadas , Descoberta de Drogas , Canais de Potássio Éter-A-Go-Go/fisiologia , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Medições Luminescentes , Potenciais da Membrana/efeitos dos fármacos , Técnicas de Patch-ClampRESUMO
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.
Assuntos
Azetidinas/química , Bloqueadores dos Canais de Cálcio/síntese química , Canais de Cálcio Tipo T/química , Piperidinas/química , Compostos de Espiro/química , Animais , Azetidinas/síntese química , Azetidinas/uso terapêutico , Bloqueadores dos Canais de Cálcio/química , Bloqueadores dos Canais de Cálcio/uso terapêutico , Canais de Cálcio Tipo T/metabolismo , Desenho de Fármacos , Humanos , Dor/tratamento farmacológico , Ligação Proteica , Ratos , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Bloqueadores dos Canais de Cálcio/química , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo T/efeitos dos fármacos , Sulfonamidas/química , Sulfonamidas/farmacologia , Animais , Cristalografia por Raios X , Desenho de Fármacos , Técnicas In Vitro , Técnicas de Patch-Clamp , Relação Estrutura-AtividadeRESUMO
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.