Receptor localization, native tissue binding and ex vivo occupancy for centrally penetrant P2X7 antagonists in the rat

BACKGROUND AND PURPOSE

The P2X7 receptor is implicated in inflammation and pain and is therefore a potential target for therapeutic intervention. Here, the development of a native tissue radioligand binding, localization and ex vivo occupancy assay for centrally penetrant P2X7 receptor antagonists is described.

EXPERIMENTAL APPROACH

Autoradiography studies using the P2X7 antagonist radioligand [³H]-A-804598 were carried out in rat brain and spinal cord. Subsequent in vitro binding and ex vivo occupancy assays were performed using rat cortex homogenate.

KEY RESULTS

P2X7 expression was shown to be widespread throughout the rat brain, and in the grey matter of the spinal cord. In binding assays in rat cortex homogenate, ∼60% specific binding was achieved at equilibrium. In kinetic binding assays, k_on and k_off values of 0.0021·min⁻¹·nM⁻¹ and 0.0070·min⁻¹ were determined, and the K_d derived from kinetic measurements was consistent with that derived from saturation analysis. Novel P2X7 antagonists inhibited the binding of [³H]-A-804598 to rat cortex P2X7 receptors with K_i values of <40 nM. In an ex vivo occupancy assay, a P2X7 antagonist dosed orally to rats caused a concentration-dependent inhibition of the specific binding of [³H]-A-804598 to rat cortex.

CONCLUSIONS AND IMPLICATIONS

The present study describes the development of an assay that allows localization of P2X7 receptors, the measurement of the binding affinity of P2X7 receptor antagonists in native tissue, and provides a means of determining central P2X7 receptor occupancy. These assays could form an important part of a P2X7 drug discovery programme.     

世界胃肠病学组织全球指南——发展中国家幽门螺杆菌感染

世界上有一半人口感染幽门螺杆菌（H．pylori）,其感染率因地理位置、种族、年龄和社会经济状况不同而存在很大差异．在发展中国家较高．发达国家较低。但总体而言,近年世界许多地区的H．pylori感染率均呈下降趋势。     

胎盘早剥产妇、新生儿血及脐血中组织因子和组织因子途径抑制物的变化

目的:检测胎盘早剥产妇血、新生儿血及脐血组织因子及组织因子途径抑制物蛋白的变化,以及这些变化对其凝血功能的影响。方法:收集2006-10/2007-04在中山大学附属第一医院产科的胎盘早剥产妇血(足月或早产不限)、分娩胎儿的脐带血及新生儿血标本各11例,4例为足月产,其余均为胎龄29～36周早产标本,男婴5份,女婴6份。正常对照15份来自同期广州市妇婴医院产妇及分娩胎儿。标本收集时间母亲血为分娩前24h内、脐血为分娩即刻、新生儿血为出生后2h内抽取,排除标准为产妇本次分娩前无血液系统疾病史,未使用过对凝血功能产生影响的药物,采用酶联免疫吸附方法测定血浆组织因子及组织因子途径抑制物抗原水平,并与正常分娩产妇对照组进行比较。病理及正常标本均经产妇及家属知情同意后获得。结果:①胎盘早剥产妇血、新生儿血及脐血血浆中组织因子测定值明显高于正常对照组(P<0.05)。②胎盘早剥产妇血、新生儿血浆及脐血中组织因子途径抑制物测定值较正常分娩产妇降低,差异具有显著性意义(P<0.01)。③胎盘早剥产妇血、新生儿和脐血组织因子途径抑制物/组织因子比值均较正常分娩产妇明显降低(P<0.01)。结论:胎盘早剥时产妇血、脐血及新生儿血促凝血活性增强,抗凝血活性降低,与其高凝状态相关。