Receptor localization, native tissue binding and ex vivo occupancy for centrally penetrant P2X7 antagonists in the rat
Receptor localization, native tissue binding and ex vivo occupancy for centrally penetrant P2X7 antagonists in the rat
复制标题
DOI：
10.1111/j.1476-5381.2010.01025.x
复制
发表时间：
2011-01-01
影响因子：
7.3
通讯作者：
Katugampola, S. D.
中科院分区：
文献类型：
--
作者：
Able, S. L.;Fish, R. L.;Katugampola, S. D.
P2 X7受体与炎症和疼痛有关，因此是治疗干预的潜在靶点。本文介绍了中枢渗透性P2 X7受体拮抗剂的天然组织放射性配体结合、定位和离体占有分析的发展。实验方法用P2 X7拮抗剂放射性配体[3 H]-A-804598在大鼠脑和脊髓中进行放射自显影研究。使用大鼠皮层匀浆进行随后的体外结合和离体占有测定。在大鼠皮质匀浆中的结合试验中，在平衡时实现了类似于60%的特异性结合。在动力学结合试验中，确定了0.0021中心点min-1中心点nM-1和0.0070中心点min-1的k（on）和k（off）值，并且从动力学测量得到的Kd与从饱和分析得到的Kd一致。新型P2 X7拮抗剂抑制[3 H]-A-804598与大鼠皮质P2 X7受体的结合，Ki值< 40 nM。在离体占有分析中，大鼠口服P2 X7拮抗剂引起[3 H]-A-804598与大鼠皮层特异性结合的浓度依赖性抑制。结论和意义本研究描述了一种允许P2 X7受体定位的分析的发展，P2 X7受体拮抗剂在天然组织中的结合亲和力的测量，并提供了测定中心P2 X7受体占有率的方法。这些分析可以成为P2 X7药物发现计划的重要组成部分。
BACKGROUND AND PURPOSEThe 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 APPROACHAutoradiography 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 RESULTSP2X7 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, similar to 60% specific binding was achieved at equilibrium. In kinetic binding assays, k(on) and k(off) values of 0.0021 center dot min-1 center dot nM-1 and 0.0070 center dot 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 IMPLICATIONSThe 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.