新型脑保护剂H004及其活性葡萄糖醛酸代谢产物的脑转运代谢机制及药代-药效学研究

H004 is a novel neuroprotective candidate with our knowledge property right. H004 was found to exhibit more potent neuroprotective activity on experimental cerebral ischemia than positive drug edaravone with relatively poor toxicity. H004-7-O-glucuronide (H004G) was identified as the main metabolite of H004 in vivo. H004G exhibited similar neuroprotective activities to those of H004 in vitro. Systemic and brain exposure of H004G were 7.7- and 2.3-fold higher than that of H004，respectively. The efficacy of CNS agents is closely related to the transport / metabolism of drugs at the blood-brain barrier and drug distribution in brain. The transport / metabolism of H004 and H004G at the blood-brain barrier remain unclear. This project intends to use humanized recombined enzyme / transporter cell models and pathological models combined with microdialysis, micro-injection and LC-MS/MS technology to study the correlations between pharmacokinetics and pharmacodynamics of H004/H004G based on the cytokine CKLF1-CCR4 axis, to analyze the relationship between the ratio of H004/H004G in different brain regions / subcellular and neuroprotective effect, to explore the main effective material of H004 in vivo, as well as the possible neuroprotective target of H004/H004G, to discuss the transport and metabolism of H004/H004G at the blood-brain barrier. The results of above studies would provide essential experimental data for the development of novel neuroprotective agents. Meanwhile, the research work would also promote the research on active glucuronide conjugates.

H004是具有我国自主知识产权的新型脑保护剂，对实验性脑缺血的保护作用明显优于阳性药依达拉奉，且毒性较小。H004在体内主要生成葡萄糖醛酸苷（H004G）。H004G的体外活性与原药相当，其血浆和脑内暴露量分别是原药的7.7倍和2.3倍。已知中枢神经系统药物的起效时间、作用强度和持续时间与药物经血脑屏障的转运及代谢特点以及靶组织分布密切相关。目前H004/H004G的脑转运代谢及分子机制尚不明了。本项目拟采用人源化高表达酶/转运蛋白细胞及体内外病理模型，结合微透析/微注射和液质联用分析技术，以CKLF1-CCR4信号轴为靶点进行药代-药效学相关研究，分析H004/H004G在脑区/亚细胞的分布特点与药效的关系，阐明H004的体内有效形式及可能的作用靶点，探讨H004/H004G脑转运/代谢的分子机制，为开发新型脑保护剂提供重要依据，同时促进活性葡萄糖醛酸苷类化合物的深入研究。

新型脑保护剂H004作用机制新颖，脑保护作用确切，且毒性较小。H004在体内主要生成H004G，H004G的脑暴露量远高于H004。本项目采用人源化高表达酶/转运蛋白细胞及体内外病理模型，结合微透析/微注射和液质联用分析技术，对H004/H004G的脑转运/代谢分子机制及药代-药效学进行相关研究，结果表明H004G在体内和体外具与H004类似的抗脑缺血作用。H004G 在大鼠血浆和脑内的暴露量明显高于 H004。人脑胶质细胞（HEB）中的UGT1A7可以介导H004的葡萄糖醛酸化，而在神经元（SH-SY5Y）和脑微血管内皮细胞（hCMEC / D3）中未生成H004G。在大鼠侧脑室注射H004后，仅在下丘脑中检测到较低浓度的H004G。推测脑内H004可以转化为H004G，但脑内代谢不是脑中H004G的主要来源。在MDCK-MDR1细胞模型中，H004的膜渗透性是H004G的70倍，并且当P-gp活性受阻时，H004的定向转运消失了。在hCMEC / D3细胞模型中，吡喃敏感性H + / OC反转运蛋白抑制剂可显着抑制H004摄取，GLUT抑制剂可显著抑制H004G的摄取，OATP抑制剂和P-gp抑制剂可显着抑制H004的外排。因此，虽然IMM-H004的渗透性较高，这有助于药理作用的快速发挥，但是，由于P-gp和OATP的外排作用 H004在脑中的暴露量不高。虽然H004G渗透性较低，但由于脑内外存在较高的浓度梯度，以及GLUT的摄取作用，H004G在大脑中具有更高且更持久的暴露，有助于药理作用的维持。本研究阐明了H004 的体内有效形式，为开发新型脑保护剂提供了重要依据，同时促进了活性葡萄糖醛酸苷类化合物的深入研究。