P-gp的分子影像探针制备及其在难治性癫痫耐药机制中作用的研究

About 30% patients with epilepsy will eventually develop refractory epilepsy because they lose the sensitivity to antiepileptic drugs (AEDs). However, the drug resistance mechanism of refractory epilepsy has not been clarified so far. Recent studies have shown that multidrug transporter protein was closely related with the drug resistance mechanism, and P-glycoprotein (P-gp)was overexpressed at the blood-brain barrier in patients with refractory epilepsy. It is believed that this P-gp overexperession plays an important role in the development of drug resistance in refractory epilepsy, in which antiepileptic drugs will be effluxed from the site of action through an active energy-dependent pump transportation process,which leads to the drug resistance effect. In our study, an in vivo 11C-Verapamil(VPM) Positron Emission Tomography (PET) study will be performed using rat models of refractory epilepsy before and after administration of the third generation P-gp inhibitor, tariquidar (TQD). In addition,the experiment on expression of P-gp in brain tissues will be performed using immunohistochemistry (IHC) tehcniques. Furthermore,the tracer distribution in different regions of brain tissues, influx rate constant (K1), the efflux rate constant (K2) and their coorelation to P-gp expression level will be estimated .A study of PET imaging on patients with refractory epilepsy will be performed based on the results obtained from the previous proposed steps. By monitoring the change of P-gp functional level in the PET imaging before and after administration of P-gp inhibitor, the accuracy of therapeutic efficiency will be evaluated and a clinic methodology will be established. Eventually, the role of P-gp overexperession on the drug resistance mechanism will be explored. It is believed that a theoretical and practical foundation will be established for drug efficacy prediction and therapeutic target exploration in refractory epilepsy.

30%左右的癫痫患者对抗癫痫药物不敏感而发展为难治性癫痫，但对于难治性癫痫的耐药机制迄今尚未完全阐明。研究表明，多药转运蛋白与耐药机制密切相关，P-糖蛋白（P-gp）在其中起着重要作用，且P-gp在血脑屏障中过度表达；我们据此推测，在难治性癫痫中，P-gp通过活跃的泵运机制将抗癫痫药物转运出作用靶位，从而导致了耐药性产生。本研究拟于难治性癫痫活体大鼠模型上，使用第三代P-gp抑制剂tariquidar前后分别行11C-Verapamil的micro PET显像，结合免疫组化技术，分析示踪剂在脑组织中分布的区域性差异、流入速率常数（K1）及解离速率常数（K2）与P-gp功能表达的相关性；在此基础上，对难治性癫痫患者进行P-gp的PET显像研究，以期初步建立预测难治性癫痫药物疗效的临床方法；进一步阐明P-gp过度表达在难治性癫痫耐药机制产生中的作用，并为治疗靶点的研究提供依据。

P-糖蛋白（P-gp）对维持机体微环境的稳态特别是脑内微环境具有重要作用，难治性癫痫及肿瘤等疾病耐药性的产生已证实与血脑屏障及肿瘤屏障上P-gp的功能及表达的改变相关。设计能显示体内P-gp功能及表达差异的探针对相关疾病的精准诊疗具有重要意义，相关二氢吡啶类（DHPs）药物是一类重要的多药耐药逆转剂，[18F]标记的DHPs有望成为一种新型的显示P-gp功能及表达的PET示踪剂本研究首先基于Hantzsch反应探索[18F]-DHPs的最佳标记条件标记，然后合成一系列的1,4-二氢吡啶类衍生物，并对其逆转P-gp引起的多药耐药性的性能进行评价，筛选出逆转效果最好的化合物作为P-gp探针，进行放射标记及PET-CT显像研究。我们成功合成了15种新型的含氟离子的1,4-二氢吡啶类衍生物，经相关实验筛，化合物2的逆转耐药功能及与P-gp的功能相关性最佳。基于Hantzsch反应的DHPs放射性化学标记中，第一步[18F]标记对氟苯甲醛的产率﹥70%，基于Hantzsch反应的第二步标记法，以乙酸铵作为氮源，在90℃下反应20min放射标记产率最佳。[18F]2在体外稳定性好，注射P-gp抑制剂后小脑内[18F]2探针摄取的浓度较对照增加了3-5倍。放射标记结果表明，基于Hantzsch反应的DHPs合成及标记方法是可行的，可用于DPHs的[18F]标记。通过常规的二氢吡啶类衍生物的合成方法，能成功合成含氟离子的1,4-二氢吡啶类衍生物，A1B1的逆转耐药功能及与P-gp的功能相关性最佳。以乙酸铵作供氮体相比于氨水，其放射化学产率高，反应时间明显缩短。[18F]2的动物显像结果达到了预期的效果，能较好的显示血脑屏障上P-gp的功能及表达。