生物钟基因Per1在芬太尼时辰药动学中的调控作用研究

The efficacy of fentanyl and other analgesic drugs displays significant circadian rhythm. Further research on the chronopharmacology of fentanyl is helpful to optimize the clinical treatment, thereby improving the therapeutic effects. Our previous study showed that the chronopharmacokinetic characteristics of fentanyl are consistent with its chronopharmacodynamic characteristics. However, the internal mechanism of circadian variation of fentanyl process in vivo remains unclear. Fentanyl is mainly metabolized by CYP3A4, and the expression of CYP3A4 also shows circadian rhythm. Our preliminary results demonstrated that the circadian expression of CYP3A4 is consistent with that of clock gene Per1. Furthermore, foreign researches suggest that Per1 plays an important role in analgesia. Therefore, we propose that Per1 can regulate the circadian expression of CYP3A4, thus determining the chronokinetics characteristic of fentanyl. By cell experiment and Per1 knockout animal study, this project intends to validate the regulation of CYP3A4 rhythmic expression on fentanyl in vivo, and on this basis, to study the effect of Per1 on the chronopharmacokinetics of fentanyl through regulating the circadian expression of CYP3A4. Moreover, we plans to investigate the molecular mechanism of the circadian expression of CYP3A4 regulated by Per1 based on nuclear receptors. These results could not only provide the theory foundation for the rational use of fentanyl, but also enriches the chronopharmacology study.

芬太尼等镇痛药物的疗效具有显著的昼夜节律性，对其时辰药理学的深入研究有助于优化临床治疗方案进而提高治疗效果。我们前期研究发现芬太尼的时辰药动学特征与其时辰药效学特征吻合，而其体内过程存在昼夜差异的内在机制尚不清楚。芬太尼主要经CYP3A4代谢，CYP3A4表达也具有昼夜节律性。我们的预实验结果显示钟基因Per1与CYP3A4的昼夜节律特征一致，同时，国外研究提示钟基因Per1在镇痛中发挥重要作用。因此我们提出假说认为Per1可调控CYP3A4的节律表达，从而最终决定芬太尼的时辰药动学特征。本项目采用体外实验和Per1敲除动物实验，在验证CYP3A4节律表达对芬太尼体内过程的调控作用基础上，研究Per1对于CYP3A4的调控进而影响芬太尼时辰药动学的作用，并基于核受体来探讨Per1调控CYP3A4节律的分子机制。研究结果不仅可以为芬太尼的合理用药提供理论依据，也丰富了时辰药理学的研究内容。

芬太尼是人工合成的强效麻醉性镇痛药，其主要在肝脏经CYP3A4代谢为无活性的去甲基芬太尼。研究表明芬太尼的镇痛效果呈现明显的昼夜节律，我们前期实验显示芬太尼时辰药动学特征与其时辰药效学保持一致，但芬太尼体内过程昼夜节律变化的内在机制目前尚未阐明。生物钟Per1在调控药物代谢和镇痛方面起着非常重要作用，其是否可以通过调控芬太尼体内过程的节律变化进而影响其药动学目前还未见报道。本研究首先利用体外肝细胞系，初步测定CYP3A4和生物钟基因Per1的昼夜节律表达特征，结果显示：CYP3A4的节律表达与Per1的表达保持一致，即CYP3A4和Per1的表达在21:00明显高于9:00。接着体内研究以CYP3A4特异性抑制剂处理作对照组，在不同时间点给予大鼠芬太尼后，测定其体内药动学变化，同时测定CYP3A4的节律表达。结果表明，LC-MS/MS测定方法特异性、灵敏度、精密度、准确度和稳定性均良好，能有效测定不同时间血浆芬太尼的药动学变化；正常组小鼠在9:00和21:00给予芬太尼后体内药动学呈现明显节律变化，其血药浓度在9:00明显高于21:00，而给予维拉帕米预处理后，在不同时间给予芬太尼后体内药动学并无明显的节律差异；同时正常组大鼠肝脏中CYP3A4表达呈现明显节律变化，其在21:00的表达明显高于9:00，而维拉帕米组大鼠CYP3A4表达明显偏低，且无明显节律变化。进一步利用Per1敲除大鼠模型，考察不同时间给予大鼠芬太尼后其药动学变化，测定Per1及CYP3A4的节律表达。初步结果显示：与正常组相比，在Per1敲除大鼠中，芬太尼在9:00和21:00给药后体内药动学过程无明显变化，且CYP3A4表达在9:00和21:00并未显示明显的节律变化。基于此，我们分析芬太尼体内过程的节律变化与CYP3A4的节律表达密切相关，生物钟基因Per1可通过调控CYP3A4的节律表达进而影响芬太尼的时辰药动学，但Per1是如何调控CYP3A4的表达还有待进一步研究。