瑞格非尼对人尿苷二磷酸葡糖醛酸转移酶活性抑制作用的体外研究

Inhibition of regorafenib against UDP-glucuronosyltransferases

应用体外人肝微粒体及重组人源代谢酶孵育体系考察了瑞格非尼(regorafenib,REG)对12种人尿苷二磷酸葡糖醛酸转移酶(UGTs)的抑制作用,通过体外-体内外推(IV-IVE)预测REG与经过UGT1A1代谢消除药物共服引发药物-药物相互作用(DDI)的风险。以混合人肝微粒体(HLM)及重组人源UGTs作为酶源,4-甲基伞形酮(4-MU)作为UGTs的非特异性探针底物,N-(3-羧丙基)-4-羟基-1,8-萘酰亚胺(NCHN)和N-(正丁基)-4-(4-羟苯基)-1,8-萘酰亚胺(NPHN)作为UGT1A1的特异性探针底物,三氟拉嗪(TFP)作为UGT1A4的特异性探针底物,评估REG对12种人UGTs的抑制作用。通过非线性回归并拟合曲线求得半数最大抑制浓度(IC_(50)),Lineweaver-Burk和Dixon作图法确定抑制类型,二次作图法求得抑制动力学常数(K_i),并基于体外抑制动力学参数预测了REG抑制UGT1A1所引发DDI的潜在可能性。体外抑制实验表明,REG对UGT1A1、UGT1A7、UGT1A9和UGT2B7具有较强的抑制作用,IC_(50)为0.15~6.6μmol·L^(-1),K_i为0.027~14μmol·L^(-1)。REG可竞争性的抑制UGT1A1催化的4-MU-O-葡糖醛酸化反应及UGT1A1催化的NPHN-O-葡糖醛酸化反应,而非竞争性的抑制UGT1A1催化的NCHN-4-O-葡糖醛酸化反应,对UGT1A7、UGT1A9和UGT2B7催化的4-MU-O-葡糖醛酸化反应呈现混合型的抑制类型。口服治疗剂量的REG(160 mg·d^(-1))可导致UGT1A1代表性底物NPHN和NCHN的AUC分别增加101%~302%和13%~109%。结果提示,REG与主要经UGT1A1代谢的底物药物联合应用时,可通过强效抑制UGT1A1进而影响其在机体内的代谢清除,在临床使用过程中需要密切关注。

This study was designed to investigate the inhibitory effects of regorafenib（REG）on the catalytic activities of 12 kinds of human UGT isoforms and human liver microsomes（HLM）in vitro.The broader potential of REG to perpetrate drug-drug interactions（DDI）arising from UGT enzyme inhibition is predicted by in vitro-vivo extrapolation（IV-IVE）.Fifty mixed HLM and 12 kinds of recombinant UGTs were utilized as enzyme sources to evaluation the inhibitory effects of REG against UGTs.4-Methylumbelliferone（4-MU）as a nonselective substrate of UGTs except for UGT1A4,N-（3-carboxypropyl）-4-hydroxy-1,8-napht-halimide（NCHN）and N-butyl-4-（4-hydroxyphenyl）-1,8-naphthalimide（NPHN）as the specific fluorescent substrate of UGT1A1,and trifluoperazine（TFP）as the specific substrate of UGT1A4.The half maximal inhibitory concentration（IC_（50））was calculated via the nonlinear regression analysis using Graphpad Prism 6.0,the inhibition kinetic types were selected and evaluated based on the intersection location of Lineweaver-Burk plot and Dixon plot,and K_i values were determined by the second plot of slopes.The potential DDI risk based on UGT1A1 inhibition was also evaluated through the in vitro parameters.The results demonstrated that REG displayed strong inhibitory effects against UGT1A1,1 A7,1A9,and 2B7.The IC_（50） values were from 0.15 to 6.6 μmol·L^（-1） and K_i values from 0.027 to 14 μmol·L^（-1）.The REG exerted competitive inhibition against UGT1A1-mediated 4-MU-O-glucuronidation and UGT1A1-mediated NPHN-O-glucuronidation,while the inhibition of NCHN-4-O-glucuronide by REG was suited to noncompetitive inhibition in both HLM and recombinant UGT1A1.Likewise,REG exhibited a mixed efficacy in inhibition of UGT1A7-,UGT1A9-,and UGT2B7-catalyzed 4-MU-O-glucuronidation.The AUC ratio of UGT1A1 specific substrates NPHN and NCHN can be increased by 101% to 302% and 13% to 109%,respectively.These results suggest that much caution should be exercised when REG is co-administered with UGT1A1 substrates.