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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。
The inhibitory effects of regorafenib (REG) on 12 human uridine diphosphate glucuronosyltransferases (UGTs) were investigated using in vitro human liver microsomes and recombinant human metabolic enzyme incubation system. Intra-extravasation (IV-IVE) predicts the risk of REGs triggering drug-drug interactions (DDIs) with co-administration of UGT1A1 elimination drugs. Using 4-methylumbelliferone (4-MU) as a nonspecific probe substrate for UGTs, mixed human liver microsomes (HLMs) and recombinant human UGTs as enzyme source, N- (3-carboxypropyl) Specificity of UGT1A1 for 4-hydroxy-1,8-naphthalimide (NCHN) and N- (n-butyl) -4- (4- hydroxyphenyl) -1,8-naphthalimide (NPHN) Probe substrate, trifluoperazine (TFP) was used as a specific probe substrate for UGT1A4 to assess the inhibitory effect of REG on 12 human UGTs. Inhibitory kinetic constants (K_i) were determined by Lineweaver-Burk and Dixon, and the inhibition kinetic constants (K_i) were calculated by quadratic mapping method based on nonlinear regression and fitted curves. In vitro inhibition of kinetic parameters predicts the potential of REGs to inhibit DDT induced by UGT1A1. In vitro inhibition experiments showed that REG had a strong inhibitory effect on UGT1A1, UGT1A7, UGT1A9 and UGT2B7 with IC 50 of 0.15-6.6μmol·L -1 and K 0 of 0.027-14μmol·L -1, . REG competitively inhibited UGT1A1-catalyzed 4-MU-O-glucuronidation and UGT1A1-catalyzed NPHN-O-glucuronidation rather than competitive inhibition of UGT1A1-catalyzed NCHN-4-O- The uronic acidification reaction showed a mixed type of inhibition for 4-MU-O-glucuronidation catalyzed by UGT1A7, UGT1A9 and UGT2B7. The oral therapeutic dose of REG (160 mg · d -1) resulted in an increase of 101% -302% and 13% -109%, respectively, in the AUC of NPHN and NCHN, which are representative substrates of UGT1A1. The results suggest that when used in combination with UGT1A1-metabolized substrate drugs, REGs may be metabolically cleared in vivo by potent inhibition of UGT1A1 and require close attention during clinical use.