金松双黄酮对尿苷二磷酸葡糖醛酸转移酶的抑制作用

Inhibition of sciadopitysin against UDP-glucuronosyltransferases

借助体外代谢孵育体系考察了金松双黄酮(sciadopitysin,SP)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)预测体内药物-药物相互作用(DDI)的风险。以混合人肝微粒体(HLM)及重组表达的人UGTs作为酶源,选用4-甲基伞形酮(4-MU)、三氟拉嗪(TFP)、N-3-羧丙基-4-羟基-1,8-萘酰亚胺(NCHN)分别为UGTs酶的广谱探针底物、UGT1A4和UGT1A1的特异性探针底物,评估金松双黄酮对12种人UGT酶的抑制作用。通过非线性拟合求得半数最大抑制浓度IC50和抑制动力学常数Ki及抑制类型;并基于体外参数预测了金松双黄酮通过抑制UGT1A1引发的潜在DDI风险。体外抑制实验表明,金松双黄酮对UGT1A1、UGT1A3、UGT1A8和UGT1A10有较强的抑制作用,当终浓度为10μmol·L^(-1)时,UGT1A1、UGT1A3、UGT1A8和UGT1A10的残余活性均小于30%。金松双黄酮对UGT1A1、UGT1A3、UGT1A8和UGT1A10的半数最大抑制浓度IC50为0.20~1.34μmol·L^(-1),抑制动力学常数Ki为0.07~2.12μmol·L^(-1)。口服金松双黄酮240 mg·d^(-1)可导致UGT1A1底物的AUC增加19%~147%。金松双黄酮可竞争性的抑制UGT1A1、UGT1A3、UGT1A8和UGT1A10催化的4-MU-O-葡糖醛酸化反应及UGT1A1催化的NCHN-O-葡糖醛酸化反应。金松双黄酮对UGT1A1的强抑制作用有可能减缓UGT1A1底物的代谢,进而引发DDI风险。上述研究提示金松双黄酮与临床药物联合应用时,应该特别注意其通过抑制UGT酶而引发的DDI。

This study was designed to investigate the inhibitory effects of sciadopitysin on the catalytic activities of human 12 kinds of UDP-glucuronosyltransferases（UGTs） in vitro.The risk of drug-drug interactions（DDI） is predicted by in vitro-in vivo extrapolation（IV-IVE）.Methods A panel of recombinant human UGT isoforms and human liver microsome（HLM） as well as a series substrates including 4-methyl umbelliferone（4-MU）,trifluoperazine（TFP） and N-3-carboxypropyl-4-hydroxy-1,8-naphthalimide（NCHN）（UGT1A1 specific fluorescent probe substrates） were used to characterize the inhibitory effects of sciadopitysin on human UGTs in vitro.The half maximum inhibitory concentration（IC50） and the constant of inhibition kinetics（Ki） were obtained by nonlinear regression using Graph Pad Prism 6.0 software.The potential risk of DDI induced by UGT1A1 was predicted based on in vitro parameters.The results demonstrated that sciadopitysin had strong inhibitory effects on UGT1A1,UGT1A3,UGT1A8 and UGT1A10,with the remaining activity being below 30% at a final concentration of 10 μmol·L^-1.For UGT1A1,UGT1A3,UGT1A8 and UGT1A10,the IC50 was 0.20 μmol·L^-1 to 1.34 μmol·L^-1,the inhibition kinetic constant Ki was 0.07 μmol·L^-1to 2.12 μmol·L^-1.The AUC ratio of UGT1A1 can be increased by 19% to 147% at the oral dose of 240 mg·d^-1The sciadopitysin competitively inhibited the formation of 4-MU-O-glucuronide by UGT1A1,UGT1A3,UGT1A8,and UGT1A10.At the same time,the inhibition of NCHN-O-glucuronidation by UGT1A1 was consistent with the competitive inhibition.The strong inhibition of sciadopitysin on UGT1A1 led to reduction of the metabolism of UGT1A1 substrates,and increased the risk of DDI.When co-administrated with other drugs,special attentions should be given to the DDI from inhibition of drug metabolism enzymes to prevent serious clinical consequences.