外排转运蛋白对异莨菪亭体内处置的调控机制研究

Effect of efflux transporters on disposition of isoscopoletin in vivo

目的采用外排转运蛋白基因敲除小鼠模型,探讨乳腺癌耐药蛋白(breastcancerresistanceprotein,BCRP)和多药耐药相关蛋白2(multidrugresistance-associatedprotein2,MRP2)对异莨菪亭及其代谢物异莨菪亭-6-O-β-葡萄糖醛酸苷(isoscopoletin-6-O-β-glucuronide,I-6-G)体内处置的影响及调控机制。方法野生型FVB小鼠、Bcrp1^(-/-)小鼠、Mrp2^(-/-)小鼠和Bcrp1^(-/-)/Mrp2^(-/-)小鼠分别ig异莨菪亭(2 mg/kg)或尾iv异莨菪亭(1 mg/kg),采用超高效液相色谱与质谱联用(UHPLCMS/MS)法测定小鼠血浆中异莨菪亭及I-6-G的浓度,分析外排转运蛋白BCRP和MRP2对异莨菪亭及I-6-G药动学特征的影响;采用小鼠在体肠灌流模型研究BCRP和MRP2对异莨菪亭及I-6-G肠道处置的调控作用;采用小鼠肝微粒体和小肠微粒体考察异莨菪亭在野生型FVB小鼠、Bcrp1^(-/-)小鼠、Mrp2^(-/-)小鼠和Bcrp1^(-/-)/Mrp2^(-/-)小鼠体内的葡萄糖醛酸化代谢特征。结果异莨菪亭在野生型FVB小鼠、Bcrp1^(-/-)小鼠、Mrp2^(-/-)小鼠和Bcrp1^(-/-)/Mrp2^(-/-)小鼠体内的生物利用度分别为7.85%、3.54%、5.76%、10.27%。ig异莨菪亭后,与野生型FVB小鼠相比,Mrp2^(-/-)小鼠和Bcrp1^(-/-)/Mrp2^(-/-)小鼠体内异莨菪亭的药-时曲线下面积(AUC_(0-t))分别增加了2.8、5.6倍(P<0.01、0.001),Bcrp1^(-/-)小鼠、Mrp2^(-/-)小鼠和Bcrp1^(-/-)/Mrp2^(-/-)小鼠体内I-6-G的AUC_(0-t)分别增加了0.6、1.0、8.2倍(P<0.05、0.001)。尾iv异莨菪亭后,与野生型FVB小鼠相比,Mrp2^(-/-)小鼠和Bcrp1^(-/-)/Mrp2^(-/-)小鼠体内异莨菪亭的AUC_(0-t)分别增加了4.0、3.8倍(P<0.05、0.01),Bcrp1^(-/-)小鼠和Bcrp1^(-/-)/Mrp2^(-/-)小鼠体内I-6-G的AUC_(0-t)分别增加了2.1、9.1倍(P<0.01)。与野生型FVB小鼠相比,Bcrp1^(-/-)小鼠、Mrp2^(-/-)小鼠和Bcrp1^(-/-)/Mrp2^(-/-)小鼠体内I-6-G外排入十二指肠肠腔和胆汁中的量均减少了55.7%~90.1%(P<0.05、0.01)。外排转运蛋白BCRP和MRP2缺失后,对小鼠肝微粒体和小肠微粒体中异莨菪亭葡萄糖醛酸化代谢物I-6-G的清除率影响较小。结论外排转运蛋白BCRP和MRP2参与调控异莨菪亭及其代谢物I-6-G的体内处置。

Objective To investigate the effect of breast cancer resistance protein(BCRP) and multidrug resistance-associated protein 2(MRP2) on disposition of isoscopoletin and isoscopoletin-6-O-β-glucuronide(I-6-G) in vivo based on efflux transporter gene knockout mouse models. Methods Wild-type FVB, Bcrp1^(-/-), Mrp2^(-/-)and Bcrp1^(-/-)/Mrp2^(-/-)mice were ig isoscopidine(2 mg/kg) or tail iv isoscopidine(1 mg/kg), UHPLC-MS/MS method was used to quantify isoscopoletin and I-6-G in plasma of mice to evaluate the role of BCRP and MRP2 on pharmacokinetics of isoscopoletin and I-6-G. Single-pass intestinal perfusion model was established to investigate the role of BCRP and MRP2 on intestinal disposition of isoscopoletin and I-6-G. Enzyme kinetics of isoscopoletin glucuronidation by liver microsomes and small intestine microsomes were characterized in wild-type FVB, Bcrp1^(-/-), Mrp2^(-/-)and Bcrp1^(-/-)/Mrp2^(-/-)mice. Results Bioavailability of isoscopoletin in wild-type FVB, Bcrp1^(-/-), Mrp2^(-/-)and Bcrp1^(-/-)/Mrp2^(-/-)mice were respectively 7.85%, 3.54%, 5.76% and 10.27%. After ig isoscopoletin, AUC_(0-t) of isoscopoletin in Mrp2^(-/-)and Bcrp1^(-/-)/Mrp2^(-/-)mice were 2.8 and 5.6-fold higher than those of in wild-type FVB mice(P < 0.01, 0.001);AUC_(0-t) of I-6-G in Bcrp1^(-/-), Mrp2^(-/-)and Bcrp1^(-/-)/Mrp2^(-/-)mice were 0.6, 1.0 and 8.2-fold higher than those of in wild-type FVB mice(P < 0.05, 0.001). After iv isoscopoletin, AUC_(0-t) of isoscopoletin in Mrp2^(-/-)and Bcrp1^(-/-)/Mrp2^(-/-)mice showed 4.0 and 3.8-fold higher than those of in wild-type FVB mice(P < 0.05, 0.01);AUC_(0-t) of I-6-G in Bcrp1^(-/-)and Bcrp1^(-/-)/Mrp2^(-/-)mice showed 2.1 and 9.1-fold higher than those of in wild-type FVB mice(P < 0.01). Compared with wild-type FVB mice, amounts of I-6-G excreted in duodenum and bile in Bcrp1^(-/-), Mrp2^(-/-)and Bcrp1^(-/-)/Mrp2^(-/-)mice were significantly decreased by 55.7%—90.1%(P < 0.05, 0.01). The absence of efflux transporters BCRP and MRP2 had little effect on clearance rate of isoscopoltin glucuronide metabolite I-6-G in mouse liver microsomes and intestinal microsomes. Conclusion BCRP and MRP2 are involved in the disposition of isoscopoletin and I-6-G in mice.