目的 从药物转运体角度探讨小白菊内酯衍生物ACT001的跨膜转运机制，并阐明ACT001可能产生的耐药性机制。方法 采用人药物转运体高表达单克隆细胞株/囊泡、人结肠腺癌细胞（Caco-2、LS-180），使用qRT-PCR、Western blotting、LC-MS/MS放射性同位素示踪等技术手段，共同研究ACT001的跨膜转运机制和耐药性。结果 小白菊内酯衍生物ACT001能够抑制外排转运体BCRP的转运活性，其半数抑制浓度（half inhibitory concentration，IC₅₀）为48.6μmol/L。ACT001在Caco-2细胞单层上的外排率为2.95，添加乳腺癌耐药蛋白（breast cancer resistance protein，BCRP）抑制剂Ko143可使外排率降至0.807；添加P糖蛋白（P-glycoprotein，P-gp）抑制剂盐酸维拉帕米对ACT001的外排率没有影响。在LS-180细胞中添加10 μmol/L的ACT001诱导72 h，可使P-gp和BCRP的mRNA表达水平分别提高8.89、8.21倍，蛋白相对表达量分别提高3.76、2.92倍，表明ACT001能诱导BCRP和P-gp的表达。结论 小白菊内酯衍生物ACT001是外排转运体BCRP的底物，BCRP在血脑屏障和肿瘤细胞中高表达，ACT001到达血脑屏障时，会被BCRP识别外排，导致跨血脑屏障能力下降，不能在靶点位置达到有效的血药浓度从而药效降低。ACT001是BCRP和P-gp的诱导剂，能上调LS-180细胞中2种转运体的mRNA和蛋白表达水平；BCRP和P-gp在血脑屏障和肿瘤组织高表达，对2种蛋白的诱导作用会进一步导致药效下降并产生耐药性，使治疗失败。另外，BCRP和P-gp在体内分布广泛，当ACT001与2种蛋白的抑制剂或底物联用时，由于竞争和诱导作用，会对其他药物的转运产生影响，发生药物-药物相互作用。

Objective To explore the transmembrane transport mechanism of parthenolide derivative ACT001 from perspective of drug transporters and clarify the possible drug resistance mechanism of it. Methods In vitro human transporter-transfected cell models/vesicles and Caco-2, LS-180 cells, combined with qRT-PCR, Western blotting, LC-MS/MS and radioisotope tracing techniques were used to study the transmembrane transport mechanism and drug resistance of ACT001. Results Parthenolide derivative ACT001 could inhibit the transport activity of efflux transporter breast cancer resistance protein (BCRP) with half inhibitory concentration (IC₅₀) of 48.6 μmol/L. The efflux rate of ACT001 on monolayer of Caco-2 cells was 2.95, and was reduced to 0.807 by addition of BCRP inhibitor Ko143. P-glycoprotein (P-gp) inhibitor verapamil hydrochloride had no effect on efflux rate. In LS-180 cells, 10 μmol/L ACT001 increased mRNA expression levels of P-gp and BCRP by 8.89-fold and 8.21-fold after induction for 72 h, respectively. Under the same conditions, protein amount of two transporters was increased by 3.76-fold and 2.92-fold, respectively, indicating ACT001 could induce the expressions of BCRP and P-gp. Conclusion Parthenolide derivative ACT001 is a substrate of BCRP. BCRP is highly expressed in blood-brain barrier and tumor cells. When ACT001 reaches the blood-brain barrier, it will be recognized by BCRP for efflux, resulting in a decrease in ability to cross the blood-brain barrier. The effective blood drug concentration cannot be reached at the target site and drug effect is reduced. ACT001 is an inducer of BCRP and P-gp, which can up-regulate mRNA and protein expression levels of two transporters in LS-180 cells; BCRP and P-gp are highly expressed in blood brain barrier and tumor tissues, and induction of two proteins will further lead to the decline of drug efficacy and development of drug resistance, making the treatment fail. In addition, BCRP and P-gp are widely distributed. When ACT001 is used in combination with inhibitors or substrates of the two proteins, it will affect the transport of other drugs and cause drug-drug interactions due to competition and induction.

R285.62

中国医学科学院医学与健康科技创新工程项目（2019-I2M-5-020）；天津市“项目＋团队”重点培养专项（创新类）（XC202030）