目的 通过大鼠原代肝细胞模型，明确小檗碱、巴马汀和药根碱的肝细胞摄取特征。方法 Seglen两步法分离大鼠原代肝细胞并悬浮培养，应用已知底物瑞舒伐他汀评价转运体的功能，将药物在4和37 ℃分别与肝细胞孵育不同时间，UPLC-MS/MS测定肝细胞摄取量，考察温度和时间对药物摄取的影响，并计算转运参数。通过浓度依赖的摄取实验，计算得到Km、Vmax和主动摄取率等参数。在肝细胞模型上，将转运体的特异抑制剂与小檗碱、巴马汀和药根碱共孵育，观察阳性抑制剂对受试药物转运的影响。结果 小檗碱、巴马汀和药根碱在37 ℃条件下的摄取量随着底物浓度的增高而增加，4 ℃条件下摄取量较低且变化很小，3种生物碱进入肝细胞的CLactive/CLuptake分别为85.26%、74.90%和57.74%。阳性抑制剂布洛芬、地高辛、甘草酸和盐酸哌唑嗪能显著降低小檗碱和巴马汀的摄取，甘草酸和盐酸哌唑嗪能减少药根碱的摄取。结论 小檗碱、巴马汀和药根碱进入肝细胞均以主动转运为主，Oatp1a1、Oatp1a4、Oatp1b2和Oct1可能介导了小檗碱和巴马汀的肝脏摄取，Oatp1b2和Oct1参与了药根碱的主动转运。
Abstract
OBJECTIVE To evaluate the uptake characterizations of berberine, palmatine and jateorhizine through hepatic uptake model with rat primary hepatocytes. METHODS Rat hepatocytes were isolated by Seglen′s two-step method and cultured in suspension. The activity of transporters was assessed using rosuvastatin which is known as the substrate of the transporters. The tested drugs were incubated with rat hepatocytes at 4 or 37 ℃ for various periods of time and the drugs were determined using UPLC-MS/MS method to calculate the uptake amounts. The effect of cultural time and temperature on the hepatic uptake of these compounds were assessed. The kinetic parameters such as Km, Vmax and CLactive/CLuptake were calculated from concentration dependent uptake experiments. The effects of positive inhibitors on the uptake of berberine, palmatine and jateorhizine were also surveyed by pre-incubation of the inhibitors with the rat hepatocytes, followed by co-incubation with berberine, palmatine and jateorhizine. RESULTS The hepatic uptake of berberine, palmatine and jateorhizine increased along with the increase of concentration at 37 ℃ while the change of hepatic uptake was very little at 4 ℃. The value of CLactive/CLuptake was 85.26%, 74.90% and 57.74% for berberine, palmatine and jateorhizine. Ibuprofen, digoxin, glycyrrhizin and prazosin which were the known inhibitors of transporters could remarkably reduce the hepatic uptake of berberine and palmatine, respectively. Glycyrrhizin and prazosin could reduce the uptake of jateorhizine. CONCLUSION The transport of berberine, palmatine and jateorhizine into hepatocytes is mainly through an active process. Berberine and palmatine are the substrates of Oatp1a1, Oatp1a4, Oatp1b2 and Oct1, and the uptake of jateorhizine might be associated with Oatp1b2and Oct1.
关键词
原代肝细胞 /
转运 /
小檗碱 /
巴马汀 /
药根碱
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Key words
primary hepatocyte /
transport /
berberine /
palmatine /
jateorhizine
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中图分类号：
R944
R965
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参考文献
[1] SHITARA Y, HORIE T, SUGIYAMA Y. Transporters as a determinant of drug clearance and tissue distribution. Eur J Pharm Sci, 2006, 27(5):425-446.
[2] LIU H, SAHI J. Role of hepatic drug transporters in drug development. J Clin Pharmacol, 2016, 56(7): 11-22.
[3] ZHANG F, WEI Y H, LI B X, et al. Progress in liver transporter-mediated drug interactions with oral antidiabetic agents. Chin Pharm J(中国药学杂志), 2014, 49(13): 1099-1103.
[4] NIEMI M, PASANEN M K, NEUVONEN P J. Organic anion transporting polypeptide 1B1: a genetically polymorphic transporter of major importance for hepatic drug uptake. Pharmacol Rev, 2011, 63(1):157-181.
[5] EVERS R, CHU X Y. Role of the murine organic anion-transporting polypeptide 1b2(Oatp1b2) in drug disposition and hepatotoxicity. Mol Pharmacol, 2008, 74(2):309-311.
[6] SEGLEN P O. Preparation of isolated rat liver cells. Methods Cell Biol, 1976,13:29-83.
[7] LIANG R F, ZHANG F, SHI L F, et al. Tissue distribution of berberine, palmatine and jateorhizine in rat determined by UPLC-MS/MS. Chin J Pharm Anal(药物分析杂志), 2016, 36(8):1421-1427.
[8] YABE Y, GALETIN A, HOUSTON J B. Kinetic characterization of rat hepatic uptake of 16 actively transported drugs. Drug Metab Dispos, 2011, 39(10):1808-1814.
[9] JEON Y J, KIM H S, SONG K S, et al. Protective effect of dieckol against chemical hypoxia-induced cytotoxicity in primarycultured mouse hepatocytes. Drug Chem Toxicol, 2015, 38(2):180-187.
LU Y, SLIZGI J R, BROUWER K R, et al. Hepatocellular disposition and transporter interactions with tolvaptan and metabolites in sandwich-cultured human hepatocytes. Drug Metab Dispos, 2016, 44(6):867-870.
TETSUKA K, GERST N, TAMURA K, et al. Glucuronidation and subsequent biliary excretion of mycophenolic acid in rat sandwich-cultured hepatocytes. Drug Metab Pharmacokinet, 2014, 29(2):129-134.
ZHANG A, WANG C, LIU Q, et al. Involvement of organic anion-transporting polypeptides in the hepatic uptake of dioscin in rats and humans. Drug Metab Dispos,2013, 41(5):994-1003.
MICO B A, FEDEROWICZ D A, RIPPLE M G, et al. In vivo inhibition of oxidative drug metabolism by, and acute toxicity of, 1-aminobenzotriazole(ABT). A tool for biochemical toxicology. Biochem Pharmacol, 1988, 37(13):2515-2519.
MENOCHET K, KENWORTHY K E, HOUSTON J B, et al. Simultaneous assessment of uptake and metabolism in rat hepatocytes: a comprehensive mechanistic model. J Pharmacol Exp Ther, 2012, 341(1):2-15.
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脚注
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基金
国家自然科学基金资助项目(81403124)
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