穿心莲二萜内酯类化合物跨膜转运机制研究
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摘要
目的：通过采用Caco-2细胞模型对穿心莲内酯、脱水穿心莲内酯和新穿心莲内酯在体外的吸收特征进行系统研究,为深入探讨穿心莲二萜内酯类化合物的药代动力学特征及其药理学作用机制提供试验和理论依据。
方法：①采用摇瓶法测定穿心莲内酯、脱水穿心莲内酯和新穿心莲内酯在正辛醇-水和王辛醇-缓冲液体系中的表观油水分配系数。
②建立液相色谱串联质谱检测方法,测定Caco-2细胞外穿心莲内酯、脱水穿心莲内酯和新穿心莲内酯的浓度；建立及全面评价Caco-2单层细胞特性；用Caco-2细胞模型研究穿心莲内酯、脱水穿心莲内酯和新穿心莲内酯的双向转运：考察时间、浓度、体系温度、抑制剂对三者跨膜转运的影响,探究其在Caco-2细胞转运试验中的吸收机制和外排现象。
③研究了穿心莲内酯和新穿心莲内酯不同配比条件下在Caco-2细胞模型中的转运情况。固定穿心莲内酯浓度,改变新穿心莲内酯的剂量,观察新穿心莲内酯对穿心莲内酯转运的影响。
结果：①37℃下穿心莲内酯、脱水穿心莲内酯和新穿心莲内酯的表观油水分配系数分别为3.90(log Papp=0.29)、19.75(log Papp=1.30)和1.75(log Papp=0.24),穿心莲内酯和新穿心莲内酯在pH6磷酸盐缓冲液时表观油水分配系数最大,而脱水穿心莲内酯则在pH5磷酸盐缓冲液时最大。
②Transwell板上培养21天后的Caco-2细胞单层结构紧密、TEER值>500Ω·cm2、碱性磷酸酶已经大部分集中于刷状缘一侧(AP侧)、模型通透性较好。
③穿心莲内酯在Caco-2细胞模型中,随时间和浓度的增加,药物吸收呈饱和趋势,且受温度和碘乙酰胺影响,但不受外排抑制剂维拉帕米和MK-571的影响。
④脱水穿心莲内酯在Caco-2细胞模型中,随着浓度的增加和时间的延长,转运量呈线性增加,不受温度、维拉帕米和MK-571的影响。
⑤新穿心莲内酯在Caco-2细胞模型中,存在浓度和时间依赖性,而温度和碘乙酰胺显著的影响转运结果,不受维拉帕米和MK-571的影响。
⑥穿心莲内酯和新穿心莲内酯结合给药,随着新穿心莲内酯浓度的增加,穿心莲内酯从肠腔侧(AP)到基底侧(BL)的转运量减少。
结论：①pH对穿心莲内酯、脱水穿心莲内酯和新穿心莲内酯的表观油水分配系数有一定影响。在一定范围内,pH值增加可使穿心莲二萜内酯类成分的表观油水分配系数减小。
②本研究建立的完整Caco-2细胞单层模型符合药物转运试验的要求。
③穿心莲内酯在Caco-2细胞中的吸收主要是由载体介导的主动转运,且该主动转运的载体可能位于AP侧。
④脱水穿心莲内酯主要以被动扩散为主要方式经由Caco-2细胞单层吸收和转运。
⑤新穿心莲内酯在Caco-2细胞中的吸收转运主要由被动转运和主动转运共同参与,主要转运的载体可能位于AP侧。
⑥穿心莲内酯的吸收转运可被新穿心莲内酯竞争性抑制,二者的吸收转运可能由同-转运载体介导。
Objective:The present study was to investigate the absorptive mechanism of andrographolide, dehydroandrographolide and neoandrographolide and to refer the experimental and theories gist for a deep study of the pharmacokinetics and pharmacological mechanism, using Caco-2 cells as models and determing apparent octanol-water partition coefficient.
Methods:①The apparent octanol-water/buffer partition coefficient of andrographolide, dehydroandrographolide and neoandrographolide was measured by shaking flask method.
②An LC/MS/MS method was developed for analyzing the Caco-2 cell assay samples. Establish and assess the characteristic of Caco-2 cell monolayer. Caco-2 cell monolayer model was used to investigate the bidirectional transport of andrographolide, dehydroandrographolide and neoandrographolide. The effect of time, drug concentration, system temperature and inhabitors on the absorption of them was observed for exploring the absorptive characteristic and transcellular efflux.
③The transport of andrographolide and neoandrographolide was studied while two compound were co-administrated. The effect of neoandrographolide on transport of andrographolide was tested across Caco-2 cell monolayers.
Results:①The apparent octanol-water partition coefficient (Papp) of andrographolide, dehydroandrographolide and neoandrographolide were 3.90 (log Papp=0.29),19.75 (log Papp=1.30) and 1.75 (log Papp=0.24) in water at 37℃respectively. Andrographolide and neoandrographolide have the highest partition coefficient in pH6, and dehydroandrogapholide in pH5.
②Caco-2 cells cultured on transwell plate had formed a tight structure with a TEER value of Caco-2 monolayers being greater than 500Ω·cm2. Permeability and the expression of alkaline phosphatase in the cell model were superior.
②Time and concentration saturation were observed for the absorptive transport of andrographolide across Caco-2 monolayers. The transport of andrographolide was influenced by the change of temperature and the presence of iodoacetamide, but not Verapamil or MK-571.
④The amount of dehydrographolide which was transported increased linearly with the time and concentration. And it was not influenced by the change of temperature and the presence of Verapamil or MK-571.
⑤The amount of dehydrographolide which was transported increased linearly with the time and concentration. But it was influenced by the change of temperature and the presence of iodoacetamide, but not Verapamil or MK-571.
⑥Co-administration of andrographolide and neoandrographolide leaded to a decrease transport of andrographolide from apical side to basolateral side when concentration of neoandrographolide increased.
Conclusions:①The Papp of andrographolide, dehydroandrographolide and neoandrographolide was influenced by pH, and the higher pH could decrease Papp of them.
②The study established an integrated Caco-2 cell monolayers model, which met the requirement of the drug transport experiment.
③The absorption and transport of andrographolide in Caco-2 cell monolayer was an active transportation mediated by transporter, which mainly located in the apical side of Caco-2 cell monolayers.
④The absorption and transport of dehydroangrapholide was passive diffusion as the dominating process in Caco-2 cell monolayers.
⑤The absorption and transport of dehydroangrapholide was passive diffusion and active transport. And the transporter maybe located in the apical side of Caco-2 cell monolayers.
⑥The absorption and transport of andrographolide could be inhibited by neoandrographolide. They may be mediated by the same transporter.
方法：①采用摇瓶法测定穿心莲内酯、脱水穿心莲内酯和新穿心莲内酯在正辛醇-水和王辛醇-缓冲液体系中的表观油水分配系数。
②建立液相色谱串联质谱检测方法,测定Caco-2细胞外穿心莲内酯、脱水穿心莲内酯和新穿心莲内酯的浓度；建立及全面评价Caco-2单层细胞特性；用Caco-2细胞模型研究穿心莲内酯、脱水穿心莲内酯和新穿心莲内酯的双向转运：考察时间、浓度、体系温度、抑制剂对三者跨膜转运的影响,探究其在Caco-2细胞转运试验中的吸收机制和外排现象。
③研究了穿心莲内酯和新穿心莲内酯不同配比条件下在Caco-2细胞模型中的转运情况。固定穿心莲内酯浓度,改变新穿心莲内酯的剂量,观察新穿心莲内酯对穿心莲内酯转运的影响。
结果：①37℃下穿心莲内酯、脱水穿心莲内酯和新穿心莲内酯的表观油水分配系数分别为3.90(log Papp=0.29)、19.75(log Papp=1.30)和1.75(log Papp=0.24),穿心莲内酯和新穿心莲内酯在pH6磷酸盐缓冲液时表观油水分配系数最大,而脱水穿心莲内酯则在pH5磷酸盐缓冲液时最大。
②Transwell板上培养21天后的Caco-2细胞单层结构紧密、TEER值>500Ω·cm2、碱性磷酸酶已经大部分集中于刷状缘一侧(AP侧)、模型通透性较好。
③穿心莲内酯在Caco-2细胞模型中,随时间和浓度的增加,药物吸收呈饱和趋势,且受温度和碘乙酰胺影响,但不受外排抑制剂维拉帕米和MK-571的影响。
④脱水穿心莲内酯在Caco-2细胞模型中,随着浓度的增加和时间的延长,转运量呈线性增加,不受温度、维拉帕米和MK-571的影响。
⑤新穿心莲内酯在Caco-2细胞模型中,存在浓度和时间依赖性,而温度和碘乙酰胺显著的影响转运结果,不受维拉帕米和MK-571的影响。
⑥穿心莲内酯和新穿心莲内酯结合给药,随着新穿心莲内酯浓度的增加,穿心莲内酯从肠腔侧(AP)到基底侧(BL)的转运量减少。
结论：①pH对穿心莲内酯、脱水穿心莲内酯和新穿心莲内酯的表观油水分配系数有一定影响。在一定范围内,pH值增加可使穿心莲二萜内酯类成分的表观油水分配系数减小。
②本研究建立的完整Caco-2细胞单层模型符合药物转运试验的要求。
③穿心莲内酯在Caco-2细胞中的吸收主要是由载体介导的主动转运,且该主动转运的载体可能位于AP侧。
④脱水穿心莲内酯主要以被动扩散为主要方式经由Caco-2细胞单层吸收和转运。
⑤新穿心莲内酯在Caco-2细胞中的吸收转运主要由被动转运和主动转运共同参与,主要转运的载体可能位于AP侧。
⑥穿心莲内酯的吸收转运可被新穿心莲内酯竞争性抑制,二者的吸收转运可能由同-转运载体介导。
Objective:The present study was to investigate the absorptive mechanism of andrographolide, dehydroandrographolide and neoandrographolide and to refer the experimental and theories gist for a deep study of the pharmacokinetics and pharmacological mechanism, using Caco-2 cells as models and determing apparent octanol-water partition coefficient.
Methods:①The apparent octanol-water/buffer partition coefficient of andrographolide, dehydroandrographolide and neoandrographolide was measured by shaking flask method.
②An LC/MS/MS method was developed for analyzing the Caco-2 cell assay samples. Establish and assess the characteristic of Caco-2 cell monolayer. Caco-2 cell monolayer model was used to investigate the bidirectional transport of andrographolide, dehydroandrographolide and neoandrographolide. The effect of time, drug concentration, system temperature and inhabitors on the absorption of them was observed for exploring the absorptive characteristic and transcellular efflux.
③The transport of andrographolide and neoandrographolide was studied while two compound were co-administrated. The effect of neoandrographolide on transport of andrographolide was tested across Caco-2 cell monolayers.
Results:①The apparent octanol-water partition coefficient (Papp) of andrographolide, dehydroandrographolide and neoandrographolide were 3.90 (log Papp=0.29),19.75 (log Papp=1.30) and 1.75 (log Papp=0.24) in water at 37℃respectively. Andrographolide and neoandrographolide have the highest partition coefficient in pH6, and dehydroandrogapholide in pH5.
②Caco-2 cells cultured on transwell plate had formed a tight structure with a TEER value of Caco-2 monolayers being greater than 500Ω·cm2. Permeability and the expression of alkaline phosphatase in the cell model were superior.
②Time and concentration saturation were observed for the absorptive transport of andrographolide across Caco-2 monolayers. The transport of andrographolide was influenced by the change of temperature and the presence of iodoacetamide, but not Verapamil or MK-571.
④The amount of dehydrographolide which was transported increased linearly with the time and concentration. And it was not influenced by the change of temperature and the presence of Verapamil or MK-571.
⑤The amount of dehydrographolide which was transported increased linearly with the time and concentration. But it was influenced by the change of temperature and the presence of iodoacetamide, but not Verapamil or MK-571.
⑥Co-administration of andrographolide and neoandrographolide leaded to a decrease transport of andrographolide from apical side to basolateral side when concentration of neoandrographolide increased.
Conclusions:①The Papp of andrographolide, dehydroandrographolide and neoandrographolide was influenced by pH, and the higher pH could decrease Papp of them.
②The study established an integrated Caco-2 cell monolayers model, which met the requirement of the drug transport experiment.
③The absorption and transport of andrographolide in Caco-2 cell monolayer was an active transportation mediated by transporter, which mainly located in the apical side of Caco-2 cell monolayers.
④The absorption and transport of dehydroangrapholide was passive diffusion as the dominating process in Caco-2 cell monolayers.
⑤The absorption and transport of dehydroangrapholide was passive diffusion and active transport. And the transporter maybe located in the apical side of Caco-2 cell monolayers.
⑥The absorption and transport of andrographolide could be inhibited by neoandrographolide. They may be mediated by the same transporter.
引文
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53.胡晓渝,姚彤炜,曾苏.Caco-2细胞系及其在药物吸收、代谢中的应用.中国现代应用药学杂志,2002,19(2)：88-90.
54. Dokladny K, Moseley PL, Ma TY. Physiologically relevant increase in temperature causes an increase in intestinal epithelial tight junction permeability. Am. J. Physiol. Gastrointest. LiverPhysiol.2006,290(2):204-212.
55. Yamashita S, Furubayashi T, Kataoka M, et al. Optimized conditions for prediction of intestinal drug permeability using Caco-2 cells. Eur. J. Pharm. Sci.2000,10(3):195-204.
56. BartelsH. Korsiak E, Daniel H. Transport activity of the MDRl-gene product in monolayers of Caco-2 cells. Z. Gastroent.1994,32:15-18.
57. Roepe PD. The P-glycoprotein effux pump:how does it transport drug? J. Membrane Biol. 1997:160(3):161-175.
58. Artursson P, Palm K, Luthman K. Caco-2 monlayers in experimental and theoretical prediction of drug transport. Adv. Drug Deliv. Rev.2001,46(1-3):27-43.
59. Yee S. In vitro permeability across Caco-2 cells (colonic) can predict in vivo (small intestinal) absorption in man-fact or myth. Pharm. Res.1997,14(6):763-766.
60. Gan LL, Dhiren RT. Applications of the Caco-2 model in the design and development of orally active drugs:elucidation of biochemical and physical barriers posed by the intestinal epithelium. Adv. Drug Deliv. Rev.1997,23(1):77.
61. Jezyk N, Li C, Stewart BH, et al. Transport of pregabalin in rat intestine and Caco-2 monolayers. Pharm. Res.1999,16(4):519.
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63.关溯,毕惠嫦,陈孝,等.隐丹参酮在Caco-2细胞模型中的吸收机制.中国临床药理学杂志,2005,21(4)：268-271.
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