Identification of esterases expressed in Caco-2 cells and effects of their hydrolyzing activity in predicting human intestinal absorption
Identification of esterases expressed in Caco-2 cells and effects of their hydrolyzing activity in predicting human intestinal absorption
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DOI：
10.1124/dmd.105.004226
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发表时间：
2005-08-01
影响因子：
3.9
通讯作者：
Hashimoto, M
中科院分区：
文献类型：
--
作者：
Imai, T;Imoto, M;Hashimoto, M
采用Caco-2细胞研究替莫普利的吸收特性，并对Caco-2细胞表达的酯酶进行鉴定。在Caco-2细胞中，替莫普利几乎完全水解为替莫卡普拉。替莫普利在Caco-2细胞9000 g上清液（S9）和刷状缘膜囊泡中的水解实验表明，替莫普利摄取后主要在细胞内水解，然后形成的替莫普利拉被转运到细胞顶面和基底面。在非变性聚丙烯酰胺凝胶电泳中，Caco-2细胞S9的1-萘丁酸酯水解酶活性检测显示一条酶活性高的带和另一条酶活性极低的带。主带和次带中的蛋白被鉴定为羧酸酯酶-1（hCE-1）和羧酸酯酶-2（hCE-2）。逆转录-聚合酶链反应证实hCE-1在Caco-2细胞中有高表达。在正常人小肠中，hCE-2大量存在，尽管人肝脏表达高得多的hCE-1水平和低水平的hCE-2。Caco-2细胞中羧酸酯酶的表达模式与人小肠中的完全不同，但与人肝脏中的非常相似。由于hCE-1的底物特异性与hCE-2不同，因此建议在含酯和酰胺的药物（如前药）的情况下，应仔细使用Caco-2细胞单层预测人体肠道吸收。
The absorption characteristics of temocapril were investigated using Caco-2 cells, and the esterases expressed in Caco-2 cells were identified. Temocapril was almost completely hydrolyzed to temocaprilat during transport across Caco-2 cells. Hydrolysis experiments of temocapril in Caco-2 cell 9000g supernatant (S9) and brush-border membrane vesicles showed that temocapril was mainly hydrolyzed within the cells after uptake, after which the temocaprilat formed was transported to both the apical and basolateral surfaces. In native polyacrylamide gel electrophoresis by detection of hydrolase activity for 1-naphthylbutyrate, Caco-2 cell S9 showed a band with high esterase activity and another band with extremely low activity. The proteins in the major and minor bands were identified as carboxylesterase-1 (hCE-1) and carboxylesterase-2 (hCE-2). The abundant expression of hCE-1 in Caco-2 cells was supported by reverse transcription-polymerase chain reaction. In the normal human small intestine, hCE-2 is abundantly present, although the human liver expresses much higher levels of hCE-1 and lower levels of hCE-2. The expression pattern of carboxylesterases in Caco-2 cells is completely different from that in human small intestine but very similar to that in human liver. Since the substrate specificity of hCE-1 differs from that of hCE-2, it is suggested that the prediction of human intestinal absorption using Caco-2 cell monolayers should be performed carefully in the case of ester- and amide-containing drugs such as prodrugs.