Development of a Caco-2 Cell Line Carrying the Human Intestine-Type CES Expression Profile as a Promising Tool for Ester-Containing Drug Permeability Studies

• Published in: Biological & pharmaceutical bulletin Vol. 41; no. 5; pp. 697 - 706
• Main Authors: Ishizaki, Yuma, Furihata, Tomomi, Oyama, Yusuke, Ohura, Kayoko, Imai, Teruko, Hosokawa, Masakiyo, Akita, Hidetaka, Chiba, Kan
• Format: Journal Article
• Language: English
• Published: Japan The Pharmaceutical Society of Japan 01.05.2018; Pharmaceutical Society of Japan; Japan Science and Technology Agency
• Subjects: Caco-2 cell; Carboxylesterase; Colon; Drug development; Drug metabolism; drug permeability; Enterocytes; Gene expression; Hydrolase; mRNA; Permeability; prodrug; Serine; Serine hydrolase; Small intestine; prodrug; carboxylesterase; small intestine; drug permeability; Caco-2 cell
• ISSN: 0918-6158; 1347-5215
• DOI: 10.1248/bpb.b17-00880

Carboxylesterase 2 (CES2), which is a member of the serine hydrolase superfamily, is primarily expressed in the human small intestine, where it plays an important role in the metabolism of ester-containing drugs. Therefore, to facilitate continued progress in ester-containing drug development, it is crucial to evaluate how CES2-mediated hydrolysis influences its intestinal permeability characteristics. Human colon carcinoma Caco-2 cells have long been widely used in drug permeability studies as an enterocyte model. However, they are not suitable for ester-containing drug permeability studies due to the fact that Caco-2 cells express CES1 (which is not expressed in human enterocytes) but do not express CES2. To resolve this problem, we created a new Caco-2 cell line carrying the human small intestine-type CES expression profile. We began by introducing short-hairpin RNA for CES1 mRNA knockdown into Caco-2 cells to generate CES1-decifient Caco-2 cells (Caco-2CES1KD cells). Then, we developed Caco-2CES1KD cells that stably express CES2 (CES2/Caco-2CES1KD cells) and their control Mock/Caco-2CES1KD cells. The results of a series of functional expression experiments confirmed that CES2-specific activity, along with CES2 mRNA and protein expression, were clearly detected in our CES2/Caco-2CES1KD cells. Furthermore, we also confirmed that CES2/Caco-2CES1KD cells retained their tight junction formation property as well as their drug efflux transporter functions. Collectively, based on our results clearly showing that CES2/Caco-2CES1KD cells carry the human intestinal-type CES expression profile, while concomitantly retaining their barrier properties, it can be expected that this cell line will provide a promising in vitro model for ester-containing drug permeability studies.