Yike Cai1, Chenshu Xu2, Peiyi Chen3, Jinqing Hu4, Rong Hu4, Min Huang4, Huichang Bi5. 1. Laboratory of Drug Metabolism and Pharmacokinetics, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, Guangdong 510006, China; Guangdong Food and Drug Administration Center for Evaluation and Certification, Guangzhou, Guangdong 510080, China. 2. Department of Pharmacy, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong 510080, China. 3. Guangdong Food and Drug Administration Center for Evaluation and Certification, Guangzhou, Guangdong 510080, China. 4. Laboratory of Drug Metabolism and Pharmacokinetics, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, Guangdong 510006, China. 5. Laboratory of Drug Metabolism and Pharmacokinetics, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, Guangdong 510006, China. Electronic address: bihchang@mail.sysu.edu.cn.
Abstract
INTRODUCTION: Monolayers of Caco-2 cells have been widely accepted as one of the well-established in vitro models to predict intestinal drug permeability and absorption in humans. However, the procedure for culturing the traditional 21-day Caco-2 model is labor intensive and time consuming, which limits its wide application in drug development. The objective of the present study was to develop a rapid Caco-2 model with a 7-day cell culture process. METHODS: A few modifications of the BIOCOAT® HTS Caco-2 Assay System were introduced including changing the cell seeding density, the composition of cell culture media, and the interval to change media. The monolayer structure was visualized through confocal microscopy. The transepithelial electrical resistance (TEER), apparent permeability coefficients (Papp), and functional activity of P-glycoprotein (P-gp) were determined and compared with the 21-day model. Moreover, pharmacokinetic studies were performed in animal models to evaluate the absolute bioavailability of oral doses (Foral) of different compounds. RESULTS: Our newly developed 7-day Caco-2 model displayed comparable cellular morphology and integrity with the traditional 21-day model. No significant difference in paracellular and transcellular permeability was observed between the two systems. The efflux ratios of transporting digoxin, the prototypical substrate of P-gp in 21- and 7-day systems were 16.04 and 24.92, respectively. When the P-gp inhibitor verapamil was present, the efflux ratios of 21- and 7-day systems were 1.37 and 0.86, respectively, suggesting the comparability of the P-gp functional activity in both systems. Furthermore, pharmacokinetic studies of several compounds performed in animal models revealed that the absolute bioavailability of oral doses in vivo was well correlated with the Caco-2 permeability in vitro. DISCUSSION: The novel system provides a rapid and economical option for assessing the drug permeability, and is applicable to the studies of intestinal drug absorption.
INTRODUCTION: Monolayers of Caco-2 cells have been widely accepted as one of the well-established in vitro models to predict intestinal drug permeability and absorption in humans. However, the procedure for culturing the traditional 21-day Caco-2 model is labor intensive and time consuming, which limits its wide application in drug development. The objective of the present study was to develop a rapid Caco-2 model with a 7-day cell culture process. METHODS: A few modifications of the BIOCOAT® HTS Caco-2 Assay System were introduced including changing the cell seeding density, the composition of cell culture media, and the interval to change media. The monolayer structure was visualized through confocal microscopy. The transepithelial electrical resistance (TEER), apparent permeability coefficients (Papp), and functional activity of P-glycoprotein (P-gp) were determined and compared with the 21-day model. Moreover, pharmacokinetic studies were performed in animal models to evaluate the absolute bioavailability of oral doses (Foral) of different compounds. RESULTS: Our newly developed 7-day Caco-2 model displayed comparable cellular morphology and integrity with the traditional 21-day model. No significant difference in paracellular and transcellular permeability was observed between the two systems. The efflux ratios of transporting digoxin, the prototypical substrate of P-gp in 21- and 7-day systems were 16.04 and 24.92, respectively. When the P-gp inhibitor verapamil was present, the efflux ratios of 21- and 7-day systems were 1.37 and 0.86, respectively, suggesting the comparability of the P-gp functional activity in both systems. Furthermore, pharmacokinetic studies of several compounds performed in animal models revealed that the absolute bioavailability of oral doses in vivo was well correlated with the Caco-2 permeability in vitro. DISCUSSION: The novel system provides a rapid and economical option for assessing the drug permeability, and is applicable to the studies of intestinal drug absorption.