Category: Preclinical Development
Purpose: In vitro drug absorption and permeability experiments are widely used in the discovery and development of orally administered drugs. The standard method is to use human intestinal epithelial Caco-2 cells cultured on cell culture plates or Transwell® plates. The transport efficiency of drugs obtained from these in vitro studies are well correlated with in vivo bioavailability. However, there are contrasting reports indicating that the in vitro permeability of some drugs are not in accordance with those obtained from in vivo experiments. This would be due to the difference of the thickness of the unstirred water layer (UWL) on the surface of intestinal epithelial cells between in vitro and in vivo experiments. Therefore, the construction of an effective method for stirring at the cell surface is necessary to reduce the thickness of the UWL for the accurate evaluation of in vitro drug absorption. In this study, we developed an artificial intestinal tract system which can easily form the tubular structure through the application of air pressure using pneumatic balloon actuators (PBA). Since this system enables study of drug absorption under flow conditions, we evaluated in vitro absorption of drugs with varying lipophilicity in Caco-2 cells using this system.
Methods: The artificial intestinal tract consists of PBA formed by polydimethylsiloxane films, two seal areas, and two air inlets (Fig. 1A). The PBA transitions from a flat plane to a circular tube in response to the air pressure (Fig. 1B,C). Caco-2 cells were seeded on the PBA of the artificial intestinal tract and were cultured for 14 d. Then, the artificial intestinal tract was actuated from a flat plane to a circular tube by constant air infusion into PBA. Hanks’ balanced salt solution (HBSS) containing 50 μM of imipramine, 50 μM of chlorpromazine, 1 mM of 5-aminosalicylic acid, or 100 μM of clonidine was perfused in the artificial intestinal tract for 1 h at flow rates of 0.05 and 0.5 mL/min. After washing the cells with ice-cold HBSS, the cells were collected in acetonitrile, and drug concentration was measured by HPLC.
Results: The cultivated Caco-2 cells on the PBA of the artificial intestinal tract form the tight junction and its structure was maintained intact following perfusion. In addition, the cell viability was not changed by the perfusion. These results indicate that the actuation of the intestinal tract and the perfusion do not affect the characteristics of Caco-2 cells. In order to determine whether the artificial intestinal tract can decrease the effect of a UWL on in vitro drug absorption studies, we evaluated the uptake of two lipophilic drugs into Caco-2 cells on the artificial intestinal tract. Cumulative amount of imipramine and chlorpromazine in Caco-2 cells were approximately 6.5 times higher with internal flow than those without internal flow (Fig. 2A,B). In contrast, the cumulative accumulation of these drugs in Caco-2 cells seeded on the cell culture plates were only 1.5 times increased by mechanical shaking. These results suggest that the internal flow of the artificial intestinal tract effectively agitates the surface of Caco-2 cells, resulting in reduction of the UWL thickness. Transport across a UWL is a rate-limiting process in the intestinal absorption of lipophilic drugs, and therefore the artificial intestinal tract could achieve a 6.5-fold reduction in the UWL thickness on the surface of Caco-2 cells. As the UWL in the intestine under static conditions has been reported to be over 6 times thicker than that under physiological dynamic conditions,1) our system would enable the study of in vitro absorption by simulating conditions similar to those of in vivo physiological conditions, particularly with regard to the presence of a UWL. In addition, we also evaluated the accumulation of drugs with poor lipophilicity, 5-aminosalicylic acid and clonidine, in Caco-2 cells. The cumulative amount of both drugs in Caco-2 cells was not changed with internal flow of the artificial intestinal tract (Fig. 2C,D). Since hydrophilic drugs are hardly restricted by a UWL, it is to be expected that absorption of 5-aminosalicylic acid and clonidine in Caco-2 cells would be unaffected by internal flow. These results further indicate that the internal flow of our artificial intestinal tract reduces the effect of a UWL on drug absorption in Caco-2 cells.
Conclusion: We successfully demonstrated increased drug absorption by Caco-2 cells through reduction of the UWL thickness using our artificial intestinal tract. This system could contribute to the development of a precise in vitro method for estimating drug permeability in the human small intestine.
1. Korjamo T et al., J Pharm Sci. 98, 4469-4479 (2009).