Methodology for Quantitating Rapid Transport Processes in Monolayer Cell Cultures.

Abstract

The investigation of drug transport and bioconversion in cultured cell systems is a rapidly growing area of research. Application of the physical model approach has provided basic insight into the kinetics of drug activity in several cell systems. While significant advances have been made in kinetic studies involving suspension cell cultures, a general lack of kinetic oriented methodology has retarded comparable progress with monolayer cell cultures. The goal of this investigation was the development of methodology which would facilitate the evaluation of rapid kinetic processes in monolayer cell cultures. To achieve this goal, a falling film of saline wash procedure was developed to study rapid transport processes with monolayer cultures of human embryonic lung cells WI-38 (VA 13A). Due to the technique's simplicity, it has general application to any monolayer cell culture. Furthermore, kinetic processes with half-lives as short as 5 to 10 seconds may be monitored with precision of 10 to 20%. Initial kinetic investigations were conducted on cardiac glycosides: ouabain, digoxin and digitoxin. The latter two compounds bound rapidly to the cell membrane with half lives in the order of one to two minutes. Subsequent membrane permeation and intracellular binding occurred very slowly with half-lives of approximately 20 hours. Although ouabain failed to reveal a rapid membrane binding step, the slower membrane permeation process resulted in an extremely large partition coefficient. In comparison to the other cardiac glycosides, this large partition coefficient suggested that a kinetically indistinguishable binding process had been included in the apparent membrane permeation step of ouabain. In addition, the membrane permeability coefficients measured in this study correlated well with the lipophilicity of the respective compounds, thus supporting the importance of lipophilicity in drug transport across cell membranes. The methodology resulting from this investigation should have many applications in drug research and development. In particular, this methodology may be used to quantitate the changes in membrane binding and permeation produced by structural modification of drug molecules. Due to the availability of several monolayer human cell cultures derived from tissues associated with disease states, it is hoped that basic transport studies employing this methodology could lead to the development of better therapeutic agents.