TACROLIMUS (FK-506) BIOTRANSFORMATION IN PRIMARY RAT HEPATOCYTES DEPENDS ON EXTRACELLULAR-MATRIX GEOMETRY

Established in vitro models for studies of hepatic drug biotransformat ion include the use of primary hepatocytes. In normal liver the space of Disse provides the possibility of bilateral attachment to extracell ular matrix for each hepatocyte. This configuration is disrupted by th e cell isolation procedure of normal liver tissue, which delivers susp ensions of round shaped cells. In standard culture configurations this unphysiologic cell shape terminates in a morphological dedifferentiat ion and inability to biotransform drugs. This study analyses the relev ance of extracellular matrix geometry in hepatocyte monolayer configur ations for expression and activity of cytochrome P450 3A. This enzyme is involved in the biotransformation of a large number of pharmaceutic als including the immunosuppressants tacrolimus and sirolimus. Morphol ogical analysis of primary rat hepatocytes cultured with and without o verlay of collagen type I was performed by transmission and scanning e lectron microscopy. Expression and activity of cytochrome P450 3A was studied by Western blot and the use of two model drugs specific for th is enzyme. To this purpose the immunosuppressive drugs tacrolimus and sirolimus were used. Metabolites were analyzed by HPLC and HPLC/MS. Tw o sided attachment to extracellular matrix induces profound changes of the hepatocellular morphology in vitro resulting in the reconstitutio n of a polyhedric cell shape. This phenomenon is paralleled by an enha nced expression of cytochrome P450 3A and corresponding metabolic acti vity. As shown for tacrolimus biotransformation, the model may be usef ul to study complex metabolic patterns. In addition this model may fac ilitate studies of the kinetics of hepatocellular drug biotransformati on in a setting with prolonged stability.