Transport and metabolic characterization of Caco-2 cells expressing CYP3A4and CYP3A4 plus oxidoreductase

Purpose. To further characterize CYP3A4-transfected Caco-2 cells with regar d to morphological, transport, and metabolic properties, and to evaluate a different Caco-2 cell strain transfected with both CYP3A4 and oxidoreductas e (OR). Methods. Transfected Caco-2 cells, Caco-2 TC7 cells, and wild-type Caco-2 c ells grown onto Millicell(TM) were used. We determined the morphological ch aracteristics of transfected cell monolayers using light and transmission e lectron microscope. We determined the transport and metabolic capabilities of the transfected cells, TC7 cells, and wildtype cells with a variety of d rugs, nutrients, and marker compounds. Results. The transfected Caco-2 cells formed a tight monolayer with TEER va lues and mannitol transport similar to the untransfected parent cell strain (wild type). However, the transfected cells (grown onto Millicell(TM) reac hed maturity approximately 33% faster than the wildtype cells. Permeabiliti es of propranolol, nifedipine, testosterone, linopirdine, mannitol, and cep halexin were similar in transfected and wildtype Caco-2 cells. On the other hand, the transfected cells of early passages were much more metabolically active, and metabolized standard CYP3A4 substrates (e.g., testosterone and nifedipine) as much as 100 times faster than untransfected cells. in addit ion, metabolism of standard substrates was inhibitable by ketoconazole and TAO. Using comparable data, the transfected cells metabolized testosterone the fastest, followed by linopirdine and nifedipine (approximate ratio: 10: 6:2). The metabolites of standard substrates were generally preferably excr eted to the apical membrane. Conclusion. The monolayers of newly transfected cells (CYP3A4 + OR) have a significantly increased level of CYP3A4 activities compared to untransfecte d cells. These cell monolayers also have desirable morphological and transp ort characteristics that are similar to untransfected cells.