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Date of Award
Thesis - Pacific Access Restricted
Master of Science (M.S.)
Pharmaceutical and Chemical Sciences
First Committee Member
Purpose. The fate of an orally administered drug is dependent on many parameters before it can reach the systemic circulation, including drug absorption and first-pass metabolism in the gut and the liver. Mammalian cells lines such as MDCK and Caco-2 are commonly employed to assess drug permeability but they lack or have low expression level of drug metabolism enzyme expression such as CYP3A4, which contributes to significant first-pass gut and liver metabolism for many drugs. Consequently, these cell lines are not sufficient to integrate metabolism when assessing drug absorption. Here, we tested MDCK and IEC-18 cells transiently over-expressing CYP3A4 as models that can simultaneously assay a compound's permeability and metabolism potential in a single experiment. Method. A recombinant adenovirus carrying the hCYP3A4 cDNA was constructed according to Stratagene's AdEasy XL Adenoviral system. This adenovirus was used to transiently transfect hCYP3A4 into MDCK and IEC-18 cells. Western blot was performed to assess the level of hCYP3A4 expression in the wild type and CYP3A4 over-expressing IEC-18 and MDCK cells. In situ metabolism and transport studies were performed with wild-types and IEC-18-3A4 or MDCK-3A4 cells. Results. The amount of CYP3A4 present in MDCK-3A4 cells was 250 times to that of wild type cells which 1/4th the amount present in human liver microsomes. The amount of CYP3A4 present in IEC-18-3A4 cells was 150 times to that of wild type cells which 1/6th the amount present in human liver microsomes. In metabolism studies, there was higher formation of metabolites in cells transfected with hCYP3A4 compared to controls. In addition, apical to basal transport studies of several drugs in IEC-18-3A4 and MDCK-3A4 showed increased appearance of metabolites compared to the wild-type cells. Conclusions. This model may be a useful to assess the extent of drug absorption into systemic circulation after oral administration.
Vangala, Swathi. (2013). Human Cytochrome P450 3A4 Over-Expressing IEC-18 and MDCK Cell Lines as an In-Vitro Model to Assess Gut Permeability and the Enzyme Metabolism. University of the Pacific, Thesis - Pacific Access Restricted. http://scholarlycommons.pacific.edu/uop_etds/273
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