Novel in vitro cell culture models for studying pharmacokinetic drug-drug interactions following oral administration
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In this study different in vitro models were developed to study the long term and short term drug- drug interactions occurring upon oral administration. MDCK cells were stably transfected for the simultaneous expression of P-gp and CYP3A4 (MMC) as well as MRP2 and CYP3A4 (MMrC). These were characterized by using expression and activity studies. Cellular transport study of 200 μM cortisol, was performed to determine their combined activity. The study was carried across control, single transfected and dual transfected cell lines. Similar studies were also carried out in the presence of 50 μM naringin (herb) and 3 μM morphine (drug of abuse). A dual chamber model with transfected MDCK on apical and transfected HEPG2 cell line in basal chamber was also used to study similar interactions. For long term interactions LS180 cells were treated with combinations of HIV protease inhibitors (PIs: Lopinavir, Indinavir, Saquinavir and Amprinavir) and mRNA expression of efflux transporters and CYP3A4 was quantified. These expression levels were further correlated to PXR levels. Uptake of florescent and radioactive substrates was used to study activity of the proteins. Cytotoxicity and ATP assays were used stress markers. The MMC and MMrC cell lines were characterized for stable expression of both proteins. Decreased Apical-Basolateral transport of cortisol and enhanced metabolite formation was observed in dual transfected cells as compared to non-transfected and single-transfected controls. Transport of cortisol increased fivefold in presence of naringin in MMC and threefold in presence of morphine. The dual chamber model showed greater in vitro - in vivo correlation to rat model than single chamber models. Binary combinations of PIs showed greater induction of efflux proteins (5-15 fold) as compared to ternary combinations (1-4 fold) or individual PIs. The altered mRNA expression correlated with expression levels of PXR but not CAR. Expression levels also correlated well with protein expressions and activity assays. No cytotoxicity or ATP activity was observed. These new cell lines can serve as an invaluable model to study drug interactions as they incorporate the combined contribution of efflux transporters and CYP3A4. Also use of drug combinations can significantly alter their bioavailability and disposition resulting in failure during chronic therapy.
Table of Contents
Introduction -- Literature Review -- Screening of gatifloxacin interaction with efflux transporters using transfected MDCK cell lines: a possible mechanism for drug resistance -- Transfected MDCK cell line with enhanced expression of CYP3A4 and p-glycoprotein as a model to study their interactions in drug detoxification -- Development of transfected MDCK cell line with enhanced expression of CYP3A4 and multidrug resistance protein-2 (MRP-2) as a model to study their interactions in drug detoxification -- Development of an integrated simultaneous absorption and metabolisn in-vitro cell culture model to study the drug efflux and metabolism simultaneously during first pass effect -- Binary and ternary combinations of anti-HIV protease inhibitor: effect of gene/protein expression and functional activity of CYP3A4 and efflux transporters -- Summary and recommendations