Genetic variation in the multidrug resistance gene (MDR1): Impact on drug delivery and disposition

The aim of my thesis research is to evaluate the impact of genetic variation in the multidrug resistance gene (MDR1) on the drug delivery and disposition of (P-glycoprotein) P-gp substrates. To facilitate this, a novel set of tools were developed to determine MDR1 mRNA levels and to generate recombinant cells expressing MDR1 genetic variants. The development of a semi-quantitative full-length MDR1 mRNA assay allowed for the estimation of mRNA as an indicator of P-gp levels in cell lines and leukemia patients. The full-length cDNA generated from the semi-quantitative assay was used to generate a series of MDR1 mammalian expression plasmids each containing an individual SNP or haplotype of MDR1. These plasmids permitted the development of a recombinant expression system consistently expressing MDR1 variants in polarized epithelial cells, allowing for functional studies to evaluate changes in P-gP activity.; The G → A polymorphism at nucleotide position 1199 in MDR1 , resulting in a serine(400)asparagine modification, was evaluated with this recombinant system. Alterations in the P-gp-mediated transepithelial permeability and cellular chemoresistance were observed for the anticancer agents vinblastine and vincristine, but not for doxorubicin, indicating that G1199A may influence penetration of anticancer agents into the brain. The G1199A polymorphism also had an impact on the transepithelial permeability of HIV protease inhibitors. For amprenavir, indinavir, lopinavir, ritonavir, and saquinavir, cells expressing the MDR1 1199A polymorphism displayed significantly higher transepithelial efflux than wild-type cells. However, it appeared that the observed differences were due primarily to changes in permeability in the apical-to-basolateral direction in these polarized cells. Therefore, the impact of G1199A may be most significant on absorption of HIV protease inhibitors from the intestine and penetration into the brain.; Collectively, my dissertation research has provided direct evidence that the G1199A polymorphism leads to functional changes in P-gp activity at the cellular level. The impact on pharmacokinetics and therapeutic effects remain to be evaluated. The unique assays and recombinant cell expression system developed as a part of this thesis will be pivotal in evaluating the impact and defining the role of MDR1 genetic polymorphisms in interindividual variability and drug-drug interactions.