Functional Expression of ABC Transporters at Blood-tissue Barriers: Relevance to Antiretroviral Drug Tissue Distribution

Although combination antiretroviral therapy is successful in reducing HIV-1 viral loads in plasma, several tissue compartments i.e., the brain and male genital tract have been identified as viral reservoirs which can harbor actively replicating HIV-1 virus (Chan, 2005;Dahl, et al., 2010;Smith, et al., 2007;Solas, et al., 2003). In addition to harboring the virus, several studies have observed antiretroviral drugs present at sub-therapeutic concentrations in these tissue compartments that may contribute to the formation of drug resistance and allow systemic viral repopulation. ATP-binding cassette drug efflux transporters i.e., P-glycoprotein and breast cancer resistance protein, have been previously shown to efflux antiretroviral drugs at the level of blood-brain barrier (Dallas, et al., 2004a;Lee, et al., 2007;Lee and Bendayan, 2004;Ronaldson, et al., 2004;Ronaldson, et al., 2007); however, few studies have examined the role of these transporters at the blood-testis barrier. The overall goal of this thesis was to investigate the functional expression of ATP-binding cassette drug efflux transporters at tissue-plasma barriers i.e., the blood-brain barrier and the blood-testis barrier, and provide insight into the role that these transporters play in limiting the distribution of antiretroviral drugs into these potential sanctuary tissues. We demonstrated that P-glycoprotein, breast cancer resistance protein and multidrug resistance associated protein-1 are localized and expressed at the plasma membrane in primary human Sertoli cells and mouse Sertoli cell culture systems using quantitative polymerase chain reaction, immunoblotting and confocal immunofluorescence microscopy, respectively. In mouse Sertoli cell culture systems, using radiolabeled and fluorescent substrates of P-glycoprotein, breast cancer resistance protein and multidrug resistance associated proteins; we demonstrated that these transporters are not only expressed, but also functional in this cell system. We observed that several of the antiretroviral drugs can serve as both, substrates and inhibitors of these transporters at clinically relevant concentrations, including several of the protease inhibitors. We further examined the role of drug transporters in the tissue distribution of atazanavir and observed that P-glycoprotein/breast cancer resistance protein (Mdr1a/1b-/-. Abcg2-/-) knockout mice and wild-type mice pre-treated with the P-glycoprotein and breast cancer resistance protein inhibitor, elacridar, have enhanced accumulation of atazanavir in brain and testes tissues compared to control mice. In addition, we investigated the role of ritonavir as a potential inhibitor of P-glycoprotein and breast cancer resistance protein, using Cyp3a-/- (8-gene) knockout mouse model, and demonstrated that ritonavir can modestly enhance the accumulation of atazanavir in the brain and testes tissues. To better understand the role of HIV-1 viral proteins on the mRNA expression of ATP-binding cassette drug efflux transporters, we examined their expression in a HIV-1 transgenic rat model, and showed that HIV-1 viral transgene expression can affect the expression of ATP-binding cassette drug efflux transporters in both a tissue- and age-dependent manner. The results from these studies have generated new information on the mechanisms of transport and distribution of antiretroviral drugs at the blood-testis barrier and the blood-brain barrier, and could better guide more effective antiretroviral therapy to reduce potential sexual transmission of HIV-1 and prevention of HIV-1 associated neurological complications.

Keywords: Antiretroviral drugs
atazanavir
distribution
Drug transporters
HIV
knockout mouse model
Advisor: Reina, Bendayan