HIV protease inhibitors, such as, ritonavir and saquinavir, are substrates for xenobiotic efflux pumps, e.g., P-glycoprotein and Mrp2 and thus penetrate the blood-brain barrier poorly. To map the extracellular and intracellular signals that regulate these transporters, we use 1) pharmacological tools, 2) intact brain capillaries from rats and mice (including transgenics and knockouts), 2) fluorescent substrates, 3) confocal imaging to measure transport function, 4) Western blotting to measure transporter expression, and 5) brain perfusion in rats and mice to validate signaling-based changes in blood-brain barrier transporter function in vivo. Our recent studies have focused on identifying signals that modify efflux pump activity at the barrier. One strategy for improving CNS delivery of drugs that are P-glycoprotein substrates involves activating protein kinase C (PKC) isoform beta1 or sphingolipid signaling at the blood-brain barrier. This causes a rapid and reversible reduction in basal P-glycoprotein transport activity in isolated brain capillaries and in intact rats. We have now identified a drug, Fingolimod (FTY720) currently in use in the clinic, that will target this signaling system and reduced P-glycoprotein tranport activity in vitro and in vivo. In vivo, such signaling increases brain uptake of drugs that are P-glycoprotein substrates. Thus, targeting signals that regulate basal activity of this transporter increases delivery of therapeutic drugs to the brain, including HIV protease inhibitors. These in vitro and in vivo experiments with animal models suggest a specific strategy for modifying this barrier to improve drug delivery, but also potentially important complications of polypharmacy related to xenobiotic upregulation of efflux transporter expression. A second project involves understanding in glial cells how the activity of efflux transporters and their handling of HIV protease inhibitors are affected by inflammation. Active HIV infection within the central nervous system (CNS) is confined primarily to microglia. The glial cell compartment acts as a viral reservoir behind the blood-brain barrier. It provides an additional roadblock to effective pharmacological treatment via expression of multiple drug efflux transporters, including P-glycoprotein. HIV/AIDS patients frequently suffer bacterial and viral co-infections, leading to deregulation of glial cell function and release of pro-inflammatory mediators including cytokines, chemokines, and nitric oxide. To better define the role of inflammation in decreased HIV drug accumulation into CNS targets, we measured accumulation of the antiretroviral saquinavir in purified cultures of rodent microglia exposed to the prototypical inflammatory mediator lipopolysaccharide (LPS). LPS did not directly inhibit saquinavir transport, and did not affect P-glycoprotein protein expression. LPS exposure did not alter RNA and/or protein expression of other transporters including multidrug resistance-associated protein 1 and several solute carrier uptake transporters. LPS exposure decreased saquinavir accumulation in microglia by a complex mechanism involving NF-κβand the MEK1/2. These data provide new pharmacological insights into why microglia act as a difficult-to-treat viral sanctuary site.

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Miller, D S (2015) Regulation of ABC transporters at the blood-brain barrier. Clin Pharmacol Ther 97:395-403
Miller, David S (2014) Sphingolipid signaling reduces basal P-glycoprotein activity in renal proximal tubule. J Pharmacol Exp Ther 348:459-64
Emmert, Dana; Campos, Christopher R; Ward, David et al. (2014) Reversible dimers of the atypical antipsychotic quetiapine inhibit p-glycoprotein-mediated efflux in vitro with increased binding affinity and in situ at the blood-brain barrier. ACS Chem Neurosci 5:305-17
Wang, Xueqian; Campos, Christopher R; Peart, John C et al. (2014) Nrf2 upregulates ATP binding cassette transporter expression and activity at the blood-brain and blood-spinal cord barriers. J Neurosci 34:8585-93
Cartwright, Tara A; Campos, Christopher R; Cannon, Ronald E et al. (2013) Mrp1 is essential for sphingolipid signaling to p-glycoprotein in mouse blood-brain and blood-spinal cord barriers. J Cereb Blood Flow Metab 33:381-8
Dallas, Shannon; Block, Michelle L; Thompson, Deborah M et al. (2013) Microglial activation decreases retention of the protease inhibitor saquinavir: implications for HIV treatment. J Neuroinflammation 10:58
Miller, David S; Cannon, Ronald E (2013) Signaling Pathways that Regulate Basal ABC Transporter Activity at the Blood-Brain Barrier. Curr Pharm Des :
Namanja, Hilda A; Emmert, Dana; Davis, David A et al. (2012) Toward eradicating HIV reservoirs in the brain: inhibiting P-glycoprotein at the blood-brain barrier with prodrug abacavir dimers. J Am Chem Soc 134:2976-80
Zhang, Dan; Hu, Xiaoming; Qian, Li et al. (2011) Microglial MAC1 receptor and PI3K are essential in mediating ýý-amyloid peptide-induced microglial activation and subsequent neurotoxicity. J Neuroinflammation 8:3
Wang, Xueqian; Hawkins, Brian T; Miller, David S (2011) Activating PKC-ýý1 at the blood-brain barrier reverses induction of P-glycoprotein activity by dioxin and restores drug delivery to the CNS. J Cereb Blood Flow Metab 31:1371-5

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