Use of antiviral drugs to treat HIV infection is often limited by the inability of compounds to access sites of infection. In this regard, the brain provides a sanctuary for HIV. Three barriers can confound treatment of virus within the brain: the blood-brain barrier at the capillary endothelium (blocks drug entry), the blood-CSF barrier at the choroid plexus (facilitates drug efflux) and the plasma membranes of the glial cells that harbor HIV (limits access to virus).? ? Blood-Brain Barrier: HIV protease inhibitors penetrate the blood-brain barrier poorly and our experiments with isolated brain capillaries from rat and pig show that ATP-driven drug export pumps, e.g., p-glycoprotein and Mrp2, handle ritonavir and saquinavir. Our in vitro and in vivo experiments with animal models suggest 3 strategies for circumventing this barrier: specific inhibition of p-glycoprotein activity, transient opening of brain capillary tight junctions and rapid and reversible reduction in p-glycoprotein transport function signaled by the brain's innate immune response. However, our experiments also show three potentially important complications. First, we found increased protein expression of and p-glycoprotein and Mrp2 transport activity in brain capillaries of rodents dosed with drugs that activate the nuclear receptor, PXR. Since patients are rarely given only one drug at a time and since many commonly prescribed drugs (including HIV protease inhibitors) are PXR ligands, these findings raise the possibility of a further tightening of the barrier in patients receiving polypharmacy. Second, chronically exposing rat brain capillaries to low levels of the proinflammatory cytokine, TNF-alpha, substantially increased p-glycoprotein (but not Mrp2) activity and expression. This increase was signaled through TNF-R1, endothelin A/B receptors, NOS, PKC and NF-kB. Thus, low level activation of the brain's innate immune response, a common finding in many CNS diseases including HIV, caused a tightening of the specific blood-brain barrier, which would impede entry of drugs that are substrates for p-glycoprotein, including HIV protease inhibitors. Finally, we are now extending these studies to include effects of TLR3 activation, since this receptor is expressed in brain capillaries where it recognizes and responds to viral products. ? ? Glial Cells: In the CNS, both astrocytes and microglia harbor HIV and microglia are the site of productive infection. Although astrocytes are known to express several xenobiotic efflux pumps, little is known about efflux pump expression in microglia or about how pump expression in glia is regulated. We are using primary cultures of rat microglia and astrocytes to examine changes in accumulation of saquinavir and expression of p-glycoprotein and Mrp1 after exposure to inflammogens (LPS and double-stranded RNA, a viral product) and proinflammatory cytokines (IL-1, TNF-alpha). In both cells, specific inhibition of p-glycoprotein or Mrp1 activity increased saquinavir accumulation, consistent with a role for both transporters in drug efflux. Exposure to low levels of LPS or to double-stranded RNA further reduced saquinavir accumulation, primarily a result of increased p-glycoprotein activity. Western blots showed LPS increased p-glycoprotein expression in astrocytes but not in microglia. Neither Mrp1 activity nor Mrp1 expression was affected in either cell. Inhibitor studies implicated IL-1 and iNOS in signaling from TLR4 (the LPS receptor) to p-glycoprotein. These studies show upregulation of p-glycoprotein expression in primary astrocytes and microglia occurs by distinct, cell-specific mechanisms.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Intramural Research (Z01)
Project #
1Z01ES080060-09
Application #
7734500
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
9
Fiscal Year
2008
Total Cost
$577,189
Indirect Cost
City
State
Country
United States
Zip Code
Hartz, Anika M S; Bauer, Bjorn; Block, Michelle L et al. (2008) Diesel exhaust particles induce oxidative stress, proinflammatory signaling, and P-glycoprotein up-regulation at the blood-brain barrier. FASEB J 22:2723-33
Miller, David S; Bauer, Bjorn; Hartz, Anika M S (2008) Modulation of P-glycoprotein at the blood-brain barrier: opportunities to improve central nervous system pharmacotherapy. Pharmacol Rev 60:196-209
Bauer, Bjorn; Hartz, Anika M S; Lucking, Jonathan R et al. (2008) Coordinated nuclear receptor regulation of the efflux transporter, Mrp2, and the phase-II metabolizing enzyme, GSTpi, at the blood-brain barrier. J Cereb Blood Flow Metab 28:1222-34
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Wielgus, Albert R; Chignell, Colin F; Miller, David S et al. (2007) Phototoxicity in human retinal pigment epithelial cells promoted by hypericin, a component of St. John's wort. Photochem Photobiol 83:706-13
Bauer, Bjorn; Hartz, Anika M S; Miller, David S (2007) Tumor necrosis factor alpha and endothelin-1 increase P-glycoprotein expression and transport activity at the blood-brain barrier. Mol Pharmacol 71:667-75
Zhang, Wei; Dallas, Shannon; Zhang, Dan et al. (2007) Microglial PHOX and Mac-1 are essential to the enhanced dopaminergic neurodegeneration elicited by A30P and A53T mutant alpha-synuclein. Glia 55:1178-88
Bauer, Bjorn; Yang, Xiaodong; Hartz, Anika M S et al. (2006) In vivo activation of human pregnane X receptor tightens the blood-brain barrier to methadone through P-glycoprotein up-regulation. Mol Pharmacol 70:1212-9
Wang, Tongguang; Zhang, Wei; Pei, Zhong et al. (2006) Reactive microgliosis participates in MPP+-induced dopaminergic neurodegeneration: role of 67 kDa laminin receptor. FASEB J 20:906-15
Dallas, Shannon; Miller, David S; Bendayan, Reina (2006) Multidrug resistance-associated proteins: expression and function in the central nervous system. Pharmacol Rev 58:140-61

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