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 show potentially important complications. These include increased expression of and p-glycoprotein and Mrp2 transport activity in brain capillaries of rodents dosed with drugs that activate the nuclear receptor, PXR. Similar increases were found in transgenic mice expressing human PXR and exposed to the antibiotic, rifampin, a specific hPXR ligand. Rifampin-induced mice exhibited a tightened blood-brain barrier to the p-glycoprotein substrate, methadone, as evidenced by a 75% reduction in its antinociceptive effect. Since patients are rarely given only one drug at a time and since many commonly prescribed drugs are hPXR 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 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. ? ? 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.
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