Modulation of P-glycoprotein at the blood-brain barrier: opportunities to improve central nervous system pharmacotherapy

Abstract

Pharmacotherapy of central nervous system (CNS) disorders (e.g., neurodegenerative diseases, epilepsy, brain cancer, and neuro-AIDS) is limited by the blood-brain barrier. P-glycoprotein, an ATP-driven, drug efflux transporter, is a critical element of that barrier. High level of expression, luminal membrane location, multispecificity, and high transport potency make P-glycoprotein a selective gatekeeper of the blood-brain barrier and thus a primary obstacle to drug delivery into the brain. As such, P-glycoprotein limits entry into the CNS for a large number of prescribed drugs, contributes to the poor success rate of CNS drug candidates, and probably contributes to patient-to-patient variability in response to CNS pharmacotherapy. Modulating P-glycoprotein could therefore improve drug delivery into the brain. Here we review the current understanding of signaling mechanisms responsible for the modulation of P-glycoprotein activity/expression at the blood-brain barrier with an emphasis on recent studies from our laboratories. Using intact brain capillaries from rats and mice, we have identified multiple extracellular and intracellular signals that regulate this transporter; several signaling pathways have been mapped. Three pathways are triggered by elements of the brain's innate immune response, one by glutamate, one by xenobiotic-nuclear receptor (pregnane X receptor) interactions, and one by elevated beta-amyloid levels. Signaling is complex, with several pathways sharing common signaling elements [tumor necrosis factor (TNF) receptor 1, endothelin (ET) B receptor, protein kinase C, and nitric-oxide synthase), suggesting a regulatory network. Several pathways include autocrine/paracrine elements, involving release of the proinflammatory cytokine, TNF-alpha, and the polypeptide hormone, ET-1. Finally, several steps in signaling are potential therapeutic targets that could be used to modulate P-glycoprotein activity in the clinic.

Figure 1

Localization of efflux transporters at the blood-brain barrier. Transporters shown have been demonstrated to be expressed in the brain capillary endothelium at the protein level. Arrows indicate direction of substrate transport; ABC transporters are marked with ATP/ADP hydrolysis. P-glycoprotein, multidrug resistance protein (Mrp) isoforms 1, 2, and 4, and breast cancer resistance protein (BCRP) are expressed in the luminal membrane. However, localization of Mrps within the brain capillary endothelium is still controversial (see, e.g., (Dallas et al., 2006; Loscher and Potschka, 2005)). We have localized Mrp2 and Mrp4 to the luminal membrane of rat brain capillaries (Bauer et al., 2008) and thus placed them there in the diagram. The organic anion transporting polypeptide 2, Oatp2, is localized to both luminal and abluminal membranes (Gao et al., 1999); Oatp3 has been detected in the abluminal membrane (Ohtsuki et al., 2004).

Figure 2

Expression of P-glycoprotein at the blood-brain barrier. (A) Western blot showing enrichment of P-glycoprotein from whole rat brain homogenate to capillary lysate to isolated capillary membranes. Note that P-glycoprotein expression levels are highest in capillary membranes (1 μg total protein loading) compared to brain and renal brush border membranes (10 μg total protein loading). (B) Immunostaining of an isolated rat brain capillary for P-glycoprotein (green). Nuclei are stained with propidium iodide (red). Note the “rail road track”-like P-glycoprotein staining of the luminal membrane that defines the capillary lumen and outlines trapped red blood cells. (C) Genetically knocking out P-glycoprotein increases drug levels in the brain. Plasma levels are not changed substantially, indicating a key role for P-glycoprotein at the blood-brain barrier. Data shown for each drug is the per cent increase in the two compartments calculated from data for wild-type and P-glycoprotein-null mice (graphed from data generated by Schinkel and coworkers and compiled in (Mizuno et al., 2003)).

Figure 3

Proinflammatory signaling to P-glycoprotein in rat brain capillaries. (A) Time course showing changes in P-glycoprotein-mediated transport activity in isolated rat brain capillaries exposed to TNF-α and ET-1. Note the rapid reduction in activity and the delayed increase over control levels. The inset shows Western blots of capillary membranes after 1 h and 6 h of exposure. Note the absence of change in P-glycoprotein expression after 1 h and the dramatic change after 6 h (Bauer et al., 2007a). These blots were processed using different exposure times and expression levels cannot be compared across blots. We showed constant P-glycoprotein expression in capillaries over 6 h of incubation in control medium (Bauer et al., 2007a). (B) LPS, TNF-α and ET-1 signaling to P-glycoprotein in the short-term (rapid reduction of transport activity (Hartz et al., 2004; 2006)). (C) TNF-α and ET-1 signaling to P-glycoprotein in the long-term (increased transport activity and transporter expression (Bauer et al., 2007a)).

Figure 4

Network of signaling mechanisms that modulate P-glycoprotein activity in rodent brain capillaries.. Shown is a compilation of signaling pathways disclosed over the past several years by our studies on rat and mouse brain capillaries. Each step has been validated using pharmacological tools or knockout mice. At this time, two of these pathways, PXR and glutamate, have been validated in vivo. For details see: (Bauer et al., 2004; Bauer et al., 2007a; Bauer et al., 2007b; Bauer et al., 2006; Hartz et al., 2004; 2006).