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Improvement of central nervous system pharmacotherapy by modulation of Pglycoprotein at the blood-brain barrier

David S. Miller1*
  • 1 NIH/NIEHS, Research Triangle Park, United States

One defining feature of the brain capillary phenotype is the expression of ATP-driven, drug efflux pumps (ATP-binding cassette (ABC) transporters) on the luminal, blood-facing plasma membrane of the endothelial cells. These are members of the ABC B, C and G families of transporters that collectively restrict uptake of a large number of lipophilic xenobiotics that, based on structure, should readily diffuse across endothelial cell membranes. Of these transporters, we have the most complete picture of function and regulation for P-glycoprotein (ABCB1), which handles a surprisingly large number of therapeutic drugs (polyspecificity) and is expressed at high levels in the brain capillary endothelium [1]. P-glycoprotein has proven to be a primary obstacle to delivery of chemotherapeutics, anti-epileptics and HIV protease inhibitors to the brain. P-glycoprotein knockout mice have been available for over a decade and for many drugs that are P-glycoprotein substrates these animals exhibit large increases in brain to plasma concentration ratios over wild-type controls. In addition, Animal studies have shown that reductions in P-glycoprotein transport activity have the potential to increase effectiveness of chemotherapeutics against implanted human tumors [2, 3] and antiepileptic drugs against seizures. Thus, it is important to appreciate the physiological and pathophysiological mechanisms that regulate this transporter, with a view towards 1) modulating transporter activity to improve drug delivery, 2) understanding the factors contributing to patient-to-patient variability in response to CNS-acting drugs, and 3) augmenting CNS protection in the face of challenges from the periphery. Here I review recent work largely from my laboratory focused on and the mechanisms that underlie changes in P-glycoprotein transporter expression and activity at the blood-brain barrier.
Regulation of Expression – In barrier and excretory tissues, xenobiotics, including commonly prescribed drugs and toxicants, increase P-glycoprotein expression and transport activity by acting through the pregnane-X receptor (PXR) and the constitutive androstane receptor (CAR). Recent studies show expression of both PXR and CAR in brain capillaries or brain capillary endothelial cells from mouse, rat, pig and human [4-7]. Exposing rat or mouse brain capillaries to PXR, CAR or glucocorticoid receptor (GR) ligands increases transport activity and protein expression of Pglycoprotein ([4, 8] and Wang and Miller unpublished data; Fig. 1). Dosing rodents with PXR and CAR ligands increases transport activity and protein expression of P-glycoprotein in brain capillaries. Importantly, dosing hPXR transgenic mice with rifampin, at a dose that resulted in free plasma drug levels equivalent to those seen in patients undergoing a course of rifampin treatment, increased Pglycoprotein in brain capillaries 2-3-fold. In these rifampin-dosed hPXR mice, the antinociceptive effects of injected methadone were reduced by 70% [8]. Thus, two master regulators of xenobiotic defense, PXR and CAR, function in brain capillaries to tighten the selective blood-brain barrier.
Inflammation and the generation of reactive oxygen (and nitrogen) species are cofactors in nearly every CNS disease and there is substantial evidence that severe inflammatory and oxidative stress can acutely disrupt the blood-brain barrier at the level of the tight junctions. Recent studies with brain capillary endothelial cells and intact capillaries have mapped in detail several stress-related signaling pathways that target nuclear transcription factors to increase P-glycoprotein expression in brain capillaries and brain endothelial cells [9, 10]. These pathways involve elements of the barrier’s response to inflammation and oxidative stress. Not surprisingly, these pathways converge on the transcription factors Nf-κB and AP-1, which have been implicated in responses leading to both cell protection and cell death (Fig. 1).
Altered expression of P-glycoprotein at the blood-brain barrier accompanies multiple neuropathologies. Reduced transporter expression is associated with Alzheimer’s disease [11], Jacob- Creutzfeld disease [12], Parkinson’s disease [13], HIV infection [14] and normal aging [15] and increased expression of P-glycoprotein, Mrp1, Mrp2 and Bcrp is associated with epileptic seizures [16]. Evidence connecting efflux transporter overexpression with pharmacoresistance to antiepileptic drugs is strongest for P-glycoprotein [16] and recent studies with animal models suggest both a mechanistic basis for such resistance and a therapeutic strategy for overcoming it. Bauer et al [17] demonstrated that the neurotransmitter, glutamate, signals through an NMDA receptor, cyclooxygenase- 2 (COX-2), prostaglandin E2 and Nf-κB to increase expression of P-glycoprotein in brain capillaries (Fig. 1). In vivo studies [18] have shown that targeting NMDA receptors, COX-2 and a prostaglandin E2 receptor (EP-1) blocks seizure-induced upregulation of P-glycoprotein, thus validating the major elements of the signaling system (Fig. 1).
Regulation of Transport Activity - In spite of the success of specific transport inhibitors in improving drug delivery to the brain in animal studies, these results have not translated well to the clinic. Thus, an understanding of the mechanisms that regulate basal transporter activity is even more critical. Recent studies have disclosed two signaling pathways that modulate basal activity of Pglycoprotein. First, exposing capillaries to low levels of LPS, TNF-α or endothelin-1 (ET-1) causes a rapid and fully reversible loss of P-glycoprotein transport function with no change in protein expression; inhibitors of protein synthesis are without effect and neither Mrp2-mediated transport nor tight junctional permeability is altered [4, 19]. As shown in Fig. 1, signaling involves ligand binding to toll-like receptor-4 (TLR4), TNF-R1 or ETBR, followed by activation of NOS and PKC. All of these steps occur in series on or in capillary endothelial cells. In the first publication describing these findings we speculated that this rapid and reversible loss of specific transport activity in the capillary endothelium could provide the window in time needed to deliver P-glycoprotein-excluded drugs to the CNS with minimal loss of protection [20]. To that end, we have used pharmacological tools to identify PKCβI as a downstream signal in the pathway (Rigor et al, in press). The latter result was validated in vivo using brain perfusion. Treatment of rats with a specific PKCβI agonist, rapidly and specifically increased brain uptake of the P-glycoprotein substrate, verapamil, without altering uptake of sucrose, a sensitive measure of changes in tight junction permeability (Rigor et al, submitted). Thus, targeting signaling through PKCβI has the potential to enhance delivery of therapeutic drugs to the brain.
The second distinct pathway that signals rapid, reversible loss of P-glycoprotein activity in brain capillaries is initiated by vascular endothelial growth factor (VEGF). Increased brain expression of VEGF is associated with neurological disease, brain injury and blood-brain barrier dysfunction [21]. Exposing isolated rat brain capillaries to VEGF acutely and reversibly decreases P-glycoprotein transport activity without decreasing transport protein expression or opening tight junctions (Hawkins et al, in press; Fig. 1). This effect is blocked by inhibitors of the VEGF receptor, flk-1, and Src kinase. In vivo, intracerebroventricular injection of VEGF increases brain distribution of the P-glycoprotein substrates, morphine and verapamil, but not the tight junction marker, sucrose; this effect is blocked by a Src kinase inhibitor (Hawkins et al, in press). These findings imply that P-glycoprotein activity can be acutely diminished in pathological conditions associated with increased brain VEGF expression. They also imply that once the more downstream elements of VEGF signaling to P-glycoprotein are identified, they could provide additional accessible targets that could be used to acutely modulate Pglycoprotein activity and thus improve brain drug delivery.

Conclusions -It is now clear from studies with animal models and patient samples that expression/activity of P-glycoprotein at the blood-brain barrier can be a “moving target,” affected by disease, pharmacotherapy, diet and genetics (single nucleotide polymorphisms and likely epigenetics). Thus, an understanding of signaling could provide opportunities to both selectively fine tune barrier function up or down and begin to identify the barrier-based and external factors that contribute to patient-to-patient variability in response to CNS-acting drugs. This work was supported by the Intramural Research Program of the National Institute Environmental Health Sciences, NIH.

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References

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Conference: Pharmacology and Toxicology of the Blood-Brain Barrier: State of the Art, Needs for Future Research and Expected Benefits for the EU, Brussels, Belgium, 11 Feb - 12 Feb, 2010.

Presentation Type: Oral Presentation

Topic: Presentations

Citation: Miller DS (2010). Improvement of central nervous system pharmacotherapy by modulation of Pglycoprotein at the blood-brain barrier. Front. Pharmacol. Conference Abstract: Pharmacology and Toxicology of the Blood-Brain Barrier: State of the Art, Needs for Future Research and Expected Benefits for the EU. doi: 10.3389/conf.fphar.2010.02.00010

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Received: 24 Feb 2010; Published Online: 24 Feb 2010.

* Correspondence: David S Miller, NIH/NIEHS, Research Triangle Park, North Carolina, United States, miller28@mail.nih.gov