P-glycoprotein trafficking as a therapeutic target to optimize CNS drug delivery

Abstract

The primary function of the blood-brain barrier (BBB)/neurovascular unit is to protect the central nervous system (CNS) from potentially harmful xenobiotic substances and maintain CNS homeostasis. Restricted access to the CNS is maintained via a combination of tight junction proteins as well as a variety of efflux and influx transporters that limits the transcellular and paracellular movement of solutes. Of the transporters identified at the BBB, P-glycoprotein (P-gp) has emerged as the transporter that is the greatest obstacle to effective CNS drug delivery. In this chapter, we provide data to support intracellular protein trafficking of P-gp within cerebral capillary microvessels as a potential target for improved drug delivery. We show that pain-induced changes in P-gp trafficking are associated with changes in P-gp's association with caveolin-1, a key scaffolding/trafficking protein that colocalizes with P-gp at the luminal membrane of brain microvessels. Changes in colocalization with the phosphorylated and nonphosphorylated forms of caveolin-1, by pain, are accompanied by dynamic changes in the distribution, relocalization, and activation of P-gp "pools" between microvascular endothelial cell subcellular compartments. Since redox-sensitive processes may be involved in signaling disassembly of higher-order structures of P-gp, we feel that manipulating redox signaling, via specific protein targeting at the BBB, may protect disulfide bond integrity of P-gp reservoirs and control trafficking to the membrane surface, providing improved CNS drug delivery. The advantage of therapeutic drug "relocalization" of a protein is that the physiological impact can be modified, temporarily or long term, despite pathology-induced changes in gene transcription.

Keywords: ATP-binding cassette transporter; Blood–brain barrier; Drug delivery; Neurovascular unit; P-glycoprotein; Peripheral inflammatory pain; Protein trafficking; Reactive oxygen species; Redox-sensitive protein pools.

Figure 1. Transporters expressed in cells comprising the neurovascular unit (NVU)

A large number of transporters are expressed on capillary endothelial cells, astrocytes, microglia, and neurons. Transporter systems aid in transport of nutrients, peptides, drugs and ions into the brain parenchyma and as well as efflux of waste and potentially neurotoxic substance out of the brain. Arrows indicate the proposed direction of substrate transport. (Adapted from (Sanchez-Covarrubias et al., 2014)

Figure 2. Methods of drug transport across the blood - brain barrier

This figure describes the various routes of delivery of xenobiotics across the BBB from transcytosis to paracellular delivery to various types of transporter-based delivery. (Adapted from (Sanchez-Covarrubias et al., 2014)

Figure 3. P-glycoprotein transporter – can it be targeted?

Although preclinical evidence suggests that P-glycoprotein transport activity can be modulated with small molecule inhibitors, clinical evidence indicates that this approach cannot work. Use of small molecule inhibitors to block P-glycoprotein in clinical settings has resulted in significant toxicity associated with increased deposition of drug in peripheral tissues or due to high concentrations of the inhibitor itself. Morphine is a good example of the perils of blocking P-glycoprotein to increase drug delivery to the brain. In the setting of functional P-glycoprotein, only 0.02% of systemic morphine is able to permeate the blood-brain barrier. Blockade of P-glycoprotein at the blood-brain barrier would significantly increase this amount and lead to clinically significant adverse drug reactions (i.e., seizures).

Figure 4. P-glycoprotein (P-gp) at the Blood-Brain Barrier: the Greatest Molecular Challenge to CNS Drug Delivery

Note the significant cross section of drugs and xenobiotics that are all substrates for P-gp.

Figure 5. P-glycoprotein (P-gp) Trafficking within Caveolin-Enriched Domains modulated by Peripheral Inflammatory Pain

These data show that the previously characterized increase, in vivo, of P-glycoprotein functional activity, at 3 h post inflammatory pain, was associated with a dynamic redistribution of P-glycoprotein between micro-vascular, endothelial cells, subcellular compartments. The top panel shows the typical OptiPrep density gradient profile of fractions from previously intact rat microvessels. P-gp and the key scaffolding protein caveolin were both shown to be associated with luminal membrane enriched fractions 15 and 16 (Bottom panel) and dynamically trafficked to higher density fractions after the pain stimulus. No trafficking change was noted for the brain efflux transporter Mrp4 after a peripheral pain stimulus to the rats. (Adapted from (McCaffrey et al., 2012)

Figure 6

Model Indicating Proposed Molecular Targets of Peripheral Inflammatory Pain-Induced Reactive Oxygen Species (ROS) that Account for the Compromised Blood-Brain Barrier Observed During Peripheral Inflammatory Pain.