Despite the success of specific transport inhibitors in improving drug delivery to the brain in animal studies, the results have not been translated into the clinic. One problem is that we possess incomplete knowledge about transport function in the human BBB in situ. Indeed, it has been suggested that P-glycoprotein is present in excess in brain capillaries. As a result, more complete inhibition of transport activity would be needed in vivo
than one would predict based in vitro
dose response curves. Consistent with this view, limited experiments using positron emission spectroscopy in primates and human subjects showed more modest increases in brain levels than expected when the labeled drugs are given with P-glycoprotein inhibitors [63
]. In addition, preliminary results from clinical trials suggest that systemic toxicity of specific P-glycoprotein inhibitors limits the ability to achieve free plasma concentrations sufficient to abolish transporter activity. Thus, for humans, the full impact of P-glycoprotein inhibition on drug accumulation in the brain is not known for any drug, making an understanding of the mechanisms that regulate basal transporter activity even more critical.
Rapid (minutes) and reversible changes in transport mediated by certain ABC transporters occurs in hepatocytes and renal proximal tubules [64
]. In rat brain capillaries, similar modulation of transport activity occurs in response to three separate signaling pathways. In the first pathway, 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 [14
]. As shown in , signaling involves ligand binding to toll-like receptor-4 (TLR4), tumor necrosis factor −α receptor 1 (TNFR1) or ETB
R, followed by activation of NOS and PKC. All of these steps occur on or in capillary endothelial cells.
Figure 2 Signals that rapidly decrease P-glycoprotein transport activity at the BBB. Two signaling pathways (proinflammatory and vascular endothelial growth factor (VEGF) – induced) were initially described in rat brain capillaries through the use of pharmacological (more ...)
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 time window needed to deliver P-glycoprotein-excluded drug to the CNS [25
]. To that end, we have used pharmacological tools to identify the specific signals in the pathway downstream of ETB
R. Recent experiments show that iNOS activation is the penultimate identified step in the pathway (Rigor and Miller, unpublished data) and that activation of PKCβI is the final identified step [66
]. The latter result was validated in vivo
using brain perfusion. Treatment of rats with 12-deoxyphorbol 13-phenylacetate 20-acetate (dPPA), a specific PKCβI agonist, rapidly and specifically increased cyclosporine A-sensitive brain uptake of 14
C-verapamil, indicating loss of P-glycoprotein activity. dPPA did not affect uptake of 14
C-Sucrose, a sensitive measure of changes in tight junction permeability [66
]. Thus, targeted signaling through PKCβI has the potential to enhance delivery of therapeutic drugs to the brain. Although there would certainly be problems using phorbol ester derivatives like dPPA in the clinic, this PKCβI activator provided excellent proof-of-principle. One could envision an engineered peptide or small molecule PKCβI activator to enhance delivery of pharmacotherapeutics to treat brain tumors and other CNS diseases.
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 this growth factor is associated with neurological disease, brain injury and BBB dysfunction [67
]. Exposing isolated rat brain capillaries to VEGF acutely and reversibly decreases P-glycoprotein transport activity without decreasing transport protein expression or opening tight junctions [68
]. This effect is blocked by inhibitors of the VEGF receptor, Flk-1 and Src kinase, but not by inhibitors of phosphatidylinostitol-3 (PI3)-kinase or PKC. VEGF also increases Tyr-14 phosphorylation of caveolin-1 and this phosphorylation is blocked by a Src kinase inhibitor (PP2). Previous studies using brain capillary endothelial cells suggested a role for caveolin-1 in regulation of P-glycoprotein activity [69
]. 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 systemic PP2 [68
]. These findings imply that P-glycoprotein activity is 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 modulate P-glycoprotein activity acutely and thus improve brain drug delivery.
The third pathway involves estrogen regulation of Bcrp activity. Estrogen regulates BCRP expression in multiple tissues, where transporter expression is higher in males than in females [71
]. In brain capillaries from rats and mice, there are no such differences, presumably due to estrogen production by brain aromatase [73
]. Brain capillaries from male and female rats and mice respond identically to nanomolar concentrations of 17-β-estradiol (E2) by rapidly and reversibly reducing transport mediated by Bcrp [73
]. Such effects are independent of transcription and translation. Experiments with estrogen receptor (ER) agonists and antagonists and with brain capillaries from ER knockout mice indicate non-genomic E2 signaling through both ERα and ERβ. Initial experiments implicate PI3-kinase and AKT as downstream elements of ER signaling and as potential therapeutic targets [73
]. On the one hand, these findings suggest a simple strategy to increase brain penetration of chemotherapeutics that are Bcrp substrates, e.g., topotecan and imatinib [7
]. On the other hand, they identify one potential mechanism by which potent environmental estrogens could alter BBB function, an area in need of further study.
Rapid and reversible loss of P-glycoprotein and Bcrp transport activity indicates modulation of transporter function over a time scale of minutes. Although the mechanistic basis for this is unclear, one could envision two general classes of underlying mechanism: first, trafficking of the transporter away from the cell surface and second, modification of the transporter or its immediate microenvironment in the plasma membrane. In hepatocytes, there is direct evidence for rapid trafficking of P-glycoprotein and other ABC transporters between intracellular sites and the canalicular membrane [64
]. Both caveoli and lipid rafts have been implicated in regulation of P-glycoprotein in endothelial cells [49
] and BCRP in tumor cells [74
]. Our unpublished experiments with isolated rat brain capillaries suggest an essential cytoskeletal contribution to both TNF-α and VEGF signaling, it is not yet certain whether the cytoskeleton-dependent step is proximal to the loss of activity or whether it indicates translocation.