Brain Shuttle Neprilysin reduces central Amyloid-ß levels.

Reducing Amyloid ß (Aß) in the brain is of fundamental importance for advancing the therapeutics for Alzheimer`s disease. The endogenous metallopeptidase neprilysin (NEP) has been identified as one of the key Aß-degrading enzymes. Delivery of NEP to the brain by utilizing the Brain Shuttle (BS) transport system offers a promising approach for clearing central Aß. We fused the extracellular catalytic domain of NEP to an active or inactive BS module. The two BS-NEP constructs were used to investigate the pharmacokinetic/pharmacodynamics relationships in the blood and the cerebrospinal fluid (CSF) in dose-response and multiple dosing. As previously shown, NEP was highly effective at degrading Aß in blood but not in the CSF compartment after systemic administration. In contrast, the NEP with an active BS module led to a significant CSF exposure of BS-NEP, followed by substantial Aß reduction in CSF and brain parenchyma. Our data show that a BS module against the transferrin receptor facilitates the transport of an Aß degrading enzyme across the blood-brain barriers to efficiently reduce Aß levels in both CSF and brain.

PPAR-α, a lipid-sensing transcription factor, regulates blood-brain barrier efflux transporter expression.

Lipid sensor peroxisome proliferator-activated receptor alpha (PPAR- α) is the master regulator of lipid metabolism. Dietary release of endogenous free fatty acids, fibrates, and certain persistent environmental pollutants, e.g. perfluoroalkyl fire-fighting foam components, are peroxisome proliferator-activated receptor alpha ligands. Here, we define a role for peroxisome proliferator-activated receptor alpha in regulating the expression of three ATP-driven drug efflux transporters at the rat and mouse blood-brain barriers: P-glycoprotein (Abcb1), breast cancer resistance protein (Bcrp/Abcg2), and multidrug resistance-associated protein 2 (Mrp2/Abcc2). Exposing isolated rat brain capillaries to linoleic acid, clofibrate, or PKAs increased the transport activity and protein expression of the three ABC transporters. These effects were blocked by the PPAR- α antagonist, GW6471. Dosing rats with 20 mg/kg or 200 mg/kg of clofibrate decreased the brain accumulation of the P-glycoprotein substrate, verapamil, by 50% (in situ brain perfusion; effects blocked by GW6471) and increased P-glycoprotein expression and activity in capillaries ex vivo. Fasting C57Bl/6 wild-type mice for 24 h increased both serum lipids and brain capillary P-glycoprotein transport activity. Fasting did not alter P-glycoprotein activity in PPAR- α knockout mice. These results indicate that hyperlipidemia, lipid-lowering fibrates and exposure to certain fire-fighting foam components activate blood-brain barrier peroxisome proliferator-activated receptor alpha, increase drug efflux transporter expression and reduce drug delivery to the brain.

Effects of deep hypothermic circulatory arrest on the blood brain barrier in a cardiopulmonary bypass model--a pilot study.

BACKGROUND: Neurologic injury is common after cardiac surgery and disruption of the blood brain barrier (BBB) has been proposed as a contributing factor. We sought to study BBB characteristics in a rodent model of cardiopulmonary bypass (CPB) and deep hypothermic circulatory arrest (DHCA). METHODS: Adult rats were subjected to CPB/DHCA or to sham surgery. Analysis included Western blotting of relevant BBB proteins in addition to in vivo brain magnetic resonance imaging (MRI) with a clinically used low-molecular contrast agent. RESULTS: While quantitative analysis of BBB proteins revealed similar expression levels, MRI showed evidence of BBB disruption after CPB/DHCA compared to sham surgery. CONCLUSIONS: Combining molecular BBB analysis and MRI technology in a rodent model is a highly translatable approach to study adverse neurologic outcomes following CPB/DHCA.

Nrf2 upregulates ATP binding cassette transporter expression and activity at the blood-brain and blood-spinal cord barriers.

Activation of nuclear factor E2-related factor-2 (Nrf2), a sensor of oxidative stress, is neuroprotective in animal models of cerebral ischemia, traumatic brain injury, subarachnoid hemorrhage, and spinal cord injury. We show here that Nrf2 activation with sulforaphane (SFN) in vivo or in vitro increases expression and transport activity of three ATP-driven drug efflux pumps at the blood-brain barrier [P-glycoprotein, ATP binding cassette b1 (Abcb1); multidrug resistance-associated protein-2 (Mrp2), Abcc2; and breast cancer resistance protein (Bcrp), Abcg2]. Dosing rats with SFN increased protein expression of all three transporters in brain capillaries and decreased by 50% brain accumulation of the P-glycoprotein substrate verapamil. Exposing rat or mouse brain capillaries to SFN increased P-glycoprotein, Bcrp, and Mrp2 transport activity and protein expression; SFN increased P-glycoprotein activity in mouse spinal cord capillaries. Inhibiting transcription or translation abolished upregulation of P-glycoprotein activity. No such effects were seen in brain capillaries from Nrf2-null mice, indicating Nrf2 dependence. Nrf2 signaled indirectly to increase transporter activity/expression. The p53 inhibitor pifithrin abolished the SFN-induced increase in transporter activity/expression, and the p53-activator nutlin-3 increased P-glycoprotein activity. SFN did not alter P-glycoprotein transport activity in brain and spinal cord capillaries from p53-null mice. Inhibitors of p38 MAPK and nuclear factor κB (NF-κB) blocked the effects of SFN and nutlin-3 on P-glycoprotein activity. These results implicate Nrf2, p53, and NF-κB in the upregulation of P-glycoprotein, Bcrp, and Mrp2 at blood-CNS barriers. They imply that the barriers are tightened selectively (efflux transporter upregulation) by oxidative stress, providing increased neuroprotection, but also reduced penetration of many therapeutic drugs.

Reversible dimers of the atypical antipsychotic quetiapine inhibit p-glycoprotein-mediated efflux in vitro with increased binding affinity and in situ at the blood-brain barrier.

The multidrug resistance transporter P-glycoprotein (P-gp) is highly expressed in the capillary endothelial cells of the blood-brain barrier (BBB) where it functions to limit the brain penetration of many drugs, including antipsychotic agents used to treat schizophrenia. Therefore, in an effort to inhibit the transporter, we designed dimers of the antipsychotic drug and P-gp substrate quetiapine (QT), linked by variable length tethers. In P-gp overexpressing cells and in human brain capillary endothelial hCMEC/D3 cells, the dimer with the shortest tether length (QT2C2) (1) was the most potent inhibitor showing >80-fold better inhibition of P-gp-mediated transport than monomeric QT. The dimers, which are linked via ester moieties, are designed to revert to the therapeutic monomer once inside the target cells. We demonstrated that the addition of two sterically blocking methyl groups to the linker (QT2C2Me2, 8) increased the half-life of the molecule in plasma 10-fold as compared to the dimer lacking methyl groups (QT2C2, 1), while retaining inhibitory potency for P-gp transport and sensitivity to cellular esterases. Experiments with purified P-gp demonstrated that QT2C2 (1) and QT2C2Me2 (8) interacted with both the H- and R-binding sites of the transporter with binding affinities 20- to 30-fold higher than that of monomeric QT. Using isolated rat brain capillaries, QT2C2Me2 (8) was a more potent inhibitor of P-gp transport than QT. Lastly, we showed that QT2C2Me2 (8) increased the accumulation of the P-gp substrate verapamil in rat brain in situ three times more than QT. Together, these results indicate that the QT dimer QT2C2Me2 (8) strongly inhibited P-gp transport activity in human brain capillary endothelial cells, in rat brain capillaries, and at the BBB in an animal model.

Mrp1 is essential for sphingolipid signaling to p-glycoprotein in mouse blood-brain and blood-spinal cord barriers.

At the blood-brain and blood-spinal cord barriers, P-glycoprotein, an ATP-driven drug efflux pump, is a major obstacle to central nervous system (CNS) pharmacotherapy. Recently, we showed that signaling through tumor necrosis factor-α (TNF-α), sphingolipids, and sphingosine-1-phosphate receptor 1 (S1PR1) rapidly and reversibly reduced basal P-glycoprotein transport activity in the rat blood-brain barrier. The present study extends those findings to the mouse blood-brain and blood-spinal cord barriers and, importantly, identifies multidrug resistance-associated protein 1 (Mrp1, Abcc1) as the transporter that mediates S1P efflux from brain and spinal cord endothelial cells. In brain and spinal cord capillaries isolated from wild-type mice, TNF-α, sphingosine, S1P, the S1PR agonist fingolimod (FTY720), and its active, phosphorylated metabolite, FTY720P, reduced P-glycoprotein transport activity; these effects were abolished by a specific S1PR1 antagonist. In brain and spinal cord capillaries isolated from Mrp1-null mice, neither TNF-α nor sphingosine nor FTY720 reduced P-glycoprotein transport activity. However, S1P and FTY720P had the same S1PR1-dependent effects on transport activity as in capillaries from wild-type mice. Thus, deletion of Mrp1 alone terminated endogenous signaling to S1PR1. These results identify Mrp1 as the transporter essential for S1P efflux from the endothelial cells and thus for inside-out S1P signaling to P-glycoprotein at the blood-brain and blood-spinal cord barriers.

1α,25-Dihydroxyvitamin D3-liganded vitamin D receptor increases expression and transport activity of P-glycoprotein in isolated rat brain capillaries and human and rat brain microvessel endothelial cells.

Induction of the multidrug resistance protein 1 (MDR1)/P-glycoprotein (P-gp) by the vitamin D receptor (VDR) was investigated in isolated rat brain capillaries and rat (RBE4) and human (hCMEC/D3) brain microvessel endothelial cell lines. Incubation of isolated rat brain capillaries with 10 nM of the VDR ligand, 1α,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] for 4 h increased P-gp protein expression fourfold. Incubation with 1,25(OH)(2)D(3) for 4 or 24 h increased P-gp transport activity (specific luminal accumulation of NBD-CSA, the fluorescent P-gp substrate) by 25-30%. In RBE4 cells, Mdr1b mRNA was induced in a concentration-dependent manner by exposure to 1,25(OH)(2)D(3). Concomitantly, P-gp protein expression increased 2.5-fold and was accompanied by a 20-35% reduction in cellular accumulation of the P-gp substrates, rhodamine 6G (R6G), and HiLyte Fluor 488-labeled human amyloid beta 1-42 (hAß(42)). In hCMEC/D3 cells, a 3 day exposure to 100 nM 1,25(OH)(2)D(3) increased MDR1 mRNA expression (40%) and P-gp protein (threefold); cellular accumulation of R6G and hAß(42) was reduced by 30%. Thus, VDR activation up-regulates Mdr1/MDR1 and P-gp protein in isolated rat brain capillaries and rodent and human brain microvascular endothelia, implicating a role for VDR in increasing the brain clearance of P-gp substrates, including hAß(42), a plaque-forming precursor in Alzheimer's disease.

Targeting blood-brain barrier sphingolipid signaling reduces basal P-glycoprotein activity and improves drug delivery to the brain.

P-glycoprotein, an ATP-driven drug efflux pump, is a major obstacle to the delivery of small-molecule drugs across the blood-brain barrier and into the CNS. Here we test a unique signaling-based strategy to overcome this obstacle. We used a confocal microscopy-based assay with isolated rat brain capillaries to map a signaling pathway that within minutes abolishes P-glycoprotein transport activity without altering transporter protein expression or tight junction permeability. This pathway encompasses elements of proinflammatory- (TNF-α) and sphingolipid-based signaling. Critical to this pathway was signaling through sphingosine-1-phosphate receptor 1 (S1PR1). In brain capillaries, S1P acted through S1PR1 to rapidly and reversibly reduce P-glycoprotein transport activity. Sphingosine reduced transport by a sphingosine kinase-dependent mechanism. Importantly, fingolimod (FTY720), a S1P analog recently approved for treatment of multiple sclerosis, also rapidly reduced P-glycoprotein activity; similar effects were found with the active, phosphorylated metabolite (FTY720P). We validated these findings in vivo using in situ brain perfusion in rats. Administration of S1P, FTY720, or FTY729P increased brain uptake of three radiolabeled P-glycoprotein substrates, (3)H-verapamil (threefold increase), (3)H-loperamide (fivefold increase), and (3)H-paclitaxel (fivefold increase); blocking S1PR1 abolished this effect. Tight junctional permeability, measured as brain (14)C-sucrose accumulation, was not altered. Therefore, targeting signaling through S1PR1 at the blood-brain barrier with the sphingolipid-based drugs, FTY720 or FTY720P, can rapidly and reversibly reduce basal P-glycoprotein activity and thus improve delivery of small-molecule therapeutics to the brain.

ABC transporter function and regulation at the blood-spinal cord barrier.

We present here an initial characterization of ATP binding cassette (ABC) transporter function and regulation at the blood-spinal cord barrier. We isolated capillaries from rat spinal cords and studied transport function using a confocal microscopy-based assay and protein expression using western blots. These capillaries exhibited transport function and protein expression of P-glycoprotein (Abcb1), multidrug resistance protein 2 (Mrp2, Abcc2), and breast cancer-related protein (Bcrp, Abcg2). Exposing isolated capillaries to dioxin (activates aryl hydrocarbon receptor) increased transport mediated by all three transporters. Brain and spinal cord capillaries from dioxin-dosed rats exhibited increased P-glycoprotein-mediated transport and increased protein expression for all three ABC transporters. These findings indicate similar ABC transporter expression, function, and regulation at the blood-spinal cord and blood-brain barriers.

Nociceptive inhibition prevents inflammatory pain induced changes in the blood-brain barrier.

Previous studies by our group have shown that peripheral inflammatory insult, using the lambda-carrageenan inflammatory pain (CIP) model, induced alterations in the molecular and functional properties of the blood-brain barrier (BBB). The question remained whether these changes were mediated via an inflammatory and/or neuronal mechanism. In this study, we investigated the involvement of neuronal input from pain activity on alterations in BBB integrity by peripheral inhibition of nociceptive input. A perineural injection of 0.75% bupivacaine into the right hind leg prior to CIP was used for peripheral nerve block. Upon nerve block, there was a significant decrease in thermal allodynia induced by CIP, but no effect on edema formation 1 h post-CIP. BBB permeability was increased 1 h post-CIP treatment as determined by in situ brain perfusion of [(14)C] sucrose; bupivacaine nerve block of CIP caused an attenuation of [(14)C] sucrose permeability, back to saline control levels. Paralleling the changes in [(14)C] sucrose permeability, we also report increased expression of three tight junction (TJ) proteins, zonula occluden-1 (ZO-1), occludin and claudin-5 with CIP. Upon bupivacaine nerve block, changes in expression were prevented. These data show that the lambda-carrageenan-induced changes in [(14)C] sucrose permeability and protein expression of ZO-1, occludin and claudin-5 are prevented with inhibition of nociceptive input. Therefore, we suggest that nociceptive signaling is in part responsible for the alteration in BBB integrity under CIP.

Comparative changes in the blood-brain barrier and cerebral infarction of SHR and WKY rats.

Hypertension is involved in the exacerbation of stroke. It is unclear how blood-brain barrier (BBB) tight-junction (TJ) and ion transporter proteins critical for maintaining brain homeostasis contribute to cerebral infarction during hypertension development. In the present study, we investigated cerebral infarct volume following permanent 4-h middle cerebral artery occlusion (MCAO) and characterized the expression of BBB TJ and ion transporter proteins in brain microvessels of spontaneously hypertensive rats (SHR) compared with age-matched Wistar-Kyoto (WKY) rats at 5 wk (prehypertension), 10 wk (early-stage hypertension), and 15 wk (later-stage hypertension) of age. Hypertensive SHR show increased infarct volume following MCAO compared with WKY control rats. BBB TJ and ion transporter proteins, known to contribute to edema and fluid volume changes in the brain, show differential protein expression patterns during hypertension development. Western blot analysis of TJ protein zonula occludens-2 (ZO-2) showed decreased expression, while ion transporter, Na(+)/H(+) exchanger 1 (NHE-1), was markedly increased in hypertensive SHR. Expression of TJ proteins ZO-1, occludin, actin, claudin-5, and Na(+)-K(+)-2Cl(-) cotransporter remain unaffected in SHR compared with control. Selective inhibition of NHE-1 using dimethylamiloride significantly attenuated ischemia-induced infarct volume in hypertensive SHR following MCAO, suggesting a novel role for NHE-1 in the brain in the regulation of ischemia-induced infarct volume in SHR.

Nicotine increases in vivo blood-brain barrier permeability and alters cerebral microvascular tight junction protein distribution.

The blood-brain barrier (BBB) is critical to the health of the central nervous system. The BBB is formed primarily by the presence of tight junctions (TJ) between cerebral microvessel endothelial cells. In light of the known effects of nicotine on endothelial cell biology, the specific effects of nicotine on the in vivo BBB were examined. Using in situ brain perfusion, it was found that continuous administration of nicotine (4.5 mg free base x kg(-1) x day(-1)) for 1 and 7 days led to increased permeability of the BBB to [14C]-sucrose without significant changes in its initial volume of distribution. The expression and distribution of the TJ-associated proteins actin, occludin, claudin-1, -3, and -5, and ZO-1 and -2 were analyzed by Western blot and immunofluorescence microscopy. Though no changes in total protein expression were observed, nicotine treatment was associated with altered cellular distribution of ZO-1 and diminished junctional immunoreactivity of claudin-3. It is proposed that nicotine leads to changes in BBB permeability via the modulation of TJ proteins.

Effect of lambda-carrageenan-induced inflammatory pain on brain uptake of codeine and antinociception.

This study investigated the potential clinical implications of lambda-carrageenan-induced inflammatory pain on brain uptake of a commonly used analgesic, codeine, in relation to the fundamental properties of the blood-brain barrier (BBB) correlated to its antinociceptive profile over a 168-h time course. BBB uptake of [14C]sucrose (a membrane impermeant marker) and [3H]codeine were investigated using an in situ brain perfusion model in the rat. Results demonstrated a significantly increased brain uptake of [14C]sucrose at 1, 3, 6 and 48 h (139+/-9%, 166+/-19%, 138+/-13% and 146+/-7% compared with control, respectively) and [3H]codeine at 3 and 48 h (179+/-6% and 179+/-12% compared with control, respectively). Capillary depletion analyses ensured that increased radioisotope associated with the brain was due to increased uptake rather than trapping in the cerebral vasculature. Antinociception studies using a radiant-heat tail flick analgesia method demonstrated that lambda-carrageenan-induced inflammatory pain enhanced the in vivo antinociceptive profile of i.p.-administered codeine (7 mg/kg) at 3 and 48 h (144+/-11% and 155+/-9% compared with control, respectively). This study demonstrated that brain uptake and antinociception of codeine are increased during lambda-carrageenan-induced inflammatory pain, suggesting that the presence of inflammatory pain may be an important consideration in therapeutic drug dosing, potential adverse effects and/or neurotoxicity.

Effect of guanidino modification and proline substitution on the in vitro stability and blood-brain barrier permeability of endomorphin II.

Endomorphin II (ENDII), an endogenous ligand for the mu-opioid receptor, was investigated as a possible analgesic with fewer side effects than morphine. To improve CNS entry of END II, structural modification was also examined to determine whether Pro(4) substitution and cationization affected physico-chemical characteristics, blood-brain barrier (BBB) transport, and analgesic profile. END II and its Pro(4)-substituted analog, Morphiceptin (MOR), were cationized by guanidino (GU)-addition. MOR was seven times less lipophilic than END II, whereas GU-addition decreased lipophilicity of both peptides. MOR did not affect in vitro BBB permeability; however, GU-addition increased permeability of MOR by 31%. MOR decreased protein binding by 23% compared to END II, whereas GU-addition increased protein binding of both peptides by 71 and 113%, respectively. MOR increased brain t(1/2) compared to END II. GU-addition significantly increased t(1/2) of MOR and END II in both brain (sixfold and 10-fold, respectively) and serum (over 10-fold). Pro(4)-substitution and GU-addition enhanced the in vivo analgesia profiles of i.v. administered END II and MOR, but decreased i.c.v. analgesia profiles. This study demonstrates Pro(4)-substitution decreases protein binding and enhances brain stability while cationization enhances both brain and serum stability with variable effects on BBB permeability. The analgesic profiles show that both Pro(4)-substitution and cationization enhance i.v. analgesia and thus, are promising structural modifications for the development of successful opioid drugs.