Enhanced neuroprotective effects of basic fibroblast growth factor in regional brain ischemia after conjugation to a blood-brain barrier delivery vector

Basic fibroblast growth factor (bFGF) has minimal pharmacological effects in the central nervous system in the absence of blood-brain barrier (BBB) disruption. BBB transport of bFGF occurs via an absorptive-mediated transcytosis mechanism, which is relatively inefficient. To enhance the BBB transport of bFGF, this neurotrophin was reformulated to enable receptor-mediated transport across the BBB via the transferrin receptor. bFGF was monobiotinylated and coupled to a BBB drug-delivery vector comprised of streptavidin (SA) and the OX26 monoclonal antibody to the rat transferrin receptor. The entire conjugate of biotinylated bFGF bound to the OX26-SA is designated bio-bFGF/OX26-SA. The bFGF retains receptor-binding affinity and has increased brain uptake following conjugation to OX26-SA. The bio-bFGF/OX26-SA conjugate protects cortical cell cultures against hypoxia/reoxygenation insult in a dose-dependent manner in vitro. A single intravenous injection of bio-bFGF/OX26-SA, equivalent to a dose of 25 microg/kg bFGF, produces an 80% reduction in infarct volume in the brain of rats subjected to permanent occlusion of the middle cerebral artery in parallel with a significant improvement of neurologic deficit. The neuroprotection is time-dependent, and there is a 67% reduction in stroke volume if the conjugate is administered at 60 min after arterial occlusion, whereas no significant reduction in stroke volume is observed if treatment is delayed 2 h. In conclusion, neuroprotection in regional brain ischemia is possible following the delayed intravenous injection of low doses of bFGF providing the neurotrophin is conjugated to a BBB drug-targeting system.     

Systemic combinatorial peptide selection yields a non-canonical iron-mimicry mechanism for targeting tumors in a mouse model of human glioblastoma

The management of CNS tumors is limited by the blood-brain barrier (BBB), a vascular interface that restricts the passage of most molecules from the blood into the brain. Here we show that phage particles targeted with certain ligand motifs selected in vivo from a combinatorial peptide library can cross the BBB under normal and pathological conditions. Specifically, we demonstrated that phage clones displaying an iron-mimic peptide were able to target a protein complex of transferrin and transferrin receptor (TfR) through a non-canonical allosteric binding mechanism and that this functional protein complex mediated transport of the corresponding viral particles into the normal mouse brain. We also showed that, in an orthotopic mouse model of human glioblastoma, a combination of TfR overexpression plus extended vascular permeability and ligand retention resulted in remarkable brain tumor targeting of chimeric adeno-associated virus/phage particles displaying the iron-mimic peptide and carrying a gene of interest. As a proof of concept, we delivered the HSV thymidine kinase gene for molecular-genetic imaging and targeted therapy of intracranial xenografted tumors. Finally, we established that these experimental findings might be clinically relevant by determining through human tissue microarrays that many primary astrocytic tumors strongly express TfR. Together, our combinatorial selection system and results may provide a translational avenue for the targeted detection and treatment of brain tumors.     

Targeting rat anti-mouse transferrin receptor monoclonal antibodies through blood-brain barrier in mouse

Drug targeting through the brain capillary endothelium, which forms the blood-brain barrier (BBB) in vivo, may be achieved with peptidomimetic monoclonal antibodies that target peptide transcytosis systems on the BBB in vivo. Murine monoclonal antibodies to the rat transferrin receptor, such as the OX26 monoclonal antibody, are targeted through the BBB on the transferrin receptor in the rat. However, the present studies show the OX26 monoclonal antibody is not an effective brain delivery vector in mice. The emergence of transgenic mouse models creates a need for brain drug-targeting vectors for this species. Two rat monoclonal antibodies, 8D3 and RI7-217, to the mouse transferrin receptor were evaluated in the present studies. Both the RI7-217 and the 8D3 antibody had comparable permeability-surface area products at the mouse BBB in vivo. However, owing to a higher plasma area under the concentration curve, the mouse brain uptake of the 8D3 antibody was higher, 3.1 +/- 0.4% of injected dose [(ID)/g] compared with the brain uptake of the RI7 antibody, 1.6 +/- 0.2% ID/g, at 60 min after i.v. injection. Conversely, the mouse brain uptake of the OX26 antibody, which does not recognize the mouse transferrin receptor, was negligible, 0.06 +/- 0.01% ID/g. The RI7-127 antibody was more selective for brain because this antibody was not measureably taken up by liver. The capillary depletion technique demonstrated transcytosis of the RI7-217 antibody through the mouse BBB in vivo. The brain uptake of the 8D3 antibody was saturable, consistent with a receptor-mediated transport process. In conclusion, these studies indicate rat monoclonal antibodies to the mouse transferrin receptor may be used for brain drug-targeting studies in mice such as transgenic mouse models.     

<Emphasis Type="Italic">In Vitro</Emphasis> and Initial <Emphasis Type="Italic">In Vivo</Emphasis> Evaluation of <Superscript>68</Superscript>Ga-Labeled Transferrin Receptor (TfR) Binding Peptides as Potential Carriers for Enhanced Drug Transport into TfR Expressing Cells

Purpose  

The transferrin receptor (TfR) is one of the most attractive targets to overcome the blood–brain barrier (BBB). It has recently been shown that THRPPMWSPVWP binds to the TfR and is subsequently internalized into TfR-expressing cells. Here, we evaluated the ability of THRPPMWSPVWP to become internalized into human TfR-expressing cells via endocytosis to determine its potential to act as a carrier system for the transport of small molecules across the BBB.     

Organic anion-transporting polypeptides mediate transport of opioid peptides across blood-brain barrier

Organic anion-transporting polypeptides (Oatps) are a rapidly growing gene family of polyspecific membrane transporters. In rat brain, Oatp1 (gene symbol Slc21a1) and Oatp2 (Slc21a5) are localized at the apical and basolateral domains, respectively, of the choroid plexus epithelium. Furthermore, Oatp2 is strongly expressed at the rat blood-brain barrier (BBB). This study localizes the human OATP (now called OATP-A; SLC21A3) at the BBB in humans. Furthermore, with the Xenopus laevis oocyte system the delta-opioid receptor agonists [D-penicillamine(2,5)]enkephalin (DPDPE) and deltorphin II were identified as new transport substrates of OATP-A. This OATP-A-mediated DPDPE and deltorphin II transport exhibited apparent K(m) values of approximately 202 and 330 microM, respectively, and OATP-A-mediated deltorphin II transport was inhibited by the mu-opioid receptor agonist Tyr-D-Ala-Gly-N-methyl-Phe-glycinol, the endogenous peptide Leu-enkephalin, and the opiate antagonists naloxone and naltrindole. DPDPE also was transported by rat Oatp1 (K(m) approximately 48 microM) and Oatp2 (K(m) approximately 19 microM), whereas deltorphin II was only transported by Oatp1 (K(m) approximately 137 microM). These results demonstrate that OATP-A can mediate transport of the analgesic opioid peptides DPDPE and deltorphin II across the human BBB. Furthermore, because rat Oatp1 and Oatp2 exhibit similar but not identical transport activities as OATP-A, the results generally indicate that members of the Oatp/OATP gene family of membrane transporters play an important role in carrier-mediated transport of opioid peptides across the BBB and blood-cerebrospinal fluid barrier of the mammalian brain.     

Enhanced delivery of doxorubicin into the brain via a peptide-vector-mediated strategy: saturation kinetics and specificity

Doxorubicin delivery to the brain is often restricted because of the poor transport of this therapeutic molecule through the blood-brain barrier (BBB). To overcome this problem, we have recently developed a technology, Pep:trans, based on short natural-derived peptides that are able to cross efficiently the BBB without compromising its integrity. In this study, we have used the in situ mouse brain perfusion method to evaluate the brain uptake of free and vectorized doxorubicin. Doxorubicin was coupled covalently to small peptide vectors: L-SynB1 (18 amino acids), L-SynB3 (10 amino acids), and its enantio form D-SynB3. We first confirmed the very low brain uptake of free radiolabeled doxorubicin, which is most likely due to the efflux activity of the P-glycoprotein at the level of the BBB. Vectorization with either L-SynB1, L-SynB3, or D-SynB3 significantly increased the brain uptake of doxorubicin (about 30-fold). We also investigated the mechanism of transport of vectorized doxorubicin. We show that vectorized doxorubicin uses a saturable transport mechanism to cross the BBB. The effect of poly(L-lysine) and protamine, endocytosis inhibitors, on the transport across the brain was also investigated. Both inhibitors reduced the brain uptake of vectorized doxorubicin in a dose-dependent manner. These studies indicate that the transport of vectorized doxorubicin appears to occur via an adsorptive-mediated endocytosis.     

Blood-brain barrier permeability of novel [D-arg2]dermorphin (1-4) analogs: transport property is related to the slow onset of antinociceptive activity in the central nervous system

To clarify the pharmacological characteristics of Nalpha-amidino-Tyr-D-Arg-Phe-betaAla-OH (ADAB) and Nalpha-amidino-Tyr-D-Arg-Phe-MebetaAla-OH (ADAMB), mu1-opioid receptor-selective [D-Arg2]dermorphin tetrapeptide analogs, the plasma pharmacokinetics, and the in vivo blood-brain barrier (BBB) transport of these peptides were quantitatively evaluated. The mechanism responsible for the BBB transport of these peptides was also examined. The in vivo BBB permeation influx rates of 125I-ADAB and 125I-ADAMB after an i.v. bolus injection into mice were determined to be 0.0515 +/- 0.0284 microl/(min.g of brain) and 0.0290 +/- 0.0059 microl/(min.g of brain), respectively, both rates being slower than that of 125I-Tyr-D-Arg-Phe-betaAla-OH (125I-TAPA), a [D-Arg2]dermorphin tetrapeptide analog. To elucidate the BBB transport mechanism of ADAB and ADAMB, a conditionally immortalized mouse brain capillary endothelial cell line (TM-BBB4) was used as an in vitro model of the BBB. The internalization of both 125I-ADAB and 125I-ADAMB into cells was concentration-dependent with half-saturation constant (Kd) values of 3.76 +/- 0.83 and 5.68 +/- 1.75 microM, respectively. The acid-resistant binding of both ADAB and ADAMB was significantly inhibited by dansylcadaverine (an endocytosis inhibitor) and poly-l-lysine and protamine (polycations), but it was not inhibited by 2,4-dinitrophenol, or at 4 degrees C. These results suggest that ADAB and ADAMB are transported through the BBB with slower permeation rates than that of TAPA, and this is likely to be a factor in the slow onset of their antinociceptive activity in the central nervous system. The mechanism of the BBB transport of these drugs is considered to be adsorptive-mediated endocytosis.     

Molecular architecture determines brain delivery of a transferrin receptor–targeted lysosomal enzyme

Delivery of biotherapeutics across the blood–brain barrier (BBB) is a challenge. Many approaches fuse biotherapeutics to platforms that bind the transferrin receptor (TfR), a brain endothelial cell target, to facilitate receptor-mediated transcytosis across the BBB. Here, we characterized the pharmacological behavior of two distinct TfR-targeted platforms fused to iduronate 2-sulfatase (IDS), a lysosomal enzyme deficient in mucopolysaccharidosis type II (MPS II), and compared the relative brain exposures and functional activities of both approaches in mouse models. IDS fused to a moderate-affinity, monovalent TfR-binding enzyme transport vehicle (ETV:IDS) resulted in widespread brain exposure, internalization by parenchymal cells, and significant substrate reduction in the CNS of an MPS II mouse model. In contrast, IDS fused to a standard high-affinity bivalent antibody (IgG:IDS) resulted in lower brain uptake, limited biodistribution beyond brain endothelial cells, and reduced brain substrate reduction. These results highlight important features likely to impact the clinical development of TfR-targeting platforms in MPS II and potentially other CNS diseases.     

Transport of nerve growth factor encapsulated into liposomes across the blood-brain barrier: in vitro and in vivo studies.

A nerve growth factor (NGF) was encapsulated into liposomes in order to protect it from the enzyme degradation in vivo and promote it permeability across the blood-brain barrier (BBB). RMP-7, a ligand to the B2 receptor on brain microvascular endothelial cells (BMVEC), was combined with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-n-[poly(ethylenegly-col)]-hydroxy succinamide (DSPE-PEG-NHS) to obtain DSPE-PEG-RMP-7. Then DSPE-PEG-RMP-7 was incorporated into the liposomes' surface to target sterically stabilized liposomes (SSL-T) to the brain. The highest percent of NGF encapsulated into liposomes was about 34%, and the average size of liposomes was below 100 nm. A primary model of BBB was established and evaluated by morphological, permeability, and transendothelial electrical resistance (TEER). The BBB model was employed to study the permeability of NGF liposomes in vitro. The results indicated that the liposomes could enhance transport of NGF across the BBB. The best transport rate was received with NGF-SSL-T. The brain distribution of NGF liposomes was studied in vivo, the amount of NGF in the brain was increased in the order: NGF-SSL-T>NGF-SSL+RMP-7>NGF-SSL>NGF-L. The maximum concentration of NGF was recorded in 30 min following the intravenous injection. In particular, a majority of NGF was distributed in striatum, hippocampus and cortex, and the concentration of NGF was relatively lower in olfactory bulb, cerebellum and brain stem. There was a close relationship between P(e) (permeability coefficient on in vitro BBB model) and T(e) (brain targeted coefficient in vivo) for NGF encapsulated into the liposomes.     

Transport of vasopressin fragments across the blood-brain barrier: in vitro studies using monolayer cultures of bovine brain endothelial cells

A well established in vitro blood-brain barrier (BBB) model, consisting of bovine cerebrovascular endothelial monolayers from primary cultures, was used to study the transport profile of vasopressin and its fragments across the BBB and to assess the metabolic properties of the BBB for the behaviorally active vasopressin fragment arginine vasopressin (AVP)1-8 (desglycinamide-AVP). All vasopressin fragments crossed the in vitro BBB to a measurable extent. Endothelial permeabilities were (in 10(-3) cm/min): AVP1-6, 3.0 +/- 0.2; AVP1-7, 4.6 +/- 0.4; AVP1-8, 2.0 +/- 0.5 and AVP1-9, 2.4 +/- 0.4. A significant effect of molecular size on endothelial permeability was seen. Transport rate of AVP1-8, expressed as BBB-clearance, was not affected by luminal concentration change and proved to be symmetrical. These findings suggest that, in the concentration range studied, vasopressin-like peptides can cross the BBB mainly by paracellular transport and that no relevant carrier mediation is involved. AVP1-8 was metabolized slowly (half-life, 6.5 hr) by a 60 cm2 confluent monolayer to AVP1-7, which was not broken down further, suggesting that carboxypeptidases are responsible for AVP1-8 metabolism in the BBB.     

Biotin delivery to brain with a covalent conjugate of avidin and a monoclonal antibody to the transferrin receptor.

The OX26 mouse monoclonal antibody to the rat transferrin receptor undergoes transcytosis through the brain capillary endothelial wall, which makes up the blood-brain barrier (BBB) in vivo, owing to high concentrations of transferrin receptor on the BBB. This property allows the OX26 antibody to serve as a brain drug transport vector. To simplify coupling of therapeutics to the OX26 antibody, the present studies examine the use of the avidin/biotin system to promote coupling of biotin and biotinylated drugs to brain transport vectors. The OX26 antibody was affinity purified from ascites fluid and was covalently coupled via a thioether linkage to avidin, and the conjugate was purified to homogeneity by gel filtration fast protein liquid chromatography. The biotin binding capacity of the avidin-OX26 conjugate was measured, and 2.3 biotin binding sites per avidin-OX26 (1:1) conjugate were detected. Transcytosis through the BBB of [3H]biotin bound to either unconjugated avidin or to the avidin-OX26 conjugate was measured with an internal carotid artery perfusion/capillary depletion technique. [3H]Biotin bound to the avidin-OX26 conjugate was transported through the BBB at rates that equaled the rate of transcytosis of the unconjugated OX26 antibody. The clearance from plasma of [3H]biotin bound to the avidin-OX26 conjugate approximated the rate of clearance of the unconjugated OX26 antibody, and not the rate of clearance of [3H]biotin bound to avidin, which was cleared from plasma at much faster rates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of multiple transporters in the efflux of 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors across the blood-brain barrier

Statins, 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors, are frequently used for the treatment of hypercholesterolemia. The present study aimed to examine the involvement of organic anion transporters in the efflux transport of pravastatin and pitavastatin across the blood-brain barrier (BBB). Transport studies using cDNA-transfected cells revealed that these statins are substrates of multispecific organic anion transporters expressed at the BBB (rOat3:Slc22a8 and rOatp2:Slco1a4). The efflux of these statins across the BBB was characterized using the brain efflux index method. The efflux clearance of pitavastatin across the BBB, obtained from the elimination rate constant and the distribution volume in the brain, was greater than that of pravastatin (364 versus 59 microl/min/g brain). The efflux of pravastatin and pitavastatin was saturable (apparent Km values: 18 and 5 muM, respectively) and inhibited by probenecid but unaffected by tetraethylammonium. Furthermore, an inhibitor of the efflux pathway for hydrophilic organic anions across the BBB (p-aminohippurate), and inhibitors of the efflux pathway for amphipathic organic anions (taurocholate and digoxin) inhibited the efflux of both statins. The degree of inhibition by p-aminohippurate was similar and partial for the efflux of pravastatin and pitavastatin. Taurocholate and digoxin completely inhibited the efflux of pitavastatin, whereas their effect was partial for the efflux of pravastatin. The results of the present study suggest the involvement of multiple transporters, including rOat3 and rOatp2, in the efflux transport of pravastatin and pitavastatin across the BBB, each making a different contribution.     

Glycosylated neuropeptides: A new vista for neuropsychopharmacology?

The application of endogenous neuropeptides (e.g., enkephalins) as analgesics has been retarded by their poor stability in vivo and by their inability to effectively penetrate the blood-brain barrier (BBB). Effective BBB transport of glycosylated enkephalins has been demonstrated in several labs now. Analgesia (antinociception) levels greater than morphine, and with reduced side effects have been observed for several glycopeptides related to enkephalin. Somewhat paradoxically, enhanced BBB transport across this lipophilic barrier is achieved by attaching water-soluble carbohydrate groups to the peptide moieties to produce biousian glycopeptides that can be either water-soluble or membrane bound. Transport is believed to rely on an endocytotic mechanism (transcytosis), and allows for systemic delivery and transport of the water-soluble glycopeptides. Much larger endorphin/dynorphin glycopeptide analogs bearing amphipathic helix address regions also have been shown to penetrate the BBB in mice. This holds forth the possibility of transporting much larger neuropeptides across the BBB, which may encompass a wide variety of receptors beyond the opioid receptors.     

GHB (gamma-hydroxybutyrate) carrier-mediated transport across the blood-brain barrier

gamma-Hydroxybutyrate (sodium oxybate, GHB) is an approved therapeutic agent for cataplexy with narcolepsy. GHB is widely abused as an anabolic agent, euphoriant, and date rape drug. Recreational abuse or overdose of GHB (or its precursors gamma-butyrolactone or 1,4-butanediol) results in dose-dependent central nervous system (CNS) effects (respiratory depression, unconsciousness, coma, and death) as well as tolerance and withdrawal. An understanding of the CNS transport mechanisms of GHB may provide insight into overdose treatment approaches. The hypothesis that GHB undergoes carrier-mediated transport across the BBB was tested using a rat in situ brain perfusion technique. Various pharmacological agents were used to probe the pharmacological characteristics of the transporter. GHB exhibited carrier-mediated transport across the BBB consistent with a high-capacity, low-affinity transporter; averaged brain region parameters were V(max) = 709 +/- 214 nmol/min/g, K(m) = 11.0 +/- 3.56 mM, and CL(ns) = 0.019 +/- 0.003 cm(3)/min/g. Short-chain monocarboxylic acids (pyruvic, lactic, and beta-hydroxybutyric), medium-chain fatty acids (hexanoic and valproic), and organic anions (probenecid, benzoic, salicylic, and alpha-cyano-4-hydroxycinnamic acid) significantly inhibited GHB influx by 35 to 90%. Dicarboxylic acids (succinic and glutaric) and gamma-aminobutyric acid did not inhibit GHB BBB transport. Mutual inhibition was observed between GHB and benzoic acid, a well known substrate of the monocarboxylate transporter MCT1. These results are suggestive of GHB crossing the BBB via an MCT isoform. These novel findings of GHB BBB transport suggest potential therapeutic approaches in the treatment of GHB overdoses. We are currently conducting "proof-of-concept" studies involving the use of GHB brain transport inhibitors during GHB toxicity.     

Identification and design of peptides as a new drug delivery system for the brain

By controlling access to the brain, the blood-brain barrier (BBB) restricts the entry of proteins and potential drugs to cerebral tissues. We demonstrate here the transcytosis ability of aprotinin and peptides derived from Kunitz domains using an in vitro model of the BBB and in situ brain perfusion. Aprotinin transcytosis across bovine brain capillary endothelial cell (BBCEC) monolayers is at least 10-fold greater than that of holo-transferrin. Sucrose permeability was unaffected by high concentrations of aprotinin, indicating that transcytosis of aprotinin was unrelated to changes in the BBCEC monolayer integrity. Alignment of the amino acid sequence of aprotinin with the Kunitz domains of human proteins allowed the identification and design of a family of peptides, named Angiopeps. These peptides, and in particular Angiopep-2, exhibit higher transcytosis capacity and parenchyma accumulation than aprotinin. Overall, these results suggest that these Kunitz-derived peptides could be advantageously used as a new brain delivery system for pharmacological agents that do not readily enter the brain.     

Normalization of striatal tyrosine hydroxylase and reversal of motor impairment in experimental parkinsonism with intravenous nonviral gene therapy and a brain-specific promoter

The goal of this work was to normalize striatal tyrosine hydroxylase (TH) activity with intravenous nonviral TH gene therapy and at the same time eliminate ectopic TH gene expression in peripheral organs such as liver in the rat. TH-expression plasmids, containing either the SV40 promoter or the glial fibrillary acidic protein (GFAP) gene promoter, were globally delivered to the brain across the blood-brain barrier (BBB) after intravenous administration of pegylated immunoliposomes (PILs). The GFAP-TH- or SV40-TH-expression plasmids were encapsulated in the interior of 85-nm PILs, which were targeted across both the BBB and the neuronal cell membrane with a monoclonal antibody (mAb) to the transferrin receptor (TfR). Striatal TH activity was 98% depleted with the unilateral intracerebral injection of 6-hydroxydopamine. TH in the striatum ipsilateral to the lesion was normalized 3 days after the intravenous injection of 10 microg per rat of either the SV40-TH or the GFAP-TH plasmid DNA. Whereas the SV40-TH gene caused a 10-fold increase in hepatic TH activity, there was no increase in liver TH with the GFAP-TH gene. The GFAP-TH gene therapy caused an 82% reduction in apomorphine-induced rotation in the lesioned rats. Confocal microscopy using antibodies to TH, GFAP, and neuronal nuclei (NeuN) showed the GFAP-TH gene was selectively expressed in nigra-striatal neurons, with no expression in either cortical neurons, or astrocytes. These studies demonstrate that global delivery of exogenous genes to the brain is possible with intravenous nonviral gene transfer, and that ectopic gene expression is eliminated with the use of brain-specific gene promoters.     

Transport across the blood-brain barrier: stereoselectivity and PET-tracers.

This article reviews positron emission tomography (PET) studies of labeled enantiomers of different PET-tracers from the early 1980s. Comparative studies on stereoselective behavior of the transport of tracers across the blood-brain barrier (BBB) are discussed. Tracers are transported through the BBB into the brain, via diffusion or via several transport systems. These transport systems are able to transport endogenous and exogenous compounds from the blood into the brain, and visa versa. A clear difference exists in BBB transport of the enantiomers of several tracers. In addition, in most cases, binding of these labeled enantiomers to receptors/transporters is stereoselective. Finally, use of the biological inactive labeled enantiomer for the measurement of nonspecific binding is discussed. Given the differences in transport and binding, it is concluded that quantitative PET studies can only be performed using pure enantiomers.     

Investigation of efflux transport of dehydroepiandrosterone sulfate and mitoxantrone at the mouse blood-brain barrier: a minor role of breast cancer resistance protein

Breast cancer resistance protein (Bcrp/Abcg2) is a new efflux transporter found at the blood-brain barrier (BBB) of humans and pigs. Since it has been hypothesized that Bcrp may act as a new type of efflux transporter at the BBB, we investigated the involvement of Bcrp in the efflux transport of typical substrates, dehydroepiandrosterone sulfate (DHEAS) and mitoxantrone, across the mouse BBB. The expression of Bcrp in mouse brain capillaries was confirmed by quantitative polymerase chain reaction, Western blot, and immunohistochemical analysis. The role of Bcrp as an efflux transporter was evaluated using the in situ brain perfusion method in wild-type and P-glycoprotein (P-gp) knockout mice with or without treatment with GF120918 (Elacridar), an inhibitor of both Bcrp and P-gp. The increased brain uptake of [(3)H]DHEAS and [(3)H]mitoxantrone by GF120918 in wild-type and P-gp knockout mice suggested the existence of a GF120918-sensitive and P-gp-independent efflux transporter for DHEAS and mitoxantrone across the BBB. However, the brain uptake of [(3)H]DHEAS in Bcrp knockout mice was comparable with that in wild-type mice, and the effect of GF120918 was still observed in Bcrp knockout mice. In addition, the brain uptake of [(3)H]mitoxantrone was also similar in wild-type and Bcrp knockout mice. These results suggest that although BCRP is expressed at the BBB it plays a minor role in active efflux transport of DHEAS and mitoxantrone out of brain and that one or more GF120918-sensitive efflux transporters distinct from BCRP or P-gp contributes to the brain efflux of DHEAS and mitoxantrone.     

Clearance of Alzheimer's amyloid-ss(1-40) peptide from brain by LDL receptor-related protein-1 at the blood-brain barrier

Elimination of amyloid-ss peptide (Ass) from the brain is poorly understood. After intracerebral microinjections in young mice, (125)I-Ass(1-40) was rapidly removed from the brain (t(1/2) 相似文献