P-glycoprotein mediates brain-to-blood efflux transport of buprenorphine across the blood-brain barrier

The involvement of P-glycoprotein (P-gp) in buprenorphine (BNP) transport at the blood-brain barrier (BBB) in rats was investigated in vivo by means of both the brain uptake index technique and the brain efflux index technique. P-gp inhibitors, such as cyclosporin A, quinidine and verapamil, enhanced the apparent brain uptake of [3H]BNP by 1.5-fold. The increment of the BNP uptake by the brain suggests the involvement of a P-gp efflux mechanism of BNP transport at the BBB. [3H]BNP was eliminated with an apparent elimination half-life of 27.5 min after microinjection into the parietal cortex area 2 regions of the rat brain. The apparent efflux clearance of [3H]BNP across the BBB was 0.154 ml/min/g brain, which was calculated from the elimination rate constant (2.52 x 10- 2 min- 1) and the distribution volume in the brain (6.11 ml/g brain). The efflux transport of [3H]BNP was inhibited by range from 32 to 64% in the presence of P-gp inhibitors. The present results suggest that BNP is transported from the brain across the BBB via a P-gp-mediated efflux transport system, at least in part.     

Recent advances in the brain-to-blood efflux transport across the blood-brain barrier

Elucidating the details of the blood-brain barrier (BBB) transport mechanism is a very important step towards successful drug targeting to the brain and understanding what happens in the brain. Although several brain uptake methods have been developed to characterize transport at the BBB, these are mainly useful for investigating influx transport across the BBB. In 1992, P-glycoprotein was found to act as an efflux pump for anti-cancer drugs at the BBB using primary cultured bovine brain endothelial cells. In order to determine the direct efflux transport from the brain to the circulating blood of exogenous compounds in vivo, the Brain Efflux Index method was developed to characterize several BBB efflux transport systems. Recently, we have established conditionally immortalized rat (TR-BBB) and mouse (TM-BBB) brain capillary endothelial cell lines from transgenic rats and mice harboring temperature-sensitive simian virus 40 large T-antigen gene to characterize the transport mechanisms at the BBB in vitro. TR-BBB and TM-BBB cells possess certain in vivo transport functions and express mRNAs for the BBB. Using a combination of newly developed in vivo and in vitro methods, we have elucidated the efflux transport mechanism at the BBB for neurosteroids, excitatory neurotransmitters, suppressive neurotransmitters, amino acids, and other organic anions to understand the physiological role played by the BBB as a detoxifying organ for the brain.     

Drug transport across the blood-brain barrier

This review describes various aspects of the transport of drugs across the blood-brain barrier and comprises three parts. In this first part, the anatomical and physiological aspects of blood-brain transport are discussed. It appears that the blood-brain barrier has an anatomical basis at the endothelium of the capillary wall. This endothelium is characterized by the presence of very tight junctions. As a result, the transport by passive diffusion of drugs with a low lipophilicity, is restricted. For certain classes of closely related relatively hydrophilic compounds, however, the presence of specialized carrier systems has been demonstrated which may facilitate transport. Also evidence is presently available, that the permeability of the blood-brain barrier may be under active regulatory control. It is expected that improved knowledge of the anatomical and physiological aspects of the blood-brain barrier and its regulation will provide a scientific basis for the development of strategies to improve the transport of drugs into the central nervous system.     

Drug transport across the blood-brain barrier

This is the third part of a review on the transport of drugs across the blood-brain barrier. In the first two parts, the anatomical and physiological aspects and the various techniques that can be used to study blood-brain transport have been discussed and reviewed. This third part focuses specifically on the mechanisms that are involved in drug transport across the blood-brain barrier. In addition, the opportunities to improve drug transport into the brain will be reviewed. Emphasis is on the transport of peptides.     

Blood-brain barrier transport of H1-antagonist ebastine and its metabolite carebastine

The transport mechanism of the non-sedative H1-antagonist ebastine and its first-pass carboxylic acid metabolite carebastine at the blood-brain barrier (BBB) was studied. In rats, the brain uptake index (BUI) value of [14 C]carebastine was significantly lower than that of [14 C]ebastine. The BUI value of [14 C]carebastine was greatly increased by the addition of non-labeled carebastine. The steady-state uptake of [14 C]carebastine by P-glycoprotein-overexpressing K562/ADM cells was significantly lower than that by their parental drug-sensitive cell line K562. The decreased steady-state uptake of [14 C]carebastine by K562/ADM cells was reversed by verapamil. Steady-state uptake of [14 C]carebastine by primary cultured bovine brain capillary endothelial cells (bovine BCECs) was increased in the presence of metabolic inhibitors and verapamil. Non-labeled carebastine increased the steady-state uptake of a P-glycoprotein substrate, [3 H]vincristine, by bovine BCECs. The initial uptake of [3 H]mepyramine by bovine BCECs and RBEC1 (an immortalized cell line from rat brain capillary endothelial cells) was strongly inhibited by ebastine, while zwitterionic carebastine was slightly inhibitory. The values of brain-to-plasma unbound concentration ratio (Kp,f) in mdr1a(-/-) mice were increased 5.3-fold and 4.2-fold for [14 C ebastine and for [14 C]carebastine, respectively, compared with those in mdr1a(+/+) mice. Non-radiolabeled carebastine increased the Kp,f values of [14 C]carebastine in both types of mice. In conclusion, carebastine was shown to be a substrate for P-glycoprotein-mediated efflux from the brain at the BBB. A second efflux system may also be involved. The relatively low affinity of the uptake transport system for carebastine also limits the brain distribution of ebastine/carebastine.     

Contribution of P-glycoprotein to bunitrolol efflux across blood-brain barrier

In this study, we investigated the mechanism of the blood-brain barrier (BBB) transport of bunitrolol (BTL), as a model of beta-blocker, in vivo and in vitro. In order to define the contribution of P-glycoprotein (P-gp) to the active efflux of BTL from brain to blood, we examined the in vivo brain distribution of BTL in mdr1a(-/-) mice with a disrupted mdr1a gene. After intravenous administration of BTL to mdr1a(-/-) mice, the brain concentration and Kp value of BTL were significantly increased as compared with those in mdr1a(+/+) mice. Next, the contribution of the mdr1a P-gp to in vitro uptake of BTL was compared in LV500 cells and L cells (mouse mdr1a-expressing cells and host cells, respectively). The intracellular accumulations of [3H]vinblastine and BTL by LV500 cells were lower than those by L cells, but were significantly increased by verapamil, a P-gp inhibitor. Furthermore, the BTL uptake by KB-VJ300 cells, which express human P-gp, was also significantly lower than that by KB host cells, and was increased by verapamil. The steady-state uptake of BTL by LLC-GA5-COL300 cells, expressing human P-gp, was significantly increased in the presence of 20 microM cyclosporin A (another P-gp inhibitor), which had no effect in the LLC-PK1 host cells. On the other hand, the steady-state intracellular accumulation of BTL by MBEC4 cells, which express mdr1b P-gp instead of mdr1a P-gp, was not significantly changed in the presence of verapamil. This finding suggested that BTL is not a good substrate for mdr1b P-gp. In conclusion, our results suggest that BTL is transported from brain to blood by mdr1a P-gp in mice and by MDR1 in humans, and this presumably accounts for the low brain distribution of BTL.     

Apolipoprotein-mediated transport of nanoparticle-bound drugs across the blood-brain barrier

Recent studies have shown that drugs that are normally unable to cross the blood-brain barrier (BBB) following intravenous injection can be transported across this barrier by binding to poly(butyl cyanoacrylate) nanoparticles and coating with polysorbate 80. However, the mechanism of this transport so far was not known. In the present paper, the possible involvement of apolipoproteins in the transport of nanoparticle-bound drugs into the brain is investigated. Poly(butyl cyanoacrylate) nanoparticles loaded with the hexapeptide dalargin were coated with the apolipoproteins AII, B, CII, E, or J without or after precoating with polysorbate 80. In addition, loperamide-loaded nanoparticles were coated with apolipoprotein E alone or again after precoating with polysorbate 80. After intravenous injection to ICR mice the antinociceptive threshold was measured by the tail flick test. Furthermore, the antinociceptive threshold of polysorbate 80-coated dalargin-loaded nanoparticles was determined in ApoEtm1Unc and C57BL/6J mice. The results show that only dalargin or loperamide-loaded nanoparticles coated with polysorbate 80 and/or with apolipoprotein B or E were able to achieve an antinociceptive effect. This effect was significantly higher after polysorbate-precoating and apolipoprotein B or E-overcoating. With the apolipoprotein E-deficient ApoEtm1Unc mice the antinociceptive effect was considerably reduced in comparison to the C57BL/6J mice. These results suggest that apolipoproteins B and E are involved in the mediation of the transport of drugs bound to poly(butyl cyanoacrylate) nanoparticles across the BBB. Polysorbate 80-coated nanoparticles adsorb these apolipoproteins from the blood after injection and thus seem to mimic lipoprotein particles that could be taken up by the brain capillary endothelial cells via receptor-mediated endocytosis. Bound drugs then may be further transported into the brain by diffusion following release within the endothelial cells or, alternatively, by transcytosis.     

P-糖蛋白限制经血脑屏障的尼莫地平转运(英文)

目的:研究P-糖蛋白(P-gp)是否限制尼莫地平(NMD)从血液循环进入脑内。方法:当原代培养的鼠脑微血管内皮细胞(BCEC)长至互相连接成片时,加入含有NMD 10mg/L的Hanks’液37℃温孵,记录细胞摄取NMD的时间过程,将含有NMD和各种不同受试药物的Hanks’液分别加入到不同培养孔中,检测90min时细胞对NMD的摄取量,在细胞摄取NMD 90min后,分别加入红霉素,克拉霉素和环孢素A以检测P-糖蛋白抑制剂对原代培养的脑微血管内皮细胞外排NMD的影响。结果:细胞对NMD的摄取呈时间依赖性,P-gp抑制剂或代谢抑制剂的加入会增加稳态时NMD在细胞内的浓度,P-gp抑制剂的加入也使NMD的外排受到了抑制。结论:P-gp限制NMD进入脑内,P-gp抑制剂的加入可增加NMD的进入。     

In vivo evidence for the efflux transport of pentazocine from the brain across the blood-brain barrier using the brain efflux index method

The efflux transport of pentazocine (PTZ) from the brain across the blood-brain barrier (BBB) was investigated using the Brain Efflux Index method. PTZ was eliminated with the apparent elimination half-life of 13.0 min after microinjection into the parietal cortex area 2 region of the rat brain. The apparent efflux clearance of PTZ across the BBB was 137 microl/min/g brain, which was calculated from the elimination rate constant (5.35 x 10(-2) min(-1) and the distribution volume in the brain (2.56 ml/g brain). The efflux transport of PTZ was decreased in the presence of unlabeled PTZ, suggesting that PTZ is eliminated by a carrier-mediated transport system across the BBB. To characterize the efflux transport of PTZ from the brain in vivo, the effects of several compounds on the efflux transport of PTZ were investigated. P-glycoprotein (P-gp) inhibitors (verapamil and quinidine) reduced the PTZ efflux transport. In addition, the efflux transport of PTZ was inhibited by organic cations such as l-carnitine and tetraethylammonium (TEA), whereas organic anions such as p-aminohippuric acid, probenecid and taurocholate did not affect the PTZ efflux transport. The present results suggest that PTZ is transported from the brain across the BBB via l-carnitine/TEA-sensitive carrier-mediated efflux transport system(s) in addition to P-gp.     

Molecular mechanisms of drug influx and efflux transport at the blood-brain barrier

The blood-brain barrier (BBB) segregates the circulating blood from interstitial fluid in the brain and restricts drug permeability into the brain. Recent studies have revealed that the BBB exhibits not only blood-to-brain influx transport for the supply of nutrients, but also brain-to-blood efflux transport to excrete drugs and endogenous compounds. The influx transport system allows drugs to enter the brain. (L)-DOPA is transported into the brain by the large neutral amino acid transport system, system L. A cationic mu-opioid peptide analogue enters the brain by adsorptive-mediated endocytosis. In contrast, efflux transport limits the distribution of drugs in the brain. The ATP binding cassette transporter B1 (ABCB1) mediates the efflux transport of lipophilic drugs at the BBB by using ATP energy. Furthermore, organic anion transporter 3 (OAT3) is expressed at the BBB and mediates the efflux transport of homovanillic acid, a dopamine metabolite. This efflux transport is also likely to be involved in the transport of anionic drugs such as 6-mercaptopurine and acyclovir. Clarifying the BBB transport could give us important information allowing the development of better CNS drugs and improving our understanding of the relationship between CNS diseases and BBB functions.     

Improving the transport of chemotherapeutic drugs across the blood-brain barrier

The successful treatment of brain tumors or metastases in the brain is still hampered by the very efficient blood-brain barrier, which prevents the cerebral accumulation of a pharmacologically sufficient amount of a drug. Beside the possibility of disintegrating the functionality of this effective working barrier, a nanocarrier-mediated transport is presently an interesting and promising method to increase the drug concentration in the brain. Nanocarriers are small vesicles (<200 nm) and can be prepared by polymerization, resulting in nanoparticles, or by producing superficial lipid structures to incorporate the drug. In this context, liposomes are of importance owing to their ability to adapt their properties to the pharmacological requirements. In this article, we will give an overview of current possibilities of enhancing anticancer drug transport across the blood-brain barrier, based on its structure and functionality. Special consideration will be given to recent liposomal approaches that use active targeting for receptor-mediated transport across this physiological barrier.     

Carrier-mediated or specialized transport of drugs across the blood-brain barrier

In the drug development process, it remains a difficult task to regulate the entry of the drugs. However, recent progress in studies of the transporter-mediated influx and efflux of endogenous and exogenous compounds, including synthetic drugs, across the blood-brain barrier (BBB) is beginning to provide a rational basis for controlling drug distribution to the brain. This paper describes mechanisms established in the last decade for carrier-mediated influx and efflux of drugs and endocytosis of biologically active peptides across the BBB. The transport systems at the BBB described here are the uptake transporters for nutrients, such as amino acids and hexoses, monocarboxylates, amines, carnitine and glutathione and efflux transporters, such as P-glycoprotein and multiple organic anion transporters. Delivery of cationized peptides across the BBB via adsorptive-mediated endocytosis is also described. By utilizing such highly specific transport mechanisms, it should be possible to establish strategies to regulate the entry of candidate drugs, including peptides, into the brain.     

Effect of bioflavonoids on vincristine transport across blood-brain barrier

Several grapefruit juice bioflavonoids, including quercetin, are reported to stimulate P-glycoprotein-mediated drug efflux from cultured tumor cells. To see whether these bioflavonoids alter the permeation of vincristine across the blood-brain barrier, we conducted experiments with cultured mouse brain capillary endothelial cells (MBEC4 cells) in vitro and ddY mice in vivo. The steady-state uptake of [3H]vincristine by MBEC4 cells was decreased by 10 microM quercetin, but increased by 50 microM quercetin. Similarly, the in vivo brain-to-plasma concentration ratio of [3H]vincristine in ddY mice was decreased by coadministration of 0.1 mg/kg quercetin, but increased by 1.0 mg/kg quercetin. Kaempferol had a similar biphasic effect on the in vitro uptake of [3H]vincristine. Other aglycones tested (chrysin, flavon, hesperetin, naringenin) increased [3H]vincristine uptake in the 10-50 microM range, and glycosides (hesperidin, naringin, rutin) were without effect. We then addressed the mechanism of the concentration-dependent biphasic action of quercetin. Verapamil, a P-glycoprotein inhibitor, inhibited the efflux of [3H]vincristine from MBEC4 cells, while 10 microM quercetin significantly stimulated it. The uptake of [3H]vincristine by MBEC4 cells was increased by inhibitors of protein kinase C, but decreased by phorbol 12-myristate-13-acetate (PMA), as well as by 10 microM quercetin. The phosphorylation level of P-glycoprotein was increased in the presence of 5 microM quercetin or 100 nM PMA, but decreased by the protein kinase C inhibitor H7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine, 30 microM). We conclude that low concentrations of quercetin indirectly activate the transport of [3H]vincristine by enhancing the phosphorylation (and hence activity) of P-glycoprotein, whereas high concentrations of quercetin inhibit P-glycoprotein. Our results indicate that patients taking drugs which are P-glycoprotein substrates may need to restrict their intake of bioflavonoid-containing foods and beverages, such as grapefruit juice.     

Putative proteins involved in manganese transport across the blood-brain barrier

Manganese (Mn) is an essential nutrient required for proper growth and maintenance of numerous biological systems. At high levels it is known to be neurotoxic. While focused research concerning the transport of Mn across the blood-brain barrier (BBB) is on-going, the exact identity of the transporter(s) responsible is still debated. The transferrin receptor (TfR) and the divalent metal transporter-1 (DMT-1) have long been thought to play a role in brain Mn deposition. However, evidence suggests that Mn may also be transported by other proteins. One model system of the BBB, rat brain endothelial (RBE4) cells, are known to express many proteins suspected to be involved in metal transport. This review will discuss the biological importance of Mn, and then briefly describe several proteins that may be involved in transport of this metal across the BBB. The latter section will examine the potential usefulness of RBE4 cells in characterizing various aspects of Mn transport, and basic culture techniques involved in working with these cells. It is hoped that ideas put forth in this article will stimulate further investigations into the complex nature of Mn transport, and address the importance as well as the limitation of in vitro models in answering these questions.     

Beta-adrenergic regulation of amine precursor amino acid transport across the blood-brain barrier.

Beta-Adrenoceptor agonists were administered i.p. into rats and amino acid levels in brain and plasma were then determined to assess the effects on transport across the blood-brain barrier. Isoproterenol (10 mumol/kg) caused significant increases in aromatic amino acid (tyrosine, phenylalanine and tryptophan) levels in cerebral cortex and decreases in almost all amino acid concentrations in plasma. This effect of isoproterenol on brain tyrosine level was dose-dependent with an ED50 of 0.25 mumol/kg. Salbutamol (beta 2-adrenoceptor agonist, 10 mumol/kg) showed similar effects, but dobutamine (beta 1-adrenoceptor agonist, 50 mumol/kg) failed to increase brain amino acid levels. When 1-threo-3,4-dihydroxyphenylalanine (L-DOPA, 100 mumol/kg) was i.p. loaded, beta-adrenoceptor agonists promoted the transport of L-DOPA into brain without increasing the clearance rate of plasma L-DOPA. Moreover, significant increases in dopamine and its metabolites were observed in rat brain. These findings suggest that the transport of aromatic amino acids across the blood-brain barrier may be regulated through beta 2-adrenoceptors and that co-administration of beta 2-adrenoceptor agonists with L-DOPA may enhance the therapeutic efficacy of L-DOPA.     

脑缺血再灌注后（^3H）GABA通过大鼠血脑屏障的外排转运

目的 研究脑缺血／再灌后（＾３Ｈ）ＧＡＢＡ通过大鼠血脑屏障的外排运是否增强及其机制。方法 将（＾３Ｈ）ＧＡＢＡ或ＧＡＢＡ（或丙磺舒）与其联合注射到缺血／再灌大在脑皮层顶二区后,测定（＾３Ｈ）ＧＡＢＡ的脑外排指数（ＢＥＩ）及ｉｖ依文思蓝（ＥＢ）后ＥＢ的脑摄取量。结果：１０ｍｉｎ缺血／再灌３０ｍｉｎ,２ｈ,６ｈ和２４ｈ大鼠的ＢＥＩ分别为６７％,８３％,９２％和８７％,显著高于对照值（５８％）,ＥＢ脑报     

泊洛沙姆在药物穿越血脑屏障中的重要作用

泊洛沙姆是一种具有药理活性的多功能药用辅料，在药剂学中应用广泛。近年来，研究发现泊洛沙姆可以通过多种作用机制帮助药物穿越血脑屏障，抑制血脑屏障上的P-糖蛋白、多药耐药相关蛋白等外排泵系统；吸附血浆中的不同载脂蛋白后，通过与血脑屏障上相应受体的结合，使泊洛沙姆包被的纳米粒主动转运入脑；连接各种配体及单克隆抗体等导向性分子，使其通过受体介导的转运进入脑部。本文综述了泊洛沙姆在促进药物穿越血脑屏障的重要作用，对设计脑靶向药物传递系统具有重要意义。     

Experimental methods and transport models for drug delivery across the blood-brain barrier

The blood-brain barrier (BBB) is a dynamic barrier essential for maintaining the micro-environment of the brain. Although the special anatomical features of the BBB determine its protective role for the central nervous system (CNS) from blood-born neurotoxins, however, the BBB extremely limits the therapeutic efficacy of drugs into the CNS, which greatly hinders the treatment of major brain diseases. This review summarized the unique structures of the BBB, described a variety of in vivo and in vitro experimental methods for determining the transport properties of the BBB, e.g., the permeability of the BBB to water, ions, and solutes including nutrients, therapeutic agents and drug carriers, and presented newly developed mathematical models which quantitatively correlate the anatomical structures of the BBB with its barrier functions. Finally, on the basis of the experimental observations and the quantitative models, several strategies for drug delivery through the BBB were proposed.     

In vitro model for evaluating drug transport across the blood-brain barrier

The passage of substances across the blood-brain barrier (BBB) is regulated in the cerebral capillaries, which possess certain distinct different morphological and enzymatic properties compared with the capillaries of other organs. Investigations of the functional characteristics of brain capillaries have been facilitated by the use of cultured brain endothelial cells, but in most studies some characteristics of the in vivo BBB are lost. To provide an in vitro system for studying brain capillary functions, we have developed a process of coculture that closely mimics the in vivo situation by culturing brain capillary endothelial cells on one side of a filter and astrocytes on the other. In order to assess the drug transport across the blood-brain barrier, we compared the extraction ratios in vivo to the permeability of the in vitro model. The in vivo and the in vitro values showed a strong correlation. The relative ease with which such cocultures can be produced in large quantities facilitates the screening of new centrally active drugs. This model provides an easier, reproducible and mass-production method to study the blood-brain barrier in vitro.