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.     

New approaches to in vitro models of blood-brain barrier drug transport

The pharmaceutical industry has been searching for an in vitro blood-brain barrier (BBB) model that preserves in vivo transporter functions in CNS drug discovery and development. The application of conditionally immortalized cell lines derived from transgenic animals harboring temperature-sensitive SV40 large T-antigen gene, is a rational and promising approach to such a workable in vitro BBB model. The established brain capillary endothelial cell lines retain the in vivo transport rate of several compounds and various forms of gene expression. Furthermore, this new approach has enabled the development of stable and reproducible co-culture models with a pericyte cell line and/or an astrocyte cell line.     

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.     

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.     

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.     

转运蛋白在药物血脑屏障转运中的重要作用

血脑屏障上存在多种转运蛋白,能够影响药物透过血脑屏障的行为及在脑内的分布程度。对转运蛋白在药物血脑屏障转运中的重要作用进行研究,可深入地解析药物在血脑屏障上的动态并更有效地调控药物转运行为,有助于了解血脑屏障上转运蛋白与底物药物的相互作用,指导脑靶向与非脑靶向药物的研发设计。本文对转运蛋白在药物血脑屏障转运中的重要作用进行了综述。     

Novel models for assessing blood-brain barrier drug permeation

INTRODUCTION: The blood-brain barrier (BBB) is a selectively permeable micro-vascular unit which prevents many central nervous system (CNS)-targeted compounds from reaching the brain. A significant problem in CNS drug development is the ability to model BBB permeability in a timely, reproducible and cost-effective manner. Through the years, several models have been used such as artificial membranes, cell culture and animal models. AREAS COVERED: In this focused review, the authors cover novel models which have been developed or are in the process of being developed which can be used in modeling BBB. These models can either be used to determine BBB permeability or whether a compound may be disrupting the BBB. Many of these models lend themselves to high-throughput screening. The main model organisms covered here are the grasshopper (Locusta migratoria), fruit fly (Drosophila melanogaster) and zebrafish (Danio rerio). EXPERT OPINION: Many of the models covered here have only recently been utilized for BBB studies and still needs to be fully studied for its impact on reducing costs during drug development. The strength of these models lay in the fact that a whole organism experiment can be done in high throughput fashion as compared with classical vertebrate models such as micro-dialysis.     

Influx and efflux transport of H1-antagonist epinastine across the blood-brain barrier.

We investigated influx and efflux transporters involved in blood-brain barrier transport of the nonsedative H1-antagonist epinastine. The basal-to-apical transport of [14C]epinastine was markedly higher than that in the opposite direction in LLC-GA5-COL150 cells stably transfected with human multidrug resistance (MDR)1 gene. The brain-to-plasma concentration ratio of [14C]epinastine in mdr1a/b(-/-) mice was 3.2 times higher than that in wild-type mice. The uptake of both [3H]mepyramine and [14C]epinastine into immortalized rat brain capillary endothelial cells (RBEC)1 showed temperature and concentration dependence. The kinetic parameters, K(m), V(max), and uptake clearance (V(max)/K(m)), of the initial uptake of [3H]mepyramine and [14C]epinastine by RBEC1 were 150 microM, 41.8 nmol/min/mg protein, and 279 microl/min/mg protein for mepyramine and 10.0 mM, 339 nmol/min/mg protein, and 33.9 microl/min/mg protein for epinastine, respectively. The uptake of [3H]mepyramine and [14C]epinastine by RBEC1 was inhibited by organic cations such as quinidine, amantadine, and verapamil, but not by other organic cations, tetraethyl ammonium, guanidine, and carnitine. Organic anions such as benzoic acid, estrone-3-sulfate, taurocholate, and neutral digoxin were not inhibitory. Furthermore, some cationic H1 antagonists (chlorpheniramine, cyproheptadine, ketotifen, and desloratadine) inhibited the [3H]mepyramine and [14C]epinastine uptake into RBEC1. In conclusion, the present study demonstrated that the combination of efficient efflux transport by P-glycoprotein and poor uptake by the influx transporter, which is identical with that responsible for the uptake of mepyramine, account for the low brain distribution of epinastine.     

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。     

The ABC of the blood-brain barrier - regulation of drug efflux pumps

According to the World Health Organization Central nervous system disorders are the major medical challenge of the 21st Century, yet treatments for many CNS disorders are either inadequate or absent. One reason is the existence of the blood-brain barrier, which strictly limits the access of substances to the brain. A key element of the barrier function is the expression of ABC export proteins in the luminal membrane of brain microvessel endothelial cells. Understanding the signaling cascades and the response to endogenous and exogenous stimuli, which lead to altered expression or function of the transporters as well as subsequent modulation of the transporters, may offer novel strategies to overcome the barrier and to improve drug delivery to the brain. This review gives a short overview about structure of the key elements of the blood-brain barrier with emphasis on ABC transporters. An insight into regulation of function and expression of these transport proteins is given and the involvement of these transporters in CNS diseases is discussed.     

Pluronic block copolymers as modulators of drug efflux transporter activity in the blood-brain barrier

Drug efflux transporters can influence the absorption, tissue distribution and elimination of many therapeutic agents. Modulation of drug efflux transporter activity is being explored as a means for improving the pharmacokinetic and pharmacodynamic properties of various drugs. In this regard, several polymer formulations have been shown to inhibit drug efflux transporters such as P-glycoprotein (P-gp). The current review will focus on Pluronic block copolymers in particular, the mechanisms involved in the effects of Pluronic on drug efflux transporters, and the optimal polymer compositions required for inhibition of drug efflux transporters. Special emphasis will be placed on the potential applications of Pluronic in enhancing the blood-brain barrier (BBB) penetration of drugs.     

Blood-brain barrier efflux transport

Efflux transport at the blood-brain barrier (BBB) limits the brain tissue exposure to a variety of potential therapeutic agents, including compounds that are relatively lipophilic and would be predicted to permeate the endothelial lining of the brain microvasculature. Recent advances in molecular and cell biology have led to identification of several specific transport systems at the blood-brain interface. Refinement of classical pharmacokinetic experimentation has allowed assessment of the structural specificity of transporters, the impact of efflux transport on brain tissue exposure, and the potential for drug-drug interactions at the level of BBB efflux transport. The objective of this minireview is to summarize efflux transporter characteristics (location, specificity, and potential inhibition) for transport systems identified in the BBB. A variety of experimental approaches available to ascertain or predict the impact of efflux transport on net brain tissue uptake of substrates also are presented. The potential impact of efflux transport on the pharmacodynamics of agents acting in the central nervous system are illustrated. Finally, general issues regarding the role of identifying efflux transport as part of the drug development process are discussed.     

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.     

New experimental models of the blood-brain barrier for CNS drug discovery

Introduction: The blood-brain barrier (BBB) is a dynamic biological interface which actively controls the passage of substances between the blood and the central nervous system (CNS). From a biological and functional standpoint, the BBB plays a crucial role in maintaining brain homeostasis inasmuch that deterioration of BBB functions are prodromal to many CNS disorders. Conversely, the BBB hinders the delivery of drugs targeting the brain to treat a variety of neurological diseases.

Area covered: This article reviews recent technological improvements and innovation in the field of BBB modeling including static and dynamic cell-based platforms, microfluidic systems and the use of stem cells and 3D printing technologies. Additionally, the authors laid out a roadmap for the integration of microfluidics and stem cell biology as a holistic approach for the development of novel in vitro BBB platforms.

Expert opinion: Development of effective CNS drugs has been hindered by the lack of reliable strategies to mimic the BBB and cerebrovascular impairments in vitro. Technological advancements in BBB modeling have fostered the development of highly integrative and quasi- physiological in vitro platforms to support the process of drug discovery. These advanced in vitro tools are likely to further current understanding of the cerebrovascular modulatory mechanisms.     

Comparison of drug permeabilities across the blood-retinal barrier, blood-aqueous humor barrier, and blood-brain barrier

Drugs vary in their ability to permeate the blood-retinal barrier (BRB), blood-aqueous humor barrier (BAB), and blood-brain barrier (BBB) and the factors affecting the drug permeation remain unclear. In this study, the permeability of various substances across BRB, BAB, and BBB in rats was determined using the brain uptake index (BUI), retinal uptake index (RUI), and aqueous humor uptake index (AHUI) methods. Lipophilic substances showed high permeabilities across BBB and BRB. The RUI values of these substances were approximately four-fold higher than the BUI values. The AHUI versus lipophilicity curve had a parabolic shape with AHUI(max) values at log D(7.4) ranging from -1.0 to 0.0. On the basis of the difference on the lipophilicities, verapamil, quinidine, and digoxin showed lower permeability than predicted from those across BBB and BRB, whereas only digoxin showed a lower permeability across BRB. These low permeabilities were significantly increased by P-glycoprotein inhibitors. Furthermore, anion transporter inhibition increased the absorption of digoxin to permeate into the retina and aqueous humor. In conclusion, this study suggests that efflux transport systems play an important role in the ocular absorption of drugs from the circulating blood after systemic administration.     

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.