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.     

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.     

The blood-brain barrier efflux transporters as a detoxifying system for the brain

The role played by efflux transport systems across the blood-brain barrier (BBB) in the disposition of xenobiotics in the brain is described. Several drugs and organic anions are transported across the BBB via P-glycoprotein and other carrier-mediated efflux transport systems. Studies using in vitro cultured brain capillary endothelial cells, kinetic analysis, and mdr1a gene knock-out mice have shown that P-glycoprotein, located on the BBB, restricts the entry of vincristine and quinidine to the brain. Brain microdialysis studies have demonstrated that the brain interstitial fluid (ISF) concentrations of quinolone antibiotics are significantly lower than their corresponding unbound serum concentrations. A distributed model analysis supports the finding that efflux transport systems on the BBB restrict distribution of 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxyinosine (DDI), and quinolone antibiotics. A brain efflux index (BEI) method has been developed to provide direct evidence of an efflux transport system for carrying substrates from the cerebrum to the circulating blood across the BBB. The BEI method revealed the existence of carrier-mediated efflux organic anion transport systems for compounds such as p-aminohippuric acid, AZT, DDI, taurocholic acid, BQ-123, and estron sulfate. Moreover, cerebral neurotransmitters such as gamma-aminobutyric acid, L-glutamic acid, and L-aspartic acid are transported from brain to the circulating blood in the intact form via a carrier-mediated efflux transport system. The BBB not only restricts nonspecific permeation from the circulating blood to the brain, but also functions as an active efflux transport system for xenobiotics. Accordingly, the BBB plays a very important role by pumping xenobiotics and some endogenous compounds out of the brain, acting as a central nervous system (CNS)-specific detoxifying system supporting and maintaining normal cerebral function.     

Efflux transport systems for drugs at the blood-brain barrier and blood-cerebrospinal fluid barrier (Part 2)

Penetration of the blood-brain barrier or blood-cerebrospinal fluid barrier is necessary if a drug is to achieve the required concentration for a desired pharmacological effect. Efflux transport systems at such barriers provide protection for the CNS by removing drugs from the brain or cerebrospinal fluid, and transferring them to the systemic circulation. In Part 2 of this review, in vivo and in vitro studies of efflux transport via these barriers are discussed, with reference to the transporters previously described in Part 1(1).     

Drug transport at the blood-brain barrier and the choroid plexus

The blood-brain barrier (BBB) and blood-CSF barrier (BCSFB) represent the main interfaces between the central nervous system (CNS) and the peripheral circulation. Drug exposure to the CNS is dependent on a variety of factors, including the physical barrier presented by the BBB and the BCSFB and the affinity of the substrate for specific transport systems located at both of these interfaces. It is the aggregate effect of these factors that ultimately determines the total CNS exposure, and thus pharmacological efficacy, of a drug or drug candidate. This review discusses the anatomical and biochemical barriers presented to solute access to the CNS. In particular, the important role played by various efflux transporters in the overall barrier function is considered in detail, as current literature suggests that efflux transport likely represents a key determinant of overall CNS exposure for many substrates. Finally, it is important to consider not only the net delivery of the agent to the CNS, but also the ability of the agent to access the relevant target site within the CNS. Potential approaches to increasing both net CNS and target-site exposure, when such exposure is dictated by efflux transport, are considered.     

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.     

Drug transport to the brain: key roles for the efflux pump P-glycoprotein in the blood-brain barrier

1. The blood-brain barrier (BBB) contributes to brain homeostastis and fulfills a protective function by controlling the access of solutes and toxic substances to the central nervous system (CNS). The efflux transporter P-glycoprotein (P-gp) is a key element of the molecular machinery that confers special permeability properties to the BBB. 2. P-gp, which was initially recognized for its ability to expel anticancer drugs from multidrug-resistant cancer cells, is strongly expressed in brain capillaries. Its expression in the BBB limits the accumulation of many hydrophobic molecules and potentially toxic substances in the brain. 3. The purpose of this review is to summarize the current state of knowledge about the expression of P-gp, its cellular localization as well as its possible functions in the BBB.     

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.     

Hydrophilic anti-migraine triptans are substrates for OATP1A2, a transporter expressed at human blood-brain barrier

1. OATP1A2 is expressed in the luminal membrane of human blood-brain barrier (BBB). The human tissue with the highest OATP1A2 mRNA expression is the brain.

2. We have established a robust BacMam2-OATP1A2 transduced HEK293 system. Among the 36 central nervous system (CNS) marketed drugs tested, hydrophilic triptans, 5-HT_1B/1D receptor agonists for the treatment of migraine attacks, were identified as OATP1A2 substrates. Kinetics (K_m and V_max) were determined for six marketed triptans.

3. Structure-activity relationship (SAR) obtained from 18 triptan structural analogs revealed that the positively charged basic amine atom was essential for efficient OATP1A2-mediated triptan uptake and uptake rate was in the order of tertiary > secondary > primary.

4. Preliminary quantitative SAR analysis of the triptan analogs demonstrated positive correlation between OATP1A2-mediated uptake rate and van der Waals volume (vdw_vol).

5. OATP1A2 was specifically expressed on the apical side of MDCKII monolayer after BacMam2-OATP1A2 transduction and can facilitate transport of triptans across the MDCKII monolayer from apical to basolateral side. Involvement of OATP1A2 for brain penetration of triptans in human requires further investigation.

     

Hydrophilic anti-migraine triptans are substrates for OATP1A2, a transporter expressed at human blood-brain barrier

OATP1A2 is expressed in the luminal membrane of human blood-brain barrier (BBB). The human tissue with the highest OATP1A2 mRNA expression is the brain. We have established a robust BacMam2-OATP1A2 transduced HEK293 system. Among the 36 central nervous system (CNS) marketed drugs tested, hydrophilic triptans, 5-HT(1B/1D) receptor agonists for the treatment of migraine attacks, were identified as OATP1A2 substrates. Kinetics (K(m) and V(max)) were determined for six marketed triptans. Structure-activity relationship (SAR) obtained from 18 triptan structural analogs revealed that the positively charged basic amine atom was essential for efficient OATP1A2-mediated triptan uptake and uptake rate was in the order of tertiary > secondary > primary. Preliminary quantitative SAR analysis of the triptan analogs demonstrated positive correlation between OATP1A2-mediated uptake rate and van der Waals volume (vdw_vol). OATP1A2 was specifically expressed on the apical side of MDCKII monolayer after BacMam2-OATP1A2 transduction and can facilitate transport of triptans across the MDCKII monolayer from apical to basolateral side. Involvement of OATP1A2 for brain penetration of triptans in human requires further investigation.     

Efflux transport systems for drugs at the blood-brain barrier and blood-cerebrospinal fluid barrier (Part 1)

Penetration through the blood-brain barrier (BBB) and blood-cerebrospinal fluid barrier (BCSFB) is necessary if a drug is to achieve the required concentration for a desired pharmacological effect. Efflux transport systems at the BBB and BCSFB provide a protective barrier function by removing drugs from the brain or cerebrospinal fluid and transferring them to the systemic circulation, respectively; several transporters at the BBB and BCSFB have been identified. Efflux transport should be taken into consideration during drug development to improve brain penetration and to avoid drug-drug interactions involving these transporters and subsequent side effects.     

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.     

Effects of aripiprazole and its active metabolite dehydroaripiprazole on the activities of drug efflux transporters expressed both in the intestine and at the blood-brain barrier

The inhibition potencies of aripiprazole and its active metabolite, dehydroaripiprazole, on the activities of human multidrug resistance protein 1 (MDR1/ABCB1; P‐glycoprotein), breast cancer resistance protein (BCRP/ABCG2) and multidrug resistance‐associated protein 4 (MRP4/ABCC4), that are drug efflux transporters expressed both in the intestine and at the blood–brain barrier (BBB), were investigated. Aripiprazole and dehydroapripiprazole showed relatively strong inhibitory effects on human MDR1 with IC₅₀ values of 1.2 and 1.3 µm in human MDR1‐transfected Mardin‐Darby canine kidney (MDCKII‐MDR1) cells, respectively. The inhibition potencies of other atypical antipsychotics (risperidone, paliperidone, olanzapine and ziprasidone) for human MDR1 were also evaluated using the same in vitro experimental system and IC₅₀ values were more than 10‐fold higher than those of the two compounds. Aripiprazole and dehydroaripiprazole also had inhibition potencies against human BCRP with IC₅₀ values of 3.5 and 0.52 µm , respectively. The ratios of steady‐state unbound concentrations of aripiprazole and dehydroaripiprazole to their IC₅₀ values against human MDR1 and BCRP activities were less than 0.1, whereas the theoretically maximum gastrointestinal concentration of aripiprazole ([I]₂) to its IC₅₀ values was much higher than the cut‐off value of 10, proposed by the International Transporter Consortium (ITC) and the Food and Drug Administration (FDA). In contrast, aripiprazole and dehydroaripiprazole showed almost no inhibitory effect against the activity of human MRP4. These findings indicate that aripiprazole is unlikely to cause drug–drug interactions (DDIs) at the BBB when co‐administered with substrate drugs of these drug transporters investigated. However, interactions at the intestinal absorption process may be of concern. Copyright © 2012 John Wiley & Sons, Ltd.     

Opioid transport by ATP-binding cassette transporters at the blood-brain barrier: implications for neuropsychopharmacology

Some of the ATP-binding cassette (ABC) transporters like P-glycoprotein (P-gp; ABCB1, MDR1), BCRP (ABCG2) and MRPs (ABCCs) that are present at the blood-brain barrier (BBB) influence the brain pharmacokinetics (PK) of their substrates by restricting their uptake or enhancing their clearance from the brain into the blood, which has consequences for their CNS pharmacodynamics (PD). Opioid drugs have been invaluable tools for understanding the PK-PD relationships of these ABC-transporters. The effects of morphine, methadone and loperamide on the CNS are modulated by P-gp. This review examines the ways in which other opioid drugs and some of their active metabolites interact with ABC transporters and suggests new mechanisms that may be involved in the variability of the response of the CNS to these drugs like carrier-mediated system belonging to the solute carrier (SLC) superfamily. Exposure to opioids may also alter the expression of ABC transporters. P-gp can be overproduced during morphine treatment, suggesting that the drug has a direct or, more likely, an indirect action. Variations in cerebral neurotransmitters during exposure to opioids and the release of cytokines during pain could be new endogenous stimuli affecting transporter synthesis. This review concludes with an analysis of the pharmacotherapeutic and clinical impacts of the interactions between ABC transporters and opioids.     

Influence of P-glycoprotein on brucine transport at the in vitro blood-brain barrier

Brucine is a central agonist that can pass through the blood-brain barrier (BBB). The goal of this study is to examine whether brucine is one of the substrates of the drug transporter P-glycoprotein (P-gp) and to examine the effects of P-gp on the brucine transport at the in vitro BBB model. The P-gp ATPase assay was utilized to investigate the in vitro affinity of P-gp to brucine. Results suggested that K(m) of brucine (11.4 μmol/l) was smaller than the positive control, verapamil (16.4 μmol/l). In this study, we developed an in vitro BBB model, comprising a co-culture of primary rat brain microvessel endothelial cells and astrocytes for the transport study. The validated model was correct and available. Transendothelial electrical resistance reached (283.78 ± 18.85) Ω cm(2). The model displayed limited permeability to fluorescein sodium and [(125)I]albumin, with the apparent permeability coefficient Papp of (10.36 ± 0.86) × 10(-6) cm/s and (6.00 ± 0.78) × 10(-6)cm/s, respectively. The quantity of the bidirectional transport of brucine was determined by ultra-performance liquid chromatography-tandem mass spectrometry. In the absence of verapamil, the transport of brucine from basolateral compartment to apical compartment (BL-AP) was higher than from AP to BL at low, middle, and high concentrations (P<0.05). The excretion rate was 1.32, 1.56, and 1.54, respectively. However, following exposure to verapamil, the excretion rate at three different concentrations was decreased (P<0.05). All the results suggest that P-gp prevented brucine from passing through the in vitro BBB model.     

Analgesia and the blood-brain barrier transport system for Tyr-MIF-1/enkephalins: evidence for a dissociation

The blood-brain barrier is capable of transporting peptides with anti-opiate (Tyr-MIF-1) and opiate (enkephalins) activity out of the central nervous system. The relationship of this transport system to the various actions of opiates remains unexplored. This study examined the relationship between the rate of transport and opiate-induced analgesia. Both restraint, a stress that provokes an opiate-mediated analgesia, and the administration of morphine (12 mg/kg, i.p.) each induced an inhibition in the rate of transport. Such inhibition exhibited specificity, since the saturable, brain to blood transport of iodide remained unaltered. However, it was possible to dissociate analgesia and inhibition of transport. The onset and peak of analgesia, as measured by tail-flick latency induced by morphine, preceded the onset and peak of the inhibition of transport. Naltrexone, which blocks opiate-mediated analgesia, also induced inhibition of transport without any significant effect on tail-flick latency. (-) Naloxone but not (+) naloxone also weakly inhibited transport. Deprivation of food and water, associated with analgesia possibly mediated by the opiate, beta-endorphin, which is not transported out of the brain by this system, did not alter transport. These results suggest that while inhibition of transport and analgesia may occur together, these events probably represent two separate aspects of the action of opiates, that may even be mediated by separate receptor sites or peptides in the opiate family.     

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.