Comparison of blood-brain barrier permeability assays: in situ brain perfusion, MDR1-MDCKII and PAMPA-BBB

Permeability data from MDR1-MDCKII and PAMPA-BBB assays were compared to data from in situ brain perfusion to evaluate the accuracy of in vitro assays in predicting in vivo blood-brain barrier (BBB) permeability. PAMPA-BBB significantly correlated to in situ brain perfusion, however, MDR1-MDCKII had no correlation with in situ brain perfusion. PAMPA-BBB also significantly correlated with MDR1-MDCKII. The differential correlation of PAMPA-BBB and MDR1-MDCKII to in situ brain perfusion appears to be mainly due to the difference in membrane characteristics rather than binding to brain tissue. The MDR1-MDCKII cell membrane has lower ratios of: phospholipid to cholesterol, unsaturated to saturated acyl chains, and phosphatidyl-choline (PC) to sphingomyelin (SM) than brain endothelial cells, making it a poor passive permeability model for BBB. The BBB is more hydrophobic, rigid, and less fluidic than MDR1-MDCKII cell membrane. PAMPA-BBB more closely matches the BBB membrane in these characteristics and is a more accurate passive diffusion permeability model for BBB than MDR1-MDCKII. PAMPA-BBB is high throughput, low cost and has good prediction of in vivo BBB permeability, and therefore, it is a valuable tool in drug discovery to screen compounds for the rate of brain penetration.     

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.     

Synthesis and biochemical evaluation of 3-fluoromethyl-1,2,3, 4-tetrahydroisoquinolines as selective inhibitors of phenylethanolamine N-methyltransferase versus the alpha(2)-adrenoceptor.

A series of 3-fluoromethyl-1,2,3,4-tetrahydroisoquinolines (3-fluoromethyl-THIQs) was proposed, and their phenylethanolamine N-methyltransferase (PNMT) and alpha(2)-adrenoceptor affinities were predicted through the use of comparative molecular field analysis (CoMFA) models. These compounds were synthesized and evaluated for affinity at PNMT and the alpha(2)-adrenoceptor. It was discovered that these compounds are some of the most selective inhibitors of PNMT versus the alpha(2)-adrenoceptor known. To determine the ability of these compounds to penetrate the blood-brain barrier (BBB), a series of THIQs possessing a variety of calculated partition coefficients (Clog P) were assayed using an in vitro BBB model. This study found a good correlation between lipophilicity (Clog P) and BBB permeability, which indicated that THIQs possessing Clog P values of at least 0.13-0.57 should have some penetration into the brain. Two compounds [3-fluoromethyl-7-N-(4-chlorophenyl)aminosulfonyl-THIQ (18) and 3-fluoromethyl-7-cyano-THIQ (20)] possess calculated partition coefficients greater than 0.57 and display selectivities (alpha(2)-adrenoceptor K(i)/PNMT K(i)) greater than 200 and thus represent promising leads in the development of highly selective inhibitors of PNMT with the ability to penetrate the BBB.     

Molecular anatomy of the brain endothelial barrier: an overview of the distributional features

The blood-brain barrier (BBB) impedes the influx of intravascular compounds from the blood to the brain. The elements composing the BBB are endothelial cells, pericytes and the end-feet of astrocytes. Among them, the endothelial cell barrier line is the most critical for preventing toxic substances from entering the brain. In this review, we focus on the ultrastructural distribution of important components in the intracellular junction and cytoplasm of brain endothelial cells. The ultrastructural distribution of tight junction-specific integral membrane proteins such as occludin, junctional adhesion molecules, claudin, peripheral zonula occludens protein-1 (ZO-1), adherens junction-specific transmembrane protein cadherin, and adherens junction-associated peripheral proteins alpha-catenin, beta-catenin, and p120 catenin is reviewed. P-glycoprotein and some other transporters recently discovered in endothelial cells prevent several compounds from entering the brain parenchyma. It is likely that the transient inhibition of P-glycoprotein by antidepressants enables other medicines to enter the brain. Vesicular transport with clathrin-mediated or adsorptive endocytosis through endothelial cells is also critical for transportation of blood-born substances from the bloodstream to the brain. How medicines pass the BBB to reach the brain parenchyma is discussed.     

Blood-Brain Barrier Permeability of Asiaticoside,Madecassoside and Asiatic Acid in Porcine Brain Endothelial Cell Model

Neurotherapeutic potentials of Centella asiatica and its reputation to boost memory, prevent cognitive deficits and improve brain functions are widely acknowledged. The plant's bioactive compounds, i.e. asiaticoside, madecassoside and asiatic acid were reported to have central nervous system (CNS) actions, particularly in protecting the brain against neurodegenerative disorders. Hence, it is important for these compounds to cross the blood-brain barrier (BBB) to be clinically effective therapeutics. This study aimed to explore the capability of asiaticoside, madecassoside and asiatic acid to cross the BBB using in vitro BBB model from primary porcine brain endothelial cells (PBECs). Our findings showed that asiaticoside, madecassoside and asiatic acid are highly BBB permeable with apparent permeability (P_app) of 70.61 ± 6.60, 53.31 ± 12.55 and 50.94 ± 10.91 × 10^?6 cm/s respectively. No evidence of cytotoxicity and tight junction disruption of the PBECs were observed in the presence of these compounds. Asiatic acid showed cytoprotective effect towards the PBECs against oxidative stress. This study reported for the first time that Centella asiatica compounds demonstrated high capability to cross the BBB, comparable to central nervous system drugs, and therefore warrant further development as therapeutics for the treatment of neurodegenerative diseases.     

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.     

Altered brain penetration of diclofenac and mefenamic acid, but not acetaminophen, in Shiga-like toxin II-treated mice

It is well accepted that bacterial and virus infections elevate the levels of cytokines in serum and cerebrospinal fluids. Such high levels of cytokines might alter the integrity of the blood-brain barrier (BBB) and/or blood-cerebrospinal fluid barrier (BCSFB), subsequently affecting brain penetration of drugs. However, few reports have addressed this issue. Thus, we investigated brain penetration of cyclooxygenase (COX) inhibitors, commonly used as antipyretics, in mice treated with Shiga-like toxin II (SLT-II) derived from E. coli O157:H7, which significantly elevates cytokine levels. As antipyretics, we used diclofenac, mefenamic acid, and acetaminophen. We found that SLT-II significantly increased the brain-to-plasma concentration ratio (Kp) of diclofenac and mefenamic acid, but not of acetaminophen. Moreover, the Kp of diclofenac and mefenamic acid was increased by probenecid, an anionic compound. These results suggest that efflux anion transporters might be involved in the transport of diclofenac and mefenamic acid. Western blot analysis revealed that SLT-II decreased the expression of organic anion transporter-3, an efflux transporter located on the BBB and/or BCSFB. Taken together, these results suggest that SLT-II and/or SLT-II-stimulated cytokines might change brain penetration of drugs and could possibly increase the risk of their side-effects by altering the expression of transporters.     

A comparison of the effects of p-glycoprotein inhibitors on the blood-brain barrier permeation of cyclic prodrugs of an opioid peptide (DADLE)

The objective of this study was to elucidate the role of P-glycoprotein (P-gp) in restricting the blood-brain barrier (BBB) permeation of cyclic prodrugs of the opioid peptide DADLE (H-Tyr-D-Ala-Gly-Phe-D-Leu-OH). The BBB permeation characteristics of these prodrugs and DADLE were determined using an in situ perfused rat brain model and in vitro cell culture model (MDCK-MDR1 cells) of the BBB. The activities of P-gp in these models were characterized using a known substrate (quinidine) and known inhibitors [cyclosporine A (CyA), GF-120918, PSC-833] of P-gp. Cyclic peptide prodrugs exhibited very poor permeation in both models. Inclusion of GF-120918, CyA, or PSC-833 in the brain perfusion medium or the cell culture medium significantly increased the permeation of these cyclic prodrugs. The order of potency of these P-gp inhibitors, as measured using the cyclic prodrugs as substrates, was, by in vitro MDCK-MDR1 cells: GF-120918 = CyA >or= PSC-833; and by in situ rat brain perfusion: GF-120918 > CyA = PSC-833. In conclusion, P-gp in the BBB is the major factor restricting the brain permeation of these cyclic prodrugs. MDCK-MDR1 cells can predict the order of potencies of the investigated P-gp inhibitors to enhance the rat BBB permeation of quinidine and the cyclic prodrugs.     

Antiemetic specificity of dopamine antagonists

Twelve antagonists of apomorphine-induced emesis in dogs were studied in different tests to evaluate their antiemetic specificity. Ten of these antagonists were neuroleptics: benzquinamide, clebopride, bromopride, prochlorperazine, haloperidol, chlorpromazine, thiethylperazine, metoclopramide, droperidol, and pimozide blocked conditioned responding in dogs and apomorphine-induced stereotyped behavior in rats. The use of these compounds as anti-emetics entails a risk of neurological side effects. Metopimazine and domperidone were devoid of neuroleptic activity. Metopimazine, however, showed potent -adrenergic blocking activity, showed histamine H₁ antagonism, and induced palpebral ptosis. Therapeutic doses of metopimazine are, therefore, likely to produce sedation and side-effects related to autonomic blockade. Domperidone showed potent antiemetic activity and, up to high doses, no other central or peripheral effects. Therefore, domperidone is the only specific antiemetic known.     

Evidence of carrier‐mediated transport in the penetration of donepezil into the rat brain

The objective of this study was to characterize the mechanism that controls the transport of donepezil into the brain. The apparent brain uptake clearance (CL_app,br) was decreased as a function of the dose of donepezil, suggesting an involvement of a saturable transport process via transporter(s) in the penetration across the blood–brain barrier (BBB). Consistent with in vivo results, the uptake of substrates for organic cation transporters was significantly reduced by donepezil in both MBEC4 cells (i.e., a model for BBB) and HEK 293 cells expressing the transporters found in the brain, indicative of the involvement of organic cation transporters in the transport of the drug. Furthermore, donepezil transport was enhanced (p < 0.01) in HEK 293 cells expressing rOCNT1, rOCTN2, or rCHT1. The CL_app,br was reduced up to 52.8% of the control in rats that had been pretreated with choline, while the CL_app,br was unaffected with pretreatments with organic cations other than choline, suggesting that choline and donepezil share a common transport mechanism in the penetration across the BBB in vivo. Taken together, these observations suggest that the transport of donepezil across the BBB is mediated by organic cation transporters such as choline transport system(s). © 2009 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 99: 1548–1566, 2010     

Pharmacokinetics and pharmacodynamics of some oximes and associated therapeutic consequences: a critical review

Undoubtedly, the use of oximes represents real progress in counteracting intoxications with organophosphates (OP), through potentiating antidotal effects of atropine. The penetration extent of these compounds through the blood–brain barrier (BBB) to significantly reactivate phosphorylated or phosphonylated acetylcholinesterase (AChE) in the brain still remains a debatable issue. Penetration of biological barriers by oximes was investigated mainly through determination of several quantitative parameters characterizing digestive absorption and BBB penetration. A weak penetration of biological barriers could be concluded from the available experimental data. The functional parameters/therapeutic effects following the penetration of oximes through BBB, more precisely the antagonism of OP‐induced seizures and hypothermia, prevention of brain damage and respiratory center protection, leading to the final end‐point, the survival of intoxicated organisms, are of high interest. It seems obvious that oximes are weakly penetrating the BBB, with minimal brain AChE reactivation (<5%) in important functional areas, such as the ponto‐medullar. The cerebral protection achieved through administration of oximes is only partial, without major impact on the antagonism of OP‐induced seizures, hypothermia and respiratory center inhibition. The antidotal effects probably result from synergic effects of other PD properties, different from the brain AChE reactivation process. Oxime structures especially designed for enhanced BBB penetration, through potentiating the hydrophobic characteristics, more often produce neurotoxic effects. Certainly, obtaining oximes with broad action spectrum (active against all OP types) would make a sense, but certainly, such a target is not achievable only through the increase in their penetrability in the brain.     

Endocytosis at the blood–brain barrier: From basic understanding to drug delivery strategies

The blood–brain barrier (BBB) protects the central nervous system (CNS) from potentially harmful xenobiotics and endogenous molecules. Anatomically, it comprises the brain microvasculature whose functionality is nevertheless influenced by associated astrocyte, pericyte and neuronal cells. The highly restrictive paracellular pathway within brain microvasculature restricts significant CNS penetration to only those drugs whose physicochemical properties afford ready penetration into hydrophobic cell membranes or are capable of exploiting endogenous active transport processes such as solute carriers or endocytosis pathways. Endocytosis at the BBB is an essential pathway by which the brain obtains its nutrients and affords communication with the periphery. The development of strategies to exploit these endocytic pathways for the purposes of drug delivery to the CNS is still an immature field although some impressive results have been documented with the targeting of particular receptors. This current article initially provides an overview of general endocytosis processes and pathways showing evidence of their functional existence within the BBB. Subsequent sections provide, in an entity-specific manner, comprehensive reviews on BBB transport investigations of endocytosis involving: transferrin and the targeting of the transferrin receptor; hormones; cytokines; cell penetrating peptides; microorganisms and toxins, and nanoparticles aimed at more effectively delivering drugs to the CNS.     

Endocytosis at the blood-brain barrier: from basic understanding to drug delivery strategies

The blood-brain barrier (BBB) protects the central nervous system (CNS) from potentially harmful xenobiotics and endogenous molecules. Anatomically, it comprises the brain microvasculature whose functionality is nevertheless influenced by associated astrocyte, pericyte and neuronal cells. The highly restrictive paracellular pathway within brain microvasculature restricts significant CNS penetration to only those drugs whose physicochemical properties afford ready penetration into hydrophobic cell membranes or are capable of exploiting endogenous active transport processes such as solute carriers or endocytosis pathways. Endocytosis at the BBB is an essential pathway by which the brain obtains its nutrients and affords communication with the periphery. The development of strategies to exploit these endocytic pathways for the purposes of drug delivery to the CNS is still an immature field although some impressive results have been documented with the targeting of particular receptors. This current article initially provides an overview of general endocytosis processes and pathways showing evidence of their functional existence within the BBB. Subsequent sections provide, in an entity-specific manner, comprehensive reviews on BBB transport investigations of endocytosis involving: transferrin and the targeting of the transferrin receptor; hormones; cytokines; cell penetrating peptides; microorganisms and toxins, and nanoparticles aimed at more effectively delivering drugs to the CNS.     

Discovery Studio软件在分析中药成分透过血脑屏障中的应用

血脑屏障是药物能否进入脑组织发挥作用的重要屏障。中药有效成分的结构与其透过血脑屏障的能力有一定的关系。Discovery Studio软件对非糖苷类成分通过血脑屏障的分析结果与文献报道基本吻合,而对糖苷类成分则无法准确预测。     

Effect of P-glycoprotein-mediated efflux on cerebrospinal fluid/plasma concentration ratio.

The ratio of drug levels in cerebrospinal fluid (CSF) to plasma (CSF/plasma) at equilibrium has been viewed as in vivo free fraction (fp) in plasma [CSF/plasma = fp], if no active transport is involved in brain penetration. We determined the CSF/plasma level following oral administration in rats and in vitro rat plasma protein binding for 20 compounds that were synthesized in our institute and have similar physicochemical properties. However, results indicated that the CSF/plasma was not only poorly correlated with fp but remarkably lower than fp in most of the compounds tested, suggesting that certain transporters such as P-glycoprotein (P-gp) located in blood-brain barrier (BBB) may decrease the unbound drug concentration in the brain. We evaluated P-gp-mediated transport activity of the 20 compounds with P-gp (mdr1a)-transfected LLC-PK1 cells and calculated P-gp efflux index (PEI), indicating the extent of P-gp-mediated transport. A plot of the CSF/plasma versus fp/PEI showed a strong correlation (r = 0.93), and the absolute values were almost identical [CSF/plasma = fp/PEI]. These results suggest that P-gp quantitatively shifts the equilibrium of unbound drugs across the BBB. Although we cannot rule out the possibility that endogenous transporters other than P-gp on BBB and/or blood-CSF barrier may affect CSF levels of compounds, the present study indicated that fp and PEI measurements may be useful in predicting in vivo CSF/plasma fractions for central nervous system-targeting drugs.     

Assessment of Blood–Brain Barrier Permeability Using the <Emphasis Type="BoldItalic">In Situ</Emphasis> Mouse Brain Perfusion Technique

Purpose  To assess the blood–brain barrier (BBB) permeability of 12 clinically-used drugs in mdr1a(+/+) and mdr1a(−/−) mice, and investigate the influence of lipophilicity, nonspecific brain tissue binding, and P-gp-mediated efflux on the rate of brain uptake. Methods  The BBB partition coefficient (PS) was determined using the in situ mouse brain perfusion technique. The net brain uptake for 12 compounds, and the time course of brain uptake for selected compounds ranging in BBB equilibration kinetics from rapidly-equilibrating (e.g., alfentanil, sufentanil) to slowly-equilibrating (fexofenadine), was determined and compared. Results  There was a sigmoidal relationship in mdr1a(−/−) mice between the log-PS and clogD_7.4 in the range of 0–5. The brain uptake clearance was a function of both permeability and blood flow rate. The brain unbound fraction was inversely proportional to lipophilicity. Alfentanil achieved brain equilibrium approximately 4,000-fold faster than fexofenadine, based on the magnitude of PS×fu,brain. Conclusions   In situ brain perfusion is a useful technique to determine BBB permeability. Lipophilicity, ionization state, molecular weight and polar surface area are all important determinants for brain penetration. The time to blood-to-brain equilibrium varies widely for different compounds, and is determined by a multiplicity of pharmacokinetic factors.     

Case study: adapting in vitro blood-brain barrier models for use in early-stage drug discovery

Several parameters influencing the brain distribution of compounds must be considered when designing potential neuropharmaceuticals in early-stage drug discovery. The blood-brain barrier (BBB) represents an obstacle for drug penetration into the brain. Many in vitro BBB models have proven useful for predicting the BBB permeation rate, but do not meet all criteria for use in early-stage drug discovery: feasibility, rapidity, reliability and a low requirement for human resources. To meet this demand, we have developed a robust, higher-throughput, cell-based model exhibiting BBB features (low paracellular permeability, functional efflux pumps and the correct endothelial phenotype). This system comes in a ready-to-use, frozen format, appropriate for in-house use by large pharmaceutical firms and small biotech companies during early-stage drug discovery.