Understanding and circumventing the blood-brain barrier.

UNLABELLED: The blood-brain barrier presents a challenging obstacle to effective drug delivery to the central nervous system (CNS). Although biologically intended to protect the brain and spinal cord and provide a very stable fluid environment, the presence of a blood-brain barrier makes treatment of many CNS diseases difficult to achieve, as the required therapies cannot be delivered across the barrier in sufficient quantities or at all. Until relatively recently the blood-brain barrier was viewed largely as a physical barrier to diffusion, and the presence of tight junctions between endothelial cells simply prevented the passive diffusion of solutes from blood into the brain. Recent advances in cell and molecular biology have provided new insights into the function of the blood-brain barrier and it is now appreciated that, in addition to being a physical barrier, it is a complex transport and metabolic barrier and is a highly reactive and dynamic endothelium. Advances in understanding of the cell biology of the blood-brain barrier have opened new avenues and possibilities for improved drug delivery to the CNS. The challenges posed by the blood-brain barrier and the possibilities for overcoming them are reviewed. CONCLUSION: Increased understanding of the molecular biology of the blood-brain barrier is now opening the way for new strategies to deliver drugs to the CNS.

Glial induction of blood-brain barrier-like L-system amino acid transport in the ECV304 cell line.

The blood-brain barrier (BBB) is formed by the presence of tight junction complexes between brain endothelial cells that restrict paracellular permeability. As a consequence, a number of transport proteins are expressed on cerebral endothelial cells to facilitate the transport of nutrients into the brain. Although the modulation of barrier tight junction properties by glial-conditioned medium and by second messengers is well established, little is known about the effects of these factors on carrier-mediated BBB transport processes. The ECV304 cell line shows an endothelial phenotype and can be induced to upregulate certain BBB features in the presence of glial factors. In the present study, we have examined the effect of conditioned medium derived from rat C6-glioma cells (C6CM) on the function of the L-system amino acid transporter in ECV304 cells, using L-leucine as the model substrate, and have determined whether the changes observed can be mimicked by modulating intracellular cAMP levels. ECV304 cells exposed to C6CM exhibited a significant increase in both the affinity of leucine transport and the diffusional constant (Michaelis-Menten), while the maximal transport capacity remained unchanged. Conversely, acute exposure to modulators of the PKA and PKC second messenger pathways was found to reduce significantly the maximal transport capacity and diffusion constants, while transport affinity remained unchanged. In both cases, the maximal flux of leucine was increased, indicating transport of greater efficiency. This study indicates that exposure of ECV304 cells to C6CM provides an influence inducing L-system transport properties characteristic of brain endothelial cells. Furthermore, it appears that L-system-mediated transport of amino acids can be modulated by several distinct pathways.

S-adenosylmethionine is substrate for carrier mediated transport at the blood-brain barrier in vitro.

S-adenosylmethionine (SAM) is the sole methyl donor in the CNS where it is involved in a multitude of biochemical reactions. Peripherally administered SAM has been shown to increase SAM levels in cerebrospinal fluid and is reported to be effective in the treatment of numerous neurological disorders suggesting SAM crosses the blood-brain barrier (BBB). The mechanism of SAM entry into the brain remains unknown, but the presence of adenosyl and methionine residues in the molecule suggests probable entry via carrier mediated transport. We have investigated whether SAM utilises endogenous transport systems in cerebral endothelial cells, using RBE4 cells, an in vitro model of the BBB. SAM did not influence the transport of [(3)H]-methionine and only marginally reduced the uptake of [(3)H]-leucine in RBE4 cells. The inhibition constant for the latter was 2.11+/-0.29 mM (mean+/-S.E.M.). However, increasing concentrations of SAM strongly inhibited the transport of [3H]-adenosine in RBE4 cells in both the presence and the absence of sodium in the medium, with K(i) values of 199+/-32 and 139+/-8.4 microM, respectively. Lineweaver-Burk plots suggest a competitive mode of inhibition. The findings suggest that SAM is not recognised by the L-system transporter for large neutral amino acids at the brain endothelium. A significant interaction with the transport of adenosine, however, indicates that SAM has affinity for the nucleoside carrier systems; this is within the range of K(m) values of natural substrates and suggest that SAM may enter the CNS via the Na(+)-independent nucleoside carrier systems at the brain capillary endothelium.

Interaction of poly(butylcyanoacrylate) nanoparticles with the blood-brain barrier in vivo and in vitro.

Poly(butylcyanoacrylate) nanoparticles were produced by emulsion polymerisation and used either uncoated or overcoated with polysorbate 80 (Tween 80). [3H]-dalargin bound to nanoparticles overcoated with polysorbate 80 or in the form of saline solution was injected into mice and the brain concentrations of radioactivity determined. Statistically significant, three-fold higher brain concentrations with the nanoparticle preparations were obtained after 45 minutes, the time of greatest pharmacological response assessed as analgesia in previous experiments. In addition the brain inulin spaces in rats and the uptake of fluoresceine isothiocyanate labelled nanoparticles in immortalised rat cerebral endothelial cells, (RBE4) were measured. The inulin spaces after i.v. injection of polysorbate 80-coated nanoparticles were significantly increased by 1% compared to controls. This is interpreted as indicating that there is no large scale opening of the tight junctions of the brain endothelium by the polysorbate 80-coated nanoparticles. In in vitro experiments endocytic uptake of fluorescent nanoparticles by RBE4 cells was only observed after polysorbate 80-overcoating, not with uncoated particles. These results further support the hypothesis that the mechanism of blood-brain barrier transport of drugs by polysorbate 80-coated nanoparticles is one of endocytosis followed by possible transcytosis. The experiments were conducted in several laboratories as part of an EEC/INTAS collaborative program. For various procedural and regulatory reasons this necessitated the use of both rats and mice as experimental animals. The brain endothelial cell line used for the in vitro studies is the rat RBE4.

Affinity for the P-glycoprotein efflux pump at the blood-brain barrier may explain the lack of CNS side-effects of modern antihistamines.

First generation H1 receptor antagonists are often associated with adverse CNS effects such as sedation, whereas modern, second generation antihistamines are generally non-sedating. The difference in therapeutic profile is mainly due to the poor CNS penetration of the modern derivatives. Current explanations for the differential ability of classical and modern antihistamines to cross the blood-brain barrier (BBB), based on differences in lipophilicity or protein binding, are inadequate. We have tested the hypothesis that non-sedating antihistamines fail to enter the CNS due to recognition by the P-glycoprotein (Pgp) drug efflux pump expressed on the luminal surface of cerebral endothelial cells forming the BBB in vivo. The ability of several sedating and non-sedating antihistamines to affect the uptake of the Pgp model substrate [3H]-colchicine was examined using the immortalised rat brain endothelial cell line, RBE4, an established in vitro model of the BBB expressing Pgp. All second generation antihistamines tested, significantly increased net accumulation of [3H]-colchicine to a level similar to that caused by the Pgp inhibitor verapamil. By contrast, the first generation antihistamines showed no affinity for Pgp. The results indicate that differences in the ability of classical and modern antihistamines to interact with Pgp at the BBB may determine their CNS penetration and as a consequence the presence or absence of central side-effects.

Determinants of passive drug entry into the central nervous system.

1. The blood-brain barriers restrict the passive diffusion of many drugs into the brain and constitute a significant obstacle in the pharmacological treatment of central nervous system diseases and disorders. The degree of restriction they impose is variable, with some lipid-insoluble drugs effectively excluded from the brain, while many lipid-soluble drugs do not appear to be subject to any restriction. 2. The ease with which any particular drug diffuses across the blood-brain barrier is determined largely by the number and strength of intermolecular forces "holding" it to surrounding water molecules. By quantifying the molecular features that contribute to these forces, it is possible to predict the in vivo blood-brain barrier permeability of a drug from its molecular structure. Dipolarity, polarizability, and hydrogen bonding ability are factors that appear to reduce permeability, whereas molecular volume (size) and molar refraction are associated with increased permeability. 3. Increasing the passive entry of "restricted" drugs into the central nervous system can be achieved by disrupting the blood-brain barrier (increased paracellular diffusion) or by modifying the structure of "restricted" drugs to temporarily or permanently increase their lipid solubility (increased transcellular permeability). 4. Competitive inhibition of outwardly directed active efflux mechanisms (P-glycoprotein and MRP, the multidrug resistance-related protein) can also significantly increase the accumulation of certain drugs within the central nervous system.

Carrier-mediated delivery of metabotrophic glutamate receptor ligands to the central nervous system: structural tolerance and potential of the L-system amino acid transporter at the blood-brain barrier.

The brain endothelial large neutral amino acid carrier (L-system) is well suited for facilitated drug transport to the brain because of its high transport capacity and relatively broad structural substrate tolerance. The authors have examined the potential of this transporter for central nervous system (CNS) delivery of a new family of compounds derived from the large neutral amino acid phenylglycine. These compounds are highly selective for specific isoforms of metabotropic glutamate receptors (mGluRs) but will only become effective therapeutics for CNS diseases such as ischemic disorders, stroke, and epilepsy if they can effectively cross the blood-brain barrier. Using the immortalized rat brain endothelial cell line RBE4 as in vitro blood-brain barrier model, the authors have studied the interaction of phenylglycine and selected derivatives with the L-system-mediated transport of L-[3H]-histidine. The transport of L-histidine was characteristic of the L-system in vivo with the following kinetic parameters: Km 135 +/- 18 micromol/L, Vmax 15.3 +/- 1.13 nmol/min/mg protein, and K(D) 2.38 +/- 0.84 microL/min/mg protein. The affinities of the L-system for phenylglycine and the derivatives investigated increased in the order S-4-carboxyphenylglycine (Ki = 16 mmol/L) < R-phenylglycine (2.2 mmol/L) < S-3-hydroxy-phenylglycine (48 micromol/L) < S-phenylglycine (34 micromol/L), suggesting that a negative charge at the side chain or R-configuration is detrimental for carrier recognition, whereas neutral side chain substituents are well tolerated. The authors have further shown (1) that the mode of interaction with the L-system of S-phenylglycine and S-3hydroxy-phenylglycine is competitive, and (2) that the transporter carries these two agents into the cell as shown by high-performance liquid chromatography (HPLC) analysis of the RBE4 cell contents. The study provides the first evidence for the potential of S-phenylglycine derivatives for carrier-mediated delivery to the CNS and outlines the substrate specificity of the L-system at the blood-brain barrier for this class of mGluR ligands. As the affinities of S-phenylglycine and S-3-hydroxy-phenylglycine for the L-system carrier are even higher than those of some natural substrates, these agents should efficiently enter CNS via this route. Possible strategies for a synergistic optimization of phenylglycine-derived therapeutics with respect to desired activity at the CNS target combined with carrier-mediated delivery to overcome the blood-brain barrier are discussed.

Potential of immobilized artificial membranes for predicting drug penetration across the blood-brain barrier.

PURPOSE: The present study evaluates immobilized artificial membrane (IAM) chromatography for predicting drug permeability across the blood-brain barrier (BBB) and outlines the potential and limitations of IAMs as a predictive tool by comparison with conventional methods based on octanol/water partitioning and octadecylsilane (ODS)-HPLC. METHODS: IAM-and ODS-HPLC capacity factors were determined in order to derive the hydrophobic indices log kIAM nad log kW for two sets of compounds ranging from very lipid soluble (steroids) to more hydrophilic agents (biogenic amines). The uptake of the compounds across the in vivo BBB expressed as brain uptake index (BUI) has been correlated with these HPLC capacity factors as well as octanol/ water partition (ClogP) and distribution coefficients (log D7.4). RESULTS: For both test groups log kIAM correlates significantly with the respective log BUI of the drug (r2 = 0.729 and 0.747, p < 0.05), whereas with log kW, log D7.4 and ClogP there is only a correlation for the group of steroids (r2 = 0.789, 0.659 and 0.809, p < 0.05) but not for the group of biogenic amines. There is a good correlation between log kIAM and log kW. ClogP or log D7.4 for the group of steroids (r2 = 0.945.0867 and 0.974, p < 0.01) but not for the biogenic amines. CONCLUSIONS: All physico-chemical descriptors examined in this study equally well describe brain uptake of lipophilic compounds, while log kIAM is superior over log D7.4, ClogP and log kW when polar and ionizable compounds are included. The predictive value of IAMs, combined with the power of HPLC holds thus great promise for the selection process of drug candidates with high brain penetration.

Significant entry of tubocurarine into the brain of rats by adsorption to polysorbate 80-coated polybutylcyanoacrylate nanoparticles: an in situ brain perfusion study.

The possibility of using polysorbate 80-coated polybutylcyanoacrylate nanoparticles to deliver low molecular polar hydrophilic drugs to the CNS has been studied. Tubocurarine (a quaternary ammonium salt) does not penetrate the normal intact blood-brain barrier. However, the injection of this drug directly into the cerebral ventricles of the brain provokes the development of epileptiform seizures as assessed by electroencephalogram (EEG). An in situ perfused rat brain technique was used as an experimental technique together with a simultaneous recording of the EEG. Nanoparticles were prepared by butylcyanoacrylate polymerization in an acidic medium. Fifteen minutes after the introduction of tubocurarine-loaded polysorbate 80-coated nanoparticles into the perfusate, epileptiform spikes in the EEG appeared. Intraventricular injection of tubocurarine caused the appearance of the EEG seizures 5 min after administration. Neither tubocurarine solution nor tubocurarine-loaded nanoparticles without polysorbate 80 or a mixture of polysorbate 80 and tubocurarine were able to influence the EEG. Thus only the loading of tubocurarine onto the polysorbate 80-coated nanoparticles appears to enable the transport of this quaternary ammonium compound through the blood-brain barrier.

Functional expression of P-glycoprotein in an immortalised cell line of rat brain endothelial cells, RBE4.

The presence of P-glycoprotein in the cell plasma membrane limits the penetration of many cytotoxic substances into cells that express the gene product. There is considerable evidence also to indicate that P-glycoprotein is expressed as part of the normal blood-brain barrier in the luminal membranes of the cerebral capillary endothelial cells, where it presumably performs a protective function for the brain. This report describes the functional expression of P-glycoprotein in an immortalised cell line, RBE4, derived from rat cerebral capillary endothelial cells. The expression of P-glycoprotein is demonstrated by western immunoblotting and by immunogold and fluorescent staining with monoclonal antibodies. The cellular accumulation of [3H]colchicine and [3H]-vinblastine is investigated and shown to be enhanced by the presence of azidothymidine, chlorpromazine, verapamil, cyclosporin A, and PSC 833 ([3'-keto-Bmt1]-[Val2]-cyclosporin) at 50 or 100 microM concentration. It is concluded that the RBE4 cell line is a valuable tool for investigating the mechanisms of P-glycoprotein activity both in the blood-brain barrier and in multidrug resistance in general.

Arginine vasopressin reduces the blood-brain transfer of L-tyrosine and L-valine: further evidence of the effect of the peptide on the L-system transporter at the blood-brain barrier.

Arginine vasopressin (AVP) coinjected into the carotid artery in physiological concentrations (0.1 nmol/l), with either L-[3H]tyrosine or L-[3H]valine, induced changes in the kinetic parameters of the blood-to-brain transfer of both large neutral amino acids (LNAA) without alterations in brain haemodynamics. The half-saturation constant (Km), the maximum velocity of transport (V(max)) and Kd, the nonsaturable transport constant, were estimated in 9 brain regions of male Wistar rats anaesthetized with ether. Apart from Kd, significant changes in Km and V(max) were observed in all brain regions investigated. On average Km decreased from 0.17 to 0.048 mmol/l for tyrosine, and from 0.61 to 0.059 mmol/l for valine, whereas V(max) declined from 22 to 9.9 nmol/min/g for tyrosine, and from 29 to 3.2 nmol/min/g for valine, respectively. The results provide further evidence that vasopressin-receptor interactions at the blood-brain barrier (BBB) induce changes in the properties of the common transporter, the L-system, which eventually result in a suppression of the blood-to-brain transfer of LNAA. Data analysis of the 5 LNAA tested so far reveals a significant negative correlation (R = 0.98, P < 0.05) between the respective substrate affinity for the transporter and the corresponding magnitude of transport reduction induced by circulating AVP. Calculations of the unidirectional influx (J) of the LNAA indicate that AVP (1) reduces J by approximately one-third for every LNAA, but (2) does not change the relative contribution for each single LNAA to the total influx across the BBB.

The blood-brain barrier: principles for targeting peptides and drugs to the central nervous system.

The presence of the blood-brain barrier (BBB), reduces the brain uptake of many drugs, peptides and other solutes from blood. Strategies for increasing the uptake of drugs and peptide-based drugs include; structural modifications to increase plasma half-life; improving passive penetration of the BBB by increasing the lipophilicity of the molecule; designing drugs which react with transporters present in the BBB; and reducing turnover and efflux from the central nervous system (CNS).

Changes in amino acid levels in rat plasma, cisternal cerebrospinal fluid, and brain tissue induced by intravenously infused arginine-vasopressin.

Circulating arginine-vasopressin (AVP) is known to reduce the blood-to-brain transfer of large neutral amino acids (AA). As a first step to examine whether the reduced uptake by brain endothelial cells is reflected in changes in large neutral amino acid levels of the extracellular fluid environment of cells within the nervous tissue, we measured the concentrations of amino acids in plasma, cerebrospinal fluid (CSF), and hippocampal tissue of rats before and after infusion of AVP (34 and 68 ng/min/kg, respectively) over the time period of 60 min. AA levels changed in all compartments investigated during both saline and AVP infusions. Whereas in the saline-infused controls changes in CSF AA levels paralleled those in plasma, this correlation was abolished by raising AVP concentrations. The effect of AVP was found to be i) dependent on the AA, ii) different with respect to direction and iii) magnitude of changes in AA levels, and iv) in some cases dose dependent. In summary, AVP infusion increased plasma levels of 10 AA, but decreased all 15 AA measured by some 30% in CSF. In contrast to CSF, levels of AA were slightly enhanced in the hippocampal tissue. The results are not solely explicable by a reduced blood-to-brain transfer of AA. We conclude that further mechanisms by which AVP affects the availability of AA to the brain may exist. The physiological significance of the findings might be related to brain osmoregulation, especially in situations of stress.

Peptides and the blood-brain barrier: the status of our understanding.

In this limited review, it has only been possible to highlight some of the more significant interactions of peptides with the blood-brain barrier. The literature has been reviewed extensively in recent years, and the major reviews are included in the references. Certainly one of the major outstanding problems is an elucidation of the precise mechanism(s) by which centrally active peptides produce their effects. Without question peripherally administered peptides are able to modify central nervous activity; and for a rapidly growing number of peptides, an extraction by the cerebral endothelial cells can be demonstrated. For some of these peptides, the extraction involves highly specific transporters. What is far less clear is whether this internalization of peptide into the endothelial cells is the first step in a process of transcytosis, with an eventual abluminal exocytosis into brain extracellular fluid of the intact peptide, or an active fragment or whether their entry into brain extracellular fluid is via a different route. If, on the other hand, the mechanism of central action is via the circumventricular organs, a general entry into brain extracellular fluid may not be required. Clearly for different peptides the route and mechanism of action will differ and future attention should be focused on the precise mechanisms producing the central effects of defined peptides.

Simple high-performance liquid chromatographic analysis of free primary amino acid concentrations in rat plasma and cisternal cerebrospinal fluid.

The quantitation of 16 acidic, basic, small and large neutral amino acids was performed using 10-microliters sample aliquots of cisternal cerebrospinal fluid (CSF) and blood plasma of rats. The analytical technique is based upon a two-buffer HPLC system with fluorimetric detection of pre-column derivatized primary amino acids with o-phthaldialdehyde (OPA). A modification of a well established method, the power of the present technique comes from an improved resolution and sensitivity by installing a column heater adjusted to 43 degrees C and strictly reducing any contamination by background amino acids. The analysis is simplified by separating the amino acid derivatives with a linear buffer gradient and less time consuming by the use of a short analytical column with a higher flow-rate. Analytical precision, linearity of response and reproducibility were highly acceptable at both CSF and plasma concentrations of amino acids without changing any of the separation or detection parameters.

Permeability of the blood-brain barrier to the immunosuppressive cyclic peptide cyclosporin A.

Uptake of the immunosuppressive lipophilic peptide cyclosporin A has been measured by a number of techniques. The brain uptake index (BUI) technique in the rat yields only a small BUI value that is not significantly different from that of sucrose and mannitol and is comparable to other published BUI values for this compound. Brain perfusion studies in the guinea pig produce a unidirectional cerebrovascular permeability constant (Kin) of 1.2 +/- 0.28 microliter g-1 min-1 for the hippocampus. Intravenous bolus injection techniques also in the guinea pig characteristically produce a larger Kin value of 2.53 +/- 0.38 microliter g-1 min-1 for the same brain region, even after a correction for the inulin space of the tissue has been made. Apparent penetration of cyclosporin A into the cerebrospinal fluid (CSF) determined with the intravenous bolus injection technique is small with a Kin of 0.79 +/- 0.07 microliter g-1 min-1. However it is suggested that the radioactivity present in CSF is largely tritiated water. Studies with cultured cerebral endothelial cells from the rat have also been carried out and show that the cultured cells take up and accumulate cyclosporin A in vitro, achieving a tissue-to-medium ratio of 20 after 25 min of incubation. It is suggested that cyclosporin A is primarily taken up from lipoprotein at the blood-brain interface but, because of tight junctions at the blood-brain and blood-CSF barriers, becomes effectively trapped in the cerebral endothelial cells and the choroid plexus.

Chronic amphetamine intoxication and the blood-brain barrier permeability to inert polar molecules studied in the vascularly perfused guinea pig brain.

The brain vascular perfusion method, with a multiple-time brain uptake analysis, has been employed to study the effects of chronic amphetamine intoxication on the kinetics of entry of 2 inert polar molecules, D-[14C]mannitol (mol.wt. 180) and [3H]polyethylene glycol (PEG, mol.wt. 4000) into the forebrain of the guinea pig. The unidirectional transfer constants, Kin, determined from graphic analysis 14 and 20 days after chronic amphetamine treatment (5 mg/kg daily, i.p.) showed a marked time-dependent progressive enhancement of transfer for both molecules. The kinetic features of this entry suggest the opening up of pathways through the blood-brain barrier (BBB) which allows mannitol and PEG to pass into the brain at rates which are irrespective of their molecular size and/or lipophilia and these changes cannot be attributed to simple mechanical factors such as hypertension. This opening of the BBB was associated with changes in behaviour (increased locomotor activity, stereotypy, hypervigilance, social withdrawal, and loss of weight) seen in 14- and 20-day amphetamine-treated animals. At 7 and 28 days after the withdrawal of the amphetamine treatment, the behavioural manifestations were absent, and the Kin values for both molecules were not significantly different from those measured in normal control animals which had been treated with placebo injections. The present results suggest a reversible dysfunction of the BBB as a consequence of the chronic amphetamine intoxication which correlates with the behavioural syndrome induced in the guinea pig.