Current Medicinal Chemistry - Central Nervous System Agents - Volume 2, Issue 3, 2002

The blood-brain barrier (BBB) represents a functional interface between the blood stream and the neuronal microenvironment. Distinct cellular and molecular features of brain microvessel endothelial cells result in barrier and carrier functions that guarantee exclusion of adverse components such as neurotoxic metabolites on the one hand and selective passage of essential nutrients on the other hand. Circumventricular organs are brain structures that lack BBB characteristics, allowing for hormone-mediated interactions between e.g. the pituitary and distant organs. The retina, as an integral part of the central nervous system, is enclosed by the pigment epithelium, which functions as a barrier interface between the systemic blood vessels of the neighboring choroid and the retina. Various BBB-specific markers, tight junction components, and carrier systems including amino acid and saccharide transporters have been cloned. P-glycoprotein has been of special interest because this efflux pump counteracts entry of numerous therapeutically relevant drugs into the nervous system. Various in vitro systems of the retinal pigment epithelium (RPE) have been established and employed to analyze pharmacological aspects and pathological cell interactions. The most advanced systems are organotypic cultures and acute preparations of the RPE, i.e. fully intact tissue sheets that can be used as in vivo-like BBB models for transport studies and drug profiling.

To be effective as therapeutic agents, centrally acting drugs must cross the blood-brain barrier (BBB). Conversely, to be devoid of unwanted CNS effects, peripherally acting drugs must show limited brain accessibility. This demonstrates clearly the need for different methods to assess the blood-brain penetration at different levels of project development in industry. Since the experimental determination of blood-brain partitioning is difficult, time consuming and expensive, also other methods like in-silico approaches mainly based on physicochemical properties like solubility, lipophilicity, molecular size, hydrogen-bonding capacity and charge are used. Approaches for drug delivery and drug modification are also reviewed in the present article.In vitro cellular models based on cell cultures growing in two-chamber systems for transport studies or isolated microvessels play an important role for compound screening. To achieve and use the full potential of these models a characterization of anatomical, physiological and biochemical properties is needed. The more strict the criteria for BBB models the better prediction of penetration and cellular mechanisms. Unfortunately, the throughput decreases often in parallel. Therefore, though high-throughput assays as the MDCK / CACO assay or artificial membrane assays are used but they still suffer from low predictability for specifically transported substances (transporters, Pglycoprotein, brain specific receptors) due to differences between peripheral epithelial cells and brain endothelial cells.The animal experiment with radiolabelled and non-radiolabelled compound not only have the highest level and the highest predictability but the highest cost and lowest throughput as well. There is no “golden rule” for approaching brain penetration in industry but models available are used often in parallel (in silico, in vitro, in vivo).

In this paper the various BBB systems have been described including the various isolation procedures of brain capillary endothelial cells (BCEC), the culture of monolayers of BCEC and endothelial cell lines, and the coculture of BCEC with various types of astrocytes. The transference of results between BBB culture systems has been discussed together with the application of the various BCEC co-culture systems in research. It is concluded that there is a need for interlaboratory validation of BBB data, particularly BBB transport data.

Recently, one of the key trends in the pharmaceutical industry has been the integration of what has traditionally been considered ‘development’ activities into the early phases of the drug discovery process. The aim of this integration is the prompt identification and elimination of candidate molecules that are unlikely to survive later phases in drug development. Combinatorial chemistry and high throughput screening techniques have enormously increased the possibility of finding new lead structures. Applying these techniques millions of compounds can be generated but most of them show poor biopharmaceutical properties. Identifying and removing compounds with poor properties at an early stage is strongly demanded to save both time and costs. Because biopharmaceutical parameters, such as the blood-brain permeation, cannot be determined for a large number of compounds, alternative evaluation methods are desirable.In the last thirty years a variety of theoretical transport and permeation models have been developed to describe mathematically how a drug is passively transported and how a compound is able to pass a membrane. Progress in understanding the role of physicochemical properties in membrane permeability relevant to important processes such as blood-brain barrier permeation,brings rational drug design more within reach. Several new methods able to estimate rapidly the biopharmaceutical properties on the basis of molecular structures have been developed recently. This article will review the most important and recent techniques in this field and will discuss their applicability in the drug discovery process.

Over the past 50 years a number of efforts have been made to relate physicochemical parameters to the ability of molecules to cross the blood-brain barrier. Predominantly drugs enter the brain cells by transcellular passive diffusion through cells while paracellular transport is not considered significant due to the tight junctions between cells. Early work focused on correlations of brain uptake with a measured value of lipophilicity. Computational models were developed to model this parameter and the important structural characteristics of molecules, e.g. size and hydrogen-bonding capacity. New molecular descriptors, such as polar surface area and solvent free energies, have been generated and used. Practical methodology for predicting passive diffusion has also diversified with the use of cell monolayers and artificial membranes. These methods need to be validated against appropriate in vivo data and there is a need to consider the brain penetration data itself. Brain uptake is often expressed as partitioning of drugs into whole brain from blood or plasma, but the usual receptor targets for drugs are in the aqueous environment, extra-cellular fluid (ECF) surrounding the cell. In the brain, ECF concentrations are generally regarded to be reasonably well represented by cerebro-spinal fluid (CSF) concentrations. However there is limited literature data on CSF concentrations. In the blood-brain barrier the transporter P-glycoprotein (P-gp) is known to limit brain-uptake of certain compounds. In addition some compounds, including sugar and amino acids, may be actively transported into the brain. Models for brain penetration in the future are likely to include a number of in silico computed parameters or a number of physical measurements to allow contributions of passive and active transport to be considered.

Poly(butyl cyanoacrylate) nanoparticles coated with polysorbate 80 (Tween 80) enable the transport of a number of drugs across the blood-brain barrier (BBB) into the brain following intravenous injection. These drugs include the hexapeptide endorphin dalargin, the dipeptide kytorphin, loperamide, tubocurarine, doxorubicin, and the NMDAreceptor antagonists MRZ 2 / 576 and MRZ 2 / 596.After binding to the polysorbate-coated particles, dalargin as well as loperamide exhibited a dose-dependent antinociceptive effect after i.v. injection as determined by the tail-flick as well as by the hot plate test. This effect was accompanied by a Straub reaction and was totally inhibited by pretreatment with naloxone, indicating that it is a central effect and not peripheral analgesia. After brain perfusion of rats with tubocurarine bound to the polysorbate-coated nanoparticles epileptic spikes were observable in the EEC of the rats but not with the controls. Other very interesting results were obtained with the NMDA-receptor antagonists MRZ 2 / 576 and MRZ 2 / 596. The very short anticonvulsive response of MRZ 2 / 576 was increased from below 30 to 300 min, and the transport across the BBB of the non-penetrating MRZ 2 / 596 was enabled after i.v. injection. Intravenous injection of polysorbate 80-coated nanoparticles loaded with doxorubicin (5 mg / kg) achieved very high brain levels of 6 μg / g brain tissue while all the controls, including uncoated nanoparticles and doxorubicin solutions mixed with polysorbate, did not reach the analytical detection limit of 0.1 μg / g. Moreover, experiments with the extremely aggressive glioblastoma 101 / 8 transplanted intracranially showed a long term survival for 6 months of 40 % of the rats after intravenous injection of the polysorbate 80-coated nanoparticle preparation (3 x 1.5 mg / kg). The surviving animals were sacrificed after this time and showed total remission by histological investigation. Untreated controls died within 10 - 20 days, the animals in the six other control groups between 10 - 50 days.The mechanism of the drug transport across the blood-brain barrier with the nanoparticles requires further elucidation. The most likely mechanism at present appears to be endocytotic uptake by the brain capillary endothelial cells followed either by release of the drugs in these cells and diffusion into the brain or by transcytosis. Endocytotic uptake of the polysorbate-coated nanoparticles but not of uncoated particles has been shown with bovine, murine, rat, and human brain capillary endothelial cells. After injection of the nanoparticles, apolipoprotein E (apo E) or apo B adsorption of the particles seems to occur as already shown in vitro, followed by interaction with the LDL receptor and endocytotic uptake. This scenario is rather likely since both apolipoproteins can interact with the LDL-receptor. They may then be taken up like the naturally occurring lipoprotein particles.