Carsten T. Beuckmann

Primary cultures of brain microvessel endothelial cells: a valid and flexible model to study drug transport through the blood–brain barrier in vitro

Studies on drug entry into the brain and permeation of the blood-brain barrier start to gain more and more importance in neuropharmaceutical research in order to develop new drugs for the therapy of central nervous system diseases. Procedures that provide quick access to permeation properties of those drugs with high throughput are difficult to achieve with animal models. Although various useful cell culture models approaching this issue have been described, results are often not comparable among each other unless determined with an equal experimental setup. Reproducibility of cell culture methods as well as corresponding findings gathered with these tools are often impeded due to the lack of details in experimental manuals. Here we present a precise manual for preparation and maintenance of porcine brain microvessel endothelial cells, serving as a culture model of the blood-brain barrier. Furthermore experimental details for blood-brain barrier transport investigations are presented. Validation of this model was carried out by determination of bioelectric properties and permeation experiments using various marker molecules reflecting paracellular and transcellular blood-brain barrier penetration. Results obtained with our model are closely resembling the in vivo-situation although astrocytes are not included. This simplification of the system is one of the major advantages towards robot derived cell cultures necessary for high throughput screening.

An improved low-permeability in vitro-model of the blood–brain barrier: transport studies on retinoids, sucrose, haloperidol, caffeine and mannitol

Primary cultures of porcine brain capillary endothelial cells grown on collagen coated polycarbonate membranes were used to build up an in vitro-model for the blood-brain barrier. Improved cultivation techniques allowed cell-storage and experiments under serum-free conditions. We employed this model to perform permeability studies in vitro with the radioactively labelled marker substances sucrose, retinoic acid, retinol, haloperidol, caffeine, and mannitol. Permeability values obtained with this blood-brain barrier model (1. 0x10-6 cm/s for sucrose, 6.2x10-6 cm/s for retinoic acid, 4.8x10-6 cm/s for retinol, 49.5x10-6 cm/s for haloperidol, 62.4x10-6 cm/s for caffeine, and 1.8x10-6 cm/s for mannitol) show a good correlation to data which are already known from in vivo-experiments. As judged by the sucrose permeability our blood-brain barrier model is less permeable than numerous other models published so far. Therefore it represents a powerful tool for in vitro-prediction of blood-brain barrier permeability of drugs and offers the possibility to scan a large quantity of drugs for their potential to enter the brain.

Orexin-A (hypocretin-1) is possibly involved in generation of anxiety-like behavior

Orexins (hypocretins) are neuropeptides expressed specifically in neurons in the lateral hypothalamic area and are known to be involved in the regulation of vigilance and feeding behavior. However, the relationship between orexin and emotional behaviors like anxiety is still poorly understood. Therefore, in this report we evaluated the effect of intracerebroventricular injection of orexin-A in two major anxiety tests, the light-dark exploration test (mouse) and the elevated plus-maze test (mouse, rat). Orexin increased time spent in the dark compartment in the light-dark test and time spent on the closed arms in the elevated plus-maze test. These results were not caused by a hypothetical sedative or activity-inducing effect of orexin-A because spontaneous locomotor activity did not alter upon orexin-A application under novel conditions. We therefore suggest an anxiogenic effect of orexin-A. To our knowledge, this is the first report about a relationship between orexin-A and anxiety.

Discovery of (1R,2S)-2-{[(2,4-Dimethylpyrimidin-5-yl)oxy]methyl}-2-(3-fluorophenyl)-N-(5-fluoropyridin-2-yl)cyclopropanecarboxamide (E2006): A Potent and Efficacious Oral Orexin Receptor Antagonist.

The orexin/hypocretin receptors are a family of G protein-coupled receptors and consist of orexin-1 (OX1) and orexin-2 (OX2) receptor subtypes. Orexin receptors are expressed throughout the central nervous system and are involved in the regulation of the sleep/wake cycle. Because modulation of these receptors constitutes a promising target for novel treatments of disorders associated with the control of sleep and wakefulness, such as insomnia, the development of orexin receptor antagonists has emerged as an important focus in drug discovery research. Here, we report the design, synthesis, characterization, and structure-activity relationships (SARs) of novel orexin receptor antagonists. Various modifications made to the core structure of a previously developed compound (-)-5, the lead molecule, resulted in compounds with improved chemical and pharmacological profiles. The investigation afforded a potential therapeutic agent, (1R,2S)-2-{[(2,4-dimethylpyrimidin-5-yl)oxy]methyl}-2-(3-fluorophenyl)-N-(5-fluoropyridin-2-yl)cyclopropanecarboxamide (E2006), an orally active, potent orexin antagonist. The efficacy was demonstrated in mice in an in vivo study by using sleep parameter measurements.

Design, synthesis, and structure-activity relationships of a series of novel N-aryl-2-phenylcyclopropanecarboxamide that are potent and orally active orexin receptor antagonists.

Herein we describe the design, synthesis, and structure-activity relationships (SARs) of a novel phenylcyclopropane series represented by 7 and 33 b as antagonists of orexin 1 and orexin 2 receptors. With 4 serving as the initial lead for the development of orexin antagonists, exploration of SAR resulted in improved binding affinity for orexin 1 and orexin 2 receptors. Among the synthesized compounds, 33 b ((-)-N-(5-cyanopyridin-2-yl)-2-[(3,4-dimethoxyphenyl)oxymethyl]-2-phenylcyclopropanecarboxamide) exhibited potent in vitro activity and oral efficacy in animal sleep measurement experiments. The results of our study suggest that compound 33 b may serve as a valuable template for the development of new orexin receptor antagonists.

A new astrocytic cell line which is able to induce a blood-brain barrier property in cultured brain capillary endothelial cells.

Astrocytes, a member of the glial cell family in the central nervous system, are assumed to play a crucial role in the formation of the blood-brain barrier (BBB) in vertebrates. It was shown that astrocytes induce BBB-properties in brain capillary endothelial cells (BCEC) in vitro. We now established an astroglial cell line of non-tumoral origin. The cloned cell line (A7) shows a highly increased proliferation rate and expresses the astrocytic marker glial fibrillary acidic protein. Furthermore, the clone A7 expresses S-100-protein and vimentin, which are also expressed by primary cultured astrocytes. This cell line therefore shows general astrocytic features. In addition, we were able to show that A7 cells re-induce the BBB-related marker enzyme alkaline phosphatase in BCEC, when these two cell types are co-cultured. Thus we have a cell line which can be readily cultured in large quantities, shows common astrocyte properties and is able to influence BCEC with respect to a BBB-related feature.

In Vitro and In Silico Characterization of Lemborexant (E2006), a Novel Dual Orexin Receptor Antagonist

Orexin (hypocretin) neuropeptides have, among others, been implicated in arousal/sleep control, and antagonizing the orexin signaling pathway has been previously demonstrated to promote sleep in animals and humans. This mechanism opens up a new therapeutic approach to curb excessive wakefulness in insomnia disorder rather than to promote sleep-related signaling. Here we describe the preclinical pharmacological in vitro and in silico characterization of lemborexant ((1R,2S)-2-{[(2,4-dimethylpyrimidin-5-yl)oxy]methyl}-2-(3-fluorophenyl)-N-(5-fluoropyridin-2-yl)cyclopropanecarboxamide)), a dual orexin receptor antagonist (DORA), as a novel experimental therapeutic agent for the symptomatic treatment of insomnia disorder and compare its properties to two other DORAs, almorexant and suvorexant. Lemborexant binds to both orexin receptors and functionally inhibits them in a competitive manner with low nanomolar potency, without any species difference apparent among human, rat, and mouse receptors. Binding and dissociation kinetics on both orexin receptors are rapid. Lemborexant is selective for both orexin receptors over 88 other receptors, transporters, and ion channels of important physiologic function. In silico modeling of lemborexant into the orexin receptors showed that it assumes the same type of conformation within the receptor-binding pocket as suvorexant, the π-stacked horseshoe-like conformation.