"In vitro" evaluation of blood-brain barrier transport

The blood-brain barrier (BBB), which is formed by brain capillary endothelial cells, effectively protects the central nervous system (CNS) from potential neurotoxic agents and maintains the brain homeostasis for synaptic signaling (Abbott, 2004). Unfortunately, the restrictive permeability properties of the BBB also exclude the majority of potential therapeutics from entering the brain and present a major challenge in the treatment of many CNS disorders such as Alzheimer’s disease and brain tumors (Pardridge, 2003). Therefore, strategies to deliver drugs across the BBB to the brain are of increasing interest. Moreover, a predictive in vitro BBB model retaining barrier-specific properties for the screening of drug candidates and for the evaluation of drug delivery strategies to the brain would be of great benefit in the development of CNS therapeutics.
Primary brain capillary endothelial cells mimic the in vivo BBB characteristics the best with respect to paracellular tightness and functional expression of transporters and receptors (Gumbleton and Audus, 2001). However, primary cells can be maintained in culture only for a limited life span before they undergo senescence. Moreover, since primary brain endothelial cells are often isolated from animals, species differences may exist (Reichel et al., 2003). Immortalized cells can be used to overcome these drawbacks of short life span and species differences. However, immortalized cell lines suffer from reduced paracellular resistance and downregulation of gene expression of most transporters (Pardridge, 2004a). A potential approach to overcome these limitations is to generate a conditionally immortalized cell line using a temperature-sensitive immortalization gene. This immortalization gene is only active at a temperature of 33°C that leads to cell proliferation. At physiological temperature of 37°C, this immortalization gene is inactivated and cell differentiation is favored (Terasaki et al., 2003).
Therefore, a focus of the present thesis was to characterize the conditionally immortalized human BBB in vitro model, termed TY09, with respect to its BBB specific characteristics and its potential application in transendothelial drug transport screening (section 3.1). This in vitro model was obtained by immortalization of primary microvascular endothelial cells isolated from a human brain tissue with a temperature-sensitive immortalization gene. The cells exhibited spindle-shaped morphology similar to primary cells, expressed von Willebrand factor and γ-glutamyl transpeptidase, and showed acetylated LDL uptake. Western blot and mRNA analysis revealed the expression of important tight junction proteins, solute carriers, and ATP efflux transporters up to a passage of 50. Transendothelial transport experiments of reference compounds with different physicochemical properties in TY09 cells showed similar transport characteristics as compared to the well-characterized human hCMEC/D3 model. However, the slightly higher paracellular tightness of TY09 model led to a lower background signal and allowed a better differentiation of compounds with low, medium and high permeability. This enhanced tightness enables mechanistic bidirectional transport studies of compounds with similar lipophilicity. Transport studies of psychoactive compounds (i.e. cathinones) in TY09 cells revealed good brain penetration for all tested cathinones. An asymmetric transport characteristic was detected for methylenedioxypyrovalerone (MDPV), indicating the potential participation of an active uptake process, which may contribute to the high potency of this compound. Section 3.2 discusses the in vitro evaluation of the BBB permeability of the psychoactive compounds.
As stated above, the poor penetration of neurotherapeutics remains a challenge for the treatment of brain diseases. Therefore, much research has been put on the development of drug delivery strategies. Utilization of endogenous receptor-mediated transport systems that are highly expressed at the brain endothelium offers an effective strategy to overcome the BBB (Pardridge, 2003). Antibodies directed against these receptors have been shown to undergo transcytosis in animals and can be used as transport vectors for brain drug delivery (Pardridge, 1997; Pardridge et al., 1995). Conjugation of nanoparticles to targeting antibodies directed against an endogenous receptor system offers the possibility to carry drugs to the brain in pharmacologically significant quantities. Hence, much research has been focused on the development of different nanoparticles for brain drug delivery. In the past years, drug delivery systems based on artificial vesicles, such as polymersomes, have attracted much attention due to their tunable carrier properties and their ability to carry hydrophilic compounds in their aqueous core and lipophilic substances in their membrane (Ahmed and Discher, 2004; Christian et al., 2009; Discher and Eisenberg, 2002).
Hence, another aim of this thesis was to evaluate the potential of antibody-targeted polymersomes for the implementation of drug targeting strategies to the brain (section 3.3). For this purpose, the anti-human insulin receptor antibody 83-14 (83 14 mAb) was used as targeting vector because this antibody has been shown to undergo transcytosis in vivo upon binding to the insulin receptor with high affinity (Pardridge et al., 1995). Polymersomes based on poly(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline) [PDMS-b-PMOXA] block copolymers were used in this study. Characterization of polymersomes confirmed their hollow sphere and vesicle-shaped morphology. Fluorescence correlation spectroscopy experiments showed the successful conjugation of the 83 14 mAb to the polymersomes. Flow cytometry analysis revealed binding and uptake of the 83 14 mAb conjugated polymersomes by brain capillary endothelial cells expressing the insulin receptor. Competitive uptake inhibition studies confirmed the specificity of this process. Intracellular trafficking analysis showed the colocalization of the 83-14 mAb with a subpopulation of early endosomes and lysosomes after incubation for 20 min. An altered intracellular localization of the 83 14 mAb conjugated polymersomes was observed. The transcytosis process of the 83 14 mAb across the BBB remains unresolved and the factors involved in the altered trafficking of 83-14 mAb conjugated polymersomes need to be elucidated. Nevertheless, these observations contribute to the further understanding of the transcytosis process of the 83 14 mAb and the intracellular pathway of 83-14 mAb conjugated nanoparticles.
With respect to screening of BBB permeability of compounds, sensitive analytical methods are required. This is particularly important for substances where only small quantities cross the BBB such as macromolecules. Capillary electrophoresis represents with its advantages (high sensitivity, low sample requirement, fast and automated measurements) a promising analytical technique for the quantification of molecules in the context of transport studies.
Therefore, another objective of this thesis was to develop a sensitive analytical method based on capillary electrophoresis equipped with laser-induced fluorescence detector (CE-LIF) for the quantification of the transport of macromolecules across the BBB in vitro (section 3.4). In this study, we aimed to quantify the BBB permeation of fluorescently labeled 83 14 mAb across monolayers of hCMEC/D3 and TY09 cells expressing the insulin receptor. The analytical method using CE-LIF obtained a low limit of quantification (LLOQ) for the antibody in the picomolar range. However, in contrast to the in vivo observation of the 83-14 mAb transcytosis to the brain (Pardridge et al., 1995), in vitro analysis of the transported amount of fluorescently labeled 83 14 mAb across human brain endothelial cell monolayers did not reveal the active process of transcytosis. Possible reasons for this observation are discussed in this section.
With regard to future applications of therapeutic antibodies, one of the topics of the present thesis was to extend the analytical method based on capillary electrophoresis for the viscosity determination of therapeutic antibody solutions (section 3.5). Therapeutic antibodies are often administered as highly concentrated solutions in order to achieve a therapeutic effect. These highly concentrated solutions show an increase in viscosity which limits their application. Therefore, in drug development, the viscosity of the protein solutions needs to be determined in order to optimize the formulation and adjust the viscosity appropriate for parenteral application. Different methods are applied for viscosity measurements. Most methods need a relatively high amount of the expensive samples and are time consuming. Therefore, in the present thesis, the possibility to employ capillary electrophoresis for viscosity measurements of protein samples was evaluated. Using CE, it was possible to estimate the viscosities (in the range of 2 to 40 mPas) of typical protein formulations with Newtonian flow behavior. The advantages of this analytical method over other methods for viscosity measurements are the low sample consumption and the fully automated viscosity determination.