Assessment of the classification abilities of the CNS multi-parametric optimization approach by the method of logistic regression.

Assessment of "CNS drugs/CNS candidates" classification abilities of the multi-parametric optimization (CNS MPO) approach was performed by logistic regression. It was found that the five out of the six separately used physical-chemical properties (topological polar surface area, number of hydrogen-bonded donor atoms, basicity, lipophilicity of compound in neutral form and at pH = 7.4) provided accuracy of recognition below 60%. Only the descriptor of molecular weight (MW) could correctly classify two-thirds of the studied compounds. Aggregation of all six properties in the MPOscore did not improve the classification, which was worse than the classification using only MW. The results of our study demonstrate the imperfection of the CNS MPO approach; in its current form it is not very useful for computer design of new, effective CNS drugs.

A TEM protocol for quality assurance of in vitro cellular barrier models and its application to the assessment of nanoparticle transport mechanisms across barriers.

We report here a protocol to characterise and monitor the quality of in vitro human cellular barrier models using Transmission Electron Microscopy (TEM), which can be applied for transport assays, mechanistic studies and screening of drug/compound (including nanoparticle) penetration across such biological barriers. Data from two examples of biological barriers are given, namely the hCMEC/D3 endothelial blood-brain barrier model, and the Caco-2 intestinal epithelial barrier model, to show the general applicability of the method. Several aspects of this method are applicable to the quality assurance of in vitro barrier models, e.g., assessment of the multi or mono-layer structure of the endothelial cells; identification of any potential "holes" in the barrier that could confound transport assay results; validation of tight junction expression; and determination of the types and amounts of key cellular organelles present in the barrier to account for any significant changes in phenotype that may occur compared to the in vivo situation. The method described here provides a key advantage in that it prevents loss of the filter membrane during monolayer sectioning, thereby preserving critical details associated with the basal cell membrane. Applicability of the protocol for other in vitro biological barriers, such as the blood-foetus, blood-testes, blood-cerebrospinal fluid (CSF) and lung alveolar-capillary barriers is also discussed. Additionally, we demonstrate the use of the method for assessment of nanoparticle transport across cellular barriers and elucidation of transcytosis mechanisms. Sequential events of cellular endocytosis, localisation and transcytosis can be described in detail by TEM imaging, revealing useful sub-cellular details that provide evidence for the mechanism of nanoparticle transport in the hCMEC/D3 blood-brain barrier model and the Caco-2 intestinal epithelial cell model. Potential artefacts resulting from the nanoparticles interacting with the Transwell membranes can also be assessed.

In vitro assessment of alkylglyceryl-functionalized chitosan nanoparticles as permeating vectors for the blood-brain barrier.

A series of O-substituted alkylglyceryl chitosans with systematically varied alkyl chain length and degree of grafting has been employed for the formulation of aqueous nanoparticulate systems, which were in turn investigated for their effects on a modeled blood-brain-barrier system of mouse-brain endothelial cells. Barrier function measurements employing electric cell-substrate impedance sensing and analyses of tight junction-specific protein profiles have indicated that the alkylglyceryl-modified chitosan nanoparticles impact upon the integrity of the model blood-brain barrier, whereas confocal microscopy experiments have demonstrated the efficient cellular uptake and the perinuclear localization of these nanoparticles. The application of nanoparticles to the model blood-brain barrier effected an increase in its permeability, as demonstrated by following the transport of the tracer molecule fluorescein isothiocyanate.