The blood-brain barrier (BBB) is crucial for complex issues such as drug delivery, pathogenesis of chronic neurological diseases involving BBB dysfunction (e.g., brain tumors, ischemia, hypoxia, brain edema, multiple sclerosis, and meningitis) and issues related to bio-defense. Since the BBB selectively (by specific transport mechanisms) excludes most blood-borne substances and xenobiotics from entering the brain, protecting it from systemic influences. Unfortunately, bio-defense systems that developed to protect the brain from potentially dangerous substances may also contribute to the phenomenon known as multiple drug resistance (MDR) during treatment of several CNS disorders, such as drug refractory epilepsy or intractable brain tumors. Every year millions of dollars are spent by pharmaceutical companies to develop alternative pharmaceutical strategies that bypass the shielding of brain parenchyma and to study new therapeutic approaches using in vivo or in vitro models of the BBB, many of which end up not working. Rational CNS drug design cannot entirely and exclusively rely upon the physical-chemical properties of putative neurotherapeutics, since lipophilicity alone is a poor predictor for drug penetration into the CNS. This is particularly true for three large families of CNS drugs, antineoplastics, antivirals and antiepileptics. Studies performed in small animals including rodents cannot be directly extrapolated to human tissue. Preliminary results from this and other laboratories have convincingly demonstrated that use of rodent brain endothelial cells and in general endothelial cell lines from non human sources as models of clinical pharmacology are flawed. We propose to: 1) To compare the permeability values of clinically relevant antiepileptic drugs in vivo versus in vitro. This will be performed by measuring the penetration of drugs into the naive rodent brain compared to DIV-BBB established with control (i.e., non-multiple drug resistant) blood-brain barrier endothelial and glial cells. 2) To compare the permeability value of antiepileptic drugs measured directly in serum and brain of pharmacoresistant patients or in our in vitro model comprising of cells from comparable multiple drug resistant subjects. 3) To develop an automated drug dosing and sampling system to allow for automated control, data collection and analysis on large number of modules at the same time. We will also assess the feasibility of the use of a microdialysis probe for on line sampling which will overcome the need of radio- or fluorescent labeled compounds for permeability studies. 4) To implement a new design of the trans-endothelial electrical resistance measurement system in order to allow for the measurement of the impedance of BBB by the use of a ramp of different frequencies. We propose to develop and validate an improved dynamic in vitro blood-brain barrier (DIV-BBB) model that reproduces the functional characteristics of the BBB in vivo, features higher predictability, and is fully scalable and customizable. As such, it will be perfectly suited for extensive pharmacological and physiological studies. In addition, the use of DIV-BBB can be extended to research applications in Neuroscience. The DIV- BBB can be successfully used to better understand the principles of brain drug delivery, to acquire relevant knowledge of the BBB anatomy and physiology, and to study the mechanism of endothelial cells differentiation into a BBB phenotype. Considering these realistic premises, the DIV-BBB can be used to study the effects of pathophysiological conditions and facilitate the design of novel CNS drugs and therapeutical approaches targeting the brain with clear benefits for the patients. ? ? ?
