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Comparison of in vitro brain tissue binding methods by evaluating the fraction of unbound drug in the brain
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. (Translational PKPD)
2015 (English)Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
Abstract [en]

Introduction: The free drug hypothesis implies that it is only the unbound drug molecules that can produce pharmacological effect at the site of the target. For central nervous system drug targets it is difficult to measure the unbound concentration at the site of action. The fraction of unbound drug in the brain (fu,brain) is used to convert the total drug concentration in the brain to the unbound concentration. fu,brain can be estimated by in vitro methods such as equilibrium dialysis (ED), which is the gold standard method. Addition to this is the newly proposed method, lipid membrane binding assay (LIMBA).

Aim: The aim of this study was to compare two in vitro brain tissue binding methods LIMBA and ED by evaluating the fu,brain of diazepam, indomethacin, memantine and donepezil in the homogenate prepared from whole brain and different brain regions. This was done to explore if LIMBA can be an alternative method for assessment of fu,brain.

Materials and Methods: Rat brain tissue was homogenized in 1:2 dodecane, added on a DIFI filter and immersed in a drug-spiked buffer. LIMBA plate was incubated overnight at 50 rpm at 23oC. Samples were analysed by LC-MS/MS. Statistical analysis was performed using GraphPad prism 6.0. Comparison of mean fu,brain values was done using one-way ANOVA and Tukey´s test.

Results: There was a good agreement (R2 = 0.63) between the fu,brain values measured using LIMBA and ED literature values. Significant differences in fu,brain of brain regions were observed for indomethacin and memantine while no regional differences in fu,brain of donepezil and diazepam were observed.

Conclusions: The results are promising and indicating that LIMBA method could be used for assessment of fu,brain. However, complementary studies with ED experiments and extended dataset of compounds are needed.

Place, publisher, year, edition, pages
2015. , 34 p.
Keyword [en]
Blood brain barrier (BBB), Equilibrium Dialysis (ED), Lipid Membrane Binding Assay (LIMBA), fraction of unbound drug in the brain (fu, brain), protein binding in brain tissue, nonspecific binding
National Category
Pharmaceutical Sciences
URN: urn:nbn:se:uu:diva-263223OAI: oai:DiVA.org:uu-263223DiVA: diva2:857424
Subject / course
Educational program
Master of Science Programme in Pharmacy
Available from: 2015-09-29 Created: 2015-09-28 Last updated: 2015-09-29Bibliographically approved

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