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Optimization of equilibrium dialysis protocol for assessment of drug brain tissue binding and comparison with novel assay
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. (Translationell PKPD, Translational PKPD)
2015 (English)Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
Abstract [en]

Introduction: Equilibrium Dialysis (ED) is a gold standard method for the estimation of fu,brain, the fraction of unbound drug in brain tissue. Assessment of a “true” fu,brain value requires optimization of several steps of ED in particular dilution of brain homogenate. Recently proposed novel method has advantages compared to ED associated with lower work burden and costs. Aim: Main purpose was to optimize ED protocol for estimation of fu,brain of diazepam, paliperidone, paroxetine, indomethacin and thioridazine by testing i) spiking with single compound or mixture of 5 compounds (cassette) approaches, ii) different dilutions of brain homogenate and iii) added ED well-volumes. Additionally, assessment of relative agreement between ED and novel assay for drug brain tissue binding was performed. Materials and Methods: Brain tissue was homogenized in phosphate saline buffer pH7.4 to 1:2, 1:4 and 1:9 (w:v) dilutions. Single compound (1µM) and cassette spiking approaches were compared. Inter-day variability was evaluated using three separate batches of brain homogenates prepared by pooling in average 4 drug-naïve rat brains. fu,brain assessed using 100 µL and 125 µL well-volumes for 1:9 dilution was evaluated. Sample analysis was done with LC-MS/MS. Results: A significant difference was observed between fu,brain values from single compound and cassette spiking approaches for all compounds and for all dilutions. A significant difference was not observed well-volumes 100 µL versus 125 µL. The inter-day variability was good. The stability (37oC, 6h) was >90% except for thioridazine (61.52%). Conclusions: Obtained results indicate potential saturation of the binding components in the homogenate or DDI between compounds in the cassette independent of dilution for all compounds when cassette approach was used. Results regarding dilution effects was inconsistente. Assessment of fu,brain values was not affected by well-volumes.

Place, publisher, year, edition, pages
2015. , 42 p.
Keyword [en]
Equilibrium dialysis, optimization, fu, brain, drug brain tissue binding, dilution, ED well-volume, spiking approach
National Category
Pharmaceutical Sciences
URN: urn:nbn:se:uu:diva-242365OAI: oai:DiVA.org:uu-242365DiVA: diva2:783355
Subject / course
Educational program
Master of Science Programme in Pharmacy
Available from: 2015-01-26 Created: 2015-01-26 Last updated: 2015-01-26Bibliographically approved

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