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Project

Zebrafish - a small-animal model for ADME studies

In the course of lead optimization and candidate selection, ADME-studies are performed to describe the disposition of pharmaceuticals within a biological system. Hereto, high throughput screenings (HTS) are performed in order to exclude unsuitable candidates in a fast way, thus sparing time and money by concentrating efforts only on the most promising chemicals. Several models are currently available for the measurement of drug uptake in the brain, however, none of these seem to be adopted in the pharmacological industry due to various disadvantages. Zebrafish (Danio rerio) can be used as a new in vivo model, avoiding problems that are typically encountered when using mammalian models and cell cultures. Especially their high fecundity and small size make zebrafish interesting for the pharmaceutical industry since only microgram-scale amounts of compounds are needed to perform HTS. In this study, zebrafish were assessed for the purpose of ADME-studies and compared to other recently developed models, while putting emphasis on the use of zebrafish as a predictive blood-brain barrier model. Therefore, a suitable extraction method for biological samples was first developed. A sample preparation procedure for 9 pharmaceuticals displaying a variety in physicochemical properties was developed using 10-day old zebrafish (TG898). For an efficient homogenization of the samples and subsequent recovery of the compounds of interest, different amounts of organic solvents in combination with acidic modifiers were added to zebrafish samples, while 1:2 (v/v) water:methanol in combination with 0.1% formic acid resulted in acceptable recoveries between 74% and 100% for all compounds. Samples were subsequently processed using a powerful bath sonicator and centrifuged. Supernatant was then removed and evaporated in a vacuum oven before being reconstituted in a mobile phase-like solvent. Samples were analyzed using ultra-high performance liquid chromatography (UHPLC). Subsequently, the whole body uptake of representative pharmaceuticals was investigated using the administration route of immersion and UHPLC-MS/MS equipment to obtain a first idea of the sensitivity requirements of the analytical method. Remarkably, only one fish was needed to measure the whole body uptake. Two additional pharmaceuticals served to compare three different routes of administration: immersion, pericardial and yolk sac microinjections. The experiments showed that body concentrations achieved via injections are much higher than via long-term immersion experiments, thus microinjections were considered to provide a reliable tool to study pharmacokinetics in zebrafish for hydrophilic pharmaceuticals. Finally, a brain dissection technique was developed to measure the brain uptake of the chosen pharmaceutical agents on capillary UHPLC-MS/MS. Therefore, zebrafish were incubated with 7 drugs separately via the route of immersion, and the concentration reaching the brain was determined by applying a novel brain extraction procedure. Sample analysis was performed utilizing capillary ultra-high performance liquid chromatography (cap-UHPLC) coupled to a variable wavelength UV detector. Quantitative experiments carried out on pooled samples of six fish (n=6) demonstrated the semipermeable nature of the blood-brain-barrier after incubating those fish at the maximum tolerated concentration for the investigated pharmaceuticals.

Date:1 Jan 2014 →  19 Dec 2017
Keywords:zebrafish, brain, pharmaceuticals, HPLC, MS
Disciplines:Analytical chemistry, Pharmaceutical analysis and quality assurance, Chemical product design and formulation, Biomaterials engineering, Biomarker discovery and evaluation, Drug discovery and development, Medicinal products, Pharmaceutics, Pharmacognosy and phytochemistry, Pharmacology, Pharmacotherapy, Toxicology and toxinology, Other pharmaceutical sciences
Project type:PhD project