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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - ChromaFish (Chromatographic micro-column development for pharmaceutical applications in zebraFish)

Teaser

Summary

The drugs targeting the central nervous system have to reach their interaction sites, which is complicated by the presence of the blood-brain barrier, a selective semipermeable membrane separating the circulating blood from the brain and extracellular fluid. To predict Absorption, Distribution, Metabolism and Excretion properties (ADME), and more specifically absorption and permeability, high throughput screening is the method of choice. Predictive in silico and in vitro models have been developed but the outcome should be confirmed by in vivo models. Murine and rat models are well established but zebrafish can be an attractive alternative that address the increasing ethical concerns about animal testing. They are less care-intensive, possess a high genetic, physiologic and pharmacologic homology to humans, and their high fecundity and small size allow to perform â€œin vitro-likeâ€ screenings in well plates using very few amount of drugs. A novel sample preparation and analytical method to measure the whole-body uptake of drugs in zebrafish was developed in the laboratory [1]. The next step was to study uptake and metabolism of new drugs in the brain, where infinite small concentrations are expected. The aim was to develop a new type of capillary columns with core-shell particles and inner diameters < 300 Âµm. Combined with mass spectrometry, it would enhance the sensitivity and in this way lead to lower limits of quantification. The second objective was to develop a novel brain dissection technique and investigate the activity of transporter proteins. Once a suitable and validated analytical methodology would be in place, it could be applied to new drug candidates, and the results compared with those obtained using more traditional in vivo models. [1] Kislyuk S. et al. Talanta, 780â€“788

Work performed

First, I followed an online course on laboratory animal handling of 40 hours (reference B-KUL-E0E33A). I gained knowledge about the different aspects of laboratory animals (housing, caretaking, genetic and microbial quality control, anesthesia and analgesia, feeding, experimental techniques, etc.). Then, I applied this knowledge during a practical session with rabbit, guinea pig, rat, mouse, and hamster. This module was very interesting and contributes to the reliability and reproducibility of the results obtained with animal experiments and to the life quality of laboratory animals that are used for research.
Subsequently, I worked on a project in collaboration with Prof. Peter de Witte, who heads the zebrafish facility at the Department of Pharmaceutical and Pharmacological Sciences to gain more insight in the typical aspects of ADME testing in zebrafish. This project focused on the development of a platform for toxicity testing of chemicals by combining high-throughput zebrafish-based toxicity tests with a pre-metabolisation step of the compounds of interest using mammalian liver microsomes. For this purpose, two test compounds were considered, malathion and parathion. While malathion is a relatively safe organophosphate insecticide, parathion is an organophosphate insecticide that is highly toxic to mammals by all routes of exposure. It was noticed that the capacity to metabolically convert xenobiotics can be low in zebrafish and hence the effect of toxic metabolites, that are typically formed in humans can easily pass unobserved in zebrafish [5], which is problematic when trying to use zebrafish as a model for HTS screening. Therefore, the idea was to investigate whether specific enzymes that typically metabolize toxic compounds in humans/mammalians are absent in zebrafish. To confirm this hypothesis, both compounds were incubated with rat microsomes, whose role was to convert the compounds into metabolites. In a first step, parathion and malathion were exposed in vitro to rat liver microsomes for 1 h at 37 Â°C. A sample preparation step was developed to subsequently extract the compounds from the microsomes, whereby different solvents and solvent ratios were tested to obtain the best recovery. Then, a quantitative and qualitative analysis method using UHPLC-UV-MS was developed to determine the recovery of the parent compound and identity of the metabolites formed. Calibration curves were built to quantify the compounds of interest. Repeatability and accuracy were checked to validate the method.
Once it was clear what the amount of remaining malathion/parathion was in the samples obtained after incubation, and which metabolites were formed, 3 days post fertilization zebrafish eleuthero embryos were exposed to these samples for 48 h to examine whether any adverse effects occurred. It was shown that when zebrafish eleuthero embryos were exposed to parathion (without previous incubation with microsomes) at concentrations up to 50 ÂµM, the adverse effects were much lower than anticipated from a highly toxic and lipophilic compound that is likely taken up readily by eleuthero embryos. Conversely, in case the parathion samples were microsomally activated, a pronounced toxic and lethal effect was obtained. This was attributed to the formation of the highly toxic metabolite paraoxon, whose presence was confirmed by mass spectrometry. These results therefore seem to indicate that parathion is limitedly or not activated by eleuthero embryos, leading to a false negative outcome when the compound is not pre-metabolized. Conversely, it was demonstrated that malathion is rapidly metabolized into a decarboxylation product, thereby outcompeting the production of the toxic metabolite malaoxon. So, compared to the toxicity effects obtained for malathion, no increased toxicity was observed for malathion samples that were exposed to rat microsomes. Even if many more compounds are necessary to further validate its applicability, the hyphe

Final results

So far, a comprehensive methodology to assess the uptake and toxicity of 2 compounds in zebrafish has been developed and the obtained results have been submitted to International Journal of Molecular Sciences by A. Giusti et al. The results of this study will be published Open Access (on condition that the study is accepted for publication).
This study clearly demonstrated how zebrafish have the potential to be used as a platform for ADME testing in early drug development, and demonstrated the added value of specific analytical tools to quantify and identify compounds of interest to explain the observed mechanisms. On a personal level, this study gave me the opportunity to become acquainted with zebrafish samples, and to learn specific aspects about zebrafish handling, sample preparation, and analysis.
Meanwhile, I have been appointed as assistant professor in France (COBRA laboratory, INSA Rouen), and therefore I have stopped my MSCA grant. Nevertheless, the collaboration between Prof. Cabooter and Prof. de Witte is still active and other possibilities to continue this project or part of this project are considered.