The zebrafish as model for translational systems pharmacology: expanding the allometric scale in vertebrates with five orders of magnitude
R.C. van Wijk, E.H.J. Krekels, V. Kantae, A.C. Harms, Y. Guo, W.J. Veneman, F.J. Verbeek, T. Hankemeier, H.P. Spaink, and P.H. van der Graaf
Systems Pharmacology Cluster,  Division of Pharmacology &  Division of Analytical Biosciences, Leiden Academic Centre for Drug Research (LACDR), Leiden University, The Netherlands  Imaging and BioInformatics Section, Leiden Institute of Advanced Computer Science (LIACS), Leiden University, The Netherlands  Division of Animal Sciences and Health, Institute of Biology Leiden (IBL), Leiden University, The Netherlands
Objectives: The objective was to develop a method to quantify pharmacokinetics (PK) in zebrafish larvae. This can improve the increasingly used screens in zebrafish larvae, which ignore influence of PK on observed effects[1-3]. Indeed, systemic exposure is required to interpret the observed effects and derive a concentration-effect relationship. Additionally, estimated PK parameters could be used for interspecies scaling and optimization of preclinical experiments. To reach our objective, three aspects were essential: 1) accurate quantification of drug amounts in extreme small samples, 2) mixed effects PK modelling, and 3) determination of the exact volume of zebrafish larvae.
Methods: Zebrafish larvae were exposed to 1 mM paracetamol at 3 days post fertilization (dpf) for 10-300 minutes. At different time points, 3 replicates of samples with 5 larvae were lysed and paracetamol amounts were quantified by UPLC – Quadrupole TOF MS. A one and two compartment model with zero order absorption and first order elimination were tested in NONMEM 7.3. Total volume of distribution was fixed to one, yielding a relative clearance estimate. The volume of a single zebrafish was determined using VAST microscopy and silhouette 3D modelling. This volume was used to calculate the absolute clearance and bodyweight of the zebrafish larvae. The absolute clearance was compared to literature values of higher vertebrates in a log-log plot of clearance versus bodyweight.
Results: In all samples, absolute paracetamol amounts could be accurately quantified. A one compartment model with proportional error model for residual variability best described the data. Relative clearance was estimated at 1.7% of larva volume/min. Residual variability was 9.73%. The volume of a single zebrafish larva at 3 dpf was estimated to be 260 nL yielding a weight of 286 ug, being five orders of magnitude lighter than the mouse. Absolute clearance was 265.2 nL/h, which correlates well with the relationship between clearance and bodyweight in higher vertebrates.
Conclusions: We successfully developed a method to quantify drug PK in zebrafish larvae, a promising model system which has the potential to improve the understanding of drug pharmacology in early drug development and replace animal testing. This method will be further developed to include metabolic clearance, with the objective to estimate (active) metabolite exposure, as well as the possibility of interspecies scaling of different elimination pathways.
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