Rob C. van Wijk(1), Elke H.J. Krekels(1), Anita K. Ordas(2), Thijs Kreling(1), Vasudev Kantae(1), Amy C. Harms(1), Thomas Hankemeier(1), Herman P. Spaink(2), Piet H. van der Graaf(1,3)
(1) Systems Biomedicine and Pharmacology, Leiden Academic Centre for Drug Research (LACDR), Leiden University, Leiden, The Netherlands (2) Division of Animal Sciences and Health, Institute of Biology Leiden (IBL), Leiden University, Leiden, The Netherlands (3) QSP, Certara, Canterbury, United Kingdom
Objectives:
The use of zebrafish larvae as model system for drug discovery and early drug development is being recognised. It has become clear that internal drug exposure, rather than external exposure, should be studied when investigating pharmacological responses in these studies [1]. Recently the first pharmacokinetic (PK) model was developed using the paradigm compound paracetamol (acetaminophen) in zebrafish larvae of 3 days post fertilization (dpf) [2]. As sampling blood from a larva 1-2 mm in size and only hundreds of nanoliters in volume was not possible at that time, this model was based on total paracetamol amounts in lysed larvae. As a result, distribution volume could not be estimated and only relative clearance values could be obtained. For extrapolation to higher vertebrates, these parameters are however essential. Our aim here is therefore to develop an experimental method to sample blood from zebrafish larvae at 5 dpf in order to estimate pharmacokinetic parameters including distribution volume and absolute clearance.
Methods:
Blood was sampled from different anatomical locations of the larval circulation [3] using a pulled needle (borosilicate glass capillary, original diameter: 0.75 mm. Sutter Instruments) in a micromanipulator, connected to a manual CellTram pump (Eppendorf) under 20x microscopic magnification (Leica). For determination of sample volume, an image was taken of each sample within the needle. To prevent coagulation, different strategies were tested, including heparin coating of needle and collection tube.
In the PK experiment, zebrafish larvae of 5 dpf were exposed to 1 mM paracetamol for 10-170 minutes after which the larvae were washed and the blood was sampled using the optimized blood sampling method. Blood concentrations of paracetamol and its major metabolites were quantified using UPLC (Waters) – MS/MS (AB Sciex). The obtained concentrations were combined with previously gathered data of paracetamol amounts from lysed larvae from an experiment in which larvae were exposed to the same paracetamol concentration for 10-180 min, or for 60 min with a washout period of 60-240 min.
Non-linear mixed effects modelling was performed in NONMEM 7.3, simultaneously fitting paracetamol amounts in lysed zebrafish larvae and paracetamol concentrations in the blood. Paracetamol absorption from the surrounding medium was parameterized as a zero order process. Both one and two compartment models were tested for distribution. For paracetamol elimination linear and non-linear models were tested. Because of destructive sampling, only residual variability could be estimated.
Results:
Blood sampling from the posterior cardinal vein was most efficient and resulted in highest yields. Injection of the sample into a drop of heparin solution (2 uL 5 IE/mL heparin, Pharmacy AZL, Leiden) prevented coagulation and enabled handling of the sample. By pooling 15-35 blood samples, detectable blood concentrations could be reached.
A one compartment model with first order elimination best fitted the data. An additive error was used for paracetamol blood concentrations and combined error for total paracetamol amounts. Volume of distribution was estimated at 1170 nL, when assuming a weight of 299.1 µg [2,4] this yields 3.9 L/kg for zebrafish larvae of 5 dpf. This estimate is in the same order of magnitude as reported distribution volumes of paracetamol in higher vertebrates. Clearance was estimated at 1.8 µL/h, well within the recently published allometric relationship of higher vertebrates’ paracetamol clearance [2].
Conclusions:
For the first time, blood samples were taken from zebrafish larvae of only millimetres in size. The development of this technique enables quantification of blood concentration in this new vertebrate model organism, which is critical for estimating distribution and absolute clearance. It shows the potential of systems pharmacology by integration of both experimental and computational innovation.
References:
[1] Van Wijk RC et al. Drug Discovery Today: Disease Models 2016; 22:27-34
[2] Kantae V et al. Zebrafish 2016; 13:504–10
[3] Isogai S et al. Developmental Biology 2001; 230:278-301
[4] Guo Y et al. Biomed Opt Express 2017; 8(5):2611-2634
Reference: PAGE 27 (2018) Abstr 8578 [www.page-meeting.org/?abstract=8578]
Poster: Drug/Disease Modelling - Other Topics