A parent-metabolite pharmacokinetic model of paracetamol in zebrafish
Rob C. van Wijk[1], Elke H.J. Krekels[1], Vasudev Kantae[2], Amy C. Harms[2], Yuanhao Guo[3], Wouter J. Veneman[4], Fons J. Verbeek[3], Thomas Hankemeier[2], Herman P. Spaink[4], and Piet H. van der Graaf[1,5]
Systems Pharmacology Cluster, [1] Division of Pharmacology & [2] Division of Analytical Biosciences, Leiden Academic Centre for Drug Research (LACDR), Leiden University, The Netherlands [3] Section Imaging and BioInformatics, Leiden Institute of Advanced Computer Science (LIACS), Leiden University, The Netherlands [4] Division of Animal Sciences and Health, Institute of Biology Leiden (IBL), Leiden University, The Netherlands [5] QSP, Certara, Canterbury, United Kingdom
Objectives: The zebrafish larva is a promising vertebrate model organism in drug discovery and development. The pharmacokinetics (PK) of the drugs or their metabolites are however often disregarded[1]. Quantifying internal drug and metabolite exposure is important as they drive the observed pharmacological (side)effects. Additionally, quantification of elimination rates for different pathways increases our understanding of the PK translation potential of the zebrafish larvae towards higher vertebrates[2]. We developed a non-linear mixed effects (NLME) model to quantify glucuronidation, sulphation and unchanged excretion of paracetamol (acetaminophen) in zebrafish larvae, extending our concept for paracetamol PK in zebrafish larvae[3,4].
Methods: Zebrafish larvae of 5 days post fertilization (dpf) were exposed to 1 mM paracetamol either continuously for 10-180 minutes, or for 60 minutes after which they were transferred to clean medium for 60-240 minutes. Paracetamol, paracetamol-sulphate, and paracetamol-glucuronide were quantified by UPLC-QTRAP (ABSciex) in 3 replicates of 5 lysed larvae or in their incubation medium.
A NLME model was developed in NONMEM 7.3, simultaneously modelling internal and excreted amount of parent and metabolites. Destructive sampling imposed fixing the total volume of distribution to total larval volume of 300 nL which was determined by 3D volume modelling[6], and prevented estimation of inter-individual variability. Paracetamol absorption was estimated as zero order process. Metabolism was tested using first order and Michaelis-Menten kinetics. Excretion of parent and metabolite was estimated as first order process.
Results: A one compartment model best described the time course of internal paracetamol and metabolite amounts. Paracetamol absorption rate was 1.64 pmol/min. Sulphation and glucuronidation clearance was 6.55 nL/min and 1.22 nL/min, respectively. A recovery fraction for the wash-out experiment was estimated to improve model fit for both parent and metabolites.
Conclusions: The observed elimination profile of paracetamol in zebrafish larvae suggests an immature metabolic capacity, with more prominent sulphation than glucuronidation, a distinguishing trait of human neonates[5]. This metabolite model may be used to interpret pharmaco- or toxicodynamic effects caused by active metabolites, like paracetamol. It also improves mechanistic understanding of drug elimination.
References:
[1] Macrae CA, Peterson RT. Zebrafish as tools for drug discovery. Nat Publ Gr 2015;14:721–31
[2] Van Wijk RC, Krekels EHJ, Hankemeier T, Spaink HP, Van der GRaaf PH. Systems pharmacology of hepatic metabolism in zebrafish larvae. (Submitted)
[3] Kantae V, Krekels EHJ, Ordas A, González O, Van Wijk RC, Harms AC, et al. Pharmacokinetic modeling of paracetamol uptake and clearance in zebrafish larvae: Expanding the allometric scale in vertebrates with five orders of magnitude. Zebrafish 2016;13:504–10
[4] Van Wijk RC, Krekels EHJ, Kantae V, Harms AC, Guo Y, Veneman WJ, et al. The zebrafish as model for translational systems pharmacology: expanding the allometric scale in vertebrates with five orders of magnitude. PAGE 25, 2016, p. Abstr 5909
[5] Krekels EHJ, Ham S Van, Allegaert K, Hoon J De, Tibboel D, Danhof M, et al. Developmental changes rather than repeated administration drive paracetamol glucuronidation in neonates and infants. Eur J Clin Pharmacol 2015;71:1075–82
[6] Guo Y, Veneman W, Spaink HP, Verbeek FJ. 3D reconstruction and measurements of zebrafish larvae from High-throughput axial-view in vivo imaging. Biomedical Optics Express 2017;8:2611-2634