Zebrafish (Danio rerio) larvae have gained attention as a valid model to study in vivo drug
metabolism and to predict human metabolism. The microinjection of compounds, oligonucleotides,
or pathogens into zebrafish embryos at an early developmental stage is a well-established
technique. Here, we investigated the metabolism of zebrafish larvae after microinjection
of methyl 2-(1-(5-fluoropentyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamido)-3,3-dimethylbutanoate
(70N-5F-ADB) as a representative of recently introduced synthetic cannabinoids. Results were
compared to human urine data and data from the in vitro HepaRG model and the metabolic
pathway of 70N-5F-ADB were reconstructed. Out of 27 metabolites detected in human urine samples,
19 and 15 metabolites were present in zebrafish larvae and HepaRG cells, respectively. The route of
administration to zebrafish larvae had a major impact and we found a high number of metabolites
when 70N-5F-ADB was microinjected into the caudal vein, heart ventricle, or hindbrain. We further
studied the spatial distribution of the parent compound and its metabolites by mass spectrometry
imaging (MSI) of treated zebrafish larvae to demonstrate the discrepancy in metabolite profiles
among larvae exposed through different administration routes. In conclusion, zebrafish larvae
represent a superb model for studying drug metabolism, and when combined with MSI, the optimal
administration route can be determined based on in vivo drug distribution