Mechanistic and Quantitative Understanding of Pharmacokinetics in Zebrafish Larvae through Nanoscale Blood Sampling and Metabolite Modeling of Paracetamol

Zebrafish larvae are increasingly used for pharmacological research, but internal drug exposure is often not measured. Understanding pharmacokinetics is necessary for reliable translation of pharmacological results to higher vertebrates, including humans. Quantification of drug clearance and distribution requires measurements of blood concentrations. Additionally, measuring drug metabolites is of importance to understand clearance in this model organism mechanistically. We therefore mechanistically studied and quantified pharmacokinetics in zebrafish larvae, and compared this to higher vertebrates, using paracetamol (acetaminophen) as a paradigm compound. A method was developed to sample blood from zebrafish larvae 5 days post fertilization. Blood concentrations of paracetamol and its major metabolites, paracetamol-glucuronide and paracetamol-sulfate, were measured. Blood concentration data were combined with measured amounts in larval homogenates and excreted amounts and simultaneously analyzed through nonlinear mixed-effects modeling, quantifying absolute clearance and distribution volume. Blood sampling from zebrafish larvae was most successful from the posterior cardinal vein, with a median volume (interquartile range) of 1.12 nl (0.676-1.66 nl) per blood sample. Samples were pooled (n = 15-35) to reach measurable levels. Paracetamol blood concentrations at steady state were only 10% of the external paracetamol concentration. Paracetamol-sulfate was the major metabolite, and its formation was quantified using a time-dependent metabolic formation rate. Absolute clearance and distribution volume correlated well with reported values in higher vertebrates, including humans. Based on blood concentrations and advanced data analysis, the mechanistic and quantitative understanding of paracetamol pharmacokinetics in zebrafish larvae has been established. This will improve the translational value of this vertebrate model organism in drug discovery and development. SIGNIFICANCE STATEMENT: In early phases of drug development, new compounds are increasingly screened in zebrafish larvae, but the internal drug exposure is often not taken into consideration. We developed innovative experimental and computational methods, including a blood-sampling technique, to measure the paradigm drug paracetamol (acetaminophen) and its major metabolites and quantify pharmacokinetics (absorption, distribution, elimination) in zebrafish larvae of 5 days post fertilization with a total volume of only 300 nl. These parameter values were scaled to higher vertebrates, including humans.