< Back to previous page

Project

Use of fluorescent probes to study pharmacokinetics in zebrafish (Danio rerio) embryos and larvae

Despite the number of tools available for detecting toxicity in the drug discovery pipeline, post-marketing withdrawal due to toxic effects continues to be of an issue in the pharmaceutical industry. Many efforts have been made over the last years to improve the predictivity of in vitro and in vivo models that preclinically determine safety profiles of chemicals. Zebrafish (Danio rerio) offer new opportunities in this regard in the future. Zebrafish embryos and larvae are nowadays recognized as an excellent alternative non-mammalian model amenable to HTS (high-throughput screening) with a highly valuable translation of results to humans. Moreover, zebrafish are easy to manipulate genetically offering an opportunity to identify cellular and genetic factors that drive pathological responses to toxicants in mechanistic studies. Consequently, several safety assessment screenings are available using zebrafish-based platforms designed to test the toxicity of pharmaceuticals, cosmetics, and environmental samples in an efficient way.

Significantly, most zebrafish-based assays use immersion as a practical exposure route to deliver the compounds. Nevertheless, details regarding absorption, distribution, and elimination of chemicals in zebrafish embryos and larvae have yet to be examined. Alternatively, researchers can deliver compounds via micro-injections but how chemical substances distribute within the body upon microinjection and how intrabody exposure compares to the one seen during and after immersion have not been investigated. Typically, intrabody exposure, bioavailability and pharmacokinetics (PK) analysis of compounds is done from a blood sample or total digested tissue of zebrafish as the most common matrix used. However, fluorescence-based approaches can be used to evaluate the exposure profile or toxicity profile of chemicals, with the advantage of a real-time evaluation, which saves time and resources and provides high sensitivity and specificity.

In the present study, the spatiotemporal distribution of seven fluorescent alkyne compounds was examined following immersion or microinjection (yolk sac - IY, pericardially - PC, and intraperitoneally - IP) of zebrafish eleutheroembryos (ZFEE), 3 to 5 days post-fertilization (dpf) and following immersion or IY microinjection of zebrafish embryos (ZFE), 2 cell-stage to 3 dpf. The concentration (10 μM) and dose (2 mg/kg) used were selected as typically applied in preclinical experiments and zebrafish studies. The compounds’ main pharmacokinetics parameter values were determined by modelling the fluorescence of whole-body contours present in fluorescence images. Performing a QSPkR (quantitative structure pharmacokinetic relationship) analysis, we identified LogDo/w (Lipophilicity experimentally determined distribution coefficient), as the only physicochemical property that explains the final uptake of the selected compounds in the whole body. In contrast, the final uptake in the ZFE yolk compartment was mainly explained by TPSA (topological polar surface area), MR (molar refractivity), and the interaction of MR with LogD.

Overall, we found that especially in the case of short incubations (1-3 h) at 3 dpf, immersion can result in limited intrabody exposure to compounds. In this case, PC and IP microinjections represent excellent alternatives. Significantly, intra-yolk microinjections did not result in a suitable intrabody distribution of the compounds at 3 dpf. Moreover, we also found that combined administration of compounds (immersion and microinjection) provides a more stable intrabody exposure in ZFEE, at least in case of a prolonged immersion and compounds with LogDo/w value > 1. The data further show that zero to low intrabody exposure was reached after exposure by immersion of embryos (ZFE) with hydrophilic compounds. In the latter case IY microinjections, a technical procedure that can be easily automated, is highly recommended.

Finally, we validated the use of specific fluorescent probes to assess the inhibitory effect of BSEP (Bile salt export pump transporter in humans)-inhibitor drugs salt export pump in zebrafish. Cholestasis is a disruption of the enterohepatic cycling of bile acids possibly resulting into hepatotoxicity. Many cholestasis-inducers are inhibitors of BSEP (bile salt export pump transporter in humans), a protein crucial for the secretion of bile acids from hepatocytes into bile. Our preliminary results using Tauro-nor as a fluorescent probe correctly identified all three BSEP-inhibitor drugs whereas the hepatotoxicants did not show a significant decrease of the uptake of Tauro-nor in the gallbladder. The BODIPY™ FL C5-based assay on the other hand erroneously classified ketorolac and acetaminophen as Bsep inhibitors. Obviously, further research is needed to better understand the interactions of the fluorescent probes with the individual efflux transporters, and to test out larger sets of training compounds, also in transporter loss-of-function mutant larvae.

Taken together, our results and the mathematical models that we developed can help to forecast the amount of compound that will be present in the zebrafish over time after administration via immersion, microinjection, or combined immersion/micro-injection, based on the physicochemical properties of the compound. Additionally, the results shown here reveal the practicalities of the fluorescent probes when applied in the zebrafish model. The fluorescent probes offer sensitivity and specificity that align with the HTS advantages of the zebrafish. Likewise, the PK outcomes provide helpful input that might help tailor future safety assessment assays using the zebrafish-based platform.

Date:12 Dec 2017 →  19 Nov 2021
Keywords:Hepatotoxicity, Cholangiocytes, Hepatocytes, Drug-induced cholestasis, Imaging MS, Transgenic zebrafish
Disciplines:Biomarker discovery and evaluation, Drug discovery and development, Medicinal products, Pharmaceutics, Pharmacognosy and phytochemistry, Pharmacology, Pharmacotherapy, Toxicology and toxinology, Other pharmaceutical sciences
Project type:PhD project