Toxicology studies have proven to be quite translatable from zebrafish to mammals. As a matter of fact, concordance between studies ran on zebrafish and rodents can reach up to 87% (1). Therefore, the use of zebrafish in the fields of pharmacology and toxicology is opening new and innovative ways to test and develop new drug compounds and understand underlying disease mechanisms.
In this article, we will briefly address the process of drug development and clinical research, ways in which zebrafish are used in the pharmaceutical industries; and the bottlenecks and issues potentially involved.
Drug testing on zebrafish
The process of drug development starting from drug discovery and leading up to drug approval is long and challenging and involves many years of research and testing. Potential drugs are developed over several well defined and regulated steps to ensure safety and minimise adverse events.
Figure 1: Drug development process.
Drug discovery begins with the selection of promising compounds among a pool of potential drug candidates according to the best achieved interaction with the aimed target. Biochemical and cell-based assays help select lead compounds for further experimentation and development. Before entering clinical trials, potential drugs are tested in a preclinical phase which is essential to assess the safety, bioavailability and efficacy of a compound in living beings. Therefore, pharmacological and toxicological properties of the therapeutic molecule are first evaluated in a chosen animal model before being tested on humans. The preclinical phase is also crucial for enhancing the drug effect by improving the physiochemical and pharmacological properties, particularly the potency and selectivity of the compound (2).
Once the compound has been optimised to achieve the best therapeutic effect and evaluated as safe the clinical trial begins. Phase I involves a first-in-man trial on a small group of volunteers and evaluates the safety of the compound in human beings. Following phase I, phases II and III test the drug on growing groups of patients to assess the efficacy and evaluate the potential side effects and adverse events which can arise. Finally, phase IV will determine in if the drug is safe and efficacious. If the trial is successful and the drug is approved, it will be launched on the market (3).
Phase I is essentially a first-in-man trial. It involves 20-100 volunteers in good health or with the disease. This phase lasts several months and will determine the safety and appropriate dosage of the drug on humans. (3)
In this phase, the drug will be tested on a group of several hundred volunteers with the disease or condition. It will last up to two years and will allow to determine the efficacy and potential side effects of the drug. (3)
Phase III further tests the drug on groups of 300 to 3000 volunteers with the disease. It can last from 1-4 years and assesses the efficacy and potential adverse reactions of the drug. (3)
Phase IV is the last phase and tests the drug on several thousands of volunteers with the disease. It is the last phase which will determine the safety and efficacy of the drug and which will determine if the drug can be approved or not. (3)
Between 2000 and 2017, 114 studies involved zebrafish for chemical screening, 56 of which were tissue specific studies, 12 metabolic screens and 10 behavioural screens (1). The zebrafish used for these studies were equally wild-type fish, as well as mutant and transgenic lines (1).
In addition to chemical screens, zebrafish are also very appreciated for developmental toxicology screens. Toxicology studies are crucial to identify potential toxic mechanisms and to assess the safety of drug candidates before human trials. Organ toxicities are complex and require in vivo testing. Relying on embryonic stem cell tests or embryo culture would not be enough to evaluate the safety of a drug at the level of a fully developed organism. At the moment, developmental toxicity studies cannot be done in vitro, nor ex vivo or in non-mammalian models. Yet it is possible that in a near future zebrafish may be used to demonstrate clinical safety and obtain regulatory approval (1).
The use of zebrafish in pharmaceutical industries
Zebrafish have indeed several properties which make them good models for toxicology studies and have already gained the interest of several pharmaceutical companies. Their rapid development, small size and transparency, make zebrafish assays less time consuming and less costly than equivalent studies ran in rodents, thus diminishing the amount of resources needed. Additionally, effects of potential drugs can be assessed over the entire organ development period, using non-invasive observation methods (4).
For instance, Roche (Swiss multinational healthcare company) states the use of zebrafish as a research model for drug discovery and early development in their firm. They mention zebrafish as a “multi-organ culture model”, valuable for safety and efficacy studies (5). They also address the 3 R’s principle as the use of zebrafish does indeed contribute to replacing, reducing and refining animal experimentation.
While clinical candidates discovered thanks to zebrafish screens are still low, at least 10 compounds are currently being tested in clinical trials (6). According to a recent article published in Nature, these numbers are set to increase over the next years as a large panel of transgenic and mutant zebrafish lines are available. Since 2000, most of the mutant lines have been derived from forward genetic screens. However, the progress made in genomic techniques over the past years are making it easier to generate zebrafish models of interest (4). Thus, for some disease areas, using zebrafish for drug development is far more interesting than human organoids or genetic mouse models (6). Although some pharmaceutical companies are still hesitant on the use of zebrafish in their laboratories, it is most likely that if zebrafish-screened-derived compounds are efficacious on patients, the mindset will change.
The following drugs are currently being tested by various research labs and pharmaceutical companies:
ProHema is a derivate of PGE2, developed by Leonard Zon and Fate therapeutics to treat Graft versus Host Disease (GvHD). It is currently in phase II. (6)
Developed by Leonard Zon to treat Diamon-Blackfan anaemia. It is currently in phase I of testing. (6)
Developed by Scott Baraban and Epygenic Therapeutics to treat Drevet syndrome. It is currently in phase I. (6)
DB-041 is a new chemical entity being developed by David Raible and Decibel Therapeutics to treat hearing loss following aminoglycoside antibiotic treatment. It is currently in phase I. (6)
ALK2 inhibitors are dorsomorphin derivates being developed by Randall Peterson and Keros Therapeutics to treat fibrodysplasia ossifiants progressiva. It is currently in phase I. (6)
Developed by Leonard Zon to treat adenoid cystic carcinoma. It is currently in phase I. (6)
Developed by Leonard Zon to treat melanomas. It is is in phase I but has been put on hold. (6)
Ddeveloped by Veronica Kinsler and Elizabeth Patton to treat atriovenous malformations. Clinical trials have not began yet but use is compassionate (allowed use of an unauthorised medicine). (6)
Developed by Hakon Hakonarson to treat lymphatic disease. Use is also compassionate so far. (6)
Developed by David Langenau to treat rhabdomyosarcomas. It is currently in a preclinical phase. (6)
Developed by Alejandro Gutierrez and Thomas Look to treat T cell acute lymphoblastic leukaemia. It is currently in a preclinical phase. (6)
Bottlenecks and issues
Although we would like to present zebrafish as an idealistic animal model for research, it is important to underline a few issues related to drug discovery and testing. One of the most obvious discrepancies between fish and mammal testing is the administration and metabolism of a chemical compound. Zebrafish larvae are bred in 96 well plates in which chemicals are solubilised for them to be absorbed. In mammals the compound must be injected or orally injested. Therefore, only highly soluble chemicals can be efficaciously tested in zebrafish. (1)
Another issue which should not be undermined is the size of zebrafish which can have an impact on the translatability of results to humans. Defining a therapeutic window between efficacious and toxic exposure, clinical dosing and the best delivery system cannot be done easily as absorption, distribution, metabolism and excretion in zebrafish is unrelated to mammalian biological systems. (1)
As zebrafish are a useful tool for pharmaceutical toxicity testing, it is worth looking into those issues and finding a way to improve translation of toxicology results. In that sense, a better prediction of embryonic medium uptake as well as a better comprehension of zebrafish morphological endpoints could help refine biological assays and improve prediction capacities. Moreover, zebrafish have the potential to offer new insights and understanding of disease and toxic pathways, adverse outcome pathways (AOPs), drug abuse, endocrine disruptions, metabolism, bioavailability, transcriptomics and proteomics. (4)
As much as zebrafish represent many advantages for research, they are also small and difficult to handle, making zebrafish embryo assays tricky and time consuming. These type of assays require manual sorting and diving of eggs, embryos or larvae into 96 well plates for screening. Thus, automising this process can relieve researchers from a lot of work and accelerate screening processes and collection of results.
Automation has been a developing field of research over the past years. Softwares have been developed to classify embryos according to different phenotypepatterns with an accuracy reaching up to 99%(7). Therefore, progress brought to automation technologies, added to the growing knowledge around zebrafish, will continue to open novel opportunities for their use in laboratories and various fields of research. Our next article will be dedicated to the advantages of automation and the ways in which it improves work and experiments utilising zebrafish.
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References
(1) Zhang, T., Peterson, T. R., (2019). The Zebrafish in Biomedical Research. Academic Press. https://doi.org/10.1016/B978-0-12-812431-4.00051-8
(2) Forum on Neuroscience and Nervous System Disorders. Board on Health Sciences Policy. Institute of Medicine. (2014) Improving and Accelerating Therapeutic Development for Nervous System Disorders: Workshop Summary. National Academies Press (US). https://www.ncbi.nlm.nih.gov/books/NBK195047/
(3) The drug development process (US Food & Drug administration). Retrieved September 16, 2020, from https://www.fda.gov/patients/learn-about-drug-and-device-approvals/drug-development-process#:~:text=Research%20for%20a%20new%20drug%20begins%20in%20the%20laboratory.&text=Drugs%20undergo%20laboratory%20and%20animal%20testing%20to%20answer%20basic%20questions%20about%20safety.&text=Drugs%20are%20tested%20on%20people,they%20are%20safe%20and%20effective
(4) Cassar, S., Adatto, I., Freeman, J. L., Gamse, J. T., Iturria, I., Lawrence, C., Muriana, A., Peterson, R. T., Van Cruchten, S., & Zon, L. I. (2020). Use of Zebrafish in Drug Discovery Toxicology. Chemical research in toxicology, 33(1), 95–118. https://doi.org/10.1021/acs.chemrestox.9b00335
(5) Zebrafish. (F. Hoffmann-La Roche Ltd). Retrieved September 15, 2020, from https://www.roche.com/research_and_development/drawn_to_science/zebrafish.htm
(6) Cully M. (2019). Zebrafish earn their drug discovery stripes. Nature reviews. Drug discovery, 18(11), 811–813. https://doi.org/10.1038/d41573-019-00165-x
(7) Schutera, M., Dickmeis, T., Mione, M., Peravali, R., Marcato, D., Reischl, M., Mikut, R., & Pylatiuk, C. (2016). Automated phenotype pattern recognition of zebrafish for high-throughput screening. Bioengineered, 7(4), 261–265. https://doi.org/10.1080/21655979.2016.1197710