Preclinical assays are a vital step in a drug’s journey to approval for use in human subjects. They allow drugs that are too toxic or unsafe to be filtered out in the Early Drug Discovery Phase prior to being tested on humans during the clinical phase. Also, Toxicology studies of the thousands of new chemical compounds there are being developed and the deeper study of others that are currently in use is of major relevance to ensure their safety. This naturally involves testing on animals, with traditional animal models having previously encountered numerous challenges and limitations – not least on account of the ethical issues at play. One important distinction between clinical and preclinical studies is that the latter is generally not subject to the same level of approval by an ethics committee, although there are of course regulatory requirements that must be fulfilled.
Irrespective of the ethical implications of animal testing, the efficacy of traditional animal models, using rodents for example, has also been called into question. While traditional methods of testing may prove useful in estimating the effect and desired dosage of a new drug, the accuracy of this approach is often insufficient. Furthermore, the costs involved in testing on larger animals that are genetically similar to humans, such as mammals, are expensive and time-consuming. As a result, various New Alternative Models (or NAMs) are currently in development that are more cost-effective, less ethically questionable, and provide effective results that can be readily translated to humans. We will now consider one particularly promising alternative model, namely the use of zebrafish.
The variety of biological models
Before discussing the advantages of zebrafish over other animals in preclinical assays and Toxicology testing, we will first discuss the various types of models and their advantages/limitations.
- In-silico models: This refers to the use of computational models and simulations and offers a cost-effective means by which to identify Drug candidates and appropriate doses. This enables complex data analysis that can help explain the "why" with regard to a Drug being unsuitable and facilitate research as to how the drug can be improved. One disadvantage of in-silico testing is that must often be accompanied by complementary in vitro or in vivo trials in order to verify the findings and prove real positive responses as it is impossible to computerize all the variables present in living organisms.
- In vitro models: In-vitro studies use cells derived from animals or cell lines which have an infinite lifespan. While relatively inexpensive and reasonably effective, the major drawback is their inability to capture the inherent complexity of organ systems that present a variety of cell and external factors such as hormones that modulate their biological responses. As a result, they are considered to be less “translatable” to humans and are therefore limited when comes to acquiring approval for use on human subjects. Nevertheless, many improvements are being studied to replicate organ complexity in vitro by producing organ analogs in artificial support known as Organs-on-a-Chips platforms.
- In vivo models: literally meaning “in the living”, in vivo testing refers to tests that are performed inside the body of a living model. This generally provides results that can be successfully translated to humans, although testing on complex mammals, and even rodents, can be costly, time-consuming, and implies ethical concerns. This has resulted in efforts to find alternative animal models such as zebrafish embryos.
The use of Zebrafish in biomedical research, preclinical assays and toxicology studies
The zebrafish has recently emerged as an incredibly effective, model in biomedical research, preclinical studies, and toxicology assessment, especially in the embryonic stage. The species is inexpensive to maintain and reproduces in large quantities. Unlike rodents and other mammals, the fish develop ex-utero. During this development stage, offspring it almost transparent flesh, enabling direct visualization of early organogenesis and physiological responses. What’s more, zebrafish have a similar genetic structure to humans, sharing more than 70 per cent of our genes. Actually 84 per cent of genes known to be associated with human disease have a zebrafish counterpart. The zebrafish also has the same major organs and tissues, with their muscle, blood, kidneys, and eyes sharing many features with human systems. Importantly, all these benefits can be applied in the embryonic stage of the animal, as many complex organ functions are acquired 5 days post-fertilization. Zebrafish embryos are considered NAM until 5-6 days port-fertilization as they have not independent feeding and depend on the vitellus. Thus, are not under animal care policies and, therefore, have fewer ethical concerns.
Numerous drug treatments that have recently entered the clinical trials have their genesis in zebrafish. In particular, zebrafish respond to drug treatments at physiologically relevant doses, recapitulating whole animal complexity at the organ and tissue level. Also, it has been proven to be an excellent model for evaluating the toxicity of chemical compounds on different organs and systems, even in behavior studies as embryos develop nervous system responses at 5 days post-fertilization.
In addition to their use in the Early Drug Discovery Phase and Toxicology studies, zebrafish are used in the biomedical study of many human diseases including cancer, neurodegenerative disorders, neuromuscular pathologies, developmental and mental disorders, or metabolic diseases. Interestingly, this model is particularly useful in eye disease research due to its capacity to regenerate retinal cells, making it particularly promising regarding the development of new treatments for them, and in the capacity to replicate numerous common human eye diseases such as cataracts, corneal dystrophy, and glaucoma.
Conclusions
Finding alternatives to mice and rats for use in preclinical trials and toxicology studies has been an ongoing pharma and chemical industry challenge for several decades. Zebrafish embryos offer numerous advantages in preclinical assays in Drug Development and Discovery and in the detection of the adverse effects of chemicals. With ethical arguments favoring the use of non-animal methods, the ability to observe the impact of genetic manipulation or pharmacological treatment using non-invasive imaging techniques (on account of the embryos and larvae being transparent) means that the zebrafish embryos may offer a more ethically attractive solution when compared to rodents and other mammals. While zebrafish cannot be used to examine the effects of chemicals on organs that they do not possess, such as the lungs, prostate, or mammary glands, the genetic similarity between zebrafish and humans is promising with regard to future research and has already proven expedient with regard to the modeling of numerous human diseases. With the numerous benefits, combined with the cost-effectiveness of the zebrafish as an alternative model, embracing NAMs can drive innovation and enhance efficiency in preclinical research and Toxicology studies combining the in vivo complexity of a whole living organism with the advantage of using a less ethically questionable model, and all in vitro tool that improve the throughput.
Sources
- Why do we need preclinical studies? (2018, X7 Research), (https://x7cpr.com/en/need-preclinical-studies/#:~:text=Preclinical%20studies%20of%20drugs%20allow,is%20a%20time%2Dconsuming%20process.)
- R Andrew G. Polsen and Reina N. Fuji, The successes and limitations of preclinical studies in predicting the pharmacodynamics and safety of cell-surface-targeted biological agents in patients (2012) – British Journal of Pharmacology, (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3419903/)
- A Critical Evaluation of the Advantages and Limitations of In Silico Methods in Clinical Research, (https://proventainternational.com/a-critical-evaluation-of-the-advantages-and-limitations-of-in-silico-methods-in-clinical-research/)
- Soodabeh Saeidnia, Azadeh Manali and Mohammad Abdollahi, The Pros and Cons of the In-silico Pharmaco-toxicology in Drug Discovery and Development (2013), International Journal of Pharmacology, (https://scialert.net/fulltext/?doi=ijp.2013.176.181)
- Candice Tang, In vitro vs. In vivo: Is One Better?, (https://www.uhnresearch.ca/news/vitro-vs-vivo-one-better#:~:text=In%20vitro%20studies%20use%20cells,inherent%20complexity%20of%20organ%20systems)
- (2010) – BMC Neuroscience, (https://bmcneurosci.biomedcentral.com/articles/10.1186/1471-2202-11-116)
- R. Richardson, D. Tracey-White, A. Webster & M. Moosajee, The zebrafish eye—a paradigm for investigating human ocular genetics (2016) – Nature Journal, (https://www.nature.com/articles/eye2016198)
- Why use the Zebrafish in research? Your Genome, https://www.yourgenome.org/facts/why-use-the-zebrafish-in-research/#:~:text=They%20share%2070%20per%20cent,many%20features%20with%20human%20systems.
- Elizabeth Patton, Leonard I. Zon, and David M. Langenau, Zebrafish disease models in drug discovery: from preclinical modeling to clinical trials (2021), Nat. Rev. Drug Discov., (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9210578/)
