New Alternative Methodologies and Their Applications in Drug Discovery and Development

Within the field of Drug Discovery and Development the term “New Alternative Methodologies” (NAMs) refers to the use of new and innovative testing methods that come to replace traditional animal models. In addition to ethical constraints and animal welfare distress regarding the use of animals such as rodents in preclinical testing, there are a number of obstacles encountered in the traditional testing methods. These include the fact that such methods can be extremely cost and time-consuming.

Where possible, NAMs seek to avoid the use of animal testing or at least to reduce it.  For example, by developing computerized models (in silico) or using models based on cell cultures (in vitro)., However, reports drawn up as recently as June 2023 still emphasize the necessity of animal testing at some point as we are still far from understanding and reproducing all the variables present in a whole organism in an in vitro or in silico model. So, which are the alternatives, and which are the advantages and disadvantages of traditional animal testing in the preclinical stage of Early Drug Discovery and Development? Computational models are some of the most promising NAMs due to their vast capacity for data integration, cost-efficiency, and a huge reduction in testing time. Nowadays, the development of informatic models and the advances in Artificial Intelligence (AI) allow accurate predictions on complex biological processes such as pharmacokinetics or vast screenings of Drug candidates, based on the prediction of their pharmacological effects. Complex biochemical interactions including drug docking predictions, protein folding, or evaluation of complex molecular pathways are possible with the AI models and advanced computational models. The use of in silico models allows the fastening of the first steps of Drug Discovery, representing an immense advance in the field. Unfortunately, these models are not able to accurately predict all biological effects a compound may present as biological variables are too complex to model them efficiently. 

On the other hand, in vitro systems that use simplified cellular models cultured in artificial devices are the other large group of NAMs. These methods allow the production of highly complex biological models that review most of the cellular responses found in an organ in a faster and more cost-effective manner, without any regulatory or ethical barrier. Nevertheless, in vitro models have important limitations, including the physiological changes the cells suffer when cultivated outside their organs of origin and the loss of the complex intercellular and hormonal communication and regulation present in body organs. These limitations hamper the result fidelity and make cellular models dependent on a final animal testing validation.

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Therefore, animal testing remains essential before a Drug candidate is tested in humans. The dependency on animal testing presents important limitations and difficulties including cost-effectiveness, being time-consuming, and carrying ethical apprehensions. 

In recent decades there has been a worldwide tendency to improve the welfare of the animals employed in preclinical testing. This has led to the implementation of the 3R Principle (Refinement, Reduction, and Replacement of animals) to control and limit the use of animals and the improvement of their living conditions, to mitigate their suffering to a minimum. 

In vivo trials being still an essential part of the preclinical research landscape, great efforts are been made to identify accurate NAMs that can be used in combination to reduce the use of animal testing. Currently, they are various consortiums, such as the Integrated Approach to Testing and Assessment (IATA) on Developmental Neurotoxicity) pushing for using in silico, in vitro, and alternative in vivo models complementing each other for more efficient outcomes. One of the most promising candidates among those NAMs is the Zebrafish.  Zebrafish embryofr are in the middle term between in vitro and in vivo models offering the complexity of a whole organism but in an organism that is not considered an experimental animal by European law (EU Directive 2010/63/EU), until the 5-6 days post fertilization (dpf) when they start feeding independently.  


Zebrafish Embryos as a New Alternative Methodology

Zebrafish offer an astonishing number of benefits in preclinical and toxicological research and their use has become a major trend within Early Drug Discovery.

Characteristics of Zebrafish that benefit their use in the preclinical setting:

- High physiological homology: In comparison to the human reference genome shows that approximately 70% of human genes have at least one obvious zebrafish orthologue. Furthermore, more than 82% of genes known to be associated with human disease have a Zebrafish counterpart.

- Fast organogenesis: Zebrafish embryos present most organ functionality at 5 dpf allowing the use of less than 6 dpf fish for preclinical assays. Older specimens are not considered embryos as start to present independent feeding and their use is regulated as other vertebrates. The use of embryos allows for time-effective and cost-effective assays with a reliable biological system.

- Neurological similarity with humans: Zebrafish show very similar neuroanatomical organization and neurotransmitter signaling pathways to those found in humans, even in the embryonic stage. Thus, zebrafish embryos are suitable for behavior and neurological evaluations such as locomotor activity, anxiety-like behaviors, learning, memory retention, spatial and object recognition, fear responses, and social preference and interaction. Therefore, this NAM is an excellent candidate for Early Drug Discovery and Development of neuroactive compounds and neurotoxicity or neurodevelopmental toxicity evaluation.

- Genetic manipulation: There are many genetically manipulated zebrafish lines of disease models that are able to express fluorescent reporters that allow an easy evaluation of many physiological responses in real-time.

- High fertility and small size: A single breeding pair is capable of producing three to five hundred fertilized eggs in one day. This, combined with their small size of the larvae means that zebrafish are particularly feasible to maintain, and feasible for High Content Screenings.

- Transparency: Zebrafish larvae are transparent, which provides incredible insight into biological mechanisms and organ formation in their development, as well as their internal response to Drugs or compounds.


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Challenges and Considerations

As with any innovative form of preclinical research, the main obstacle that researchers must overcome is the verification of the translatability of preclinical findings to human subjects. This is essential in order to ensure the safety of human clinical test subjects and future Drug users and the efficacy of the Drug candidates. 

Assay standardization and harmonization is vitally important too. The aim of standardization is to ensure that the measures and frameworks are implemented to ensure that assays of the same analyte in the same samples prepared in different locations or a different times can be replicated. This avoids unnecessary duplication of effort and accelerates a Drug’s progress on the path to approval for clinical testing and subsequent human use. 

The fundamental motivation behind any preclinical assay is to identify Drugs that are both effective and safe for human use, and consequently, models that can be more accurately translated to human systems must be identified, and techniques and procedures must be standardized, qualified and validate. In addition, regulatory requirements must be adapted and unified as New Alternative Methodologies  are discovered and developed.



There are a number of increasingly encouraging NAMs currently under development and in use within preclinical research. At present, however, none of these are sufficiently precise to be able to replace in vivo models alone. This has resulted in the development of several NAMs, such as the zebrafish embryos. This aquatic organism has produced an impressive number of promising innovations in various fields of medicine and toxicology to name a few. 

Keeping up to date with NAMs and the rapidly evolving regulatory framework is essential for the sustainability of Drug Discovery and development. In this sense, the use of zebrafish embryos represents a valuable alternative for Drug candidate selection and safety assessment to fasten and diminish the costs with high reliability and the many benefits this model offers. Therefore, the use of zebrafish embryos allows a significant reduction in animal testing use.



  • Vidya Mahalmani, Ajay Prakash, and  Bikash Medhi, Do alternatives to animal experimentation replace preclinical research? (2023) – Indian J. Pharmacol, (
  • Kerstin Howe et. al, The zebrafish reference genome sequence and its relationship to the human genome (2013) – Nature 496, 498–503, (
  • Alternatives to Animal Testing Models in Clinical and Biomedical Research (2023), ParmaNewsIntelligence, (


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