The model experimentation

The development of new and increasingly sophisticated approaches provides the impetus towards model experimentation avoiding the use of animals

13/06/2022

Clinical Trials

For a long time, experiments on products for human use have been carried out on animals: from basic toxicological tests to tests on drugs, cosmetics, chemicals. With the change in common sensitivity and with the growing respect reserved for animals, including laboratory animals, different solutions have been sought.

In the cosmetic field, legislation has provided for a ban on the use of animals since 2013 and we rely on alternative models (now no longer alternative, as the current standard method): for finished products and ingredients tested on animals there is a ban from the market.

Even Reach and other reference regulations for the chemical sector, while requiring data that highlight the degree of toxicity for humans and the environment of the substances marketed in the EU, oblige market players, even competitors to each other, to share existing data to avoid redundant testing, and call for the preferential use of in vitro, in silico and read across methods. The engine of this change is the European Union.

With regard to the protection of animals used for scientific purposes, the EU has issued Directive 63/2010, which goes in the direction of limiting animal testing only to cases where it is really necessary. Italy has transposed this directive with the Legislative Decree 26 of 4 March 2014, which establishes the procedures for the use of animals for scientific and educational purposes (for example exercises in schools or universities). In biomedical and pharmacological research, animal research is still possible.

However, when this happens, the law itself defines some basic animal rights to take into account. The legislation also states that all research projects involving the use of animals must be previously inspected and approved by the Animal Welfare Body (Opba), which in turn forwards the application for authorization to the Ministry of Health through the “Telematic database of animal experimentation”.

All research projects with the use of vertebrate animals and certain invertebrates (such as cephalopods) must in fact be authorized by the ministry. There is also a National Committee for the protection of animals used for scientific purposes, established by the ministerial decree of 24 February 2016 (again pursuant to Legislative Decree 26/2014) at the Directorate General for Animal Health and Veterinary Medicines of the Ministry of Health; its composition was renewed with a Ministerial Decree of 1 June 2021.

The main task of this committee is to advise the competent authorities and the Opba on all matters relating to the D.L. itself and in particular on the protection of animal welfare, as well as on what is related to the acquisition, breeding, accommodation, care and use of animals, ensuring the sharing of best practices. The committee carries out numerous activities and functions, including that of promoting the principle of the 3Rs.

3R

Refinement, reduction, replacement: the 3R principle was first proposed more than 60 years ago by British biologists Russel and Burch in the book The principles of humane experimental technique (1959), the result of a project led by the Universities federation for animal welfare (Ufaw), overseen by a committee that included future Nobel laureate Peter Medawar. Replacement alternatives refer to methods that avoid the use of animals, or replace it.

This includes both absolute replacements (i.e. the replacement of animals with computer models) and relative replacements (i.e. the replacement of vertebrates with animals that have a lower potential perception of pain, such as some invertebrates).

Reduction alternatives refer to any strategy that involves using fewer animals, while still obtaining sufficient data to answer the research question, or maximizing the information obtained per animal and can therefore potentially limit or avoid the subsequent use of additional animals, without however compromising the welfare of the animals.

Refinement alternatives refer to the modification of breeding or testing procedures to minimize pain and distress and to improve the well-being of an animal used in science from the moment of its birth to its death.

The pharmaceutical industry has also adhered to this point of view for some time. Efpia (European federation of pharmaceutical industries associations) already in 2019 released the report “Putting animal welfare principles and 3rs into action“, which highlights examples of good practices aimed at improving the quality of life of animals used for Research.

The report also invites associates to promote the culture of the 3Rs as well as to avoid or limit as much as possible the use of animal testing, with the aim of adhering to a total paradigm shift in the approach to testing, even going beyond the regulatory requirement. .

For example, by implementing routine practices that focus on the elementary rules for animal welfare based on the ethology of the different species. Or by developing less invasive devices or by resorting to alternative methods that in many fields have now obtained validation for research purposes.

Beyond the 3Rs

In vitro research poses substantially no problems in the public eye, is relatively inexpensive, and is susceptible to iterative cycles of trial and error using statistically very robust study designs. Therefore, with in vitro systems there is an intrinsic opportunity to conduct exploratory research at a wide range.

Conversely, in vivo experimentation is more expensive and a good percentage of the population is firmly against it (especially on higher animals such as vertebrates). The considerations are both ethical and financial. In many countries, the law now states that an animal experiment should not be performed if an alternative method is available, and some countries openly ban testing certain animals, such as apes, with growing pressure to extend bans to animals. so-called inferior species.

The use of laboratory animals in research is therefore not very agile and very expensive, and poses problems of “consensus”. Considering an ever-increasing demand for toxicological tests by the legislator (to form the dossier of the product that will be put on the market), and also considering that biomedical research is increasingly oriented towards precision medicine and biological therapies, it comes to consequently, a push from various directions towards the complete replacement of animal tests, while at the same time seeking methodologies that are more in keeping with the specifics of human physiology.

The 3R principle was (and still is) a cornerstone of this path, but there is now talk of overcoming it and taking a further step forward. Research methods without animal models represent a significant growth opportunity for the research itself: the technologies in use are at the forefront and the funding to support study projects is significant, both from the European Union and from various industrial sectors.

Through its research framework programs, such as those called Horizon, the EU has created a fabric favorable to the development of alternative strategies with EURL Ecvam (European union reference laboratory for alternatives to animal testing), a coordination center for studies of animal free methods. The aim is to integrate cell biology knowledge with “omics technologies” to obtain models suitable for defining the effect on human health following exposure to a potentially harmful substance.

EURL coordinated European research centers on topics such as the toxicity of chemical mixtures, endocrine disruptors, the assessment of acute toxicity of substances, the bioaccumulation of chemicals in aquatic and terrestrial organisms and other environmental toxicity issues.

But alternative tests are a fundamental resource also in the field of biomedical research: EURL Ecvam has been investigating and examining approaches that go beyond animal testing in various specific areas for years: from immunogenic tests for advanced therapies to cardiovascular diseases, from autoimmune diseases to respiratory problems, from breast cancer to immuno-oncological models.

EURL Ecvam is also active in the regulatory field and deals with the validation of new tests, also with peer review activities, and with the preliminary involvement of the regulatory authorities when it comes to validating new methods.

The US Food and Drug Administration (FDA) also demonstrated its commitment to deepening the development of complementary or alternative approaches to animal models when it announced the Innovative science and technology approaches for new drugs (Istand) in December 2020. The pilot program aimed to speed up the qualification and use of new drug development tools.

The announcement explicitly includes the use of tissue chips and microphysiological systems to evaluate safety or efficacy issues, the development of new non-clinical pharmacological and toxicological assays, up to the use of AI-based algorithms to evaluate patients, develop new end -point or as a basis for designing a study.

Covid models

The development of infectious disease therapies (and their prevention) depends on the availability of disease models. To remain closely current, the response of the medical community to Covid-19 depends very much on the possibilities of modeling, both in the traditional way that uses animal models and using NAMs (see box).

When the pandemic began it was not possible to establish any “good” animal model of Covid-19 and this model is still missing today. And even if a suitable animal model is identified, it must be considered that the outcomes of viral infection can vary drastically even within animals of the same species.

The bioengineering of human organoids and organs on chips has shown itself to be an extremely promising research avenue in this field.

Organoids have been used to study human nervous system involvement in Covid-19. Before studies on brain organoids, there was no evidence in animal models that Covid-19 could affect the nervous system. In particular, laboratory-grown miniature organ systems were used extensively during the pandemic to show the numerous effects of the causative agent (severe acute respiratory coronavirus 2, SARS-CoV-2) on human organs. Exposure to the virus of most animals did not cause infections, or caused only mild clinical signs, not the severe course of infection suffered by many humans.

Micro-physiological systems have therefore provided a great opportunity to study Covid-19 in vitro, in particular with regard to brain cell infection and its relevance to the effects of Covid-19 on the human brain.

The neurovirulence of SARS-CoV-2 has been reproduced in different types of organoids based on human brain cells, constantly showing the infection of a small portion of brain cells accompanied by limited viral replication. This mirrors the neurological manifestations increasingly recognized in patients with Covid-19 (virus infection and formation of brain-specific antibodies in brain tissue and cerebrospinal fluid).

Related article:

Progressive roll-out of the new In Vitro Diagnostic Medical Devices Regulation

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