Perspectives From the 12th Annual Minipig Research Forum: Early Inclusion of the Minipig in Safety Assessment Species Selection Should be the Standard Approach

Abstract

Over the last decade, the minipig has been established as a species which can be used in biomedical research, including drug development safety assessment. There are no mandatory regulatory guidelines regarding species selection strategy for safety assessment; hence, choice is at the discretion of companies responsible for drug development. A survey of member companies by IQ DruSafe (2016) highlighted inconsistent and low use of the minipig. At the 12th Annual Minipig Research Forum in 2018, presentations and a workshop examined current practices and considered if the minipig could be utilized more from earliest drug development stages. Despite the agreed utility of scientific data and validity of the minipig, especially for small molecules, each company has its own approach in nonrodent species selection, without consistent rationale. The overall objective should be to ensure the most appropriate species is selected and is scientifically based, with the minipig systematically included from early screening stages.

The use of the minipig in all aspects of biomedical research is very diverse and continues to develop. There are a large number of articles in the literature, in books, and discussed at workshops. In particular, the history, background information, and use of the minipig is well described. ^{1 –6}

Historically, much momentum in use of the minipig in research traces back to the RETHINK project, ⁷ which was funded as a Specific Support Action under the European Community 6th Framework Programme. The primary objective was to evaluate the potential impact of regulatory toxicity testing in the minipig as an alternative approach that can contribute to the replacement, refinement, and reduction of animal testing (3Rs Principles). Expert study groups were assembled to review 5 different areas relating to the use of minipigs in regulatory safety testing: ethical issues, welfare and animal care, development of new medicines and chemicals, safety testing issues, and emerging technologies in safety testing. ⁷

The Minipig Research Forum (MRF) is a nonprofit organization for everyone working with minipigs in research. Minipig Research Forum was launched in 2007 and the network has more than 500 worldwide members from industry, academia, and regulatory bodies. Every year, a 3-day MRF meeting is held, ⁸ with a comprehensive scientific program organized by a Steering Committee consisting of different organizations using the minipig. Typically, approximately 100 participants attend the annual MRF meeting, making it a unique opportunity to meet, discuss, share knowledge, and experience across all areas of minipig use in research.

One of the 4 major themes for the 12th Annual MRF in 2018 ⁸ was “Species Selection” for safety assessment of molecules in drug development. The presentations, discussions, and conclusions during a dedicated workshop on this subject are the basis of this article.

The experience and considered opinion among members of the MRF, especially those representing biopharmaceutical/pharmaceutical companies, is that although there have certainly been improvements in minipig use, increases in reference data, and development of new models, significantly more can be done. The nonrodent species to be considered for “species selection” and subsequently for toxicological assessment does not follow a mandatory regulatory process, instead is reliant on approaches of individual companies to decide which species are primarily used, and thus whether or not to incorporate the minipig at early stages for potential selection. The participants in the MRF workshop explored the current situation regarding conventions for species selection in drug development and asked questions in order to identify what more could be done in future to expand the use of minipigs in biomedical research and drug development. Furthermore, consideration was given to whether or not regulatory barriers, commercial pressures, lack of scientific knowledge, and/or other factors play a role in current held perceptions.

One reason to look at the current situation regarding minipig use for safety assessment was prompted by an industry survey of member companies conducted by IQ DruSafe ⁹ based on their experience up to late 2014; this being a follow-up from a previous survey, ¹⁰ asking member companies various questions about their use of the minipig in drug development, particularly for safety assessment. The article showed that use of minipigs was similar to the previous survey. However, the response to the survey was quite low, with only circa 50% of companies providing input, and of those responding, only a small number used the minipig in their drug development safety assessment.

In this brief communication, we will provide the main outcome of the presentations and discussions during the Species Selection Session and interactive Workshop at the 12th Annual MRF ⁸ meeting, compare with the IQ DruSafe findings, ⁹ and provide further thoughts and suggestions on the expanded use of the minipig in biomedical research and drug development.

At the 12th Annual MRF, ⁸ 4 invited speakers provided lectures on the theme of species selection and use of minipigs as nonrodent species in safety assessment for drug development, intending to give representative perspectives from pharma/biopharma companies, governmental institutions for public health (Regulators), and contract research organizations (CROs) that provide safety testing and other research and development services required for drug development and authorization. The titles of these talks were:

Nonrodent species selection to assure human relevant safety assessment (company A).

Non-rodent species selection considerations for preclinical toxicology studies (company B).

Use of minipigs to support non-clinical safety of human pharmaceuticals: update after RETHINK. Species selection—a regulatory perspective with reference to the minipig.

Species Selection—A CRO perspective.

An interactive workshop entitled “Species Selection” was held after the lecture session, with 29 attendees representing the different areas of use of minipigs in research. During the workshop, a number of basic questions were explored, inviting discussion with the audience, with the aim to provide some general comments on the evolution of use of the minipig model since the RETHINK publications ⁷ and a conclusion on current practices. Additionally, there was consideration of some new data about minipig use in safety assessment studies, from an industry completed questionnaire about which species they had used. At the time of the MRF Workshop, these data were unpublished, but it is now in the public domain. ¹¹ These new data from 172 molecules (projects from mid-2015 back to 2012 combined small, large, and other molecules) were provided by 18 companies (European Union [EU], USA, and Japan-based) and revealed only 1 molecule used the minipig as the nonrodent species (all others used dog or nonhuman primate [NHP]). This use of minipig seemed low, being 1% (1/92) of the small molecule projects.

The species selection workshop considered aspects such as:

What is the reality about frequency of use of the minipig, when perception by many is that minipig is used quite often in drug safety evaluation?

Where are we now, when reflecting on previous publications, such as RETHINK?

Is the minipig a well-defined and characterized nonrodent model for development of new drugs?

Are there any regulatory hurdles to be tackled for agencies to accept the minipig?

If any, what are the areas of knowledge about the minipig where there needs to be substantial work to close the gaps?

What are the reasons why the minipig is not consistently used/considered in drug safety assessment?

Should the minipig be a mandatory species to be screened for any drug development program—and for any molecule type?

Session Learnings

In the session of presentations from industry, both companies (A and B) confirmed that they have experience using the minipig in safety assessment, but it was clear that the scope, extent of use, and the strategy and rationale of each company were distinctly different.

One company’s (company A) approach was to fully focus on having the minipig as the primary nonrodent species (essentially a “default” nonrodent species), providing supportive scientific data confirmed it to be suitable. This approach included the minipig in species selection screening from the earliest possible stages of development for small molecules and indeed, the company now regularly uses the minipig for safety assessment omitting the use of dogs. It was emphasized that the ability to be able to utilize the minipig in this way has taken time to establish the research experience, data, and knowledge required. This company also is actively exploring opportunities to use the minipig for safety assessment of large molecules.

The contrasting approach presented by the other company (company B) was to use the minipig as a second choice to dog and also to introduce the minipig later in development, particularly to answer specific questions which were not possible using the usual default nonrodent species of the dog (or cynomolgus monkey). The continued reliance on using typical (default) nonrodent species such as the dog was justified scientifically as being related to historical control data and the assurance given that many molecules in their development tend to be aimed at known/familiar targets and for a limited range of therapeutic areas.

The CRO perspective presented views from a long history of experience in conducting various types of minipig safety assessment studies, including exploratory, standard regulatory and Good Laboratory Practice (GLP) studies of general, targeted, and reproductive toxicology assessments. Over the years, CROs have developed and used an extensive and ever-evolving range of techniques in animal care, housing and enrichment, dose administration, sampling (eg, blood samples), clinical assessments, and reporting of minipig studies. They also have developed teams of highly trained staff with suitable methodologies to properly manage diverse study conditions, which is a competency that is increasingly shifting from companies that develop new medicines and elect to outsource preclinical development more extensively than in the past. As the CRO’s role is to conduct work at the request of a sponsor company, there is typically only limited influence on selection or justification of the nonrodent species. However, the role has been evolving collaboratively due to the breadth of expertise and historical control data that can be supportive for scientific justification as well as practical feasibility and development of extensive testing capabilities when making the decision to select a relevant nonrodent species.

In the regulatory perspective presentation, the senior and well-respected presenter acknowledged that much work has been performed since RETHINK, ⁷ and some examples were given for products that included safety assessment in the minipig. It was clearly stated that there is no issue or obstacle among regulators in accepting minipig as a nonrodent species, providing that the selection process and justification is clearly stated and also supported by the reviewing regulatory authority.

Species Selection Workshop Learnings

In the workshop, the key views of participants, many of whom represent pharmaceutical/biopharmaceutical companies and nonclinical CROs conducting global development, can be summarized as follows:

Regulatory—Delegates consider there are no regulatory hurdles for using minipigs, as the use is accepted by regulatory authorities all over the world including Japan, Korea, China, India, EU, and USA. Nobody had experience with, nor knowledge of, any instances where the minipig had not been accepted by regulatory agencies.

Reasons for selection—Participants confirmed that the practice of inclusion of minipigs in safety assessment at the current time is company-specific, ranging from minipig being a “default” to those who would never include them. The reasons for such wide-ranging consideration of minipig were thought to be largely based on a priority of different perceptions and influences within companies, individual scientists, or global regions. It is well-known that views on topics such as animal ethics and public acceptance versus scientific justification/human relevance can vary globally. In some regions, expectation by national project license bodies/government such as the United Kingdom and other parts of EU can provide at least some requirement that the minipig is being considered (though still not mandatory), but there is not as yet a globally shared approach. Other general comments from participants included those such as “It is easier to do, what we normally do.”

Historical control data—Delegates were aware that there is a perceived lack of historical control data (compared to dog and NHP). However, they all agree that this is not the case and additionally the MRF has helped to ensure the sharing of experience in the use of the minipig during safety assessment is commonplace, along with readily available historical control data (especially that generated at CROs) and acceptance from regulatory authorities.

Knowledge and experience—It was also felt that CROs can perform a key role in providing more advice and/or involvement to industry, especially to those less familiar with minipig models. Contract research organizations have vast experience in technical skills, including a wide range of dose routes (intravenous, oral, ocular, dermal, etc), sampling (eg, blood), and other animal welfare, enrichment, and technical aspects that are not, generally, considered to be issues or to present barriers for use.

Test item quantities—some participants had the opinion that choosing the minipig would detrimentally increase the requirement for test item, which often has project costs increase implications. The experience is clear in that the amount required for most packages of safety studies with minipig is not significantly greater compared to using dogs, in fact being similar up to the typical 13-week toxicology studies.

Supporting assays—it was discussed that in vitro and ex vivo assays, including enzyme activity and metabolic stability, are now available for the minipig, and many of the commonly used biomarkers are validated for minipig use in research and development. Participants considered that there was certainly more work to be done in some areas of minipig testing, in particular, a focus on how there could be more application in safety assessment for large molecules, such as monoclonal antibodies. It was agreed that greater knowledge, availability, and access to further screening assays to help in species selection (eg, for large and small molecules) and biomarker development were still required to widen opportunities for the more systematic use of the minipig.

Workshop participants were very surprised by the new data from the industry questionnaire, as the use of minipig was significantly lower than many people imagined. The new data had been collected quite recently, from 18 companies, some of which are known to have or do still use the minipig. Generally, it was felt that this low figure ought to be explored further, but it also demonstrated that more should be done to widen inclusion of the minipig.

Consensus of the workshop participants was that there are no obstacles for including minipigs in the large animal species selection process, and all nonrodent species should be equally considered based on scientific justification and pharmacological relevance, also to comply with ethical and legal requirements.

Discussion

In the lectures and workshop held during the 2018 MRF, there was good and robust reflection of the current position of industry in using minipig as a nonrodent species in safety assessment. Two companies presented very clear outlines of their internal approaches and reasoning for using or not using the minipig. Both companies use the minipig, but the extent of use and reasoning was markedly different, with one company showing a very clear and well-defined strategy on the use of the minipig as first choice nonrodent species, particularly over the dog. The other company considers various reasons, including historical experience related to types/class of molecules and disease area being worked in, but still prefer the dog as first choice, followed by NHP, with minipig only considered for specific scientific reasons. Of the 2 companies, only one includes the minipig routinely (default) within the early species selection screening processes. In the workshop, comments from another company confirmed that their own approach is slightly different to that of both the presenting companies being closer to that of the company which more actively use the minipig but not to the extent that minipig is the “default” species. However, another company represented in the workshop mentioned that minipig is not routinely considered for their species selection process, as they rely on other longer established species such as dog and cynomolgus monkey. These 3 diverse ways of dealing with nonrodent species selection ranging from using minipig as a default species to almost never considering them is thought to be very representative of the industry globally. In the IQ DruSafe paper, ⁹ only approximately 50% of companies responded to the survey about minipig use, and indeed, only approximately half of those who did respond used minipig at all, with the range of experience and reasoning being quite different. Overall, the available information is not based on a summary of opinion of all pharma/biopharma companies, based on the experience of MRF members, and information shared in the MRF, it is clear that the incorporation of minipig for safety assessment is very different from company to company, and overall is not as high as it could (and perhaps ought to) be.

The reasons for the wide-ranging approach in companies are likely very multifactorial, but in particular, there are no stipulations in any local, country, or global guidelines which require mandatory inclusion of the minipig. Other factors likely include regional considerations toward ethics of animal use, public opinion, in-house experience, and so on.

Yet, in the MRF, and stated elsewhere, ⁹ when minipig is used for safety assessment, it is acceptable to regulatory agencies globally, providing of course that adequate scientific reason and data are available as support. It was acknowledged that, in general, technical challenges of the past are not an issue, and conduct of various safety studies is as straight forward as it is for other species. Historical control databases have increased appropriately in almost all aspects, and therefore, this could not be a reason for not using the minipig. For any molecule where traditional drug metabolism can be used (eg, for small molecules), there are differences in absorption, distribution, metabolism, and excretion between nonrodent species such as dog, minipig/pig, and NHP, which may imply certain unsuitability for some molecule classes, ¹² overall the assays do exist to enable any company to be able to include minipig alongside other species for in vitro and other assessments, so that a wider range of species is used from the earliest stage. For any biological system and species, there is always room to improve our understanding with time, and this would of course include building on immune profiling and trying to apply this species for assessment of biologics. ¹³

Generally, test item requirements for minipig safety assessments packages is not significantly different to that needed for dogs, and only in a small number of cases may become an area of consideration if studies required are long term (>3 months). However, test item requirements can be factored into overall development costs from early stages, if the minipig is selected early, and indeed should not be an influence on selection strategy compared with the scientific value of the species.

Conclusion

It is the view of the authors of this article that minipig use for safety assessment is still too low compared with other nonrodents such as dog or NHP. This was underlined by data from a recent industry survey ¹¹ shared in the workshop, with minipig inclusion in safety assessment of 92 small molecules only being circa 1%. Based on the MRF workshop, and drawing on other comments and experience, the minipig is now so well defined and understood, it ought to be routinely included from the earliest screening stages for species selection by all companies, particularly for small molecules. Additionally, substantial consideration ought to be given for using the minipig for large molecule safety assessment, providing suitable screening platforms are available.

The minipig can be chosen as a nonrodent species in safety assessment as they are regulatorily acceptable, technical aspects of study conduct are not a general issue, and test item requirements are not significantly greater than for dog. One company is already setting the “benchmark” for species selection by defining minipig as a “default” species instead of dog for many types of molecule, unless scientific reasons speak against it. It is not clear why other companies have not adopted a similar approach to this “benchmark,” or at least more consistently considering minipig and including it in early screen when selecting nonrodent species.

As there are no mandatory regulatory processes to include minipig, or indeed many other species/strains of animals (eg, marmoset monkey, different strains of rodents), it means that all companies developing new drugs have individual choice about the approaches to selecting species to be used for safety testing. This means that for the majority, historical experience, ease of use, cost, and other factors may be the leading reasons for not including minipig (and other species/strains) in their selection process. While all companies would be assumed to be applying scientific judgment and strategy, this is not being done in the same way by all. We suggest that perhaps the strongest scientific assessments may be best done from requiring a wider choice of species from the earliest stage. We have seen examples of some companies taking a very different approach to others and demonstrating to us all that it is possible to change. Therefore, we would encourage greater examination of the processes behind species selection, especially by industry. If there is no change from industry, we will continue to see underutilization of the minipig, and indeed other species, with species selection for all molecules being based on a limited (selective) choice of species.

Species selection for safety assessment of new drug molecules needs to be explored further and inclusion of minipig in strategy and screening ought to be the standard approach. The historical barriers such as access to historical control data, genome, size, and technical aspects are not relevant today, and therefore there seems no reason why minipig should not be included in the species selection process. Further exploration of the current approaches in industry globally, together with appropriate inclusion of regulators, is required to educate and improve awareness of the utility of the minipig and improve the species selection process.