1. Introduction
Over the last three decades, minipigs have progressed from a little-known alternative animal model for canines and nonhuman primates (NHPs) to a promising model in regulatory toxicology and drug development [Citation1–3]. Over time, this preclinical animal model has gained strength for the evaluation of drug efficacy for various clinical indications [Citation4]. Since political and societal support for the use of canines and NHPs in research has declined over time, the minipig is currently considered to be a critically important non-rodent species for the discovery and development of drugs (). The minipig has gained the attention of the scientific community as a possible alternative large animal model that may overcome challenges presented by other animal models in the area of drug discovery and development for approval by regulatory agencies.
Table 1. Minipig as a non-rodent model in drug discovery
The RETHINK project was funded as a Specific Support Action under the European Community 6th Framework Program to assess the impact of drug toxicity evaluation in the minipig model as an alternative in regulatory toxicological studies that can support the replacement, refinement, and reduction (3Rs) of animal use in research [Citation5]. Working Groups were created to review the use of minipigs in the regulatory assessment of drugs in addition to the ethical issues, animal care and welfare, development of new drugs, safety evaluation, and use in emerging technologies for safety and toxicity studies. This was another welcomed step in the right direction for promoting the minipig model in addition to canine and NHP models for use in drug discovery.
Minipigs are strains of domestic swine that are significantly smaller compared to farm varieties of pigs that make them suitable for handling as laboratory animals. The swine strongly resembles humans in several attributes of anatomy, biochemistry, genetics, and physiology. Specifically, the cardiovascular, immune, integumentary, gastrointestinal, and renal/urinary systems of minipigs are similar to humans [Citation6]. Due to these similarities, the response of minipigs to any drug or injury/trauma (e.g. irradiation, wound, combined injury, etc.) may be more analogous to human responses when compared to other laboratory animals. Additionally, because pigs are food/agricultural animals, the laboratory use of minipigs may be more acceptable to society when compared to other large animals used in research.
The use of miniature swine (Göttingen, Hanford, Sinclair, and Yucatan) as a large, non-rodent animal model in preclinical research appears to be promising for human diseases. The Göttingen breed was developed using the founder breeds including Minnesota Minipig, German Landrace, and Vietnamese Potbelly Pig at the University of Göttingen, Germany. This is the most common minipig strain used in several disciplines. Translational preclinical swine research data support the premise that the miniature swine model is the ‘model-of-choice’ for studying drug safety/toxicity, tolerance, physiologic, and pharmacokinetics of drug absorption and efficacy (). In brief, the Göttingen minipig is becoming an animal model of worldwide importance in pharmaceutical research.
Table 2. Minipig strains commonly used in drug discovery
2. Use of minipig model for drug development
During the last three decades, tremendous progress has been made in the development of the minipig model as a useful and reputable animal model for studying regulatory toxicology; this progress is mainly due to the well-documented similarities to human physiology. Although many characteristics of this animal model are common with canines and NHPs, some features are often overlooked when studying drug pharmacology [Citation7].
Minipigs have long been used as suitable animal models for numerous clinical indications such as bone disorders, cardiovascular problems, diabetes, digestive (acute and chronic intestinal inflammation) and heart disease, metabolic disorders, and skin conditions [Citation7,Citation8]. Several minipig strains have been used in a large number of studies such as atherogenesis, dyslipidemia, and the testing of intra-arterial stents [Citation9].
Although pigs have been used less frequently than canines and NHPs in the safety investigation of drugs, minipigs are invaluable when testing select types of drugs because of essentially important, metabolizing enzymes or pathways that might be absent in different species. Minipigs may not be a good choice if sulfation that relies on 3-phospho-adenosyl-5-phosphosulfate sulfotransferase that is absent in minipigs is involved because of its importance in human metabolism [Citation1]. Nevertheless, with time, minipigs are gaining acceptance by regulatory agencies in the US, the European Union, and Japan as a suitable large animal model for the preclinical evaluation of drugs.
3. Advantages and limitations of using the minipig as an animal model in drug discovery
In terms of animal welfare concerns, it is easy to see how minipigs might be more suitable to use when compared to other large animals such as canines or NHPs. Minipigs are not athletic like canines or arboreal like NHPs; therefore, their housing requirements are less stringent. The widespread use of pigs in research also provides historical data and its relatively small size provides convenience in handling. In the area of medical devices, recommendations of the International Organization for Standardization (ISO) suggest that swine are suitable research animals, largely due to hematological and cardiovascular similarities with humans [Citation10].
The potential of the minipig as a platform for future developments in emerging technology such as genomics and transgenic technologies is promising. Commercial interest in the swine as an agricultural/food animal has provided an underlying basis for the use of this animal model and has significantly driven its scientific advancement as a major biomedical research tool. The DNA sequence homology between humans and swine indicates that minipigs will be valuable for the evaluation of biotechnology products. Swine models in general and minipig models in particular are the only non-rodent animal models in which transgenic animals can easily be created. Furthermore, minipigs are amenable to various modes of drug administration such as oral intubation, dietary intake, inhalation, dermal administration including inflicting skin wounds, multiple routes of parenteral administration, and intravenous infusion [Citation7]. For studies involving the integumentary system (specifically the skin), the minipig is more similar to humans than any other preclinical animal model. The swine is considered appropriate for the drug disposition investigation as they have a large number of enzymatic proteins as well as membrane transport processes in common with humans. These attributes make the minipig model an attractive tool for drug evaluation.
The deeper location of the blood vessels in minipigs makes blood collection somewhat difficult and requires well-trained study personnel or the use of vascular catheters, also known as vascular access ports (VAPs) for serial blood draws. VAP makes blood collection easy, but their use is frequently complicated by fibrin deposition and infections. However, despite all of the positive attributes of the minipig model, NHPs are still considered the ‘species of choice’ of large animal models with 95% sequence homology with humans at the DNA level [Citation11,Citation12]. They are considered the gold standard by the US FDA. In recent years, the availability of essential biological reagents for the minipig has improved greatly; however, the full scope of all required reagents still falls short when compared to the array of reagents currently available for NHPs. It is not going to be easy for any other animal model (other than NHPs) in the near future to have the equivalent level of documentation of basic pathogenic processes in order to gain confidence and acceptability by regulatory authorities such as the USFDA.
The minipig in general and the Göttingen minipig in particular are radiosensitive animal models as compared with other animal models like canines and NHPs. The radiation-induced disseminated intravascular coagulation has been suspected to be a contributing factor to the high radiosensitivity of this species [Citation13].
4. Characterization of minipig as animal model for acute radiation syndrome (ARS) and delayed effect of acute radiation exposure (DEARE)
It is important to develop the minipig as an alternate large animal model for the study of ARS and DEARE and ultimately to develop safe and effective medical countermeasures for these serious radiation-induced pathologies in humans [Citation14]. With the availability of additional animal models, both large and small, comes the opportunity to examine basic pathobiological responses across multiple, nonhuman species that would allow reasonable projected responses for humans. Limited availability of animal models is a bottleneck for the development of medical countermeasures, particularly for chemical, biological, radiological, and nuclear (CBRN) injuries where drugs are approved following the U.S. Food and Drug Administration (FDA) Animal Rule without conducting an efficacy study in humans [Citation15]. Similar to NHPs and canines, minipigs develop hematopoietic, gastrointestinal, and cutaneous sub-syndromes, as well as DEARE symptoms that are all quite comparable to the related sub-syndromes of humans. In general, however, the minipig is relatively radiosensitive when compared to the previously mentioned species [Citation2,Citation3,Citation16,Citation17]. Because of the minipig’s relatively large size (e.g., as compared to rodents), sequential tissue and blood sample collections from the same animalcan be done without euthanizing the animal, thus providing an opportunity to minimize inter-animal variations and to better understand the injury and recovery of individual animals over time. Patterns of radiation deposition (i.e., highly penetrating, low energy transfer type irradiation) within the minipig are similar to humans since the body thickness of a minipig is roughly equivalent to that of a young adult human. The pathophysiology of ARS in respect to hematological dynamics and platelet counts in minipigs is similar to humans, canines, and NHPs. Minipigs display classic gastrointestinal-ARS following whole-body irradiation. The minipig is a monogastric and omnivorous animal with cardiovascular and gastrointestinal (GI) systems similar to humans.
Since no single animal model can precisely mimic the various human conditions that might follow acute irradiation, especially accounting for all possible sub-syndromes and symptoms, it is important to choose the most suitable model for the specific study depending on the study objectives. Over the last ten years, there has been significant progress with the characterization of minipigs as an animal model to study radiation injuries and to evaluate the efficacy of various medical countermeasures. Several radiation countermeasures for hematopoietic and cutaneous sub-syndromes, such as Neupogen [Citation18], Neulasta [Citation19,Citation20], and adipose-derived stem cells [Citation17,Citation21], have been tested for efficacy using irradiated minipigs (). Additional agents have also been investigated for beneficial effects against radiation injury such as curcumin and basic fibroblast growth factor. Furthermore, few agents (e.g., adipose-derived regenerative cell) have been evaluated against combined injury; for example, irradiation and burn. Some agents have been tested for organ-specific radiation injury using the minipig model. It is important to note that evaluating a given agent does not necessarily constitute ‘proof-of-characterization’ and that the minipig’s basic anatomical, physiological, central nervous system, genetic, etc., features are all appropriate for a given endpoint [Citation22]. In general, however, medical countermeasures demonstrating efficacy in NHPs have also shown efficacy in minipigs [Citation18–20].
Table 3. Medical countermeasures evaluated against radiation injury using minipigs
5. Expert opinion
As stated above, the ethical and social concerns over the use of canines and NHPs have led to the effort of exploring alternate animal models for drug discovery, development, and FDA approval. We do not believe, nor do we want to suggest, that canines, NHPs, and swine differ significantly with respect to the pain and suffering that they undergo when used experimentally for drug evaluation. Thus, the argument that minipigs are more acceptable test animals as compared to canines and NHPs is not logical; this is apart from the fact that the use of minipigs in research may be less offensive to some members in society. Additionally, because pigs are agricultural/food animals, the laboratory use of minipigs may be deemed more acceptable to society when compared to other large animals used in research for the discovery and development of drugs. At the same time, species selection in preclinical research for drug development needs to be made on a case-by-case basis considering all scientific advantages, disadvantages, and animal welfare issues.
Furthermore, the lack of availability of adequate comparative data with other large animal models suggests that there is a need for more thorough and rigorous assessments of the minipig model. Additional investigations are needed in order to provide essential experimental data for judging whether or not minipigs are more reflective than other animal models (e.g., canines, NHPs, etc.) in determining drug-induced toxicities in humans. Similarly, one needs more data in judging whether the minipig is an optimal animal species to investigate drug efficacy for various clinical indications. It would be of interest to the scientific community to gain further insight into the potential use of the minipig as an animal model of choice for the development of drugs and biologics for human use, especially in cases where the minipig can be substituted for animals of greater social concerns. In this regard, the minipig has been well recognized in the pharmaceutical industry as an important non-rodent model for drug safety/toxicity evaluation and is supported with compelling scientific arguments based on comparable physiologic characteristics similar to humans. Accordingly, in certain pharmaceutical research settings, the minipig is considered to be competitive with canines and NHPs. As a result, it is possible that the minipig may become a more important animal model for pharmaceutical research and development in the future. One major drawback for its utility is its lack of use in screening for drug responsiveness or pharmacokinetics of pharmaceutical agents. Another drawback is the absence of full and appropriate characterizations of this animal model for select types of injuries or insults such as injuries resulting from CBRN-types of exposure. As a result, the U.S. FDA is not inclined to accept the minipig as an animal model for approving drugs for human use under the Animal Rule for CBRN threat agents. The FDA does not appear to be comfortable accepting the efficacy data generated in minipigs alone for the approval of medical countermeasures for ARS and DEARE under the FDA Animal Rule.
At present, we lack a systematic effort to compare the minipig with other large animal models such as canines and NHPs as non-rodent models for various pharmacotherapeutics. If such comparative efforts were to be made along with a broader characterization of the minipig model in general, perhaps then a better, more prudent, and appropriate selection of the large animal model for given pharmaceutical tests and specific clinical indications might be made. The regulatory agencies in their ‘guidance documents’ are not specific in terms of describing the use of the animal species to be used for preclinical drug evaluation, but only that two species may be used: one rodent and one non-rodent [Citation15]. Furthermore, in the U.S. FDA and European Medicines Agency (EMA)/Committee for Medicinal Products for Human Use documents, the minipig is not mentioned as one of the species of choice. Mice and rats are the first choice rodents, and rabbits/canines are the typical first choice non-rodent species [Citation10]. The NHP is used for advanced drug development and is specifically required for testing the efficacy of drugs for regulatory approval from the FDA under the Animal Rule where clinical efficacy testing under Phase II and Phase III is not possible due to ethical reasons [Citation15].
During the last few years, the identification of several biomarkers and the availability of research reagents have significantly improved the prospects of minipig use in pharmaceutical preclinical research. Such advancement in the area of drug development makes this animal model more competitive with other well-characterized large animal models [Citation6].
Declaration of interest
The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
Reviewer disclosures
Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.
Acknowledgments
The opinions or assertions contained herein are the private views of the authors and are not necessarily those of the Uniformed Services University of the Health Sciences or the Department of Defense, USA. We are thankful to Ms. Alana Carpenter and Ms. Sara Nakamura-Peek for editing the manuscript.
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