Animal Models in Toxicologic Research: Pig

2.1

Breeds

All pigs used in research and agriculture are Sus scrofa. Similar to the dog, certain traits have been selected for creating different breeds. Cross-breeding with wild pigs has led to the development of pigs with smaller stature which are more manageable in a research setting. Interbreeding of domestic or agricultural pigs has allowed selection for a nonpigmented or white skin phenotype in some breeds. Selection for outward visible phenotypes has led to variability in other genetic traits such as background changes being more common in some breeds or differences among breeds in xenobiotic metabolizing enzymes. Because of these differences among breeds, it is important to examine the gene atlas from the specific breed being used if conducting a xenobiotic study ( ).

Minipigs

Several minipig breeds exist worldwide. Some are naturally occurring (e.g., Yucatan and Wuzhishan) and others such as the Göttingen minipig have been bred and selected for attributes such as size and coat color that facilitate their use in biomedical research. The predominant breeds of minipigs used in biomedical research are the Göttingen, Hanford, Sinclair, and Yucatan in North America and Europe ( ; ). In Asia, several different breeds are used including Bama, Clawn, Microminipig, Ohmini, and Wuzhishan ( ). Body weight ranges (fully grown animals) vary among the different breeds ( Figure 20.1 ). For the Yucatan micropig and the Göttingen minipig, the adult body weight range is 35–55 kg, for Sinclair and Hanford minipigs, it is 50–60 kg, and for the Yucatan minipig and the Hormel minipig, the weight range is 70–90 kg. Presently, the Göttingen is the only minipig breed that is bred and available worldwide for research purposes. The other minipig breeds may be available from local breeders or via shipping. All minipigs obtained from vendors have a defined health status. Vendors regularly test for pathogens to identify if any are present in their colonies and to confirm negative status for selected pathogens. Animals with defined flora are preferred for product registration studies, especially those that must be conducted according to GLP (Good Laboratory Practice) principles, to decrease confounding findings.

2.2

Basic Biological Characteristics

The lifespan of swine ranges from 15 to 25 years depending on the breed. Litter sizes also vary from 4 to 20 piglets, with minipigs having smaller litters (e.g., four to nine offspring) ( Table 20.1 ). Many biomedical studies use pigs of different ages to correlate with different human life stages ( Table 20.2 ).

Part of the pig's natural behavior is rooting. This may lead to foreign material or small particles being inhaled resulting in inflammation of the airways. Pigs have been reported to have nutritional deficiencies in the research setting, especially conventional breeds ( ). Conventional breed vendors may mix their own feed on the farm, and it is not tested as rigorously as the minipig vendors pelleted feed. This has resulted in Vitamin E and selenium deficiencies in at least one study ( ). Minipigs have suffered from water deprivation (salt toxicity) shortly after shipping ( ). Pigs used for research are prone to all of the diseases that occur in conventional pigs, and these diseases are occasionally seen in research settings.

Sexual maturity occurs at 3–7 months depending on the breed, with minipig breeds maturing at approximately 4–6 months. Maturity in pigs is defined by some as the age at which the first estrus or mature sperm is noted, whereas others refer to the age of physeal closure ( ). As such, it is difficult to designate an age of the onset of maturity without an agreed upon definition; however, most regulatory agencies require sexual maturity for safety studies, not physeal closure or skeletal maturity. Physeal closure happens sooner in smaller breeds ( ).

There are a few anatomical differences in the pig compared to other species. Pigs have a laryngeal diverticulum which may make intubation or gavage more difficult. They have a fibromuscular protrusion at the pyloric outflow of the stomach, known as the torus pyloricus. The colon is organized into centripetal coils in the pig, an anatomical but not functional difference ( ).

2.3

Husbandry Considerations

Not only do pigs grow very quickly, there are a wide range of breeds, all with different adult weights. This necessitates that the guide for animal care and use is consulted to ensure correct area needs are met when housing the animals ( ). Housing guidelines may vary depending on the country in which studies are conducted. There are different guidelines that can vary greatly for housing of pigs depending on whether they are being used for biomedical or agricultural research ( ).

Pigs have a tendency to root, and this rooting behavior often results in spillage of water and food. For this reason, food and water dishes are often firmly attached to the sides or floor of caging and water can be provided via an automatic system. There is currently no consensus on the best type of caging for pigs, as most facilities require flexibility for different species. Pigs require flooring that allows secure footing and it is helpful if the flooring also helps in keeping hooves from becoming overgrown.

Pigs are intelligent and can be trained using clickers and often are amenable to extended periods in a sling. Placing a pig in a sling allows prolonged humane restraint without the need for anesthesia.

Pigs are social animals and, if not group housed due to study requirements, should be allowed to see or touch snouts with neighbor animals through cage walls. Pigs can be destructive, and enrichment should be sturdy enough to not be broken or chewed into smaller pieces.

3.1

Study Design Considerations

Prior to study initiation, the animals should be acclimated at the facility for a minimum of 7–10 days, during which time they are observed clinically in order to evaluate their general health status prior to start of dosing. Baseline evaluations such as collection of blood for clinical pathological analyses may also be taken, as well as electrocardiograms to examine heart function. Training for study-related procedures, such as dermal dosing, should be started during the acclimation period to both facilitate a successful start of treatment and help familiarize the animals with their handlers by daily close contact.

Minipigs are typically 4–5 months of age at start of dosing on toxicity studies, which is considered the age of sexual maturity in minipigs. Use of sexually mature animals is essential for any nonclinical toxicity studies evaluating male reproduction (see Male Reproductive Tract, Vol 4, Chap 9). Certain study types require older (and especially larger) animals at the start of treatment. This is the case for continuous infusion studies, where the ambulatory infusion equipment is of such a size and weight that the animal must be large enough to handle the apparatus. Larger animals are also needed for embryo–fetal studies, where sows with more fully mature reproductive systems are needed, for wound healing studies where larger body surfaces will enable establishment of sufficient wounds without having to use more animals, and for certain studies investigating local tolerance. Younger pigs are required for juvenile toxicity studies.

There are some differences in the metabolizing enzymes between humans and pigs, between sexes, and even among different pig breeds. Many studies looking at sex differences in xenobiotic metabolizing enzymes (e.g., cytochrome (CYP) P540s, UGT, and SULT) have shown that once castrated, the differences between the sexes decrease, suggesting that the levels are dependent on testosterone. Different breeds have been shown to have different testosterone levels and subsequent differences in CYP levels ( ; ; ; ; ; ; ). Age-related changes in these enzymes are commonly reported as well, specifically that CYP activity increases with age in juveniles, but then decreases into adulthood. It appears that these changes coincide with puberty (increased CYP enzyme activity) and adulthood (decreased activity relative to puberty but increased from prepuberty). Due to these variables, it is recommended that there be standardization of breed, sex, and age of animals tested.

If the drug being tested is metabolized by aldehyde oxidase, N -acetyltransferase, or CYP2C9, the minipig should be the preferred large animal model as activity of these enzymes in the pig most closely mimics those in humans compared to other nonclinical large animal model options (e.g., dog, nonhuman primate [NHP]) ( ). The enzyme 3-phospho-adenosyl-5-phosphosulphate sulphotransferase is not effective in pigs ( ). As with selecting any relevant animal species for toxicity studies, it is best to determine if the test article being administered is metabolized in the pig if it is being considered.

According to guideline requirements for nonclinical toxicity studies across all species, systemic exposure [i.e., the pharmacokinetic profile] should be evaluated as well as other parameters (e.g., clinical observations, food consumption, body and organ weights, clinical chemistry and hematology, urinalysis, and macroscopic and microscopic pathology evaluation) ( ). In addition, ophthalmologic and electrocardiographic evaluation may be included, as well as more specialized evaluations such as dermatologic scoring or cytokine responses. Specific protocols for evaluating these parameters should be included in the study design. All of these procedures can easily be performed in minipigs under each of the dosing conditions described in this chapter.

For detailed evaluation of specific clinical parameters, the minipig may serve as an excellent test species. For example, in evaluation of cardiovascular function in specific safety pharmacology studies, use of implanted devices to measure heart rate, body temp, movements, and other parameters can greatly facilitate collection of key electrocardiographic data ( ). In such studies, electrodes may be implanted subcutaneously in the chest and detailed electrocardiographic recordings generated on a continuous basis or at designated intervals, without handling the animals. In addition, blood pressure may be measured using a transducer implanted into a branch of the femoral artery.

For each parameter measured, it is important to build a historical control database for comparison with information from current and future studies. This database is essential as group sizes in nonrodent studies are typically small (e.g., 4–5/sex/group). Historic data provide support for evaluation and interpretation of data and additional assurance of data quality by showing consistency among groups and across time. While there are several retrospective review papers that discuss findings in control minipigs ( ; ; ), it is still important that each facility has an ongoing internal database developed for reference purposes.

3.2

Oral Toxicity Studies

Anatomically, the upper part of the gastrointestinal tract (e.g., esophagus, stomach, and small intestine) of pigs is very similar to the same regions in humans ( ; ). Pigs have similar gastric cell types, intestinal villous structure, and intestinal secretions as do humans ( ). Furthermore, the changes in pH along the small intestine, as well as the transit time in the small intestine, are very similar in pigs when compared to humans. In contrast, the porcine large intestine is arranged in a series of coils, while in humans the large intestine is organized as a linear organ with several sharp angles. Despite these physical differences, the functions of the human and pig large intestine are comparable. These facts, combined with a high degree of similarity in metabolism and in the microbiome, make the minipig an ideal species for testing orally dosed drugs ( ).

Oral dosing of liquids (e.g., solutions or suspensions) by gavage in the minipig is a straightforward procedure that can easily be handled by two trained technicians using a swine dosing chair or sling. Volumes up to 20 mL/kg (single dose) or 10 mL/kg (multiple doses) can be administered using this procedure.

Like oral gavage dosing, administration of capsules or tablets can be accomplished with the animal in an upright position in a swine dosing chair or restrained in a sling. Alternatively, tablets can be administered hidden in a small portion of moist diet. This is also possible for capsules; however, there is a high risk that the animals will chew the capsules, resulting in an undesirable premature release of the entire dose. To minimize this risk, capsules can be placed at the caudal part of the tongue using water to help the animal to swallow the capsule.

3.3

Intravenous Toxicity Studies

Minipigs are also suitable as a nonrodent species for test articles intended for intravenous use. Intravenous dosing via both bolus and infusion is feasible in minipigs. However, access to peripheral veins is limited; the auricular (external ear) veins can be used for single intravenous bolus injections but are not recommended for repeated injections or continuous infusion. This lack of available reusable superficial vessels necessitates surgical implantation of catheters to be used when repeated intravenous dosing is required.

For repeated intravenous bolus injections or for infusion of short durations (maximum 30 min), a vascular access port (VAP) can be used. For intravenous infusions greater than 30 min, a cannula/catheter is recommended to limit irritation. The VAP is implanted subcutaneously and connected to the bloodstream via a catheter implanted in a major blood vessel. In principle, the VAP can be implanted at almost any site. However, for practical reasons it is best implanted in the region of the neck or cranial aspect of the back, with the catheter inserted into the external jugular vein allowing easy dosing in the conscious animal ( ). Dosing is performed by locating the VAP externally (accomplished by palpation of the VAP through the skin), followed by skin puncture and puncture of the silicone membrane of the VAP, through which there is direct intravenous access via the catheter. For this purpose, a hypodermic needle is used. A VAP can be implanted in a minipig beginning at 7 days after birth. Growth of the animals should be taken into account when implanting the catheters and the VAPs into younger animals, or for any study during which the animal may grow.

Due to the weight and size of the equipment used (the combined weight of the ambulatory infusion pump plus counterbalance is approximately 2 kg), animals used for continuous infusion studies should be at least 10 kg. In order to allow for free movement of the animals within their pen, the preference is to use ambulatory infusion pumps, which can be carried by the animals in specially designed jackets. However, a tethered system in which a wall or pen-top mounted pump not carried by the animal delivers the dosing solution via a long overhead tube can also be considered; in this conformation, the animals are often confined to a relatively small area for the time of dosing to prevent detachment or kinks in the catheter that might disrupt the infusion. Various infusion pumps are commercially available, and it is important to consider size and weight (including the filled infusion reservoir) when choosing equipment. The infusion pump is connected to a central vein via an implanted catheter.

Using the correct techniques, patency of catheters can be maintained for several weeks without any problems, thereby enabling continuous infusion studies of approximately 4 weeks duration.

3.4

Subcutaneous Dosing Toxicity Studies

The pig is a relevant nonrodent species to be considered for test articles intended for subcutaneous administration. The sparse hair coat and lack of pigmentation of many breeds facilitate clinical evaluation needed to detect potential reactions after dosing. Although there is firm dermal attachment to the underlying tissues, the dose volume that can be applied is generally not different than that which is applied to dogs in single or repeated dose toxicity studies. Typical dose volume that can be delivered is 0.5 mL/kg and may go as high as 2.0 mL/kg (maximum of 20 mL per site not to exceed 2 sites per animal).

Subcutaneous dosing is best performed in the lateral aspect of the neck. Due to potential local reactions caused by the injection procedure, and in some cases also by the test article, daily subcutaneous injections are usually performed at different sites, alternating between both sides of the neck. The injection sites to be used during a study can be indicated by tattooing, making it possible to rotate the injections according to a planned scheme as well as identify all sites to be collected at necropsy for subsequent histopathological evaluation.

3.5

Dermal Toxicity Studies

The similarity between the skin of humans and pigs makes the pig an ideal model for use in nonclinical dermal studies ( ). Features of swine skin that are similar to that of humans include sparse hair cover, a firm attachment to underlying structures, relatively thick epidermis, dermal to epidermal thickness ratios, similar dermal collagen and elastin content, formation of rete ridges, and variance in cutaneous pigment ( ). Other species used for dermal toxicity testing do not have all of these features, particularly including dermal to epidermal thickness ratios, sparse hair, and a firm dermal to subcutis to underlying muscle attachment. Additionally, pigs tolerate occlusive bandages well and have limited ability to manipulate, remove, or ingest material applied dorsally. There are some key differences between the pig and human skin, including a lack of eccrine sweat glands across much of the skin of pigs, decreased dermal and follicular vascularity, and variances in physiochemical response in vascular endothelium, but these are outweighed by the similarities ( ). Indeed, for pharmaceutical products intended for dermal application, it is difficult to justify using any nonrodent species other than the pig for these studies.

As a general practice for dermal toxicity studies in pigs, the test item is applied to an area corresponding to approximately 10% of the total body surface area. Normally, the skin on the back is used, as this area is protected from self-inflicted injuries and oral ingestion of test material and is easy to cover with protective bandages. Furthermore, this area is not normally subjected to external contamination with urine and feces. Doses up to 1 gram per kilogram body weight can be applied on the area described. Certainly, administration in this area bears certain limitations, as this is one of the areas of the thickest dermis and has more dense and coarse hair than the ventrum or axillae ( ). However, it is still more readily translatable than a species with a dense haircoat such as the dog, rabbit, or even NHP, in which the dermis is much thinner and less similar to humans.

Selection of an appropriate intraspecimen control site for histological evaluation is an important aspect of study design, particularly as there is pronounced variance in histologic appearance and character of the skin from site to site within a given animal ( ). The most appropriate control site is one that shares location and/or histologic features with the test article–dosed site. For example, the dermis on the flanks and shoulders of the minipig is significantly thicker than that of the ventral, inguinal, or axillary regions and even thicker than that of the mid or lower back. Additionally, the density of hair follicles, glands, and other adnexal structures varies significantly from one site to another. The epidermis overall is approximately the same thickness across most regions of minipig skin, but variance of other features, such as collagen and elastin composition or distribution of resident leukocytes, may also be a factor in evaluation of potential test article–related effects ( Figure 20.2 ).

During dosing, the treated area is usually covered by a gauze dressing, held by a net-like bandage covering the thorax and abdomen. It is of great importance to allow a daily treatment/bandage-free period, as the risk of dermal irritation and infections otherwise increases due to the moist, warm, semiocclusive environment. As a standard practice, a daily dosing period of 6 h is often used, although other durations are possible. At the termination of the dosing period, any occlusive dressing and remaining test substance are removed, and the treated area of skin is washed or swabbed to remove residual material.

3.6

Other Routes of Dose Administration

In addition to the dosing procedures described previously, other nontraditional routes of dose administration are also possible in the minipig. The dosing procedures described below should not be seen as a complete list of possible routes in the minipig, but as examples of nonroutine options which may be utilized.

Vaginal dosing may be needed for local tolerance testing of liquid or gel formulations of test articles intended for intravaginal use in humans. Alternatively, medical devices intended for intravaginal use may also be tested. Use of sexually mature animals will ensure that any potential effects on cyclic changes will be able to be evaluated. Care should be taken during dosing procedures not to induce iatrogenic damage of the vaginal mucosa. Aseptic principles should be applied at dosing in order to avoid procedure-related infections. When performing intravaginal dosing, physical restraints such as slings, in conjunction with preconditioning and training, may serve to limit potential iatrogenic effects, or animals may be sedated or anesthetized to provide chemical immobilization.

Repeated intravesicular dosing into the urinary bladder or even into the renal pelvis can be performed in female minipigs via a urinary catheter inserted through the urethra under surgical anesthesia. This route is not possible in males due to the anatomical course of the urethra. Intravesicular dosing can also be performed in both males and females by use of a pig-tail catheter introduced surgically into the bladder. The catheter may either be exteriorized through the abdominal wall or attached to an Access Port (AP), which is typically implanted on the back. Similarly, direct evaluation of intestinal absorption of liquid formulations or suspensions can be performed via a surgically implanted duodenal catheter. This route is used to avoid the impact of the stomach secretions on the test article. Again, the catheter can either be exteriorized through the abdominal wall or attached to a subcutaneous AP.

Topical, intravitreal, and subretinal dosing has been performed in the minipig eye in nonclinical safety assessment of pharmaceuticals. Diagnostic techniques include electroretinograms, optical coherence tomography, and histopathology.

3.7