Emesis in minipigs and relevance in nonclinical safety testing

Emesis (the act of vomiting) and nausea (the sensation of an urge to vomit) are amongst the most common side effects of marketed drugs for human use (Percie du Sert 2012).

Introduction

Emesis (the act of vomiting) and nausea (the sensation of an urge to vomit) are amongst the most common side effects of marketed drugs for human use (Percie du Sert 2012).

Emesis is the forceful oral expulsion of solid and/or liquid gastric contents and, is associated with contraction of the abdominal and chest wall musculature. This is an important physiological defense mechanism against intoxication. Numerous natural and synthetic chemicals can induce emesis and are thus exploited therapeutically as emetics in cases of acute poisoning in animals and humans. On the other side, anti-emetics are used to prevent or reduce drug-induced emesis such as from the widely applied chemotherapy agent cisplatin (Horn 2008; Aung and Soo 2016).

There are important species differences regarding emesis. The ferret is the most recognised model for assessing emesis (Holmes et al 2009). Several commonly used laboratory animal species such as rat, mouse, and rabbit lack a vomiting response (Horn 2008, 2013), where structural differences in the esophagus and diaphragm are considered responsible. Nonrodent species commonly used in nonclinical toxicology studies such as the dog, minipig, and nonhuman primates, i.e. marmoset and macaque monkey can vomit, but these species significantly differ to each other in their sensitivity to respond to an emetic stimulus. In addition, emesis is modifiable and can be conditioned in a number of species, including human. Such differences in emesis sensitivity can be important aspects for nonclinical safety testing. For example, dose-limiting emesis of a drug candidate may prevent the meaningful use of the dog in toxicology testing. After oral dosing, emesis can be a main contributor to highly variable plasma exposure, which may result in challenges to correlate effects to dose and respective exposure levels. Emesis can also lead to secondary clinical symptoms (e.g., abnormal body position and decreased activity), body weight loss, and anatomic side effects such as histopathological changes in the upper gastrointestinal tract, limiting the potential of a particular species to predict human relevant toxicities.

The Beagle dog has been used over decades as the primary nonrodent species in safety testing. However, Beagles are particularly sensitive for the induction of emesis. Even under therapeutic use of the sedative α2-agonist xylazine vomiting can occur in 10%-20% of treated dogs (Lumb and Jones 2007). The minipig, another nonrodent species increasingly used in toxicology, is largely resistant to emesis, although the perception that minipigs do not vomit is mistaken. Nevertheless, the fact that the minipig is significantly less prone to emesis adds to the many other physiological and biological advantages in regulatory safety testing compared to the dog. For example, minipigs tolerate NSAIDs much better and show less / less severe gastrointestinal lesions than dogs; minipigs largely tolerate vitamin D-analogues whilst the dog is hypersensitive; minipigs show less arteriopathy and other cardiovascular lesions, when dogs are often overly sensitive (McAnulty et al. 2012, Schmitt et al. 2015).

There are excellent reviews on the absence of emesis in rodents (Horn et al. 2013) and on emesis in dogs (Elwood et al, 2010). This mini review highlights emesis in minipigs and its relevance for the use of the minipig as a nonrodent species in nonclinical safety testing, mainly as opposed to the dog.

Common causes for minipigs to vomit

There are multiple causes leading to emesis in many species including the pig/minipig. For a high-level overview on emesis in minipigs see for example: https://thepigsite.com/articles/whats-wrong-with-my-pigs-vomiting. Interestingly, pregnancy-induced nausea and vomiting seen in humans during the first trimester (also called morning sickness) is not known for any other species. This unique feature is considered to be related to a particularly broad diet in humans compared to other mammals, and a developmental mechanism that helps the mother avoid substances which could be dangerous to the developing embryo (Flaxman and Sherman 2008).

It is important to reflect and triage the various possible causes for emesis in the minipig to enable conclusions to be drawn on potential test article-induced emesis in nonclinical safety studies. In the following, the main causes for emesis in the minipig/pig are discussed.

Pathogens, diseases, and physiologic conditions

Emesis is a prominent clinical sign of several infections and parasites in pigs/minipigs, including hemagglutinating encephalomyelitis, porcine epidemic diarrhea, colibacillosis (E. coli diarrhea), transmissible gastroenteritis, and swine fever (https://vetmed.iastate.edu/vdpam/FSVD/swine/index-diseases). In contrast, experimental animals such as minipigs are generally purpose-bred, grown and kept under stringent and well-controlled housing conditions. Colonies are regularly health monitored and tested for the absence of a wide range of pathogens according to FELASA guidelines (Berset Convenor et al. 2020).

Organ based diseases such as liver or kidney disease, tumors in the gastrointestinal tract, liver or pancreas, hypoglycaemic shock, constipation and intestinal obstruction as well as some central nervous system or psychiatric conditions such as post-operative pain are also known causes of vomiting in the minipig. Consequently, although rather a rare event, minipigs must be deprived of food for up to 12 hours before anesthesia to prevent vomiting (Alstrup 2000).

Motion-induced emesis has an early evolutionary origin and is present in many animal species including the minipig. Therefore, pigs/minipigs are generally not fed before and during transportation (Bradshaw et al. 1996, Randall and Bradshaw 1998).

Toxicity

Various drugs, in addition to intentionally emetic-inducing drugs (e.g. metoclopramide), can lead to emesis in the minipig. Examples include sedatives and analgesics (e.g., propofol), CNS active drugs (e.g., the dopamine-receptor agonist pramipexole), NSAIDs (e.g., meloxicam), opioids (e.g., apomorphine), and chemotherapeutic medications (e.g. cisplatin). Also nutritional components (e.g., deficiency or excess of vitamin D) or contaminations (e.g. the mycotoxin deoxynivalenol) can lead to emesis in the pig/minipig.

Irradiation

Like in many other species, including the human, whole and partial body irradiation was shown to induce emesis also in the minipig (Kaur et al 2018).

Emesis animal models in Research and Development

Anti-emetic drugs such as NK1 receptor antagonists, which block differing types of emesis (e.g., induced by drugs, motion or vagal stimulation) strongly indicate the presence of a common pathway for emesis (Andrews and Rudd 2004).

Animal emesis models exist in a number of species including ferret, cat, dog, and pig/minipig (Florczyk et al 1982; Andrews and Rudd 2004; Holmes et al 2009; Szelenyi et al 1994; Shim et al. 2014, Kaur et al. 2008). Ferret and dog were historically considered as the ‘gold standard’, likely due to the ease to induce emesis in these species, with a particular sensitivity to conditioning in dogs (Holmes et al 2009). Species differences in the emetic sensitivity to the dopamine receptor agonist apomorphine highlight the high predictive value of both dog and pig for humans (Holmes et al 2009).

Szelenyi et al. (1994) report that the sensitivity to emetogenic anticancer drugs and their susceptibility to currently known antiemetic drugs are comparable in the domestic pig and cancer patients. Retrospective data analysis of anti-cancer drugs revealed that the dog is better than the monkey in predicting vomiting in humans (Schein et al., 1970). Emesis is also used as a key indicator in a neonatal swine model for peanut allergy (Helm et al., 2002). Overall, no animal species is a universal predictor of emetic liability in human and the choice of species should be an informed decision based on the type of compound investigated and the type of emetic mechanism activated (Percie du Sert et al. 2012).

While these animal emesis models have shown utility, the value and use of regulatory safety testing in general to predict the human susceptibility to emesis can be questioned given the high percentage of drugs with emesis as side effect, sometimes limiting their therapeutic value. Dependent on the incidence and severity in toxicity studies emesis can sometimes be considered an adverse effect or indicating target organ toxicity in the CNS and/or the gastrointestinal tract. Overall, there is a good correlation for the occurrence of vomiting in preclinical studies and gastrointestinal adverse drug reactions in clinical studies (Tamaki et al 2013). The minipig in particular may provide relevant results for radiation-induced GI injury due to similar physiological parameters as humans, including transit time and pH, as well as being less prone to emesis than canines (Singh et al. 2016).

The biological basis of species differences for emesis

The exact biological reason why rodents fail to produce either retching or vomiting is unclear. While there are certain anatomical constraints (e.g., reduced muscularity of the diaphragm and long esophagus) the absent brainstem neurological component (i.e., lack of key neural circuits) has been suggested as the most likely reason (Horn et al. 2013).

From an evolutionary viewpoint, dogs are predators with a natural tendency to vomit while pigs are prey animals. Thus, the high sensitivity of dogs to emesis is related to their feeding behavior as carnivores. The preference of some dog breeds, including the Beagle, to consume large meals very rapidly reflects the competitive feeding behavior of its ancestor, the wolf. Also, dogs prefer a diet rich in fat in the attempt to maximize caloric intake whenever food is available, although large amounts of fat-rich diet can lead to digestive problems. As a result, dogs often overeat when offered excess diet and vomit in particular too much fat-rich food consumed too fast (Bradshaw 2006, WALTHAM Centre for Pet Nutrition 2012). Similarly, oral administration of large amounts or volumes of almost any test article can cause a dog to vomit (Gad 2017). A review on emesis in dogs is provided in Elwood et al. 2010. In contrast, the omnivore pig will eat almost anything while foraging for food. Therefore, diet used for minipigs generally has a high fibre content and is specifically designed to ensure that restricted feeding provides the animals with sufficient nutrients (Ellegaard et al. 2010). Overall, its biology and natural behavior makes the minipig much less prone to emesis than the dog. The minipig is consequently a preferred nonrodent species for nonclinical safety testing whenever large amounts of test item formulation need to be dosed, particularly by the oral route.

Nausea is a distinct unpleasant human sensation and thus it has been argued that animal species may not display a similar subjective experience. Yet, other gastrointestinal effects observed in animals such as hypersalivation, loss of appetite, diarrhea and vomiting can be used as indication, preferably together, to predict nausea (Elwood et al. 2010, Parker and Limebeer 2006; Parkinson 2012).

Examples comparing drug-induced emesis in regulatory nonrodent species

Minipigs and cynomolgus monkeys showed comparable emesis induction after oral gavage of a positive allosteric modulator of the metabotropic glutamate receptor 5 (mGlu5), RG7342. In a subsequent human clinical trial, tolerability of RG7342 was indeed shown to be limited by adverse events including nausea and vomiting (Sturm et al. 2018). These results demonstrate the clinical relevance of emesis findings in both minipig and monkey to predict nausea and vomiting in humans. Toxicity studies in the dog were not performed for this project.

A negative allosteric modulator of mGlu5, basimglurant, showed emesis in Beagle dogs and cynomolgus monkeys (internal data). Nausea and vomiting was amongst the observed adverse effects in humans (Quiroz et al. 2016). Toxicity studies in the minipig were not performed for this project. 

Rupniak et al. (2019) reported that the Neurokinin-2 receptor (NK2R) agonist LMN-NKA caused less emesis in minipigs than in dogs after intravenous or subcutaneous administration, concluding the minipig may be a better species for nonclinical safety testing.

Emesis seen after oral administration can be followed up and thus confirmed with intravenous dosing to reach high peak levels often associated with CNS mediated emesis.

Selection of the nonrodent species for compounds with emetogenic potential

To support the development of human pharmaceuticals, toxicity studies in a rodent and a nonrodent species are generally required. The selection of an appropriate species for nonclinical safety testing should primarily consider scientific, practical and ethical factors (Prior et al. 2020). However, comparative pharmacokinetic studies in several nonrodent species are not necessarily performed to assist in the selection of the best suitable species. This is also a reason why the dog is still the routine species in many projects. Nonhuman primates such as the cynomolgus monkey should only be used if unequivocally necessary and once all other possible avenues of potential alternatives have been explored (Schmitt et al. 2015). This species selection paradigm particularly applies to small molecule testing.

Minipigs are generally less prone to emesis than dogs, although both species tend to eat their vomit and, therefore, when not carefully and continuously monitored emesis can be overlooked. Yet, overall the minipig may often allow higher dose-levels to be tested in nonclinical safety studies, thus exploring the full range of toxicity of compounds with emetogenic potential. An additional advantage of such lower sensitivity for emesis is related to reduced issues with variable and/or undefined exposures associated with vomiting shortly after oral administration of a compound. Therefore, emesis can be a limiting factor in the selection of a nonrodent species in toxicology. Overall, the minipig offers several advantages over the dog in nonclinical safety testing, particularly with regards to the gastrointestinal system due to its greater anatomical similarity to humans and a lower sensitivity for emesis (Bode et al, 2010).

Conclusion

Similar to humans and many other species the minipig is able to vomit, yet much less sensitive than the dog. In general, minipigs can be considered human-relevant model for emesis. Based on the totality of factors to be considered for species selection, the minipig is a favorable nonrodent for the safety testing of small molecules with emetogenic potential.

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