Why the Ferret?

The earliest scientific publications on vomiting involved dogs and cats [12,13] and both species continued to dominate research for most of the twentieth century [8,14,15] although the dog predominated. They were joined by nonhuman primates after the Second World War particularly for studies of motion sickness (mainly the squirrel monkey, Saimiri sciureus, see Ref. 16 for review), possibly stimulated by the need to understand the potential impact of space flight on humans. Nonhuman primates also became used for studies of the emetic effect of radiation (e.g., Ref. 17), prompted by a need to understand the effects of ionizing radiation including in the setting of treatment for cancer [18]. Although some pharmacological studies of emesis in the 1980s used the marmoset (Callithrix jacchus, e.g., Ref. 19) and some in the 1990s used the piglet [20], neither have been widely adopted for such studies.

The predominance of the dog reflects another comment of Claude Bernard [1]: “Even to-day, many people choose dogs for experiments, not only because it is easier to procure this animal, but also because they think that experiments performed on dogs can more properly be applied to man than those performed on frogs.” In addition, it was quickly recognized that vomiting could be evoked readily in dogs by a diverse range of stimuli capable of evoking nausea and vomiting in humans [8]. The tractability of dogs as a laboratory species probably contributed to its widespread and continued use, prompted particularly by the acceptability of data from the dog by regulators in support of drug licensing. The utility of the dog was also enhanced by the development or adaptation of surgical techniques required for the study of the physiology and pharmacology of vomiting (e.g., abdominal vagotomy, chronic implantation of vascular catheters, and electrodes/transducers in the gut muscle; e.g., see Refs. 21 and 22).

To summarize, the dog, cat, and nonhuman primate were all regarded as acceptable species in which to study emesis in the 1970s. Why did studies on the ferret emerge not only from researchers in academe but also in the pharmaceutical industry? The case of the ferret is particularly interesting as research on the ferret over the last 30 years played a key role in the introduction of two new antiemetic drugs to the clinical setting. Some aspects of this history are to be reviewed later and provide an insight into why a novel species is studied and how it becomes acceptable to the research, funding, and regulatory communities. It should be noted that the dates of publication of studies do not necessarily reflect the dates of the original studies.

What Led to an Interest in Animal Models of Nausea and Vomiting in the Early 1980s and Adoption of the Ferret as a Model?

The main driver was the unmet clinical need to alleviate the nausea and vomiting associated with both radio- and chemotherapeutic treatment of cancer. Of particular concern were agents such as cisplatin, for which the high-dose incidence of emesis was close to 100% in patients not receiving antiemetic treatment. The initial demonstration of the anticancer effect of cisplatin was published in mice (a species unable to vomit) in 1969 [23], and the first clinical studies took place in the early 1970s (see Ref. 24 for some of the history of the use of cisplatin in cancer). The introduction of highly emetic but potentially curative therapies stimulated controlled clinical trials of current antiemetics (see Ref. 25 for an overview of the area prior to 5-hydroxytryptamine [5-HT₃] receptor antagonists) and stimulated interest in understanding the mechanism by which the agents induced emesis. This included not only research to identify novel antiemetics but also research to identify less emetic analogs, particularly of cisplatin [26]. Broadly speaking, these areas of research required animal models showing an emetic response to cytotoxic drugs (particularly cisplatin) and ionizing radiation. Although studies in the dog, cat, and nonhuman primate continued (primarily in the United States and Japan), the ferret rapidly became an accepted model and its use sustained for a number of years because of the translation from ferret to human in the antiemetic activity of the 5-HT₃ and neurokinin 1 (NK₁) receptor antagonists. The drivers for the adoption of the ferret are discussed in detail later but include relative cost, size, and availability (depending on country where studied) in comparison with the then more established models (dog, cat, and nonhuman primate). The scientific advantages and disadvantages of the ferret model could, of course, be assessed only once a sufficient body of data became available to allow comparison with other models. Although in the early 1980s, the ferret was a well-established species for the study of influenza, the hypothalamic pituitary portal system, and the visual system, in the broad area of gastrointestinal physiology, there was resistance and, in the experience of one of the authors (PLRA), a frequent initial comment from reviewers was: “Why use the ferret rather than cat or dog?”

In the 1970s, metoclopramide was a relatively commonly used antiemetic agent but with limited efficacy against highly emetic anticancer chemotherapy and radiotherapy at the doses normally used. However, Gralla and colleagues [27] showed that “high doses” of metoclopramide were effective although there were dystonic reactions. This observation prompted interest from a group led by Dr. Gareth Sanger at Beecham Research Laboratories in the United Kingdom. They were dissecting the pharmacology of metoclopramide, which, at that time, was only considered to be a dopamine D₂ receptor antagonist. Their preclinical research demonstrated that at high-dose metoclopramide was also an antagonist at the 5-HTM receptor, now known as the 5-HT₃ receptor, and this led them to ask whether this pharmacological effect could be responsible for the antiemetic effect of high-dose metoclopramide [28]. Other companies and individuals (e.g., John Fozard at Merell-Dow, France and Brian Richardson at Sandoz, Switzerland) had been studying the pharmacology of the neuronal 5-hydroxytryptamine (5-HT) receptor (5-HTM/5-HT₃) with a view to treating migraine and pain and had developed selective 5-HT₃ receptor antagonists (MDL7222 and ICS-205930). MDL7222 was used by Miner and Sanger [29] and ICS-205930 by Costall et al. [30] in ferrets to demonstrate that a 5-HT₃ receptor antagonist could block cisplatin-induced emesis. These initial observations in ferrets were pivotal in the development of the first 5-HT₃ receptor antagonists shown to be efficacious in the clinic against chemotherapy-induced emesis (BRL43694, Granisetron; GR38032F, Ondansetron; ICS -205930, Tropisetron). In addition, these initial results showing translation to the clinic further encouraged research using ferrets (see section ” Identifying Antiemetic Efficacy” for detailed analysis).

The Sensitivity to Key Emetic Challenges and Antiemetic Agents Was Rapidly Established

An important aspect in rapidly establishing ferrets as an “alternative” to dogs and cats was the early demonstration that it responded to a diverse range of emetic stimuli of relevance to clinical problems at the time. Within 10 years of the initial full paper in 1982 by Florczyk and colleagues [31], ferrets had been demonstrated to have an emetic response to a wide range of substances. These fall broadly into three groups.

Substances and treatments known to induce nausea and vomiting in the clinic. In this category, cytotoxic drugs were of particular interest, with responses to cisplatin (including structural analogs), Adriamycin, and cyclophosphamide [26,31]; chloromethane, emetine, and trimelamol [32]. The ferret was also demonstrated to be particularly sensitive to whole body X-irradiation with several groups publishing responses to radiation using different dose protocols [33–35]. It was subsequently demonstrated that emesis could also be induced by neutron and proton radiation [36]. The ferret radiation model has recently been used to investigate potential radioprotective agents [37].
“Standard” emetic challenges. These were stimuli routinely used at the time in cats and dogs to test substances for antiemetic activity. Included in this category are apomorphine [26,38], morphine [39,40], intragastric copper sulfate, and ipecacuanha [41].
Substances with emetic liability. A number of substances were studied because they were representative of novel chemical entities (NCEs) with the potential to treat disease but where nausea and vomiting were potential dose-limiting toxicities. An early example in the public domain was the xanthine bronchodilators such as theophylline [42], of current interest because of the therapeutic potential of phosphodiesterase inhibitors in the treatment of asthma (e.g., Ref. 43).

Of equal significance was the demonstration that the emetic response to a number of standard emetic challenges was reduced or blocked by antiemetics already known to have efficacy in humans or in dogs. In this category, of particular significance in establishing ferrets as a potential model for investigating novel antiemetic agents was the demonstration that the response to the dopamine receptor agonist apomorphine was blocked by domperidone and metoclopramide and that the latter (particularly at relatively high doses) could also diminish or block the emetic response to cisplatin and cyclophosphamide [29,44–47]. Also of relevance at the time was that the opioid agonist butorphanol reduced the emetic response to cisplatin in both dogs and ferrets [48].

Pharmaceutical Companies Were Early Adopters of the Model

A crucial aspect in establishing the ferret as an acceptable species for the study of emesis and antiemetic agents was its rapid adoption by the pharmaceutical industry in several countries. This occurred either by undertaking studies in-house and/or in close collaboration with academics already undertaking research using ferrets. It is not possible to ascertain how many studies were performed by contract research organizations but in-house and collaborative academic studies appear to be responsible for the majority of publications in the 1980s. Figure 31.1 shows the rapid rise in the number of papers studying emesis in the ferret between 1982 and 1992. Pharmaceutical companies publishing within 10 years of the original Florczyk et al. [31] paper, either alone or with an academic partner, are included in Table 31.1 in alphabetical order. Over the last 20 years, there has been a sustained flow of published studies utilizing variants of the ferret emesis model from a number of pharmaceutical companies (see Table 31.2). It is important to point out that a number of the publications were in collaboration with academic research groups and, in addition, a number of the publications with only academic authors give acknowledgments to companies for gifts of compounds and for research funding. The main academic research groups publishing on various aspects of emesis in ferrets in the 1980s were based in the United Kingdom (Bradford University (e.g., Ref. 30); London University [49], and Oxford University [50]), the United States of America (Armed Forces Radiobiology Research Institute [35]), and Canada (Calgary [51])). These were joined in the 1990s by groups in Japan (Hokkaido [52]) and Hong Kong [53,54]. A series of international meetings over the critical decade of intense research using the ferret model, between 1984 and 1994, focused primarily on the basic science aspects of nausea and vomiting. Meetings were held in Oxford in 1984 [55], Ottawa in 1988 [56], Marseille in 1992 [57], and Oxford in 1994 [58] and served to promote rapid exchange of information about the model between the major groups and to provide a forum in which others could learn about the model. These meetings were only made possible because of the interest of the pharmaceutical industry at the time in the area of anticancer therapy-induced emesis and the identification of novel antiemetic agents using the ferret (5-HT₃ receptor antagonists, e.g., Ref. 29; and NK₁ receptor antagonists, e.g., Ref. 59).

c31-fig-0001 — Fig. 31.1. Number of articles in the area of emesis research published per year per species.
The graph was constructed based on Pubmed searches using the species name combined with the MeSH terms: emesis, vomiting, retching, and nausea. (1) First publication reporting a ferret model of emesis [31]. (2) First publication reporting the efficacy of a 5-HT₃ receptor antagonist in the ferret model of cisplatin-induced emesis [29]. (3) First FDA approval for a 5-HT₃ receptor antagonist: Zofran® (ondansetron) in 1991. (4) First publication reporting the efficacy of a NK₁ receptor antagonist in the ferret model of cisplatin-induced emesis [234]. (5) First publication reporting the efficacy of ondansetron against chemotherapy-induced nausea and vomiting in the clinic [235]. (6) First publication reporting the efficacy of aprepitant against chemotherapy-induced nausea and vomiting in the clinic [236]. (7) FDA approval for Emend® (aprepitant) in 2003.

Table 31.1. Pharmaceutical Companies Publishing within 10 years of the Original Full Research Paper Publication of the Ferret Model [31]

Company	Country	Reference
Beecham Pharmaceuticals	United Kingdom	29
Boehringer Ingelheim	Italy	132
Bristol Laboratories	United States	31
Bristol-Myers Company	United States	239
Dainippon Pharmaceutical Co., Ltd.	Japan	240
Glaxo Group Research	United Kingdom	46
NOVA Pharmaceutical Corporation	United States	42
Laboratorio Almirall	Spain	241
Novo Nordisk	Denmark	242
A.H. Robins Research Laboratories	United States	243
Vita Laboratories	Spain	244
Yamanouchi Pharmaceutical Co., Ltd.	Japan	245
Yoshitomi Pharmaceutical Industries, Ltd.	Japan	246

Table 31.2. Pharmaceutical Companies that Published Studies Utilizing Variants of the Ferret Emesis Model

Company	Country	Reference
Abbott Laboratories	United States	145,201,247
Astellas Pharam Inc.	Japan	248,249
Astra Hassle	Sweden	212
Chiroscience Ltd.	United Kingdom	250
Fujisawa Pharmaceutical Co., Ltd	Japan	251,252
Kanebo Ltd.	Japan	253
Lexicon Pharmaceutical Inc.	United States	254
Merck Sharp and Dohme	United Kingdom	59
Merck Frosst Canada Inc.	Canada	199,255,256
Mitsubishi Tanabe Pharma Corporation	Japan	257
Nastech Pharmaceutical Company Inc.	United States	258
Paradigm Therapeutics Ltd.	United Kingdom	259
Pfizer Inc.	United States	85
Synthelabo Recherche	France	260
Takeda Cambridge Ltd.	United Kingdom	261
Tsumura & Co.	Japan	262

Techniques

For the ferret to be a viable model, it was necessary that techniques used in previous studies of cats and dogs could be applied to this species. A major barrier in ferrets was the lack of a convenient superficial vein for drug administration in animals which may be difficult to handle. Prior to publishing the first paper on the emetic effects of cisplatin, Florczyk et al. [7] published a method for chronic jugular catheterization in ferrets, and this was used by Davis [50] in a modified form. This method requires surgery under general anesthesia and implantation of an indwelling valve, so postoperative analgesia and antibiotics are required. The valve requires regular flushing to maintain patency but this and drug administration do not require sedation or anesthesia and is well tolerated. This method was rapidly adopted although some researchers (e.g., Ref. 60) anesthetized their animals for drug administration (see section “Route of Administration and Anesthesia”).

The classical studies of Borison and Wang [8] implicated the abdominal visceral nerves and the area postrema in mediating the emetic response to ingested and systemic agents, respectively. Techniques for abdominal vagotomy and section of the greater splanchnic nerves as well as formation of a gastric fistula were available largely because of the studies of gastric physiology and the ferret tolerated these procedures well. The surgery of the abdominal vagus was facilitated by the broadly similar arrangement of the major vagal trunks and branches to man [61], and the presence of single dorsal and ventral trunks clearly visible at the level of the diaphragm (Fig. 31.2). Techniques for ablation of the area postrema were also developed (Fig. 31.3), facilitated by a knowledge of the surgical approach used in studies recording from the brainstem and application of drugs to the brainstem of the anesthetized ferret [62,63]. Techniques for the administration of drugs into the third ventricle, developed for studies of reproductive behavior in ferrets [64], were adapted for studies of emesis to investigate central effects of peptides (e.g., ghrelin [65]).

c31-fig-0002 — Fig. 31.2. The distribution of the ventral vagal trunk in ferrets.
Reproduced with permission from Mackay and Andrews [61]. Scale bar = 1 cm. (1) ventral vagal trunk, (2) hepatic division of the vagal trunk, (3) hepatic branch of hepatic division, (4) pyloric branch of hepatic division, (5) gastric division of ventral vagal trunk, (6) ventral nerve of Latarjet, (7) dorsal vagal trunk, (8) celiac division of dorsal vagal trunk, (9) gastric division of dorsal vagal trunk, (10) dorsal nerve of Latarjet, (11) spleen, (12) stomach, and (13) liver, (14) diaphragm.

c31-fig-0003 — Fig. 31.3. Scanning electron micrographs (SEMs) and coronal hemisections of the dorsal brainstem at the level of the area postrema in a sham-operated (A, C, E) and an APX ferret (B, D, F).
Reproduced with permission from Percie du Sert et al. [237]. A dorsal view of the AP is shown in the panel on the left (A and B) and the middle panel (C and D) shows a detailed view of the caudal (central) part of the AP and fourth ventricle viewed obliquely. The black double-strike arrows are pointing toward the area postrema; note that the area postrema is intact in the sham-operated ferret (A and C) and the caudal (central) part of the AP is ablated in the APX ferret (B and D); rostral parts of the AP (the “wings”) are present and appear to be intact. Histological sections (E and F) were taken at the level of the AP “wings,” note that prior SEM processing affected the quality of the histology sections. ML, midline; CC, central canal; IV, floor of the fourth ventricle; AP, area postrema.

Relative Size and Cost

For testing NCEs in the pharmaceutical industry, the size of the animal may be an issue because of the amount of novel compound that may be needed to establish in vivo efficacy at an early stage in preclinical development. This was given by Florczyk et al. [31] (1984) working at Bristol Laboratories, Syracuse, New York, USA, as a reason for investigating the potential of the ferret as a model for testing emetic effects of the anticancer drug cisplatin (and its analogs) and potential antiemetic agents. The requirement for less novel compound was also used as one of the arguments for the use of shrews (S. murinus and Cryptotis parva) as species for the study of emesis [9,11]. Florczyk et al. [31] also pointed out that less housing space was needed, and the purchase cost was less than for dogs and cats and that ferrets were more readily available from commercial suppliers.

The size of the ferret was also a factor facilitating surgical investigation of pathways. It is large enough not to require microsurgical techniques for most procedures (e.g., abdominal vagotomy) and its size in relation to dogs also facilitated surgery by a single investigator. Additionally, in contrast to the dog and nonhuman primate, the smaller size of the ferret also simplified the procedure for exposure of the ferret to total body irradiation (to be shown later). A related factor was that although the ferret was a novel species for emetic studies, it was quite widely used in other research areas (see other chapters in this volume) and so could be obtained readily from established breeders.

The Ferret Is a Carnivore and the Digestive Tract Has Morphological Similarities to Humans

The ability to vomit is widely distributed across mammalian orders with the notable exception of the Rodentia and Lagomorpha [66,67] (Fig. 31.4). The widespread acceptability of dogs and cats as species for the study of emesis meant that it was more likely that other species for study would come from the order Carnivora. In addition, dogs and cats were commonly used as species to study the physiology of the gastrointestinal tract in view of the broad morphological similarity (but not identity) to that in humans. Of particular significance was the lack of an aglandular fore stomach, present in rodents but not carnivores or humans, and the presence of basal acid secretion as occurs in humans. These morphological and functional similarities to humans and to other accepted species will have further suggested that the ferret could perhaps “substitute” for cats and dogs in studies of digestive tract physiology including vomiting. The 1970s and 1980s saw a period when a number of papers were published from both UK- and USA-based authors establishing the ferret as a species for the study of the digestive tract. The studies in the United States focused primarily on gastric acid secretion probably prompted by continued interest in peptic ulceration (e.g., Refs. [68–70]), whereas those in the United Kingdom focused on the neural control of motility (e.g., Refs. 62 and 71–73). In both types of investigation, the ability of the ferret to retch and vomit was noted. For example, in the gastric acid secretion studies in conscious ferrets with a chronic gastric fistula, Basso and Passaro [68] reported that high doses of calcium ions (60 mg/kg, i.p.) induced vomiting. Electrical stimulation of thoracic and abdominal vagal afferents in urethane-anesthetized animals was also shown to be capable of inducing retching [74]. Vomiting in conscious ferrets was also noted incidentally in a 1983 study as a consequence of treatment with jackbean urease-producing hyperammonemia [75].

c31-fig-0004 — Fig. 31.4. Phylogenetic tree showing the presence or absence of the ability to vomit in members of the 11 major mammalian orders, representing ∼98% of mammalian species, based on key reports in the published literature.
Reproduced with permission from Sanger et al. [66]. Blue circle: functional motilin system; red circle: functional ghrelin system. Red cross: point at which it is proposed that the vomiting reflex was lost; blue cross: point at which a functional motilin system was lost. Shaded box indicates orders and species in which the vomiting reflex is absent. The arrow next to the rabbit silhouette indicates that coprophagia is a normal part of digestive behavior of this animal.

The Ferret Was Used to Study Systems Allied to Emesis

Although emesis is the focus of this chapter, researches on the physiology and pharmacology of body systems related to emesis also provided data to compliment understanding of emesis. These include the following.

Anatomical studies of the brainstem. The central projections of the vagal afferents and the area postrema together with the projections of the key brainstem nuclei (e.g., nucleus tractus solitarius) to the hypothalamus and amygdala have been studied in detail [76–79]. The central connectivity of the vagal afferents and the area postrema has also been investigated using Fos immunohistochemistry [80–82]. A number of related studies have shown the distribution of receptors and neurotransmitters in brainstem nuclei involved in emesis. These include cannabinoid 1 receptor and fatty acid amide hydrolase [83], TRPV1 receptor [84], substance P binding and displacement by NK₁ receptor antagonists [85,86], 5-HT₃ receptor [87], acetylcholine and muscarinic₁ receptors [88], calcitonin gene-related peptide-, galanin-, neuropeptide-Y-, neutotensin-, somatostatin-immunoreactivity [79]; catecholamine, GLP-1, oxytocin, vasopressin, and corticotrophin releasing factor [89].
There have been a limited number of neurophysiological studies of the dorsal brainstem in ferrets in vivo [62] and using brainstem slice preparations to record from the nucleus tractus solitarius [90] and the area postrema [91].
Physiological studies of the vagus. The in vitro grease gap preparation of the vagus used to study the pharmacology of the receptors located on the vagus was adapted from the rat (e.g., Ref. 92) to the ferret and was used to characterize 5-HT₃ [93] and prostanoid receptors [94]. In addition, studies recording from individual, functionally characterized abdominal vagal afferents continued and provided data on the sensitivity of the peripheral vagal afferent terminals to various substances including 5-hydroxytryptamine, substance P, gamma-aminobutyric acid (GABA), ghrelin, and glutamate [95–101].
Esophagus and diaphragm. Forceful contraction of the diaphragm together with the abdominal muscles provides the major expulsive force for vomiting. However, the crural muscle fibers of the diaphragm that encircle the lower portion of the esophagus and which, when contracted, contribute to the antireflux barrier between the stomach and esophagus do not contract during vomiting, thereby removing one of the barriers to the expulsion of vomit. Inhibition of the crural diaphragm also occurs during eructation (“belching”) and episodes of gastroesophageal reflux. The ferret has become established as a model for investigation of gastroesophageal reflux, particularly the control of the lower esophageal sphincter (LES) and the crural diaphragm and the genesis of transient LES relaxations implicated in episodes of gastroesophageal reflux [102–105]. Pharmacological studies have shown that the GABA_B receptor agonist baclofen, inhibited transient LES relaxations as did a cannabinoid CB₁ receptor agonist [105,106]. Interestingly, both baclofen and CB₁ receptor agonists (e.g., delta-9-tetrahydrocannabinol) have been shown to have antiemetic activity in the ferret (Andrews and Hawthorn, unpublished; [107,274]), implying that there may be a relationship between antiemetic and antireflux activity for drugs acting as receptor agonists either on the abdominal vagal afferents or in the brainstem integrative pathways (e.g., nucleus tractus solitarius [108]).
Gastroesophageal reflux is a common symptom in children with neurological impairment. It responds relatively poorly to pharmacological therapy and many children undergo surgery in the form of a “Nissen Fundoplication,” This procedure involves wrapping part of the proximal stomach around the lower esophagus to externally tighten the gastroesophageal junction [109]. Although the surgery may improve gastroesophageal reflux, it may induce retching with or without associated vomiting. The mechanism was investigated in ferrets by performing Nissen-type fundoplication and assessing the sensitivity of the emetic reflex pre- and postsurgery/sham surgery [110,111]. The surgical procedure sensitized the emetic reflex such that the threshold to a central stimulus was reduced postsurgery. Additional evidence for the plasticity of the emetic reflex in the ferret is reviewed [112].

Incidental Factors

Although there were a number of specific “drivers” leading to the use of the ferret for studies of emesis, there were also other, more serendipitous influences. One of the authors (PLRA) first encountered the ferret during the course of a PhD, investigating the vagal control of gastric motility using the urethane-anesthetized ferret. It was observed that electrical stimulation of the central end of an infracardiac thoracic branch of the vagus evoked a reflex fall in gastric pressure that was often accompanied by retching, especially when higher frequencies (>20 Hz) of afferent stimulation were used [74]. This incidental observation not only suggested that afferent fibers in the abdominal vagus could evoke the emetic response (not a novel finding) but also showed that this could be studied in ferrets rather than dogs and cats used in the previous studies and could be studied under anesthesia. A small grant (£1200) from the SmithKline Foundation in 1981 funded initial studies by PLRA to investigate the role of the vagus in cisplatin emesis in the ferret.

In 1984, PLRA was contacted for advice on research with ferrets by Dr. Chris Davis who had recently embarked on a PhD in the Department of Clinical Pharmacology at Oxford University with the working title “Neuropharmacological investigations into the mechanisms of emesis caused by cytotoxic drugs and radiation” [50]. Our initial meetings and the beginning of a collaboration fortuitously coincided with an international symposium on “Nausea and Vomiting: Mechanisms and Treatment” being organized by Dr. Davis, Dr. Lake-Bakaar (from Janssen Pharmaceuticals), and Professor D.G. Grahame-Smith (Head of Clinical Pharmacology, Oxford and Dr. Davis' lead supervisor). This meeting brought together basic scientists and clinicians to identify causes, mechanisms, and challenges, and played a critical role in stimulating research in this area.

The small size of the academic research community interested in either basic or clinical aspects of emesis in the early 1980s in the United Kingdom made it easy for the two pharmaceutical companies (Beecham and Glaxo) most interested in the area, both of which had their research effort in this area based in the United Kingdom, to identify researchers with whom to consult or collaborate.

Schurig et al. [26] commented that their own studies involved an element of serendipity. Another research group at Bristol-Myers Pharmaceutical, Co., had obtained ferrets, and because of their small size and cost, they tested if the ferrets responded to apomorphine, a widely used standard emetic challenge to which dogs and humans are particularly sensitive and which was known to act via dopamine receptors in the area postrema. The presence of a response encouraged the further study of the potential of the ferret for screening analogs of platinum as potential anticancer drugs. Three doses of apomorphine were studied, 2.5, 3.75, and 5 mg/kg, s.c. but only the highest dose evoked a response. The basis for selecting these doses is not given, but in comparison with other studies of sensitivity to apomorphine, these doses are all “high,” and this is a particular issue, as several studies have demonstrated that the emetic response to apomorphine has a “bell-shaped” dose response curve [38,41]. There is also an unresolved discrepancy in the literature on apomorphine sensitivity from ferrets originating in North America versus the United Kingdom, with the latter being more sensitive. The presence of an emetic response to apomorphine was a key factor in its development as a model because of the ability to compare responses with existing data from dogs and humans and to provide a stimulus to test the functionality of the area postrema. At the time the area postrema was widely considered to be the key site at which emetic agents in the circulation acted to induce emesis, hence its description as “the chemoreceptor trigger zone (CTZ) for vomiting” [113].

The Physiology and Pharmacology of Emesis in the Ferret

The majority of research on nausea and vomiting involving ferrets has the identification of novel antiemetics or emetic liability as a main objective. These aspects are reviewed in detail. However, these applied studies have also necessitated basic studies to understand aspects of the physiology and pharmacology of the emetic reflex in ferrets. Some of these findings are discussed in this section as they were important in establishing the model by matching data with that available for established dog and cat models. The major pathways responsible for induction of nausea and vomiting and the key physiological events comprising nausea and vomiting are illustrated in Fig. 31.5 to provide a background to the following sections.

c31-fig-0005 — Fig. 31.5. A diagrammatic summary of the main afferent pathways capable of inducing nausea and vomiting converging on the nucleus tractus solitarius (NTS): vestibular system and cranial nerve VIII; area postrema, and the abdominal vagal afferents (VA_m, vagal afferent mechanoreceptors; VA_c, vagal afferent mucosal chemoreceptors; NG,nodose ganglion).
The NTS sends outputs to the major motor nuclei (dorsal motor vagal nucleus, ventral respiratory group [VRG], Botzinger neurones, presympathetic neurones) responsible for the mechanical events of retching and vomiting (e.g., VN, abdominal vagus nerve from the dorsal motor vagal nucleus mediating lower esophageal sphincter [LES] relaxation, gastric relaxation, and giant retrograde contraction of the small intestine; P, phrenic nerve with nuclei in C₃–C₅ driven from VRG; MN, spinal motor neurones), the prodromata of vomiting often associated with nausea (mediated by sympathetic and parasympathetic nerves) and the rostral projections (predominantly via the parabrachial nucleus [PBN]) leading to vasopressin secretion (hypothalamus–posterior pituitary) and the more complex responses requiring cerebral cortical involvement including the genesis of the sensation of nausea itself. UOS, upper esophageal sphincter; EMG, electromyogam. From Stern et al. [5] modified with permission.

The Mechanics of Emesis

Retching and vomiting occur by contraction of the diaphragm, and intercostal and abdominal muscles, preceded by relaxation of the proximal stomach and a retrograde giant contraction in the small intestine. The mechanics of retching and vomiting were primarily studied in the cat and the dog [114], so an early task was to demonstrate similar mechanisms in ferrets. Proximal gastric relaxation prior to the onset of retching was an early finding in urethane-anesthetized ferrets, and it was also noted that retching was preceded by licking and swallowing, and vomiting was accompanied by mouth opening, all features observed in conscious animals. To date, the retrograde giant contraction has not been described in ferrets. Recordings of intrathoracic and abdominal pressure showed the characteristic rhythmic changes associated with retching and vomiting (Fig. 31.6), with responses evoked by electrical stimulation of abdominal vagal afferents and application of hypertonic saline to the gastric antral mucosa [115,116]. The gag reflex can also be elicited in the anesthetized ferret by gentle pharyngeal probing. These findings in anesthetized animals were confirmed by studies in conscious animals. More recent studies using telemetry of unrestrained animals enable prolonged recording of the pressure signatures associated with emesis as well as other behaviors such as defecation. Telemetry has only relatively recently been applied to the ferret for monitoring emetic activity [117,118], so the vast majority of studies have employed direct observation of the animals sometimes supplemented by video recording. In the conscious ferret, the frequency of retching is ∼1 Hz, making it possible for a trained observer to count the number of retches accurately in an emetic episode in real time; something also possible in the cat and dog but not in Suncus where the retching frequency is ∼4 Hz [119]. Independent quantification of retching and vomiting is essential to fully characterize the response to an emetic agent as the ratio of retches to a vomit is affected by the stimulus as well as the gastric volume [120]; antiemetic agents [121], as well as some surgical procedures (e.g., Nissen fundoplication [110,111]) can differentially affect retching and vomiting. A recommendation for the way in which data on retching and vomiting should be reported is given in section “Experimental Design and Reporting.”

c31-fig-0006 — Fig. 31.6. Example of intracorpus and intra-abdominal pressure traces in ferrets.
(A) The effect of application of 1 M NaCl to the antrum on intracorpus pressure in the presence of atropine, guanenthidine, and greater splanchnic nerve section. Note the initial fall in pressure interrupted by the large increase in pressure when the animal retched, after which the pressure slowly returned. (B) Conditions as earlier except that the 1M NaCl was present from the point indicated to the end of the record and the recording was made at a fast paper speed. The 1 M NaCl evoked a burst of retching (+ve intragastric and –ve intrathoracic pressure) concluding with a vomit (+ve intragastric and intrathoracic pressure). This record also illustrates the absence of effect of NaCl on blood pressure until retching intervened. Reproduced with permission from Andrews and Wood [116]. (C and D) Emetic episodes of retches and vomits in the conscious ferret recorded via (C) thoracic venous pressure and (D) intra-abdominal pressure.

Advances in telemetry including automated analysis [118] allow recording of multiple physiological parameters during studies of emesis in ferrets, enabling a detailed analysis of the microarchitecture of emetic episodes and their relation to changes in electrical activity of the stomach, cardiovascular system, core temperature, and locomotor activity (see discussion later).

The Neural Pathways for Induction and Coordination of the Emetic Reflex

It is hypothesized that the main function of the emetic reflex is to identify and eject toxins accidentally ingested with the food and which, if absorbed, could have serious consequences [122]. In view of this, the early demonstration that electrical stimulation of abdominal vagal afferents and irritant agents (e.g., hypertonic sodium chloride) in the stomach could induce an emetic response was particularly relevant [49,116,123]. These studies also demonstrated how rapidly the emetic reflex could be induced by an adequate stimulus with the latency for electrical stimulation of the vagus being 19.4 ± 1.2 seconds and hypertonic sodium chloride being 40.0 ± 4.5 seconds [49,116]. The characterization of the modality of the information signaled by the abdominal vagal afferents to the brainstem required direct recording from the afferent fibers with such studies identifying muscle mechanoreceptors [72], mucosal chemoreceptors [95,99], and demonstrating sensitivity of the mucosal afferents to 5-hydroxytryptamine and substance P [95,99,100]. These afferent recording techniques were also used in ferrets and rats to investigate the pharmacology of the receptors (particularly 5-HT₃ and NK₁) located on the peripheral terminals of the vagal afferents [95,96,100,124].

The ability to evoke an emetic response by abdominal vagal afferent stimulation in an animal under anesthesia was utilized in studies attempting to identify the sites at which particular emetic agents acted. For example, the response was blocked reversibly by the NK₁ receptor antagonist CP-99, 994, indicating a major site of action was likely to be in the brainstem [85]. This study also showed that although the emetic response was blocked, the elevation of blood pressure induced by vagal afferent stimulation was not blocked, indicating selectivity for the emetic pathways (Fig. 31.7). This was supported by the lack of effect of NK₁ receptor antagonists on the cardiorespiratory von Bezold–Jarisch reflex. This reflex is induced experimentally by rapid injection of 5-HT (or a selective 5-HT₃ receptor agonist) into the heart causing activation of cardiac vagal afferents resulting in a transient reflex bradycardia mediated by vagal efferents and accompanied by an increase in depth of respiration. This latter reflex also studied in anesthetized animals played an important role in the discovery of selective 5-HT₃ receptor antagonists. In the rat, Fozard (see Ref. 125 for review) showed that “high” doses of metoclopramide antagonized the reflex response but not the response to direct stimulation of cardiac efferents. This observation provided one of the early pieces of evidence that metoclopramide (previously only considered to be a dopamine D₂ receptor antagonist) at high doses was able to antagonize the activation of the 5-HT receptors located on the peripheral terminals of the cardiac vagal afferents. These 5-HT receptors later became defined as 5-HT₃ receptors and metoclopramide at high doses was shown be a 5-HT₃ receptor antagonist (see section “What Led to an Interest in Animal Models of Nausea and Vomiting in the Early 1980s and Adoption of the Ferret as a Model?”). The von Bezold–Jarisch reflex was one of the assays used to identify selective 5-HT₃ receptor antagonists and to demonstrate in vivo efficacy. An example of this reflex response in ferrets, and its blockade by a selective 5-HT₃ receptor antagonist (GR38032, Ondansetron), with well characterized antiemetic effects is shown in Fig. 31.8.

c31-fig-0007 — Fig. 31.7. A representative strip chart recording of intrathoracic pressure (ITP) and blood pressure (BP) responses to ventral abdominal vagus stimulation (vs) before (upper, Control), 15 minutes following (middle, CP-99,994), and 90 minutes after (lower, Recovery) administration of 1 mg/kg CP-99,994, i.v. Reproduced with permission from Watson et al. [85].

c31-fig-0008 — Fig. 31.8. The effect of GR 38032 (0.5 mg/kg, i.v.) on the cardiovascular (BP) and respiratory (intrathoracic pressure) responses to a rapid intravenous injection (jugular) of 5-HT (30 μg) in ferrets. Reproduced with permission from Andrews and Bhandari [238].

The area postrema, a circumventricular organ where the blood–brain barrier is relatively permeable, is located in the caudal part of the floor of the fourth ventricle at the obex. It was recognized by early researchers using surgical ablation as a site from which emesis could be induced by substances in the circulation [8]. The area postrema is often considered a “second line of defense,” responsible for triggering an emetic response if ingested toxins have avoided detection prior to absorption. The area postrema in ferrets is U-shaped as is the case in cats, dogs, and humans. Surgical ablation abolishes the emetic response to the dopamine receptor antagonist apomorphine, as is also the case in cat, dog, and human [113]. Apomorphine is frequently used as challenge to activate the brainstem emetic mechanisms via the area postrema in a way analogous to the use of intragastric copper sulfate as a method for activating the central pathways via the abdominal vagal afferents. These challenges are often used to demonstrate that a pathway has been lesioned when investigating the mechanism by which NCEs may cause emesis.

Utilizing a range of well defined emetic stimuli known to act via the area postrema or vagal afferents also provides insights into the spectrum and site of action of a novel antiemetic agent. For example, 5-HT₃ receptor antagonists, which are effective against stimuli acting via the abdominal vagal afferents, have no effect against apomorphine or loperamide, which are acting via the area postrema. In contrast, NK₁ receptor antagonists have efficacy against stimuli acting via both pathways [108,126].

The afferent pathways by which emesis can be evoked (area postrema and abdominal vagal afferents) and the major output motor pathways (e.g., abdominal vagal efferents to the gut and the phrenic nerves to the diaphragm) have been relatively well studied, but equally important are the central integrative pathways particularly in the brainstem. Studies of “fictive emesis” in decerebrate ferrets by Onishi et al. [127], combined with Fos immunohistochemistry and antagonist microinjection, demonstrated that at least at a general level, the brainstem circuitry coordinating emesis was the same in ferrets as previously described in dogs and cats. These similarities in the organization of the brainstem, in general, and the regions involved in processing vagal afferent and efferent information specifically are supported by both anatomical and neurophysiological studies of the ferret brain in vivo and brain slices in vitro (see section “Anatomical Studies of the Brainstem”).

An intact brainstem is essential for retching and vomiting, but the genesis of the sensation of nausea by activation of the area postrema, abdominal vagal afferents and the vestibular system requires projection to more rostral brain regions [5]. Relatively little is known in ferrets about these “higher” projections with the exception that in ferrets, as is the case in humans and the dog, emetic stimuli evoke a large increase in the plasma concentration of vasopressin (ADH), demonstrating an input to the posterior hypothalamus (see Ref. 5 for review).

The Diversity of Emetic Stimuli

Considering that the ferret has only been used for studies of emesis for ∼30 years, a diverse range of emetic stimuli has been investigated. The majority of studies have focused on chemotherapeutic agents but a substantial number have studied other emetic stimuli (Fig. 31.9A). Figure 31.9B shows more than 60 compounds in rank order of their emetic latency. Some of the stimuli, such as those activating the area postrema (e.g., apomorphine) and those stimulating gastrointestinal vagal afferents (e.g., intragastric irritants such as copper sulfate or hypertonic sodium chloride), have been used to demonstrate the existence of pathways and mechanisms present in other species. The majority of stimuli investigated reflect areas of clinical interest, particularly treatment for cancer where the requirement was to understand the mechanisms (e.g., Ref. 44), identify chemotherapy agents with lower emetogenic potential (e.g., platinum analogs [26]), and also to identify novel antiemetics (to be shown later). A particularly important feature of the ferret was that the response to cisplatin showed two phases (acute and delayed) as was the case in humans (Fig. 31.10). The importance of this development was that in humans, the acute and delayed phases were not equally susceptible to the same antiemetics, implying different pathways and pharmacology. The implications of this finding to translation from ferret to human are discussed in section “Identifying Antiemetic Efficacy.”

c31-fig-0009a — Fig. 31.9. (A and B) 266 publications were identified from Pubmed using the keywords: emesis and ferret in April 2010, 155 publications contained emetic data, which were extracted. (A) Ferrets used in emetic research 1975–2010 categorized per type of emetic inducer. Data for 142 unique compounds were extracted to construct this graph. (B) Compounds used in ferret publications 1975–2010 ordered by latency.
Latency data were extracted from the publications; for similar dose and mode of administration, data were combined by computing mean weighted by the number of animals. The data plotted on the graph represent the minimum latency following systemic administration of the compound.

c31-fig-0009b — Fig. 31.9. (A and B) 266 publications were identified from Pubmed using the keywords: emesis and ferret in April 2010, 155 publications contained emetic data, which were extracted. (A) Ferrets used in emetic research 1975–2010 categorized per type of emetic inducer. Data for 142 unique compounds were extracted to construct this graph. (B) Compounds used in ferret publications 1975–2010 ordered by latency.
Latency data were extracted from the publications; for similar dose and mode of administration, data were combined by computing mean weighted by the number of animals. The data plotted on the graph represent the minimum latency following systemic administration of the compound.

c31-fig-0010 — Fig. 31.10. Profile of emesis induced by cisplatin 10 mg/kg (A) and 5 mg/kg (B) cisplatin in ferrets.
Reproduced with permission from Percie du Sert et al. [142]. (A) Data plotted as mean vomits ± SD per 30-minute periods, collected from studies involving 34 animals and mean retches ± SD, collected from four of those studies involving 20 out of the 34 animals. (B) Data plotted as weighted mean R + V ± SD per 4-hour periods collected from nine studies involving 92 animals.

The demonstration that emesis could be induced by total body X-irradiation was also significant in establishing the utility of the ferret model. Radiotherapy was used either alone or in combination with chemotherapy for the treatment of cancer and prior to bone-marrow transplant and the resulting emesis was difficult to treat. For example, in 1978, general anesthesia was investigated as a method for preventing emesis during high-dose total body irradiation prior to bone-marrow transplantation [128]. The initial studies on X-irradiation-induced emesis were undertaken in the United Kingdom by researchers at Oxford University (C.J. Davis, see Ref. 50. PhD thesis) and London University (P.L.R. Andrews and J. Hawthorn) often working in conjunction [33] but studies were also undertaken in the United States [35] and Canada [129]. X-irradiation rapidly became included in the range of challenges used to investigate novel antiemetic agents including the 5-HT₃ receptor antagonist [34,46,130–133] and later the ultrapotent capsaicin analog resiniferatoxin [134] and NK₁ receptor antagonists [135]. Most studies investigated X-irradiation but in the United States, research at the Armed Forces Radiobiology Research Institute led by G.L. King showed that emesis could also be induced in ferrets by gamma, neutron, and proton radiation [35,36,136], and the response induced by gamma and proton radiation was reduced or blocked by 5-HT₃ receptor antagonists. One of the reasons these latter types of radiation were of interest was because of the types of exposure likely to be encountered in extended space flight (see [137] for recent studies of proton radiation in the ferret). Analysis of the dose–response sensitivity of the ferret to both X- and gamma-radiation shows that the ferret has a lower latency to the onset of emesis and is more sensitive to the emetic effects (lower ED₅₀ and ED₁₀₀) than other laboratory species and humans with an ED₁₀₀ of 125 cGy in comparison with 400 cGy (ED₈₀) for humans, 700 cGy for dogs, 800 cGy (ED₈₀) for monkeys, 800 cGy for Suncus, and >4000 cGy for cats [35,50,138].

One stimulus missing from the range of stimuli investigated in ferrets is motion. Although methods for the induction of emesis by motion have been developed in dogs, cats, and nonhuman primates (see Ref. 16 for review), these have not been adopted for the ferret. There is no a priori reason to suspect that the ferret would not demonstrate an emetic response to motion provided an adequate stimulus. There is a preliminary report of motion-induced emesis in the mink (Mustela vison), a close relative of the ferret, that has also been shown to respond to cisplatin [139]. However, there seems little justification for investigating motion sensitivity in ferrets, especially as S. murinus has become an accepted small animal model for investigating the effects of novel antiemetic agents on motion sickness, and models of motion sickness are well established in healthy human subjects.

In contrast to Suncus murinus, ferrets are not sensitive to emesis induced by exposure to the anesthetic agent isoflurane (±morphine) so may not be a suitable model for studying post operative nausea and vomiting [140].

Responses to Antiemetics

The validity of a model for identification of novel antiemetic agents relies to some extent on demonstrating that antiemetics with established efficacy in humans are effective against similar stimuli in the animal model. In part, this assumes that the emetic stimulus is acting via the same pathway as in humans and also that the same neurotransmitter(s) and receptors are involved. Comparison of human and animal model data also requires that comparably defined and detailed data are collected, and while this occurs in a limited way for vomiting, there is no direct comparator data for nausea [141]. For novel antiemetics investigated in an animal model such as the ferret, comparison will only be possible if the agent is found to be “safe,” is subsequently investigated in humans, and the data are published. Although our ability to make direct comparisons is limited, the data available from animal studies are extensive, and when examined systematically, and where possible quantitatively, can provide important insights not obvious in individual studies and generate hypotheses for novel approaches as well (see Ref. 142 and Fig. 31.11). It should also be recalled that data from antiemetics, especially when the site(s) of action is known, both enable identification of the receptors and neurotransmitters in the pathways inducing and coordinating retching and vomiting, and provide some insights into nausea. Identification of these neurotransmitters and receptors also helps understand the mechanism underlying the emetic liability of some drugs and NCEs. Space does not permit a detailed review of all the substances where published data exist for antiemetic activity in ferrets, but Table 31.3 summarizes data for substances tested in the acute cisplatin-emesis model—the model most extensively studied in ferrets. The acute emetic response to cisplatin in ferrets can be abolished by abdominal vagotomy. This observation together with other evidence led to the proposal by Andrews et al. in 1988 that anticancer chemotherapy and radiation were induced by local release of 5-HT from the enterochromaffin cells in the intestinal mucosa. Those authors posited that nausea and vomiting were triggered by 5-HT, which acted on 5-HT₃ receptors located on the abdominal vagal afferent terminals, and led to stimulation of the vagal afferents projecting to the brainstem [32]. Taking the pharmacology of the substances listed at face value, Table 31.3 provides evidence that a surprising variety of receptors and transmitters are involved at some point in the enterochromaffin cell-vagal afferent-brainstem (primarily nucleus tractus solitarius) pathway mediating the acute emetic response to cisplatin.

c31-fig-0011 — Fig. 31.11. Efficacy of 5-HT3 receptor antagonists on the daily number of retches + vomits (R + V) induced by 5 mg/kg i.p. cisplatin during the acute (day 1) and delayed (days 2 and 3) phases of emesis.
Reproduced with permission from Percie du Sert et al. [142]. Point estimates and 95% confidence intervals for each of the 5-HT₃ receptor antagonist versus control comparisons ranked by dose. The effect estimate was computed as the weighted mean difference (WMD) and expressed as the proportion of retches and vomits in the control group. An effect estimate of −1 indicates that emesis was abolished in the treatment group, 0 indicates that the treatment had no effect on the R + V response and an effect estimate >0 indicates that the treatment increased the number of R + V. The size of each square represents the weight of the comparison in the WMD calculation.

Table 31.3. A Summary of Compounds Investigated for Their Effects on the Acute Phase of Cisplatin-Induced Emesis in the Ferret

Translation from the Laboratory to the Clinic

In emesis research, ferrets can be used for three different purposes: in drug discovery to assess the efficacy of antiemetic drugs, in safety pharmacology to identify emetic liability, and to investigate the mechanism of nausea and emesis.

Identifying Antiemetic Efficacy

Several models have been used to investigate the effects of potential antiemetic compounds in ferrets. Common emetic inducers in this species include the opioids morphine [140,143,144], morphine-6-glucuronide [84], and loperamide [134], which activate opioid μ receptors in the brainstem and the nonspecific dopamine receptor agonist apomorphine [47], which triggers emetic pathways in the area postrema via the activation of D₂ and D₃ receptors [145]. Models using anticancer therapies such as cytotoxic agents and radiation [146] have also been crucial in establishing the role of the ferret in antiemetic research. The ferret models of cisplatin-induced emesis, in particular, were instrumental in bringing to the market the two main classes of antiemetic drugs currently in clinical use, the 5-HT₃ and NK₁ receptor antagonists. NK₁ receptor antagonists such as aprepitant (Emend®, Merck and Co.) are indicated for treatment of chemotherapy-induced nausea and vomiting and 5-HT₃ receptor antagonists such as ondansetron (Zofran®, GlaxoSmithKline) are additionally recommended against radiation-induced and postoperative nausea and vomiting (PONV).

Antagonism of Acute and Delayed Cisplatin: A Case Study of Translation

Two models of cisplatin-induced emesis have been developed in ferrets. The original, acute emesis model studied by Florczyk et al. [31] followed the emetic response to cisplatin over ∼6 hours during which time the response gradually declined. Studies by John Rudd at Bradford University (currently at the Chinese University Hong Kong) led to the recognition that if the cisplatin dose was adjusted and the ferrets were studied for several days, emesis recommenced (Ref. 147; see Ref. 24 for anecdotes from Professor Rudd about the development of the model). The antiemetic efficacy of the 5-HT₃ receptor antagonists was originally demonstrated in the acute emesis model, while the subsequently developed acute and delayed model was central in establishing the efficacy of NK₁ receptor antagonists. It should be noted that the piglet has a biphasic emetic response to cisplatin and has also been used to study the acute and delayed phases, as well as the effect of antiemetics [148].

In the clinic, in the absence of antiemetic prophylaxis, cisplatin induces nausea and vomiting in virtually all patients [149]; the emetic response lasts up to 5 days and is typically divided into an intense acute phase during the first 24 hours and a protracted delayed emesis phase, which peaks 2–3 days following cisplatin administration [150]. The ferret model of cisplatin-induced acute emesis was developed in the early 1980s following reports that cisplatin provoked a reliable emetic response in this species [31]. This model typically uses a high dose of cisplatin (10 mg/kg), injected intravenously or intraperitoneally, to induce emesis. The effects of potential antiemetic agents, administered either prophylactically or once the response is established, are observed over a short observation period of up to 6 hours. The acute phase model was pivotal in establishing the antiemetic efficacy of 5-HT₃ receptor antagonists and also contributed to the elucidation of the mechanisms regulating the acute emetic response (see section “Responses to Antimetics” earlier).

The delayed model of cisplatin-induced emesis was subsequently developed in the mid-1990s. This model uses a lower dose of cisplatin (5 mg/kg, i.p.), which induces in ferrets a biphasic emetic response over 3 days (see Fig. 31.10), a profile similar to the response observed in the clinic [147]. The delayed model correctly identified the potential of NK₁ receptor antagonists to reduce vomiting in patients treated with cisplatin from the second day onward following chemotherapy [4] and consistent with the clinical situation, also showed the antiemetic efficacy of dexamethasone [151], a synthetic glucocorticoid used in combination with 5-HT₃ and NK₁ receptor antagonists as part of the antiemetic regimen.

Despite these successes, illustrating the predictivity of the ferret cisplatin models and the apparent similarity in the biphasic response induced by cisplatin in ferrets and in humans, a recent systematic review and meta-analysis highlighted a few discrepancies with the clinical picture that warrant caution [142]. It revealed that the relative magnitude of the acute and delayed phases is inverted in ferrets. In humans, while virtually all unprotected patients experience vomiting during the acute phase, the delayed phase is less severe, with a reported incidence varying between studies of around 44–89% in patients treated with placebo antiemetics [150,152]. In ferrets, however, the delayed phase is more severe than the first day, with nearly twice as many retches and vomits during the peak of the delayed phase (44–56 hours postcisplatin) than during the peak of the acute phase (4–16 hours), and effectively all ferrets developing emesis during the delayed phase and a lower incidence during the acute phase [142].

While the efficacy of ondansetron during the first day following cisplatin administration is very accurately predicted with the acute emesis model, its efficacy during the delayed phase is not consistent with the clinical situation. Whereas 5-HT₃ receptor antagonists have a limited efficacy past the first day in humans [153,154], ondansetron reduces the severity of emesis in ferrets by about 60% (Fig. 31.11). The discrepancy may be explained to some extent by the disparity of outcome measures collected in humans (percentage of patients protected) and in ferrets (number of retches and vomits), but it may also reflect a difference in the mechanisms driving delayed emesis in the two species. While 5-HT-dependent mechanisms have been shown to predominate within 12 hours of cisplatin administration in humans [155], the profile of 5-HT₃ receptor antagonists efficacy in ferrets suggests a much longer involvement of 5-HT, for several days following cisplatin administration.

The ferret model correctly identified the antiemetic potential of NK₁ receptor antagonists but their efficacy appears to have been overestimated. In ferrets, aprepitant completely blocked the emetic response in both the acute and the delayed cisplatin models [156], but despite an impressive profile of efficacy against the delayed phase of emesis in patients treated with cisplatin, 28–45% still experience vomiting and aprepitant offers limited protection within the first 24 hours [157,158].

The emetic response induced by cisplatin is clearly complex and stems from the activation of multiple mechanisms. These differences likely reflect differences in the relative predominance of these pathways in the two species and their timing of activation. A small proportion of patients are still refractory to 5-HT₃ receptor antagonist therapy during the acute phase and the substance P-independent mechanisms mediating the delayed phase remain to be elucidated. Whether these pathways are implicated in the ferret emetic response and can be identified in this species is unknown.

Identifying Emetic Liability

Nausea and vomiting are common adverse side effects of drugs in clinical use. Over half of the drugs on the electronic Medicine Compendium include nausea as an undesirable side effect and a third of the drugs include both nausea and vomiting [159]. In drug discovery and development programs, nausea and vomiting interfere with the development of valuable new drugs and an analysis from Pfizer revealed that it is one of the adverse effects with the greatest impact on the development of novel therapeutics [160]. In vivo models used in safety pharmacology to detect emetic liability include the ferret and the dog emesis models, and the rat pica model, which uses kaolin consumption as a surrogate behavior for emesis [141].

Recently, the ferret emesis model was compared with emesis in the dog and pica in the rat, to assess which model was best predictive of human nausea and vomiting [141]. The predictivity of the ferret model was assessed using 10 compounds known to induce nausea and vomiting in humans. These compounds were representative of various mechanisms of action, including drugs which trigger the emetic reflex via the abdominal vagal afferents (e.g., the cytotoxic cyclophosphamide and the gastric irritant copper sulfate), drugs which directly activate receptors on the area postrema (e.g., morphine and apomorphine, which activate opioid and dopamine receptors, respectively) and drugs which induce nausea and vomiting via mechanisms that have not been fully elucidated (e.g., the antimanic agent lithium chloride and the phosphodiesterase 4 [PDE4] inhibitor rolipram).

This study showed that emesis in ferrets predicted emetic liability accurately for 9 out of 10 compounds investigated. In ferrets, the gastrointestinal hormone cholecystokinin octopeptide (CCK-8) triggered signs prodromal of emesis but vomiting was not induced at doses over two orders of magnitude higher than the ED₅₀ for vomiting in human subjects. However, the endogenous CCK releaser casein acid hydrolysate was capable of inducing emesis in ferrets with the response reduced by the CCK₁ receptor antagonist devazepide [161]. Other emetic hormone peptides have been found without overt effects in ferrets. Gastrin, angiotensin II, and vasopressin failed to induce emesis in this species [77], despite reports of pentagastrin and vasopressin-induced nausea and emesis in humans [162–164]. We found no report of human emesis induced by exogenous administration of angiotensin II but the hormone precursor induced emesis in the dog [165]. More data are warranted to formulate a definitive conclusion about the ability of systemically administered peptides to induce emesis in ferrets. Currently, those studies suggest that the ferret may not be an appropriate model to detect the emetic liability induced by circulating peptide hormones.

For the nine other compounds investigated, emesis in ferrets was a predictor of nausea and vomiting in humans but ferrets displayed a lower dose sensitivity compared with humans and doses one to two orders of magnitude higher were typically required to induce emesis with a comparable severity [141]. The ranking of compounds based on severity of the response was very similar between man and ferret, with cytotoxic agents such as cisplatin being the most emetogenic and apomorphine provoking a less intense vomiting response.

It is worth noting that while the ferret model seems to be highly predictive, the specificity of the model has not been assessed and it would be valuable to investigate whether compounds that do not induce emesis in humans induce emesis in ferrets or vice versa. To answer the question of which animal model is the best predictor of emetic liability of an NCE in humans requires comparison of all compounds for which data are available from both ferret (or other species of interest) and human (healthy volunteers and patients). Such an analysis can only be conducted using information in the public domain and hence is unlikely to reflect the full set of data available (cf. difference between the number of registered/completed clinical trials and the number published). A full assessment would require access to unpublished data held by both academic and pharmaceutical company researchers. Even if such data were available, comparison might be difficult because of different outcome measures in human and ferret studies (see section “Measuring the Emetic Response”).

Detecting Nausea

Recording Gastric Myoelectric Activity and Autonomic Nervous System Outputs

Nausea is a subjective sensation, described as a feeling of sickness, accompanied by the urge to vomit [5]. Due to its subjective nature, nausea is not directly quantifiable in nonhuman species and investigating nausea in animal models relies on surrogate behaviors and markers. Evidence suggests that nausea could arise from an imbalanced autonomic outflow, shifting toward a dominance of sympathetic activity [166]. In humans, nausea is often accompanied or preceded by physiological events, triggered by changes in the autonomic nervous outputs. Events primarily mediated via sympathetic efferents including pallor, cold sweating, and tachycardia, while salivation, inhibition of gastric peristalsis, and relaxation of the proximal stomach are predominantly under parasympathetic control [5].

Also under autonomic efferent control, and considered one of the major correlates of the human experience of nausea is a disruption of the gastric myoelectric activity (GMA). GMA is a pacesetter potential generated by the interstitial cells of Cajal (ICCs), resulting in an electrical rhythm (i.e., the slow waves), which drives gastric motility [167]. A mean frequency of 3 cycles per minutes (cpm) is considered normal in humans. Thus far, GMA has been predominantly investigated in experimental models of motion sickness, where healthy volunteers are subjected to illusory self-motion (vection) or placed in a rotating chair, and reports of nausea are associated with gastric dysrhythmia [168,169]. Dysrhythmia has also been observed in other settings, with nausea induced by chemotherapy [170], morphine [171], and vasopressin [163]. It has also been linked with idiopathic conditions associated with nausea including pregnancy [172], dyspepsia [173], gastric ulcers [174], and eating disorders [175].

GMA has been measured in conscious ferrets using telemetry. The electrical slow waves can be recorded with biopotential wires implanted in the wall of the antrum, on the serosal side. Under normal conditions, the frequency of the slow waves in ferrets is around 9 cpm, which is three times faster than in humans [117]. For comparison, cats, dogs, and rats have a slow wave frequency of around 5 cpm [176–178] and a frequency of 13 cpm has been recorded in S. murinus [179]. In ferrets, low frequency signals up to 18 cpm are isolated from the telemetry recordings and a power spectrum analysis is used to identify the dominant frequency and the percentage of power in the normal range and the bradygastric and tachygastric ranges (dysrhythmia). Emetic stimuli have been shown to induce dysrhythmia and both cisplatin and apomorphine reduced the percentage of power in the normal range, predominantly via an increase in bradygastric activity [117].

Another measurement of autonomic outflow that has been associated with the human experience of nausea is the heart rate variability, which is derived from an electrocardiogram. Following power spectrum analysis of the R-R interval data, the high frequency component represents modulation from the vagal outflow to the heart, while the low frequency component represents a mixture of sympathetic and parasympathetic outflow. In human volunteers, vection-induced nausea has been correlated with an increase in the low frequency component of the heart rate variability, suggesting a sympathetic shift in the sympathovagal balance [180]. The technique has been used to investigate autonomic outflow in dogs [181,182] and in rats [183], and it could be adapted for use in ferrets to detect this important correlate of nausea.

Measuring Plasma Vasopressin

Vasopressin (ADH) has also been associated with nausea. Vasopressin is a stress-related hormone released from the posterior pituitary into the circulation; it is an antidiuretic and a vasoconstrictor, both of which contribute to the maintenance of arterial blood pressure. In humans, nausea is accompanied by a rapid surge in plasma vasopressin, even in the absence of vomiting, to levels much higher than maximal antidiuresis. Elevated levels of the hormone have been measured in nauseated individuals challenged with chemotherapy [184], apomorphine [185], ipecacuanha syrup [186], ethanol [185], morphine [171], and vection [187].

Similar findings have been observed in ferrets, and a surge of plasma vasopressin was measured following challenges with morphine [188], apomorphine, and abdominal vagal afferent stimulation [189]. Of note, CCK-8, which failed to induce emesis in ferrets, provoked a dose-related increase in plasma vasopressin to levels two orders of magnitude higher than baseline [89].

Further research would be required to test the predictivity and specificity of plasma vasopressin in ferrets, but these initial findings indicate that this measure may be used as a surrogate for human nausea.

Behavior Analysis

Numerous studies have attempted to recognize behaviors suggestive of nausea in ferrets and other species. Models which have been proposed to have utility in investigating nausea include pica [190], conditioned taste aversion, conditioned food avoidance [191], and conditioned gaping [192] in the rat, and conditioned retching in S. murinus [193]. In addition, periemesis behaviors have been studied in cats [194], dogs [195], and piglets [148]. However, similarly to the study of behaviors in ferrets, the translational value of these models is unclear, especially with nonemetic species such as the rat, where the interpretation is further complicated by the inability to differentiate the activation of potential nauseogenic and emetic pathways (see chapter 8, Ref. 5 for review). In ferrets, behaviors such as lip licking, burrowing, and backward walking have commonly been recorded, and many emetic agents have been investigated for their potential to induce such behaviors. These include anticancer treatments such as cisplatin, cyclophosphamide, radiation and trimelamol [131,196,197], the opioid loperamide [80], copper sulfate [198], dopamine agonists [145], and PDE4 inhibitors [199].

A fair amount of discrepancy has been reported between studies. Lip licking or obtrusive licking is believed to reflect the salivation observed in nauseated humans. Several studies described an increase in the frequency of the behavior in ferrets challenged with anticancer treatments, copper sulfate, and loperamide [35,136,196,198]. Others found no difference with the behavior of control ferrets using the same emetic stimuli [196,200]. Studies investigated the frequency of burrowing and backward walking reported similarly conflicted findings; some studies describing an increase in the frequency of this naturally occurring behavior following cytotoxic drugs and radiation [35,131,136], while other studies found no changes [196,200]. Interestingly, treatment with an NK₁ receptor antagonist without any emetic stimulus was also found to reduce spontaneous lip licking and burrowing activity [200]. Other behaviors potentially representative of human nausea include gagging, mouth scratching, wet dog shake, hyperventilation, body scratching, and failure to rear. These behaviors have been investigated following a variety of emetic challenges, but once again, discrepancies are observed between studies [35,80,136,196,198–200].

The discrepancies between studies might stem from the fact that all the behaviors investigated are part of the normal repertoire of ferrets. None of these behaviors seem to be specifically associated with gastrointestinal malaise or a ferret equivalent of nausea in humans. Additionally, there is a high degree of variability between individual animals, which might be amplified by the origin or gender of the animals or, in the case of burrowing, the amount of sawdust in the cage, contributing to interstudy variability.

Another approach, which led to more consistent finding between studies, was developed to investigate ferret behavior. Rather than focusing on individual behaviors, a few studies have attempted to develop a general “nausea” index. The index is typically based on the cumulative occurrence of behaviors suggestive of malaise such as lip licking, mouth scratching, backward walking, gagging, burrowing, and belly dragging [145,197,201,202]. In addition, negative points can be included in the index, to deduct from the score behavior associated with normal healthy activity such as rearing, grooming, rolling over, sniffing, and playing with drinking bowl [197]. In ferrets challenged with dopamine receptor agonists, PDE4 inhibitors, and cyclophosphamide, “nausea” scores significantly increased [197,201,202].

Treatments with 5-HT₃ receptor antagonists and dexamethasone have been shown to reduce, but not abolish, the “nausea” index and the burrowing behavior following cyclophosphamide and radiation [136,197]. To validate the use of behavioral studies to predict human nausea, it would be crucial to demonstrate that 5-HT₃ and NK₁ receptor antagonists do not abolish the behavior changes induced by drugs such as cisplatin. Antiemetic prophylaxis with 5-HT₃ and NK₁ receptor antagonists and dexamethasone would not be expected to make a drastic change on the occurrence of these behaviors as antiemetic drugs have a limited efficacy against nausea in humans: over 50% of patients receiving cisplatin chemotherapy and antiemetic treatment still experience some degree of nausea [203].

Assessing the Translational Value of the Ferret Model

A crucial component of the translational value of a model is its predictive validity. Based on the effect of either an emetic or an antiemetic drug in ferrets, can we predict the effect of that drug in humans? The ferret emesis models have face validity in the sense that the same symptoms (i.e., retching and vomiting) are observed in both species but to enable a meaningful comparison, the same outcomes should be measured in both species. However, different outcomes are often measured in human and ferret studies.

Measuring the Emetic Response

The emetic response is characterized by its latency, intensity, and incidence. These three parameters vary depending on the emetic inducers. For example, in ferrets, cisplatin (10 mg/kg i.p.) induces emesis with a long latency (∼90 minutes) and high intensity (∼150 retches + vomits in the first 4 hours) and incidence (100% of the animals develop emesis [142]). In contrast, apomorphine (0.25 mg/kg s.c) induces emesis with a short latency of 6 minutes, a low intensity (30 retches + vomits over 30 minutes) and a high incidence (∼90%) [141].

Intensity

In ferrets, the intensity can be measured by counting the number of emetic episodes, usually defined as bouts of individual retches and/or vomits separated by intervals shorter than 5 seconds [204]. The relatively slow retching frequency in this species also enables easy quantification of the total number of retches and vomits, either via direct observation or video recording. However, distinguishing between retches and vomits on video can be difficult and direct observation is often needed to quantify them separately; this is especially relevant to studies looking at antiemetic efficacy. Cannabinoids, for example, reduce the number of retches and vomits differentially. The CB₁/CB₂ receptor agonist Win 55,212-2 has a greater impact on the number of vomits induced by apomorphine with the number of episodes culminating in a vomit reduced from 60% to 10%, while the number of retches per episode stays unchanged [121]. This finding extends to other species as apomorphine-induced vomiting was shown to be selectively blocked by the cannabinoid levonantradol in the cat [205]. This highlights the importance of recording retches and vomits separately, and quantifying retches in addition to vomits in studies looking at antiemetic efficacy, as an antiemetic drug blocking vomits but not retches would be of limited use in the clinic.

Incidence

The incidence of emesis is calculated as the number of animals developing emesis during the observation period divided by the total number of animals. The incidence is therefore modulated by the duration of the observation period if the latter is shorter than the full length of the emetic response. In studies looking at antiemetic efficacy, the incidence measure will be biased by the effect of the drug on the latency. If the antiemetic drug delays the onset of the emetic response, it will appear to reduce the incidence artificially. For example, in ferrets, the 5-HT₃ receptor antagonist ondansetron reduces the incidence of cisplatin-induced emesis by 76% over a 2-hour observation period, but only by 11% over a 6-hour period and 0% over 24 hours [142].

Latency

The latency represents the delay between the administration of the emetic agent and the onset of the emetic response (i.e., the first retch or vomit). It is arguably indicative of the mechanisms activated to trigger retching and vomiting and constitutes an important variable in the assessment of the predictive value of the ferret models and its comparison with other species. The latency is also an indication of the rate and extent of absorption to reach a given plasma exposure, and for some compounds such as cyclophosphamide, the latency reflects hepatic metabolism. The long latency to the onset of cisplatin-induced emesis, for example, reflects the time necessary to induce the formation of free radicals in the enterochromaffin cells of the upper gastrointestinal tract, and subsequently trigger the calcium-dependent release of 5-HT, which in turn activates the 5-HT₃ receptors on vagal afferent terminals [126,206]. On the other end of the spectrum, the very short latency to the onset of the response to subcutaneous apomorphine reflects the direct activation of dopamine D₂ and D₃ receptors in the area postrema [207], without the involvement of intermediate mediators.

The latency should only be reported in animals which develop emesis. To ease statistical analysis, the latency is often reported for all the animals in the group, with animals that did not develop emesis assigned a latency equivalent to the duration of the observation period. This, however, biases the latency measurement, as the incidence becomes a confounding variable. Antiemetic treatments can reduce the incidence of emesis without necessarily increasing the latency in animals that are not protected. Assigning a long latency to the animals protected from emesis will artificially increase the average latency in the group. Antiemetic drugs have different profiles of efficacy, and while 5-HT₃ receptor antagonists delay the onset of the emetic response in a dose-dependent manner (e.g., Ref. 29), NK₁ receptors and cannabinoids have a very limited effect on the latency [85,121].

Comparison with Humans

In studies of patients treated with anticancer chemotherapy, official guidelines recommend measuring all three parameters: intensity, incidence, and latency to assess antiemetic efficacy. The European Medicine Agency recommends a measure of intensity and incidence as primary endpoints, with latency as a secondary endpoint [208], while the American Society of Clinical Oncology states that counting the number of vomiting episodes after treatment is the most important clinical trial endpoint and recommends using the number of patients protected from emesis as an accurate and reliable measurement [157].

However, the human data available in the literature are scarce and incomplete. In a recent study analyzing emetic data in the public literature, while 86% of the human studies reported the incidence of emesis, only 33% and 23% reported the latency and intensity, respectively, and all three outcomes were only included in 8% of the publications [141]. The reporting of preclinical studies was marginally more comprehensive, but the preferred outcome was the intensity, reported in 95% of the publications, while the incidence and latency were found in 69% and 61% of the publications, respectively. The disparity in the reporting of human and ferret studies is problematic. The lack of common ground prevents interspecies comparison and limits the usability of the preclinical data. It also makes it difficult to assess the translational value of the ferret model accurately.

Refinement and Optimization of the Ferret Model

Husbandry and Housing

Ferrets are social, intelligent, and inquisitive animals, they need to be housed in an environment with sufficient complexity to cater for their needs. Many book chapters have been written on the subject (e.g., Refs. 201 and 209, and Chapter 6 of this text) and should be consulted before embarking on a study using ferrets.

The main points to consider include housing the animals in compatible social groups: special care should be taken with noncastrated males, which may fight during the breeding season. Sufficient height should be provided to allow animals to stand on their hind legs and there should be enough space to allow for a sleeping area, a food storage, and eating area, and a separate corner for micturition and defecation. Good ferret housing should also include environmental enrichment, which can be provided by rigid plastic tubes or containers of various materials, fabric tubes, or hammocks, and solid floor with bedding to allow for the ferret's natural burrowing behavior.

Experimental Design and Reporting

Animal research is often scrutinized to ensure that the design, analysis and reporting of animal experiments meets the highest quality standards. A recent study across various fields of research revealed that there is a significant scope for improvement in the way in vivo experiments are designed, in the statistical analysis of the data, and in the information included in the publication [210]. Specific problems with the experimental design included the lack of measures to reduce subjective bias such as random allocation of the animals into treatment groups and concealment of treatment allocation when assessing the results. Publications using the ferret model are not exempted from these weaknesses and in a systematic review, which identified 115 publications describing cisplatin-induced emesis in ferrets between 1981 and 2007 [142], only one study stated that animals had been randomized into treatment groups and one study stated that the person who observed the emetic response and associated behaviors and the person responsible for analyzing the data were both blind to the treatments. One could arguably generalize these findings to ferret studies using emetic inducers other than cisplatin and emetic studies using other species. Presumably, this could be a reporting issue rather than a problem with the way these studies were designed and conducted, but it is likely that a majority of studies using in vivo models of emesis fail to use validated methods to randomize and conceal treatment allocations. Future studies must consider these issues.

While taking into account the editorial policies of the journals where the research is published, studies using the ferret model should ideally be reported in accordance with the ARRIVE² guidelines, which were developed to improve the design and reporting of in vivo research [211]. The guidelines consist of a 20-item checklist summarizing the key information that has to be included in a manuscript to ensure that a study contains enough information to be peer reviewed or reproduced.

The study design should be clearly described, including steps taken to minimize subjective bias, which as noted earlier is often lacking in studies reporting emetic research. Studies should also include a comprehensive description of the experimental procedures and exactly how, when, and where procedures where carried out and why they were carried out this way. The experimental outcomes should also be defined properly. As mentioned in section “Assessing the Translational Value of the Ferret Model,” the emetic response is characterized by its intensity, its incidence, and the latency to its onset, all of which have to be defined clearly to enable any comparison between studies. For example, one should specify if the latency was measured as the time to the onset of the first retch or to the first vomit. The intensity of the emetic response can be measured in many different ways with the most precise being the number of retches and the number of vomits. Some studies also report the number of emetic episodes or bouts of emesis and need a clear definition for the reader to be able to determine what constitutes an episode. Are only vomiting episodes measured or is a series of retches considered an episode? What separates one episode from the next? In terms of incidence, it is crucial to specify what behavior was under investigation, be it the retching, the vomiting, or any other behavior as the findings would be completely different. In addition, any measure of intensity or incidence without specifying the duration of the observation period is pointless. A “nausea” index also needs to be explained, what behaviors were specifically recorded, how were these behaviors recognized and defined?

Factors Influencing Experimental Outcome

Experiments inherently include factors that may influence the outcome or the interpretation of the results and this applies equally to studies of emesis. It is essential that there is transparent reporting of all relevant experimental details using guidelines such as ARRIVE [211]. In addition, it is also important that the behavioral effect of a drug is described. If a candidate antiemetic agent has sedative properties, it may be difficult to separate a true antiemetic from the sedative action (see Ref. 212 for discussion of this problem). An objective assessment of potentially confounding factors can often only be made once there is an accumulated body of data using a particular model. In the case of the acute cisplatin (10 mg/kg, i.v. or i.p.) emesis model in ferrets, systematic analysis of evaluable published data over ∼30 years has revealed a number of factors to be summarized later. While the analysis is from studies of cisplatin [142], the findings serve as a caution for other studies where comparable analyses have not been undertaken.

Animals

Many clinical studies have detected a difference in the incidence of postoperative and chemotherapy-induced nausea and vomiting depending on the age and gender of the patients [213–215], and it is safe to assume that animal studies reflect these differences. While the majority of studies have used groups of male ferrets, some have used mixed male and female groups. Although there was no difference in the latency of emesis between male only and mixed groups, the magnitude of the emetic response was smaller in the mixed groups [216]. The latency of emesis was shorter in groups composed only of albino animals compared with mixed albino and fitch (pigmented) groups, but there was no difference in the magnitude of the response. The country of origin of the animals also affected the response with animals from New Zealand having a longer latency to the onset of emesis (and fewer retches and vomits) compared with those from either the United Kingdom or the United States. Although these effects are all relatively small, they all achieved statistical significance (data in Ref. 142). Of note, the use of mixed sex or mixed strain groups in emesis studies is likely to increase the variability of the response within the group, therefore decreasing the power of the statistical analysis and increasing the number of animals needed without yielding more information. A more efficient way of ensuring that results can be generalized would be to use sex or strain as an independent variable to investigate whether they influence the results.

Housing conditions may also influence the experimental outcome. Variables such as the number of cage companions, bedding material, or environmental enrichment are crucial to interpreting the findings of any behavioral study, for example, the natural burrowing behavior observed in ferrets is dependent on the presence and quantity of sawdust on the cage floor. Specifically relevant to emetic studies, the type of bedding can modulate levels of antioxidants such as ascorbic acid and glutathione in rats [217], both of which have antiemetic properties against cisplatin-induced emesis in dogs [218]. In addition, ferrets might ingest some of the bedding material, which could also have an impact on the emetic response.

Route of Administration and Anesthesia

The route of administration of a drug under study should ideally mimic the intended route of administration in humans, and this is especially the case in safety pharmacology studies [219]. Although it is possible to administer drugs intravenously in ferrets using a superficial vein in the forelimb, the more usual method is to implant a chronic jugular catheter (see section “Techniques”), which also allows serial blood sampling for pharmacokinetic studies.

When cisplatin is administered intravenously to ferrets (and other animals), it is given as rapidly as possible. In the clinic, however, chemotherapeutic agents are usually given by intravenous infusion to reduce toxicity, including emesis [220]. In smaller laboratory species such as the ferret, the intraperitoneal route if often used for administration of cytotoxic drugs although it is a very rarely used clinical route. Both intraperitoneal and intravenous routes of administration have been used to investigate the acute phase of cisplatin and cyclophosphamide-induced emesis in ferrets with the latency, pattern, and magnitude of the response being similar with both routes. However, the response induced by the intravenous route of administration is more readily blocked by abdominal visceral nerve lesions, suggesting that the abdominal cavity may become sensitized following surgery [44].

Systematic review of the route of administration showed that the latency of emesis was 1.16 ± 0.35 hours (n = 277) following intravenous cisplatin (10 mg/kg) compared with 1.51 ± 0.29 hours (n = 134) following intraperitoneal administration of the same dose (P < 0.0001). However, the route of administration is also related to the use of anesthesia. General anesthesia has been used for both chronic implantation of a jugular catheter for cisplatin administration [31] and also to enable direct jugular administration of cisplatin [45]. Anesthesia is not used prior to intraperitoneal administration. The use of anesthesia (either volatile or injectable) either 3 hours or 3 days prior to administration of cisplatin reduced the latency of the onset of emesis [216]. There may also be variation in the impact of different anesthetic regimens on the emetic response. The volatile anesthetic sevoflurane, for example, has been shown to reduce the levels of the endocannabinoid anandamide, whereas injectable agents such as propofol do not impact blood anandamide concentration [221]. Considering that endocannabinoids, and anandamide, in particular, play an important role in the physiological control of emesis [84], the anesthetic regimen used is a variable that might influence the interpretation of the effect of an antiemetic drug and should be reported.

The subcutaneous route (usually scruff of the neck) is frequently used for administration of antiemetic agents, and this route is well tolerated. It has also been used for administration of a known emetic (e.g., apomorphine, loperamide) or suspected emetic agent. Few studies have compared the two routes, however, but this should be carefully considered when investigating novel agents. As Andrews et al. [38], for example, found that it was possible to identify an ED₁₀₀ dose for apomorphine administered by the subcutaneous route while it was not possible for apomorphine administered by the intravenous route.

The intragastric (gavage) route of administration of emetic challenges such as copper sulfate, hypertonic solutions, and ipecacuanha is well established in ferrets. In our experience, the typical rigid dosing needle used for rodents is not suitable because of the short length and rigidity. The method commonly used is modification of that described by Pyle [222] and involves gently passing a flexible Portex catheter (size 0/0 or smaller, 30 cm long) through a hole in a polypropylene T- piece placed between the jaws with an assistant holding the animal vertically. The length of tube inserted is based on external measurement of the estimated location of the stomach. This method has most often been used to administer a range of emetic agents using volumes up to 50 mL (depending on animal size). Ferrets will readily drink milk at a relatively fixed rate (Ref. 223; note that this should only be given infrequently to minimize the possibility of diarrhea). While it may be possible to administer some food based toxins and drugs with emetic liability via this route, it is not really practical for mucosal irritants such as copper sulfate and hypertonic saline. Drinking should be considered as a route for administration of novel potential antiemetic drugs intended for prophylactic use.

Improving the Model, What are the Limits?

The cisplatin model in ferrets has clearly been important in the development of two antiemetic agents currently used in the clinic. Detailed analysis of the way in which the efficacy of the agents in preclinical studies of acute and delayed emesis translated to the clinic revealed some disparities. A question arising from this analysis is whether the model could be refined to improve its predictive validity for investigations of candidate novel antiemetics. To illustrate the issues, many of which have wider applicability to animal models, one needs a clear vision of that which is being modeled. In both the acute and delayed cisplatin models, the intent is to give a dose of cisplatin sufficient to induce an emetic response in all animals so that the effect of an antiemetic can be investigated. This differs in many ways from what happens in the clinic and some of the differences, together with some areas of uncertainty, between the ferret cytotoxic drug-induced emesis model and the patient in the clinic are summarized in Table 31.4. The impact of any of these factors on the emetic response to cisplatin and hence the efficacy of candidate antiemetics can be hypothesized but is not known with certainty. The issue that arises is how close one should attempt to make the ferret model of cisplatin-induced emesis to a model of a cancer patient undergoing therapy? For example, are there scientific reasons to conclude that a tumor-bearing ferret given cisplatin would be a more relevant model, and if so, could such studies be justified ethically? Some case could be made if evidence emerged that the presence of a tumor modified the emetic mechanism activated by cisplatin. While the example of chemotherapy-induced emesis is quite specific, it illustrates some of the problems studying a clinical problem in healthy animals and the extent to which we should “make” the animal more like the patient in an attempt to improve translation.

Table 31.4. A Detailed Comparison of the Ferret Model of Emesis Induced by a Chemotherapeutic Agent (or Radiation) Based Primarily on the Response to Cisplatin Compared With a Patient with Cancer Undergoing Chemotherapy

Parameter	Human patient with cancer	Ferret “model”
Class	Mammalia	Mammalia
Order	Primates	Carnivora
Species	Homo sapiens	Mustela putorius furo L.
Tumor	Present	Absent
Surgery (requiring general anesthesia)	In some patients to reduce/remove tumor	In some animals to implant catheter, telemetry devices, lesion pathways
Hydration	Infusion with chemotherapy	No infusion
Psychological status	Knowledge of disease prognosis, treatment outcome, and possible side effects	No knowledge but likely “stress” from handling and injection
Environment	Hospital ward/Day center. Depending on setting may be exposed to others undergoing treatment (sight, sound, olfaction)	Home cage/new cage to which animal is adapted.
Environment		Depending on setting may be exposed to others under study (sight, sound, olfaction)
Emetic history	Emetic pathways highly likely to have been activated by motion and food poisoning early in life and in some cases by alcohol consumption, attempts at tobacco smoking, medication other than for cancer, pregnancy and surgery/anesthesia (PONV)	Emetic pathways unlikely to have been activated in laboratory bred and reared animals not previously involved in any emetic research
Factors known to influence emetic response to chemotherapy agent treatment	Age, sex, previous emetic history	Strain, country of origin, previous surgery
Nutritional status	May be impaired status	Normal
Concomitant medication (not antiemetics)	Probable	No
Access to food and fluid	Yes to both	Yes, but depends on nature and duration of the study
Chemotherapy agent/radiation dose and rate of administration	Optimized to attempt to reduce incidence of emesis (and other side effects)	Optimised to induce emesis
Purpose	To treat/palliate cancer	To induce emesis
Number of cycles of cytotoxic administration	Multiple	Single
Cisplatin dose	50–120 mg/m² (approximates to 1–3 mg/kg)	Acute—10 mg/kg
Cisplatin dose	50–120 mg/m² (approximates to 1–3 mg/kg)	Acute and delayed—5 mg/kg
Latency to onset of acute phase of emesis	∼1.5–2.5 hours (dose dependent)	∼1.5–10.5 hours (dose dependent)
Relative severity of acute and delayed phases	Acute > delayed	Delayed > acute
Antiemetics given	Yes	No, unless part of the study
Efficacy of 5-HT₃ receptor antagonists in acute phase of cisplatin-induced emesis	Incidence reduced by 50–60%,	Incidence reduced by 30–60%, intensity reduced by 65–90%
Efficacy of 5-HT₃ receptor antagonists in delayed phase of cisplatin-induced emesis	Incidence reduced by 15%	Acute phase intensity reduced by 70% and delayed phases reduced by 60%
Efficacy of NK₁ receptor antagonists in acute phase of cisplatin-induced emesis	No placebo-controlled studies, efficacy comparable to 5-HT₃ receptor antagonists	Abolish acute phase
Efficacy of NK₁ receptor antagonists in delayed phase of cisplatin-induced emesis	Delayed phase incidence reduced by 30–50%	Abolish both acute and delayed phases
Monitoring of retching and vomiting	Occurrence noted, episodes may be quantified usually by retrospective self-reporting; some studies have used independent assessment by clinical staff	Precise quantification possible in real time by direct observation and telemetry
Monitoring of nausea	Self-reporting, usually retrospective but assumes all patients are reporting the same phenomenon.	No direct measurement but animal behavior can be quantified precisely although interpretation is very controversial
Monitoring physiological parameters	Cardiovascular (SDSD of heart rate); gastric electric activity (EGG); hormones (ADH, cortisol)	Cardiovascular (heart rate, BP); respiration (intrathoracic and abdominal pressure)
Monitoring physiological parameters		Gastric electric activity (EGG); hormones (ADH, cortisol)

While we may contemplate optimizing animal models to more closely mimic a patient, at least superficially, there are two “fixed” factors we must be mindful of in this area. First, although comparisons of the neurophysiology of the brainstem (e.g., Horn et al., 2013) and other brain regions can be made readily between the animal models, similar data are not available for humans. The extent of the comparisons is limited largely, therefore, to anatomical and clinical investigations. The key functions of the brainstem in regulation of respiratory, cardiovascular, and digestive systems are conserved in mammals (and arguably all vertebrates), so it is not unreasonable to assert that the major pathways involved in the reflex responses of retching and vomiting are likely to be the same in humans and these animal models. The projections to the higher regions of the brain likely to be involved in the sensation of nausea are as yet poorly defined in humans (but see section “Detecting Nausea”), so comparison with the very limited data on such projections in animal models, in general, and ferrets, in particular, is even more problematic. Secondly, even if we assume that the fundamental pathways (e.g., vagal afferents, area postrema, vestibular system) involved in vomiting are similar between humans and the animal models, this only provides a model in which the pathway by which, for example, a drug (e.g., cisplatin) can induce emesis. However, this assumes that the drug utilizes the same pathway to the same extent in humans and the animal model. Again, data on humans is lacking in most cases. For the model to have utility in identification of novel anti-emetic agents, both the pathway and the key neurotransmitters and associate receptor subtypes must be the same in humans and the animal model. This is particularly problematic as so little is known about the transmitters and receptors in key pathways in humans; so often the studies in animal models are making the assumption that the pathway pharmacology is comparable to humans. Most information in humans comes from studies of brain disease (e.g., Alzheimer's disease) and efficacious pharmacological therapies (e.g., dopamine receptor agonist for Parkinson's Disease) although this situation is improving with PET studies in healthy volunteers (e.g., NK₁; Hargreaves [224]). For an anti-emetic, we currently argue that an animal model is predictive of humans if it demonstrates efficacy to the same challenge in humans and non-predictive if it does not. However, if we better understood the neuropharmacology of the key pathways in humans, preclinical models could be selected and refined on a more rational basis.

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