The Effect of 5-HT2C Receptor Activation on Nausea-Induced Behaviour in Rats

Leonardo Berardo Silenieks

A Thesis presented to The University of Guelph

In partial fulfilment of requirements for the degree of Master of Science in Biomedical Science (Neuroscience)

Guelph, Ontario, Canada © Leonardo Berardo Silenieks, August, 2014

ABSTRACT

THE EFFECT OF 5-HT2C RECEPTOR ACTIVATION ON NAUSEA-INDUCED BEHAVIOUR IN RATS

Leonardo B Silenieks Advisor: University of Guelph, 2014 Professor Neil MacLusky Professor Linda A. Parker

The present study aimed at investigating the role of the 5-HT2C receptor in nausea using different models of nausea-induced behaviour in rats. Two chemically distinct 5-HT2C agonists (lorcaserin and CP-809101) were evaluated using the conditioned gaping model. At therapeutically relevant doses, lorcaserin caused rats to display conditioned gaping reactions. This effect was successfully blocked by pre-treating the animals with the 5-HT2C antagonists SB-242084, suggesting that this receptor is involved in the genesis of nausea. In contrast, CP-809101 did not produce conditioned gaping in rats administered with doses higher than its efficacy range in feeding studies. These results suggest that the relevance of this receptor in nausea may be more complex than previously considered. It invites the question of whether functional selectivity may be the reason for the differences between side effect profiles of these two serotonergic drugs.

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Acknowledgements

My master's thesis was only possible because of the support I've received along the way. I would like to formally thank my supervisor Dr. Linda Parker for not only giving me this opportunity, but also for being able to guide me through this project, from my research background through her area of expertise. Dr. Parker made my Masters experience an extremely valuable one through her insight, patience and ability to find ways to make it all possible. It was a pleasure to see her passion and dedication to scientific research and I know I will take some of that passion with me in my future endeavors. I am very grateful and feel privileged to have had her as my supervisor.

I wish to thank my other supervisor, Dr. Neil MacLusky, for his input and contribution to my thesis. Through the last two years he has showed me that with hard work it really is possible to achieve my goals, and he allowed me to find and follow my own path. I would also like to thank Dr. Guy Higgins for seeing the potential in me and being my mentor for the last five years. His guidance has really helped me understand what my passions are and how to follow them.

I would also like to thank Dr. Cheryl Limebeer for all her help, flexibility and technical expertise. There are few people that have such a large breadth of skills and knowledge combined with the will to teach them. I would also like to thank my fellow lab mates for making the Parker lab such a hospitable place, where help is available down the hallway at any time, especially Erin Rock and Martin Sticht for their help. I would also like to thank some of my other lab mates from my other lab, Winnie Lau, Amy Patrick and Sandy Thevarkunnel, for their friendship and willingness to help.

I thank my dear wife and best friend, Carlin Sweeney, who has been my partner in so many ways in our last twelve years. I would not be able to accomplish so much without her wisdom, love, inspiration and stoicism. Without her, none of this would be possible.

Lastly, I would like to thank my parents for their unending support throughout my life. They have inspired me to work hard and love science since I was young, and I know that I am only where I am because they helped me get here. I will simply never be able to thank them enough, so I dedicate this thesis to them.

Table of Contents Abstract ...... ii

Acknowledgements ...... iii

List of Abreviations ...... vi

List of Figures ...... vii

Chapter 1: General introduction...... 1

1.1 Nausea and emesis ...... 2

1.2 Serotonin and its role in nausea and emesis ...... 6

1.3 5-HT3 receptor ...... 7

1.4 5-HT1A receptor ...... 8

1.5 5-HT2 receptor ...... 9

1.6 Functional selectivity and serotonergic receptors ...... 11

1.7 Models of emesis and nausea in the rat ...... 13

1.8 Conditioned flavour avoidance ...... 15

1.9 Conditioned taste aversion and the taste reactivity test...... 16

1.10 Pica ...... 18

1.11 Present studies ...... 19

Chapter 2: Effects of 5-HT2C receptor agonists Lorcaserin and CP-809101 in the taste reactivity and conditioned flavour avoidance models in rats ...... 21

2.1 Introduction ...... 21

2.2 Methods ...... 24

Animals and housing...... 24

Surgery ...... 24

Apparatus ...... 25

Drugs and treatment regimens ...... 25

Experiment 1 – Effect of lorcaserin in the test reactivity and flavour avoidance models .... 26

Experiment 2 – Effect of CP-809101 in the taste reactivity and flavour avoidance models 27

Experiment 3 - Effect of systemic SB-242084 pretreatment on Lorcaserin-induced conditioned gaping and flavour avoidance ...... 27

Data Analysis ...... 28

2.3 Results ...... 28

Experiment 1 – Effect of lorcaserin in the test reactivity and flavour avoidance models .... 28

Experiment 2 – Effect of CP-809101 in the taste reactivity and flavour avoidance models 29

Experiment 3 – Effect of systemic SB-242084 pretreatment on Lorcaserin-induced conditioned gaping and flavour avoidance ...... 31

2.4 Discussion ...... 32

2.5 Conclusion ...... 35

2.6 Figures ...... 37

Chapter 3: Lorcaserin does not induce pica behaviour in rats ...... 44

3.1 Introduction ...... 44

3.2 Materials and Methods ...... 46

Animals and housing...... 46

Drugs and treatment regimens ...... 46

Test conditions ...... 47

Data analysis ...... 47

3.3 Results ...... 47

3.4 Discussion ...... 48

3.5 Figures ...... 50

Chapter 4: General discussion ...... 51

4.1 Conclusion ...... 56

References ...... 56

List of Abreviations

2-AG - 2-Arachidonoylglycerol 5-HT - Serotonin 8-OH-DPAT - 7-(Dipropylamino)-5,6,7,8-tetrahydronaphthalen-1-ol AA - Arachidonic acid ANOVA - Analysis of variance AP - Area postrema CBD - Cannabidiol CFA - Conditioned flavor avoidance CNS - Central nervous system CSF – Cerebrospinal fluid CTZ - Chemoreceptor trigger zone DIO - 2,5-dimethoxy-4-iodoamphetamine FDA - Food and Drug Administration GABA - Gamma-Aminobutyric acid GI - Gastrointestinal IP - Inositol phosphate LiCl - Lithium chloride LSD - D-lysergic acid diethylamide mACh - Muscarinic acetylcholine mCPP - Meta-Chlorophenylpiperazine NCE - New chemical entity NK1-r - Neurokinin 1 receptor NTS - Nucleus of the solitary tract PK - Phamacokinetics PLA2 - Phospholipase A2 PLC - Phospholipase C TFMPP - Trifluoromethylphenylpiperazine TRT - Taste reactivity test

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List of Figures

Figure 1. Schematic diagram of known neural mechanisms involved in nausea and emesis...... 5 Figure 2. Effect of saccharin-lorcaserin pairing in the taste reactivity test ...... 37 Figure 3. Effect of saccharin-lorcaserin pairing on tongue protrusions ...... 38 Figure 4. Effect of saccharin-lorcaserin pairing in a conditioned flavour avoidance test ...... 39 Figure 5. Effect of saccharin-CP-809101 pairing in the taste reactivity test ...... 40 Figure 6. Effect of saccharin-lorcaserin pairing on tongue protrusions ...... 41 Figure 7. Effect of saccharin-CP-809101 pairing in a conditioned flavour avoidance test ...... 42 Figure 8. Effect of SB-242084 on lorcaserin-induced gaping and flavour avoidance ...... 43 Figure 9. Effect of lorcaserin on rat pica behaviour ...... 50

Chapter 1: General introduction

Serotonin (5-hydroxytryptamine, 5-HT) is one of the major modulating neurotransmitters in animals. The proliferation of research into this signalling molecule has shown that serotonin is responsible for regulating a considerable number of physiological processes. For this reason research into ways of manipulating the serotonergic system has yielded a large number of drugs and therapeutic options for a variety of conditions including depression, nausea, schizophrenia and obesity (Higgins et al. 2013). As recently as 2012, a selective 5-HT2C receptor agonist, lorcaserin, was approved for the treatment of obesity. This was significant as it was the first anti- obesity drug to be approved in 13 years and it was a direct outcome of a research thread that began with early work investigating the potential for non-selective serotonergic compounds to reduce feeding (Chan et al. 2013, Thomsen et al. 2008).

Early pre-clinical studies revealed that treatment with meta-Chlorophenylpiperazine (mCPP, a non-selective serotonergic agonist) was able to reduce food intake in animals, suggesting that the serotonergic system played some role in the regulation of feeding (Samanin et al. 1979). These early findings eventually led to the development of the 5-HT releaser and reuptake inhibitor Dexfenfluramine (Redux®), which was shown to have promising potential as an anorectic drug (Guy-Grand et al. 1989). Further examination of the effects of dexfenfluramine and fenfluramine using selective antagonists such as SB-242084, revealed that the 5-HT2C receptor was likely the pharmacological target capable of modulating appetite (Vickers et al.

2001). This hypothesis was confirmed after several other 5-HT2C receptor agonists were shown to have anorectic properties that could be reversed via 5-HT2C receptor antagonism (Martin et al. 1998). Furthermore, mutant mice lacking this receptor are known to develop obesity and are insensitive to the hypophagic properties of dexfenfluramine (Tecott et al. 1995).

Several compounds that target the serotonin 2C receptor have been synthesized since these early discoveries and have been studied in the attempt to find a promising candidate drug to treat obesity, this culminated in the aforementioned approval of lorcaserin. Lorcaserin is a highly selective 5-HT2C receptor agonist with therapeutically useful anorectic properties (Thomsen et al.

2008). However, while this drug has been successful in terms of addressing obesity, both clinical and preclinical work have suggested that at high (yet relevant) doses, the drug can cause nausea and general malaise in patients and laboratory animals. In humans, it was reported that 21% of patients receiving 20 mg of locaserin experienced nausea compared to the placebo treatment group in which no subjects reported this side effect. Preclinical work with monkeys also demonstrated that lorcaserin can produce emesis at very high doses (125 mg/kg/day) while a recent study in rats has also reported behavioural signs of gastrointestinal discomfort (Arena Pharmaceuticals, 2010 FDA briefing document NDA 22-529, Higgins et al. 2013). These observations led to the question of whether lorcaserin-induced nausea is yet another effect of modulating centrally located 5-HT2C receptors. Research into the role of this receptor in nausea is virtually non-existent, therefore the principal goal of this thesis is to investigate some of the unexpected effects of systemic exposure to selective 5-HT2C receptor agonists, in particular nausea.

1.1 Nausea and emesis

Emesis (the process of vomiting) is a rather common experience in humans and many other animal species. While it is an unpleasant experience, it does arise from an evolutionary need to expel toxic and potentially dangerous materials from the digestive system. Similarly, the experience of nausea also results in the avoidance of potentially harmful foods by decreasing appetite. Evolutionarily this system was likely a very important aspect of foraging, where species in certain environments are required to experiment with novel sources of energy and sometimes with foods of inferior quality. During this process, animals may ingest food contaminated with harmful bacteria, toxins, fungi, viruses and other parasites which can be harmful and potentially fatal. Therefore the presence of this mechanism increases the fitness of the species in the evolutionary sense of the word.

While an emetic response is easily observed and quantified, nausea is not. Unlike the experience of vomiting, nausea is a much more subjective process. It tends to involve feelings of gastrointestinal discomfort, dizziness, sweating and salivation. Nausea must also have an

3 evolutionary role; it has been postulated that nausea is a sensation that becomes associated with toxic foods that have been consumed in the past, resulting in subsequent avoidance of these foods (Garcia et al. 1974; Parker, 2003). Nausea can be potentially debilitating and is one of the primary causes of patient non-compliance to drugs (Schwartzberg, 2007), for this reason it is indeed a clinically relevant area of research, particularly when considering how much is still unknown regarding this topic. The subjective nature of nausea, however, does pose an experimental challenge, since it is much more difficult to measure such an event with an animal model than it is to measure the occurrence of an emetic response.

While nausea and vomiting are related concepts to the general public, they are distinct responses that have related yet different mechanisms, allowing them to occur independently in some cases. To the scientific community, emesis and nausea pose very different challenges. Modern pharmacological treatments are very effective in managing emesis; however treating nausea has been a much more difficult endeavor (Andrews and Horn, 2006).

Nausea and emesis can occur as a response to a variety of conditions. These conditions can include the ingestion of contaminated foods, exposure to emetogenic medications, pregnancy, stress, concussions, overstimulation of the vestibular system, etc. Such diversity of potential causes suggests a multitude of input modalities that are able to trigger these two physiological responses. Inputs can originate directly from the Central Nervous System, for example, following a concussion or during a turbulent boat ride (via the vestibular apparatus), conversely inputs may be originating in the periphery, in the case of food poisoning, where these signals travel via the vagus nerve to the vomiting centre in the lateral ventricular formation of the medulla oblongata (Borison and Wang, 1948). Another key region for the induction of emesis is the area postrema (AP) near the fourth ventricle, where chemical emetic stimuli have been shown to be detected by cells in an area now known as the chemoreceptor trigger zone (CTZ), which in turn activates the vomiting centre (Barnes , 1984). The CTZ is often thought to be the area responsible for triggering vomiting caused by circulating chemicals (Andrews, 1992).

The current model suggests that all these inputs are processed in a central integrating region of the nucleus of the solitary tract (NTS), located at the base of the brainstem (Horn, 2008). Integration of inputs is then directed to the appropriate efferent pathways. In emetic events, these can be motor pathways producing gastric movements, to facilitate the expulsion of

4 gastric content, as well as respiratory motor responses, increasing abdominal pressure while decreasing thoracic pressure (Horn, 2008). Autonomic responses such as cold sweating, salivation and vasoconstriction are also activated during vomiting; however some researchers believe that they may result from the stress of nausea. (Andrews and Horn, 2006 Signals for nausea and emesis)

Rostral projections from the brainstem to the insular cortex and amygdala are involved in higher processing of inputs; these regions appear to be more directly associated with the genesis of nausea, while having little involvement with emetic processes. (Andrews and Horn 2006; Limebeer et al. 2004; Limebeer and Parker 2000). Processed output signals from these "higher" cortical regions are then responsible for producing the previously mentioned prodromal autonomic symptoms.

Figure 1. Schematic diagram of known neural mechanisms involved in nausea and emesis. Inputs from a variety of neurotransmitter systems can occur at several different sensory systems and are carried by afferent fibers to the nucleus tractus solitarus, located in the brainstem. The NTS is important in processing these inputs and orchestrating output signals responsible for the manifestation of prodromal signs of vomiting, associated autonomic responses and the recruitment of mechanical processes involved in emetic responses and nausea. Both nausea and emesis share much of this circuitry, however they can both occur independently from each other. (Andrews, 1992; Horn, 2008; Harris, 2010)

1.2 Serotonin and its role in nausea and emesis

The development of cancer treatments in the 1960's benefited tremendously from the discovery that cisplatin could regress the development of tumors in rats (Rosenberg et al. 1969). This treatment, however, produced significant side effects including severe nausea and emesis in patients giving rise to a new clinical challenge; how to administer chemotherapy agents such as cisplatin without the discouragement of its side effects. One of the first candidate drugs was metoclopramide, which is mostly a dopamine 2 (D2) receptor antagonist with some 5-HT3/5-HT4 receptor antagonist properties. Not long after, it was established that the 5-HT3 receptor antagonist properties of metoclopramide compounded its D2 antiemetic effects (Costall et al, 1986). These new findings rekindled the scientific interest in serotonin as a potential antiemetic target.

Serotonin is a monoamine neurotransmitter derived from the amino acid tryptophan. Serotonin is a major modulating neurotransmitter found in abundance in the gastrointestinal tract while also having a very important role in the central nervous system. In the GI tract, most of the serotonin is produced by enterochromaffin cells that line the gut; this accounts for the vast majority of the serotonin synthesized in the body (Gershon and Tack, 2007). In the central nervous system, serotonin is a product of neurons from the raphe nuclei, located near the midline of the brainstem and around the reticular formation (Hornung, 2003). Serotonergic projections from the raphe nucleus reach most regions of the cortex, allowing it to modulate other neurotransmitter systems such as GABA, acetylcholine, dopamine and glutamate in a variety of ways. The modulating nature of serotonin is possible due to the variety of its receptor types distributed in the nervous system. There are 7 families of 5-HT receptors (Raymond et al. 2001), which are mostly G-protein coupled receptors with the sole exception being the 5-HT3 receptor, which functions through a cys-loop ligand-gated ion channel (Lummis, 2012).

1.3 5-HT3 receptor

Research in emesis and nausea up to this point has been primarily focused on the 5-HT3 receptor subtype. Serotonin released by enterochromaffin cells in the gut activate 5-HT3 receptors which send ascending signals through the vagus nerve directed to the nucleus of the solitary tract (Horn, 2008). The area postrema (AP) and nucleus tractus solitarius (NTS) are another pertinent region for nausea and vomiting, and are also rich in 5-HT3 receptors (Pratt and Bowery, 1989). While there are still questions regarding the exact role of the AP in these processes, it does appear to be important in the emergence of emetic responses. 5-HT3 antagonism with ondansetron has been shown to inhibit cisplatin-induced vomiting in ferrets when delivered directly to the vicinity of the AP/NTS region (Higgins et al. 1989). This area may act as a chemoreceptor system able to monitor serotonergic fluctuations, and 5-HT3 receptors may serve to relay emetic signals from the bloodstream to the NTS, which is important since serotonin is unable to readily cross the blood brain barrier (Johnston et al. 2013).

While this is not the only receptor used for the treatment of emesis and nausea, 5-HT3 antagonists are currently one of the primary pharmacological targets used clinically to treat nausea and vomiting produced by chemotherapy, radiotherapy as well as postoperative nausea and emesis. Preclinical work using current animal models also demonstrate that this target is useful for the treatment of emesis from some sources; it is unable to ameliorate symptoms caused by others. Experiments using the shrews, dogs, ferrets and cats demonstrate that different antagonists for this receptor successfully inhibited emesis induced by chemotherapeutic agents (Torii et al. 1991a). The same study also showed that these antagonists are not able to block emetic responses from other emetic stimuli such as high doses of nicotine, copper sulfate and motion. This data is reflective of the fact that while these antagonists are clinically efficacious in the treatment of some types of nausea and emesis, many others require different interventions such as dopaminergic D2 antagonists, NK1 receptor antagonists or corticosteroids (Becker, 2010).

Some examples of 5-HT3 receptor antagonists used as antiemetics are Ondansetron (Zofran), Granisetron (Kytril), Dolasetron (Anzemet), Palonosetron (Aloxi) and Tropisetron (Setrovel) which are all fairly effective in treating emesis while having mixed efficacy in

8 eliminating treatment associated nausea (Parker, 2013). For this reason an emphasis in producing pharmacological solutions to treat nausea is becoming a priority over solutions to treat emesis.

1.4 5-HT1A receptor

5-HT1 receptors make up another group of serotonergic receptors that have shown to be relevant to our understanding of nausea and vomiting, in particular the 5-HT1A receptor subtype.

The 5-HT1A autoreceptor is a serotonergic regulating receptor found in dendrites and neuronal cell bodies, mostly in the dorsal and median Raphe Nuclei (Verge et al, 1985).

Studies by Lucot and Campton in the 1980's demonstrated that agonists to this receptor can block emesis from multiple input modalities. For instance, cats did not vomit during a motion sickness experiment after a pretreatment of the 5-HT1A receptor agonist Buspirone (Lucot and Crampton, 1987a). Buspirone was also able to block emetic responses generated via systemic injection of xylazine as well as cisplatin, both of which are known emetogens (Lucot and Crampton, 1987b). Following this research thread, the Lucot group and other groups also demonstrated similar antiemetic properties from 8-OH-DPAT, a selective 5-HT1A receptor agonist, commonly used to research this receptor target (Lucot and Crampton, 1989; Okada et al, 1994). Furthermore, research investigating the properties of the cannabinoid Cannabidiol (CBD), has also suggested that its antiemetic properties are likely to be associated to its affinity for the

5-HT1A receptor (Russo et al. 2005; Rock et al. 2012). Buspirone, Canabidiol and other 5-HT1A agonists with antiemetic properties are thought to block emetic signals through a decrease in serotonergic firing, reducing overall serotonergic tone in the Raphe Nuclei. Despite findings showing that 8-OH-DPAT can suppress nausea-induced behaviour in rats during taste reactivity tests, clinical application of these findings have been limited (Limebeer and Parker, 2003)

1.5 5-HT2 receptor

The serotonin 2 receptor class family has made only a few appearances in the nausea and emesis literature. While there have been empirical suggestions that these receptor subtypes may be somehow involved in these two processes, little attention has been paid mainly due to either the small nature of pharmacological effects or inconsistencies between agonists for the same receptor.

Indication that this receptor family is involved in nausea and emesis began with a study that demonstrated that LSD (D-lysergic acid diethylamide) has antiemetic properties (Dhawan and Gupta, 1961). LSD has potent dopaminergic and serotonergic agonist properties. It is an agonist for most serotonin receptors, and it has particularly high affinity for the 5-HT2A and 5-

HT2C receptors while having little 5-HT3 receptor activity (Appel et al. 2004, Gresch et al. 2005).

More recently, in an experiment where 5-HT was injected systemically into House musk shrews (Suncus murinus), vomiting was successfully blocked by tropisetron (selective 5-HT3 antagonist) as well as pindolol (partial 5-HT1A agonist/antagonist), mianserin (5-HT2 antagonist) and ketanserin (5-HT2 antagonist) (Torii et al. 1991a). Experimental data has also been published showing that the 5-HT2 receptor partial agonist DOI (2,5-dimethoxy-4-iodoamphetamine) is able to prevent house musk shrews from displaying emetic responses induced by chemotherapeutic agents as well as motion (Okada et al. 1995). A later study involving DOI and ketanserin has also demonstrated that these agonists are able to inhibit motion-induced emesis in the shrew.

This study also showed that the peripheral 5-HT2A /5-HT2C receptor agonist alpha-methyl-5-HT had no impact on motion- and cisplatin-induced vomiting in Suncus murinus, suggesting that perhaps only central 5-HT2 receptors participate in the emetic process (Javid and Naylor, 2002; Ismaiel et al. 1990, Okada et al. 1995).

A potentially unexplored mechanism in which 5-HT2 receptors may be relevant to nausea and emesis research involves changes in the concentration of circulating ghrelin. Ghrelin is a hormone mainly produced in the stomach and pancreas and is primarily involved in the stimulation of feeding behaviour. In experiments where rats were exposed to cisplatin, animals

10 displayed a significant reduction in feeding along with behavioural signs of severe malaise (Takeda et al. 2008). These effects have been partly attributed to increases of circulating serotonin produced by enterochromaffin cells (Refer to section 1.3 for more details about this process). This increase in serotonin is known to activate 5-HT3 receptors, driving one of the main pathways in the genesis of emesis and nausea. This general surge of serotonin also activates centrally located 5-HT2B and 5-HT2C receptors via the vagal input, which is known to reduce plasma levels of ghrelin, potentially resulting in a suppression of feeding. In addition, treatment with cisplatin has been shown to specifically increase 5-HT2C receptor gene expression in the hypothalamus further linking chemotherapy side effects to serotonergic receptors beyond the 5-

HT3 receptor (Yakabi et al. 2010). In a study by Takeda et al., rats that were treated with intravenously delivered ghrelin have been shown to be more resilient to decreases in food intake following exposure to cisplatin. The study also looked at the effects of 5-HT2B and 5-HT2C receptor antagonists (SB-215505 and SB-242084 respectively) and found that these drugs were not only able to increase the animal’s food intake but also increase ghrelin levels in the cisplatin treated animals (Takeda et al. 2008). Furthermore, treatment with the 5-HT3 receptor antagonist ondansetron was unable to improve food intake of cisplatin treated animals despite its antiemetic properties. While the Takeda group presumed that the 5-HT2B and 5-HT2C receptors were not involved in the emesis and nausea produced by cisplatin, this view was mostly speculative and relied on assumptions that had not been tested.

A study by Rudd et al. investigating the role of ghrelin in ferrets demonstrated that this hormone does have anti-emetic potential against cisplatin exposure (Rudd et al. 2006). These findings may therefore suggest that 5-HT2 receptor subtypes may indeed have a role in nausea and emesis. A potential mechanism for this involvement may be a decline in circulating ghrelin resulting from 5-HT2C receptor activation, which increases the organism's vulnerability to emetic stimuli. This proposed mechanism remains consistent with the findings showing that antagonism of this receptor decreases cisplatin-induced nausea via ghrelin release.

Overall however, the picture painted by studies investigating the involvement of 5-HT2 receptors in nausea and emesis is inconclusive and at times even contradictory. While publications such as the article by Torii (1991a) and his group suggest a pro-emetic involvement of 5-HT2C and 5-HT2A receptors, others like Okada et al. (1995) imply the opposite role. A safe

11 explanation to this inconsistency would be to point out that many of the compounds previously tested have mixed selectivity profiles. This limitation can certainly be eliminated by using more selective compounds in future studies as they become available. However, another possible alternative explanation for these inconsistencies may lie on ligand biased activation of these receptors and the concept of functional selectivity.

1.6 Functional selectivity and serotonergic receptors

Traditional receptor pharmacology hinges on the idea that receptors operate through precise conformational outcomes in which particular ligands are able to interact with, the commonly labeled "lock-and-key" analogy. This school of thought tends to classify ligands as having roles of full or partial agonists, antagonists or inverse agonists (Kenanin, 2009; Kenanin, 2004). Successful binding of a ligand to its respective receptor triggers one or multiple effector pathways which are associated with the activated receptor. Second messengers involved in these pathways are the ones responsible for activating intracellular mechanisms that then produce the expected response (Lounsbury, 2009).

The prevalence of these traditional ideas is reflected on the nomenclature of receptors studied in pharmacology. Ligands are generally classified based on what receptors they interact with and what general outcomes are produced as a result of this interaction. While this notion has been very useful for studying pharmacological interactions, it may not fully reflect the complexity of these processes. The suggestion that different agonists for the same receptor are able to produce different effects is relatively new. There is however a growing number of publications about functional selectivity and biased agonism that present supporting data from a biochemical, pharmacological and even behavioral viewpoint (Urban et al. 2007; Kenakin , 2011).

There are multiple possible conformations in which large transmembrane receptors can have, and perhaps properties such as efficacy and affinity are only two factors involved in the interaction between a ligand and its target. Different ligands with high affinity for a given receptor have been shown to produce selective effects through differential activation of

12 associated secondary messengers. This phenomenon has been largely shown in publications investigating the pharmacology of dopamine D1 (Ryman-Rasmussen et al. 2005), D2 (Lawler et al. 1999), and serotonin agonism (Berg et al. 1998; Stout et al. 2002).

Despite the already intricate array of serotonin receptor subtypes, it appears that many serotonin receptors may also be subjected to further outcome differentiation based on ligand biases. For example, the 5-HT2C receptor is associated with multiple intracellular effector pathways. Activation of this receptor has been shown to activate phospholipase C (PLC) in several brain regions (Wolf and Schutz, 1997). The activation of this PLC-mediated pathway leads to an accumulation of inositol phosphate in the cytoplasm which serves to open calcium ion channels in the endoplasmic reticulum. This in turn leads to an increase in Ca2+ concentration in the cytoplasm which continues the molecular cascade necessary to produce the associated outcomes (Alberts et al. 2002). Beyond the PLC mediated response, 5-HT2C agonism also activates phospholipase A2, which is a phospholipase also involved in 5-HT2A and 5-HT2B activation. PLA2 activation through G-protein intervention initiates a different molecular cascade that involves the release of arachidonic acid (AA), serving as another effector pathway for this receptor (Raymond et al. 2001).

Historically, the magnitude of the activation of signal transduction pathways has been thought to be dictated by receptor affinity and efficacy. More recent studies have shown that different ligands for the same receptors can activate these pathways differentially (Berg et al. 1998). Berg and colleagues conducted an experiment in hamster ovarian cells (CHO-1C19) expressing the 5-HT2C receptor, assessing the levels of activation of two effector pathways associated with 5-HT2C receptor agonism. This concentration-response curve measured both the amount of arachidonic acid released and the level of accumulation of inositol phosphate after 10 minutes of exposure to serotonin 2C ligands such as LSD (lysergic acid diethylamide), DOI, quipazine and TFMPP (Trifluoromethylphenylpiperazine). The experiment showed that the agonists TFMPP and quipazine produced an increase in IP accumulation with a peak comparable to the maximal response produced by serotonin. However, the response curve for AA release with these two compounds showed a weaker activation of this pathway compared to what is produced by serotonin.

The preferential activation of the PLC-IP pathway in the presence of TFMPP and quipazine can be contrasted with the response curve produced by DOI and LSD agonism. Both of these ligands increased IP accumulation, however, they were much more powerful activators of the PLA2-AA effector pathway. This contrast has become a prime example of how serotonin receptors are able to produce agonist biased molecular responses.

Recent research has extended the idea of functional selectivity to the ability of various agonists to produce varying receptor desensitization (Stout et al. 2002). Serotonin 2C receptor agonists have been compared and shown to produce different levels of tolerance after some exposure to these ligands. Presumably, different agonists are capable of differentially activating cellular desensitization mechanisms. Studies investigating these differences for the 5-HT2C receptor have shown that desensitization elicited by pretreatment with different agonists varies with which agonist is being investigated, and also appears to be unrelated to the agonist's ability to activate either the PLC-IP pathway or the PLA2-AA pathway (Berg and Clarke, 2009). This opens yet another area of research that may eventually impact drug discovery allowing researchers to further increase the specificity of future pharmacological targets.

Acceptance of the existence of biased signaling would not only have a big impact in our understanding of pharmacology, but would also have tremendous implications on how we develop new therapeutics. This concept increases the complexity of pharmacological effects of different drugs, which can also increase the specificity of targets during the development of

NCEs. In the context of this research, it is possible that the desired anorectic effects from 5-HT2C activation occurs via a distinct mechanism from the undesired side effects observed during clinical trials of lorcaserin. If this is true, it may be possible to develop another 5-HT2C agonist capable of reducing food intake in humans without the generation of nausea and other side effects.

1.7 Models of emesis and nausea in the rat

Despite the fact that animal models are so often challenged on ethical grounds, the value of using animals in research must be acknowledged. Many clinical strides made over the last

14 century would not have been impossible without these preclinical options, which direct our efforts towards research with a significantly better chance of success. Unavailability of animal models with predictive validity – the ability of an animal model to predict a particular effect in humans – within an area of study can hinder research into novel therapeutic options when compared to areas with an abundance of available animal models.

A potential example of this problem is the comparison between models of nausea versus models of emesis (Parker 2013, Yamamoto et al. 2004). Emetic episodes are easy to identify in humans and other animals capable of vomiting as they are discrete and objectively observable. This allowed for the development of good animal models of emesis with considerable predictive validity (Costall et al. 1987, Takeda et al. 1993). Several species are used to model emesis, some of the most popular ones being ferrets (Mustela putorius furo), house musk shrews (Suncus murinus), dogs, cats and different primates (Florczyk et al. 1982; Ueno et al. 1987, Percie du Sert et al. 2012). These are useful species as they are physically capable of producing emetic responses. Although rodents are some of the most useful species in medical research, working with them in this area is difficult since rats and mice are unable to vomit. Despite the fact that this reflex has been selected out of these species, they still provide useful models to study emesis that will be covered in more detail in the next section (Horn et al. 2013, Sanger et al. 2011).

Nausea, on the other hand, is a much more subjective phenomenon, where even two accounts from people suffering from nausea may differ. This creates grounds for a much more controversial scientific debate on whether our current animal models of nausea are in fact measuring different levels of nausea in animals. To some extent there is no definitive way of confirming whether these animals experience nausea, however the predictive validity of these models should not be ignored (see Parker 2013 for review).

While the evolutionary reasons why mice and rats can't vomit are unknown, much of the circuitry involved in the process of vomiting was maintained. Rats detect emetic toxins, in a manner similar to that of species that vomit. For example, the nausea-inducing chemotherapeutic drug, cisplatin, causes the release of serotonin (5-HT) from enteroendocrine cells in the gastrointestinal tract, which activates 5-HT3 receptors on vagal afferent fibers in both ferrets (that vomit) and rats (Horn et al., 2004). In both species, this vagal activation is blocked by 5-HT3 receptor antagonists (Endo et al., 1995; Horn et al., 2004). As well, the area postrema

15 detects blood-borne toxins in rats (Bernstein et al., 1992; Eckel & Ossenkopp, 1996), as it does in vomiting species. Therefore, in the rat, the detection mechanism for vomiting is present, but the motor output is missing. Because the rat cannot vomit, it in fact serves as an excellent species for the investigation of nausea.

1.8 Conditioned flavour avoidance

As a survival strategy, most animals learn to use caution when encountering novel conditions in their environment. This strategy is particularly visible when an animal encounters unfamiliar foods which can pose great threat if it is poisonous to the animal. Since rats lack the ability to vomit, it is of vital importance for their survival that they develop good strategies to avoid ingesting toxic substances which they will be unable to expel.

When a rat is exposed to a new flavour and then experiences any change in their internal homeostatic environment it learns to avoid that particular flavour in the future. This behavioral pattern that arises from a flavour association eventually led to the idea of conditioned taste aversion learning (Garcia et al. 1974). Initially this conditioned flavour avoidance (CFA) process was used to test different psychoactive drugs to measure how aversive the different treatments were. This is done by quantifying their avoidance towards the previously paired flavour. This flavour-illness association is measured by the difference in the amount of flavoured solution consumed during a test via either a one or two bottle preference test (Grill and Norgren, 1978).

While this paradigm was able to measure levels of avoidance of tastes in animals, later experiments have showed that rats display this behavior towards both aversive and rewarding conditions. For instance rats will not only avoid flavours paired with emetogens such as LiCl, but will also avoid flavours paired with rewarding drugs like amphetamine (Berridge et al. 1981, Parker 1982).

Drugs that produce rewarding effects in rats produce conditioned place preference behavior, while drugs that produce aversive effects cause rats to display conditioned place aversion (Bardo and Bevins, 2000). By comparing results of rats exposed to rewarding drugs like amphetamine, cocaine and morphine to aversive drugs like cisplatin, fenfluramine and lithium

16 chloride, it is clear that the effects of these compounds are very different (Hunt and Amit, 1987). However, as previously mentioned, conditioned flavour avoidance tests do not discriminate between these two categories of drugs. This supports the notion that CFA is not a selective measure of nausea-inducing potential of a drug, but may rather quantify an association between a drug and the magnitude of stress derived by the novelty of its effect on the animal (Parker LA, 2003). This can be thought to be a type of neophobia triggered by an unknown stimuli, and in species unable to produce a vomiting response, flavour avoidance does not discriminate between hedonic and distressing experiences. To further support this idea, house musk shrews, a vomiting species, has been shown to develop taste preference behaviour as a result of amphetamine- and morphine-saccharin pairing experiments, while avoiding saccharin as result of LiCl-saccharin pairing (Parker et al. 2002; Smith et al. 2001). This idea is still speculative, and other potential explanations have been posed in the literature (Hunt and Amit, 1987).

As well, anti-emetic drugs do not weaken either the establishment or the expression of conditioned flavour avoidance. Numerous studies have shown that clinically efficacious anti- emetics such as the D2 receptor antagonists prochlorperazine and trimethobenzamide; the histamine H1-receptor antagonist cyclizine (Rabin and Hunt, 1983) as well as the 5-HT3 receptor antagonist ondansetron (Limebeer and Parker, 2000) have little effect on conditioned flavour avoidance resulting from the pairing of a palatable flavour with an emetogen. This fact further reinforces the notion that unlike gaping reactions, flavour avoidance may not be a selective measure of nausea-induced behaviour (Parker, 2014).

1.9 Conditioned taste aversion and the taste reactivity test

In 1978, Grill and Norgren published a more refined and systematic method to measure conditioned taste aversion learning, using the taste reactivity test (Grill and Norgren, 1978). Their methodology allowed more controlled assessment of palatability assessment of gustatory stimuli. The previously mentioned bottle test relied on the spontaneous initiation of drinking behavior before the animal was able to reject the presented flavour. The taste reactivity test (TRT) involves a direct infusion of the flavoured fluid into the oral cavity of the rat which

17 increases the amount of control the experimenter has over the rat’s exposure to this flavour. Furthermore, during this test, animals are free to move in their test chambers, allowing the experimenter to measure spontaneous behaviors arising from this exposure. Generally, animals in this test are presented with highly palatable solutions, such as sucrose or saccharin, which under normal circumstances elicit hedonic reactions. Rats will make tongue protrusions and mouth movements characteristic of ingestive behavior during the infusion of these flavours. In contrast, animals presented with unpleasant flavours will produce a very different set of reactions which can include chin rubbing, gaping (wide opening of the mouth exposing the lower incisors), passive dripping, head shaking and paw treading (see Grill and Norgren, 1978 for detailed description of these behaviors). Gaping is the most reliable aversive behaviour (Parker, 2014).

In the context of nausea and emesis research, animals tested in the taste reactivity test are generally exposed to a palatable solution and then subsequently given the treatment in question. While the animal will display signs that it finds the solution palatable during the first exposure, re-exposure to the solution will produce an aversive response if the test solution is in fact noxious. These aversive responses will generally be expressed via disgust reactions including chin rubbing, gapes, paw treading and head shakes. As it becomes clear after reviewing the literature, many of these behaviors are sources of debate on what exactly they indicate, therefore often these types of experiments do focus on some of the more defined responses such as gaping reactions.

Despite their inability to vomit, rats have maintained not only some of the neurological circuitry but also some of the movements involved in producing a vomiting response. Gaping may simply be a vestigial response to similar muscular programs activated during an emetic event (Travers et al. 1986). Visually, the wide opening of the mouth observed during a gape reaction is very similar to pre-vomiting responses seen in other species such as the house musk shrew. The fact that only drugs that can produce vomiting in shrews triggers gaping in rats further supports this idea (Parker and Limebeer, 2006).

Conditioned gaping reactions can occur as a response to flavour-illness pairings and seem to consistently allow for empirical discrimination of substances that are known to be nausea- inducing in humans. This can be contrasted with the aforementioned CFA, which is a less discriminating response to psychoactive drugs. The literature is replete with studies that

18 demonstrate this difference between drugs that only produce conditioned flavour avoidance and drugs that produce conditioned gaping reactions as well as CFA (Parker, 2003).

1.10 Pica

Another commonly used model of nausea and vomiting relies on the behaviour of animals that ingest substances with no nutritional value, such as kaolin, when exposed to illness- producing substances (cisplatin, LiCl, copper sulphate) or conditions (motion exposure). This behaviour can be seen in animals such as mice and rats, which lack an emetic reflex but are also observed in species able to vomit (Yamamoto et al. 2004). It is thought that this type of behaviour occurs as a mean to help relief gastro-intestinal discomfort in animals. This discomfort can be a result of nausea, vomiting as well as diarrhea. To support this idea, some studies have also shown that dopamine D2 receptors in the CTZ as well as 5-HT3 receptors in the stomach are involved in the occurrence of this behavior in animals (Takeda et al. 1993).

While pica has been a useful model to help scientists study the neurobiology and neuropharmacology of emesis and nausea, the choice of species does appear important when using this model. Vomiting species such as the house musk shrew seem to require higher levels of noxious stimuli to begin consuming kaolin, while non emetic ones such as the rat, tend to be more sensitive to equivalent stimuli. This was demonstrated by administering different emetogens to shrews and rats. Shrews only displayed pica behavior after exposure to LiCl, but not cisplatin, nicotine or copper sulfate. Alternatively, rats consumed kaolin after exposure to all of these treatments (Yamamoto et al. 2004).

A further limiting aspect of the model is the fact that pica experiments tend to take place over a span of several hours or days in order to allow the animals a long enough period to consume measurable quantities of the clay. For this reason, it may not be a suitable model for measuring potential to induce nausea or emesis of acute treatments. For these reasons drugs with long half-life such as cisplatin (Percie du Sert et al. 2012) tend to produce much more robust effects in this model. Another limitation that arises from the design of pica experiments is the fact that little knowledge is gained in terms of temporal profile of the illness produced (Percie du

Sert et al. 2012). For these reasons, it is prudent to say that results from pica experiments may be useful for predicting the emetogenicity of novel treatments if combined with other preclinical models. Similarly to the conditioned flavour avoidance paradigm, data from pica experiments must be supported by other tests to strengthen its predictive value.

1.11 Present studies

Clinical application of selective 5-HT2C receptor agonists such as lorcaserin, is a result of scientific effort to characterize this receptor subclass. Despite recent developments in this area, our understanding of the role of this and other serotonergic receptors is still rudimentary. For instance, the involvement of serotonin receptors, other than the 5-HT3 receptor in nausea and emesis remains poorly understood. For this reason, GI related side effects of approved serotonergic pharmaceuticals cannot be isolated out of drug development. In order to systematically develop better drugs, a more comprehensive understanding of its targets is imperative.

This thesis is an effort to demonstrate that the 5-HT2C receptor has a role in the generation of nausea. Clinical trial data for lorcaserin reported nausea as the most common side effect in humans (Schram et al. 2011); early preclinical work has also shown signs that 5-HT2C receptor agonists can produce general GI related side-effects (Higgins et al. 2013). Chapter 2 documents experiments investigating two selective 5-HT2C receptor agonists in a taste reactivity test. The potential of lorcaserin to generate nausea was studied by measurements of conditioned gaping reactions as well as conditioned aversive responses. The second selective agonist used was CP-809101, which is an even more selective agonist for the 5-HT2C receptor (Siuciak et al. 2007). The rationale of this comparison is to identify whether nausea-induced behaviours from lorcaserin are also produced by more selective drugs, potentially suggesting that these side effects are indeed 5-HT2C receptor mediated. Along with the use of a more selective agonist, a selective antagonist for this receptor was also tested. Pretreatment of animals with the selective

5-HT2C receptor antagonist SB-242084 prior to conditioning sessions was used to further confirm if nausea related to the injection of lorcaserin can be attenuated by blocking this particular target

(Kennett et al. 1997). Conditioned flavour avoidance tests were also conducted, and while they may not be as selective of a tool for screening for nausea and emetic potential, they may still prove useful to highlight differences between the effects of lorcaserin and CP-809101 treatment.

Chapter 3 describes a different experiment looking at lorcaserin in a different model of nausea and emesis. In this experiment the effects of lorcaserin in a pica test were explored. Pica differs from the other tests used in chapter 2 as it is an unconditioned behaviour. This assessment of lorcaserin is important to further investigate the nausea-inducing potential of 5-HT2C receptor agonists.

Chapter 2: Effects of 5-HT2C receptor agonists lorcaserin and CP-809101 in the taste reactivity and conditioned flavour avoidance models in rats

2.1 Introduction

Indications that serotonin receptors are important pharmacological targets for the treatment of obesity have their origins in studies reporting mCPP (meta-Chlorophenylpiperazine) induced hypophagia. While mCPP is not selective for the serotonin 2C (5-HT2C) receptor, experiments using different serotonin antagonists suggested that reductions in feeding behaviour may have been mediated by this receptor (Kennett and Curzon 1991). Early evidence of the role of the 5-HT2C receptor in feeding is comprised mostly of animal work (Samanin et al. 1979), however, human trials were later conducted and showed a similar reduction in food intake (Cowen et al. 1995).

From this early research, other serotonergic compounds were investigated as potential anorectic drugs. Dexfenfluramine (Redux®) was one of the early serotonergic drugs to achieve success during clinical trials (Guy-Grand et al. 1989), eventually achieving FDA approval for the treatment of obesity. Pharmacologically, dexfenfluramine increases levels of extracellular serotonin in the CNS via augmentation of 5-HT release from synaptosomes as well as inhibition of serotonin removal from synapses. This compound also produces dexnorfenfluramine, an active metabolite that also acts as an agonist for the 5-HT2C receptor. After some years in the market, dexfenfluramine was removed due to associations with cardiac valvulopathy as well as pulmonary hypertension (Connolly et al. 1997). Since then, much of the cardiovascular side effects produced by Dexfenfluramine have been linked to its activity at peripheral 5-HT2B receptors (Halford and Harrold, 2012). Many other serotonergic drugs have emerged from this early discovery and much effort has been put into the attempt to produce comparable efficacy without the serious side effects from the non-selective increase of serotonergic tone.

The recent approval of lorcaserin (Belviq®) by the FDA for the treatment of obesity has further enhanced therapeutic interest in the serotonin 2C receptor subtype. This approval was not only the first for an anti-obesity drug in 13 years, but will likely open the doors for other

22 potential indications of this drug class. The function of this receptor has proven to be much broader than the regulation of feeding behaviour. Over a decade of research highlights the ability of this particular receptor to attenuate other compulsive behaviours (Martin et al. 1998; Fletcher et al. 2008; Higgins et al. 2012), which may eventually yield drugs aiming to help patients afflicted with nicotine dependence and substance abuse.

Consistent with the anorectic properties of the 5-HT2C receptor seen in clinical trials, rodent models have shown that lorcaserin is able to reduce food intake in rats through a palatability-induced feeding test at a dose of 1 mg/kg (Higgins et al. 2012). Palatability-induced feeding is a useful method for assessing subtle changes in feeding behaviour since unlike many other tests, animals are not food restricted but are instead presented with highly palatable food. Other selective agonists for the 2C receptor have also been tested in similar animal feeding models, and while these have failed to have clinical impact, they serve as powerful research tools. These compounds have similar pharmacological selectivity to lorcaserin, for instance Ro 60-0175, a commonly used agonist to study this receptor, has a relatively similar affinity to lorcaserin for this target. Ro 60-0175 has a 13-fold selectivity for the 5-HT2C receptor over the 5-

HT2A receptor, while lorcaserin has a 16-fold selectivity (2C/2A) using functional in vitro assays.

However, Ro 60-0175 does show more affinity than lorcaserin for the 5-HT2B receptor (Higgins et al. 2013). Perhaps the most selective 5-HT2C receptor agonist known is CP-809101, with 1585-fold selectivity for the 2C receptor over the 2A receptor. For this reason CP-809101 is also an important research drug, and much like lorcaserin and Ro 60-0175, can be used as an anorectic agent in animals. CP-809101 was able to reduce feeding in rats during a palatability- induced feeding experiment at a 3 mg/kg. While the effective dose of CP-809101 was higher than lorcaserin in this particular model, similar comparison using a motivation driven operant conditioning paradigm indicated an effect at 0.3 mg/kg of CP-809101, while the required dose of lorcaserin was 0.6 mg/kg in rats (Higgins et al. 2013).

While much of the preclinical research focuses on the therapeutic properties of lorcaserin and other selective 5-HT2C receptor agonists, it is equally important to understand potential adverse effects of these drugs. Clinical data submitted by Arena Pharmaceuticals to the FDA showed that beyond its efficacy in reducing body weight in humans, the most common adverse effects of lorcaserin were headaches, nausea and vomiting (Arena Pharmaceuticals, 2010 FDA

23 briefing document NDA 22-529, Taylor et al. 2013). There is also data from in vivo work showing early evidence that comparable adverse effects can be reproduced in rodents and monkeys (Arena Pharmaceuticals, 2010 FDA briefing document NDA 22-529, Higgins et al. 2013).

In these experiments we attempted to further investigate side effects from 5-HT2C receptor agonists by utilizing conditioned disgust reactions as a model of nausea-induced behaviour in the rat (see Parker and Limebeer, 2006 for review). Rats are unable to vomit; however, when they are exposed to a saccharin flavoured solution previously paired with an emetic drug such as lithium chloride, they display conditioned gaping reactions as well as other aversive behaviours such as paw treading and chin rubbing. Gaping reactions, wide opening of the mouth that exposes lower incisors, are only produced by chemicals able to generate emetic responses in species able to vomit and can be observed and quantified during taste reactivity (TR) tests (Parker, 2003). Additionally, a one bottle conditioned flavour avoidance (CFA) test was conducted in all animals upon completion of all taste reactivity sessions. CFA is an older paradigm used to assess the nauseating potential of drugs. While this model may still have use as a less laborious test for screening nausea-inducing compounds, it is known to be a much less selective test. While flavour avoidance does occur from flavour pairings with emetogens like lithium chloride and cisplatin, it can also occur with compounds that are not known to cause severe GI discomfort, such as amphetamine and cocaine (Berger, 1972; Zalaquett and Parker, 1989). In fact, rats self-administer amphetamine and cocaine as well as display conditioned place preference during experiments using these drugs, which indicate that these compounds are not causing distress to the animal and in fact are more likely producing pleasurable experiences, rendering the CFA paradigm much more limited (Parker, 2003; Pickens and Harris, 1968; Reicher and Holman, 1977).

The fact that ligands for the 5-HT2C receptor are known to have pleitropic behaviour (affects multiple intracellular pathways) further adds relevance to the comparison between these agonists (Urban et al. 2007). Different agonists for this receptor such as DOI (2,5-Dimethoxy-4- iodoamphetamine), TFMPP (3-Trifluoromethylphenylpiperazine) and quipazine have been shown to differentially activate 5-HT2C associated signalling cascades in vitro (Berg et al. 2009). This property of serotonergic receptors is also expressed behaviourally; several recent

24 publications comparing serotonergic agonists have demonstrated results consistent with this notion (Higgins et al. 2013, Newman-Tancredi, 2011). In this experiment, two of the most selective 5-HT2C receptor agonists (lorcaserin and CP-809101) were tested in these rat models of nausea. While the saccharin- lorcaserin pairing produced the expected nausea-induced behaviour in the form of conditioned gapes, the more selective CP-809101 did not produce a significant effect at doses of equivalent efficacy in previously published feeding assays (Higgins et al.

2013). Additionally, a 5-HT2C receptor antagonist was used as a means to further characterize the pharmacology of this receptor subtype in the production of nausea.

2.2 Methods

Animals and housing

Nine week old male Sprague-Dawley rats were used in both experiments (source: Charles River, St. Constant, Quebec, Canada). Animals weighed approximately 200-300g at the beginning of the study and were singly housed in polycarbonate caging. Standard laboratory chow (Highland Rat Chow 8640) and fresh water was provided ad libitum. The animal holding room was maintained in a 12h light-dark cycle where lights were on between 19:00 to 7:00. Temperature was kept at 21 ± 2ºC and 45 ± 20% humidity. All testing was conducted during the animals’ dark phase. Animals were handled prior to all testing and health monitoring was done throughout the duration of the experiment. Both experiments were approved by the University of Guelph Animal Care Committee and conducted in accordance with guidelines set by the Canadian Council of Animal Care (CCAC).

Surgery

All rats were surgically implanted with an intraoral cannula similar to the procedure described by Limebeer et al (2012). Surgeries were performed under inhalant anaesthetic

(isoflurane). Animals were administered with the non-steroidal anti-inflammatory carprofen (5 mg/kg) and the antibiotic procillin (100 mg/kg) 30 minutes prior to the procedure. A 15-gauge stainless steel needle was inserted at the back of the neck and guided behind and below the left ear along the mandible. The needle was inserted into the oral cavity from a point just behind the first molar. A ~10 cm polyethylene tube (PE90) with an inner diameter of 0.86mm and outer diameter of 1.27mm was passed through the needle, after which, the steel needle was removed leaving only the plastic tubing held in place by elastic disks. Animals were monitored and had their cannulas flushed with chlorhexidine daily to ensure patency and to clean the area.

Apparatus

The transparent Plexiglas® taste reactivity chamber (22.5 x 26 x 20 cm) was placed over a glass table. A mirror was positioned beneath the table top at a 45º angle allowing a tracking video camera (Sony DCR-HG28) to be placed directly in front of the apparatus in order to be able to record the rat's activity from a ventral point of view. A syringe pump (KD Scientific 780100) was used to deliver the saccharin solution at a constant rate of 1 ml/min. The pump was connected to polyethylene tubing which infused the animal's cannula inside the apparatus through an aperture located on the lid of the test chamber.

Drugs and treatment regimens

A 0.15 M lithium chloride (LiCl; Sigma-Aldrich) solution was prepared in sterile water and administered at a volume of 20 ml/kg (127 mg/kg). Lorcaserin ((1R)-8-chloro-1-methyl- 2,3,4,5-tetrahydro-1H-3-benzazepine HCl) (NPS Pharmaceuticals, Toronto, Canada) and CP- 809101 (2-(3-chlorobenzyloxy)-6-(piperazin-1-yl)pyrazine) (NPS Pharmaceuticals, Toronto, Canada) were prepared in sterile saline and 5% tween in saline, respectively, at a dose volume of 1 ml/kg. SB-242084 dyhydrochloride (6-chloro-5-methyl-1-[2-(2-methylpyridyl-3-yloxy)-pyrid-

5-yl carbamoyl] indoline) was prepared in 8% hydroxypropyl-β-cyclodextrin and 25mM citric acid at a dose volume of 1 ml/kg.

Administration of lorcaserin, CP-809101 and lithium chloride was done immediately after conditioning sessions. Onset of somatic signs from the administration of both 5-HT2C receptor agonists was determined to be equivalent (4-5 min) from an unpublished experiment using these routes of administration and formulation protocols.

Experiment 1 – Effect of lorcaserin in the test reactivity and flavour avoidance models

Following four days of recovery and post-surgical monitoring, animals were exposed to the test chamber for 5 min. During this habituation (Day 0), the infusion pump was attached in order to deliver fresh water at a flow rate of 1 ml/min. Animals were returned to their home cages immediately following this exposure to the test conditions.

The first conditioning session occurred on the following day (Day 1). After flushing the intra-oral cannula with water to remove any obstructions, animals were placed in the taste reactivity test chamber. Rats were given 1 min to habituate to the chamber, followed by a 3 min intraoral infusion of 0.1% saccharin solution. Once removed from the chamber, animals had their cannulas flushed with water and were immediately given their pseudo-randomly assigned treatment prior to returning to their home cage. Treatment groups were divided into vehicle (0.9% NaCl, n=8, s.c.), lorcaserin at 1.0 mg/kg (n=8, s.c.), 3.0 mg/kg (n=8, s.c.), 6.0 mg/kg (n=15, s.c.), and lithium chloride at 127 mg/kg (n=8, i.p.). A second equivalent conditioning session also occurred on Day 4 to ensure the formation of a strong flavour-treatment association. During conditioning sessions gaping reactions, paw treading episodes, tongue protrusions and the incidence of chin rubbing were recorded for later video analysis by an observer blind to treatments, using the video analysis software Observer (Noldus Information Technology, Leesburg, VA, USA). General activity was also monitored during the experiment and consisted of accumulated ambulatory period and time spent rearing in seconds.

The taste reactivity test was conducted three days (Day 7) after the second conditioning session. During the TR test, the animals were once again placed in the test chamber attached to an infusion pump. After a minute of habituation, animals were infused with the saccharin solution for 3 min, in which time their behaviour was recorded for later video analysis.

Upon completion of the TR test, animals returned to their respective home cages and were deprived of water for 16 hours. On Day 8, a one bottle flavour avoidance test was conducted, where the water deprived rats were presented with a single graduated bottle of 0.1% saccharin solution. The volume of saccharin ingested by the animals was recorded at 60, 120 and 360 minutes and the total intake was calculated from these values.

Experiment 2 – Effect of CP-809101 in the taste reactivity and flavour avoidance models

Rats in this experiment underwent identical surgical and experimental procedure as animals in Experiment 1, with the following exceptions. During both conditioning trials, animals received one of the pseudo-randomly assigned treatments which included vehicle (5% tween 80 in 0.9% saline, n=8, s.c.), CP-809101 at 3.0 mg/kg (n=9, s.c.), 6.0 mg/kg (n=8, s.c.), 12mg/kg (n=8, s.c.), lorcaserin at 6.0 mg/kg (n=15, s.c.).

Experiment 3 - Effect of systemic SB-242084 pretreatment on lorcaserin-induced conditioned gaping and flavour avoidance

Identical surgical and experimental procedures were used in this experiment with the following exceptions. During both conditioning trials, animals received one of the pseudo- randomly assigned treatments which included vehicle (5% tween 80 in 0.9% saline, n=8, s.c.), lorcaserin at 6.0 mg/kg (n=15, s.c.) and lorcaserin at 6.0 mg/kg pretreated with SB-242084 at 0.5 mg/kg (n=8, SB-242084 was administered i.p. 20 minutes prior to conditioning). The selected

28 dose for SB-242084 is based on previous data demonstrating successful antagonism against various 5-HT2C agonist-induced responses (Kennett et al. 1997)

Data Analysis

Because the variability within each group for taste reactivity behaviours of gaping, total aversive reactions and tongue protrusions, significantly differed (showed non-homogeneity), these data were transformed into logarithmic scores and were entered into a one-way Analysis of Variance (ANOVA), with treatment groups as the between-subjects factor. The volume of saccharin consumed at 360 min during the conditioned flavour avoidance tests was entered into a between factor ANOVA. Bonferroni post hoc comparisons were conducted to determine statistical significance of effects. Statistical significance was defined as P < 0.05

2.3 Results

Experiment 1 – Effect of lorcaserin in the test reactivity and flavour avoidance models

After the two conditioning sessions, animals that received lorcaserin produced gaping reactions to the saccharin flavoured solution. An overall main effect of treatment with lorcaserin was seen from a one-way ANOVA of transformed gaping responses, F(4,42)=10.12; p<0.001. As evident in Figure 2A, groups that received lorcaserin at 3 mg/kg (p =0.011), lorcaserin at 6 mg/kg (p < 0.001) and LiCl (p< 0.001) gaped significantly more than the vehicle group. As well the group receiving lorcaserin at 6 mg/kg (p=0.022) and LiCl (p < 0.001) gaped significantly more than the lorcaserin 1 mg/kg group. Groups administered with lorcaserin 6 mg/kg and LiCl did not significantly differ.

A comparison of total aversive responses between groups also indicates a similar pattern with an overall main effect emerging from a one-way ANOVA of the transformed scores, F(4,42)=19.69; p<0.001. When all aversive reactions are assessed, each treatment produced a greater aversion than group Vehicle (p’s<0.05). As well, the lorcaserin 1 mg/kg group displayed fewer aversive reactions than lorcaserin 3 mg/kg (p=0.047), lorcaserin 6 mg/kg (p=0.004) and LiCl (p=0.001).

The frequency of tongue protrusions significantly differed among the groups (Figure 3), F(4,42)=12.56; p<0.01. All treatment groups showed a decrease in tongue protrusions when compared to the vehicle group (p’s < 0.001). No other groups significantly differed from one another.

General activity was also analysed and according to a one-way ANOVA, the various treatment groups had no overall effect on this measure during the taste reactivity test. Animals that received vehicle injections exhibited active behaviour for an average of 42±4.5s during the taste reactivity test, compared to an average of 41.9±4.3s of activity in the lithium chloride treated animals and 48.1±5.3s in the animals treated with 6 mg/kg of lorcaserin.

As is depicted in Figure 4, during a 6 h conditioned flavour avoidance test, rats conditioned with lorcaserin 3 mg/kg (p<0.001) , lorcaserin 6 mg/kg (p =0.001) and LiCl (p<0.001) drank less saccharin than those conditioned with vehicle (p<0.01); with a significant effect of dose in the one-way ANOVA, F(4,43)=11.7, p<0.01. Group lorcaserin 1 mg/kg drank more saccharin then groups receiving lorcaserin at 3 mg/kg (p =0.007) or LiCl (p = 0.002), but not lorcaserin 6 mg/kg or vehicle. However, animals administered with lorcaserin 6 mg/kg, lorcaserin 3 mg/kg and LiCl did not significantly differ.

Experiment 2 – Effect of CP-809101 in the taste reactivity and flavour avoidance models

Taste reactivity tests from Experiment 2 showed no change in gaping reactions from all doses of CP-809101 when compared to vehicle group, as illustrated in Figure 5A. An overall

30 effect of treatment was seen using a one-way ANOVA of transformed gaping data , F(4,43)=11.34, p<0.001. Bonferroni post-hoc comparison tests revealed that only the positive control, administered with 6 mg/kg of lorcaserin, significantly differed from vehicle group (p<0.001). As well, group lorcaserin 6 mg/kg also differed significantly from groups administered with CP-809101 at 3 mg/kg (p<0.001) and 6 mg/kg (p=0.003), but not CP-809101 at 12 mg/kg (p=0.124).

In this experiment, the mean number of total aversive responses was also monitored, indicating that the treatment with 6 mg/kg of lorcaserin produced aversive responses (p<0.01) while none of the groups treated with CP-809101 showed an increase in total aversive responses when compared to vehicle (Figure 5B). The single-factor ANOVA of the mean revealed a significant treatment group effect, F (4,43)=10.45; p < 0.001. Bonferroni post-hoc comparison tests revealed that only group treated with lorcaserin at 6 mg/kg significantly differed from the vehicle group (p<0.001). As well, lorcaserin 6 mg/kg also differed significantly from CP-809101 at 3 mg/kg (p<0.001) and CP-809101 at 6 mg/kg (p=0.01), but not CP-809101 at 12 mg/kg (p=0.174).

The incidence of tongue protrusions was significantly attenuated by all treatments during the taste reactivity test, producing an overall significant group effect of the transformed data, F(4,43)=10.79, (p<0.001). As is evident in Figure 6, all treatment groups displayed less tongue protrusions than Group Vehicle (p’s < 0.001). No other groups significantly differed from one another. Activity measurements revealed no effect from drug treatments. F (4,43)=1.65.

Data collected from the conditioned flavour avoidance test indicates that even at the lowest dose of CP-809101 a decrease in saccharin ingestion relative to vehicle occurred (Figure 7; p<0.01). A one-way ANOVA indicated that after 360 minutes there was an overall effect on the cumulative saccharin intake, F(4,44)=27.34; p<0.001, and all treatments reduced voluntary intake when compared to vehicle treated rats (p’s<0.001). Group lorcaserin 6 mg/kg also drank less saccharin than rats administered with CP-809101 at 3 mg/kg (p = 0.01), but did not significantly differ from CP-809101 at 6 mg/kg or 12 mg/kg.

Experiment 3 – Effect of systemic SB-242084 pretreatment on lorcaserin-induced conditioned gaping and flavour avoidance

The taste reactivity test from Experiment 3 indicated that pretreatment of animals with SB-242084 attenuated the incidence of lorcaserin-induced gaping reactions. A one-way ANOVA of the transformed gaping scores revealed a significant overall effect, F(2,28)=16.47, p<0.001. Bonferroni post-hoc tests revealed that rats receiving lorcaserin at 6 mg/kg displayed significantly more gaping than either vehicle group (p < 0.001) or group receiving both SB-242084 and lorcaserin 6 mg/kg (p = 0.011). Group treated with SB-242084 and lorcaserin 6 mg/kg did not significantly differ from vehicle group, suggesting that the antagonist completely blocked the effect of the 5-HT2C agonist.

More broadly, analysis of the total number of aversive reactions also revealed that the effect of lorcaserin was mediated by its action at the 5-HT2C receptor. The ANOVA of the transformed aversive reaction data revealed a significant effect , F(2,28)=19.23, p<0.001. Bonferroni comparison test revealed that the group receiving lorcaserin 6 mg/kg displayed more aversive reactions than either the vehicle group (p<0.001) or group receiving SB242084 and lorcaserin 6 mg/kg (p=0.01), the latter two groups did not differ.

Pretreatment with the 5-HT2C antagonist, however, did not block the suppressive effect of lorcaserin on tongue protrusions. The ANOVA of the transformed tongue protrusion data revealed a significant effect, F(2,28)=15.45, (p<0.001). The frequency of tongue protrusions was significantly reduced in both treatment groups when compared to the saline control animals (p’s<0.001). Comparison between rats treated with lorcaserin and rats treated with lorcaserin as well as SB-242084 produced an equivalent number of tongue protrusions that did not significantly differ.

Drug treatments produced no significant change in general activity measurements according to a one-way ANOVA (F2,28=1.22). Lorcaserin treated rats on average displayed active behaviour for 48.1±5.4s while SB-242084 only reduced the average period of activity to 43.6±7.2s.

Pretreatment with SB-242084 did not prevent lorcaserin-induced flavour avoidance, cumulative intake of saccharin from the one-bottle test showed a decreasing effect from both treatment groups when compared to control animals (Figure 8B). ANOVA analysis showed a significant overall effect was present F(2,28)=51.53; p<0.01. Comparison between animals dosed only with lorcaserin 6 mg/kg and animals that received both lorcaserin and SB-242084 injections shows no difference between these groups in terms of saccharin intake.

2.4 Discussion

In Experiment 1, at a dose of 3 mg/kg and 6 mg/kg, lorcaserin produced conditioned gaping reactions in rats following two pairings with saccharin solution; this effect was comparable to the effect produced by the LiCl pairing (Figure 2). This dose of lorcaserin is higher than efficacious doses used in models of feeding using rats. In vivo data published by Higgins et al. (2013) indicates that lorcaserin begins to reduce feeding behaviour at doses as low as 0.6 mg/kg in a progressive ratio schedule in an operant conditioning test using food pellets as rewards. A less sensitive palatability-induced feeding test in rats showed efficacy only at the 1 mg/kg dose of lorcaserin (Higgins et al. 2012). During the same palatability experiment, behavioural satiety sequence measurements only indicated disruptions to normal feeding patterns at the 3 mg/kg, a dose consistent with the results from this study. Results from these animal tests are also consistent with clinical findings that lorcaserin can cause nausea in humans exposed to supratherapeutic doses of the drug (Arena Pharmaceuticals, 2010 FDA briefing document NDA 22-529).

Given the differences between lorcaserin and CP-809101, one of the objectives of the current study was to determine if the nausea-induced behavior of gaping was mediated by the action of lorcaserin at the 5-HT2C receptor, rather than an effect through a non-5-HT2C receptor mediated mechanism. This was accomplished by investigating whether the pretreatment of a selective 5-HT2C receptor antagonist can block this effect. Indeed, the 5-HT2C receptor antagonist SB-242084 successfully blocked lorcaserin-induced conditioned gaping reactions as well as total aversive reactions. As it is shown in Figure 8(A), the occurrence of gaping reactions was

33 significantly reduced by the antagonist pretreatment in comparison to the group only treated with 6 mg/kg of lorcaserin. These results provide compelling evidence that gaping reactions associated with the lorcaserin pairing are mediated by 5-HT2C receptor activity.

When comparing different 5-HT2C agonists, CP-809101 is considered to be one of the most selective drugs available (Siuciak et al. 2007). While lorcaserin is very selective for the this receptor, in vitro data shows that it has a much lower pEC50 when compared with CP-809101 (Siuciak et al. 2007, Thomsen et al. 2008). Despite lorcaserin’s relatively lower affinity, there are studies found in the literature comparing the affinity of structurally distinct

5-HT2C receptor agonists, which has shown that in vivo profiles may not be directly correlated to receptor affinity (Higgins et al. 2013). These differences are not only expressed by the intensity of produced behaviours but also in what effects are actually produced. Results from the present experiment further indicate that while similar, these two 5-HT2C ligands can produce distinct behavioural outcomes.

While the lorcaserin data obtained in Experiment 1 is consistent with human trials, Experiment 2 highlights a very different pattern when substituting lorcaserin for the more selective CP-809101. Palatability feeding experiments, similar to the ones mentioned with lorcaserin, showed efficacy of this drug at 3 mg/kg compared to a much lower needed dose of 0.3 mg/kg in the progressive ratio test (Higgins et al. 2013). During the present taste reactivity test, CP-809101 was much better tolerated than lorcaserin, producing a small, yet not significant, increase in gaping reactions at the 12 mg/kg dose (Figure 5). This is appreciably higher than its in vivo efficacy range (0.3 – 3 mg/kg). Doses of CP-809101 can be contrasted with lorcaserin, where conditioned gapes and aversive responses occurred at 3 mg/kg and 1 mg/kg respectively, which is much closer to its efficacy range (0.6 – 1 mg/kg). It is relevant to note that CP-809101 did produce a significant decrease in saccharin intake during CFA tests, even at the lowest dose (3 mg/kg, s.c.), giving some assurance that the selected doses of CP-809101 were sufficient to produce perceivable somatic effects in the rats.

This considerable difference in efficacy range compared to tolerance between lorcaserin and CP-809101 suggests that the nausea produced by lorcaserin is not mediated by the 5-HT2C receptor, therefore side effects from the more selective agonist are not as pronounced. This interpretation however, clearly contradicts the results from Experiment 3, where a 5-HT2C

34 antagonist successfully blocked the nausea-induced behaviour produced by lorcaserin. Therefore an alternative hypothesis is needed to explain these results.

Serotonin receptors are known to be coupled to multiple effector pathways and perhaps this seemingly inconsistent pharmacological behaviour is a result of ligand biased activation of the 5-HT2C receptor. (Kenakin 2010, Schmid et al. 2008). While the concept of ligand biased agonism or functional selectivity (Urban et al. 2007 for review) challenges some fundamental concepts in receptor pharmacology, strong evidence has been published showing that 5-HT receptor agonists are capable of activating these pathways to differing degrees (Berg et al. 1998,

2009). The relative efficacy of 5-HT2C ligands is dependent on the activation of two known effector pathways, one involving phospholipase A2-mediated release of arachidonic acid and another one through a phospholipase C-mediated accumulation of inositol phosphates (Stout et al. 2002). A suggested hypothesis poses that a receptor is able to exist in different active conformations, which can trigger intracellular pathways in a biased manner depending on its interaction with a particular agonist. However, the number of conformations possible for any given receptor is still a topic of debate (Kenakin 1995, Leff et al. 1997).

It seems reasonable to attribute differences between the effects of these two drugs to functional selectivity. Lorcaserin may be initiating the effector pathway responsible for producing nausea while the effects of CP-809101 may be a result of a different activation ratio. There are other potential explanations for these results, and differences in drug absorption could also explain this inconsistency. Recently published pharmacokinetic data shows that plasma levels of lorcaserin in Sprague-Dawley rats exposed to therapeutic doses of the drug, is similar to reported human plasma concentrations from phase III obesity studies (Arena Pharmaceuticals, 2010 FDA briefing document NDA 22-529, Higgins et al. 2013). No data is available for plasma and CSF exposure of CP-809101 at this time, leaving room for the possibility that CP-809101 may simply not have the same absorption potential as lorcaserin. Since nausea is a centrally controlled process, measurements of plasma and brain exposure would also be an important measurement to obtain. Much of the differences between these two 5-HT2C agonists could simply be a result of higher lorcaserin exposure in the brain. Results from the conditioned taste avoidance test do however, show another contrast between the two 5-HT2C agonists. Lorcaserin effects occurred at similar doses in both the TR test as well as the CFA test. Unlike lorcaserin,

CP-809101 TRT effects were not significant even at the 12 mg/kg dose, while effects in the CFA test were measured at all doses. Data from the CFA test with CP-809101 does suggest that there is brain penetration; however the comparison between the CFA and the TR data may be an indication that this drug is not as aversive to the animals. Inconsistencies between CFA and TR results are commonly reported, and often CFA results are interpreted to be a fear based learning response to novel interoceptive changes rather than a selective measure of nausea-induced behaviour. Therefore, this inconsistency may be another piece of evidence of how functional selectivity might be producing different behavioural outcomes, even though these compounds are acting on the same receptor target.

Lastly, despite the fact that the taste reactivity test showed that SB-242084 is capable of blocking lorcaserin-induced gaping, CFA results made no distinction between lorcaserin treated rats and rats pretreated with the antagonist prior to lorcaserin injection (Figure 8). As previously suggested, flavour avoidance is a less reliable measure of the potential for a compound to induce nausea or emesis (Parker et al. 2008). Flavour avoidance occurs in the presence of anti-emetics such as ondansetron and cannabinoids which are able to prevent nausea-induced behaviour from emetogens, but still allow other changes to be generated in the rat's internal milieu. As a result, these changes become associated with the paired flavour and are physiologically treated as a sign of danger (Parker et al. 2009). Therefore, the fact that SB-242084 did not prevent conditioned flavour avoidance from lorcaserin, may either be an indication that there are other changes being perceived by the animals which are not affected by the antagonist, or that the antagonist itself produces such effects.

2.5 Conclusion

Findings from these experiments raise further questions regarding 5-HT2C receptor pharmacology. Differences in receptor selectivity may not be sufficient to explain the effects of lorcaserin and CP-809101 in these models of nausea. While functional selectivity may appear to be a compelling explanation, only by further exploring the differences between signalling pathways after 5-HT2C receptor activation by these two agonists, will we reach a convincing

rationalization of these results. A potential next step would be to investigate whether a 5-HT2C receptor antagonist, such as SB-242084, displays further anti-emetic properties against gaping reactions elicited by a different mechanism. This would further consolidate the ideas presented in our discussion, and possibly even suggest that the 5-HT2C receptor may be involved in the generation of gaping reactions or even nausea. While it is well known that serotonin is a key neurotransmitter in nausea and emesis, very little of what we known involves serotonin receptors beyond the 5-HT3 receptor. The involvement of other serotonin receptors in nausea and emesis is possible and further characterizing their role may lead to new receptor targets for pharmacological management of nausea and vomiting. Lastly, testing other 5-HT2C agonists in these models could provide a more complete picture of its role in nausea, as well as providing more information about how different 5-HT2C receptor ligands may produce varying pharmacological outcomes, potentially helping identify 5-HT2C receptor agonists with better tolerability profiles and potentially better therapeutic value.

2.6 Figures

Figure 2. Effect of saccharin-lorcaserin pairing in the taste reactivity test Effect of saccharin-lorcaserin (1-6 mg/kg, s.c.) and saccharin-lithium chloride (127 mg/kg, i.p.) pairing during a 3 minute taste reactivity test. (A) Mean (±SEM) number of conditioned gaping reactions elicited by 0.1% saccharin solution following two conditioning sessions. (B) Mean (±SEM) number of aversive reactions during a 3 minute taste reactivity test after two conditioning sessions. *P<0.05 vs vehicle pretreatment (Bonferroni's test)

Figure 3. Effect of saccharin-lorcaserin pairing on tongue protrusions Effect of saccharin-lorcaserin (1-6 mg/kg, s.c.) and saccharin-lithium chloride (127 mg/kg, i.p.) pairing on the incidence of tongue protrusions during a 3 minute taste reactivity test. *P<0.05 vs vehicle pretreatment (Bonferroni's test)

Figure 4. Effect of saccharin-lorcaserin pairing in a conditioned flavour avoidance test Effect of saccharin-lorcaserin (1-6 mg/kg, s.c.) and saccharin-lithium chloride (127 mg/kg, i.p.) pairing during a one-bottle flavour avoidance test after two conditioning sessions. Mean cumulative (±SEM) intake of 0.1% saccharin solution after 360 min. *P<0.01 vs vehicle treatment (Bonferroni's test)

Figure 5. Effect of saccharin-CP-809101 pairing in the taste reactivity test Effect of CP-809101 (3-12 mg/kg, s.c.)-saccharin and lorcaserin (6 mg/kg, s.c.)-saccharin pairings during a 3 minute taste reactivity test. (A) Mean (±SEM) number of conditioned gaping reactions elicited by 0.1% saccharin solution following two conditioning sessions. (B) Mean (±SEM) number of aversive reactions during a 3 minute taste reactivity test after two conditioning sessions. *P<0.01 vs vehicle pretreatment (Bonferroni's test)

Figure 6. Effect of saccharin-lorcaserin pairing on tongue protrusions Effect of saccharin-CP-809101 (3-12 mg/kg, s.c.) and saccharin-lorcaserin (6 mg/kg, s.c.) pairing on the incidence of tongue protrusions during a 3 minute taste reactivity test. *P<0.05 vs vehicle pretreatment (Bonferroni's test)

Figure 7. Effect of saccharin-CP-809101 pairing in a conditioned flavour avoidance test Effect of saccharin-CP-809101 (3-12 mg/kg, s.c.) and saccharin-lorcaserin (3 mg/kg, s.c.) pairing during a one-bottle flavour avoidance test after two conditioning sessions. Mean cumulative (±SEM) intake of 0.1% saccharin solution after 360 min. *P<0.01 vs vehicle treatment (Bonferroni's test)

Figure 8. Effect of SB-242084 on lorcaserin-induced gaping and flavour avoidance (A) Effect of SB-242084 (0.5 mg/kg, i.p.) pretreatment on lorcaserin- induced gaping reactions to a saccharin in the taste reactivity test. The figure expresses mean (±SEM) number of conditioned gaping reactions elicited by 0.1% saccharin solution following two conditioning sessions. (B) Effect of SB-242084 (0.5 mg/kg, i.p.) pretreatment on saccharin-lorcaserin pairing during a one-bottle flavour avoidance test. Mean cumulative (±SEM) intake of 0.1% saccharin solution after 360 min. *P<0.01 vs vehicle treatment (Bonferroni's test)

Chapter 3: Lorcaserin does not induce pica behaviour in rats

3.1 Introduction

The consumption of non-nutritive substances is generally referred to as pica. This is a behaviour that can manifest in many species in response to a variety of triggers. In animals, this behaviour is often seen as a consequence of gastro-intestinal discomfort and malaise, and it is perceived as a defense mechanism against the ingestion of harmful substances. Supporting evidence comes from the fact that animals such as rats, consume non-nutritive materials (kaolin being a commonly used one) in a dose dependent manner in response to an exposure to emetogens, presumably as a response to purge the emetogen from the animal (Liu et al. 2005). To further support the idea that pica is a response to illness, it has also been reported to occur when these species are exposed to illness-inducing motion (Mitchell et al. 1977).

Anti-emetic interventions used with this model are also capable of attenuating pica as it is demonstrated by the administration of 5-HT3 receptor antagonist ondansetron as well as the NK1 receptor antagonists HSP-117 ((2S,3S)-3-[(5-isopropyl-2,3-dihydrobenzofuran-7- yl)methyl]amino-2-phenylpiperidine dihydrochloride), which decrease kaolin intake at differing degrees (Saeki et al. 2001). The ability of these treatments to block this behaviour also suggests that 5-HT3 receptors located in the stomach are important in the manifestation of pica in rats as is demonstrated by the effects of ondansetron. In addition, the HSP-117 intervention suggests NK1 receptors are also involved in the emergence of pica behaviour in rats. Furthermore, evidence also exists to show that dopamine D2 receptors in the chemoreceptor trigger zone may also be part of the mechanism in which GI distress can induce rats to consume kaolin clay (Takeda et al. 1993). The involvement of all these receptors in events of pica behaviour strengthens the notion that pica in rats involves analogous processes to the ones involved in nausea and emesis. As the availability of reliable and predictive models of nausea and emesis is lacking, pica has been an important research tool, in particular pica in rats. While other species such as shrews may also display such behavior, these species often require significantly harsher treatments in order to display the behaviour, if they are present at all (Liu et al. 2005). This contrast between species

45 may be related to the fact that rats are incapable of producing emetic responses, and therefore must rely on alternative defense mechanisms to protect themselves from consuming harmful substances. Pica therefore is a much more important response in rats, which explains why this mechanism has been amplified in this species through natural selection. For this reason, pica is considered to be a useful screening tool to reveal the potential of different treatments to produce nausea in the rat, which translates into similar effects in humans.

In recent years the serotonin 2C receptor has become a focus of many research labs as a promising mechanism in the treatment of several disorders involving compulsive behaviors. The most important development in this area of research was perhaps the approval by the FDA of a

5-HT2C receptor agonist lorcaserin (Belviq®) for the treatment of obesity (Arena Pharmaceuticals, 2010 FDA briefing document NDA 22-529, Fidler et al. 2011). A growing number of publications have also been reporting that this particular receptor target may be reducing food intake via a decrease in compulsive feeding behaviour. This notion is supported by the fact that lorcaserin is also able to reduce nicotine self-administration, discrimination and reinstatement in rats (Higgins et al. 2011). While clinical trials suggest only a few side effects of lorcaserin in humans, one of the most prevalent side effects was nausea, which occurred in 8% of subjects that received the therapeutic daily dose of 20mg, while a higher incidence was seen in subjects receiving 40mg (Arena Pharmaceuticals, 2010 FDA briefing document NDA 22-529). In the current experiment, we attempted to examine whether lorcaserin produces gastro-intestinal malaise in rats by quantifying pica behaviour induced by this compound and comparing it to the known emetogen rolipram.

3.2 Materials and Methods

Animals and housing

Adult male Sprague-Dawley rats weighing 450g to 700g were used in this experiment (source: Charlse River, St. Constant, Quebec, Canada). Animals were singly housed in polycarbonate caging with sawdust bedding. Water and chow (Purina LabDiet - 5001 Standard Rodent Diet) were freely available. Housing was maintained at a constant temperature of 22 ± 2ºC while humidity was maintained at 45 ± 20%. Lighting was set to a 12-h light-dark cycle (lights on at 0600-1800 hours) while measurements were taken during the light phased of the light/dark cycle. Experiments were conducted at the InterVivo Solutions test facility in Toronto, Canada. All experiments complied with the appropriate Animal Care and Use committee and conducted in accordance with the guidelines established by the Canadian Council of Animal Care.

Drugs and treatment regimens

Rolipram (source: Sigma-Aldrich, Oakville, Canada) was prepared as a finely suspended solution in 0.9% saline containing 5% Tween 80. Rolipram was administered via i.p. route in a dose volume of 2 mL/kg. Lorcaserin ((1R)-8-chloro-1-methyl-2,3,4,5-tetrahydro-1H-3- benzazepine HCl) (source: NPS Pharmaceuticals, Toronto, Canada) was prepared in sterile 0.9% saline and was administered subcutaneously at a dose volume of 1 mL/kg. All doses are expressed in terms of the free base.

Test conditions

China clay (Kaolin) was used as the non-nutritive material during the pica experiment. The kaolin was purchased as a large block and was cut into cubes of 2 x 2 x 2 cm. Once cut, the kaolin cubes were separated and allowed to dry in a well-ventilated environment, at room temperature for 3 days. Tests were done in each individual animal's home cage (45 x 24 x 19 cm) and a 3 day habituation period was given to the animals in the presence of the kaolin. Kaolin intake during the habituation period was measured and used to balance groups for equivalent intakes.

On the test day (day 4) animals received one of 4 treatments (lorcaserin 1 mg/kg, lorcaserin 3 mg/kg, rolipram 3mg/kg or saline) at 12:00h and kaolin, food and water intakes were also measured at 6h and 24h post dosing. Animals had ad lib access to food and water during the duration of the experiment.

Data analysis

Mean intakes were measured in grams and were analyzed by a one-way ANOVA (IBM SPSS 22). The accepted level of significance used was P<0.05.

3.3 Results

Treatment of rats with both doses of lorcaserin (1 mg/kg and 3 mg/kg, s.c.) produced no change to the consumption of kaolin clay over the course of the experiment when compared to the vehicle treated group (6 hour and 24 hours). A one-way ANOVA indicated no overall effect of drug treatments at 6 hours and at 24 hours post treatment (respectively F18,3=0.51 and

F18,3=2.05). Despite the lack of a statistically measurable effect, intraperitoneal administration of

3 mg/kg of rolipram produced a four-fold increase in the amount of kaolin consumed by the rats compared to the amount consumed by vehicle treated animals.

3.4 Discussion

The majority of animal research today uses rats and mice as the preferred species. In fact rodents make up over 80% of all animal testing in North America and Europe (Report by the Commission of the European Communities, 2007). Due to this fact, developing reliable and predictive animal models in these species is useful. For this reason, several models of nausea and emesis use rats, despite the fact that they are unable to vomit. Pica experiments are a good example of this, using a behaviour not normally exhibited by rats as a sign of gastrointestinal discomfort (nausea and vomiting). The ingestion of non-nutritive materials to relieve GI discomfort is observed both in species capable of vomiting as well as species unable to do so. It is hypothesized that pica behaviour may be more prominent in species like rats that are unable to vomit, since it must compensate for the lack of other defense mechanisms available to vomiting species such as shrews and ferrets. However, despite this fact, lorcaserin exposure at 1 mg/kg and 3 mg/kg did not produce pica behaviour in the rats used in this experiment and neither did the known emetogen, rolipram (Rock et al. 2009).

Lorcaserin has been shown to produce gastrointestinal discomfort in rats at doses (3-6 mg/kg, s.c.) comparable to the ones used in this experiment (Higgins et al. 2011). It is however possible that the selected doses for this experiment were simply not sufficiently robust to produce enough nausea in these rats to induce pica. Another study investigating the potential for lorcaserin to induce nausea in rats using the conditioned gaping model indicated that lorcaserin is only capable of producing nausea in rats at 6 mg/kg. A 3 mg/kg dose of lorcaserin did not produce significant increases in conditioned gapes, suggesting the possibility that a higher dose of lorcaserin may induce pica. The results obtained in this study may simply reflect the less sensitive nature of this model in identifying emetogenic doses of different drugs compared to other animal tests.

Previous studies conducted by the author have shown that rolipram can produce statistically significant increases in rat pica behaviour (0.3 - 1 mg/kg). Despite the fact that there was an increase in kaolin ingestion in the group treated with rolipram, the effect was not deemed statistically significant. It is quite possible that this lack of statistical significance is simply a consequence of the variability seen in the data collected from rolipram treated rats. Furthermore, it is important to note that pica behaviour as a model of nausea has some limitations, including the fact that these experiments take place over a relatively long period, therefore this study design is better suited to test drugs with long pharmacokinetic profiles. This is the reason why drugs like cisplatin tend to serve as a much better positive control for these types of studies compared to rolipram which has a much shorter half-life (Percie du Sert et al. 2012).

3.5 Figures

3.0 6h 24h 2.5

2.0

1.5 Clay intake (g) intake Clay 1.0

0.5

0.0 Saline Lor (1) Lor (3) Rol (3)

Figure 9. Effect of lorcaserin on rat pica behaviour Effect of lorcaserin (1 mg/kg and 3 mg/kg, s.c.) and rolipram (3 mg/kg, i.p.) treatment on male Sprague-Dawley rats in a pica test. Mean (±SEM) amount of kaolin consumed over 6 and 24 hours after drug administration.

Chapter 4: General discussion

During clinical trials, the 5-HT2C receptor agonist lorcaserin generated nausea as one of the more prevalent side effects in patients, especially in non-diabetic subjects (Arena Pharmaceuticals, 2010 FDA briefing document NDA 22-529). Despite the fact that this side effect did not pose significant concern to the FDA, it raised the question of drug compliance (a dose limiting factor) and secondly whether or not the 5-HT2C receptor is involved in processes of generating nausea and vomiting. The main goal of the present set of experiments was to examine the role of this serotonin receptor subtype in nausea through a pharmacological investigation of two 5-HT2C selective agonists to produce nausea-induced behaviours in rats.

In order to accomplish this, two selective 5-HT2C agonists were tested in three different assays, the taste reactivity test, flavour avoidance and pica. While all these assays are thought to model aspects of nausea and emesis in rats, they do provide different perspectives. During the pica experiment, lorcaserin did not cause animals to consume the non-nutritive material available during the test. This suggests that the drug did not produce significant gastrointestinal distress in the animals, however when considering the results from the other two assays, the data suggests that perhaps the doses chosen for the pica experiment may have been too low. The less sensitive nature of pica behaviour as a model of emesis may also be part of the reason why no effect was seen with lorcaserin at these doses. Furthermore, the temporal nature of drug effects is also important when investigating emesis and nausea in this model (Percie du Sert et al. 2012). Generally, drugs with long half-lives that produce lasting GI disturbances tend to generate more robust pica than drugs that produce acute effects. For this reason pica may not be the most suitable method to investigate the nauseating potential of lorcaserin.

While the results obtained from the pica experiment did not support the gastrointestinal side effects observed in the clinical, in contrast the data obtained from the taste reactivity test and flavour avoidance test are more supportive of our hypotheses. Systemic exposure of rats to the selective serotonin 2C agonist lorcaserin was shown to produce conditioned aversive reactions, predominantly gaping reactions and chin rubbing following two conditioning episodes. Lorcaserin also produced a robust flavour avoidance in these animals, which is another indication that they may have experienced gastrointestinal discomfort after exposure to the drug.

A subcutaneous dose of 6 mg/kg of lorcaserin was sufficient to induce gaping reactions which is believed to be a behavioural expression of nausea in this non-vomiting species (Parker and Limebeer, 2008). These results are consistent with our expectations based on the clinical trials conducted with lorcaserin and also confirms the observations previously published that lorcaserin can produce gastrointestinal distress in rats at supratherapeutical doses of the drug (Higgins et al. 2013). While the literature provides very little discussion regarding potential mechanisms for these GI disturbances, some previously published data supports the idea that 5-

HT2C receptor activation causes a decrease in levels of circulating ghrelin which may at least partially contribute to both the anorectic effects of lorcaserin as well as the GI discomfort (Takeda et al. 2008). Increases in the amount of circulating ghrelin has been shown to have antiemetic properties in animals, therefore it is possible that at high enough doses of lorcaserin, the antiemetic tone of ghrelin may be diminished, increasing the organism's vulnerability to nausea and other GI disturbances (Rudd et al. 2006). This view is mostly speculative, therefore further investigation of the role ghrelin plays in nausea and emetic processes is important to further establish our understanding of this mechanism.

The results from the taste reactivity test and the flavour avoidance test were not surprising based on what has already been published about lorcaserin. Despite this, these results only confirm that rats also experience nausea after being exposed to this compound. More information was obtained from pretreating rats with the selective 5-HT2C antagonist SB-242084, which was able to prevent rats from expressing nausea-induced behaviours observed in animals treated with lorcaserin alone. Interestingly, the antagonist SB-242084 was only able to block the aversive behaviours produced during the taste reactivity test while having little effect on the effects measured during the CFA test. This inability to block emetic effects during CFA tests has been well documented and does challenge the effects observed during the TRT. Many antiemetic compounds are incapable of blocking flavour avoidance from known emetogens (more information in section 1.8), however these results do suggest that SB-242084 only inhibits some of the effects experienced by the rats under the influence of lorcaserin.

Due to the fact that SB-242084 is a highly selective antagonist for the 5-HT2C receptor, the TRT results provide compelling evidence that the nausea produced by lorcaserin is in fact a

5-HT2C mediated effect. This gives us some confidence that lorcaserin-induced nausea is not

53 simply a result of indiscriminate agonism of other serotonergic receptors at higher doses of the drug. In this light, it would be reasonable to predict that a more selective agonist for the serotonin 2C receptor would also be able to generate nausea in rats. However, our experiments with the highly selective CP-809101 indicated that at supratherapeutic doses (12 mg/kg), CP- 809101 does not produce gaping reactions in rats. This series of experiments appears to produce paradoxical results when considered under the fundamentals of classical receptor pharmacology. However, interpretation of these results considering our current understanding of functional selectivity suggests a more cohesive story. While both lorcaserin and CP-809101 have a very selective affinity profile (more information in section 2.4), they may differentially activate the effector pathways associated with 5-HT2C receptor agonism. It is possible that comparatively, CP-809101 preferentially activates one of the effector pathways while the molecular response to lorcaserin may present a different molecular activation profile. This suggestion also accounts for the fact that CP-809101, much like lorcaserin, produced significant flavour avoidance during the CFA test. In fact, CP-809101 produced a much more robust effect in the CFA test when comparing the relative dose requirements for the two drugs. Once again, these inconsistencies in terms of effects of these two highly selective 5-HT2C agonists would be possible due to differences in activation profiles.

No data is available in the literature to suggest which effector pathways are being activated in the presence of these two 5-HT2C agonists. However, both the PLA2-AA and the PLC-IP pathways may be involved in nausea and emetic processes. It is possible that CP-809101 may be preferentially activating the PLA2-AA pathway over the PLC-IP pathway. The PLA2 mediated pathway has been previously linked to anti-emetic processes. During a study investigating the antiemetic potential of the endocannabinoid 2-AG , the non selective COX inhibitor indomethacin was administered systemically and was shown to prevent suppression of conditioned gaping from 2-AG (Sticht et al. 2012). Furthermore, systemic administration of AA suppressed conditioned gaping in the same species, which effect was also attenuated by indomethacin, suggesting that downstream metabolites of arachidonic acid play a role in the anti- nausea properties 2-AG. Therefore the PLA2 mediated effector pathway involving increased levels of AA is more likely to be the pathway involved in the mechanism in which CP-809101 generates its 5-HT2C effects, as this particular drug did not produce nausea-induced behaviours in rats during our taste reactivity experiments. It is possible to verify this claim by designing an

54 experiment aimed at blocking the PLA2-AA pathway and testing whether CP-809101 effects on feeding are affected, if in fact biased activation of this pathway is responsible for the effects on feeding and not involved in GI side effects.

Given the plausibility that the serotonin 2C effects seen with CP-809101 may occur via a biased profile for the PLA2-AA pathway, it is also possible to conjecture that lorcaserin may be producing some of its behavioural effects via the other major secondary messenger system. Under the same assumption that the inconsistencies between the effects of these two drugs occurs largely because of biased agonism, lorcaserin may be preferentially activating the PLC- IP3 pathway instead. While limited, there is evidence in the literature that suggests that inositol phosphate levels are involved in processes of nausea and emesis. Reduction of IP3 formation via

PLC inhibition has been shown to be part of the mechanism in which the 5-HT3 antagonist ondansetron produces some of its effects (Liu et al. 2012). Therefore, the nausea generated by lorcaserin during preclinical as well as clinical trials, could be mediated by increases in inositol triphosphate levels. This claim could be verified through experiments looking at whether lorcaserin-induced nausea can be attenuated through IP3 level reductions or through the blockade of the IP3 receptor.

Confirmation that functional selectivity accounts for the differences in the potential of lorcaserin and CP-809101 to produce nausea would have a significant impact in the development of future therapeutics targeting the 5-HT2C receptor as well as other receptors subjected to this phenomenon. The possibility of increasing the specificity of drug targets has the potential to change drug discovery tremendously. Functional selectivity opens the possibility that future drugs can target specific effector pathways associated with a receptor, this way it may be possible to find compounds capable of reducing feeding behaviour without undesired side effects associated with other effector pathways. Research into functional selectivity has also been able to show that receptor desensitization is another pharmacological aspect that may be subjected to agonist biases (Hayashi et al. 2005). Therefore, beyond just increasing outcome specificity during drug development, it may be possible develop NCEs that are less susceptible to system desensitization. Medications that require chronic use may be selected not only for specific outcomes but also for their ability to elicit these outcomes consistently in chronic treatment regimens.

The research contained in this report opens the opportunity for a variety of future experiments. The fact that SB-242084 was able to inhibit gaping reactions in response to lorcaserin is a strong indication that this effect may be 5-HT2C mediated; however, there is still value in further investigating the relationship between activation of this receptor subtype and nausea. Results from the taste reactivity test using CP-809101 could certainly be used to challenge this notion; therefore obtaining pharmacokinetic measurements of this drug in rats would be useful. In addition to obtaining PK data for CP-809101, it would be valuable to measure what levels of this drug actually cross the blood-brain barrier to reach the central nervous system. Equivalent data for lorcaserin is available in the literature and a comparison could potentially help explain the fact that even at high doses, CP-809101 treated rats did not produce gaping reactions.

Beyond confirming that the 5-HT2C receptor has the potential to produce nausea, it would also be useful to understand the possible mechanisms in which activation of this receptor may be triggering nausea in humans and animals. As previously discussed, the hormone ghrelin may be involved in this process, therefore investigating the potential of using ghrelin as a way to prevent lorcaserin-induced nausea could help us understand how this receptor subclass may be involved in the genesis of nausea.

Lastly, there may be experiments available to explore the previously mentioned possibility that functional selectivity is the reason for the inconsistencies between the effects of CP-809101 and lorcaserin. It would be extremely valuable to further understand how both of these drugs activate the 5-HT2C receptor beyond affinity measures. A study similar to what was done by Berg and her colleagues (Berg et al. 1998) could be conducted using modern selective

5-HT2C agonists such as lorcaserin and CP-809101. Further understanding of the molecular mechanisms involved in the effects of both of these compounds could facilitate the interpretation of our results, and perhaps confirm that it is indeed functional selectivity which is producing varying effects from the activation of the same receptor.

4.1 Conclusion

The findings in this thesis provide further information on another potential area in which the serotonin 2C receptor is involved, in addition to its role in feeding and mood regulation. While results from the taste reactivity experiments consistently reproduced reported side effects of lorcaserin during its clinical trials and provided strong evidence for the involvement of the serotonin 2C receptor in nausea, it still did not provide definitive proof that activation of this receptor subtype produces nausea. However, these results may lead future research into trying to understand how the idea of functional selectivity may be a crucial consideration during development of modern pharmacological therapeutics.

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