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Isovaline : a new analgesic Wang, Tanche 2008

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ISOVALINE: A NEW ANALGESIC by TANCHE WANG B.Sc., The University of British Columbia, 2006 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Pharmacology and Therapeutics) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April 2008 © Tanche Wang, 2008 ABSTRACT There is a great need for new analgesics. The current problem in treatment of severe pain is that side effects limit the effectiveness of therapy. Glycine receptors are important in modulation of nociception, suggesting a novel class of analgesics. Previous studies in rats show that intrathecal administration of glycine agonists and amino acids structurally similar to glycine have antinociceptive effects. The effects of isovaline, a unique, non-proteogenic glycine-like amino acid, have not been studied. Isovaline is absorbed from the gut and transported across the blood- brain-barrier. We examined the hypothesis that isovaline produces antinociception in mice. Administration of strychnine, an antagonist at glycine receptors, into the cisterna magna or lumbar intrathecal space resulted in allodynia, localized to the somatotopic distribution of the trigeminal and lumbar nerves. These findings provided a basis for models of lumbar and trigeminal neuralgia. Racemic isovaline blocked strychnine induced allodynia in both models without apparent side effects. We next investigated the antinociceptive effects of glycine-like amino acids in formalin foot assay, a conventional rodent model of acute and chronic pain. Antinociceptive effects were demonstrated on intrathecal administration of glycine, beta-alanine, and isovaline. Intravenous isovaline produced significant antinociceptive effects in the formalin foot model. The toxicity of isovaline and related amino acids were determined. Exploratory behavior, gait, and responses to stimuli were used to assess sedation. The rotarod test was used to examine central nervous system (CNS) and neuromuscular toxicities of intravenous isovaline. Lumbar ii administration of glycine and beta-alanine caused scratching and/or lower body weakness. Isovaline at 7-times intrathecal ED 50 produced lower body weakness in some animals. None of the amino acids produced sedation comparable to morphine. At 6-times ED 50 , beta-alanine produced weakness. Both glycine (ED 50) and beta-alanine (3x ED 50) but not isovaline produced local nerve irritation. Intracisternal injection of glycine did not reverse allodynia and resulted in death. Neither R nor S enantiomers of isovaline impaired performance on the rotarod test. Isovaline has significant antinociceptive properties. Given the absence of apparent CNS or motor toxicity, isovaline has potential as a clinical analgesic. iii TABLE OF CONTENTS ABSTRACT^ ii TABLE OF CONTENTS^ iv LIST OF TABLES viii LIST OF FIGURES^ ix LIST OF ABBREVIATIONS^ xi ACKNOWLEDGEMENTS xii Chapter 1. Introduction^  1 1.1. Scope of the thesis  1 1.2. Pain^  2 1.3. Allodynia 3 1.4. Pain Pathway^ 7 1.5. NMDA Receptor  10 1.6. GABA^ 12 1.7. Glycine  14 1.8. Chemistry of Glycine and Related Compounds^ 20 1.9. Rationale^ 23 Chapter 2. Materials 24 2.1. Animals^ 24 2.2. Drugs 24 2.3. Equipment^ 26 2.3.1. Anesthesia 26 2.3.2. Intrathecal Injection Mouse House Holder & Observation Chamber^ 26 iv 2.3.3. Intracisternal and Intrathecal Injection Syringe and Needle^ 28 Chapter 3. Methods^ 29 3.1. Randomization & Blinding^ 29 3.2. Allodynia: Intracisternally Administered Strychnine Induced Allodynia^ 29 3.2.1. Intracisternal Injection^ 29 3.2.2. Intracisternal Administration of Strychnine Induced Trigeminal Allodynia^ 31 3.2.3. Pilot Test for Intracisternal Administration of Glycine^ 32 3.2.4. Effect of Morphine and Isovaline on Intracisternal Strychnine Induced Allodynia^ 32 3.3. Allodynia: Lumbar Intrathecal Administered Strychnine Induced Allodynia^ 32 3.3.1. Lumbar Intrathecal Injection^ 32 3.3.2. Lumbar Allodynia^ 34 3.4. Determining Analgesia (Formalin Foot Assay)^ 34 3.4.1. Paw Formalin Assay^ 34 3.4.2. Intrathecal Administration of Isovaline and Glycine-like Compounds on Formalin Foot Assay of Analgesia^ 36 3.4.3. Intravenous Administration with Formalin Foot Assay^ 36 3.5. Determining Toxicity^ 37 3.5.1. Rotarod Test 37 3.5.2. Sedation Assay^ 37 3.5.3. Observation of Behavior and General Appearance & Allodynia Assay^ 38 3.6. Statistical analysis^ 38 Chapter 4. Results 39 4.1. Allodynia^ 39 v 4.1.1. Intracisternal Administration of Strychnine Induced Allodynia^ 39 4.1.2. Effect of Intracisternal Injection of Carbamazepine-10,11-epoxide, Morphine, Racemic Isovaline and Glycine on Strychnine Induced Allodynia^ 39 4.1.3. Lumbar Intrathecal Administration of Strychnine Induced Allodynia^ 42 4.2. Analgesia^ 43 4.2.1. Lumbar Intrathecal Injection of Glycine^ 43 4.2.2. Lumbar Intrathecal Injection of Beta-alanine 46 4.2.3. Lumbar Intrathecal Injection of Racemic Isovaline^ 46 4.2.4. Lumbar Intrathecal Injection of Controls^ 49 4.2.5. Intravenous Analgesia of Racemic Isovaline 51 4.2.6. Toxicity: Rotarod of Intravenous Administration of Racemic Isovaline^ 54 4.2.7. Optical Activity^ 56 4.2.8. Preliminary Results with ACBC^ 58 Chapter 5. Discussion^ 59 5.1. Summary of the results 59 5.2. Allodynia^ 59 5.1.1. Intracisternal Strychnine Induced Allodynia^ 59 5.1.2. Intracisternal Injection of Glycine^ 61 5.1.3. Intracisternal Isovaline Against Strychnine Induced Allodynia^ 61 5.1.4. Intracisternal Morphine Against Strychnine Induced Allodynia 61 5.1.5. Intracisternal Carbamazepine Against Strychnine Induced Allodynia^ 62 5.1.6. Lumbar Intrathecal Injection of Strychnine Induced Allodynia 62 5.1.7. Lumbar Intrathecal Injection of Isovaline Against Strychnine Induced Allodynia^ 63 vi 5.3. Analgesia^  63 5.3.1. Lumbar Intrathecal Injection Glycine-like Compounds^ 63 5.3.2. Structure Activity Relationship of Glycine-like Compounds 65 5.3.3. Toxicity of Lumbar Intrathecal Injection of Glycine^ 66 5.3.4. Toxicity of Lumbar Intrathecal Injection of Isovaline 67 5.3.5. Toxicity of Lumbar Intrathecal Injection of Beta-alanine^ 68 5.3.6. Toxicity of Lumbar Intrathecal Injection of Controls 68 5.3.7. Intravenous Analgesia of Isovaline^ 69 5.3.8. Toxicity of Intravenous Injection of Isovaline^ 70 5.4. Potential Mechanisms^ 70 5.5. Future Directions 73 Chapter 6. Conclusions^ 76 REFERENCES 78 APPENDIX^ 89 vii LIST OF TABLES Table 1. Intracisternal strychnine and carbamazepine^ 40 Table 2. Intracisternal strychnine and racemic isovaline 41 Table 3. Lumbar intrathecal strychnine and racemic isovaline^ 42 Table 4. Sedation score for lumbar intrathecal injection of glycine-like compounds^ 48 viii LIST OF FIGURES Figure 1. Diagram showing the distribution of the sensory branches of the trigeminal nerve. ^ 6 Figure 2. Trigeminal distribution in mice.^ 6 Figure 3. Neurophysiology of pain. 9 Figure 4. Model of N-terminal region of the al G1yR and organization of ligand-binding sites in heteromeric GlyRs ^  16 Figure 5. Glycinergic innervation of the spinal cord dorsal horn ^  19 Figure 6. Glycine-like compounds ^ 20 Figure 7. Chemical structure of potential analgesics ^ 25 Figure 8. Observation chamber set up ^ 27 Figure 9. Lumbar intrathecal injection in mouse ^ 28 Figure 10. Diagram of intracisternal injection 30 Figure 11. Cisterna Magna — Site of Injection of intracisternal administration ^ 30 Figure 12. Diagram of needle insertion into the intervertebral space between L5 and L6. ^ 33 Figure 13. Experimental method timeline for dose response of intrathecal injection of glycine- like compounds^  35 Figure 14. Formalin induced pain behaviours during phase I and II for various doses of glycine- like compounds.^ 44 Figure 15. Scratching in 5 min bins for various doses of glycine, beta-alanine, and isovaline^ 45 Figure 16. Formalin induced pain behaviours during phase I and II for various doses of P-alanine and isovaline. ^ 47 Figure 17. Scratching behaviours for aCSF, mannitol, beta-alanine and racemic isovaline. ^ 50 Figure 18. Respiratory rate for mice injected intravenously with 500 mg/kg isovaline or saline 52 ix Figure 19. Effects of intravenous racemic isovaline on acute and chronic phases of formalin- induced pain.^ 53 Figure 20. Comparison of time on rotarod before and after intravenous injection of 500 mg/kg S- isovaline or saline control.^  55 Figure 21. Comparison of time on rotarod before and after intravenous injection of 500 mg/kg R- isovaline or saline control.^ 55 Figure 22. Effects of intrathecal racemic, R- and S -isovaline on phase I and II of formalin- induced pain.^ 57 Figure 23. Effects of 250 mM ACBC on phase I and II of formalin induced pain^ 58 x LIST OF ABBREVIATIONS ACBC^ 1 -Aminocyclobutane- 1 -carboxylic acid aCSF Artificial Cerebrospinal Fluid bpm^ Breaths Per Minute Ca2+^Calcium Ions CNS Central Nervous System CSF^ Cerebrospinal fluid FDA Food and Drug Administration GABA^ y-aminobutyric acid GIyR Glycine Receptors IASP^ International Association for the Study of Pain K+^Potassium Ions mg2+ Magnesium Ions Na+^Sodium Ions NMDA N-methyl d-aspartate NSAID^ Non-steroidal Anti-inflammatory Drugs PE Polyethylene PGE2^ Prostaglandin E2 PKA Protein Kinase A s^ Seconds xi ACKNOWLEDGEMENTS I offer my enduring gratitude and appreciation to Dr. Bernard MacLeod for his guidance and teaching. I am very grateful to Drs. Ernest Puil, David Mathers, Stephan Schwarz, Craig Ries, Richard Wall, and Michael Walker for their valuable advice. I thank visiting scholars Dr. Il-Ok Lee and Dr. Sang Mook Lee, undergraduate student Ms. Helen Cheung, and graduate students Ms. Cheryl Chung and Dr. Terence Gilhuly for their support. I thank all the members of the Hugill Centre for Anesthesia Research and the Department of Anesthesiology, Pharmacology & Therapeutics for their support and interest. Special thanks to Mr. Christian Caritey and Mr. Andy Jeffries for their technical assistance and Ms. Wynne Leung for her administrative support. This work was supported by the Natural Sciences and Engineering Research Council of Canada, Hugill Anesthesia Research Centre, and the University of British Columbia Entrance Scholarship. Special thanks are owed to my family who have supported me morally and emotionally throughout my life. xii Chapter 1. Introduction 1.1. Scope of the thesis Pain is one of the most common reasons for seeking medical attention (Schappert, 1992) and can have significant personal, social, and economic consequences(Turk & Rudy, 1988). Chronic pain affects a third of the world population (Breivik et al., 2006; Gureje et al., 1998) with major impact on quality of life including mood, physical and social functioning, (Turk & Rudy, 1988) and costs more to society than cancer and cardiovascular diseases combined. Acute pain is distressing. Chronic pain is persistent and difficult to treat and often intractable (Eisenberg et al., 2005). Current analgesics for acute and chronic pain are limited by side effects. The difficulty in treating intractable chronic pain coupled with the need for new analgesics with fewer side effects has prompted us to seek novel analgesics. In the University of British Columbia, Department of Anesthesiology, Pharmacology & Therapeutics, Centre for Anesthesia and Analgesia, Dr. E. Puil and Dr. D. Mathers' neurophysiology laboratory established the functional presence of glycine receptors in the thalamus (Ghavanini et al., 2005; Ghavanini et al., 2006), gateway for pain to the brain. This led us to investigate treatments of acute and chronic pain. This thesis will examine the effects of glycine-like compounds, in particular isovaline as novel analgesics. Glycine is involved in respiratory depression (Ballanyi et al., 1999; Holtman et al., 1982) and cardiac toxicities (Olsson et al., 1995; Olsson et al., 1999). Glycine itself has undesirable side effects and is inappropriate as a clinical drug because of its pervasive presence and many functions in the body. In particular, glycine is a major building block in the synthesis of protein and glycine an inhibitory neurotransmitter found in various areas of the central nervous system. Isovaline is 1 structurally similar to glycine but unlike glycine, it is not naturally found in the body or in the biosphere and would be unlikely to interact with protein synthesis to induce side effects. Isovaline also has limited metabolism and would be unlikely to produce toxic metabolites. Hence, isovaline is a potential analgesic with minimal side effects. 1.2. Pain The International Association for the Study of Pain (IASP) defines pain as "an unpleasant sensory and emotional experience associated with actual or potential tissue damage or described in terms of such damage. (IASP Sub-Committee on Taxonomy, 1979)" However, since pain is a subjective human experience, it is difficult to define in animals (Merskey & Bogduk, 1994). We define nociception as processing of information about damaging stimuli up to and including the point of where perception presumably occurs, in the cerebral cortex. Pain can be divided into chronic pain and acute pain. Acute pain is usually temporary and results from something specific such as surgery, injury or infection and is generally treated with therapies such as non-steroidal anti-inflammatory drugs (NSAID's) (Merskey & Bogduk, 1994). Acute pain is better managed than chronic pain, but the current treatment is still limited by side effects. The most commonly used drugs for acute pain are NSAID's which cause side effects that include gastric bleeding, renal toxicity, and allergy. Chronic pain lasts for more than three months and can disrupt daily life. (Merskey & Bogduk, 1994). An estimated 50 million Americans have chronic pain, and 41% of patients with chronic pain report that their pain is not controlled (Nicholson et al., 2006a). Chronic pain is the most common cause of long-term 2 disability and is associated with reduced physical, psychological, and social well-being with resultant increased use of health services (Longo et al., 2000; Nicholson et al., 2006b; Reid et al., 2002). The most common sites for chronic pain are in the head, neck, knees, and lower back (Breivik et al., 2006). Chronic pain is associated with many signs and symptoms including, immobility (and consequent wasting bones and joints), depression of the immune system (causing increased susceptibility to disease), disturbed sleep, poor appetite and nutrition, dependence on medication, overdependence on caregivers and family, overuse and inappropriate use of healthcare providers and systems, poor performance on the job, isolation from family or society, anxiety and fear, bitterness, frustration, depression and suicide(World Health Organization, 2007). Current therapies for chronic pain are limited by poor efficacy (Catheline et al., 2001; Eisenberg et al., 2005). The best current therapy for chronic pain is morphine but it is limited by side effects including respiratory depression, constipation, itching, somnolence, sweating, and nausea and vomiting and less commonly, pulmonary edema, confusion, cardiac arrhythmias, hypogonadism, and sensorineural hearing loss. There is a need for pain medications for the treatment of chronic pain and that have fewer side effects (Ballantyne & Mao, 2003; Eisenberg et al., 2005). 1.3. Allodynia Allodynia is a painful behavioural response to normally innocuous stimuli. There are different kinds or types of allodynia: (1) Mechanical allodynia (also known as tactile allodynia) is a painful response from light touch/pressure applied to the skin. Mechanical allodynia can be 3 dynamic or static. (2) Thermal (heat or cold) allodynia is a painful response from normally mild skin temperatures to afflicted area. Allodynia is often associated with neuropathic pain due to its chronic nature; it can be associated with chronic pain. Generally, we perceive pain's evolutionary role as a warning to protect us from behaviours that are potentially harmful, but allodynia appears to be a dysfunctional pain system with no apparent evolutionary benefit. Allodynia that is localized to the trigeminal system is known as trigeminal allodynia, and is apparent in the disorder, trigeminal neuralgia (tic douloureux). The trigeminal nerve or fifth cranial nerve carries sensation from the face to the brain (Figs. 1 and 2). The fifth nerve has three branches: The ophthalmic branch mediates sensation in the eye, upper eyelid and forehead. The maxillary branch mediates sensation in the lower eyelid, cheek, nostril, upper lip and upper gum. The mandibular branch mediates sensations in the jaw, lower lip, lower gum and some of the muscles used for chewing. The mandibular branch also has a motor function to activate muscles of the mandible. The motor fibers originate in the motor nucleus of the fifth nerve, located near the main trigeminal nucleus in the pons, and have bilateral cortical representation. The trigeminal nerve nucleus is the largest cranial nerve nuclei and is found in the medulla, midbrain, and brainstem. The trigeminal nucleus is divided into three parts and from caudal to rostral are the spinal trigeminal nucleus, main trigeminal nucleus, and mesencephalic trigeminal nucleus. The three parts of the trigeminal nucleus receives different types of sensory information. The spinal trigeminal nucleus receives pain and temperature sensations from the face carried by cranial nerves V, VII, IX, and X. The main trigeminal nucleus receives touch and position sensations from the face and mouth via cranial nerves V, VII, IX, and X. The mesencephalic trigeminal nucleus receives position sensations from the jaw and touch sensations from the teeth. The touch and position information from the face is sent to the ventral posteromedial nucleus (VPN) of the 4 thalamus before being sent to the cortex. Trigeminal allodynia is characterized by episodes of sharp, stabbing pain in the cheek, lips, gums, or chin on one side of the face. Trigeminal allodynia is paroxysmal with unpredictable sudden periods of pain between painless periods. The sudden bouts of pain are excruciatingly painful and can often be debilitating. Autopsies of the trigeminal allodynia patients reveal no visible pathologies with the trigeminal nerve and the etiology of trigeminal allodynia is not well known. There are limited medications to treat trigeminal allodynia and even opioids' effectiveness are limited. The lack of medications and understanding of the disorder has prompted the Food and Drug Administration (FDA) to issue specialized grants for research of trigeminal allodynia models and therapeutics. Rational development of analgesics for chronic and recurrent pain depends on the availability of animal models that mimic symptoms or pathophysiology of the disorder. This remains a challenge in trigeminal allodynia where the etiology is not well known. Relatively few animal models have reproduced behavioural changes associated with trigeminal allodynia. Lumbar intrathecal administration of strychnine to antagonise of glycine receptors produced allodynia in rats. The strychnine induced allodynia was blocked by glycine and other glycine-like compounds administered similarly (Beyer et al., 1988; Jiang et al., 2004; Khandwala & Loomis, 1998). Along with the finding of glycine receptors in the thalamus and brain stem, we investigated the role of glycine receptors and glycine-like compounds in a new rodent model of strychnine induced trigeminal neuralgia. 5 Figure 1. Diagram showing the distribution of the sensory branches of the trigeminal nerve. Figure 2. Trigeminal distribution in mice. 6 1.4. Pain Pathway Our search for novel pain medications is based on the concept of modulating the pain pathway to minimize the transmission of nociceptive afferent input. The propagation of pain is initiated with the activation of nociceptors in the periphery by a noxious stimulus. The nociceptors correspond to the distal part of the primary afferent fiber. Primary afferent fibers are classified in terms of structure, diameter and conduction velocity. C-type fibers are unmyelinated, ranging in diameter from 0.4 to 1.2 pm and have a velocity of 0.5 — 2 m/s. M fibers are barely myelinated, ranging in diameter from 2.0 to 6.0 pm and have a velocity of 12-30 m/s. C fibers present thermosensitive receptors reacting to heating and cooling, mechanoreceptors of low threshold and specific receptors for algogenic substances such as potassium ions, acetylcholine, proteolytic enzymes, serotonin, prostaglandin, substance P and histamine. AS fibers are classified into two groups. The first group, Type I, corresponds to fibers of high threshold mechanoreceptors and respond weakly to thermal or chemical stimuli, and after being sensitized, to harmful heat. Type II, presents fibers with mechanothermal receptors for high temperatures (45-53 °C) and some receptors for intense cold (-15 °C) and later sensitized to vigorous mechanical stimuli at nonnoxious thresholds. Noxious stimuli in the periphery stimulate nociceptors and results in a signal propagated along the primary afferent fiber to the spinal cord (Wall et al., 2006) (Fig. 3). At the spinal cord, the primary afferent fibers terminate in the dorsal horn of the spinal cord to form synapses with a secondary neuron that may terminate on other neurons such as interneurons before forming synapses with projection fibers. There are several nociceptive pathways of the spinal cord that relays the nociceptive signal from the primary afferent in the spinal cord and to the brain including spinothalamic, spinoreticular, spinomesencephalic, spinoparabrachial, spinohypothalamic, spinocervical tract, and postsynaptic pathway of the spinal column (Almeida 7 et al., 2004). The spinothalamic tract decussates at the spinal cord anterolaterally via the anterior white commissure and ascends to terminate at the thalamus. The spinoreticular tract ascends mainly through the anterolateral contralateral funiculus of the spinal cord and is composed of two components with one component projecting to the lateral reticular formation, a precerebellar nucleus involved in motor control and the other component projects to the medial, pontomedullary reticular formation, and then onto thalamocortical circuits. The spinomesencephalic tract ascends through the anterolateral contralateral funiculus of the spinal cord and involves to bundles with the spinoannular tract projecting to the periaqueductal gray and the spinotectal tract projecting to the deep superior colliculus. The spinoparabrachial tract ascends through the contralateral dorsolateral funiculus of the spinal cord and to the parabrachial nucleus in the brain stem and is differentiated into two pathways with the spinoparabrachial amygdaloid pathway which projects to the amygdala and stria terminalis and the spinoparabrachial hypothalamic pathway which projects to the ventromedial nucleus of the hypothalamus. The spinohypothalamic tract ascends through the contralateral anterolateral funiculus of the spinal cord and to the hyopothalamus with collaterals to the supraoptic decussation that travel to the contralateral brain and caudally to innervate the hypothalamus, pons, thalamus, and limbic structures such as the amygdala, septum and striatum. The spinocervical tract ascends ipsilaterally from the dorsolateral funiculus to the lateral cervical nucleus in the medullary segments Cl-C3, the site of the first relay, from which it crosses the midline and establishes second order projections to the posterior and medial thalamus. Finally, the postsynaptic pathway of the spinal column ascends ipsilateraly in the spinal cord to the first relay in the nucleus of the spinal column and then project to the lateral thalamus and superior colliculus. 8 Central Nucleus of Arrygdala Ventromedial Nucleus of Hypothalamus Spinal Nucleus Of Nerve V V VII IX !Dorsal Column' Spin oparabrachial Hypothalamic Pathway Spin oparabrachial Amygdaloid Pathway Spinocervical Tract Polysynaptic Pathway Reticular Formation SpinoreticularTract SpinomesencephalicTract tr Spinothalamic Tract 111 Spinohypothalamic Tract In general the pain pathways ascend the spinal cord through the projection fibers ipsilaterally or cross the spinal cord anterolaterally and ascend to terminate at the thalamus directly or indirectly through the reticular activating system (RAS). The RAS synapses with extrathalamic neurons to the neocortex. The projection fiber forms a synapse with tertiary neurons at the thalamus. The tertiary neuron from the thalamus transmits the nociceptive signal to the cortex for conscious perception of pain. Figure 3. Neurophysiology of pain. Sensation from peripheral receptors travels along specific pain nerves, and is modulated throughout the spinal cord and brain. 9 1.5. NMDA Receptor The pain pathway is mediated by neurotransmitters glutamate, y-aminobutyric acid (GABA), glycine and a number of peptides such as neurokinins, calcitonin gene related peptide (CGRP), somatostatin, and galanin (Dickenson, 1995). There are currently three types of neurokinin or tachykinin receptors: neurokinin-1, neurokinin-2, neurokinin-3. Substance P (SP) is one of a family of neurokinins and is the preferred neurokinin at the neurokinin-1 receptor. The neurokinin receptors have been shown to be postsynaptic to the afferent fiber terminals, located in laminae I, II, and X of the dorsal horn of the spinal cord (Otsuka & Yoshioka, 1993). Calcitonin gene-related peptide is a primary afferent peptide that is released by noxious stimuli and excites dorsal horn neurones. Somatostatin is present in small diameter cells in the dorsal root ganglion and afferent terminals in the substantia gelatinosa of the spinal cord. Noxius thermal stimulation increases the release of somatostatin within the dorsal horn of the spinal cord and application of somatostatin results in hyperpolarization of dorsal horn neurones and a reduction in spontaneous firing suggesting somatostatin has an inhibitory role in the dorsal horn. However, spinal application of somatostatin results in motor dysfunction and paralysis at doses just above antinociceptive doses. Galanin is a putative inhibitory peptide which is colocalized with SP and CGRP in a large proportion of primary afferents. Studies with thermal nociception indicates galanin has antinociceptive but there is also evidence that galanin has pronociceptive effects (Dray et al., 1994). Glycine, is a well established inhibitory neurotransmitter- in the spinal cord and brainstem and is involved in antinociception (Werman et al., 1968). The NMDA receptor. is ionotropic and is activated by glutamate as well as the selective agonist, NMDA, to open an ion channel that allows the flow of Na+ and small amounts of Ca2+ into the 10 cell and K+ out of the cell. An excitatory inward current is generated from the flow of Na + and small amounts of Ca2+ ions into the cell and IC' out of the cell. The NMDA receptor requires glycine as a co-agonist with NMDA agonists to activate the NMDA receptor channel. D-serine is a more potent co-agonist than glycine for the NMDA receptor. NMDA receptors are also voltage dependently blocked by Mg2+, and a depolarization in transmembrane potential will enhance the opening of the ion channels in NMDA receptors. The NMDA receptor is a heterodimer composed of two subunits, NR1 and NR2, with NR1 existing in eight variants including NR1a, NR1b, NR1c, NR1d, NR2a, NR2b, NR2c, NR2d, and NR2 existing in four variants ranging from NR2A to NR2D. A third related gene family of NR3 A through B subunits have inhibitory effect on receptor activity. Glycine elicits excitatory currents in NMDA receptors (NR1a and NR3A or NR3B subunits expressed in Xenopus oocytes) independent of glutamate or NMDA (Chatterton et al., 2002). NMDA and glutamate cannot evoke glycine independent response. NMDA receptors have been identified in unmyelinated and myelinated axons in peripheral somatic tissues (Carlton et al., 1995; Coggeshall & Carlton, 1998) and local injections of glutamate or NMDA result in nociceptive behaviour that can be attenuated by NMDA receptor antagonists (Jackson et al., 1995; Lawand et al., 1997; Zhou, Bonasera, & Carlton, 1996). Peripheral administration of MK-801, a noncompetitive NMDA receptor antagonist, produces local anesthetic-like effects (Ushida et al., 1999). Hyperalgesia and spontaneous pain behaviour observed in formalin test or after inflammation can be inhibited by peripheral administration of NMDA receptor antagonists, including those clinically available and by NR2B-selective compounds (Davidson & Carlton, 1998; Jackson et al., 1995; Leem et al., 2001; Taniguchi et al., 1997; You et al., 2002). The nociceptive responses induced by injection of glutamate into the mouse paw appear to involve not only peripheral but also spinal and supraspinal NMDA 11 receptors and are largely mediated by release of nitric oxide (NO) (Beirith et al., 2002). Central NMDA receptors are involved in central sensitisation or a state of increased dorsal horn excitability leading to facilitation of sensory input which results in low intensity stimulus acting via low threshold afferents generating pain (the phenomenon of allodynia) and noxious inputs resulting in a pain response of augmented amplitude and duration (hyperalgesia). Changes in NMDA receptor subunit expression is altered in the rat lumbar spinal cord by inducing pain with formalin injection into the hind paw (Petrenko et al., 2003). Central sensitization in the spinal cord dorsal horn is mediated by presynaptic and postsynaptic NMDA receptors. Small diameter primary afferent fibers terminating in the dorsal horn express NMDA receptors and activation of the presynaptic NMDA receptors causes the release of substance P from primary afferents (Liu et al., 1997). In addition, glutamate released from the presynaptic terminal and potentially enhances its own release in a feed forward manner in response to subsequent stimuli. Supraspinal NMDA receptor activation and receptor subunit expression is up-regulated in the brainstem after inflammation (Miki et al., 2002; Terayama et al., 2000). 1.6. GABA y-Aminobutyric acid (GABA) is the main inhibitory transmitter in the brain. GABA is released by spinal cord interneurons and inhibits transmitter release by primary afferent terminals in the dorsal horn. GABA acts on three types of receptors, GABAA GABAB and GABAc receptors. GABAA and GABA.c receptors are ionophores that when activated result in an increase in chloride conductance to typically stabilize the transmembrane potential near the resting membrane potential and hence decrease excitability. A pharmacological distinction of GABA A 12 receptors is a benzodiazepine binding site that enhances the activity of endogenous GABA (Hevers & Luddens, 1998). GABAA receptors are found in the dorsal root ganglion, and superficial dorsal horn terminals and cell bodies and mirrors the high concentration of GABA found in the dorsal horn of the spinal cord. GABA B receptors are G-protein coupled receptors that serve to diminish the opening of voltage-gated calcium channels on nerve terminals to decrease Ca2+ conductance, and postsynaptically to increase K + conductance which hyperpolarizes the membrane (Hammond, 2001). GABA B binding is maximal in laminae II with half of the binding lost after capsaicin or rhizotomy (Price et al., 1987). GABA has been implicated in modulating pain and a number of experiments show the importance of GABA in the modulation of pain (Hammond, 2001). Intrathecal administration of GABAA receptor antagonists bicuculline or picrotoxin produced allodynia and hyperalgesia in the rat (McGowan & Hammond, 1993; Sivilotti & Woolf, 1994; Yaksh, 1989). Similarly, GABAB receptor antagonist CGP35348 produced mechanical allodynia in the rat (Hao, Xu, & Wiesenfeld-Hallin, 1994). In the formalin foot test, GABA A receptor agonists muscimol and isoguvacine suppressed the first- and second- phase responses to formalin, indicating that GABAA receptor agonists are effective against persistent inflammatory pain, as well as acute pain (Dirig & Yaksh, 1995; Kaneko & Hammond, 1997). A GABA A receptor agonist, THIP, produced analgesia in humans at doses of 10 to 20 mg in an open — label, single-blind trial of sever chronic cancer pain (Kjaer & Nielsen, 1983) and in a double-blind, placebo-controlled trial of experimental pain after tooth pulp stimulation in human volunteers (Lindeburg et al., 1983). However, between 30 and 80% of patients reported sedation and dizziness. The small therapeutic index between the antinociceptive and sedative properties of THIP precluded further clinical development of this GABA A receptor agonist and no other such drugs have been brought 13 forward since. In an attempt to discovery analgesics based on imitating the action of GABA, different analogues of GABA have been produced including gabapentin and baclofen. These attempts were successful in producing new analgesics that paradoxically act through non- GABAergic systems (Bryans & Wustrow, 1999; Mao & Chen, 2000)At the present time, there are no clinical analgesics with effects due to GABA or glycine receptor agonism. 1.7. Glycine Recently, the functional presence of glycine receptors in the ventrobasal thalamus was established (Ghavanini et al., 2005; Ghavanini et al., 2006). The thalamus is important in receiving pain transmission from the spinal cord pain pathways. This suggests the possibility of modulating pain through glycine receptors in the brain. There have also been immunohistochemistry studies indicating relatively high levels of glycine receptors in the substantia gelantinosa (Todd & Sullivan, 1990), which is important in nociception. These findings suggest the importance of glycine receptors in the pain pathway. Glycine and similar amino acids act on two types of receptors: glycine receptors (GlyR or glycineA site) that are sensitive to strychnine, an antagonist that originated as a convulsive alkaloid from the Indian tree Strychnos mix vomica. Glycine also acts on the glycine site (glycineB) on the NMDA (N-methyl-D-aspartate) receptor which is insensitive to strychnine (Danysz & Parsons, 1998; McBain & Mayer, 1994). The strychnine sensitive glycine receptor is a pentameric ionotropic receptor with chloride conductance and is composed of a and subunits together with gephyrin, an anchoring protein 14 required for the postsynaptic clustering of glycine receptors and major GABA A receptor subtypes (Fig. 4) (Kneussel & Betz, 2000). Biochemical studies show that a subunits are essential for assembly of functional GlyR (Kneussel & Betz, 2000; Meyer et al., 1995). The stoichiometry of the synaptic glycine receptor subunits is 2a:313 (Grudzinska et al., 2005). There are 4 types of a subunits (al, a2, a3, and a4) and so far a single type of 13 subunit (Laube et al., 2002b; Lynch, 2004). Biochemical and in situ hybridization studies have shown that GlyRa 1 mRNA is prominently expressed in spinal cord, brainstem and colliculi of adult rodents. a2 subunits mRNA are abundant at birth and found only in low levels in adult hippocampus, cerebral cortex and thalamus (Malosio et al., 1991; Sato, Kiyama, & Tohyama, 1992). Moderate levels of a3 subunits mRNA are detected in spinal cord, cerebellum and olfactory bulb (Malosio et al., 1991). Presumably because of the low level of a4 subunit mRNA, it has so far escaped localization in the mammalian central nervous system (Harvey et al., 2000) but have been found in the retina (Heinze et al., 2007). There are many isoforms for the glycine receptors based on the different subunit composition (Betz & Laube, 2006). The neonatal form of the GlyR is thought to be a homopentamer of a2 subunits (Hoch et al., 1989; Takahashi, 2005) found mainly extrasynaptically in vivo, whereas functional adult synaptic GlyR are mainly composed of al and 13 subunits (Meyer et al., 1995). However, recent studies show that both al and a2 are present in young animals (Betz & Laube, 2006). 15 (b) Figure 4. Model of N-terminal region of the al GIyR and organization of ligand-binding sites in heteromeric GlyRs.(a) View of the extracellular domain (ECD) of the pentameric al glycine receptor (G1yR) modeled after the structure of the acetylcholine-binding protein (AChBP) (Brejc et al., 2001; Laube et al., 2002a). Individual al subunit backbones are coloured differently. Glycine indicated by capped sticks is bound at the interface of adjacent subunits (Grudzinska et al., 2005). (b) Schematic drawing of the pentameric arrangement of GIyR subunits in heterooligomeric a 1 R GlyRs. Binding sites for glycine are indicated in yellow, and glycine sites also capable of binding strychnine are shown by a red surround. Note the non-equivalence of glycine- and strychnine- binding sites in the hetero-oligomeric receptor. The binding properties of the 1313 interface are unknown (indicated by "?"). 16 Glycine receptors are found throughout the spinal cord. Immunocytochemistry studies suggest that the majority of glycinergic interneurons are found in the deeper laminae (III-VI) of the spinal cord. Glycine is often colocalized in nerve terminals with GABA in the superficial layers (Todd & Sullivan, 1990). Recently, a specific subtype of glycine receptor with the a3 subunit was found to be localized in the substantia gelatinosa or laminae II of the dorsal horn which is known to be the main region for nociceptive afferent fibers to synapse at the dorsal horn (Hosl et al., 2006). While al subunits are expressed throughout the grey matter, only a few synaptic GlyR a3 clusters co-localize with GlyR al subunits. It was later elucidated that prostaglandin E2 (PGE2) acts on the prostaglandin, eicosanoid (EP2) receptor to increase intracellular protein kinase A (PKA) which binds to the glycine receptor a3 subunit to cause a decrease in chloride current, resulting in dis-inhibition (Harvey et al., 2004). Mice deficient in the GlyR a3 subunit were indistinguishable from wild type mice in the chronic constriction model of chronic pain, and the formalin assay, a model of acute and chronic pain (Hosl, 2006). In the spinal cord, glycine receptors are activated by glycine, taurine, 13—alanine and D- or L- serine (Tokutomi et al. , 1989) in the following order of potency: glycine >13-alanine > taurine >> serine (Curtis et al., 1968). Glycine binds to loop A, 11e93, Ala101 and Asn102 in the glycine receptor agonist-binding pocket (Vafa et al., 1999). The (3-amino acids bind to loop A, in the Ala101-Thr112 region of the glycine receptor agonist-binding pocket (Han et al., 2001). The 13— amino acid binding site is thus structurally close to, but distinct from, the glycine binding site. Noxious information is carried through fine myelinated A6 and unmyelinated C spinal nerve fibres to the superficial laminae (Light & Perl, 1979; Yoshimura & Jessell, 1989), particularly 17 substantia gelatinosa (SG; laminae II) of the spinal cord where the information is modulated. Numerous glycinergic neurons are found in the dorsal horn of the spinal cord (Zeilhofer et al., 2005). Stimulation of rat skin with brushing and pinching elicits a barrage of glycinergic IPSCs in the glycinergic synaptic connections in the superficial dorsal horn (Narikawa et al., 2000). Relief of glycinergic and GABAergic inhibition by their respective blockers of glycine and GABAA receptors in the dorsal horn can elicit and exaggerate nociceptive responses (Cronin et al., 2004; Loomis et al., 2001; Sherman et al., 1997; Sivilotti & Woolf, 1994). Inhibitory glycinergic innervation of the spinal cord dorsal horn originates from three sources (Fig. 5) (Zeilhofer, 2005). First, spinal glycinergic interneurons can be directly activated by input from mechanosensory primary afferent nerve fibers (Narikawa et al., 2000). Second, descending antinociceptive fibers can activate local glycinergic interneurons (Baba et al., 2001; Tambeli et al., 2003). Third, direct descending glycinergic neurons originating from the ventral rostromedial medulla forms synapses in the dorsal horn (Antal et al., 1996; Zeilhofer et al., 2005). Noxious information is modulation is partly through inhibitory glycinergic circuitry (Narikawa et al., 2000) and is further projected through spinothalamic neurons to the thalamus which also contains inhibitory glycinergic synapses. Therefore, glycine agonists may be potential analgesics and may act in the spinal cord or the brain. 18 thalamus brainstem nuclei • Descending ^ antinociceptive fibertracts mechanoreceptive AS or Ari fiber ^//̂ nociceptive C fiber  Figure 5. Glycinergic innervation of the spinal cord dorsal horn. Inhibitory glycinergic and excitatory neurons are represented by open and filled circles, respectively. First, spinal glycinergic interneurons can be directly activated by input from mechanosensitive primary afferent nerve fibers. Second, descending antinociceptive fibers can activate local glycinergic interneurons. Third, glycinergic input to the dorsal horn can originate directly from descending glycinergic neurons originating from the ventral rostromedial medulla. 19 1.8. Chemistry of Glycine and Related Compounds Chemically, glycine is the simplest of amino acids with no substituents on the alpha carbon, the methyl group on the backbone of amino acids in between the amino and carboxyl groups (Fig. 6). The glycine receptor agonist, beta-alanine is structurally similar to glycine except with an extension of a methyl group on the glycine amino acid backbone to result in two intervening methyl groups between the amino and carboxyl groups. Another extension on the amino acid backbone with three intervening methyl groups between the amino and carboxyl groups will result in GABA. Isovaline is related to glycine and GABA in possessing the amino acid backbone but is substituted with a methyl and ethyl group at the alpha carbon which results in a stereocenter at the alpha carbon. 0 H2Njt■OH Common Name: Glycine CAS No.:^[56-40-6] IUPAC Name: Aminoethanoic acid 0 H2N Common Name: B-alanine CAS No.:^[107-95-9] IUPAC Name: 3-aminopropanoic acid 0 H2N HO OH  4').12 Common Name: GABA CAS No.:^[56-12-2] IUPAC Name: y-aminobutyric acid 0 OH Common Name: R-isovaline CAS No.:^[3059-97-0] IUPAC Name:^(R)-2-amino-2-methylbutanoic acid Common Name: D-serine CAS No.:^[312-84-5] IUPAC Name: D-2-Amino-3-hydroxypropanoic acid 0 OH NH2 Common Name: S-isovaline CAS No.:^[595-40-4] IUPAC Name:^(S)-2-amino-2-methylbutanoic acid Figure 6. Glycine-like compounds 20 Glycine is an endogenous inhibitory neurotransmitter that is found throughout the central nervous system. It was once used as a bladder irrigation fluid until the realization of toxicity limited its use (Hahn, 2006; Olsson et al., 1995; Olsson & Hahn, 1999). Glycine depresses respiration (Ballanyi et al., 1999; Holtman et al., 1982) and effects on the heart include bradycardia and hypotension (Olsson et al., 1995; Olsson & Hahn, 1999). Other glycine-like compounds such as fl-alanine and taurine also cause respiratory depression, bradycardia and hypotension (Wessberg et al., 1983). Other glycine analogues and glycine receptor agonists may be antinociceptive and have fewer side effects. The glycine analogue would need to be more selective for the analgesic effect and act less pervasively than the omnipresent glycine. An exogenous agent would be less likely to be incorporated into protein and cause unwanted side effects. Isovaline was a logical choice because it is chemically similar to glycine, non- proteinogenic and almost never found in the biosphere (Bruckner et al., 1991; Gabrys & Konecki, 1981; Kvenvolden et al., 1971; Mita & Shimoyama, 1998; Schiell et al., 2001). We also believe isovaline possesses the clinically important feature of being able to cross the blood brain barrier and the gut. Isovaline may be able to cross the blood brain barrier because of the presence of transporters for amino acids similar to isovaline such as valine transporters. Moreover, a similar amino acid, alpha-aminoisobutyric acid (AIB) is a reference indicator for crossing the blood brain barrier. Isovaline was found to be able to cross the gut by active transport (Evered et al., 1967). The origin and the most abundant natural source of isovaline are from the Murchison meteorite (Pizzarello & Weber, 2004). Murchison meteorite is 4.5 billion years old, about the same age as Earth, and isovaline was found in this carbonaceous meteorite in relatively larger quantities than 21 other substances and contained enantiomeric excess of S-isovaline. The presence of enantiomeric excess of a-methyl amino acids such as isovaline may be due to racemization of these amino acids is made difficult by methyl substitution of the a-hydrogen atom and as a result, they have retained their original enantiomeric excess even though exposed to conditions that over time allowed racemization of the a-amino acids with an a-hydrogen atom. The predominance of one stereoisomer over the other distinguishes the meteorite isovaline from man-made isovaline and other amino acids. The stability of the isovaline stereoisomers has been implicated to be the template for sugars and DNA leading to homochirality in the origin of life but was never incorporated into life (Pizzarello & Weber, 2004). Isovaline is a stable molecule having survived the atmosphere on its entry to Earth and is definitely an exogenous molecule to the human body despite being molecularly similar to glycine. 22 1.9. Rationale Current analgesics are limited by side effects or are not effective in chronic pain (Ballanyi et al., 1999; Holtman et al., 1982; Olsson et al., 1995; Olsson & Hahn, 1999). The discovery of glycine receptors in areas important in the pain pathway has prompted our interest in discovering novel analgesics based on glycine (Ghavanini et al., 2005; Ghavanini et al., 2006). Based on the finding of glycine receptors in the thalamus and brain stem, we investigated the role of glycine- like compounds supraspinally and spinally in acute and neuropathic pain. Glycine administered lumbar intrathecally in rats blocked strychnine induced allodynia (Beyer et al., 1988; Jiang et al., 2004; Khandwala & Loomis, 1998). Glycine injected intracisternally by our laboratory was also found to cause respiratory depression followed by death. Glycine has toxicities and inefficient transport systems in the blood brain barrier and is inappropriate as a clinical analgesic. Hence, an analogue of glycine, isovaline, was tested in reducing a form of neuropathic pain, allodynia. Isovaline would potentially have fewer side effects because isovaline is not proteogenic, completely exogenous to the biosphere, and is very stable (Pizzarello & Weber, 2004). 23 Chapter 2. Materials 2.1. Animals On approval by the Animal Care Committee of the University of British Columbia, we studied female CD-1 mice weighing 20 — 30 g. Twelve mice were housed in each cage in an environmentally controlled room with ambient temperature, 21 ± 1 °C, relative humidity 55 ± 5%, reversed 12:12 h dark-light cycle and lights on at 07:00 h. Food and water were freely available. 2.2. Drugs All solutions administered intrathecally were prepared in artificial cerebrospinal fluid (aCSF) containing (in mM): 124 NaCI, 26 NaHCO3, 1.25 NaH2PO4, 2.5 KC1, 2 MgC1 2 , and 2 CaC1 2 , and 10 dextrose. The pH of the solution was 7.3-7.4 and the average osmolality was — 305 mosmols measured with a freezing point osmometer (Advanced Instruments, Norwood, USA). Peripherally administered drugs were prepared in 0.9% saline. Glycine, fl-alanine, mannitol, strychnine, carbamazepine-10,11-epoxide and formalin were purchased from Sigma Chemical Company (St. Louis, USA) (Fig. 7). Formalin was diluted to 5% with distilled water. Morphine sulfate was purchased from the British Drug House (London, England). Racemic isovaline hydrochloride and base were synthesized by Biofine International (Vancouver, BC). R — Isovaline and S - isovaline monohydrate were purchased from ACROS Organics (Geel, Belgium). Glycine was prepared in a concentration of 60, 125, and 250mM and fl-alanine was prepared in a concentration of 60, 125, 250, and 500 mM, racemic isovaline in 60, 125, 250 and 500 mM, and morphine 30 mM. Lidocaine hydrochloride 2% was obtained from AstraZeneca 24 0H2N` Common Name: R-isovaline CAS No.:^[3059-97-0] IUPAC Name:^(R)-2-amino-2-methylbutanoic acid OH Canada, Mississauga, Ontario. The drugs were injected intracisternally and lumbar intrathecally at a volume of 5 p.1 (Sakurada et al., 1995). Intravenous administration of the racemic isovaline was injected at a dose of 500 mg/kg and volume of 3 ml/kg. Sevoflurane was obtained from Abbott Laboratories Limited, Saint-Laurent, Quebec. The pH of the administered drug solutions was measured and for all cases was 7 ± 1. A solution of highest dose isovaline, 500 mM, the hydrochloride form had a pH of —2. The osmolality of 500 mM isovaline base, isovaline hydrochloride and beta-alanine were 1826, 1534 and 753 mosmols, 250 mM isovaline, beta- alanine and glycine were 830, 472, and 510 mosmols, 125 mM isovaline, beta-alanine and glycine were 543, 408, and 364 mosmols, 60 mM isovaline, beta-alanine and glycine were 471, 352, and 358 mosmols, 30 mM morphine was 297 mosmols and 500 mM mannitol was 1240 mosmols. 0 H2N')(OH Common Name: Glycine CAS No.:^[56-40-6] IUPAC Name: Aminoethanoic acid H2N^OH Common Name: il-alanine CAS No.:^[107-95-9] IUPAC Name: 3-aminopropanoic acid 0 OH NH2 Common Name: S-isovaline CAS No.:^[595-40-4] IUPAC Name:^(S)-2-amino-2-methylbutanoic acid Figure 7. Chemical structure of potential analgesics 25 2.3. Equipment 2.3.1. Anesthesia Sevoflurane (0.2 ml) was administered in to a 750 ml Tupperware container to induce anesthesia, this was followed by administration through a mask and non-rebreathing circuit. This resulted in 4% sevoflurane sufficient to induce and maintain anesthesia. Mice were anesthetized for four minutes before taken out of the container for intrathecal injection. An anesthetic circuit was used to maintain anesthesia during the intrathecal injection (sevoflurane in air, 0.01 ml/s). The anesthetic circuit was constructed from 60 ml syringe filled with 0.05 ml (1.5%) sevoflurane at a volume of 50 ml was attached to a plastic head piece with 10 cm of tubing. 2.3.2. Intrathecal Injection Mouse House Holder & Observation Chamber A holder for the mice during the intrathecal injection was made from plasticine moulded to fit the mouse. A rotund extrusion was moulded in the lumbar area to facilitate in opening the spaces between vertebras and enhance the ease of injection (Fig. 6). An observation chamber made from a transparent plastic cage (30 cm long, 15 cm high and 15 cm wide divided by an opaque internal wall) was used for all experiments including the side-by-side observation in the trigeminal allodynia model. All experiments were recorded with a video camera mounted over the observation chamber (Fig. 8). 26 Figure 8. Observation chamber set up 27 2.3.3. Intracisternal and Intrathecal Injection Syringe and Needle Intracisternal injections of the drug were conducted with a 26 gauge, noncoring bevelled tip, 2" removable needle fitted with a 4 mm depth stop made of polyethylene tubing (PE), attached to a standard 10 ul Hamilton microliter gas tight syringe(Hamilton co. NV, USA) at a volume of 5 ul (Figs. 12). Lumbar intrathecal injections were done with a 251.11 Hamilton syringe (Hamilton co. NV, USA) attached by luer lock to a disposable 30 gauge, 1" needle (BD, Franklin Lakes, New Jersey) (Fig. 9). Sevoflurane in Figure 9. Lumbar intrathecal injection in mouse 28 Chapter 3. Methods 3.1. Randomization & Blinding The order of drug injection was randomized by computer (http://www.randomizer.org ). The order of injection was blinded to the experimenters. The code was broken only after the completion of the experiment. 3.2. Allodynia: Intracisternally Administered Strychnine Induced Allodynia 3.2.1. Intracisternal Injection The mouse was anesthetized as above, and placed in a prone position with their neck draped onto a 10 mm diameter cylindrical headrest (Figs. 11 and 12). The head was immobilized with the thumb and forefinger, with the index finger palpating the space between the occiput and atlas (Reijneveld, Taphoorn, & Voest, 1999). The needle inserted at an angle between 45 to 55°. The syringe was held in position for a few seconds then slowly removed to minimize any outflow of the drug. This procedure, including anesthesia, took approximately three minutes. 29 Figure 10. Diagram of intracisternal injection. ^ Cerebellum  Cisterna  Medulla^Magna Oblongata Figure 11. Cisterna Magna — Site of Injection of intracisternal administration. Injection site in mouse brain. 30 3.2.2. Intracisternal Administration of Strychnine Induced Trigeminal Allodynia The reactions of each pair of mice to stroking both sides of the face, behind the ears, back and hind paws were observed. The investigators injecting and observing the animals were blinded. Doses of strychnine, 25, 50, 100, 200, and 400 µM, were tested (3 mice per dose) to determine the maximal doses of strychnine that did not evoke movement disorder or convulsions. This was found to be 200 [I,N4 strychnine at a volume of injection of 50. This effect lasted for at least 15 minutes. A similar procedure was used to determine the maximal dose of carbamazepine (carbamazepine-10,11-epoxide) (Johannessen et al., 1976) by testing 0.8 and 3.2 04 carbamazepine. The lower dose was ineffective and the upper dose was the maximal soluble dose. The doses used in the experiments were 3 x le gm of strychnine (200 JAM) and 4 x 1 0-9 gm of carbamazepine-10,11-epoxide (3.2 1A,M) injected in a volume of 5^Due to limited solubility of carbamazepine, the active soluble metabolite of carbamazepine, carbamazepine- 10,11-epoxide was used. The experiments were performed in a blinded side by side comparison of pairs. The incidence and severity of allodynia resulting from stroking each of a pair of mice with 8 cm PE tubing within the trigeminal nerve distribution (cheeks, behind the ears) and outside the nerve distribution (flanks, hind paws and tail) were determined every two minutes for 14 minutes. Allodynia for the trigeminal distribution was demonstrated by eye squinting, backward folding of the ears, scratching of the face, with withdrawal of the face after the stimulus. As a control, allodynia was tested with the body and includes withdrawal of the body after the stimulus and tail flick upon application of mild pressure. At the end of the testing period, the blinded observer decided which animal in the pair had the severe allodynia in the trigeminal nerve distribution. 31 3.2.3. Pilot Test for Intracisternal Administration of Glycine As an initial pilot test, glycine was administered intracisternally at three concentrations 20, 200 and 1600 pM co-injected with 200 µM strychnine at a volume of injection of 5 pl. There was no change in allodynia at 20 (n=1) or 200 pM (n=5). At 1600 p.M (n=5) death preceded by respiratory depression occurred in three animals for consecutive injections. The remaining 2 animals planned for this group were not tested because of these deaths. 3.2.4. Effect of Morphine and Isovaline on Intracisternal Strychnine Induced Allodynia In the trigeminal allodynia model, the order of injections was randomized for each pair of drug and control for six pair wise comparisons. Morphine hydrochloride (3 mg/kg, 10 ml/kg volume of injection) or saline control was injected subcutaneously, 30 minutes prior to intracisternal injection of strychnine. A similar procedure was used to determine the effects if isovaline on the trigeminal allodynia model with intracisternal administration of racemic isovaline (5 mM) co- injected with 200 gM strychnine at a volume of injection of 5 pl. 3.3. Allodynia: Lumbar Intrathecal Administered Strychnine Induced Allodynia 3.3.1. Lumbar Intrathecal Injection The mouse was placed onto the mouse holder for the intrathecal injection. Intrathecal injections were performed as described by Hylden and Wilcox (Hylden & Wilcox, 1980). The mouse was held firmly by the pelvic girdle in one hand, while the syringe was held in the other hand at an angle of about 20° above the vertebral column. The 30 gauge needle was inserted into the tissue 32 spinous process tronsverSe prOCOSS spinal corC to one side of the L5 or L6 spinous process such that the needle slipped into the groove between the spinous and transverse processes. The needle was then carefully advanced forward 5 mm to the intervertebral space as the angle of the syringe was decreased to about 10°. The tip of the needle was inserted so that approximately 0.5 cm was within the vertebral column (Figs. 10 and 13). Intrathecal location of the needle tip was confirmed by a characteristic flick of the tail. The site of injection was chosen to be between L5 and L6 — near to where the spinal cord ends and the cauda equina begins (Sidman, Angevine, & Pierce, 1971) . This site represents a compromise to maximize intervertebral accessibility and to minimize the possibility of spinal damage. In the pilot study, a volume of 5 ill of 2% lidocaine hydrochloride was injected intrathecally into seven mice which elicited immediate hind limb paralysis, lasting for ten minutes in all animals. Figure 12. Diagram of needle insertion into the intervertebral space between L5 and L6 in the mouse. 33 3.3.2. Lumbar Allodynia The mouse was anesthetized and injected lumbar intrathecally with either 5 gl of 100 p.M strychnine or a mixture of 2.5111 of 200 p.M strychnine and 2.5 microliters of 16 mM racemic isovaline. A pair wise comparison protocol similar to the strychnine induced trigeminal allodynia model was used. The animals were tested and recorded for the incidence and severity of allodynia by touching the lumbar region of the animals with 8 cm PE tubing every two minutes for a total of 14 minutes. Allodynia for the lower body was determined by observing for stiff tail, biting of the tail, scratching of the lower body and hind paw sensitivity to light touch. At the end of the testing period, the observer made a decision of which animal in the pair had the most severe allodynia in the lower body. A total of five pairs were done. 3.4. Determining Analgesia (Formalin Foot Assay) 3.4.1. Paw Formalin Assay The formalin assay was used to measure pain in mice after anesthesia and intrathecal injection of the drug or control (Figs. 14). The formalin assay involves 20 of formalin injected subcutaneously into the hind paw using a 50 pi Hamilton syringe and 30 gauge, 1/2" needle (Kolesnikov, Cristea, Oksman, Torosjan, & Wilson, 2004). The drug or vehicle was injected intrathecal and then five minutes later the formalin assay was conducted. The animals were then placed in a clear plexiglass container (28 x 18 cm) for observation. The pain behavior was quantified by determining the amount of time (s) the mouse spent licking the injected paw every five minutes 40 min. Two phases of spontaneous licking behavior were observed after the formalin injection. The interval from 0 to 15 mM has been defined as Phase I, and the interval 34 from 15 to 40 min has been defined as Phase II (Abbott, Franklin, & Westbrook, 1995). Time- response data were presented as the mean ± S.E.M. of 5 min bins over 40 min. Anesthesia Pre-formalin Begin Observation 4 min^5 min  Observation Period 3 min^1 min 1 min Intrathecal Injection I^I^I^I^I^I^I 5^10^15^20 25^30^35 40 Anesthetize Animal Sedation Test Formalin Injection Allodynia Test Figure 13. Experimental method timeline for dose response of intrathecal injection of glycine- like compounds 35 3.4.2. Intrathecal Administration of Isovaline and Glycine-like Compounds on Formalin Foot Assay of Analgesia The effect of intrathecal administration of glycine-like compounds on formalin foot assay of analgesia was determined. Glycine, beta-alanine, and racemic isovaline were randomly injected in lines of eleven consisting glycine (60, 125, and 250 mM), beta-alanine (60, 125, and 250 mM), racemic isovaline (125, 250, and 500 mM), aCSF vehicle control, and morphine positive control (nine mice per group). In a separate experiment beta-alanine 500 mM, isovaline base (Biofine International, Vancouver, BC) 60 and 500 mM and 500 mM mannitol and aCSF controls were tested (nine per group). The effect of different isomers of isovaline, R, S and racemic isovaline hydrochloride compared to aCSF controls was determined by administration into the lumbar intrathecal space, (ten mice per isomer). A preliminary study was performed with five mice injected lumbar intrathecally with 250 mM ACBC. 3.4.3. Intravenous Administration with Formalin Foot Assay The mouse was placed into a mouse holder and the tail warmed with a heat lamp for 2 minutes or swabbed with ethanol soaked gauze to reveal the veins on the tail. A 29 gauge, 1/2" needle, 0.3 ml insulin syringe was used to inject the drug into the vein at a volume of 3 ml/kg. The effect of intravenous racemic isovaline on formalin foot assay of analgesia compared to control was examined (ten mice per group). 36 3.5. Determining Toxicity 3.5.1. Rotarod Test Ataxia in mice was measured using the rotarod procedure of Dunham and Miya (1957) with the modification of Kalir et at. (1978) (Dunham & Miya, 1957; Kalir et al., 1978). The 27-mm diameter rod rotated at 5 rpm. At least 1 day before testing, mice were trained three times on the rod for two consecutive 5-min trials separated by .a 5-min rest period. However, during tests each trial on the rotarod was limited to a 120 s maximum. For all experiments two control trials obtained 6 min apart preceded the tail vein i.v. injection (500 mg/kg, 3 ml/kg) of the drug. Each mouse was then given 2 trials at with 6 min between trials after the injection. (Vaupel et al., 1984). The effect of R - isovaline and S - isovaline monohydrate and saline control was determined in the rotarod test (ten mice per group). 3.5.2. Sedation Assay In order to determine whether administration of the amino acids produced a sedative effect, sedation as defined as a lack of voluntary movement was recorded. Another test was done where each mouse was placed on a rubber stopper (6 cm in diameter, 2.5cm high) four minutes after drug or vehicle administration (Porreca et al., 1987). The latency to stepping off the stopper with the hind paw was recorded. The responses were evaluated by the analysis of variance. 37 3.5.3. Observation of Behavior and General Appearance & Allodynia Assay Each animal was placed in a plexiglass observation chamber and three minutes after intrathecal injection, mechanical sensitivity was measured as the occurrence of flinching or avoiding to the repetitive application of a von Frey filament 2 (10 times, 0.2 Hz) to the middle of the back of the mouse. The mouse showing greater than 40% response were considered as having mechanical allodynia (Gwak et al., 2006). Signs of toxicity were also observed including abnormal gait and feet flinch or avoidance to touch. 3.6. Statistical analysis The pair-wise comparisons in the strychnine induced allodynia tests and sedation were statistically analyzed by the binomial test because the data failed the D'Agostino-Pearson normality test and required the use of non-parametric statistics. The behavioural data are presented as mean ± standard error of mean in n experiments. The data were examined by analysis of variance (ANOVA) for multiple comparisons to a single control group. Student's test two-tailed was used when the analysis was restricted to two means. Level of significance was set to 5% (p < 0.05). Data were statistically analyzed using GraphPad Prism 5.0 (San Diego, CA). 38 Chapter 4. Results 4.1. Allodynia 4.1.1. Intracisternal Administration of Strychnine Induced Allodynia Within each pair, one animal was injected with aCSF control and the other injected with strychnine and the mouse which had the most severe allodynia was determined. The most severe response to stimulation occurred after strychnine in eight of eight pairs (Table 1). 4.1.2. Effect of Intracisternal Injection of Carbamazepine-10,11-epoxide, Morphine, Racemic Isovaline and Glycine on Strychnine Induced Allodynia Within each pair, one animal was injected with strychnine and the other co-injected with strychnine and carbamazepine-10,11-epoxide, morphine and isovaline. The mouse which had the most severe allodynia was determined. All mice with intracisternal administration of strychnine developed allodynia while none of the aCSF controlled mice developed allodynia. Intracisternal co-injection of carbamazepine-10,11-epoxide and strychnine produced less allodynia than intracisternal injection of strychnine alone in all six pairs. Intracisternal injection of morphine with strychnine resulted in less allodynia than intracisternal injection of strychnine in three out of five pairs of animals. Intracisternal injection of glycine caused respiratory depression and eventually death in three animals. Intracisternal injection of racemic isovaline with strychnine produced less allodynia than intracisternal injection of strychnine alone in all eight pairs of animals tested (Table 2). 39 Table 1. Intracisternal strychnine and carbamazepine Pairs of animals were injected intracisternally with one animal receiving strychnine (200 1.tM, 5 injection volume) and the other aCSF in the top rows and the following rows with animals receiving strychnine in one animal and the other strychnine with carbamazepine-10,1 1-epoxide (2.4 1.IM, 5^injection volume). The lowest rows are pairs of animals injected with strychnine and morphine (3 mg/kg, 10 ml/kg) or saline were injected subcutaneously. Total number of mice Number of mice with maximal allodynic response Strychnine vs. aCSF n= 16 aCSF 8' 0 Strychnine 8 8 Strychnine vs. Strychnine + Carbamazepine-10,11- epoxide n= 12 Strychnine 6 6 Strychnine + Carbamazepine- 10,11-epoxide 62 0 Strychnine vs. Strychnine + Morphine n = 10 Strychnine 5 5 Strychnine + Morphine 53 2 'Indicates p<0.01 for strychnine vs. aCSF 2 Indicates p < 0.05 for strychnine vs. carbamazepine-10,11-epoxide 3 Indicates p = 0.563 for strychnine vs. morphine; binomial test 40 Table 2. Intracisternal strychnine and racemic isovaline Pairs of animals were injected intracisternally with one animal receiving strychnine (200 [tM, 5 [II injection volume) and the other racemic isovaline (5 mM, 5 p.1 injection volume) Strychnine vs. Racemic Isovaline n= 16 Total number of mice Number of mice with maximal allodynic response Strychnine 8 8 Strychnine + Racemic Isovaline 81 0 'Indicates p < 0.01 for strychnine vs. racemic isovaline; binomial test 41 4.1.3. Lumbar Intrathecal Administration of Strychnine Induced Allodynia Lumbar intrathecal administration of strychnine with racemic isovaline resulted in a reduction in strychnine induced allodynia in the lower body (Table 3). All five pairs of animals tested with lumbar intrathecal injection of racemic isovaline with strychnine resulted in less allodynia than mice with lumbar intrathecal injections of strychnine. All five mice with lumbar intrathecal injections of strychnine produced lower body allodynia. Table 3. Lumbar intrathecal strychnine and racemic isovaline Pairs of animals were lumbar intrathecally injected with one animal receiving strychnine (100 µM, 5 1,11 injection volume) and the other racemic isovaline (8 mM, 5^injection volume) Lumbar Strychnine vs. Racemic Isovaline n= 10 Total number of mice Number of mice with maximal allodynic response Strychnine 5 5 Strychnine + Racemic Isovaline 51 0 •^•indicates p < 0.05 for strychnine vs. racemic isovaline; binomial test 42 4.2. Analgesia 4.2.1. Lumbar Intrathecal Injection of Glycine In the formalin foot test, the time course of the formalin licking generally produces a peak between 0 to 10 minutes with a quiescent phase from 10 to 20 between another peak from approximately 20 to 40 minutes and each peak is regarded to be sensitive to analgesics against acute and chronic pain respectively. The peaks are observed in all formalin foot tests but the exact demarcation for each peak varies between papers. All the formalin foot tests produced the characteristic two peaks separated by a quiescent phase. The first peak was most pronounced at 5 minutes while the second peak was most pronounced from 20 to 40 minutes with a quiescent period from 10 to 20 minutes. Hence, the time points were chosen to represent the phase I (acute phase) and phase II (chronic phase) respectively. In phase I, glycine at 250 mM and 125 mM and 60 mM was statistically significant in reducing the licking time, and all three glycine doses were also effective in reducing licking time in phase II (Fig. 14). A trend for dose dependent reduction in formalin induced licking by glycine exists but is not statistically significant. There was no sedation noted at any dose of glycine. High dose glycine (250 mM) produced irritation and bursts of scratching of the head, neck and back. This was rarely seen at other doses of glycine or any of the other compounds (Fig. 15). The amount of scratching at 250 mM glycine in phase I but not phase II was statistically significant compared to control. The duration of scratching for 125 and 60 mM glycine were not statistically significant compared to control for either phase. 43 Glycine Phase I *** *** ** 4 Ai% AI'o- (0- o- $4* O b^(43 0 . e . %4 o c)^0(% 4Ce C.0 CO CO t9 *** Zs 200 0)C t.) 100 zEz0 Phase II e c0 a.% re cl."0... 20V SS'ti3^ccv-.. * Beta-alanine 80 40 ** 0 c. e^.. c4e e e^eCPS` ^e4 ...er^Nts''qP ‘tfr 4/-1‘ft<;3■•■ o^30 200 100 0 ....sse .05 .4se .<se CPv4IP^e4 o N.V6k(43 Qfr) cc,* $11% $%.1 (CVVb0 <0 re r5 Isovaline  # # 4%cP 04 4 4 'QC0 r* ‘43 4) & 4 $4' "e#4‘. 200 100 0 e^ 0$34,,, c46cp 04 .4 .4^,,44 ,PcV cc# (1,443tZ"' Figure 14. Formalin induced pain behaviours during phase I and II for various doses of glycine- like compounds. Phase I and II are cumulative licking from 0-5 minutes and 20-40 minutes, respectively. Each block represents the mean of nine animals, and the vertical bars indicate the SEM. *P < 0.05, **P <0.01, ***P < 0.001 vs control gfoup; ANOVA. 44 A10^20^30 Time (min) 20 10 300 U) C)C 1E0 200 cv U) a) E 100 0 F- 10^20^30^40^50 Time (min) * * * -e- 250 mM Glycine * aCSF Control • 30 mM Morphine -0- 125 mM Glycine 60 mM Glycine - 250 mM Beta-alanine -0- 125 mM Beta-alanine 60 mM Beta-alanine i:81 500 mM Isovaline • 250 mM Isovaline -e 125 mM Isovaline 50 B^ • aCSF Control Figure 15. Scratching in 5 min bins for various doses of glycine, beta-alanine, and isovaline. A. Glycine, beta-alanine, and isovaline scratching time course. B. 250 mM Glycine scratching time course. Each point represents the mean of nine animals, and the vertical bars indicate the SEM. ***P < 0.001 vs control group; ANOVA. 45 4.2.2. Lumbar Intrathecal Injection of Beta-alanine Administration of beta-alanine displayed a trend for dose dependent reduction in formalin induced pain behavior (Fig. 14) but was not statistically significant. The formalin induced pain behavior in phase I and II was reduced at 250 and 125 but not 60 mM of beta-alanine (Fig. 14). The scratching response was not significant at 60, 125, and 250 mM beta-alanine tested. Sedation produced by 250, 125, and 60 mM beta-alanine was not significant. 4.2.3. Lumbar Intrathecal Injection of Racemic Isovaline Isovaline at doses 500, 250 and 125 mM reduced formalin induced licking in phase I and II (Fig. 14). Seven out of the nine animals tested with 500 mM isovaline displayed lower body weakness that abated with time, while two out of the nine animals had nearly abolished formalin induced licking without lower body weakness. None of the doses tested with isovaline displayed significant scratching or sedation. Mice injected lumbar intrathecally with morphine displayed sedation as defined as a lack of voluntary motion. While all other mice injected lumbar intrathecally with isovaline, glycine, or beta-alanine did not show sedation and were comparable to mice injected with aCSF control (Table 4). 46 Isovaline 80 40- 0 Beta-alanine 80- U) C)C LT a) ▪ 40 - a ▪ s E 0 Phase I Phase II 0 4.3\^e0 oco- te, c.(\ co Goan ^.c,0 00" 0°c.)4 \43 \43 <•‘ Ito\-\`‘b.^cP cP R-12" Sao\ 0 o <o() c& c‘ 0,c) c, \e. N0"z,c.•3 4. ,o cc\ Z\)\<3^(\et c5c\ 4q) c, 4) c,\c")K•^ccO ccO cP „co ,..„so(ocs Figure 16. Formalin induced pain behaviours during phase I and II for various doses of 0-alanine and isovaline. Phase I and II are cumulative licking from 0-5 minutes and 20-40 minutes, respectively. Each block represents the mean of nine animals, and the vertical bars indicate the SEM. **P <0.01, ***P < 0.001 vs aCSF control group; ANOVA. 47 Table 4. Sedation score for lumbar intrathecal injection of glycine-like compounds Sedation score for mice with lumbar intrathecal injection of compounds followed by formalin injection into hind paw. Sedation defined as lack of voluntary movement. Total number of mice Number of mice with sedation aCSF Control 9 0* Isovaline 9 0* Glycine 9 0* Beta-alanine 9 0* Morphine 9 9 *Significantly different from morphine P < 0.01; binomial test 48 4.2.4. Lumbar Intrathecal Injection of Controls The formalin induced licking observed with 500 mM mannitol was not significant (Fig. 16). Formalin induced licking observed with 500 mM and 60 mM racemic isovaline was significant compared to aCSF control in the second phase. Finally, the formalin induced licking observed with 500 mM beta-alanine was significant in both the first and second phase compared to aCSF control. The scratching behavior was significant with 500 mM beta-alanine in both phase I and II (Fig. 17). Six out of the nine mice injected with 500 mM beta-alanine developed lower body weakness preceded by licking, biting and kicking of the lower body. Four out of nine mice injected with 500 mM racemic isovaline developed lower body paralysis or weakness that abated with time and were preceded by licking, biting and kicking of the lower body. The sedation assay and allodynia assay did not produce results for 60 or 500 mM racemic isovaline, 500 mM beta- alanine or the mannitol or aCSF controls. 49 -e- aCSF Control -- 500 mM Mannitol Control -o- 500 mM Beta-alanine -0- 60 mM Racemic Isovaline -A- 500 mM Racemic Isovaline 10^20^30 ^ 40 ^ 50 Time (min) Figure 17. Scratching behaviours for aCSF, mannitol, beta-alanine and racemic isovaline. Each point represents the mean of nine animals, and the vertical bars indicate the SEM. *P < 0.05 and ***P < 0.001 vs control group; ANOVA. 50 4.2.5. Intravenous Analgesia of Racemic Isovaline In the first phase, the total time of formalin induced licking was not different in the isovaline 250 mM group compared to the control group (Fig. 19) (isovaline group 77 ± 7 (SEM) s vs control group 89 ± 8 s; P > 0.05). In the second phase, the total time of nociceptive behavior was significantly less in the isovaline 250 mM group than in the control group (isovaline 250 mM group 110 ± 29 s vs control group 199 ± 25 s; P < 0.01). In addition, the isovaline group mice did not show any differences except for pain-related behaviors compared to the control mice. Intravenous racemic isovaline did not produce significant decrease in respiratory rate compared to control (isovaline 500 mg/kg group 126 ± 12 bpm vs control group 126 ± 12; P > 0.05) (Fig. 18). 51 E 150 a) Cti 100 co IX 50 0 0 cna)^Saline^500 mg/kg Isovaline Figure 18. Respiratory rate for mice injected intravenously with 500 mg/kg isovaline or saline control. Values are mean ± SEM for 10 mice; Student t-test 52 ):2 100 172 50 E 0 Isovaline Phase I Phase II  0 Nse^,pcNe'^N.co\^- o ecFP^c..94^c."^4....f)<(^. \- <c ee .2prfo e ikt. ci-if 200 - 100 - Figure 19. Effects of intravenous racemic isovaline on acute and chronic phases of formalin- induced pain. The cumulative licking activity over 0-5 min (Phase I) and 20-40 min (Phase II) was plotted for the aCSF control and 500 mg/kg isovaline. The bar amplitude represents the mean of 10 animals ± SEM. ANOVA, ** p < 0.01. 53 4.2.6. Toxicity: Rotarod of Intravenous Administration of Racemic Isovaline Mice did not show significant difference between control and either R or S - isovaline as well as between pre- and post-injection of either saline or drug in the rotarod test (Figs. 21 and 22). There were no obvious signs of toxicity and the mice injected with R or S- isovaline and visually not different from saline injected control mice. 54 loo 0 03 O c 500 a) E i= 0 ED Control Group N. S-Isovaline Group  Figure 20. Comparison of time on rotarod before and after intravenous injection of 500 mg/kg S- isovaline or saline control. Values are mean ± SEM for 10 mice; ANOVA. (I) 100 Control Group E. R-Isovaline Group T-o 0 ca -16 50 0 E 0 _ G,‹C CP •ec" gel •^ O4 ▪^ e, Figure 21. Comparison of time on rotarod before and after intravenous injection of 500 mg/kg R- isovaline or saline control. Values are mean ± SEM for 10 mice, ANOVA. 55 4.2.7. Optical Activity To examine the effects of enantiomers of isovaline on the analgesic effect as measured on the formalin foot assay, R, S and racemic isovaline were administered lumbar intrathecally and tested with the formalin foot assay. In the first phase, the total time formalin induced licking was significantly different in the racemic isovaline 250 mM group compared to the control group (racemic isovaline group 28 ± 12 (SEM) s vs control group 66 ± 11 s; P < 0.05). In the second phase, the total time was significantly less in the racemic isovaline 250 mM group than in the control group (racemic isovaline 250 mM group 34 ± 16 s vs control group 211 ± 37 s; P < 0.001). Aside from the decrease in licking times, the racemic isovaline group mice were indistinguishable from the control mice. R and S isovaline both significantly decreased licking time (Fig. 24). There was no difference between the R- and S- formalin induced licking. (Fig. 22). Both R- and S- isovaline significantly decreased the total time of formalin induced licking during first phase I (R - isovaline group 40 ± 10 (SEM) s; S-isovaline group 31 ± 5 s; vs control group 75 ± 14 s; *P < 0.05, **P < 0.01) and II (R - isovaline group 83 ± 30 (SEM) s; S-isovaline group 96 ± 30 s; vs control group 203 ± 18 s; ***P < 0.001). The formalin induced licking was reduced by greater than 50% by R- and S- isovaline. Aside from the decrease in licking times, the isovaline group mice were indistinguishable from the control mice. There was no evidence of agitation, sedation, unusual grooming, and respiratory depression in mice injected with racemic, R or S isovaline. 56 80- 40 - 80- 40- _se^ z cs,<\ c., 200 200- *** ** 100 100 - 0 0 ti'so 0C.)0 .4 JC;((^g": 200 Ccs) Phase II =1 100 E Racemic Isovaline R - Isovaline^S - Isovaline 80- Ccs) Phase I^a.) ct 40- 0 Figure 22. Effects of intrathecal racemic, R- and S -isovaline on phase I and II of formalin- induced pain. The cumulative licking activity over 0-5 min (Phase I) and 20-40 min (Phase II) was plotted for the aCSF control and 250 mM racemic, R - and S - isovaline. The bar amplitude represents the mean of 10 animals per group ± SEM. ANOVA, *P < 0.05, **P <0.01, *** p < 0.001. 57 4.2.8. Preliminary Results with ACBC A pilot test with five animals injected lumbar intrathecally with 250 mM ACBC in the formalin assay showed that at the same dose of ACBC and isovaline, ACBC reduced the formalin induced licking substantially greater than isovaline (Fig. 23). CY) Phase I^Phase II 80- c a) t,a 300- 200- 100 - ■ 4°^c,(4c.P^ cp IC° (-P " r.co sr it". Figure 23. Effects of 250 mM ACBC on phase I and II of formalin induced pain. The cumulative licking activity over 0-5 min (Phase I) and 20-40 min (Phase II) was plotted for the aCSF control and racemic, R - and S - isovaline. The bar amplitude represents the mean of 5 animals per group ± SEM. ANOVA, *P < 0.05. v.) 58 Chapter 5. Discussion 5.1. Summary of the results The anti-allodynic and analgesic effects of glycine receptor agonists were examined. The hypothesis was that isovaline and other glycine receptor agonists and analogues will exert an inhibitory effect in the central nervous system and block nociception. 5.2. Allodynia It was postulated that the removal of glycinergic inhibition by intracisternal administration of strychnine would result in allodynia. Intracisternal administration of strychnine, a glycine receptor antagonist, produced trigeminal allodynia. Co-injection of strychnine and, carbamazepine-10,11-epoxide, a water soluble active metabolite of carbamazepine, the current clinical treatment for trigeminal allodynia validates the trigeminal allodynia model by reducing the strychnine induced allodynia (Scrivani et al., 2005). Co-injection of racemic isovaline and strychnine also reduced strychnine induced allodynia. 5.1.1. Intracisternal Strychnine Induced Allodynia Allodynia is a relatively rare but distressing symptom of neural injury or neuropathy that is characterized by the inappropriate perception of light tactile stimuli as being painful. Under normal circumstances, light touching or stroking of the hair of conscious mice elicits no more than an orientation response. However, after intracisternal strychnine, an identical light touch 59 stimulus evoked a vigorous scratching and flinching at the stimulation site. These behavioral responses are usually elicited only by high-intensity and potentially nociceptive stimuli, not with light touch. Thus the temporary removal or antagonism of glycinergic inhibition with strychnine results in a disturbance of sensation, resembling the allodynia observed with neuropathic pain in the trigeminal area (Beyer et al., 1985). In other cases of models of trigeminal neuralgia signs of abnormal spontaneous pain related behavior, mechanical allodynia (Vos & Maciewicz, 1991), heat hyperalgesia (Imamura et al., 1997) and inflammatory hypersensitivity(Anderson et al., 2003) have been used as animal models; however, it is unknown if this resemblance extends to the tactile allodynia observed in the model presented here. This aspect seems particularly relevant as enhanced responsiveness to innocuous stimuli is frequently observed in patients with trigeminal neuralgia and other forms of neuropathic pain. For example, weak and innocuous stimuli such as light touch, hair movement, or chewing are among the most effective triggers for eliciting attacks of trigeminal neuralgia. Intracisternal administration of strychnine into the cisterna magna and would result in diffusion to the spinal trigeminal nucleus. Strychnine is often used to identify glycine receptor activity and is a selective glycine receptor antagonist (Young & Snyder, 1974). The trigeminal nucleus receives sensory information from the trigeminal nerve which subserves the facial region. Administering strychnine onto the trigeminal nucleus would oppose the glycinergic inhibition and enhance sensory input producing a response identical to that of a painful input from even an innocuous stimulus. The result was allodynia localized to the trigeminal distribution. Indeed, injecting strychnine into the cisterna magna results in trigeminal allodynia. 60 5.1.2. Intracisternal Injection of Glycine Glycine is an endogenous agonist for the glycine receptor. Intracisternal injection of glycine at a quarter of the dose of isovaline (5 mM) caused respiratory depression and eventually death. This demonstrates glycine toxicity when injected supraspinally and hence, glycine would be unsuitable as a clinical drug. However, structural analogues of glycine may retain the analgesic property but not the toxic side effects. 5.1.3. Intracisternal Isovaline Against Strychnine Induced Allodynia Intracisternal administration of racemic isovaline in combination with strychnine resulted in alleviation of strychnine induced trigeminal allodynia. Isovaline may be acting as a glycine receptor agonist to counteract strychnine's effect on glycine receptors. However isovaline may act through other mechanisms to oppose strychnine's effects. It may be possible that isovaline may be enhancing inhibition by acting on GABA receptors or reducing excitation by antagonizing the glycine site on the NMDA receptor to counteract strychnine's effects. Nonetheless, isovaline is at least acting supraspinally and the analgesic effects implicate glycine receptors. 5.1.4. Intracisternal Morphine Against Strychnine Induced Allodynia Trigeminal neuralgia is very resistant to opioid therapy, except for extremely high doses, which cause marked sedation. Opioids are, therefore, not too useful for the treatment of trigeminal neuralgia (Loeser, 1985). In the present study, an analgesic dose based on literature (subcutaneous dose of 3 mg/kg) of morphine administered acutely produced less allodynia than mice in the strychnine only group in three mice (Umans & Inturrisi, 1981). The strychnine model 61 of trigeminal neuralgia thus seems rather insensitive to morphine, but further studies are needed to clarify the effects of simultaneous intracisternal injection of strychnine and morphine. 5.1.5. Intracisternal Carbamazepine Against Strychnine Induced Allodynia Carbamazepine is the first line drug for long-term treatment of trigeminal neuralgia. As more than 80% of patients with trigeminal neuralgia respond to anticonvulsant medication, particularly carbamazepine, it is the initial drug of choice (Rappaport & Devor, 1994). Intracisternal administration of the more water soluble active metabolite of carbamazepine, carbamazepine- 10,11-epoxide significantly relieved the strychnine induced trigeminal allodynia. This result is consistent with clinical results where carbamazepine is currently the most clinically effective treatment. 5.1.6. Lumbar Intrathecal Injection of Strychnine Induced Allodynia Lumbar intrathecal administration of strychnine induced allodynia in the lower body, consistent with the literature (Beyer et al., 1988; Jiang et al., 2004; Khandwala & Loomis, 1998). Previous lumbar intrathecal administration of strychnine was done in rats and the present results are unique for the mouse. The benefits of using mice for this method is that it circumvents problems associated with canulating the rats for intrathecal administration such as potential spinal damage and relatively labor and time intensive procedure. Using mice is also a more economical and easier to handle. 62 5.1.7. Lumbar Intrathecal Injection of Isovaline Against Strychnine Induced Allodynia The co-injection of racemic isovaline and strychnine resulted in a reduction in strychnine induced allodynia. This result is consistent with the literature that glycine and its related compounds block strychnine induced allodynia (Beyer et al., 1988; Jiang et al., 2004; Khandwala & Loomis, 1998). 5.3. Analgesia 5.3.1. Lumbar Intrathecal Injection Glycine-like Compounds We have shown for the first time, that glycine, the endogenous glycine receptor agonist, beta- alanine and isovaline are effective in the conventional model of pain, formalin foot assay. Previous work with glycine receptor agonist was related to allodynia involving the antagonism of glycinergic inhibition with strychnine. Our work is consistent with previous studies that included removal of glycinergic inhibition by strychnine in rats (Sherman, 1995) or calves genetically deficient in strychnine binding sites (Gundlach et al., 1988) and strychnine overdose in humans resulting in allodynia. Glycine receptor agonists have also been shown to counteract strychnine induced allodynia (Beyer et al., 1988). Infusion of glycine was effective in a neuropathic rats with unilateral partial ligation of the sciatic nerve (Simpson et al., 1996; Simpson et al., 1997). These results indicate glycine receptors have an important role in nociception and sensory processing and may have clinical potential in the treatment of allodynia and pain. 63 Formalin injected in the hind paw in the mouse has two phases of pain-related behaviours which involve a complex series of events. The first phase lasts between five to ten minutes and is attributed to direct chemical stimulation of chemosensitive nociceptors (Lutfy & Weber, 1998; Tjolsen et al., 1992) and reflects acute pain. The second phase starts from 15 to 20 minutes after injection and is believed to be associated with the release of local inflammatory mediators that lead to sensitization of neurons in the spinal dorsal horn and subsequent activation of nociceptors, as believed to occur in chronic pain (Hunskaar & Hole, 1987; Porro & Cavazzuti, 1993). All the amino acids tested, including glycine, beta-alanine, and isovaline possess analgesic properties at certain doses. These results are consistent with previous findings of short chain amino acids possessing analgesic properties when administered intrathecally in rodents (Beyer et al., 1988; Jiang et al., 2004; Khandwala & Loomis, 1998). Lumbar intrathecal administration of glycine, beta-alanine, and isovaline lower formalin induced licking. Glycine lowers the formalin induced licking at 60, 125 and 250 mM for both phase I and II. Beta-alanine at 250 and 125 mM lowers the formalin induced licking in phase I and II while 60 mM beta-alanine was ineffective in both phase I and II. Finally, 250 and 125 mM isovaline was effective in lowering formalin induced licking in phase I and II. The lowest dose of isovaline tested, 60 mM neutralized isovaline base was statistically significant in lowering formalin induced licking in phase II and not phase I. The highest dose of isovaline at 500 mM isovaline when administered lumbar intrathecally, virtually abolished the formalin induced licking in two out of nine animals with no visible side effects while seven out of nine animals displayed lower body weakness that abated with time. The amino acids glycine, beta-alanine and isovaline exert 64 antinociceptive effects with equal potency and efficacy (Fig. 22). This indicates glycine, beta- alanine and isovaline appear to act similarly on the glycine receptor. 5.3.2. Structure Activity Relationship of Glycine -like Compounds This study provides insight on the structure activity relationship of the analgesic effect of glycine receptor agonists. Glycine is the simplest amino acid and does not contain substituents on the alpha carbon and does not contain a stereocenter or have stereoisomers. Beta-alanine's molecular structure is very similar to glycine but with a single carbon extension on the alpha carbon adjacent to the nitrogen group and also does not have a stereocenter. It appears that lengthening the carbon backbone of the amino acid by a single carbon does not change the analgesic effect compared to glycine. Stereoisomerism does not seem essential for the analgesic affect. Isovaline is distinct from beta-alanine and glycine by containing two substituents on the alpha carbon including a methyl and ethyl group. This addition of simple substituents introduces stereoisomerism into the molecule with isovaline existing as R and S isomers of isovaline. The racemate of isovaline produced analgesia with similar potency and efficacy as glycine and beta- alanine. This indicates that a racemic mixture of isovaline has comparable analgesic effects to non-racemic glycine or beta-alanine solutions. Administration of the individual isomers of isovaline intrathecally in the same animal preparation also induced analgesia with equal potency and efficacy. This indicates that the site of action of isovaline is not stereoselective. The manipulation of the alpha carbon by the addition of two small substituents did not interfere with the analgesic effect. 65 Beta-alanine also produces analgesia and is the result of the extension of the carbon backbone of glycine. Further manipulation of the carbon backbone by extending the carbon backbone by an additional carbon would result in GABA which is known to act on the GABA receptor. Extension of the carbon backbone and addition of substituents to the beta-carbon of beta-alanine could be possible future areas to explore. Lumbar intrathecal administration of racemic isovaline reduced formalin induced licking in the formalin test. Both R- and S- isovaline decreased the formalin induced licking by greater than twice than control (Fig. 22). R and S — isovaline injected lumbar intrathecally equipotently reduced the licking in the formalin test. This suggests that stereoisomerism of isovaline was not a significant factor in the analgesic effect as measured in the formalin foot test. 5.3.3. Toxicity of Lumbar Intrathecal Injection of Glycine Glycine reduced the formalin induced licking with no observable side effects such as sedation or respiratory depression. The lack of respiratory depression was expected because the drug was administered lumbar intrathecally would not likely act on brain stem respiratory centers. However, glycine is known to be toxic and to cause respiratory depression when administered systemically (Ballanyi et al., 1999; Holtman et al., 1982). Glycine is also neurotoxic by activating NMDA receptors (Barth et al., 2005). Glycine at 250 mM was the only amino acid tested other than 500 mM beta-alanine that caused significant scratching. The scratching is an irritation of the animal and suggests an agitation of the sensory afferent fibers. Glycine at lower doses produces analgesia without scratching. The 66 higher dose of glycine inducing scratching indicates a side effect of elevated dose of glycine. The higher dose of glycine may cause irritation of the sensory nerves leading to scratching. This is consistent with the literature in which glycine was once used clinically in an irrigation fluid until the realization of toxicity (Olsson & Hahn, 1999). Isovaline at 250 mM did not produce any scratching or any visible side effects while still producing analgesia with equal potency and efficacy to glycine and suggest more clinical potential than glycine. An increase in dose to 500 mM produced lower body weakness revealing a local anesthetic effect. These observations may be indicative of approaching toxic doses. The higher toxic dose of isovaline is intrinsic with a greater potential for safe clinical analgesia. 5.3.4. Toxicity of Lumbar Intrathecal Injection of Isovaline The lowest effective dose of isovaline was 125 mM which produced analgesia in normal mice without visible adverse effects. At 500 mM isovaline lower body weakness was observed, a sign of adverse effects. Hence, the minimal therapeutic index for lumbar intrathecal administration of isovaline was 500 mM isovaline/125 mM isovaline or 4 and suggests that isovaline is a relatively safe analgesic for lumbar intrathecal administration. A possible reason for the margin of safety of is that isovaline is either unknown or of limited occurrence in the biosphere so is not found or used biologically in animals or humans, and it is unlikely to be incorporated into proteins to produce side effects. Another possible reason could be selectivity to isoforms of the glycine receptors. Alternatively, isovaline may be an antagonist at the glycine B site to dampen glutamate excitation while glycine would be a co-agonist for the glycine B site and enhance glutamate excitation to induce toxicity. 67 5.3.5. Toxicity of Lumbar Intrathecal Injection of Beta-alanine 500 mM beta-alanine was the highest dose tested; resulting with lower body weakness in 6 out of 9 animals. The toxic dose for beta-alanine administered lumbar intrathecally is in the vicinity of 500 mM and is higher than glycine but similar to isovaline. Beta-alanine at 500 mM induced significant scratching in both phase I and II of the formalin test. At 500 mM beta-alanine, lower body weakness was also observed in addition to scratching in 6 out of 9 animals. 500 mM beta- alanine resulted in more toxicity than 500 mM isovaline base which produced lower body weakness in fewer animals with and no significant scratching. 5.3.6. Toxicity of Lumbar Intrathecal Injection of Controls It has been estimated that 3% of a spinally injected dose may reach the brain within a 10 minute test period (Hylden & Wilcox, 1980). Therefore, diffusion to the brain cannot account for the antinociceptive response of intrathecally injected substance. This indicates the analgesia was spinally mediated. As for the specific site of action, agents which are active spinally may be affecting neurons or synapses directly, but may also have a nonspecific effect. Lumbar intrathecal injection of an osmolality mannitol control with an osmolality in the range of the high doses of the amino acids tested did not produce adverse effects, and was indistinguishable from aCSF injected into control mice. This indicates that osmolality was not a significant factor in the drug effects. The pH of the administered drug solutions was measured and for all cases was 7 ± 1. A solution of highest dose isovaline, 500 mM, the hydrochloride form had a pH of —2. On injection seven out of nine mice had lower body weakness. The injections in the remaining two mice nearly abolished formalin induced licking. Base isovaline had a pH of —7. On injection four out of nine mice developed lower body weakness. The injections in the remaining five mice had 68 formalin induced licking that was lower than 250 mM but greater than the 500 mM isovaline hydrochloride. Neutralizing 500 mM isovaline showed a tendency to lower the toxicities observed by reducing the number of animals with lower body weakness which resulted in an increase in formalin induced licking. However, the formalin induced licking and number of mice with lower body weakness of neutralized 500 mM isovaline was not statistically significant different compared to acidic 500 mM isovaline hydrochloride. Morphine was injected lumbar intrathecally as a positive control and significantly reduced formalin induced licking both phase I and II. However, morphine induced obvious sedation as defined as the lack of voluntary movement (Table 4). All the mice injected with morphine displayed sedation while none of the animals injected with glycine, beta-alanine, or isovaline displayed sedation. This suggests that glycine-like compounds possess analgesic properties without the sedation observed in morphine. 5.3.7. Intravenous Analgesia of Isovaline Intravenous administration of racemic isovaline significantly lowered the formalin induced licking in the formalin test. This observation demonstrates that isovaline penetrates the blood- brain-barrier. Although isovaline is exogenous to the human body and the biosphere, isovaline may cross the blood brain barrier via the glycine transporters due to a structural similarity to other amino acids like glycine. There also is a possibility that isovaline may act peripherally. Future experiments should investigate whether isovaline acts peripherally: isovaline with formalin. 69 The administered dose of racemic isovaline intravenously was 500 mg/kg at a volume of 3 mL/kg or 1.4 M. Assuming isovaline is distributed throughout the body and two-thirds of the mouse is water, the dose in the mouse would be 750 mg/kg or 4.88 mM. The administered minimal effective dose lumbar intrathecally was 60 mM. The total CSF volume of a mouse is 100 IA and assuming that isovaline is localized to a few spinal segments and dissolved in a volume of —20 CSF, the intrathecal concentration would be — 15 mM. The effective dose intrathecally is about three times greater than the effective intravenous dose. If isovaline acts in the spinal cord and the brain, then intravenous isovaline presumably acts mainly in the brain. 5.3.8. Toxicity of Intravenous Injection of Isovaline The rotarod is a sensitive test that can detect central nervous system and neuromuscular toxicities for a broad range of drugs ranging from sedatives, neuromuscular blockers, and analgesics including morphine (Vaupel et al., 1984). Intravenously administered racemic isovaline (500 mg/kg) tested negative on the rotarod test and indicates lack of central nervous system or motor side effects. Intravenous racemic isovaline did not significantly decrease respiratory rate compared to saline control. Morphine administered systemically decreases respiratory rate in mice (Shimoyama et al., 2005). 5.4. Potential Mechanisms Trigeminal allodynia is a form of chronic pain that has limited effective treatments. The etiology is uncertain but is thought to be due to compression of the trigeminal nerve but surgical alleviation of the compression has limited success. Glycine receptors are found in the trigeminal 70 nucleus in the brain stem and intracisternal administration of glycine receptor antagonist, strychnine, induced allodynia localized in the trigeminal nerve distribution. This model of trigeminal allodynia was validated with the current treatment of trigeminal allodynia, carbamazepine. The model can be used as a drug screen for potential analgesics effective for trigeminal allodynia. Trigeminal allodynia may be mediated by a loss of glycinergic inhibition in the trigeminal nucleus. Hence glycine receptor agonists and agents that enhance glycinergic inhibition have potential in the treatment of trigeminal allodynia. Administration of racemic isovaline into the cisterna magna is effective in reducing strychnine induced allodynia. This finding suggests that isovaline is a glycine receptor agonist and that glycine receptor agonists may act supraspinally in the brain stem region, providing effective treatment for trigeminal allodynia. The ability to penetrate the blood brain barrier is important for drug delivery. Initially we were uncertain that isovaline would penetrate the blood brain barrier. Although early studies showed that isovaline is actively transported in the intestines indicating potential for oral administration (Evered et al., 1967). Amino acid transporters transport amino acids across the blood brain barrier (Hawkins et al., 2006). Although the physicochemical properties of isovaline do not appear to allow isovaline to penetrate the blood brain barrier directly, intravenous administration of isovaline resulted in significant analgesic effects. Hence isovaline penetrates the blood brain barrier, likely due to amino acid transporters. Both R and S isovaline administered lumbar intrathecally produced equipotent analgesia in the formalin foot test. This finding indicates that in the whole animal R and S isovaline seem to act 71 on a non-stereospecific site, in contrast to many drugs. Barring no stereoisomeric related toxicities, the lack of stereospecific analgesia will be a benefit in the ease and economic production of the drug. The rotarod is sensitive to a broad range of adverse effects including central nervous system side effects observed with sedatives, anti-epileptics and tranquilizers. Intravenous administration of isovaline (500 mg/kg, 1.4 M) did not produce any side effects as measured with the rotarod. There were also no visible signs of side effects, comparable to a normal mouse. Intravenous administration of isovaline at 500 mg/kg (1.4 M) did not produce side effects and at the same dose, produced analgesia. At 250 mM lumbar intrathecal administration of isovaline produced analgesia with no observable side effects. But at 500 mM isovaline administered lumbar intrathecally, there were lower body weakness, an indication of an adverse effect. By combining the intravenous and lumbar intrathecal observation, we can deduce that when we administer 1.4 M of isovaline intravenously, definitely less than 500 mM isovaline is entering the spinal cord because we do not see lower body weakness and probably less than 250 mM is entering the spinal cord because the 1.4 M intravenously administered isovaline has less antinociceptive effects than the 250 mM isovaline administered lumbar intrathecally in the formalin test. Approximately, less than 15% of the isovaline administered intravenously is entering the central nervous system, assuming that isovaline is not acting peripherally. A future experiment is to test if isovaline acts peripherally, as described later. Lumbar intrathecal administration of racemic isovaline significantly reduced the formalin induced licking in the second phase and displayed a trend for reduction in the first phase. This 72 suggests that racemic isovaline has antinociceptive properties for chronic pain with a potential for the treatment of acute pain. This finding indicates that isovaline is acting at least in the spinal cord and presumably at the dorsal horn to cause antinociception. Glycine receptors are found in the dorsal horn of the spinal cord and coupled with our finding that isovaline reverses strychnine induced allodynia, it is likely that isovaline is acting through glycine receptors in the spinal cord to induce antinociception. Beta-alanine has antinociceptive properties as indicated by the formalin test and is structurally related to glycine by the extension of a single carbon on the glycine backbone. An additional extension of the amino acid backbone of the beta-alanine with another carbon would result in GABA, an inhibitory neurotransmitter widely found in the central nervous system. The chemical structure of isovaline is an amino acid and differs from beta- alanine and GABA by only a few carbons and it would be possible that isovaline may also have GABAergic activity as well. S.S. Future Directions Future experiments can investigate the mechanism of action, as it seems that isovaline may act through the strychnine sensitive glycine receptor, glycine site on the NMDA receptor or the GABA receptor. A method for distinguishing the receptors that isovaline may be acting on could be to express glycine receptors on the human embryonic kidney cells (HEK) and record the response to isovaline application. Different glycine receptor isoforms can be expressed on the HEK cells leading to further distinction of glycine receptor isoform specificity which may lead to analgesics with fewer side effects. Similarly, NMDA receptors can be expressed on the HEK cell and tested with isovaline. Again, different NMDA receptor isoforms can be experimented to find isoform specificity or subunit specificity. In addition, different antagonists for the NMDA receptor, channel, or the glycine site of the NMDA receptor can be administered with isovaline 73 to specifically identify the NMDA receptor site of action. Finally, GABA receptors can also be expressed on the HEK cells. Different types of GABA receptors including GABA A,GABAB , and GABA.c can be expressed and the subsequently different isoforms of each of the GABA receptors can also be investigated. These in vitro studies can determine which receptor and receptor subunit, subtype, or isoform isovaline is acting on. Sorting out whether isovaline acts on each of the individual receptors, GABA, NMDA, or glycine receptor or on a multiple of these receptors would provide information on the mechanism of action of isovaline. If isovaline acts on multiple receptors such as both as an antagonist on the glycine site of the NMDA receptor and an agonist at the strychnine sensitive glycine receptor then, isovaline could be a lead drug in designing analgesics that act on two distinct mechanisms. With specific information gleaned from in vitro studies, we can return to the whole animal to ensure the drug will act as predicted in the integrated biological system. Other structural analogues that can be tested include 1-aminocyclobutane-l-carboxylic acid or ACBC. ACBC is an antagonist at the glycine site of the NMDA receptor (Popik et al., 1995) and is structurally similar to isovaline with the cyclization of the methyl and ethyl substituents at the alpha carbon to result in a cyclic four carbon substitution of the alpha carbon. Unlike isovaline, ACBC does not have a stereocenter and have been shown in preliminary results to be analgesic in the formalin assay and more potent than isovaline. It indicates that the cyclic substituent on the alpha carbon may result in greater analgesic activity than the non-cyclic alpha carbon substituent. Another compound that can be tested is alpha - aminoisobutyric acid or AIB which is chemically similar to isovaline except the alpha carbon contains two methyl substituents. 74 Isovaline is shown to act in the central nervous system both in the spinal cord and the brain but it is uncertain whether isovaline may also act in the periphery. A future experiment would be to test if isovaline also acts in the periphery. A possible experiment would be to co-inject isovaline and formalin subcutaneously into the hindpaw of the mouse in the formalin test. If isovaline acts in the periphery, the co-injection of isovaline and formalin should reduce the formalin induced nociceptive behavior. In addition, co-injection of glycine with formalin in the formalin test would provide information as to the presence of glycine receptors in the peripheral nerves. Co- injection of ACBC, an NMDA receptor glycine site antagonist, with formalin in the formalin test would also provide information of whether glycine site on the NMDA also have analgesic actions in the periphery. The discovery of glycine receptors acting in the trigeminal nucleus to elicit trigeminal allodynia has resulted in a model and screen for trigeminal allodynia. Future studies can include testing other glycine receptor agonists as potential analgesics for trigeminal allodynia. GABA is a major inhibitory neurotransmitter that is also found in the trigeminal nucleus, another possible model for trigeminal allodynia may be to investigate if loss of GABAergic inhibition in the trigeminal nucleus will develop trigeminal allodynia. A model of trigeminal allodynia based on intracisternal administration of GABA antagonists such as GABAA antagonist, bicuculline or GABAB antagonist, baclofen, can be developed using similar methods to the strychnine induced model of trigeminal allodynia. 75 Chapter 6. Conclusions The purpose of this thesis was to investigate the effects of strychnine intrathecally and isovaline and related glycine receptor agonists and analogues. We found that lumbar and cisternal intrathecal injection of strychnine induced allodynia and isovaline has non-stereospecific antinociceptive properties. Intracisternal injection of glycine causes respiratory depression and death at doses lower than the isovaline doses used in present experiments. Intracisternal morphine does not decrease strychnine induced allodynia while carbamazepine is effective in decreasing strychnine induced allodynia, in accordance to the clinical effects of these drugs. Furthermore, intracisternal administration of strychnine produces allodynia localized to the trigeminal distribution. The mechanism of action may be through the strychnine sensitive glycine receptor, but isovaline also may act on other receptors such as the glycine site of the NMDA receptor and the GABA receptor. Lumbar intrathecal administration of glycine like compounds including glycine, beta-alanine, and isovaline produces analgesia in models of acute and chronic pain. Lumbar intrathecal administration of glycine produced signs of toxicity such as scratching at 250 mM whereas beta-alanine and isovaline elicited local anesthetic effects at 500 mM. Since at a high concentration of 500 mM isovaline, there are adverse effects of lower body weakness when administered intrathecally and is effective at 125 mM, isovaline has a therapeutic index of at least four for lumbar intrathecal administration. 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The glycine synaptic receptor: Evidence that strychnine binding is associated with the ionic conductance mechanism. Proceedings of the National Academy of Sciences of the United States of America, 71, 4002-4005. Zeilhofer, H. U. (2005). The glycinergic control of spinal pain processing. Cellular and Molecular Life Sciences, 62, 2027-2035. Zeilhofer, H. U., Studler, B., Arabadzisz, D., Schweizer, C., Ahmadi, S., Layh, B.,(2005). Glycinergic neurons expressing enhanced green fluorescent protein in bacterial artificial chromosome transgenic mice. The Journal of Comparative Neurology, 482, 123-141. Zhou, S., Bonasera, L., & Carlton, S. M. (1996). Peripheral administration of NMDA, AMPA or KA results in pain behaviors in rats. Neuroreport, 7, 895-900. 88 THE UNIVERSITY OF BRITISH COLUMBIA APPENDIX UBC tfy ANIMAL CARE CERTIFICATE Application Number: A06-0215 Investigator or Course Director: Bernard A. Macleod Department: Pharmacology & Therapeutics Animals:^Mice CD 1 36 Start Date:^August 1, 2006 ApprovalDate: July 17, 2006 Funding Sources: Funding UBC Centre for Anesthesia and AnalgesiaAgency: Funding Title: Treatment of Trigeminal Neuralgia Unfunded title: N/A The Animal Care Committee has examined and approved the use of animals for the above experimental project. This pilot project certificate is valid for three months from the above start or approval date (whichever is later) provided there is no change in the experimental procedures. A copy of this certificate must be displayed in your animal facility. Office of Research Services and Administration 102, 6190 Agronomy Road, Vancouver, BC V6T 1Z3 Phone: 604-827-5111 Fax: 604-822-5093 89 THE UNIVERSITY OF BRITISH COLUMBIAUBC liF ANIMAL CARE CERTIFICATE Application Number: A06-0428 Investigator or Course Director: Bernard A. Macleod Department: Anesthesiology, Pharmacology & Therapeutics Animals:^Mice CD-1 48 Start Date:^September 1, 2006 ApprovalDate: October 12, 2006 Funding Sources: Funding Agency: Funding Title: UBC Centre for Anesthesia and Analgesia A mouse model of transient allodynia Unfunded title: N/A The Animal Care Committee has examined and approved the use of animals for the above experimental project. This pilot project certificate is valid for three months from the above start or approval date (whichever is later) provided there is no change in the experimental procedures. A copy of this certificate must be displayed in your animal facility. Office of Research Services and Administration 102, 6190 Agronomy Road, Vancouver, BC V6T 1Z3 Phone: 604-827-5111 Fax: 604-822-5093 90 UBC THE UNIVERSITY OF BRITISH COLUMBIA ANIMAL CARE CERTIFICATE Application Number: A06-1450 Investigator or Course Director: Bernard A. Macleod Department: Anesthesiology, Pharmacology & Therapeutics Animals:^Mice CD1 216 Start Date:^October 1, 2006 ApprovalDate: October 12, 2006 Funding Sources: Funding UBC Centre for Anesthesia and AnalgesiaAgency: Funding Title: Teatment of Trigeminal. Neuralgia in Mice with Glycine and Cogeners Unfunded title: N/A The Animal Care Committee has examined and approved the use of animals for the above experimental project. This certificate is valid for one year from the above start or approval date (whichever is later) provided there is no change in the experimental procedures. Annual review is required by the CCAC and some granting agencies. A copy of this certificate must be displayed in your animal facility. Office of Research Services and Administration 102, 6190 Agronomy Road, Vancouver, BC V6T 1Z3 Phone: 604-827-5111 Fax: 604-822-5093 91 UBC THE UNIVERSITY OF BRITISH COLUMBIA ANIMAL CARE CERTIFICATE Application Number: A07-0152 Investigator or Course Director: Bernard A. Macleod Department: Anesthesiology, Pharmacology & Therapeutics Animals:  Mice CD1 801 Start Date: October 1, 2007 Approval Date: October 11, 2007 Funding Sources: Funding Agency: Funding Title: Funding Agency: Funding Title: Various Sources Jean Templeton Hugill Anesthesiology Research Fund Various Canadian Foundations (non-HS) Jean Templeton Hugill Anesthesiology Research Fund Unfunded title:^N/A The Animal Care Committee has examined and approved the use of animals for the above experimental project. This certificate is valid for one year from the above start or approval date (whichever is later) provided there is no change in the experimental procedures. Annual review is required by the CCAC and some granting agencies. A copy of this certificate must be displayed in your animal facility. Office of Research Services and Administration 102, 6190 Agronomy Road, Vancouver, BC V6T 1Z3 Phone: 604-827-5111 Fax: 604-822-5093 92 Application Number: A07-0590 Investigator or Course Director: Bernard A. Macleod Department: Anesthesiology, Pharmacology & Therapeutics Animals: Mice CD1 40 Start Date:^November 1, 2007 Funding Sources: Funding Agency: Funding Title: Approval Date: December 14, 2007 Various Sources Jean Templeton Hugill Anesthesiology Research Fund Funding Agency: Various Canadian Foundations (non-HS) Funding Title:^Jean Templeton Hugill Anesthesiology Research Fund Unfunded title:^N/A IJBC^ THE UNIVERSITY OF BRITISH COLUMBIA ANIMAL CARE CERTIFICATE The Animal Care Committee has examined and approved the use of animals for the above experimental project. This certificate is valid for one year from the above start or approval date (whichever is later) provided there is no change in the experimental procedures. Annual review is required by the CCAC and some granting agencies. A copy of this certificate must be displayed in your animal facility. Office of Research Services and Administration 102, 6190 Agronomy Road, Vancouver, BC V6T 1Z3 Phone: 604-827-5111 Fax: 604-822-5093 93

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