Carolyn A. Fairbanks - US grants

DESCRIPTION: (Provided by Applicant) This application is for a Mentored Research Scientist Development Award (K01). Recently, agmatine (decarboxylated arginine) has been isolated from mammalian brain and spinal cord. Agmatine both antagonizes the NMDA receptor and inhibits nitric oxide synthase (NOS). Because glutamate is thought to drive synaptic plasticity by activating both NMDA receptors and NOS in series, it is conceivable that agmatine participates in control of synaptic plasticity and related behavioral phenomena (e.g. learning and memory, opioid tolerance, chronic pain) as a neuromodulator of glutamate. Exogenously administered agmatine has been shown to be neuroprotective in a model of cerebral ischemia and to prevent development of opioid-induced tolerance, both thought to be processes requiring plasticity. Preliminary data presented here demonstrates that exogenous agmatine reverses nerve injury- and inflammation-induced allodynia and hyperalgesia. The primary goal of the proposed study is whether endogenous agmatine participates in the control of spinally derived pain. The proposed studies will address that goal through pursuit of the following specific aims. 1). Determine the effect of endogenous agmatine in the spinal cord on nerve injury- and inflammation-induced hyperalgesia. The induction of behaviorally assessed allodynia and hyperalgesia will be compared across strains and conditions with differing agmatine concentration in spinal cord extracts. 2). Determine whether endogenous agmatine can be released from the spinal cord. Agmatine concentration in spinal cord dialysate will be compared prior to and following exposure to K+ and other depolarizing substances. 3). Determine how localization of agmatine in spinal cord and dorsal root ganglia (DRG) is related to that of other neurochemical markers. Antisera directed against agmatine will be used in laser confocal and electron microscopy studies to determine its regional, cellular, and ultrastructural localization in the spinal cord and DRG as well as its relationship to the localization of pain-related signaling molecules and proteins. 4.) Determine the relationship between agmatine and glutamate-mediated excitation in the spinal cord. Electrophysiological experiments will explore whether previously observed NMDA receptor channel blockade or other action accounts for agmatine's block of neuronal excitation and behavioral plasticity in spinal cord. The results of these studies will clarify the role of endogenous agmatine in nociception, its localization in relation to neurochemical markers of nociception, and its relationship to the excitatory nociceptive transmitter glutamate. The studies will also explore its participation as a novel neuromodulator of central sensitization. Elucidation of the role of the endogenous agmatine during hyperalgesia and allodynia may lead to the development of a novel class of analgesic drugs or the identification of therapeutic targets.

DESCRIPTION: (provided by applicant) This proposal is for an Exploratory/Developmental Grant Application (R21), specifically addressing RFA # PAR-01-047, the Cuffing Edge Basic Research Award (CEBRA) program. Recently, agmatine (decarboxylated arginine) has been isolated from mammalian brain & spinal cord and found to antagonize NMDA receptors and inhibit nitric oxide synthase (NOS). Because glutamate is thought to drive synaptic plasticity by activating both NMDA receptors and NOS in series, agmatine may participate in control of synaptic plasticity and related behavioral phenomena (e.g. learning, memory, chronic pain, opioid tolerance and self-administration) as a neuromodulator of glutamate. Exogenously administered agmatine is neuroprotective in models of cerebral ischemia (Gilad, 1996) and spinal cord injury (Yu et. al., 2000) and prevents development of opioid analgesic tolerance (Kolesnikov 1996; Fairbanks, 1997), all considered to be processes requiring plasticity. In addition to these published reports, preliminary data presented here demonstrates that exogenous agmatine prevents fentanyl self-administration. The primary goal of the proposed study is to determine whether endogenous agmatine modulates opioid-induced analgesic tolerance and self-administration. That objective will be addressed by determining the relationship of agmatine levels to opioid tolerance and self-administration. First, agmatine concentration will be systematically measured in brain and spinal cord regions thought to be involved in opioid addiction and analgesic tolerance. These measurements will be compared across multiple strains of mouse known to have differential sensitivities to induction of opioid analgesic tolerance and self-administration. If, as exogenous agmatine findings predict, endogenous agmatine protects against induction of opioid analgesic tolerance and self -administration, then mouse strains with relatively low concentrations of central nervous system agmatine will be more sensitive to induction than those with high concentrations of CNS agmatine, an inverse correlation. The second component of the project will determine whether manipulating levels of CNS agmatine changes analgesic tolerance and self-administration also through an inverse relationship. The results of these proposed Phase I CEBRA R21 studies will determine whether or not there exists a modulatory relationship between endogenous agmatine and the glutamatergic mechanisms underlying opioid tolerance and self-administration. Such a finding would provide a rationale for pursuing in depth mechanistic clarification of the role of endogenous agmatine in opioid analgesic tolerance and self-administration, a strategy that would comprise the subsequent PHASE 11 CEBRA R01 application. Elucidation of a role for endogenous agmatine in modulation of the development of opioid tolerance and self-administration may lead to the development of a novel class of drugs or alternative methods to treat addiction, or to the identification of new therapeutic targets.

[unreadable] DESCRIPTION (provided by applicant): Over the past five decades, the neuroscientific community has made significant progress in our understanding of the organization and function of the spinal cord dorsal horn and dorsal root ganglia (DRG). Many new proteins have been identified as important in the pain signal conduction pathway. Novel gene therapy approaches may offer new opportunities for long-term pain management strategies. The adeno-associated virus (AAV) serotype has been shown to be useful for transduction of neurons in cortex, brainstem, and cerebellum. Its utility for transduction of neurons in the spinal cord and dorsal root ganglia has also been explored; attempts to deliver the AAV vectors to the spinal cord or DRG have only been successful with direct intraparenchymal or intraneural injections. Less invasive and more clinically therapeutic direct lumbar puncture approaches have not successfully transduced the spinal cord. In contrast, we have now succeeded in achieving widespread spinal cord and DRG transduction of the marker green fluorescent protein (GFP) by direct lumbar puncture injection of an AAV serotype 5 (AAV5) in conscious mouse and rat. These preliminary studies support our proposal to optimize delivery and characterize potential function of AAV5 based vectors targeting specific genes known to modulate chronic pain signaling within spinal cord or DRG. Efficient AAV5-mediated genetic manipulation offers substantial opportunities to 1) further study mechanisms underlying chronic pain and 2) develop novel gene-based therapies for the treatment and management of chronic pain using a non-invasive delivery route with established safety margins. The proposed research will assess the utility of delivery of AAV-vector by lumbar puncture as a useful tool for basic scientific study of chronic pain as well as a potential therapeutic delivery option. The primary objectives of the project are: 1) To optimize delivery to the spinal cord and DRG and characterize the distribution of the AAV5-GFP construct. 2) To validate this approach, in a system well established in the pain signal conduction pathway. 3) To apply the approach to a novel non-opioid system that may exert control on the development of chronic opioid tolerance and maintenance of chronic pain. Future applications of intrathecal delivery of AAV5 constructs will enhance study of other novel targets participating in chronic pain at the level of the spinal cord and DRG and may enable translational developments of chronic pain therapies. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Opioids remain the most effective pharmacotherapeutics for the treatment of many chronic pain conditions. However, the potential for misuse of opioids can impact the decision to use them, particularly in the treatment of non-cancer pain. Opioid tolerance, dependence, addiction and some chronic pain conditions all share common mechanisms of neuronal adaptation. Consequently, distinguishing between these responses in an individual is challenging. Central drug delivery methods (e.g. intrathecal) enable some separation of chronic pain mechanisms (in large part spinally mediated) from those driving motivated behavior (supraspinally mediated). The proposed research program will use these techniques in experimental murine models of chronic pain to separate fentanyl self-administered (orally) for relief of chronic hyperalgesia from opioid self- administered in excess of that required for relief of hyperalgesia (anti-hyperalgesia). Experimental subjects with induced chronic neuropathic or cancer-evoked hyperalgesia of the hindpaw will be allowed to self-administer opioid orally under operant control daily for four weeks;the resulting anti-hyperalgesia will be determined after each operant session. Rescue anti-hyperalgesic agents will be administered intrathecally on various days after induction of hyperalgesia to assess the impact of spinal anti-hyperalgesia on opioid self-administration. Determining the effect of spinally administered rescue analgesic on opioid self-administration will enable an assessment of the amount of opioid administered for anti-hyperalgesia independent of that self-administered for supraspinally mediated reward. Those subjects continuing to self-administer opioid in the presence of adequate spinal analgesic will be further evaluated to determine supraspinal mechanisms that pre-dispose them to opioid self-administration in excess of that required for anti-hyperalgesia. Conversely, experimental subjects that reduce opioid self-administration in response to spinal analgesic (for alleviation of mechanical hyperalgesia) will be evaluated for factors that protect them from excess opioid self-administration. Clarification of the conditions under which opioids are misused when given for treatment of chronic pain may better inform the decision-to-treat process. PUBLIC HEALTH RELEVANCE: Chronic pain sufferers are thought to take opiate analgesics more for pain relief than in search of reward;however, fear of abuse liability often limits their use, resulting in undertreatment of chronic pain. The relationship between opiate self-administration and chronic pain intensity is rarely studied in experimental animals. Modeling opioid self- administration in mice experiencing chronic pain will provide insight into this concept and ultimately improve patient access to opioid therapy for their pain.

DESCRIPTION (provided by applicant): Acupuncture and electroacupuncture have been increasingly adopted in North America as a complement to pharmacotherapy for chronic pain over the last twenty years (1997 NIH Consensus Statement on Acupuncture). At least thirty years of research indicate a role for opioidergic systems in the mechanisms underlying acupuncture-evoked analgesia. However, the mechanism(s) underlying the effectiveness of acupuncture for pain management remains only partially understood. In recent years a role for NMDA receptor antagonists to enhance opioid analgesia by inhibition of the development of analgesic tolerance has been clearly defined. However, the contribution of endogenous modulators of the NMDA receptor system to analgesia induced by acupuncture has been minimally explored. Agmatine (decarboxylated arginine) is an endogenous substance that antagonizes the NMDA receptor and inhibits nitric oxide synthase. Like synthetic NMDA receptor antagonists, agmatine also inhibits development of opioid tolerance and dependence. Since agmatine is expressed in the CNS, strategies could be used to optimize its activity in CNS regions where electroacupuncture exerts therapeutic benefit. The objective of this application is to determine the mechanism by which agmatine contributes to electroacupuncture-evoked analgesia. The central hypothesis is that endogenous agmatine exerts its effects through inhibition of the NMDA receptor at the NR2B receptor subunit. The rationale for the proposed research is that, with demonstration of endogenous agmatine participation in electroacupuncture-evoked analgesia and identification of the mechanism, endogenous agmatine could be regulated in site-specifically to optimize electroacupuncture therapy. Two specific aims are proposed: #1: Determine the extent to which and the mechanism by which endogenous agmatine contributes to electroacupuncture-induced pain control. #2: Determine the ability of electroacupuncture to release endogenous agmatine. This contribution will be significant because understanding how agmatine manifests its effects is an important step toward optimizing its therapeutic application in both non-pharmacological and pharmacological pain management approaches. The research proposed in this application is innovative because it represents a new mechanism to explain analgesia evoked by electroacupuncture, a commonly applied non-pharmacological pain management therapy that is not well understood. The successful completion of this research is expected to position both electroacupuncture and agmatine as complementary approaches to improve pain management and therapy that may translate to other therapeutic indications arising from maladaptive neuroplasticity. PUBLIC HEALTH RELEVANCE: The technique of acupuncture for analgesia is a system known to activate natural (endogenous) pain control systems. Endogenous pain control has motivated a multi-decade global research effort focused primarily, though not exclusively, on opioid neurotransmitters (e.g., endorphins). The proposed research would assess the potential contributions of an entirely novel spinal pain control system (agmatine) to acupuncture effects. Agmatine blocks the long-term effects of a well characterized nerve stimulant, glutamate. The goal of the present research program is to determine whether agmatine acts concurrently or synergistically with other natural pain control systems to provide the pain relief achieved through application of acupuncture.

DESCRIPTION (provided by applicant): We propose to hold a conference on the role of Complementary and Alternative Medicine (CAM) in pain management and research following the annual meeting of the American Pain Society (APS) in May 2013. The APS is an interdisciplinary community comprised of scientists, clinicians and other professionals committed to the pursuit of furthering knowledge of pain mechanisms and pain relief. The purpose of the APS satellite conference, Complementary and Alternative Medicine: Roles in Chronic Pain Management and Research, is to raise awareness of CAM pain management approaches and specific challenges for CAM pain research scientists and clinicians. The conference will highlight the state-of-science in chronic pain research as it relates to CAM. The primary goal of the conference is to create interdisciplinary exchange and encourage potential collaborations among chronic pain clinicians and researchers in CAM. The conference will address the multiple challenges and opportunities associated with current CAM pain research. Sessions are planned to promote interactions between clinicians, scientists, and trainees with interest in applying CAM to chronic pain. Poster sessions and a symposia are also specifically planned to highlight contributions made by early career scholars and investigators. These submissions will be peer-reviewed prior to acceptance. CAM approaches for pain management is an emerging emphasis area within APS; the development of this conference is intended to strengthen the presence of CAM as a research interest area within APS.

DESCRIPTION (provided by applicant): Analgesic tolerance arises with chronic opioid pharmacotherapy. Blockade of NMDA receptors reduces the development of opioid tolerance, but delivery of NMDA receptor antagonists can result in motor and/or cognitive toxicity. Agmatine is a decarboxylated form of L-arginine produced in the central nervous system (CNS) that significantly reduces opioid analgesic tolerance. Although evidence suggests that agmatine antagonizes the NMDA receptor, unlike many synthetic NMDA receptor antagonists delivered spinally, it does not elicit motor deficits. This pharmacological profile suggests that spinally delivered agmatine may act at the NR2B subunit of the NMDA receptor, the expression of which is restricted to the sensory region of the spinal cord and not in the ventral horn. The objective o this application is to define the mechanism by which CNS agmatine modifies behavioral neuroplasticity, evaluate its role as a neuromodulator, and determine the potential of targeting the agmatinergic system as a gene therapy to improve opioid analgesia. The central hypothesis is that CNS agmatine inhibits opioid analgesic tolerance through antagonism of the NMDA receptor and in particular the NR2B-containing NMDA receptors. Three specific aims are proposed: Specific Aim #1: Delineate the contribution of CNS-derived agmatine to inhibition of opioid tolerance. We will address this aim through evaluation of induction of spinal opioid tolerance under conditions of genetic alteration of agmatine's synthetic and degradative enzymes in vivo. Specific Aim #2: Define the mechanism by which CNS-derived agmatine inhibits opioid tolerance. We will address this aim with pharmacological, physiological, and molecular approaches that will test the relationship of agmatine to the glutamatergic system. Specific Aim #3: Determine the impact of opioid tolerance on the agmatinergic system and its target receptor(s). To address this aim, we will use anatomical and molecular approaches. The experimental approach is innovative because it will uncover a largely understudied inhibitory system that opposes maladaptive neuroplasticity. The information acquired will indicate the extent to which endogenous agmatine can be accessed to maximize inhibition of pathological neuroadaptation (e.g. opioid tolerance). The knowledge gained is expected to be useful for management and treatment of chronic pain and directly applicable to other CNS dysfunctions.