Robert W. Gereau - US grants

The hippocampus is critically involved in normal physiological processes such as learning and memory and in various pathological states such as temporal lobe epilepsy. The hippocampus has a well-defined trisynaptic circuit, and each synapse in this circuit uses glutamate as its neurotransmitter. Synaptically released glutamate exerts its actions on neurons via activation of ionotropic glutamate receptors (iGLuRs) and metabotropic glutamate receptors (mGluRs). iGluRs are ligand gated ion that mediate fast synaptic transmission. The mGluRs comprise a family of receptors coupled to intracellular second messengers through G proteins, and these receptors mediate intrinsic modulation in glutamatergic circuits. The mGluRs have a variety of neuromodulatory actions in hippocampus, including excitatory effects on neurons and modulation of synaptic transmission. 8 mGluRs have been cloned (mGlu1-8), and genetic studies have shown that one of these (mGlu5) is critical for hippocampus-dependent learning, indicating that mGlu5 is critical in hippocampal function. However, the cellular actions of mGIu5 have not been well defined. Because of the important role of mGIu5 in hippocampus-dependent learning, it is important to know how mGlu5 modulates the function of this structure, and further how mGlu5 may itself be modulated. We have shown that mGlu5 undergoes protein kinase C (PKC)-mediated desensitization and phosphorylation in expression systems. However, we have not yet unequivocally identified the sites of phosphorylation or determined whether this desensitization occurs in the brain. The studies proposed here will address the specific roles of mGlu5 in hippocampal function using genetically altered mice. Furthermore, we will test the hypothesis that mGIu5 undergoes PKC-mediated desensitization in response to physiological and pathological stimuli and that this desensitization requires PKC phosphorylation of specific sites in mGlu5. We will accomplish these goals using patch clamp physiology in wt and knockout mice and direct protein sequencing to identify sites of PKC phosphorylation. Finally, we will generate antibodies that recognize these sites of mGlu5 phosphorylation for use in determining whether these sites are phosphorylated in vivo. These studies will help identify the specific functions and mechanism of modulation of mGlu5 in hippocampus.

DESCRIPTION (provided by applicant): 'Physiological pain' is a first line defense necessary for the preservation of life. The intensity of this pain is correlated with the noxious stimulus intensity and lasts only as long as the stimulus. 'Pathological' or 'chronic pain', on the other hand, is associated with an altered sensitivity to stimuli manifested as allodynia (pain sensations to non-painful stimuli) or hyperalgesia (increased sensitivity to painful stimuli). These pathological pain sensations are not directly correlated to stimulus intensity and commonly outlast the duration of the stimulus. Key to the effective treatment persistent pain is having an understanding of the underlying mechanisms. One molecule that is intimately involved in thermal nociception is VR1. This receptor is almost exclusively expressed in cells involved in nociceptive transmission, and is activated by heat, low pH and capsaicin, the pungent component of hot peppers. Studies of knockout mice show that VR1 is critical for thermal hyperalgesia. Other studies show that inflammation-induced thermal hypersensitivity involves kinase-dependent amplification of capsaicin- or heat-evoked currents, suggesting that VR1 function is upregulated by phosphorylation in response to activation of G protein-coupled receptors for various inflammatory mediators. One such inflammatory mediator is glutamate (Glu), which is released into peripheral tissues during inflammation. We have found that G protein-coupled receptors for Glu (known as metabotropic Glu receptors or mGluRs) are expressed in sensory nerve endings. Peripheral application of mGluR agonists leads to thermal hypersensitivity, whereas antagonists reduce inflammatory hyperalgesia, suggesting that Glu released in the periphery following inflammation is a key mediator of inflammation-evoked hyperalgesia. To gain more understanding of the mechanisms underlying peripheral mGluR modulation of thermal sensitivity, we are studying the phosphorylation and modulation of VR1 by different protein kinases involved in thermal hyperalgesia. These studies will include electrophysiological recordings and Ca2+ imaging to determine the molecular basis of mGluR modulation of VR1, and phosphorylation studies will identify functionally relevant sites of phosphorylation. Finally, we will generate and utilize antibodies that specifically recognize the phosphorylated form of VR1 to determine the conditions under which VR1 is phosphorylated in vivo. These studies will provide valuable insights into the molecular basis for persistent pain states, and may suggest new targets for the development of novel analgesic agents.

Abstract Chronic pain represents an immense clinical problem, with over 100 million Americans afflicted and an annual price tag exceeding half a trillion dollars, according to a recent report from the Institute of Medicine. Studies in our lab are designed to identify molecular, cellular, and circuit mechanisms of sensitization in pain pathways with the goal of identifying novel targets for analgesic intervention. Studies performed in our lab previously identified a critical signaling cascade in neurons of the central nucleus of the amygdala (CeA) that underlies central pain sensitization. This pathway is initiated by metabotropic glutamate receptor subtype 5 (mGlu5) activation of extracellular signal-regulated kinase/ERK signaling, leading to increased firing of CeA neurons. This increase in excitability likely contributes to central sensitization associated with persistent pain. Our prior work, and that of several other groups, suggests that neurons in the CeA represent a critical node of neuromodulation underlying the development of chronic pain. An important finding from our prior studies was that this maladaptive plasticity in the CeA leading to persistent pain sensitization is specific to the right hemisphere. That is, no matter the sight of the injury, plasticity in the right (and not left) CeA was responsible for bilateral pain hypersensitivity. Furthermore, manipulation of neural activity only in the right CeA was found to produce bilateral pain sensitization. The mechanisms generating this hemispheric lateralization are completely unknown. In the present application, we will conduct a series of studies aimed at understanding the circuit context of CeA neurons that are activated by acute pain sensitization. We will perform studies aimed at identifying critical inputs, the type of plasticity that occurs at these synapses, and the major outputs of pain-responsive CeA neurons. We will test whether CeA neurons activated in the context of pain sensitization are necessary and sufficient for the development of pain sensitization, ongoing pain and comorbid disorders. By specifically targeting pain- activated neurons in this study, we may be able to determine if they possess unique neurochemical properties that represent novel therapeutic targets, or genetic signatures that would enable future studies to more precisely determine their function. In vivo 2-photon imaging and microendoscope cameras will be used to monitor activity of these neurons using genetically-encoded Ca2+ sensors, over days to weeks, to determine how the properties of these neurons change during the transition from acute to persistent pain. We will ask whether the population of neurons responsive to heat, cold, or touch change over time, and whether altered activity of these neurons in persistent pain conditions can be normalized using treatments that reduce pain or comorbid anxiety. These studies employ a host of modern techniques including advanced viral tracing, genetic mapping, in vivo calcium imaging, and optogenetic approaches, together with technologies developed in our lab for wireless optogenetic studies to address these important questions.

It is well-established that the amygdala, a forebrain multinuclear structure, plays a crucial role in emotional[unreadable] behaviors such as fear, anxiety, and stress. It has recently been proposed that the amygdala, in particular the[unreadable] central nucleus (CeA), is also involved in the modulation of pain sensation. Evidence from anatomical,[unreadable] behavioral, and physiological studies support the hypothesis that the amygdala serves as a neural pain center[unreadable] that integrates noxious sensory information and emotions. Preliminary results from our lab and others have[unreadable] demonstrated that electrophysiological changes occur in the central nucleus of the amygdala during periods of[unreadable] persistent pain. For this reason, we have been performing studies aimed at elucidating the signaling cascades[unreadable] involved in the modulation of pain sensation by the amygdala. Our preliminary studies indicate that[unreadable] inflammation of one hindpaw induces acute pain in the injected paw, and after a period of several hours, also[unreadable] in the uninjured contralateral paw. Interestingly, the timing of the onset of this contralateral hypersensitivity[unreadable] coincides with the activation of the extracellular signal regulated kinase (ERK) in the right amygdala,[unreadable] specifically in the laterocapsular subdivision of the central nucleus (CeC). Our preliminary data support the[unreadable] hypothesis that ERK activation in the right (but not left) CeC underlies the development of generalized pain[unreadable] hypersensitivity after inflammation. However, there are still a number of unanswered questions regarding the[unreadable] role of amygdala ERK activation in pain modulation. The present application will address some of these issues.[unreadable] In the first aim, we will test whether the right lateralized ERK activation in the CeC is physiologically relevant in[unreadable] multiple pain models. The second aim of the proposal utilizes patch clamp recordings from brain slices to test[unreadable] the hypothesis that ERK activation leads to acute modulation of neuronal excitability and/or synaptic[unreadable] transmission in the CeC. These studies will lay important groundwork for future investigations of the[unreadable] importance of amygdala ERK signaling in pain sensitization.

[unreadable] DESCRIPTION (provided by applicant): Our research addresses the cellular and molecular mechanisms that underlie pain hypersensitivity associated with injury and disease. We have been studying the role of the neurotransmitter glutamate (Glu) in the modulation of pain sensitivity in the periphery. Glu is a key inflammatory mediator that is released into peripheral tissues during inflammation, and we have found that G protein-coupled Glu receptors known as metabotropic Glu (mGlu) receptors are expressed in peripheral terminals of nociceptors. In the previous term of this grant, our studies showed that activation of peripheral mGluS in nociceptor terminals induces hypersensitivity to thermal and mechanical stimuli. The thermal hyperalgesia can be explained, at least in part, by PKA-dependent sensitization of the heat-sensing protein, TRPV1. We showed that PKA and PKC directly phosphorylate TRPV1, and identified phosphorylation sites critical for modulation of TRPV1 by PKA and PKC in heterologous systems. However, differences in the PKA and PKC modulation of TRPV1 in heterologous systems relative to natively expressed TRPV1 call into question whether the same phosphorylation sites are involved. While this TRPV1 modulation can account for regulation of thermal nociception by mGluS, it cannot explain the induction of mechanical hypersensitivity. Preliminary data show that mGluS enhances excitability of nociceptors, and we suggest that this may underlie the induction of mechanical sensitization by mGluS. Our work and the work of others points to an important role of mGluS is mediating pain hypersensitivity, and as a consequence mGluS antagonists are being pursued as a novel class of analgesics. However, recent work has called into question whether mGluS antagonists reduce pain by blocking mGluS or by an "off target" action. Studies in the present proposal will address the following open questions: 1) What phosphorylation sites mediate sensitization of TRPV1 in native DRG neurons? 2) What is the relative importance of central and peripheral mGluS in pain hypersensitivity? 3) What are the cellular mechanisms that underlie mechanical hypersensitivity induced by activation of peripheral mGluS? These studies will reveal the cellular and molecular mechanisms by which mGluS modulates thermal and mechanical pain sensation, and will clearly define the role of central and peripheral mGluS in the modulation of pain. They may also help promote the development of mGluS antagonists as analgesics. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The Midwest Pain Interest Group Meeting is an annual meeting of basic scientists, clinicians, and their trainees that work in the pain field. The meeting has as its core mission the exchange of knowledge about pain research and treatments and the development of young pain scientists. The meeting rotates between a number of Midwest institutions, and for 2007 the meeting is being hosted by Washington University School of Medicine in St. Louis on June 8 and 9. At this year's meeting, a main focus will be to increase basic science/clinical science interactions as part of an overall goal of promoting research translation. The meeting takes place over two days, with an afternoon poster session and reception incorporating both basic science and clinical research on day one. This is followed by a dinner and social event, which would not be supported by NIH funds. The following morning sees two concurrent sessions, the first is a continuing medical education session for clinicians; this will include multiple topics related to state of the art pain management, and the second morning session is oral presentations by trainees and junior faculty from the represented institutions. Presentations by women and members of underrepresented minorities are particularly encouraged. After these talks, both sessions come together for a luncheon with a plenary speaker that is of broad interest to clinicians and basic scientists. This year the plenary speaker will be Dr. Jeff Mogil from McGill, who has agreed to come and will be speaking on the general topic of genetic influences on pain and analgesia. This meeting provides an annual forum where we emphasize extensive informal interaction between basic scientists and clinicians working in the pain field. The emphasis is always on trainees. Having this meeting on an annual basis helps establish a consistent network of pain research peers in the region, and has been a real driver for new and innovative collaborations amongst member institutions. [unreadable] [unreadable] [unreadable]

2H-1,3,2-Oxazaphosphorin-2-amine, N,N-bis(2-chloroethyl)tetrahydro-, 2-oxide; 2H-1,3,2-oxazaphosphorin-2-amine, N,N-is(2-chloroethyl)tetrahydro-,2-oxide; Abdomen; Abdominal; Abdominal Muscles; Acute; Address; Allergy; Animal Model; Animal Models and Related Studies; Animals; Antimorphic mutation; Autonomic pain; Behavior; Behavioral; Biochemical; Bladder; Bladder Diseases; Bladder Disorder; CTX; CYCLO-cell; Carloxan; Cell Communication and Signaling; Cell Signaling; Central Nervous System; Characteristics; Chronic; Ciclofosfamida; Ciclofosfamide; Cicloxal; Clafen; Claphene; Cycloblastin; Cycloblastine; Cyclophospham; Cyclophosphamide; Cyclophosphamidum; Cyclophosphan; Cyclophosphane; Cyclophosphanum; Cyclostin; Cyclostine; Cytophosphan; Cytophosphane; Cytoxan; Dominant Negative; Dominant-Negative Mutant; Dominant-Negative Mutation; Dorsal Horn Cells; Dorsal Horn of the Spinal Cord; Dose; ERK 1; ERK1; ERK1 Kinase; ERK2; ERT1; Endoxan; Endoxana; Enduxan; Extracellular Signal-Regulated Kinase 1; Fosfaseron; Genoxal; Genuxal; Goals; Hyperalgesia; Hyperalgesic Sensations; Hypersensitivity; INFLM; Inflammation; Inflammatory; Injury; Interstitial Cystitis; Intracellular Communication and Signaling; Ion Channels, Potassium; Isoforms; K channel; Knock-out; Knockout; Knockout Mice; Laboratories; Ledoxina; Localized; MAP Kinase 3; MAP-ERK Kinase; MAPK ERK Kinases; MAPK1; MAPK1 gene; MAPK2; MAPK3; MAPK3 Mitogen-Activated Protein Kinase; MAPK3 gene; MEKs; Mammals, Mice; Measurement; Mediating; Medulla Spinalis; Meiosis-Activated Myelin Basic Protein Kinase p44(mpk); Methods; Methods and Techniques; Methods, Other; Mice; Mice, Knock-out; Mice, Knockout; Microtubule-Associated Protein-2 Kinase; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase 3 Gene; Mitoxan; Modeling; Molecular; Murine; Mus; Neosar; Nervous System, CNS; Neuraxis; Neurons, Dorsal Horn; Neurons, Posterior Horn; Nociception; Nociceptive Impulse; Nociceptive Stimulus; Null Mouse; P41MAPK; P42MAPK; P44ERK1; P44MAPK; PRKM1; PRKM2; PSTkinase p44mpk; Pain; Pain Disorder; Pain Research; Painful; Patients; Phosphorylation; Posterior Horn Cells; Potassium Channel; Process; Procytox; Protein Isoforms; Protein Phosphorylation; Protein-Serine-Threonine Kinase p44(mpk); Research; Role; Sendoxan; Signal Transduction; Signal Transduction Systems; Signaling; Spinal; Spinal Cord; Spinal cord posterior horn; Syklofosfamid; Syndrome; Techniques; Testing; Time; Transgenic Animals; Urinary System, Bladder; Visceral; Visceral pain; Zytoxan; allodynia; biological signal transduction; central pain; central sensitization; cortical pain; dorsal horn; experience; experiment; experimental research; experimental study; frontier; hyperalgia; inflammatory pain; model organism; mouse model; neuronal excitability; new therapeutics; next generation therapeutics; nociceptive; novel therapeutics; p44 (MAPK); p44 MAPK; research study; response; social role; urinary bladder; urinary bladder disorder

DESCRIPTION (provided by applicant): Chronic pain, depression, and addiction represent immense health problems of epidemic proportions. The 2011 Institute of Medicine (IOM) report on Relieving Pain in America states that over 116 million Americans suffer from chronic pain with an annual price tag exceeding half a trillion dollars. Similarly, the National Institute of Mental Heath and National Institute of Drug Abuse have reported that mood disorders and addiction affect greater than 10% of the total US population. The mammalian nervous system is built from hundreds of different neuronal and glial cell types. This incredibly diverse array of cells has made dissecting brain function and treating neuropathogical states such as pain, depression, and addiction one of the most difficult challenges facing medical research. Understanding how these neural circuits communicate with one another is one of the major goals of neuroscience, and discoveries in this arena open new avenues for therapeutic intervention. As nanotechnology and materials engineering have evolved, there has been an increasing need and potential for neural micropolymeric interfaces to be developed that could be used for the study and treatment of neurological and psychiatric diseases. In this transformative research application we have assembled a multidisciplinary collaborative team between materials scientists and neurobiologists. Together we propose to: (i) Develop novel biocompatible, multimodal micro-ILED devices suitable for stable integration with the central and peripheral nervous system, (ii) use a combination of these micro-ILED devices with optogenetics to dissect the neural circuits involved in and develop treatments for neuropathic pain (iii) employ these micro-ILED devices for dissecting neural circuits and signal transduction in stress and affective disorders. In an integrated team approach, we will test, develop, and optimize this novel technology. The ultimate goal will be to develop multifunctional nanomaterial micro-ILED wireless devices for full integration with diverse neural circuits. In this project we using a combination of light-sensitive channel activation and light-activation of intracellular signal transduction cascades using engineered G-protein coupled receptors (GPCRs) within peripheral neural circuits involved in pain and central neural circuits involved in stress and negative affect including the locus ceoruleus (LC) and ventral tegemental areas (VTA). Using these novel micro-ILED devices we will dissect the heterogeneous populations of sensory nociceptors, stress, and reward neurocircuitry. Together this research will not only provide a foundation for the integration of nanoscale devices with mammalian neural circuits, but also it will guide future efforts to interface and interact with selected neural circuits in clinical settigs with respect to pain and psychiatric diseases.

Bladder pain and voiding dysfunction are the cardinal signs of IC/BPS. Patients with IC/BPS report enhanced pain on bladder filling (distension), and we recently found that these patients also demonstrate segmental referred hyperalgesia, reflecting the presence of central sensitization in IC/BPS. Referred hyperalgesia and pain on bladder distension seen in patients is also observed in animal models of IC/BPS, providing a laboratory model system for us to pursue the development of novel therapies for IC/BPS. Studies in animal models are needed to gain a clear understanding of the mechanisms of pain and voiding dysfunction in the context of bladder injury and IC/BPS, and this knowledge is necessary to develop novel mechanism-based therapies. Much regarding these mechanisms remains to be determined, and key among these unknowns is knowledge of what sensory neurons carry signals from the bladder to the central nervous system and what are the key receptors mediating bladder pain transmission. In this project, we will address these critical knowledge gaps. In Aim 1 we use optogenetic approaches to identify subpopulations of afferent neurons innervating the bladder that are responsible for carrying signals that regulate bladder pain and voiding dysfunction. This may form the basis for nonpharmacologic approaches to the management of IC/BPS. In Aims 2 and 3, we propose a translational research study aimed at a novel pharmacologic approach to managing pain and voiding dysfunction: inhibition of metabotropic glutamate receptor 5 (mGlu5). This includes preclinical studies identifying the site of action of the mGlu5 antagonist fenobam in Aim 2, and in Aim 3, we propose a proof of concept clinical study to test the efficacy of fenobam in treating bladder pain and voiding dysfunction in patients with IC/BPS. Together, the studies proposed here will provide key insights into the cells and signaling molecules that mediate bladder pain and voiding dysfunction in mice and in IC/BPS patients. This will be the first directly translational study of its kind, and may provide evidence supporting an entirely new therapeutic approach to the management of IC/BPS, as well as paving the way for new non-pharmacologic approaches using optogenetics as a therapeutic approach in the future.

? DESCRIPTION (provided by applicant): Bladder pain and dysfunction are sources of profound debilitation for millions of people in the United States with interstitial cystitis/bladde pain syndrome, overactive bladder, and neurogenic bladder. Current treatments for these disorders are ineffective and do not address the underlying pathology. A substantial barrier to the development of improved therapeutics is an insufficient understanding of mechanisms by which the bladder is controlled. Progress will depend critically on the development of new technologies in neural interfaces. In particular, a combination of optogenetic approaches along with new, wireless optoelectronic systems and electronic hardware for nerve recording and electrical stimulation will provide a unique set of tools for enhanced insights into peripheral organ control. Additionally, the ability to measure, in real-time, neural inflammation and to programmably deliver local anti-inflammatory agents at the neural interface will not only allow robust, high performance chronic integration, but also further the understanding of maintenance at nerve- device interfaces in other applications. In this proposal, we develop a suite of soft, fully-implantable microscale devices with advanced design features specifically configured to minimize and mitigate inflammation for essentially permanent integration with the targeted nerves. In Aim 1, we propose to integrate optogenetic technologies with fully-implantable wireless systems for inhibition of peripheral neurons innervating the bladder. In Aim 2, we propose to integrate ultra-thin, flexible electrode technology and novel neuroinflammatory monitors to the wireless control of bladder peripheral nerves. In Aim 3, we integrate a fully-implantable, wirelessly programmable microfluidic platform for closed-loop maintenance of the chronic nerve interface. Our collaborative team has extensive success merging technologies into unified multi-modal devices and subsequently applying them to the mechanistic study of neuronal subpopulations. The technology produced by these aims will be optimally positioned to study mechanisms of control of end-organ function, and to do so in a way that is minimally invasive.

Abstract Interstitial cystitis/Bladder Pain Syndrome (IC/BPS) is a serious and painful condition of unknown etiology that affects 3-6% of women in the United States. The major clinical symptoms of IC/BPS are pain on bladder filling and increased urinary urgency and frequency. The majority of IC/BPS patients (90%) also suffer from comorbid anxiety and/or depression, contributing to a poor quality of life. The high rate of comorbid affective disorders in IC/BPS patients suggests that a common supraspinal neural circuit may be responsible for both enhanced pain and negative affect in patients with IC/BPS. Based on a large body of work in the neurosciences, we hypothesize that the central nucleus of the amygdala (CeA) is a crucial hub of neuronal activity that regulates both bladder pain and negative affect. In this project, we propose a series of studies that seeks to determine the necessity and sufficiency of neuronal subpopulations in the CeA in the induction of voiding dysfunction, pain sensitization, and comorbid anxiety and depression in models of cystitis. Does activation of this circuit lead only to hypersensitivity to stimulation, or is this also critical for ongoing or spontaneous pain? Does the same population of neurons mediate both pain sensitization and increased anxiety following injury? What are the critical inputs and projections from these neurons that lead to these debilitating consequences of cystitis? We employ a multidisciplinary approach including viral anatomical tract tracing, optogenetics, chemogenetics, and in vivo imaging of neural activity in awake, freely moving mice to address these questions. These studies will provide new insights into the critical role of the CeA in bladder pain and comorbid affective disorders in the context of bladder pain syndrome, and provide the basis for future studies building on this to gain insights into circuit, cellular and synaptic mechanisms of voiding dysfunction, chronic pain and comorbid anxiety and depression.

Abstract The current epidemic of opioid-related deaths ravaging the nation demands innovative new approaches to treat opioid use disorders and prevent deaths resulting from accidental overdose. Patients with a history of opioid use followed by a period of sobriety are at particularly high risk for overdose. This increased risk stems from the development of tolerance during prolonged periods of use. Tolerance can quickly fade during a period of abstinence, so if a patient relapses and takes the same dose used prior to the period of abstinence, the dose will be high enough to precipitate an acute respiratory crisis, leading to injury or death. Current treatment requires administration of naloxone by first responders. This treatment requires timely identification of the overdose and need for a rescue injection, as well as the immediate availability of the medication. The development of a fail- safe treatment that would provide a life-saving dose of naloxone without the need for intervention by another party could significantly reduce mortality. In the present application, we propose the development of a new medical device comprising an implantable, closed-loop system that senses the presence of an opioid overdose, and automatically administers a life-saving bolus injection of naloxone, and simultaneously alerts first responders. This proposal builds on technologies that the investigative team has developed over the past several years.

ABSTRACT Opioid use disorders (OUD) are responsible for a major health and socioeconomic crisis in the US, resulting in more than $500B burden on the economy and more than 47,000 deaths a year due to opioid overdose. More than 80% of OUD cases started from the use of prescription opioid painkillers, which is currently the most effective (and often the only available) option for treatment of severe pain. The current analgesics target ?-Opioid receptor (MOR), which mediates not only analgesia but also dependence, addiction leading to OUD, as well as respiratory depression and death. Diversion and misuse of prescription opioid drugs in US is the key reason for the skyrocketing opioid epidemic. Development of a new generation of safe and effective analgesics with diminished addiction and abuse potential is desperately needed. We propose to target peripheral cannabinioid receptor subtype 1 (CB1) as a mechanism to develop pain relievers devoid of the addiction potential associated with opioid receptors as well as centrally active CB1 agonists. We propose a bitopic approach targeting the orthosteric site of CB1 to achieve potency and efficacy and allosteric sodium binding pocket to achieve peripheral over central activity in vivo. Our long term goal is to develop an orally active CB1 selective agonist with nM potency, poor brain penetration with optimal drug like properties like protein binding, metabolic stability, no hERG, CYP liability and oral activity, a goal we will seek to achieve through the U19 mechanism this R34 feeds into. ADME and PK fine tuning on leads obtained through R34 will be a part of the U19 phase of development. For this R34 planning grant we bring together a multidisciplinary team with the aim to test if the bitopic approach can lead to compounds with efficacy in animal models of pain and highly restricted peripheral activity while showing selectivity for CB1 receptors. Our optimal compound to be synthesized through this R34 phase will be have the following characteristics: 1) In vitro profile: CB1-agonist with ?50 nM potency and 100 fold selectivity over other >350 other targets. 2) DMPK profile: Protein binding<5% free at 10 µM, metabolic stability>2h, hERG>10 µM, CYP inhibition/activation <20-30% at 10µM and brain:plasma <0.03. 3) In vivo profile: IP/Oral CB1 mediated analgesic, potency ED50?5-10 mg/mg with >2.5h analgesic time course and lacking central side-effects like abuse potential and other liabilities upto 15xED50 doses.