Tony Yaksh - US grants

DESCRIPTION (ADAPTED FROM THE APPLICANT'S ABSTRACT) Hyperalgesic states result from the release of primary afferent transmitter release e.g., substance P (SP) from capsaicin-sensitive C fibers which activate postsynaptic systems through an NK1 receptor. This activation leads to the spinal release of excitatory amino acids and prostaglandins (PGE2), which evokes a facilitated state through an action on NMDA and prostanoid receptors. This hormonal input leads to phosphorylation of receptors and channels which can enhance nociceptive processing. Spinal mu opioid and alpha2 adrenergic agonists produce antinociception, in part by blockade at a site presynaptic to the primary afferent leading to a block of SP release and by an action at receptors postsynaptic to the primary afferent. Continued presence of the agonists results in an increase in the dose required by a probe drug to produce a given effect. When an antagonist is delivered to a tolerant system, withdrawal is observed which is associated with hyperalgesia. Accumulating evidence suggested that these effects, injury evoked hyperalgesia, spinal tolerance and withdrawal-evoked hyperalgesia are mediated through parallel changes in spinal NMDA receptor activity and changes in the phosphorylation state of spinal neurons induced by kinases/phosphatases activated during chronic afferent input and exposure to opiates. These observations provoke several hypotheses. 1) Inhibition by mu or alpha2 agonists of capsaicin-evoked release of SP from afferent terminals or release of glutamate and PGE2 evoked by local NK1 receptor activation from nonafferent spinal terminals will be diminished by continuous exposure to mu/alpha2; 2) After chronic exposure to spinal mu or alpha2 agonists, antagonists (withdrawal) will evoke hyperalgesia and enhance release of spinal SP, PGE2/ glutamate. 3) Concurrent spinal NMDA antagonism will prevent loss of effect by mu or alpha2 agonists on evoked release and prevent release observed in antagonist-evoked withdrawal. 4) mu PKC) and phosphatases (e.g. calcineurin), which may decrease (by phosphorylation) or increase (by dephosphorylation) the ability of the agonist to reduce C transmitter release and nociception after chronic exposure. 5) Blockade of synthesis of specific kinases (PKCalpha or PKCgamma) and phosphatases (calcineurin) will reduce/enhance, respectively, tolerance and withdrawal-hyperalgesia. These studies will define i) the role of primary afferents in antinociception in the face of chronic opiate or alpha2 exposure; ii) linkages at which NMDA receptor antagonism alters spinal mu and alpha2 tolerance, and iii) the role of spinal isozymes (kinases and phosphatases) which regulate phosphorylation in the development of tolerance and hyperalgesia.

Substance P (SP) is contained in small dorsal root ganglia, is transported peripherally, and is present in peripheral nerve terminals in knee joint and skin. The comparable actions of local SP and the antidromic stimulation of somatic afferents suggest that these peripheral stores of SP may be releasable, a likelihood specifically supported by a currently modest literature. In the proposed studies, all performed in terminally anesthetized cats, perfusates of the synovial space of the knee joint (KJ) and the intradermal space of a skin bleb or the spontaneous outflow of the saphenous lymph duct will be assayed for SP-LI. We will address four specific hypothesis: 1) "SP-like immunoreactivity (SP-LI) is released in a Ca++ dependent fashion into the interstitial space from the peripheral nerve terminals of small, unmyelinated sensory afferents". Specifically, we will examine the SP-LI secretion evoked by antidromic stimulation of the sciatic nerve, with regard to effective stimulus intensity, frequency dependency, apparent relative refractory period; Ca++ dependency and the local releasing and desensitizing effects of capsaicin. 2) "SP-LI release from the peripheral terminal occurs as a function of local thermal and chemical stimulation". Specifically, the effects of 35 degrees C and 60 degrees C stimuli; and the dose dependent effects of locally applied bradykinin, histamine, prostaglandins E2 and capsaicin will be examined. A corollary to this hypothesis is that manipulations which result in sensitization of the peripheral terminal of the afferent will facilitate the peripheral release of SP-LI. This will be examined in the KJ using an acute inflammatory response evoked by intrasynovial injection of kaolin and carrageenan. 3) "The antidromic evoked release of peripheral SP-LI is subject to modulation by several classes of local receptors, including those for mu, delta, kappa opioid, alpha 1 and alpha 2 adrenergic agonists, adenosine, GABA, and somatostatin". 4) "Pharmacological agents which modulate the peripheral secretion of SP-LI will predictably alter a physiologic effect produced by antidromic activation of the somatic afferents, notably an increase in capillary permeability". These studies will thus describe the pharmacology and physiology of the peripheral terminals of primary afferents which secrete SP-LI. Current evidence clearly suggests that such antidromic release may play a prominent role not only in local inflammatory reactions, but in a variety of important pathological states including arthritis.

We have previously demonstrated that afferent input will result in a Ca++ dependent increase in the levels of met-enkephalin like immunoreactivity in spinal and mesencephalic periaqueductal (PAG) perfusates. A principal source of metenkephalin, proenkephalin A, is, however, known to possess several carboxy extended forms of met-enkephalin and a variety of large sequences (greater than 2000 Da). The fact that these forms, do gain access to the brain and spinal cord extracellular space and do have biological activity emphasizes their possible function as neurohormones. Though the several roles played by these enkephalin-secreting systems is not known, the physiological effect of activating receptors in these regions suggests these systems may play a prominent role in the processing of nociceptive information. Given the differential receptor preferences of the extended opioid peptides and the different systems associated with the several opioid receptors, it might be hypothesized that the release of the extended forms may reflect distinguishable functions. To investigate this issue we will assess in halothane anesthetized cats the release from spinal cord and PAG of: YGGFM, YGGFMR, YGGFMRF, YGGFMRGL and metorphamide. The concurrent assay of these several fragments will be made using a sequential protocol we have quantitatively characterized involving: a) ultrafiltration, b) HPLC, c) trypsin/carboxypeptidase B cleavage, and d) met-enkephalin specific radioimmunoassay. The concurrent release of these several opioid peptides from brain and spinal cord will be examined as a function of: the activation of somatic and visceral afferents; direct spinal stimulation using putative afferent transmitters (glutamate, substance P, vasoactive intestinal polypeptide) or agents which depolarize afferent terminals/ganglion cells (capsaicin). Electrical stimulation thresholds and frequency dependency of the release and the effects of opioid agonists and antagonists on release will be examined. Where practical, the effects of these manipulations on enkephalin secretion will be correlated with the effects on a thermally evoked nociceptive reflex. These studies will thus provide insight regarding the physiology and pharmacology of PAG and spinal cord systems, activity in which results in the local secretion of opioid peptides. Given the powerful effects produced by the action of exogenously administered opioids in these same regions, we must conclude that stimuli producing such enkephalin secretion also activate a powerful modulation.

Peripheral nerve or spinal cord injuries can result in pain states characterized by sharp spontaneous noxious sensations (dysesthetic pain) or pronounced pain generated by low threshold mechanical stimulation (allodynia). Such pain states are frequently observed after trauma (as in the vicinity of a bullet wound or a bone fracture), intentional surgical resection (as with thoracotomies or post mastectomy) or in states of chronic compression (as with a tumor). These states are associated with clinical syndromes, such as causalgia and reflex sympathetic dystrophy. Though controversial, these syndromes frequently are comparatively less sensitive to conventional analgesic therapies, such as opiates. Pre- clinical studies have revealed comparable changes in sensitivity following peripheral nerve compression injuries and with protracted afferent input (as with the injection of formalin into the paw). Importantly, in a wide variety of studies it has been shown that these allodynic and hyperalgesic states are mediated by spinal glutamate receptors of the N-methyl-D- Aspartate (NMDA) type. Thus, even in neurogenic pain models where opiates have little effect upon the allodynia, there is a powerful suppression produced by the spinal delivery of NMDA. These observations form part of a very large convergent body of literature convincingly showing a prominent role for spinal glutamate release and a subsequent action at NMDA receptors in mediating these anomalous pain states evoked by otherwise innocuous stimuli. Given these parallels, it is appropriate to consider the role of NMDA receptor antagonist in pain states that are poorly controlled, if at all, by other therapeutic modalities. Because NMDA antagonists have unacceptable supraspinal actions (dysphoria, loss of organized thinking processes), and because the prevailing data emphasizes the role of the spinal NMDA receptor, it is necessary to consider the spinal delivery of these agents in man. No agents of this class currently exist which have been appropriately examined for safety to allow their spinal delivery in man. We have chosen to examine three agents: ketamine, dextrorphan and memantine which bind to the NMDA channel; which have been shown to have antinociceptive actions in allodynic pain models and which are available commercially as GMP preparations. Using two well defined models (rat and dog) for chronic spinal drug delivery, we will examine the spinal toxicology of these agents. Specifically, in rats, we will examine the effects of daily repeated intrathecal injections of these agents on behavior and histopathology; and determine the effects on spinal cord blood flow. In dogs, we will examine the effects of 28 days of continuous intrathecal infusion of two of the three NMDA antagonists on behavior and histopathology; and, define the kinetics of the intrathecally delivered drugs. These studies will provide the essential basis for supporting subsequent studies examining the effects of spinal NMDa antagonists in human pain states that reflect therapeutic dilemmas.

DESCRIPTION: (Applicant's abstract) Relatively brief periods of ongoing (sec - min) afferent nociceptive stimulation, as evoked with subcutaneous injection of formalin, will (i) evoke an acute afferent barrage which correlates with the acute behavioral response and ii) trigger a long lasting facilitation of spinal nociceptive processing in which a minimum stimulus yields an exaggerated pain state (hyperalgesia). Specific pharmacological interventions with spinal agents have suggested that the hyperalgesic component may be mediated by activation of spinal excitatory amino acid (EAA) and prostanoid (PG) receptors and the production of nitric oxide (NO). The observed pharmacological correlates of the behavioral consequences of acute injury yields several specific hypotheses: 1) Tissue injury (as with formalin) leads to the acute activation of spinal substance P (SP) and glutamate receptors. 2) Activation of glutamate receptors of the NMDA and nonNMDA type and SP receptors of the NK1 type evoke the subsequent formation of COX (cyclo-oxygenase) and NOS (nitric oxide synthase) products. 3) Spinal COX and NOS products enhance the release of EAA. This cascade suggests that activation of glutamate or SP receptors by endogenous release will evoke NO and PG release from spinal cord and that event will subsequently enhance glutamate release. 4) Over longer intervals of afferent activation (min-hrs) as generated by inflamed knee joints, spinal COX and NOS, under the control of circulating corticoids, may display induction, yielding enzymes with a distinct inhibitor-drug profile. Such induction would yield increased PG and NO, an increased release of spinal glutamate and a hyperalgesia with a pharmacology distinct from that observed with short term (sec-min) stimulation. To address these hypotheses, we will investigate the extra-cellular levels of EAA, NO and PGs, using in vivo lumbar intrathecal dialysis in the unanesthetized rat and correlate these with changes in "pain" behavior. The pharmacology of this release and concurrent changes in behavior will be characterized by the spinal delivery of receptor-preferring agonists/antagonists and inhibitors. This work elucidates spinal systems mediating the profound changes in processing noted in a post injury state. Aside from an appreciation of the role played by novel spinal systems in pain processing and their plasticity in the face of afferent activation, protracted afferent drive is a pervasive component of a post-injury pain state. Elucidation of these mechanisms has relevance to the evolution of pharmacotherapy for its control in humans.

there is ample evidence based on preclinical work indicating that spinal delta opioid receptors can modify the processing of nociceptive information. Modest amounts of data with poorly selective delta agonists have confirmed this possibility in humans. The development of highly selective agonists for the delta receptor raises the possibility of finally confirming the role of spinal delta receptors in humans. Thus, the compound DPDPE is such an agent and assessment of its actions in humans after spinal delivery would be an important theoretical and clinically relevant advance. The present studies are aimed at defining the safety of DPDPE given intrathecally in rat and dog models. Existing data in a rat model has provided an initial confirmation of the safety of DPDPE. The next step prior to moving to humans is to further characterize the safety of DPDPE prior to assessing its action in humans. Specifically, the present studies will I) Define the effects of spinal DPDPE on spinal blood flow in the rat; and ii) Define the toxicology/histopathology of 28 day spinal infusion of vehicle or two doses of DEPDPE in a well defined dog model with chronically placed intrathecal catheters. Based on the extensive experience with these models, should the dog model display safety, it would be appropriate to subsequently consider the development of human studies protocols for examining the spinal effects of this delta selective agonist.

The Cancer Symptom Control Program consists of 17 Participating Members, representing total peer-reviewed funding for $4.14 Million annual direct costs ($5.65 Million in total costs). During the last two years, its members were responsible for a total of 100 cancer-relevant, peer-reviewed publications, 29% of which were intra- and inter- programmatic collaborations. This Program, formerly called "Palliative Care", is dedicated to research on the impact of cancer on human consciousness and sensory experience. It is comprised of members who are conduct highly interlocking studies on the consequences of cancer and its treatment on the quality of life of the patient. These examine pain, fatigue, anxiety, depression and sleep fragmentation, caused by cancer and how these can best be ameliorated to improve quality of life and satisfaction with medical care. The studies also examine the efficience of communications between doctors and their patients and the effect of ethnic and cultural characteristics of the patients of the subjective experience of the disease. This Program brings to bear both basic and clinical research on mitigating the physical and mental anguish associated with cancer. Of course, the cancer patients themselves are the main beneficiaries of this research on symptom control, but their families with severe illnesses. Members of this Program have developed a series of studiers highly specific to cancer patients in terms of: (1) cancer pain syndromes; (2) fatigue and sleep in cancer patients, and; (3) quality of life in cancer patients.

oncology; neoplasm /cancer epidemiology; neoplasm /cancer pharmacology; health science research support; molecular pathology;

DESCRIPTION (provided by applicant): Spinal galanin systems play a role in the regulation of afferent processing that results in pain behavior after tissue and nerve injury. The spinal modulation appears to be mediated by activation of one or more of three cloned and expressed galanin receptors (Ga1R1,2,3) some of which are present in spinal cord. The focus of this proposal, characterizing these several aspects of galanin activity and pharmacology is underscored by five specific aims. Specific Aim 1: Systematically define the antinociceptive profile of intrathecal galanin and homologues in rats on models of acute nociceptive processing (thermal escape), post tissue injury pain states (formalin and carrageenan hyperalgesia), and in post nerve injury pain states (Chung tactile allodynia Vincristine evoked tactile allodynia). Specific Aim 2: Define the spinal receptor mediating the spinal action of galanin by examining the dose dependent activity of intrathecal galanin, homologues and fragments in rats treated with intrathecally delivered antisenses/mis-senses for the Gal 1, 2 and 3 receptors. Specific Aim 3: As galanin has a presynaptic locus in several systems and can block opening of calcium channels, determine if, in accord with its antinociceptive profile, these agonists modulate the evoked release of spinal glutamate and prostaglandin in vivo. Specific Aim 4 examine the behavioral characteristics and response to intrathecally delivered galanin agonists and antagonists in mice prepared to be over expressors of galanin. Specific aim 5 systematically characterize and compare displacement of I125 galanin binding in Gal-r specific expressing cell lines, suppression of adenylate cyclase and antinociceptive actions after intrathecal delivery of combrnatorially synthesized candidate families of peptidic (Galp fragments) and non-peptidic (galnon) molecules which are known to bind at the Ga1R sites. These studies will thus systematically define the actions of spinal galanin-ergic systems in regulating spinal nociceptive function. We believe the outcome of these studies will provide direct evidence for the role played by this spinal peptidergic system in pain and point to the development of novel anti-hyperpathic agents.

DESCRIPTION (provided by applicant): Continuous intrathecal infusion of concentrated morphine is widely used in pain therapy. Surprisingly, until recently there has been no study of the safety of such infusions. We investigated the effects of 28-day intrathecal morphine infusion in a canine model. Unexpectedly, at high morphine concentrations (as used in humans), we noted an aseptic mass of inflammatory cells (granuloma) arising from the dura-arachnoid, not the parenchyma, proximal to the catheter tip. Granulomas were not seen with vehicle or a variety of non-opioid agents. The alpha2 adrenergic agonist clonidine suppressed the granuloma. These observations lead to four hypotheses. 1. Granuloma induction by morphine is proportional to local concentration in cerebrospinal fluid and not simply total dose. 2: Effect is mediated by an opioid agonist action and is not limited to morphine. 3. The granuloma results from a local degranulation of dural mast cells leading to movement of inflammatory cells from the dural vessels. Accordingly, granuloma-inducing potency will be proportional to the ability to degranulate dural mast cells in ex vivo dural preparations. 4. Granuloma-inducing effects and dural mast cell activation are suppressed by local alpha2 receptor agonists and by a mast cell stabilizer. We will address these hypotheses using the canine model to examine the effects of continuous intrathecal infusion of equipotent doses of mu opioid agonists (morphine, morphine-6-glucuronide, L-methadone, hydromorphone, fentanyl or DAMGO) or equimolar concentrations of inactive opioid molecules (naloxone, morphine-3-glucronidc, D-methadone). In vivo treatment with a mast cell stabilizer, nedocromil sodium, will be examined for its effect on granuloma formation. In parallel studies, kinetics studies will permit comparisons based on measured CSF concentrations. Interaction between morphine and alpha2 agonists (clonidine, dexmedetomidine) will be studied by co-delivery. Granuloma formation and local mast cell degranulation and cytokines will be assessed histochemically and by CSF analysis. In summary, our initial work, provides the first definitive preclinieal data defining the effect, the attenuation by clonidine, and a novel mechanistic hypothesis for drug-induced degranulation of dural mast cells which suggests a novel method for the ex vivo screening of new agents. These studies are significant: 1) increasing incidence of reports of morphine-granulomas emphasize it is not rare; 2) our investigation of other opioids provide the first time assessment of the spinal safety of agents which are now in wide clinical use; and 3) this issue impacts on all agents targeted for intrathecal delivery. Accordingly, data obtained here regarding the role of local CSF concentration, the safety of non-morphine agents and the potential ameliorating effects of adjuvant agents all provide novel information to refine the utility of this important therapeutic regime.

DESCRIPTION (provided by applicant): Persistent small afferent input generated by tissue injury yields a facilitated state of nociceptive processing. The associated pain behavior is blocked by spinal delivery of agonists for Gi/o protein coupled receptors, such as 5 opiates. Analgesic efficacy occurs in part by a presynaptic inhibition of small afferent (e.g. substance P, sP). Continued intrathecal (IT) infusion of 5 opiates results in a loss of analgesic potency and a concurrent loss of suppression of sP. Enabling data suggest that with tissue injury and chronic opiate exposure the evolution of the hyperalgesic state and loss of efficacy reflects upon activation of links which involve the spinal Akt signaling cascade. These studies will undertake the following: 1. Determine expression and phosphorylation of Akt and its downstream substrates, specifically, GSK32, mTOR in spinal dorsal horn and DRG in control and in evocative pain states over time in rat after local tissue injury (Intraplantar formalin, intraplantar carrageenan) and in mice with KBxN serum-induced arthritis. 2. Define cellular localization of pAkt, pGSK3 and pmTOR in spinal DRG and dorsal horn in control animals and after peripheral inflammation. 3. Determine change in phosphorylation state of dorsal horn Akt, GSK/2 and mTOR after intraplantar formalin with the acute spinal delivery of: i) 5 agonist; ii) blockers of NK1, glutamatergic ionotropic or metabotrophic excitatory receptors. 4. Define role of the spinal Akt cascade in pain processing by examining the effects of IT inhibitors of Akt, GSK32 or mTOR in models of hyperalgesia in rat: i) acute flinching and chronic tactile allodynia after intraplantar formalin, ii) tactile allodynia and thermal hyperalgesia after intraplantar carrageenan and iii) the centrally initiated thermal hyperalgesia after IT delivery of agonists for NK1 and group I mGlu receptors; and with IP drug delivery in the mouse model of KBxN arthritis. 5. Examine role of the spinal Akt cascade in spinal opioid tolerance and dependence produced by chronic IT morphine infusion and during the withdrawal produced by antagonism of the respective receptor (e.g. naloxone), wherein the respective enzyme inhibitor is co-infused with opiate agonist. These studies will accordingly define the role of this Akt cascade in pain and opiate tolerance. PUBLIC HEALTH RELEVANCE: Hyperalgesia after injury or inflammation involves spinal signaling links, one of which we hypothesize is the Akt cascade. Akt may also mediate opioid tolerance. Our work thus targets this link for these two critical issues in pain.

DESCRIPTION (provided by applicant): Neuropathic pain (NP) is 'pain arising as a direct consequence of a lesion or disease affecting the somatosensory nervous system' that has certain features of a neuroimmune disorder. In response to peripheral nerve damage, T helper (Th) lymphocyte trafficking and induction of major histocompatibility complex (MHC) II (essential to the capture and antigen presentation for Th cell recognition) in the injured nerve and the segmental spinal cord are 'necessary and sufficient' for the development of NP. MHCII has been implicated in differential induction of NP but not inflammatory pain, but the antigens and the processes of their formation remain elusive. Our groundbreaking program centers on the organizing thesis that immunodominant antigens, formed in damaged myelinated nerves, are at the source of low-threshold mechanical hyperpathia. Touch is signaled by non-nociceptive large myelinated (Ab) afferents, which after injury begin interpreting light touch as devastating pain (mechanical allodynia). Our cutting-edge preliminary data have determined a fundamentally novel mechanism explaining this phenomenon: selective proteolysis of myelin basic protein (MBP) by matrix metalloproteinases (MMPs) releases the cryptic immunodominant epitopes, MBP84-104 and MBP69-86, normally sheltered from immunosurveillance. We clearly demonstrate that immunodominant MBP peptides produce robust allodynia upon injection into intact nerve. An intriguing finding is that endogenous immunodominant MBP69-86 epitope co-localizes with MHCII in antigen-presenting Schwann cells of the injured nerve, in close proximity to the action potential-generating nodes of Ranvier. Using state-of-the-art biochemical and molecular tools (e.g. Illumina BeadChip gene arrays, MALDI-TOF mass spectrometry, Ingenuity, NextBio, GeneGo and proprietary MMP substrate database) and fundamental (neuropathological, behavioral, systems) approaches, we will characterize the T cell-dependent and -independent actions of MBP residues in nociception (Aim 1). The molecular domains of myelinated fibers responsible for MBP action in T cell homing and locomotion, and also, the receptors for MBP residues in nerve will be determined (Aim 2). Importantly, we determined that voltage gated sodium channel expression and the spinal release of a glutamate receptor subunit depend on MMP activity. The concluding focus of the proposal are the novel catalytic and non- catalytic MMP targets in regulating T cell trafficking and integration into the spinal neuro-glial signaling network, facilitating maladaptive central plasticity and aberrant neuroimmune synaptogenesis (Aim 3). This multi- disciplinary program merges the leading expertise and utmost resources in biochemistry of metalloproteolysis (Alex Strongin, Ph.D., Sanford-Burnham Medical Research Inst.), neurobiology of MMPs in peripheral myelination and neuroinflammation (Veronica Shubayev, M.D., UCSD), and pain pathways (Tony Yaksh, Ph.D., UCSD) with innovative study designs, which we believe are bound to transform our mechanistic understanding diagnostic and therapeutic practices predicting and preventing the transition to the NP state.

DESCRIPTION (provided by applicant): Botulinum neurotoxins (BoNTs) are widely used in medicine to treat a variety of neuromuscular disorders, taking advantage of their inhibitory effect on motor-endplate transmission after local injection and leading to localized muscle paralysis. It has become apparent that BoNTs can also enter other neuronal cells including sensory and CNS neurons. A robust block of neuraxial primary afferent and second order neurons processing can be achieved by direct spinal intrathecal delivery of the toxin. We (and others) have shown that such intrathecal delivery of BoNTs can produce a potent and persistent, but reversible, block of central nociceptive processing in a variety of pain models. However, the therapeutic utility of this approach is limited by the fact that the targeting and uptake of holotoxins is not specific to neuronal subtype. Thus the possibility of accompanying muscle paralysis and facilitated sensory processing due to block of inhibitory interneurons is of paramount importance. General effects would result in a heightened pain state rather than relief from pain, and has in fact once been described after an accidental injection of BoNT/A into the intrathecal space of a patient. We are proposing to overcome this detriment by targeting the enzymatic portion of the BoNT light chain (LC) specifically to terminals and neurons involved in pain processing. We hypothesize that stable and targeted BoNT derivatives can be created by coupling of recombinant truncated version of the BoNT LC to substance P or the peptide DAMGO, and that these derivatives will provide a long lasting and yet reversible block of nociceptive processing. Past efforts to create such constructs have been hindered by the postulate that BoNT LC cannot enter cells without possessing regions of BoNT heavy chain. Our innovative approach uses G- protein coupled receptor driven internalization after specific binding of the sensory neuron ligands to their respective receptors, such that the heavy chain is not needed for internalization. Our preliminary data and recently that of others with sP demonstrates the feasibility of this approach. We propose here to create a series of constructs using light chains of different BoNT serotypes (which differ in their longevity enabling regulation of duration of drug action) and at least two different ligands (substance P and DAMGO), which bind to the NK1and mu opioid receptors, respectively. These receptors are distributed on spinal terminals /cell bodies known to play a central role in pain processing. Cell models will examine selective entry into neurons expressing these receptors, and animal models will assess effects of the constructs on nociception and establish therapeutic ratios (e.g. analgesic /side effect doses). The use of intrathecal toxins has been validated with agents such as sP-saporin and resiniferatoxin, which are in clinical trials for chronic pain patients. But, unlike these agents, he targeted LCs can produce blocks of high selectivity and of varying durations of action. The results from this project have the potential to improve treatment options for chronic pain. The constructs created in this project will also provide tools for future studies of pain processing pathways. 1

Arthralgia broadly impacts quality of life because of joint dysfunction and associated pain. Current therapeutics improve management of the arthritic joint but may not satisfactorily address the associated pain. The K/BxN serum transfer model of arthritis produces a long lasting, but reversible inflammation of the joint in mice accompanied by an early onset allodynia that surprisingly persists long after the resolution of inflammatory indices. In the early phase of the model, pain behavior responds to nonsteroidal anti-inflammatory drugs (NSAIDs) and agents that block spinal sensitization (e.g. Gabapentin), while in the post-inflammatory late phase, pain only responds to the latter agents. This behavioral profile is accompanied by a persistent activation of dorsal horn microglia and the appearance of activation transcription factor 3 (ATF3), a marker of afferent injury in the dorsal root ganglia (DRG), in males and females, suggesting a transition in both sexes from an inflammatory to a neuropathic phenotype. Unexpectedly, the female, despite evidence of nerve injury, does not display a comparable late phase pain state. Pharmacological studies and studies with mutant mice have revealed several issues. 1) Transition to a neuropathic phenotype is modulated by spinal Toll-like receptor 4 (TLR4) signaling and T and/or B cells evidenced by resolution of pain in relevant knock out strains. In females that lack T and B cells, resolution of allodynia is largely unaffected. 2) Our work indicates that TLR4 signaling, largely through MyD88 is associated with concurrent activation of proinflammatory (TNF) signaling in males leading to a persistent post inflammatory neuropathic pain state. 3) TLR4 also signals though TRIF and interferon (IFN), which we found to attenuate the algesic effects of TLR4-MyD88 signaling. We speculate that this component accounts for the lack of a late phase allodynia in the female. 4) Based on current evidence, we argue that with persistent, but reversible inflammation, sprouting and neuroma like structures in primary afferents and postganglionic sympathetic efferents occur at the peripheral terminals of the joint and the dorsal root ganglion of the K/BxN male and female. This sprouting is driven by TLR4, which activates inflammatory cells and DRG satellite cells to release growth factors, which trigger/sustain sprouting and promote migration of nerve fibers into the DRG and spinal cord. 5) Involvement of spinal TLR4 in pain processing was suggested to be male specific, and females to preferentially use adaptive immune cells (T/B lymphocytes). We believe however, that both sexes use adaptive immune cells and TLR4 signaling, but to varying degrees. The effects of sex on this transition and the underlying sprouting have not hitherto been characterized. Specifically, these studies using the K/BxN model in males and females will characterize time dependent changes in pain, sprouting (afferent and sympathetic in DRG and ankle), inflammatory cell migration into DRG and spinal cord, glial activation and the role played by sex, TLR4 signaling and T/B cells in these endpoints.

Project Summary Persistent pain states, arising from inflammatory conditions, such as in arthritis, diabetes, HIV, and chemotherapy among others, have an extraordinary negative impact on quality of life. A common feature of these initiating events is the release of damage-associated molecular pattern (DAMP) molecules, which can activate Toll-like receptor-4 (TLR4). Our previous studies suggest that TLR4 is critical in mediating the transition from acute to persistent pain. TLR4 as well as other inflammatory receptors localize to lipid raft microdomains on the plasma membrane. Lipid rafts, enriched with cholesterol and sphingomyelin, facilitate ligand-mediated receptor dimerization and downstream signaling. Removal of cholesterol from the plasma membrane reduces lipid rafts and often results in inhibition of receptor function. We have found that the secreted apoA-I binding protein (AIBP) accelerates cholesterol removal, disrupts lipid rafts, prevents TLR4 dimerization and inhibits microglia inflammatory responses to LPS. Furthermore, because AIBP binds to TLR4 and its affinity increases when TLR4 is activated by an agonist, we propose that AIBP targets cholesterol removal to lipids rafts harboring activated TLR4. In our recent work, we have also found that this mechanism is relevant to regulation of neuropathic pain states. Intrathecal injections of recombinant AIBP prevented LPS-induced tactile allodynia and, remarkably, reversed established cisplatin-induced allodynia. Based on these findings, we propose that targeted, AIBP- mediated disruption of lipid rafts and its effects upon TLR4 signaling can be a potential therapeutic strategy in treating neuropathic pain states. The Specific Aims of this proposal are: (1) to test the hypothesis that AIBP targets lipid rafts harboring activated TLR4; (2) to test the hypothesis that AIBP reduces glial activation and neuroinflammation in mouse models of neuropathic pain; and (3) to identify the origin and function of endogenous AIBP in the spinal cord. To test these hypothesis, we propose experiments, in vivo and in isolated glial cells, to elucidate the AIBP-TLR4 binding and lipid raft mechanism, and to characterize glial activation and neuroinflammation states. We will also make AIBP variants with mutated apoA-I or TLR4 binding interfaces to validate the proposed mechanism. Using our unique AIBP knockout and AIBP flox/flox mice, we will identify the role of endogenous AIBP in lipid raft regulation and neuroinflammation. In summary, our proposed experiments will elucidate the mechanisms by which AIBP reduces neuroinflammation and alleviates neuropathic pain. Our studies may also suggest that raising AIBP levels in the CNS may be a novel therapeutic approach to treat persistent pain states.

Project Summary Peripheral nerve injury can lead to anomalous sensations referred to as neuropathic pain. Nerve injury may result from a variety of insults ranging from frank injury to the nerve though sectioning or compression, and is particularly prevalent following chemotherapies used in oncology. Symptoms often include continuous burning pain and abnormal sensory sensations such as allodynia (pain as a result of non-noxious stimuli) and hyperalgesia (an increased response to a normally painful stimulus) Persistent pain after resolution of clinically appreciable signs of injury poses a therapeutic challenge and current therapies do not meet this medical need. Accordingly, novel treatment options that afford additional benefit in prevention or relief of pain are needed. In this proposal, a collaboration initiated between the University of California San Diego (UCSD) and Epigen Biosciences Inc. seeks to fully evaluate the apoA-I binding protein (AIBP), an agent that interferes with inflamed lipid rafts in spinal glia and has been found to reverse facilitated pain states in several mouse models. The goals of this proposal during the R21 phase include the AIBP protein manufacture and characterization, pharmacokinetics studies, design and refinement of experimental animal protocols to assess efficacy and pharmacodynamics of AIBP in mouse models of polyneuropathy (induced by chemotherapeutic agents) and mononeuropathy (L5 nerve ligation), and optimization of pharmacodynamics assays to evaluate AIBP engagement of spinal glia and its anti-inflammatory effects. Contingent upon the successful completion of a set of proposed milestones, the R33 phase will commence to establish dose-dependent efficacy profile for AIBP treatment of neuropathic pain and to correlate it with AIBP pharmacokinetics and pharmacodynamics. The proposal is designed to advance the AIBP project to the point where it can meet the entry criteria for NINDS Cooperative Research to Enable and Advance Translational Enterprises (CREATE) program, with the ultimate goal to develop a novel biologic therapeutics for management of persistent pain states.