Frank Porreca - US grants

The influence of the spinal cord on gastrointestinal function has only recently been recognized. Drugs can influence the gut at 4 different levels: the neurons of the enteric nervous system, the smooth muscle of the gut, the brain and as only recently shown, the spinal cord. The understanding of the relative contributions of the brain and spinal cord to the central modulation of gastrointestinal function is potentially of major importance in clinical practice. Preliminary data have indicated that opioids that effectively alter gut motility at the level of the cord are not necessarily effective at the level of the brain. The aim of this proposal is to elucidate the opioid mechanisms involved in control of gut function at the level of the spinal cord and within the brain, and the relative importance of these central structures. The studies will be on the gastrointestinal pharmacology of selective opioid receptor agonists and specific receptor antagonists. The approach involves 3 tactics: (1) the concurrent administration of threshold and subthreshold doses at both the brain and cord levels in order to determine possible additive or multiplicative effects, i.e., site-site interaction; data from these experiments will be subjected to isobolographic analysis in order to reveal the nature of the brain-cord interaction; (2) administration of agonists and antagonists by the same, or different systemic, i.c.v. or i.t. routes; and (3) the use of tolerance and cross-tolerance techniques to functionally eliminate the participation of a particular site or receptor subclass from producing the effect. The endpoints chosen focus on gastroin-testinal transit in the mouse for the first half of the proposed funding period and shift to the study of gastric emptying, small and large intestinal transit and gastric and intestinal motility in the rat as the understanding of brain-cord interaction is achieved. Additionally, animal models will include normal and spinally-transected animals so that the neural and cerebrospinal fluid connections between the brain and cord can be interrupted. The presence of high concentrations of enkephalins within the brain, the spinal cord and the gut, suggests that an understanding of the relative contributions of the brain and spinal cord and the specific opioid receptor mechanisms involved in the opioid modulation of gut function will provide new insights into the CNS-gut interface.

The intimate sequence of events occurring during the formation of the drug-receptor complex are characterized by fundamental constants which include the dissociation constant (KA, KB, reciprocals of affinity for agonists and antagonists), and the thermodynamic parameters associated with the reaction, i.e., changes in Gibbs free energy reflected as changes in enthalpy and entropy. Agonists and antagonists may bind to receptors differently, with only agonists able to possibly induce a conformational change in the receptor; this change is assumed responsible for the information transfer which initiates events leading to a measureable effect. This possible agonist-induced change in conformation is reflected by the reaction thermodynamics, with agonist and antagonists having different characteristics. It is now accepted that opioids interact with at least three types of opioid receptor, the mu, delta and kappa. While the fundamental parameters of drug-receptor interaction have been studied for many classes of pharmacological agents (e.g. alpha & beta adrenergics, nicotinics, muscarinics, benzodiazepines) surprisingly few studies have been attempted with opioids. Selective opioid agonists and antagonists, together with a non-surmountable antagonist, with affinity for all receptors, have recently been developed; these novel tools now allow the determination of opioid constants. Additionally, opioid specific bioassays have been defined with only one type of receptor (i.e. rabbit vas deferens for the kappa receptor) or created through alkylation of non-relevant receptor leaving only one functional opioid receptor. This proposal suggests the determination of individual opioid receptor constants, and the thermodynamic parameters of opioid agonists and antagonists in multiple bioassays (guinea-pig ileum, mouse vas deferens, rabbit vas deferens, hamster vas deferens). The approach focuses on receptor constants at different temperatures. Such information allows calculation of thermodynamic parameters of agonist and antagonist binding. The specific questions which will be addressed include: (a) do opioid agonists and antagonists bind to the same type of opioid receptor in different tissues (i.e. are mu receptors in the GPI the same as mu receptors in the MVD); (b) are the three opioid receptors affected differently by temperature; (c) are there differences in opioid agonist and antagonist binding, and are peptide agonists and antagonists different from non-peptides; (d) how are the binding parameters affected by tolerance, or supersensitivity. Knowledge of these fundamental opioid parameters should be instrumental in the process of rational drug design, particularly for antagonists.

It is recently evident that the central nervous system (CNS) exerts important regulatory influences on gastrointestinal (GI) motility. CNS neuropeptides appear to be especially important in central regulation of motility. Certain of the centrally-distributed "brain-gut" neuropeptides administered exogenously have dramatic effects in the motility and reflexes of the GI tract. The specific sites or mechanisms through which these peptides affect the gut have not been determined. Neuropeptides can act at a discrete sites in affecting GI motor function: the brain the neurons of the enteric nervous system, the smooth muscle of the gut, and, as only very recently demonstrated, the spinal cord. It is the hypothesis of this proposal that the "brain-gut" peptides act through chemosensitive sites in the brain and spinal cord to serve as physiological or pharmacological modulators of GI motility. We propose to focus our studies on the central effects of mammalian bombesin agastrin releasing peptide; GRP), corticotropin releasing hormone (CRF) and certain opioid neuropeptides on (a) ongoing and (b) evoked reflex activity of the gut. Our approach will initially involve administration of neuropeptides into the brain and into the spinal subarachnoid space to define the chemosensitive sites fundamentally involved in the control of GI motility. The endpoints to be studied are all standard in our laboratory and include determination of gastric emptying and small and large bowel transit, stomach and intestinal intraluminal pressure in the rat as will as GI transit in the mouse. In addition, a recently developed model proposed for study is the evoked gastrocolic reflex in the rat. The major techniques that will be employed to dissect the peptidergic pathways that control the gut include: (a) microinjections into specific brain locations and cord levels and (b) transection of the spinal cord at various levels so that the influence of the sympathetic or parasympathetic gut innervation can be determined. Our recent work provides evidence that peptide sensitive sites in the spinal cord can be distinguished anatomically, pharmacologically and functionally from those in the brain. Knowledge of the sites and functions of the neuronal "brain-gut" peptides will provide new information about central nervous system-gut interactions.

This program project grant requests continuation of funding for the development of novel, non-addicting analgesics which cross the blood brain barrier. Our original goals remain unchanged. Our approach has been, and continues to be, emphasis on therapeutic applications of opioid delta (delta) receptor pharmacology. Progress has been rapid and rewarding. At the time of the original application, our understanding of opioid delta receptors and the therapeutic potential of compounds acting at these sites was limited to two concepts - (a) that compounds with selectivity for opioid delta receptors produced antinociception with reduced addiction liability, and (b) that compounds which acted at opioid delta receptors could positively or negatively modulate the antinociceptive actions of opioid mu (mu) agonists. During the initial funding period (i.e., approximately two years at the time of submission of this application) we have identified three potentially novel mechanisms of action for compounds with opioid delta receptor activity. Exploration of these mechanisms in addition to the concomitant evaluation of addiction liability and other side effects of the novel compounds which act by these mechanisms, is the main Specific Aim the aim of this application. These three novel mechanisms may offer significant opportunities for the development of therapeutic agents which will have limited, or no, physical dependence liability. First, we have demonstrated pharmacologically that subtypes of opioid delta receptors exist, and that these can be studied by novel agonists and antagonists. We have classified these opioid delta receptors as the delta1 receptor (DPDPE/DALCE-sensitive), and the delta2 receptor ([D-Ala2]deltorphin II/5'-NTII); the pharmacology of these delta receptor subtypes is one of the aims of this proposal. Second, a series of compounds with a novel, "self-potentiating" mechanism has been identified which may involve actions at a putative opioid mu-delta receptor complex; one of these peptides readily produces antinociception after i.p. administration and produces significantly lower levels of physical dependence when compared to morphine. Third, we have discovered a lead compound (SNF 9007) which shares high affinity at opioid delta receptors and at the CCKB receptor, offering an opportunity to explore the long- established link between opioid delta antinociception and the CCK system. with the continued discovery of novel compounds from the chemistry section of this project, each of these approaches will represent progress towards new modalities for pain relieving substances which will be clinically important.

The specific goals of the proposed work are to (a) directly identify opioid delta receptor subtypes in brain and other tissues using radioligand binding techniques in wholebrain control membranes, in membranes pretreated with selective and irreversible ligands, in membranes from different strains of mice which have been reported to show differences in populations of opioid receptors (e.g., the mu-receptor deficient CXBK mouse), and in membranes from specific brain regions; (b) investigate possible differences in the regulation of interactions of the receptor subtypes with their respective effector systems by evaluation of radioligand binding in the presence of guanine nucleotides, cations (or both), or following pretreatment with pertussis toxin in vitro or in vivo; (c) use radioligand binding techniques to evaluate possible mechanism of tolerance and supersensitivity following repeated/continuous exposure of the brain to compounds with selectivity for the opioid delta receptor subtypes, and (d) use some of the above approaches in an effector system by measuring adenylyl cyclase activity in NG 108-15 cells. Such approaches will provide insights into opioid delta subtypes, and their mechanisms which may correlate with substantial data accumulated in vivo, and may be important in establishing the potential therapeutic utility of delta subtype selective drugs.

The objectives of the Biological Evaluation Core are to provide fundamental information on the nature of each of the new compounds synthesized. The procedure will allow for the identification of fundamental characteristics of each compound in the most efficient manner. Information which will be obtained for each compound includes the affinity of the compound at opioid eta, delta, kappa receptors in mouse brain homogenate. The affinity of these analogues will be measured using standard competition assays against reference radiolabelled ligands at each of these receptors types. The data from the binding assays will allow for the measurement of receptor selectivity in binding. The activity of these compounds will be determined using standard isolated bioassay tissues, including the guinea-pig isolated ileum (GPI, eta bioassay) and the mouse isolated vas deferens (MVD, delta bioassay). These assay tissues will be used to assess determined using dose-response curves in each of the bioassay tissues- these data will allow for the estimate of receptor selectivity (GPI/MVD ratio). If the compound is not active as an agonist, it will be tested in vitro as an antagonist against submaximal doses of reference compounds which have already been characterized in these assays. This will provide a complete prolife in vitro of each compound tested. Additionally, the agonist analogues will be screened following i.c.v. administration to mice in vivo using standard antinocipeptive tests such as the fail-flick test. If the analogue is an agonist in the bioassay, but does not procure antinociception in the tail-flick test following i.c.v. administration. Although dose-response studies will be emphasize for the central (i.e.,i.c.v or i.th) routes, only single doses will be used as a screen for systemic studies in the Biological Evaluation Core in order to reduce the quantity of analogue initially synthesized. Dose-response studies by systemic routes will be performed for interesting analogues in the individual projects. The data obtained in the Biological Evaluation Core will be essential for the appropriate design of new molecules by the Chemistry group and will allow the PI's to choose interesting molecules for further study in their individual assays.

The specific goals of the proposed work are to (a) directly identify opioid delta receptor subtypes in brain and other tissues using radioligand binding techniques in wholebrain control membranes, in membranes pretreated with selective and irreversible ligands, in membranes from different strains of mice which have been reported to show differences in populations of opioid receptors (e.g., the mu-receptor deficient CXBK mouse), and in membranes from specific brain regions; (b) investigate possible differences in the regulation of interactions of the receptor subtypes with their respective effector systems by evaluation of radioligand binding in the presence of guanine nucleotides, cations (or both), or following pretreatment with pertussis toxin in vitro or in vivo; (c) use radioligand binding techniques to evaluate possible mechanism of tolerance and supersensitivity following repeated/continuous exposure of the brain to compounds with selectivity for the opioid delta receptor subtypes, and (d) use some of the above approaches in an effector system by measuring adenylyl cyclase activity in NG 108-15 cells. Such approaches will provide insights into opioid delta subtypes, and their mechanisms which may correlate with substantial data accumulated in vivo, and may be important in establishing the potential therapeutic utility of delta subtype selective drugs.

This Project application is a broad collarative effort on the part of seven investigator in four institutions to study the hypothesis that opioid delta (delta) receptor subtype-selective agonist, partial agonist and antagonists may be feasible candidates for development as therapeutic agents related to the treatment of pain and addiction. These investigator bring expertise ranging from extensive experience in synthetic chemistry and molecular modeling, in pharmacological analysis of molecules in vitro and in vivo, in application of molecular techniques, to evaluation of behavior in several species including primates. Emphasis in this effort will be two-fold. First, the investigator will attempt to discover, and evaluate in several species, highly receptor subtype-selective delta ligands through a synthetic/pharmacological collaboration in order to test the underlying hypothesis. Second, the novel ligands, and other available pharmacological tools, will be employed to explore in greater detail the pharmacology and physiology of subtypes of opioid delta receptors. Strong collaborative interaction already exists and will be emphasized. The specific aims are to: (a) synthesize novel, non-peptidic opioid delta subtype-selective ligands using state-of-the-art synthetic, analytical and modeling technology with emphasis on development of a structure- activity relationship for delta subtypes, creation of affinity ligands for characterization and localization of the receptors, and radio ligands for PET and SPECT applications; (b) to synthesize sufficient quantities of these compounds for the biological projects; (c) to evaluate the novel non-peptidic molecules for their receptors selectively in vitro and to characterize them in the Biological Evaluation Core; (d) to evaluate the pharmacology of selective novel non-peptidic agonist using approaches in vivo in antinociceptive, gastrointestinal and behavioral endpoints in mice, rats, pigeons and monkeys; (e) to determine the affinity and activity of the new compounds at the cloned opioid delta receptor(s) following transfection of the receptors to cell lines, with particular emphasis on the human delta receptor(s); (f) to determine the second messenger systems involved in the mechanisms of action of compounds acting selectively at delta receptor subtypes in the naive and opioid- exposed tissue; (g) to evaluate the pharmacology of the selective novel non-peptidic antagonists following repeated exposure alone or in combination with opioids or cocaine using place pairing and self- administration paradigms; (h) to perform detailed pharmacological studied of the possible development of physical dependence via the delta receptor; and (i) to collaborate with outside consultants to obtained any data necessary to assess the central hypothesis of potential clinical importance of opioid delta ligands. It is expected that this multidisciplinary effort will result (a) in a realistic assessment of the therapeutic potential of opioid delta receptors subtype-selective ligands, (b) in the discovery of molecules with potential for development as therapeutically useful substances, and (c) in the advancement of our understanding of the role of opioid delta receptors and their subtypes in normal and pathological physiology.

Recent evidence has provided support for the view that multiple opioid delta receptors exists in the brain, in the spinal cord and in the periphery. While the identification of subtypes of opioid delta receptors provides an opportunity to explore further the physiological and pharmacological importance of the delta receptor system, progress towards this end has been hampered by the lack of highly selective, and systemically active, delta receptor agonist, partial agonist and antagonists. This portion of the Program Project application will focus on two main objectives: first, a detailed pharmacological analysis of novel delta subtype selective ligands synthesized by the chemistry section and identified in the Biological Analysis Core will be carried out. This analysis will focus on the identification of receptor subtype selectivity using methods in vitro and in vivo for analogues deemed interesting from the result of the Core. A second goal of this section of the Program Project is to explore the pharmacology of the delta receptor system in naive and delta-agonist or antagonist exposed animal, tissues and cells. A particular goal of this effort is to established whether occupation of opioid delta receptors by agonist, partial agonist, or antagonist will prevent the development of expression of the opiate abstinence syndrome. Further, studies will be carried out to determine whether a delta receptor abstinence syndrome can be demonstrated in rodents and if so, its characteristics and relationship to one or more of the delta receptor subtypes. The objective of these studies is to continue to add to our fundamental understanding of the opioid delta receptor, and its subtypes. The results of both of these efforts will be closely tied to the synthetic effort, not only in evaluating molecules already synthesized, but also in order to further aid the synthesis of new highly delta-subtype selective (i.e., 1,000-fold) agonist and antagonists. The information gained in these objectives will help in testing the hypothesis that opioid delta receptor agonist, partial agonist or antagonists will eventually have therapeutic utility in management of pain and addiction.

DESCRIPTION (Applicant's Abstract) One of the most significant health problems in our country relates directly to the consequences of pain. Estimates suggest that as many as 30 million Americans suffer from chronic pain which is resistant to medical treatment. One of the chronic abnormal pain states which is extremely difficult to treat is commonly referred to as "neuropathic pain." Our understanding of this condition is limited, but has been increased in recent years by development of animal models in which injuries are made to peripheral nerves to produce signs which reflect some aspects of human neuropathic pain states. A striking result of these injuries is a substantial increase in the expression of spinal dynorphin. It is known that dynorphin may rapidly be degraded to des-Tyr fragments which do not interact with opioid receptors and that dynorphin can produce a significant number of effects which are non-opioid in nature. This proposal is aimed at testing the hypothesis that the spinal dynorphin is intimately involved in either the initiation or maintenance of nerve-injury related pain through non-opioid mechanisms. This hypothesis will be systematically tested by (a) measurement of whether and when dynorphin expression occurs in the spinal cord following specific nerve injuries; (b) determination of the functional consequences of spinal dynorphin and its role in maintenance of the post-injury state; (c)determination of the consequences of preemptive inhibition of dynorphin activity to evaluate its role in initiation of the nerve-injury consequences; (d) identification of a mechanism by which dynorphin may produce such pathological actions via interaction with a novel binding site on the NMDA receptor complex; and (e) direct evaluation of the effects of dynorphin on the viability of neurons. The present studies should reveal much about the role of dynorphin in post-injury processes in the spinal cord. It is expected that treatments that prevent the expression of spinal dynorphin or that block the actions of pathological levels of dynorphin at a specific site on the NMDA receptor complex should provide additional therapeutic benefit in the treatment of neuropathic pain.

One of the most significant health problems in our country is the inadequate treatment of chronic abnormal pains such as those associated with nerve injury or pathology (neuropathic pain). Inextricably linked to such chronic pains are the difficulties associated with the use of opioids for prolonged periods, where tolerance limits effectiveness. An unexplained clinical and experimental paradox is that the use of spinal opioids for chronic pain results not only in analgesic tolerance, but also in the development of abnormal pain (i.e., hyperesthesias including hyperalgesia and allodynia). Opioid tolerance and the post-nerve injury state have been suggested to be (mechanistically similar, at least at the spinal level. Blockade of the NMDA receptor has been shown to both prevent the development of, and reverse established, opioid tolerance as well as many of the behavioral consequences of experimental neuropathic pain. While such studies underscore the importance of the NMDA receptor in these processes, these pharmacological investigations have not identified the underlying endogenous spinal mechanisms which may promote opioid tolerance and the consequences of nerve injury through direct or indirect actions at the NMDA receptor. Like others, we have noted many similarities between the post-nerve injury state and spinal opioid tolerance and have discovered a multisegmental elevation of spinal dynorphin in both conditions. In both situations, blockade of the actions of elevated spinal dynorphin with antiserum to the peptide reestablishes opioid antinociceptive potency and efficacy and blocks abnormal pain. These observations lead us to hypothesize that (a) spinal opioid antinociceptive tolerance and the abnormal pain seen following spinal opioid administration are due, at least in part, to the non-opioid actions of elevated (i.e., pathological) levels of spinal dynorphin, and (b) prevention of dynorphin action, or expression, will reverse or prevent opioid tolerance and spinal opioid associated pain. This hypothesis will be tested by (i) evaluating intrathecal (i.th.) opioid agonist or antagonist- induced regulation of spinal dynorphin, (ii) pharmacological elevation of spinal dynorphin with i.th. infusion of dynorphin or the non-opioid peptide dynorphin(2-17), and (iii) prevention of the actions, or expression of, elevated levels of spinal dynorphin elicited by i.th. opioid infusion using antiserum to the peptide or antisense oligodeoxynucleotides to prodynorphin. It is hypothesized that enhancing spinal dynorphin levels either physiologically, or pharmacologically, will result in features common to opioid tolerance and to peripheral nerve injury (i.e., reduced antinociceptive actions of spinal opioids and abnormal response to sensory input including hyperalgesia and tactile allodynia), while blockade of endogenous dynorphin action or prevention of dynorphin expression will reverse or prevent opioid tolerance and related pain. Such experiments will establish whether the action or expression of dynorphin is critical for the development or maintenance of opioid antinociceptive tolerance and the abnormal pain produced by sustained spinal administration of opioids. The data from these studies will provide new insight on the possibility that dynorphin may be a crucial link in promoting opioid tolerance, reveal the regulation of spinal dynorphin expression by opioids, and provide a mechanistic basis for the paradoxical pain seen following spinal opioids in animals and in humans.

This Program Project application represents a comprehensive collaborative effort by six senior investigators in two Departments of the University of Arizona to carry out a systematic investigation for the development of highly opioid receptor selective and efficacious peptide ligands that will provide nonaddictive opioid analgesics, and new modalities for the treatment of pain, drug dependence and opioid withdrawal. We emphasize studies that will provide new insights for the design of peptide, and peptide conjugate structures that will lead to new modalities for treatment of pain and drug abuse. Consortium and local arrangements have been made that will provide the preclinical data that for the first time will lead to human clinical trials for delta agonists. A highly cooperative multi-disciplinary approach involving computer aided drug design, biophysics, asymmetric synthesis, macrocyclic synthesis, binding studies, in vitro and in vivo assays, and examination of second messenger andother aspects of signal transduction has been established. Strong collaborative interactions will be emphasized with the following major specific aims: 1) to further develop a systematic approach to computer aided design of delta opioid receptor potent and selective peptide ligands that have unique profiles of biological activity, are biostable, can penetrate through the blood-brain barrier (BBB), and are highly efficacious agonists with analgesic activity; 2) prodrug approaches, and carrier mediated mechanisms, which favor passage of peptides and peptide conjugates through the BBB will be examined; 3) to utilize site specific mutagenesis, chimeric structures, second messenger studies, etc. in conjunction with modeling of the human clone delta and other opioid receptors to provide new understanding of the structural basis for agonist and antigonist activity of opioid ligands and the mechanisms of action of opioids; 4) to utilize state of the art pharmacological studies including, antisense deoxyribonucleotides, models of nonciception, and physical dependence including self-administration to obtain new insights into the mechanisms of action of biphalin, [Phe6]DPDPE and other ligands that have unique biological activity profiles including unprecedented efficacy enhancement and minimal side effects; 5) to perform the necessary preclinical toxicity studies needed to eventually file an investigator sponsored investigational New Drug Application (INDA) for human trials for DPDPE and biphalin; 6) to comprehensively examine peptide analogues stability, chemical-physical properties, distribution, bioconversion, and pharmacokinetics in relation to their ability to cross the BBB; 7) to utilize core facilities to prepare quantities of compounds needed for in vitro and in vivo bioassays to obtain more potent, efficacious peptides that can cross the BBB; 8) to develop an understanding of the molecular, dynamic and conformational properties of peptides, and peptide conjugate ligands thatmaximize penetration of the BBB.

DESCRIPTION:(from applicant's abstract): One of the most significant health problems in our country is the inadequate treatment of pain, especially the chronic abnormal pains often associated with nerve injury (neuropathic pain). Injuries to nerves in experimental models of neuropathic pain elicit peripheral, spinal and supraspinal neural plasticity characterized in part by increased expression of spinal dynorphin. Progress made in our previous funding period suggests that enhanced expression of dynorphin resulting from experimental nerve injury acts in a non-opioid fashion to promote pain. Some of the relevant observations which support a pronociceptive role of dynorphin in the post-nerve injury state include: (a) spatial and temporal correlation of increased spinal dynorphin expression across multiple, anatomically relevant spinal segments following nerve injury, (b) inhibition of nerve-injury induced pain by antiserum to dynorphin, but not control serum, and (c) demonstration of sustained nerve-injury induced pain in wild-type (WT), but not in prodynorphin "knock-out" (KO) mice. Our studies show that dynorphin (2-17) (which does not bind to opioid receptors) promotes calcium accumulation in DRG cells in culture and activates protein kinase C (PKC) in spinal cord. Moreover, des-Tyr fragments of dynorphin enhance capsaicin-stimulated CGRP release in DRG cells and in spinal cord tissue preparations. The overall hypothesis of our work is that spinal dynorphin is pronociceptive and it a critical mediator which serves to maintain pathological post-nerve injury pain. Specifically we propose a mechanism by which dynorphin maintains post-nerve injury pain by increasing spinal excitability, in part, by enhancing the release of excitatory transmitters. Our aims are designed to systematically test this hypothesized mechanism. Aim 1 will determine whether nerve-injury induced changes in dynorphin expression are the result of modulatory influences from supraspinal sites. Aim 2 will focus on measurement of nerve-injury induced release of dynorphin at the spinal level. Aim 3 will use cultured DRG cells to test if dynorphin enhances evoked CGRP release through activation of PKC. Aim 4 will use spinal cord tissue preparations taken from sham- or nerve-injured rats, as well as from sham- or nerve-injured WT and prodynorphin KO mice to determine the role of endogenous, pathological levels of dynorphin on basal and evoked CGRP release. Aim 5 will focus on the role of dynorphin in the release of excitatory amino acids in sham-operated and nerve-injured rats. The experiments in Aim 5 will be carried out in the laboratory of Dr. Tony Yaksh through a Consortium Arrangement. All of these studies focus on the central hypothesis of pronociceptive actions of dynorphin in the post nerve-injury state and test a proposed mechanism which addresses how dynorphin may act under conditions of nerve-injury to maintain pain. As most clinical conditions of neuropathic pain are treated in the post-injury (i.e., maintenance) state, such information may allow approaches to limit or reverse the pathological actions of dynorphin in maintaining neuropathic pain.

DESCRIPTION (provided by applicant): Substantial evidence supports the possibility that different neural mechanisms may underlie nerve injury- induced tactile and thermal hypersensitivity. Our recent preliminary data, for example, show that nerve-injury induced thermal hypersensitivity resolves at approximately 45 days post-injury, while tactile hypersensitivity persists apparently indefinitely (i.e., for at least more than 200 days). Lesions of the dorsal columns (DC) or microinjection of lidocaine into the n. gracilis on the side ipsilateral to the nerve-injury block nerve-injury induced tactile, but not thermal, hypersensitivity, supporting the possibility that tactile hyperresponsiveness may be mediated by large, myelinated fibers. In the absence of nerve injury, cells in the dorsal root ganglion (DRG) and in the n. gracilis either do not express neuropeptide Y (NPY), or express the peptide at very low levels. Following nerve injury, however, NPY -ir is markedly upregulated particularly in medium and large diameter DRG cells. Increased NPY -ir is also seen in the n. gracilis, as well as in the spinal dorsal horn. We propose testing the hypothesis that sustained nerve-injury induced tactile hypersensitivity results from the actions of upregulated NPY in afferent fibers which project to n. gracilis. Our preliminary data show that (a) nerve-injury induced upregulation of NPY -ir occurs in DRG cells and in projections to the ipsilateral n. gracilis through the DC, (b) NPY microinjected into the n. gracilis of uninjured animals produces ipsilateral tactile, but not thermal, hypersensitivity, (c) anti-NPY antiserum given into the n. gracilis ipsilateral to the side of nerve-injury reverses tactile, but not thermal, hypersensitivity and (d) microinjection of either of two NPY receptor antagonists into the n. gracilis ipsilateral to the side of nerve injury reverses tactile, but not thermal, hypersensitivity. Our hypothesis will be tested in Aim I by characterizing the time-course of NPY expression and determining if such expression and activity is consistent with early and/or late aspects of tactile hypersensitivity. Aim 2 will evaluate whether blockade of DC pathways or NPY expression can prevent the development of, or reverse existing, experimental neuropathic pain. Aim 3 will characterize the expression of NPY receptors in the DRG and in n. gracilis before and after nerve injury. Aim 4 will determine the possible contribution of post-synaptic dorsal column cells which project to n. gracilis to nerve injury- induced tactile hypersensitivity by determining if they receive inputs from NPY expressing fibers, and if they express NPY -ir and/or NPY receptors. Understanding the neural mechanisms underlying tactile hypersensitivity is highly significant as tactile allodynia in humans is the most devastating and difficult to manage symptom of clinical neuropathic pain. Selective reversal of injury-induced tactile hypersensitivity by NPY antagonists would have significant implications for treatment of the neuropathic condition.

DESCRIPTION (provided by applicant): An unexplained clinical paradox is that opioids can produce unexpected abnormal pain (i.e., hyperesthesias including hyperalgesia and allodynia) during their use for pain relief. Opioid-induced hyperalgesia (i.e., increased sensitivity to noxious or non-noxious sensory stimuli) is also reliably demonstrated in animals while opioids are continuously delivered suggesting that this phenomenon is not the result of opioid withdrawal. In our previous funding period, we demonstrated that continuous opioid administration to animals over a period of several days elicits remarkable, pronociceptive neuroplastic adaptations in both the peripheral and central nervous systems, which are likely to underlie the observed hyperalgesia. In spite of the potential clinical significance of such opiate-induced changes in the nervous system (i.e., the possibility that these may produce deleterious effects in patients), the mechanisms of opiate-induced hyperalgesia remain unknown. Hyperalgesia resulting from injury are characterized by a state of "central sensitization" which results from repetitive inputs to the central nervous system from injury-induced enhanced discharge of afferent fibers. Opioid-induced hyperalgesic states also might be characterized by a state of "central sensitization" or an analogous state of "sensitization" in the spinal dorsal horn. The hyperalgesia associated with opiates, however, develops in the absence of an injury with no known basis for sustained afferent discharge to the spinal cord. Opioid-induced hyperalgesia may well share some common mechanisms with injury-induced hyperalgesic states but could equally have other unique sustaining mechanisms. This proposal is designed to explore the hypothesis that opioids induce a state of "spinal sensitization" which may occur as consequence of (1) activation of descending pain facilitation mechanisms arising in the rostral ventromedial medulla (RVM) through enhanced release of RVM CCK;(2)upregulation of spinal dynorphin as a consequence of descending facilitation;and (3) the dynorphin-dependent increase in basal and/or evoked spinal PGE2 release which acts to sensitize primary afferent fibers to peripheral stimuli. This hypothesis will be tested, using behavioral, neurochemical and electrophysiological approaches. Given the prevalent reliance of our society on opiates for treatment of pain, understanding of the fundamental biological mechanisms associated with exposure to these drugs is essential in developing approaches that prevent the neurobiological changes which may promote pain.

DESCRIPTION (provided by applicant): Opiate-induced hyperalgesia has been reported in humans and in animals. Continuous opiate administration for several days produces pronociceptive neuroplastic adaptations in both the peripheral and central nervous systems which likely underlie the observed hypersensitivity. Despite the potential clinical significance of such changes, specific mechanisms of opiate- induced hypersensitivity are unknown. Injury to tissues can result in }sensitization} of nociceptors, resulting in enhanced response to noxious and normally non-noxious stimuli (i.e., hyperalgesia and allodynia, respectively). We hypothesize that opiate-induced hyperalgesia and allodynia may result from sensitization of nociceptors. Importantly, we hypothesize that sensitization of nociceptors by opiates can occur in the absence of tissue injury. Two specific questions are addressed by the experiments proposed in this application: 1) can opiates induce nociceptor sensitization without tissue injury? 2) is opiate-induced nociceptor sensitization the result, in part, of an NGF-dependent process? Behavioral, neurochemical, immunohistochemical and electrophysiological studies will test the hypothesis that opiates (a) act at opiate receptors to produce hypersensitivity and an increase in expression of NGF in peripheral tissues; (b) increase NGF-dependent phosphorylation of p38 MAPK (pp38 MAPK) in TrkA-positive cells, (c) increase NGF-dependent and pp38 MAPK-dependent trafficking of the TRPV1 channel to the periphery, (d) upregulate CGRP and substance P (SP) expression in TrkA-positive cells in an NGF-dependent, and pp38 MAPK-dependent fashion, and (e) produce NGF-, pp38 MAPK- and TRPV1-dependent hypersensitivity. PUBLIC HEALTH RELEVANCE: The consequences of opiate-induced neuroplasticity raise questions of whether unintended harm to patients might actually occur. Given the prevalent reliance on opiates for treatment of severe pain, understanding of the fundamental biological mechanisms associated with prolonged exposure to these drugs is essential. Additionally, mechanisms underlying possible nociceptor sensitization occurring in the absence of tissue injury may ultimately lead to insights into clinical conditions of prominent pain without apparent tissue injury including, for example fibromyalgia, IBS, CRPS-1 and perhaps migraine. The consequences of opiate-induced neuroplasticity raise questions of whether unintended harm to patients might actually occur. Given the prevalent reliance on opiates for treatment of severe pain, understanding of the fundamental biological mechanisms associated with prolonged exposure to these drugs is essential. Additionally, mechanisms underlying possible nociceptor sensitization occurring in the absence of tissue injury may ultimately lead to insights into clinical conditions of prominent pain without apparent tissue injury including, for example fibromyalgia, IBS, CRPS-1 and perhaps migraine.

The positive reinforcement provided by opiates have been considered as primary factors in promoting drug abuse and dependence. However, opiates also produce a host of "negative" effects which can manifest both during opiate administration and which are particularly expressed during withdrawal. Withdrawal from opiates is unpleasant and may provide negative reinforcement and contribute to drug dependence. Thus, both positive and negative reinforcement are likely to contribute to the compulsive use of opioids, a critical feature of "opioid addiction". The mechanisms by which opiates produce negative effects are unknown. Our preliminary data indicate that blockade of descending facilitation in the rostral ventromedial medulla (RVM) prevents expression of somatic and autonomic signs of opiate withdrawal indicating that facilitatory outflow from the RVM is critical in mediating both nociceptive (i.e., opiate-induced hyperalgesia) and many nonnociceptive features of the withdrawal syndrome. We hypothesize that opiate-induced descending facilitation from the RVM and subsequent upregulation of spinal dynorphin are essential for the expression of many of the negative symptoms which comprise withdrawal from opiates. Mechanisms which underlie the expression of the withdrawal syndrome thus also represent mechanisms likely promote negative reinforcement which may contribute to opiate dependence, the continued use and abuse of opiates and drug seeking behavior. This hypothesis will be explored with four Specific Aims: First, we will characterize pronociceptive transmitters in the RVM in which can mediate naloxone-precipitated withdrawal. Second, we will determine whether prevention of descending facilitation from the RVM prevents opiate-induced physical dependence (naloxone-induced or spontaneous withdrawal). Third, we will determine whether blockade of the pronociceptive actions of spinal dynorphin, or the bradykinin B2 receptor will prevent withdrawal. Fourth, we will determine if blockade of descending facilitation blocks withdrawal-induced aversion/negative reinforcement. These studies assess mechanisms which may contribute to negative reinforcement, drug dependence and abuse.

DESCRIPTION (provided by applicant): Current options for treatment of neuropathic pain remain unsatisfactory in part because of an inadequate understanding of mechanisms of this abnormal pain condition. Experimental neuropathic pain is characterized by the presence of enhanced behavioral responses to noxious or normally non-noxious sensory stimuli (i.e., allodynia). The presence of such enhanced responses is generally accepted as a translational and validating feature of the experimental pain condition and modulation of enhanced sensory thresholds is commonly used to explore mechanisms relevant to potential therapy. Studies in humans, however, have shown that changes in evoked thresholds (i.e., allodynia) frequently do not correlate with reductions in pain scores. Rather, it is the spontaneous aspects of the human pain experience that lead patients to seek treatment for their neuropathic pain. Thus, a critical shortcoming with regard to experimental evaluation of neuropathic pain is the reliability and predictive value of studies involving modulation of evoked reflexive responses to sensory stimuli and subsequent interpretation of mechanism. Experimental measurement of spontaneous pain following peripheral nerve injury has been difficult. Whether mechanisms which mediate spontaneous neuropathic pain may be distinct from those mediating enhanced evoked responses following injury is not known. We have recently demonstrated that microinjection of lidocaine into an area of the brain that mediates descending modulation of pain (i.e., the rostral ventromedial medulla or RVM) produces preference in a conditioned place pairing (CPP) paradigm in nerve injured, but not in sham-operated, rats. Additionally, we have shown that spinal administration of drugs that are known to produce relief of neuropathic pain clinically (i.e., clonidine, -conotoxin), will produce place preference only in nerve injured rats. The demonstration of place preference in nerve-injured, but not sham-operated, rats following administration of drugs which are known to activate reward pathways and in areas of the nervous system (i.e., brainstem and spinal cord) which are not a part of the reward pathway suggests the presence and modulation of spontaneous neuropathic pain. While nerve injury-induced evoked hypersensitivity (i.e., allodynia/hyperalgesia) and spontaneous neuropathic pain are likely to involve some common mechanisms, we hypothesize that evoked and spontaneous neuropathic pain can also be distinguished mechanistically (as shown by our preliminary data). The experiments proposed in this application will explore the mechanisms mediating spontaneous neuropathic pain by determining the role of (a) specific pronociceptive transmitters from primary afferent fibers innervating the spinal cord or brainstem nuclei (Aim 1), (b) subtypes of sodium channels which important in ectopic discharge (Aim 2) and (c) mediators of the descending pain modulatory pathway from the RVM and at the level of the spinal cord. At present, almost no information is known about mechanisms of human spontaneous pain beyond the limited information gained from the activity of currently employed medications that have complex pharmacology. This proposal seeks new insights into potential mechanisms of one of the most important symptoms of the human neuropathic state. Discoveries related to specific mechanisms of spontaneous pain will increase opportunities for clinical translation.

DESCRIPTION (provided by applicant): Migraine, perhaps the most common neurological condition, is a debilitating disorder characterized by episodic pain and other symptoms (i.e., nausea, vomiting, photophobia or phonophobia with or without aura). While the pathophysiology of this disorder remains unknown, an important theory suggests that migraine may occur as a consequence of cortical spreading depression (CSD). Migraine, and perhaps CSD events, may be precipitated by a variety of triggers including stress and provocative chemical agents such as donors of nitric oxide (NO). Migraine is often treated with triptan drugs which are effective in some patients but which are also associated with an increase in the incidence of headache frequency, i.e., medication overuse headache. We reasoned that transformation of episodic headaches into MOH, and perhaps CM, by triptans may result, in part, from medication-induced neural adaptations that enhance responses to stimuli that can trigger migraine attacks (i.e., migraine triggers). For this reason, we hypothesize that a period of triptan exposure results in persistent hypersensitivity to migraine triggers that are associated with increased cortical spreading depression (CSD) events and activation of primary afferent fibers that innervate the dura. Three specific aims test this hypothesis. First, are enhanced behavioral and neurochemical responses observed in rats pre-exposed to a period of triptan treatment accompanied by spontaneous (i.e., non-evoked) or increased CSD events following an evoking stimulus? (Aim 1); second, do the persistent pronociceptive changes elicited in dural primary afferent neurons by triptans result in subsequent activation and potentially increased excitability of dural afferent neurons in response to stimuli known to promote migraine attack in humans or after a CSD event? (Aim 2); and third, what are the roles of nNOS and CGRP in promoting enhanced sensitivity to migraine triggers, and/or CSD events and excitation of dural afferents? (Aim 3). A significant problem in preclinical efforts to study migraine mechanisms and pain is that animal models of headache generally require induction of tissue injury whereas migraineurs suffer from pain in the absence of injury (i.e., dysfunctional pain). Our approach mimics the human state by inducing neural adaptations using medications without causing tissue injury. Our goal is to use the triptan-induced sensitized state to investigate possible mechanisms underlying MOH and by which episodic pain may transform into CM. Critically, the events that may promote enhanced pain signaling following challenge of rats with latent sensitization with migraine triggers are also likely to yield insights into mechanisms relevant to pathophysiological states of underlying migraine. The data from the proposed experiments will result in mechanistic insights that can be tested clinically.

DESCRIPTION (provided by applicant): The University of Arizona (UA) High School Student NeuroResearch Program (HSNRP) will introduce, train, and nurture a growing cadre of diverse talented Arizona high school students including under-represented minorities in basic, translational, and clinical research on the normal and abnormal nervous system, neurological disorders, and stroke as well as encourage pursuit of advanced research experiences and health/ science-related careers. We will leverage the strong infrastructure, effective recruitment strategies, high level of student/faculty mentor participation, esprit-de-corps, and outstanding trainee productivity of our long-standing federally funded multidisciplinary/multispecialty disadvantaged high school student and medical student summer research programs to develop the training model for this new specialized NeuroResearch (NR) program. Twelve to 14 full-time summer high school trainees annually for the next 5 years will be offered an expanding menu of closely mentored NeuroResearch(NR) experiences; 2-4 will be reappointed as under- graduates for advanced NR. These will be integrated into an innovative inquiry-based Summer Institute on Medical Ignorance (SIMI) interweaving biomedical Knowns and Unknowns (what we know we don't know, don't know we don't know, and think we know but don't) with featured NR topics and sustained by periodic enrichment activities year round. SIMI emphasizes translating translation and includes a high school level brief Introduction to Pathobiology, topical seminars, laboratory/leadership/multimedia skill workshops, clinical correlations, social networking, and career advising. A unique Virtual Clinical Research Center/Questionarium forms a centerpiece for training and national/international networking. Within basic and clinical departments and specialized Centers of Excellence and overseen by an energetic HSNRP Leadership Team and Advisory Committee, high school student research will encompass cross-cutting themes and in vivo, in vitro, in situ, in silico, and modeling approaches to neurobiology/disorders including Parkinson, Alzheimer, Niemann-Pick C diseases, ALS, epilepsy, HIV encephalopathy, head trauma, hydrocephalus, muscular dystrophy, pain/ addiction pharmacology, molecular psychiatry, cognition, brain development, senescence, mental retardation, blood-brain barrier/neuroprotection, neuroimaging, neuro-genomics/proteomics, neuroengineering, deep brain stimulation, brain cancer, cerebrovascular disease, stroke, and rehabilitation. Based on our ~25-year track record and established access to large diverse pools of disadvantaged Arizona students reflected in 474 SIMI- trained high school students followed to date with many in basic/clinical NeuroResearch, we expect HSNRP to: cultivate an expanding network of diverse researchers, physicians and other health professionals; contribute to the NeuroResearch pipeline and enterprise; and improve neurohealth literacy through community engagement. Ongoing short-term and long-term evaluation including surveys, database registry and career portfolios will document efficacy of the training model and promote networking.

DESCRIPTION (provided by applicant): Pain is a subjective, multi-dimensional experience with sensory, affective and cognitive components. The affective (aversive) dimensions of pain are the main complaint of patients. We have attempted to capture and mechanistically evaluate affective dimensions of ongoing pain in preclinical settings. The aversiveness of pain provides strong motivational drive to seek relief. Thus, treatments known to relieve pain clinically produce conditioned place preference selectively in injured animals, inferring activation of brain reward pathways. This possibility is consistent with human data that relief of pain is rewarding. In this application, we ask whether, and how, relief of ongoing pain might modulate brain reward circuits. Additionally, we explore whether manipulations that selectively target affective, rather than sensory, components of pain also activate reward circuits. We will use a rat model of time-dependent incisional pain to explore the potential activation of reward pathways by pain relief resulting from either peripheral nerve block (block of afferent input) or manipulations within the rostral anterior cingulate cortex (rACC, i.e., modulation of aversiveness without altering nociceptive input). We will use anatomical (IHC), neurochemical (in vivo microdialysis), behavioral and imaging (animal fMRI) analyses to characterize the functional activation of the nucleus accumbens (NAc)(Aim 1) and/or ventral tegmental area (VTA)(Aim 2) by pain relief or the expectation of relief. Functional connectivity between the rACC and the NAc that underlies motivated behavior to seek relief will be studied in Aim 3. Understanding circuits that reflect relief of ongoing pain will help to identify new molecular targets that may be exploited for discovery of therapies directly targeting affective dimensions of pain that may have increased translational relevance and ultimately validating a biomarker of pain relief (i.e., analgesia).

? DESCRIPTION (provided by applicant): Chronic pain is fundamentally different from acute nociceptive pain in its underlying mechanisms, symptoms and especially, in response to treatment. There is a high comorbidity of chronic pain with diseases such as anxiety and depression. Some studies suggest that during chronification of pain, the affective/emotional features of pain become more dominant. Critically, achieving a positive clinical outcome with current analgesic therapies appears to be negatively correlated with pain chronicity. Neuroimaging studies in chronic pain patients have identified brain regions with altered opioid and dopamine function, suggesting that impairments in these neurotransmitter systems could underlie the transition from acute to chronic pain. We have recently demonstrated in animal pain models that relief of ongoing pain is rewarding and requires dopamine signaling in the nucleus accumbens (NAc). Moreover, these behavioral and neurochemical measures of pain relief depend on endogenous opioid neurotransmission in the cingulate cortex (ACC), an area encoding aversiveness of pain. Chronic pain may produce sustained opioid signaling in the ACC resulting in progressive changes in opioid transmitter-receptor function in this region, and over time, in reduced analgesic efficacy. Determining whether there is a causal relationship between pain, brain neuronal maladaptations and analgesic effectiveness of pain therapies in humans is difficult and potentially unethical. Therefore, we will employ a rat model of chronic neuropathic pain to perform longitudinal studies over a six months period to more closely mimic the human chronic pain condition. In Aim 1 we will investigate the temporal changes in opioid and dopamine neurotransmission in the brain regions encoding affective and motivational aspects of pain. We will use fast scan cyclic voltammetry (FSCV) and fast scan controlled adsorption voltammetry (FSCAV) techniques pioneered in our laboratories that allow measurements of phasic and tonic levels of dopamine in awake behaving animals with unprecedented temporal and spatial resolution. The phasic release of dopamine may be influenced by chronic pain-related changes in tonic dopamine levels. Additionally, we will use a novel in vivo microdialysis method involving online preservation coupled with LC-MS3 detection to measure the low levels of endogenous opioid peptides in the ACC in behaving animals in control and pain conditions. Aim 2 will investigate whether pain chronicity is related to reduced efficacy of opioid and dopamine signaling. Our laboratory demonstrated that in animals, relief of pain aversiveness produces negative reinforcement that can be assessed behaviorally using conditioned place preference (CPP). CPP can be viewed as the animal's self- report of analgesic efficacy that encompasses learning as well as motivational and affective features of ongoing pain and therefore provides information that is likely of translational relevance to the human pain experience. We will use the CPP paradigm to evaluate progression of ongoing neuropathic pain and to assess the efficacy of opioid and non-opioid analgesics over time. Aim 3 will establish if normalization of dopamine and opioid function can be achieved with reversal of chronic pain using methods currently in clinical trials. The proposed studies address gaps in our knowledge that include causality, chronicity and reversibility of pain- related brain dopamine and opioid function. Additionally, these studies may allow for objective quantification of chronicity and serv as a biomarker of effective pain relieving treatments that may speed translation and discovery of new pain therapeutics.

Summary The Administrative Core will be directed by the Co-Project Directors/Principal Investigators, Drs. Victor J. Hruby and Frank Porreca. This core serves as the focal point for the Program Project Grant's scientific, financial and information aspects. It coordinates the interdisciplinary, multi-PI and multi-site interactions to assure maximum scientific progress and success. The functions of the administrative core fall into four main categories: (a) communication and sharing of information; (b) maximizing and efficient use of resources to fulfill needs for the Cores and for the Projects; (c) record keeping and data sharing to guide investigations within the investigative team and with the outside world; and (d) financial and regulatory oversight. The Administrative Core provides a formal and centralized mechanism to accomplish these functions that will insure that the activities of the Projects and the Cores are carried out efficiently and that the benefits of the information that results from these activities is shared within the Program Project team and with the scientific community in compliance with local and Federal regulations. The main goal of the Administrative Core is to insure that the goals of the Program Project are achieved and to take advantage of the strengths and creativity of the individual investigators so that the impact of this work is maximal.

DESCRIPTION (provided by applicant): While much has been learned about the neurobiology of pain, it is disappointing that an insufficient number of novel therapies have been introduced into clinical practice. Moderate to severe pain is still treated mechanistically with opiate mu agonists and while some new mechanisms and improved formulations have improved patient care, it is widely acknowledged that the armamentarium available to physicians for the treatment of chronic pain is inadequate and that there is a large unmet medical need. The significance of our application is to address the clinical need for medications for injury or disease-induced chronic non-malignant pain with new mechanisms of action. Our goals in this PPG proposal are driven by the need of patients for new therapies to control pain as well as issues relevant to society including addiction and drug abuse. The proposals we have developed are comprehensive, multidisciplinary, and emphasize novel research hypotheses. This application brings together state-of-the-art chemistry, pharmacology, biophysics, biochemistry, and molecular biology all of which are necessary for success. Our central hypothesis is that drug design can consider known neural adaptations and mechanisms that may be relevant to specific pain conditions in order to develop improved pain therapeutics. We propose to design, synthesize and biologically validate multivalent drugs that can act as analgesics for the prolonged treatment of chronic non-malignant pain. The multifunctional characteristics of these single molecules are hypothesized to demonstrate diminished likelihood of drug addiction, drug seeking behavior, and tolerance without, or with greatly reduced, side effects that are present in currently available drugs and that diminish quality of life. Project A will be directed by Professo Victor Hruby and will focus on the discovery of multivalent opioid mu agonist-delta agonist and mu agonist/NK1 antagonist peptidic molecules with drug like characteristics and penetration across the blood brain barrier for delivery by systemic administration. Project B will be directed by Professors Frank Porreca and Ed Roberts and will aim to discover orally available and brain penetrant opioid mu/CCK antagonist molecules with drug like characteristics. Project C will be directed by Professors Alex Makriyannis, Todd Vanderah and Frank Porreca and will explore multifunctional cannabinoid agonists for improved treatment of HIV neuropathic pain. These projects will be supported by an Administrative Core (Hruby/Porreca), a synthetic core (Hruby) and a Biochemical Core (Vanderah/Streicher) that will allow maximal synergy and progress. Our goals are to discover single molecules with multiple receptor characteristics that have drug-like properties allowing for advancement to human trials for improved treatment of pain.

SUMMARY (Project B): Opiates are medically appropriate for the treatment of acute (nociceptive) or cancer pain. It is not known, however, whether these drugs provide long-term benefit in chronic non-malignant (e.g., neuropathic) pain. Some studies suggest an overall decrease in quality of life of patients receiving opiates for chronic pain. Additionally, opiates can produce physical dependence, and promote addiction, drug abuse and death due to overdose. Eric Holder, Attorney General of the United States, recently stated ?right now, few substances are more lethal than prescription opiates and heroin? (NY Times, March 10, 2014). There is a high unmet medical need for the discovery of treatments that allow management of chronic non-malignant pain, that sustain efficacy for extended periods while maintaining quality of life and that diminish the possibility of addiction and abuse. In this sub-project (Project B), and consistent with the overall aims of the Program Project grant, we consider the adaptive changes that occur in the brain as a consequence of chronic pain, and from the opiates themselves, in the design of novel therapies. Preclinical research has demonstrated that experimental neuropathic pain, or sustained exposure to opiates such as morphine, produces upregulation of cholecystokinin (CCK) in descending pain modulation circuits as well as in reward/motivation circuits. CCK elicits both pro-nociceptive and anti-opioid actions. Together these neural adaptations diminish the analgesic actions of opiates so that more drug is needed to elicit the same effect on pain (i.e., tolerance). Higher doses of drugs are associated with increased side-effects and increased likelihood of addiction and abuse. CCK produces hyperalgesia in animals and is associated with the nocebo response in humans. Blockade of CCK receptors (a) enhances opiate potency and promotes sustained efficacy in experimental chronic non-malignant pain; (b) diminishes opiate-induced sedation and gastrointestinal slowing, common side- effects that decrease quality of life with chronic use; (c) decreases the naloxone-precipitated withdrawal syndrome in morphine-dependent rats; and (d) decreases morphine-induced dopamine release in the reward pathway. Thus, blockade of CCK receptors could allow for improved opiate-mediated control of chronic non- malignant pain while maintaining quality of life. Additionally, CCK receptor blockade could diminish opiate dependence, as well as addiction and possibly drug craving providing benefits both to individuals and society. We hypothesize that we can discover a single small molecular weight, non-peptidic molecule with high affinity CCK antagonist and mu opioid agonist activity. We additionally hypothesize that such orally availability and CNS penetrant molecules will show enhanced and sustained activity in chronic pain, as well as reduced liability for dependence, addiction and craving. Such compounds with dual functionality may achieve pain-related efficacy at decreased mu opioid receptor engagement resulting in decreased side-effects, improved quality of life and diminished dependence and addiction liability. We will build on strong preliminary lead compounds with dual functionality to optimize desired interactions of a single molecule with opiate and CCK receptors, ADME and pharmacokinetics (Aim 1); characterize these molecules for effects on experimental chronic (e.g., neuropathic) non-malignant pain (Aim 2); evaluate the effects of sustained administration of these new molecules alone, or with new delivery methods, for overall liabilities that may impact quality of life from side- effects (Aim 3); and evaluate the liability of these molecules for physical dependence, addictive liability, and craving (Aim 4). This proposal is based on the optimization of novel chemical entities that have been specifically designed to address the established neural consequences that result from opiate use for chronic pain, i.e., drug design for disease.

Project Summary Many patients suffer from chronic pain in the absence of identifiable injury. Such pains are termed ?functional? and include irritable bowel syndrome, temporomandibular joint disorder, fibromyalgia, migraine and others. Functional pain patients experience pain free periods that are interrupted by attacks of pain that can persist for variable periods of time. The chronification of these pain disorders has been linked to the number and frequency of attacks suggesting that repeated nociceptive episodes promote and maintain a state of central sensitization that reflects increased vulnerability to future attacks. Functional pain patients commonly identify stress as a key trigger of pain episodes but neurobiological mechanisms remain to be determined. In this project, we test the novel hypothesis that in sensitized states, stress-induced kappa opioid receptor (KOR) signaling in the amygdala promotes functional pain responses. We have developed an injury-free rodent model of stress-related functional pain based on hyperalgesic priming with opioids. Opioids have been shown to produce opioid-induced hyperalgesia (OIH) in humans and in animals. OIH is characterized by generalized tactile and thermal hyperalgesia, decreased nociceptive thresholds, increase temporal summation, and a loss of descending noxious inhibitory controls (DNIC). Following resolution of OIH, and in the absence of stress, animals have normal pain responses. Hyperalgesic priming, however, produces a state of latent sensitization so that animals previously exposed to morphine are now prone to stress-induced hyperalgesia and a loss of DNIC that is prevented by blockade of KOR signaling within the central nucleus of the amygdala (CeA). Our electrophysiological data support a KOR-mediated disinhibition of CeA neurons that promote pain. We will use advanced behavioral and electrophysiological approaches with optogenetic and chemogenetic methods to demonstrate that activation of CeA KOR neurons in control, unprimed mice promotes pain-related responses (Specific Aim 1). These studies will establish the neural circuitry within the amygdala that may underlie a novel KOR-mediated pronociceptive CeA output that is engaged through disinhibition. Specific Aim 2 will determine if exogenous activation of the CeA KOR circuit results in amplified pain responses following priming- induced latent sensitization. In Specific Aim 3 we will determine whether blockade of stress-induced endogenous CeA KOR signaling reduces pain responses following priming-induced latent sensitization. The proposed studies will characterize a previously unknown stress-related KOR mediated hyperalgesic circuit from CeA and determine how this circuit may promote decreased resilience to stress. Importantly, these studies may unravel mechanisms for therapeutic interventions in stress-related functional pain disorders through an actionable molecular target. KOR antagonists are currently in development.

Post-traumatic headache (PTH) commonly occurs following mild traumatic brain injury (mTBI), also known as concussion. PTH is a secondary headache that often presents with a migraine-like phenotype and is subdivided as acute or persistent (PPTH) depending on whether it resolves within 3 months after injury. The pathophysiology of PTH and PPTH is not understood and no evidence-based treatments exist for these conditions. Critically, PPTH might differ from PTH, not only in the duration but also in underlying mechanisms and responsiveness to treatment. The reasons for emergence of PPTH in some patients remain unclear but may be related to risk factors including pre-existing migraine and the experience of a previous mTBI. We have developed an approach to investigate the mechanisms of PTH and PPTH as well as potential strategies for treatment. Using a weight drop method in male and female mice that recapitulates biomechanical properties and clinical features of mTBI, we have shown that a single mTBI is sufficient to induce clinically relevant PTH symptoms including an acute period of allodynia, elevated CGRP blood levels and lowered thresholds for induction of cortical spreading depression (CSD). Additionally, we have explored the concept that the transition from acute to chronic pain states may rely on a ?pain memory? that can be studied using the ?two-hit? model of hyperalgesic priming where a prior insult confers vulnerability to a subsequent provocative stimulus. Thus, following resolution of acute allodynia, mTBI mice transition into a long-lasting persistent phase (PPTH) where, remarkably, allodynia can be reinstated by physiologically relevant and common migraine triggers, including stress. CGRP is established in migraine pathogenesis and our data also suggest an important role in promoting PTH. Treatment with either a CGRP antibody or with onabotulinum toxin A (botox) prevents mTBI-related allodynia (PTH) as well as subsequent provoked allodynia representative of PPTH. However, blockade of CGRP after mTBI sensitization is established is ineffective in blocking provoked allodynia, while botox still maintains efficacy. We have hypothesized that mTBI results in CGRP release from meningeal afferents promoting PTH and central sensitization that underlies the development of PPTH, but that PPTH may be maintained in a CGRP-independent fashion. Additionally, we hypothesize that existing sensitization prior to a mTBI event will promote vulnerability to the development of CGRP-independent PPTH. We explore these hypotheses with two related but, independent, aims using behavioral, neurochemical, immunohistochemical and electrophysiolgical analyses. Aim 1 will determine whether, and when currently available therapies can block mTBI-related outcomes relevant to PTH and if these treatments can prevent the expression of PPTH. Aim 2 will determine if prior sensitization promotes more severe, long-lasting and CGRP-resistant PPTH. Our studies will fill in significant knowledge gaps about the role of CGRP in promoting PTH and the importance of pre-existing sensitization in establishing CGRP- independent PPTH. Such information will influence treatment as well as guide the discovery of new therapies.

We propose to establish a Center of Excellence for Addiction Studies (CEAS) that will offer core services allowing users to develop projects that will lead to new research in addiction. Addiction and relapse are characterized by dysregulation of brain circuitry that involves diminished activity of brain reward circuits, increased responsiveness of stress circuits and impaired functioning of executive cortical circuits. Neural changes are observed in the basal ganglia, extended amygdala and prefrontal cortical regions and encompass a wide range of endogenous neurotransmitters including dopamine, opioid peptides, endocannabinoids, corticotropin releasing factor (CRF), dynorphin, glutamate and others. While chronic pain and addiction are different disorders, there is a remarkable overlap between the influence of drugs of abuse and chronic pain on these circuits. Our faculty has broad expertise in evaluation of mechanisms that underlie the maladaptations promoted by pain in these circuits. The CEAS will be composed of four Cores and a Pilot Research Project. The Administrative Core will provide the structural elements that will allow efficient functioning of the CEAS. The Genetic Targeting of Neural Circuits Core will allow users to employ cutting edge genetic techniques including CRISPR/Cas9 gene editing, chemogenetics and optogenetics to produce cell and circuit-specific manipulations to evaluate potential mechanisms relevant to addiction. The Neuroanalytical Core will provide users with advanced methods of measuring neurotransmitters with temporal resolution spanning milli-seconds to days and with spatial specificity through advanced detection methods. The Behavioral Core will allow users to explore questions relevant to addiction using behavioral assays that evaluate addictive processes including the influence of addictive drugs on cognitive function. Investigators in the CEAS have worked together for many years and have shared and individual research funding. Additionally, the CEAS will offer opportunities for other investigators at University of Arizona as well as Arizona State University, Northern Arizona University, The University of New Mexico and Texas Tech University Health Sciences Center at Lubbock and El Paso establishing a Southwestern region engaged in addiction sciences. The CEAS will promote increased diversity in addiction research by recruiting investigators and students from under-represented populations in neuroscience and addiction. The impact of the CEAS will be to leverage established funding to develop new research on addiction research. In addition, the impact of funds from the CEAS will be amplified by commitments of matching funds from the University of Arizona and from a recently established Comprehensive Center of Pain and Addiction. The CEAS will provide key services to its users that correspond with the goals of the NIDA to enhance addiction research with a goal of development of therapies that can stem the opioid epidemic as well as impacting other substance abuse disorders. The close collaboration between the Cores ensures high expertise in all areas of the addiction research and will permit outcome measures emphasizing scientific rigor and reproducibility.

Summary ? Administrative Core This NIDA supported Center of Excellence aims to stimulate the University of Arizona?s research on the neurobiology of addiction and relapse. The Center of Excellence in Addiction Studies (CEAS) consists of three Research Cores (Genetic Targeting, Neuroanalytical, and Behavioral), a Pilot Research Project (PRP), and the Administrative Core (AC). The AC functions as the focal point for coordination and implementation of the activities of the Cores and PRP, providing financial and regulatory oversight, a data repository, and community information sharing. The main goals of the AC are to operate the CEAS seamlessly promoting maximal synergy and impact between the Cores and PRP for the research of the users that will lead to new proposals for addiction research. The AC oversees all activities of the Center and coordinates the interdisciplinary, multi-PI and multi-core interactions along with providing administrative support. The functions of the AC fall into four main categories: (1) coordinate meetings, invited lectures, consulting, retreat, internship program, travels; (2) coordinate requests from users for experiments; (3) establish a database for experimental data and data sharing; and (4) prepare yearly progress reports, and provide financial and regulatory oversight. The AC provides a formal and centralized mechanism to accomplish these functions that will ensure that the activities of the Cores and PRP are carried out efficiently, as well as insuring that the benefits of the information that result from these activities are shared within the Center and with the scientific community in compliance with local and federal regulations. The coordination of all of these activities is what the AC will do to enable success.

Project Summary: Many patients suffer from chronic pain in the absence of identifiable injury. Such pains are termed ?functional? and include irritable bowel syndrome, temporomandibular joint disorder, fibromyalgia, migraine and others. For reasons that are not understood, almost all functional pain syndromes (FPS) are female prevalent. FPS patients experience pain-free interictal periods punctuated by attacks of pain. The frequency of attacks is predictive of risk of chronification. Pain episodes thus produce a priming effect, establishing a state of increased vulnerability to future attacks, likely reflecting peripheral and central sensitization. FPS patients commonly identify stress as a key trigger of pain. Repeated stress may thus promote vulnerability and pain in a sexually dimorphic fashion. We have developed an injury-free rodent model of FPS based on hyperalgesic priming with repeated stress. Hyperalgesic priming produces a pain-free state of increased vulnerability that has been termed ?latent sensitization? (LS). Following induction of LS, normally subthreshold triggers can produce pain attacks, modeling the interictal and ictal periods of FPS. We will use this model to test the novel hypothesis that repeated stress activates kappa opioid receptor (KOR) signaling in the hypothalamus resulting in release of prolactin (PRL) and dysregulation of prolactin receptor (PRLR) isoform expression selectively in female nociceptors. PRL signals through homodimers of PRLR long and short (i.e., PRLR-L and PRLR-S) isoforms that respectively regulate transcription and pain. Repeated stress down-regulates PRLR-L promoting female-selective pain through stress-induced PRL/PRLR-S signaling. The balance of PRLR isoforms may therefore ?tune? female nociceptors to promote LS and pain from normally subthreshold stimuli. We will use genetic and chemogenetic manipulations along with anatomical, neurochemical, electrophysiological, pharmacological and behavioral studies in male and female mice to evaluate the role of dorsal root ganglion (DRG) PRLR-L down-regulation and stress-related hypothalamic KOR activation as essential mechanisms of LS and stress-related pain in females. Aim 1 will establish the effects of repeated stress on hypothalamic KOR signaling and PRL release. Aim 2 will establish a potential causal relationship of repeated stress or hypothalamic KOR activation on DRG PRLR isoform expression, neural excitability, LS and stress-related pain. Aim 3 will determine if KOR antagonists, DA agonists or a PRL antibody will prevent LS and FPS-like pain selectively in females. The proposed studies will characterize a previously unknown stress-related neuroendocrine link between hypothalamic KOR and PRL/PRLR signaling to promote female selective functional pain. Importantly, these studies will advance knowledge about previously unknown biological mechanisms and may unravel mechanisms for therapeutic interventions allowing improved therapy of FPS in women.