Frank Porreca

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• Porreca, F. (2015). Opioid Analgesics and Antagonists. In: Pharmacology and Therapeutics for Dentistry. In Pharmacology and Theapeutics for Dentistry, 7th Ediion.

Journals/Publications

• Navratilova, E., Qu, C., Ji, G., Neugebauer, V., Guerrero, M., Rosen, H., Roberts, E., & Porreca, F. (2023). Opposing Effects on Descending Control of Nociception by µ and κ Opioid Receptors in the Anterior Cingulate Cortex. Anesthesiology, 140(2), 272-283.
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  The efficiency of descending pain modulation, commonly assessed with the conditioned pain modulation procedure, is diminished in patients with chronic pain. The authors hypothesized that the efficiency of pain modulation is controlled by cortical opioid circuits.
• Lee, G. J., Porreca, F., & Navratilova, E. (2023). Prolactin and pain of endometriosis. Pharmacology & therapeutics, 247, 108435.
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  Women experience chronic pain more often than men with some pain conditions being specific to women while others are more prevalent in women. Prolactin, a neuropeptide hormone with higher serum levels in women, has recently been demonstrated in preclinical studies to sensitize nociceptive sensory neurons in a sexually dimorphic manner. Dysregulation of prolactin and prolactin receptors may be responsible for increased pain especially in female predominant conditions such as migraine, fibromyalgia, and pelvic pain. In this review, we focus on the role of prolactin in endometriosis, a condition characterized by pelvic pain and infertility that affects a large proportion of women during their reproductive age. We discuss the symptoms and pathology of endometriosis and discuss how different sources of prolactin secretion may contribute to this disease. We highlight our current understanding of prolactin-mediated mechanisms of nociceptor sensitization in females and how this mechanism may apply to endometriosis. Lastly, we report the results of a systematic review of clinical studies conducted by searching the PubMed and EMBASE databases to identify association between endometriosis and blood levels of prolactin. The results of this search strongly indicate that serum prolactin levels are increased in patients with endometriosis and support the possibility that high levels of prolactin may promote pelvic pain in these patients and increase vulnerability to other comorbid pain conditions likely by dysregulating prolactin receptor expression. Targeting of prolactin and prolactin receptors may improve management of pain associated with endometriosis.
• Maciuba, S., Bowden, G. D., Stratton, H. J., Wisniewski, K., Schteingart, C. D., Almagro, J. C., Valadon, P., Lowitz, J., Glaser, S. M., Lee, G., Dolatyari, M., Navratilova, E., Porreca, F., & Rivière, P. J. (2023). Discovery and characterization of prolactin neutralizing monoclonal antibodies for the treatment of female-prevalent pain disorders. mAbs, 15(1), 2254676.
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  Prolactin (PRL) has recently been demonstrated to elicit female-selective nociceptor sensitization and increase pain-like behaviors in female animals. Here we report the discovery and characterization of first-in-class, humanized PRL neutralizing monoclonal antibodies (PRL mAbs). We obtained two potent and selective PRL mAbs, PL 200,031 and PL 200,039. PL 200,031 was engineered as human IgG1 whereas PL 200,039 was reformatted as human IgG4. Both mAbs have sub-nanomolar affinity for human PRL (hPRL) and produce concentration-dependent and complete inhibition of hPRL signaling at the hPRL receptor (hPRLR). These two PRL mAbs are selective for hPRL as they do not inhibit other hPRLR agonists such as human growth hormone or placental lactogen. They also cross-react with non-human primate PRL but not with rodent PRL. Further, both mAbs show long clearance half-lives after intravenous administration in FcRn-humanized mice. Consistent with their isotypes, these mAbs only differ in binding affinities to Fcγ receptors, as expected by design. Finally, PL 200,019, the murine parental mAb of PL 200,031 and PL 200,039, fully blocked stress-induced and PRL-dependent pain behaviors in female PRL-humanized mice, thereby providing preclinical proof-of-efficacy for PRL mAbs in mechanisms relevant to pain in females.
• Porreca, F., & Dodick, D. W. (2023). Considering Patient Sex in Prescribing CGRP Receptor Antagonists for Short-Term Treatment of Migraine. JAMA neurology, 80(9), 885-886.
• Ikegami, D., Navratilova, E., Yue, X., Moutal, A., Kopruszinski, C. M., Khanna, R., Patwardhan, A., Dodick, D. W., & Porreca, F. (2022). A prolactin-dependent sexually dimorphic mechanism of migraine chronification. Cephalalgia : an international journal of headache, 42(3), 197-208.
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  Determination of possible sex differences in mechanisms promoting migraine progression and the contribution of prolactin and the prolactin long (PRLR-L) and short (PRLR-S) receptor isoforms.
• Ito, H., Navratilova, E., Vagnerova, B., Watanabe, M., Kopruszinski, C., Moreira de Souza, L. H., Yue, X., Ikegami, D., Moutal, A., Patwardhan, A., Khanna, R., Yamazaki, M., Guerrero, M., Rosen, H., Roberts, E., Neugebauer, V., Dodick, D. W., & Porreca, F. (2022). Chronic pain recruits hypothalamic dynorphin/kappa opioid receptor signalling to promote wakefulness and vigilance. Brain : a journal of neurology.
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  Increased vigilance in settings of potential threats or in states of vulnerability related to pain is important for survival. Pain disrupts sleep and conversely, sleep disruption enhances pain, but the underlying mechanisms remain unknown. Chronic pain engages brain stress circuits and increases secretion of dynorphin, an endogenous ligand of the kappa opioid receptor (KOR). We therefore hypothesized that hypothalamic dynorphin/KOR signaling may be a previously unknown mechanism that is recruited in pathological conditions requiring increased vigilance. We investigated the role of KOR in wakefulness, non-rapid eye movement (NREM) sleep and rapid eye movement (REM) sleep in freely moving naïve mice and in mice with neuropathic pain induced by partial sciatic nerve ligation (PSNL) using EEG/EMG recordings. Systemic continuous administration of U69,593, a KOR agonist, over 5 days through an osmotic minipump decreased the amount of NREM and REM sleep and increased sleep fragmentation in naive mice throughout the light-dark sleep cycle. We used KORcre mice to selectively express a Gi-coupled designer receptor activated by designer drugs (Gi-DREADD) in KORcre neurons of the hypothalamic paraventricular nucleus (PVN), a key node of the hypothalamic-pituitary-adrenal (HPA) stress response. Sustained activation of Gi-DREADD with clozapine-N-oxide (CNO) delivered in drinking water over 4 days disrupted sleep in these mice in a similar way as systemic U69,593. Mice with chronic neuropathic pain also showed disrupted NREM and total sleep that was normalized by systemic administration of two structurally different KOR antagonists, norbinaltorphimine (nor-BNI) and NMRA-140, currently in phase II clinical development, or by CRISPR/Cas9 editing of PVN KOR, consistent with endogenous KOR activation disrupting sleep in chronic pain. Unexpectedly, REM sleep was diminished by either systemic KOR antagonist or by CRISPR/Cas9 editing of PVN KOR in sham-operated mice. Our findings reveal previously unknown physiological and pathophysiological roles of dynorphin/KOR in eliciting arousal. Physiologically, dynorphin/KOR signaling affects transitions between sleep stages that promote REM sleep. Furthermore, while KOR antagonists do not promote somnolence in the absence of pain, they normalized disrupted sleep in chronic pain, revealing a pathophysiological role of KOR signaling that is selectively recruited to promote vigilance, increasing chances of survival. Notably, while this mechanism is likely beneficial in the short-term, disruption of the homeostatic need for sleep over longer periods may become maladaptive resulting in sustained pain chronicity. A novel approach for treatment of chronic pain may thus result from normalization of chronic pain-related sleep disruption by KOR antagonism.
• Kopruszinski, C. M., Porreca, F., & Chichorro, J. G. (2022). Editorial: Chronic orofacial pain. Frontiers in pain research (Lausanne, Switzerland), 3, 1086256.
• Kopruszinski, C. M., Vizin, R., Watanabe, M., Martinez, A. L., de Souza, L. H., Dodick, D. W., Porreca, F., & Navratilova, E. (2022). Exploring the neurobiology of the premonitory phase of migraine preclinically - a role for hypothalamic kappa opioid receptors?. The journal of headache and pain, 23(1), 126.
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  The migraine premonitory phase is characterized in part by increased thirst, urination and yawning. Imaging studies show that the hypothalamus is activated in the premonitory phase. Stress is a well know migraine initiation factor which was demonstrated to engage dynorphin/kappa opioid receptors (KOR) signaling in several brain regions, including the hypothalamus. This study proposes the exploration of the possible link between hypothalamic KOR and migraine premonitory symptoms in rodent models.
• Kopruszinski, C. M., Watanabe, M., Martinez, A. L., de Souza, L. H., Dodick, D. W., Moutal, A., Neugebauer, V., Porreca, F., & Navratilova, E. (2022). Kappa opioid receptor agonists produce sexually dimorphic and prolactin-dependent hyperalgesic priming. Pain.
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  Repeated stress produces hyperalgesic priming in preclinical models, but underlying mechanisms remain uncertain. As stress engages kappa opioid receptors (KORs), we hypothesized that repeated administration of KOR agonists might mimic, in part, stress-induced hyperalgesic priming. The potential contribution of circulating prolactin (PRL) and dysregulation of the expression of PRL receptor (PRLR) isoforms in sensory neurons after KOR agonist administration was also investigated. Mice received 3 daily doses of U-69593 or nalfurafine as a "first-hit" stimulus followed by assessment of periorbital tactile allodynia. Sixteen days after the first KOR agonist administration, animals received a subthreshold dose of inhalational umbellulone, a TRPA1 agonist, as the second-hit stimulus and periorbital allodynia was assessed. Cabergoline, a dopamine D2 receptor agonist, was used to inhibit circulating PRL in additional cohorts. Prolactin receptor isoforms were quantified in the V1 region of the trigeminal ganglion after repeated doses of U-69593. In both sexes, KOR agonists increased circulating PRL and produced allodynia that resolved within 14 days. Hyperalgesic priming, revealed by umbellulone-induced allodynia in animals previously treated with the KOR agonists, also occurred in both sexes. However, repeated U-69593 downregulated the PRLR long isoform in trigeminal neurons only in female mice. Umbellulone-induced allodynia was prevented by cabergoline co-treatment during priming with KOR agonists in female, but not male, mice. Hyperalgesic priming therefore occurs in both sexes after either biased or nonbiased KOR agonists. However, a PRL/PRLR-dependence is observed only in female nociceptors possibly contributing to pain in stress-related pain disorders in females.
• Martin, L., Ibrahim, M., Gomez, K., Yu, J., Cai, S., Chew, L. A., Bellampalli, S. S., Moutal, A., Largent-Milnes, T., Porreca, F., Khanna, R., Olivera, B. M., & Patwardhan, A. (2022). Conotoxin contulakin-G engages a neurotensin receptor 2/R-type calcium channel (Cav2.3) pathway to mediate spinal antinociception. Pain, 163(9), 1751-1762.
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  Intrathecal application of contulakin-G (CGX), a conotoxin peptide and a neurotensin analogue, has been demonstrated to be safe and potentially analgesic in humans. However, the mechanism of action for CGX analgesia is unknown. We hypothesized that spinal application of CGX produces antinociception through activation of the presynaptic neurotensin receptor (NTSR)2. In this study, we assessed the mechanisms of CGX antinociception in rodent models of inflammatory and neuropathic pain. Intrathecal administration of CGX, dose dependently, inhibited thermal and mechanical hypersensitivities in rodents of both sexes. Pharmacological and clustered regularly interspaced short palindromic repeats/Cas9 editing of NTSR2 reversed CGX-induced antinociception without affecting morphine analgesia. Electrophysiological and gene editing approaches demonstrated that CGX inhibition was dependent on the R-type voltage-gated calcium channel (Cav2.3) in sensory neurons. Anatomical studies demonstrated coexpression of NTSR2 and Cav2.3 in dorsal root ganglion neurons. Finally, synaptic fractionation and slice electrophysiology recordings confirmed a predominantly presynaptic effect. Together, these data reveal a nonopioid pathway engaged by a human-tested drug to produce antinociception.
• Navratilova, E., Oyarzo, J., Anderson, T., Broide, R. S., Subramaniam, S. R., Vazquez-Cintron, E. J., Brin, M. F., Schwedt, T. J., Dodick, D. W., & Porreca, F. (2022). Preclinical assessment of onabotulinumtoxinA for the treatment of mild traumatic brain injury-related acute and persistent post-traumatic headache. Cephalalgia : an international journal of headache, 42(11-12), 1194-1206.
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  Investigation of onabotulinumtoxinA in a murine model of acute and persistent post-traumatic headache.
• Sato, D., Narita, M., Hamada, Y., Mori, T., Tanaka, K., Tamura, H., Yamanaka, A., Matsui, R., Watanabe, D., Suda, Y., Senba, E., Watanabe, M., Navratilova, E., Porreca, F., Kuzumaki, N., & Narita, M. (2022). Relief of neuropathic pain by cell-specific manipulation of nucleus accumbens dopamine D1- and D2-receptor-expressing neurons. Molecular brain, 15(1), 10.
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  Emerging evidence suggests that the mesolimbic dopaminergic network plays a role in the modulation of pain. As chronic pain conditions are associated with hypodopaminergic tone in the nucleus accumbens (NAc), we evaluated the effects of increasing signaling at dopamine D1/D2-expressing neurons in the NAc neurons in a model of neuropathic pain induced by partial ligation of sciatic nerve. Bilateral microinjection of either the selective D1-receptor (Gs-coupled) agonist Chloro-APB or the selective D2-receptor (Gi-coupled) agonist quinpirole into the NAc partially reversed nerve injury-induced thermal allodynia. Either optical stimulation of D1-receptor-expressing neurons or optical suppression of D2-receptor-expressing neurons in both the inner and outer substructures of the NAc also transiently, but significantly, restored nerve injury-induced allodynia. Under neuropathic pain-like condition, specific facilitation of terminals of D1-receptor-expressing NAc neurons projecting to the VTA revealed a feedforward-like antinociceptive circuit. Additionally, functional suppression of cholinergic interneurons that negatively and positively control the activity of D1- and D2-receptor-expressing neurons, respectively, also transiently elicited anti-allodynic effects in nerve injured animals. These findings suggest that comprehensive activation of D1-receptor-expressing neurons and integrated suppression of D2-receptor-expressing neurons in the NAc may lead to a significant relief of neuropathic pain.
• Starling, A. J., Cortez, M. M., Jarvis, N. R., Zhang, N., Porreca, F., Chong, C. D., & Schwedt, T. J. (2022). Cutaneous heat and light-induced pain thresholds in post-traumatic headache attributed to mild traumatic brain injury. Headache, 62(6), 726-736.
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  The purpose of this study was to characterize cutaneous heat and light-induced pain thresholds in people with post-traumatic headache (PTH) compared with healthy controls (HCs).
• Watanabe, M., Kopruszinski, C. M., Moutal, A., Ikegami, D., Khanna, R., Chen, Y., Ross, S., Mackenzie, K., Stratton, J., Dodick, D. W., Navratilova, E., & Porreca, F. (2022). Dysregulation of serum prolactin links the hypothalamus with female nociceptors to promote migraine. Brain : a journal of neurology, 145(8), 2894-2909.
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  Migraine headache results from activation of meningeal nociceptors, however, the hypothalamus is activated many hours before the emergence of pain. How hypothalamic neural mechanisms may influence trigeminal nociceptor function remains unknown. Stress is a common migraine trigger that engages hypothalamic dynorphin/kappa opioid receptor (KOR) signalling and increases circulating prolactin. Prolactin acts at both long and short prolactin receptor isoforms that are expressed in trigeminal afferents. Following downregulation of the prolactin receptor long isoform, prolactin signalling at the prolactin receptor short isoform sensitizes nociceptors selectively in females. We hypothesized that stress may activate the kappa opioid receptor on tuberoinfundibular dopaminergic neurons to increase circulating prolactin leading to female-selective sensitization of trigeminal nociceptors through dysregulation of prolactin receptor isoforms. A mouse two-hit hyperalgesic priming model of migraine was used. Repeated restraint stress promoted vulnerability (i.e. first-hit priming) to a subsequent subthreshold (i.e. second-hit) stimulus from inhalational umbellulone, a TRPA1 agonist. Periorbital cutaneous allodynia served as a surrogate of migraine-like pain. Female and male KORCre; R26lsl-Sun1-GFP mice showed a high percentage of KORCre labelled neurons co-localized in tyrosine hydroxylase-positive cells in the hypothalamic arcuate nucleus. Restraint stress increased circulating prolactin to a greater degree in females. Stress-primed, but not control, mice of both sexes developed periorbital allodynia following inhalational umbellulone. Gi-DREADD activation (i.e. inhibition through Gi-coupled signalling) in KORCre neurons in the arcuate nucleus also increased circulating prolactin and repeated chemogenetic manipulation of these neurons primed mice of both sexes to umbellulone. Clustered regularly interspaced short palindromic repeats-Cas9 deletion of the arcuate nucleus KOR prevented restraint stress-induced prolactin release in female mice and priming from repeated stress episodes in both sexes. Inhibition of circulating prolactin occurred with systemic cabergoline, a dopamine D2 receptor agonist, blocked priming selectively in females. Repeated restraint stress downregulated the prolactin receptor long isoform in the trigeminal ganglia of female mice. Deletion of prolactin receptor in trigeminal ganglia by nasal clustered regularly interspaced short palindromic repeats-Cas9 targeting both prolactin receptor isoforms prevented stress-induced priming in female mice. Stress-induced activation of hypothalamic KOR increases circulating prolactin resulting in trigeminal downregulation of prolactin receptor long and pain responses to a normally innocuous TRPA1 stimulus. These are the first data that provide a mechanistic link between stress-induced hypothalamic activation and the trigeminal nociceptor effectors that produce trigeminal sensitization and migraine-like pain. This sexually dimorphic mechanism may help to explain female prevalence of migraine. KOR antagonists, currently in phase II clinical trials, may be useful as migraine preventives in both sexes, while dopamine agonists and prolactin/ prolactin receptor antibodies may improve therapy for migraine, and other stress-related neurological disorders, in females.
• Yakhnitsa, V., Ji, G., Hein, M., Presto, P., Griffin, Z., Ponomareva, O., Navratilova, E., Porreca, F., & Neugebauer, V. (2022). Kappa Opioid Receptor Blockade in the Amygdala Mitigates Pain Like-Behaviors by Inhibiting Corticotropin Releasing Factor Neurons in a Rat Model of Functional Pain. Frontiers in pharmacology, 13, 903978.
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  Functional pain syndromes (FPS) occur in the absence of identifiable tissue injury or noxious events and include conditions such as migraine, fibromyalgia, and others. Stressors are very common triggers of pain attacks in various FPS conditions. It has been recently demonstrated that kappa opioid receptors (KOR) in the central nucleus of amygdala (CeA) contribute to FPS conditions, but underlying mechanisms remain unclear. The CeA is rich in KOR and encompasses major output pathways involving extra-amygdalar projections of corticotropin releasing factor (CRF) expressing neurons. Here we tested the hypothesis that KOR blockade in the CeA in a rat model of FPS reduces pain-like and nocifensive behaviors by restoring inhibition of CeA-CRF neurons. Intra-CeA administration of a KOR antagonist (nor-BNI) decreased mechanical hypersensitivity and affective and anxiety-like behaviors in a stress-induced FPS model. In systems electrophysiology experiments in anesthetized rats, intra-CeA application of nor-BNI reduced spontaneous firing and responsiveness of CeA neurons to peripheral stimulation. In brain slice whole-cell patch-clamp recordings, nor-BNI increased feedforward inhibitory transmission evoked by optogenetic and electrical stimulation of parabrachial afferents, but had no effect on monosynaptic excitatory transmission. Nor-BNI decreased frequency, but not amplitude, of spontaneous inhibitory synaptic currents, suggesting a presynaptic action. Blocking KOR receptors in stress-induced FPS conditions may therefore represent a novel therapeutic strategy.
• Bannister, K., Bannister, K., Kucharczyk, M. W., Kucharczyk, M. W., Graven-Nielsen, T., Graven-Nielsen, T., Porreca, F., & Porreca, F. (2021). Introducing descending control of nociception: a measure of diffuse noxious inhibitory controls in conscious animals. Pain, 162(7), 1957-1959.
• Bharadwaj, V. N., Porreca, F., Cowan, R. P., Kori, S., Silberstein, S. D., & Yeomans, D. C. (2021). A new hypothesis linking oxytocin to menstrual migraine. Headache, 61(7), 1051-1059.
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  To highlight the emerging understanding of oxytocin (OT) and oxytocin receptors (OTRs) in modulating menstrual-related migraine (MRM).
• Hein, M., Hein, M., Ji, G., Ji, G., Tidwell, D., Tidwell, D., D'Souza, P., D'Souza, P., Kiritoshi, T., Kiritoshi, T., Yakhnitsa, V., Yakhnitsa, V., Navratilova, E., Navratilova, E., Porreca, F., Porreca, F., Neugebauer, V., & Neugebauer, V. (2021). Kappa opioid receptor activation in the amygdala disinhibits CRF neurons to generate pain-like behaviors. Neuropharmacology, 185, 108456.
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  Recent evidence suggests that kappa opioid receptors (KOR) in limbic brain regions such as the amygdala contribute to pain conditions, but underlying mechanisms remain to be determined. The amygdala is an important player in averse-affective aspects of pain and pain modulation. The central nucleus (CeA) serves output functions through projection neurons that include corticotropin releasing factor (CRF) expressing neurons. The CeA is also rich in KOR. Here we tested the novel hypothesis that KOR activation in the CeA generates pain-like behaviors through a mechanism that involves inhibition of synaptic inhibition (disinhibition) of CRF neurons. Intra-CeA administration of a KOR agonist (U-69,593) increased vocalizations of naïve rats to noxious stimuli, and induced anxiety-like behaviors in the open field test (OFT) and avoidance in the conditioned place preference test, without affecting mechanosensory thresholds. Optogenetic silencing of CeA-CRF neurons blocked the facilitatory effects of systemically applied U-69,593 in naïve rats. Patch-clamp recordings of CRF neurons in rat brain slices found that U-69,593 decreased feedforward inhibitory transmission evoked by optogenetic stimulation of parabrachial afferents, but had no effect on monosynaptic excitatory transmission. U-69,593 decreased frequency, but not amplitude, of inhibitory synaptic currents, suggesting a presynaptic action. Multiphoton imaging of CeA-CRF neurons in rat brain slices showed that U-69,593 increased calcium signals evoked by electrical stimulation of presumed parabrachial input. This study shows for the first time that KOR activation increases activity of amygdala CRF neurons through synaptic disinhibition, resulting in averse-affective pain-like behaviors. Blocking KOR receptors may therefore represent a novel therapeutic strategy.
• Kopruszinski, C. M., Kopruszinski, C. M., Turnes, J. M., Turnes, J. M., Swiokla, J., Swiokla, J., Weinstein, T. J., Weinstein, T. J., Schwedt, T. J., Schwedt, T. J., Dodick, D. W., Dodick, D. W., Anderson, T., Anderson, T., Navratilova, E., Navratilova, E., Porreca, F., & Porreca, F. (2021). CGRP monoclonal antibody prevents the loss of diffuse noxious inhibitory controls (DNIC) in a mouse model of post-traumatic headache. Cephalalgia : an international journal of headache, 41(6), 749-759.
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  Determine the role of calcitonin-gene related peptide in promoting post-traumatic headache and dysregulation of central pain modulation induced by mild traumatic brain injury in mice.
• Lançon, K., Qu, C., Navratilova, E., Porreca, F., & Séguéla, P. (2021). Decreased dopaminergic inhibition of pyramidal neurons in anterior cingulate cortex maintains chronic neuropathic pain. Cell reports, 37(9), 109933.
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  Pyramidal neurons in the anterior cingulate cortex (ACC), a prefrontal region involved in processing the affective components of pain, display hyperexcitability in chronic neuropathic pain conditions, and their silencing abolishes hyperalgesia. We show that dopamine, through D1 receptor (D1R) signaling, inhibits pyramidal neurons of mouse ACC by modulation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Activation of G-coupled D1R by dopamine induces the opening of HCN channels at physiological membrane potentials, driving a significant decrease in input resistance and excitability. Systemic L-DOPA in chronic neuropathic mice rescues HCN channel activity, normalizes pyramidal excitability in ACC, and blocks mechanical and thermal allodynia. Moreover, microinjection of a selective D1R agonist in the ACC relieves the aversiveness of ongoing neuropathic pain, while an ACC D1R antagonist blocks gabapentin- and lidocaine-evoked antinociception. We conclude that dopaminergic inhibition via D1R in ACC plays an analgesic role in physiological conditions and is decreased in chronic pain.
• Lee, Y. S., Remesic, M., Ramos-Colon, C., Wu, Z., LaVigne, J., Molnar, G., Tymecka, D., Misicka, A., Streicher, J. M., Hruby, V. J., & Porreca, F. (2021). Multifunctional Enkephalin Analogs with a New Biological Profile: MOR/DOR Agonism and KOR Antagonism. Biomedicines, 9(6).
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  In our previous studies, we developed a series of mixed MOR/DOR agonists that are enkephalin-like tetrapeptide analogs with an N-phenyl-N-piperidin-4-ylpropionamide (Ppp) moiety at the C-terminus. Further SAR study on the analogs, initiated by the findings from off-target screening, resulted in the discovery of (, Dmt-DNle-Gly-Phe(-Cl)-Ppp), a multifunctional ligand with MOR/DOR agonist and KOR antagonist activity (GTPγS assay: IC = 52 nM, I = 122% cf. IC = 59 nM, I = 100% for naloxone) with nanomolar range of binding affinity ( = 1.3 nM cf. = 2.4 nM for salvinorin A). Based on its unique biological profile, is considered to possess high therapeutic potential for the treatment of chronic pain by modulating pathological KOR activation while retaining analgesic efficacy attributed to its MOR/DOR agonist activity.
• Mehr-Un-Nisa, ., Munawar, M. A., Rankin, D., Hruby, V. J., Porreca, F., & Lee, Y. S. (2021). C-terminal modified Enkephalin-like tetrapeptides with enhanced affinities at the kappa opioid receptor and monoamine transporters. Bioorganic & medicinal chemistry, 51, 116509.
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  A new series of enkephalin-like tetrapeptide analogs modified at the C-terminus by an N-(3,4-dichlorophenyl)-N-(piperidin-4-yl)propionamide (DPP) moiety were designed, synthesized, and tested for their binding affinities at opioid receptors and monoamine transporters to evaluate their potential multifunctional activity for the treatment of chronic pain. Most ligands exhibited high binding affinities in the nanomolar range at the opioid receptors with a slight delta-opioid receptor (DOR) selectivity over mu-opioid receptor (MOR) and kappa-opioid receptor (KOR) and low binding affinities in the micromolar range at the monoamine transporters, SERT and NET. Ligands of which the positions 1 and 4 were substituted by Dmt and Phe(4-X) residues, respectively, showed the excellent binding affinities at three opioid receptors. Among them, Dmt-d-Tic-Gly-Phe(4-F)-DPP was the most promising considering its excellent opioid affinities, particularly unexpected high binding affinity (Ki = 0.13 nM) at the KOR, and moderate interactions with serotonin/norepinephrine reuptake inhibitors (SNRIs). Docking studies revealed that the ligand was a good fit for the KOR binding pocket (binding score = 8,750).
• Navratilova, E., Fillingim, R. B., & Porreca, F. (2021). Sexual dimorphism in functional pain syndromes. Science translational medicine, 13(619), eabj7180.
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  Injury-free pain conditions, defined as functional pain syndromes, are more prevalent and more disabling in women. Mechanisms of sexual dimorphism in functional pain are now emerging from preclinical studies, suggesting an opportunity to advance the development of sex-specific therapies that may improve treatment of pain in women.
• Phelps, C. E., Navratilova, E., & Porreca, F. (2021). Chronic Pain Produces Reversible Memory Deficits That Depend on Task Difficulty in Rats. The journal of pain, 22(11), 1467-1476.
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  Cognitive impairment associated with chronic pain remains relatively poorly understood. Use of analgesic drugs and often present co-morbidities in patients can preclude conclusions of causative relationships between chronic pain and cognitive deficits. Here, the impact of pain resulting from spinal nerve ligation (SNL) injury in rats on short and long-term memory was assessed in the novel object recognition task. To understand if chronic pain seizes the limited cognitive resources that are available at any given time, task difficulty was varied by using either very different (ie, easy task) or similar (ie, difficult task) pairs of objects. Nerve-injured, male rats exhibited no short or long-term memory deficits under easy task conditions. However, unlike sham-operated controls, injured rats showed deficits in both short and long-term memory by failing to differentiate similar objects in the difficult task version. In SNL rats, duloxetine produced anti-allodynic effects and ameliorated long-term memory deficits in the difficult task suggesting benefits of pain relief possibly complemented by noradrenergic mediated cognitive enhancement. Together these data suggest chronic pain reversibly takes up a significant amount of limited cognitive resources, leaving sufficient available for easy, but not difficult, tasks. PERSPECTIVE: Memory deficits in a rat model of chronic pain were only seen when the cognitive load was high, ie, in a difficult task. Acute treatment with duloxetine was sufficient to relieve memory deficits, suggesting chronic pain induces memory deficits by seizing limited cognitive resources to the detriment of task-related stimuli.
• Phelps, C. E., Phelps, C. E., Navratilova, E., Navratilova, E., Porreca, F., & Porreca, F. (2021). Cognition in the Chronic Pain Experience: Preclinical Insights. Trends in cognitive sciences, 25(5), 365-376.
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  Acutely, pain is protective. It promotes escape from, and future avoidance of, noxious stimuli through strong and often lifetime associative memories. However, with persistent acute pain or when pain becomes chronic, these memories can promote negative emotions and poor decisions often associated with deleterious behaviors. In this review, we discuss how preclinical studies can provide insights into the relationship between cognition and chronic pain. We also discuss the concept of pain as a cognitive disorder and new strategies for treating chronic pain that emphasize inhibiting the formation of pain memories or promoting 'forgetting' of established pain memories.
• Porreca, F., Phelps, C. E., & Navratilova, E. (2021). Chronic Pain Produces Reversible Memory Deficits That Depend on Task Difficulty in Rats.. The journal of pain. doi:10.1016/j.jpain.2021.04.016
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  Cognitive impairment associated with chronic pain remains relatively poorly understood. Use of analgesic drugs and often present co-morbidities in patients can preclude conclusions of causative relationships between chronic pain and cognitive deficits. Here, the impact of pain resulting from spinal nerve ligation (SNL) injury in rats on short and long-term memory was assessed in the novel object recognition task. To understand if chronic pain seizes the limited cognitive resources that are available at any given time, task difficulty was varied by using either very different (ie, easy task) or similar (ie, difficult task) pairs of objects. Nerve-injured, male rats exhibited no short or long-term memory deficits under easy task conditions. However, unlike sham-operated controls, injured rats showed deficits in both short and long-term memory by failing to differentiate similar objects in the difficult task version. In SNL rats, duloxetine produced anti-allodynic effects and ameliorated long-term memory deficits in the difficult task suggesting benefits of pain relief possibly complemented by noradrenergic mediated cognitive enhancement. Together these data suggest chronic pain reversibly takes up a significant amount of limited cognitive resources, leaving sufficient available for easy, but not difficult, tasks. Perspective: Memory deficits in a rat model of chronic pain were only seen when the cognitive load was high, ie, in a difficult task. Acute treatment with duloxetine was sufficient to relieve memory deficits, suggesting chronic pain induces memory deficits by seizing limited cognitive resources to the detriment of task-related stimuli.
• Salloum, M. M., Porreca, F., Patwardhan, A. M., Martin, L. F., Killgore, W. D., Khanna, R., Jones-macfarland, F. N., Jain, S. V., Ibrahim, M. M., Goel, V., Gannala, P., & Freeman, J. (2021). Evaluation of green light exposure on headache frequency and quality of life in migraine patients: A preliminary one-way cross-over clinical trial.. Cephalalgia : an international journal of headache, 41(2), 135-147. doi:10.1177/0333102420956711
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  Pharmacological management of migraine can be ineffective for some patients. We previously demonstrated that exposure to green light resulted in antinociception and reversal of thermal and mechanical hypersensitivity in rodent pain models. Given the safety of green light emitting diodes, we evaluated green light as a potential therapy in patients with episodic or chronic migraine..We recruited (29 total) patients, of whom seven had episodic migraine and 22 had chronic migraine. We used a one-way cross-over design consisting of exposure for 1-2 hours daily to white light emitting diodes for 10 weeks, followed by a 2-week washout period followed by exposure for 1-2 hours daily to green light emitting diodes for 10 weeks. Patients were allowed to continue current therapies and to initiate new treatments as directed by their physicians. Outcomes consisted of patient-reported surveys. The primary outcome measure was the number of headache days per month. Secondary outcome measures included patient-reported changes in the intensity and frequency of the headaches over a two-week period and other quality of life measures including ability to fall and stay asleep, and ability to perform work. Changes in pain medications were obtained to assess potential reduction..When seven episodic migraine and 22 chronic migraine patients were analyzed as separate cohorts, white light emitting diodes produced no significant change in headache days in either episodic migraine or chronic migraine patients. Combining data from the episodic migraine and chronic migraine groups showed that white light emitting diodes produced a small, but statistically significant reduction in headache days from (days ± SEM) 18.2 ± 1.8 to 16.5 ± 2.01 days. Green light emitting diodes resulted in a significant decrease in headache days from 7.9 ± 1.6 to 2.4 ± 1.1 and from 22.3 ± 1.2 to 9.4 ± 1.6 in episodic migraine and chronic migraine patients, respectively. While some improvement in secondary outcomes was observed with white light emitting diodes, more secondary outcomes with significantly greater magnitude including assessments of quality of life, Short-Form McGill Pain Questionnaire, Headache Impact Test-6, and Five-level version of the EuroQol five-dimensional survey without reported side effects were observed with green light emitting diodes. Conclusions regarding pain medications reduction with green light emitting diode exposure were not possible. No side effects of light therapy were reported. None of the patients in the study reported initiation of new therapies..Green light emitting diodes significantly reduced the number of headache days in people with episodic migraine or chronic migraine. Additionally, green light emitting diodes significantly improved multiple secondary outcome measures including quality of life and intensity and duration of the headache attacks. As no adverse events were reported, green light emitting diodes may provide a treatment option for those patients who prefer non-pharmacological therapies or may be considered in complementing other treatment strategies. Limitations of this study are the small number of patients evaluated. The positive data obtained support implementation of larger clinical trials to determine possible effects of green light emitting diode therapy.This study is registered with clinicaltrials.gov under NCT03677206.
• Salloum, M., Porreca, F., Patwardhan, A. M., Mata, E. I., Martin, L. F., Killgore, W. D., Khanna, R., Jones-macfarland, F. N., Jain, S. V., Ibrahim, M. M., Gunnala, P., & Goel, V. (2021). Green Light Exposure Improves Pain and Quality of Life in Fibromyalgia Patients: A Preliminary One-Way Crossover Clinical Trial.. Pain medicine (Malden, Mass.), 22(1), 118-130. doi:10.1093/pm/pnaa329
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  Fibromyalgia is a functional pain disorder in which patients suffer from widespread pain and poor quality of life. Fibromyalgia pain and its impact on quality of life are not effectively managed with current therapeutics. Previously, in a preclinical rat study, we demonstrated that exposure to green light-emitting diodes (GLED) for 8 hours/day for 5 days resulted in antinociception and reversal of thermal and mechanical hypersensitivity associated with models of injury-related pain. Given the safety of GLED and the ease of its use, our objective is to administer GLED as a potential therapy to patients with fibromyalgia..One-way crossover clinical trial..United States..We enrolled 21 adult patients with fibromyalgia recruited from the University of Arizona chronic pain clinic who were initially exposed to white light-emitting diodes and then were crossed over to GLED for 1 to 2 hours daily for 10 weeks. Data were collected by using paper surveys..When patients were exposed to GLED, but not white light-emitting diodes, they reported a significant reduction in average pain intensity on the 10-point numeric pain scale. Secondary outcomes were assessed by using the EQ-5D-5L survey, Short-Form McGill Pain Questionnaire, and Fibromyalgia Impact Questionnaire and were also significantly improved in patients exposed to GLED. GLED therapy was not associated with any measured side effects in these patients..Although the mechanism by which GLED elicits pain reduction is currently being studied, these results supporting its efficacy and safety merit a larger clinical trial.
• Swiokla, J., Porreca, F., Navratilova, E., Kopruszinski, C. M., Dodick, D. W., & Chessell, I. P. (2021). A novel, injury-free rodent model of vulnerability for assessment of acute and preventive therapies reveals temporal contributions of CGRP-receptor activation in migraine-like pain.. Cephalalgia : an international journal of headache, 41(3), 305-317. doi:10.1177/0333102420959794
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  Development and characterization of a novel injury-free preclinical model of migraine-like pain allowing mechanistic assessment of both acute and preventive treatments..A "two-hit" hyperalgesic priming strategy was used to induce vulnerability to a normally subthreshold challenge with umbellulone, a transient receptor potential ankyrin 1 (TRPA1) activator, in uninjured female and male C57BL/6 mice. Priming (i.e. the first hit) was induced by three consecutive daily episodes of restraint stress; repeated umbellulone was also evaluated for potential priming effects. Sixteen days after the first restraint stress, mice received inhalational umbellulone (i.e. the second hit) to elicit migraine-like pain. Medications currently used for acute or preventive migraine therapy including propranolol (a beta blocker) and sumatriptan (5HT1B/D agonist), as well as olcegepant, an experimental calcitonin gene related peptide (CGRP) receptor antagonist and nor-Binaltorphimine (nor-BNI), an experimental long-acting kappa opioid receptor (KOR) antagonist, were investigated for their efficacy to block priming and prevent or reverse umbellulone-induced allodynia in primed animals. To assess migraine-like pain, cutaneous allodynia was determined by responses to periorbital or hindpaw probing with von Frey filaments..Repeated restraint stress, but not umbellulone exposure, produced transient cutaneous allodynia that resolved within 16 d. Restraint stress produced long-lasting priming that persisted beyond 16 d, as demonstrated by reinstatement of cutaneous allodynia following inhalational umbellulone challenge. Pretreatment with propranolol or nor-BNI prior to restraint stress prevented both transient cutaneous allodynia and priming, demonstrated by a lack of umbellulone-induced cutaneous allodynia. Following establishment of restraint stress priming, olcegepant, but not propranolol or nor-BNI, prevented umbellulone-induced cutaneous allodynia. When administered 1 h after umbellulone, sumatriptan, but not olcegepant, reversed umbellulone-induced cutaneous allodynia in restraint stress-primed rats..We have developed a novel injury-free model with translational relevance that can be used to study mechanisms relevant to migraine-like pain and to evaluate novel acute or preventive treatments. Restraint stress priming induced a state of vulnerability to a subthreshold stimulus that has been referred to as "latent sensitization". The development of latent sensitization could be prevented by blockade of stress pathways with propranolol or with a kappa opioid receptor antagonist. Following establishment of latent sensitization, subthreshold stimulation with umbellulone reinstated cutaneous allodynia, likely from activation of meningeal TRPA1-expressing nociceptors. Accordingly, in restraint stress-primed animals, sumatriptan reversed umbellulone-induced cutaneous allodynia, supporting peripheral sites of action, while propranolol and nor-BNI were not effective. Surprisingly, olcegepant was effective in mice with latent sensitization when given prior to, but not after, umbellulone challenge, suggesting time-dependent contributions of calcitonin gene-related peptide receptor signaling in promoting migraine-like pain in this model. Activation of the calcitonin gene-related peptide receptor participates in initiating, but has a more limited role in maintaining, pain responses, supporting the efficacy of small molecule calcitonin gene-related peptide antagonists as preventive medications. Additionally, the effectiveness of sumatriptan in reversal of established pain thus suggests modulation of additional, non-calcitonin gene-related peptide receptor-mediated nociceptive mechanisms. Kappa opioid receptor antagonists may represent a novel preventive therapy for stress-related migraine.
• Taylor, P. C., Simon, L. S., Sebba, A., Quebe, A., Porreca, F., Knopp, K. L., & Choy, E. H. (2021). The Jak/STAT pathway: A focus on pain in rheumatoid arthritis.. Seminars in arthritis and rheumatism, 51(1), 278-284. doi:10.1016/j.semarthrit.2020.10.008
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  Pain is a manifestation of rheumatoid arthritis (RA) that is mediated by inflammatory and non-inflammatory mechanisms and negatively affects quality of life. Recent findings from a Phase 3 clinical trial showed that patients with RA who were treated with a Janus kinase 1 (Jak1) and Janus kinase 2 (Jak2) inhibitor achieved significantly greater improvements in pain than those treated with a tumor necrosis factor blocker; both treatments resulted in similar changes in standard clinical measures and markers of inflammation. These findings suggest that Jak1 and Jak2 inhibition may relieve pain in RA caused by inflammatory and non-inflammatory mechanisms and are consistent with the overarching involvement of the Jak-signal transducer and activator of transcription (Jak/STAT) pathway in mediating the action, expression, and regulation of a multitude of pro- and anti-inflammatory cytokines. In this review, we provide an overview of pain in RA, the underlying importance of cytokines regulated directly or indirectly by the Jak/STAT pathway, and therapeutic targeting of the Jak/STAT pathway in RA. As highlighted herein, multiple cytokines directly or indirectly regulated by the Jak/STAT pathway play important roles in mediating various mechanisms underlying pain in RA. Having a better understanding of these mechanisms may help clinicians make treatment decisions that optimize the control of inflammation and pain.
• Wu, Z., Tymecka, D., Streicher, J. M., Remesic, M., Ramos-colon, C., Porreca, F., Molnar, G., Misicka, A., Lee, Y. S., Lavigne, J., & Hruby, V. J. (2021). Multifunctional Enkephalin Analogs with a New Biological Profile: MOR/DOR Agonism and KOR Antagonism.. Biomedicines, 9(6), 625. doi:10.3390/biomedicines9060625
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  In our previous studies, we developed a series of mixed MOR/DOR agonists that are enkephalin-like tetrapeptide analogs with an N-phenyl-N-piperidin-4-ylpropionamide (Ppp) moiety at the C-terminus. Further SAR study on the analogs, initiated by the findings from off-target screening, resulted in the discovery of LYS744 (6, Dmt-DNle-Gly-Phe(p-Cl)-Ppp), a multifunctional ligand with MOR/DOR agonist and KOR antagonist activity (GTPγS assay: IC50 = 52 nM, Imax = 122% cf. IC50 = 59 nM, Imax = 100% for naloxone) with nanomolar range of binding affinity (Ki = 1.3 nM cf. Ki = 2.4 nM for salvinorin A). Based on its unique biological profile, 6 is considered to possess high therapeutic potential for the treatment of chronic pain by modulating pathological KOR activation while retaining analgesic efficacy attributed to its MOR/DOR agonist activity.
• Chen, Y., Moutal, A., Navratilova, E., Kopruszinski, C., Yue, X., Ikegami, M., Chow, M., Kanazawa, I., Bellampalli, S. S., Xie, J., Patwardhan, A., Rice, K., Fields, H., Akopian, A., Neugebauer, V., Dodick, D., Khanna, R., & Porreca, F. (2020). The prolactin receptor long isoform regulates nociceptor sensitization and opioid-induced hyperalgesia selectively in females. Science translational medicine, 12(529).
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  Pain is more prevalent in women for reasons that remain unclear. We have identified a mechanism of injury-free nociceptor sensitization and opioid-induced hyperalgesia (OIH) promoted by prolactin (PRL) in females. PRL signals through mutually inhibitory long (PRLR-L) and short (PRLR-S) receptor isoforms, and PRLR-S activation induces neuronal excitability. PRL and PRLR expression were higher in females. CRISPR-mediated editing of PRLR-L promoted nociceptor sensitization and allodynia in naïve, uninjured female mice that depended on circulating PRL. Opioids, but not trauma-induced nerve injury, decreased PRLR-L promoting OIH through activation of PRLR-S in female mice. Deletion of both PRLR-L and PRLR-S (total PRLR) prevented, whereas PRLR-L overexpression rescued established OIH selectively in females. Inhibition of circulating PRL with cabergoline, a dopamine D2 agonist, up-regulated PRLR-L and prevented OIH only in females. The PRLR-L isoform therefore confers protection against PRL-promoted pain in females. Limiting PRL/PRLR-S signaling pharmacologically or with gene therapies targeting the PRLR may be effective for reducing pain in a female-selective manner.
• Chen, Y., Navratilova, E., Dodick, D. W., & Porreca, F. (2020). An Emerging Role for Prolactin in Female-Selective Pain. Trends in neurosciences, 43(8), 635-648.
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  Women experience many pain conditions more frequently when compared with men, but the biological mechanisms underlying sex differences in pain remain poorly understood. In particular, little is known about possible sex differences in peripheral nociceptors, the fundamental building blocks of pain transmission. Emerging evidence reveals that prolactin (PRL) signaling at its cognate prolactin receptor (PRLR) in primary afferents promotes nociceptor sensitization and pain in a female-selective fashion. In this review, we summarize recent progress in understanding the female-selective role of PRL/PRLR in nociceptor sensitization and in pathological pain conditions, including postoperative, inflammatory, neuropathic, and migraine pain, as well as opioid-induced hyperalgesia. The clinical implications of the peripheral PRL/PRLR system for the discovery of new therapies for pain control in women are also discussed.
• Dickenson, A. H., Navratilova, E., Patel, R., Porreca, F., & Bannister, K. (2020). Supraspinal Opioid Circuits Differentially Modulate Spinal Neuronal Responses in Neuropathic Rats. Anesthesiology.
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  Descending control from supraspinal neuronal networks onto spinal cord neurons can modulate nociceptionEndogenous opioids in these brain circuits participate in pain modulationA differential opioidergic role for brain nuclei involved in supraspinal pain modulation has not been previously reported WHAT THIS ARTICLE TELLS US THAT IS NEW: In vivo electrophysiologic recordings from the dorsal horn of the spinal cord in male rats reveal differential effects of morphine at the anterior cingulate cortex, right amygdala, and the ventromedial medulla on evoked pain responsesThese data differentiate supraspinal opioid circuit regulation of spinal nociceptive processing and suggest that the regulation of sensory and affective components of pain are likely separate BACKGROUND:: The anterior cingulate cortex and central nucleus of the amygdala connect widely with brainstem nuclei involved in descending modulation, including the rostral ventromedial medulla. Endogenous opioids in these circuits participate in pain modulation. The hypothesis was that a differential opioidergic role for the brain nuclei listed in regulation of spinal neuronal responses because separable effects on pain behaviors in awake animals were previously observed.
• Gee, T. A., Weintraub, N. C., Lu, D., Phelps, C. E., Navratilova, E., Heien, M. L., & Porreca, F. (2020). A pain-induced tonic hypodopaminergic state augments phasic dopamine release in the nucleus accumbens. Pain, 161(10), 2376-2384.
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  Diseases and disorders such as Parkinson disease, schizophrenia, and chronic pain are characterized by altered mesolimbic dopaminergic neurotransmission. Dopamine release in the nucleus accumbens influences behavior through both tonic and phasic signaling. Tonic dopamine levels are hypothesized to inversely regulate phasic signals through dopamine D2 receptor feedback inhibition. We tested this hypothesis directly in the context of ongoing pain. Tonic and phasic dopamine signals were measured using fast-scan controlled-adsorption voltammetry and fast-scan cyclic voltammetry, respectively, in the nucleus accumbens shell of male rats with standardized levels of anesthesia. Application of capsaicin to the cornea produced a transient decrease in tonic dopamine levels. During the pain-induced hypodopaminergic state, electrically evoked phasic dopamine release was significantly increased when compared to baseline, evoked phasic release. A second application of capsaicin to the same eye had a lessened effect on tonic dopamine suggesting desensitization of TRPV1 channels in that eye. Capsaicin treatment in the alternate cornea, however, again produced coincident decreased dopaminergic tone and increased phasic dopamine release. These findings occurred independently of stimulus lateralization relative to the hemisphere of dopamine measurement. Our data show that (1) the mesolimbic dopamine circuit reliably encodes acute noxious stimuli; (2) ongoing pain produces decreases in dopaminergic tone; and (3) pain-induced decreases in tonic dopamine correspond to augmented evoked phasic dopamine release. Enhanced phasic dopamine neurotransmission resulting from salient stimuli may contribute to increased impulsivity and cognitive deficits often observed in conditions associated with decreased dopaminergic tone, including Parkinson disease and chronic pain.
• Kopruszinski, C. M., Navratilova, E., Swiokla, J., Dodick, D. W., Chessell, I. P., & Porreca, F. (2020). A novel, injury-free rodent model of vulnerability for assessment of acute and preventive therapies reveals temporal contributions of CGRP-receptor activation in migraine-like pain. Cephalalgia : an international journal of headache, 333102420959794.
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  Development and characterization of a novel injury-free preclinical model of migraine-like pain allowing mechanistic assessment of both acute and preventive treatments.
• Kopruszinski, C. M., Navratilova, E., Vagnerova, B., Swiokla, J., Patwardhan, A., Dodick, D., & Porreca, F. (2020). Cannabinoids induce latent sensitization in a preclinical model of medication overuse headache. Cephalalgia : an international journal of headache, 40(1), 68-78.
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  Evaluation of cannabinoid receptor agonists in a preclinical model of medication overuse headache.
• Kopruszinski, C. M., Swiokla, J., Lee, Y. S., Navratilova, E., VanderVeen, L., Yang, M., Liu, Y., Miyazaki, T., Schmidt, W. K., Zalevsky, J., & Porreca, F. (2020). Preclinical Assessment of the Analgesic Pharmacology of NKTR-181 in Rodents. Cellular and molecular neurobiology.
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  Pharmacological evaluation of the mu-opioid receptor (MOR) agonist properties of NKTR-181 in rodent models.
• Kopruszinski, C. M., Thornton, P., Arnold, J., Newton, P., Lowne, D., Navratilova, E., Swiokla, J., Dodick, D. W., Dobson, C., Gurrell, I., Chessell, I. P., & Porreca, F. (2020). Characterization and preclinical evaluation of a protease activated receptor 2 (PAR2) monoclonal antibody as a preventive therapy for migraine. Cephalalgia : an international journal of headache, 40(14), 1535-1550.
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  Migraine pain is thought to result from activation of meningeal nociceptors that might involve dural mast cell degranulation and release of proteases and pronociceptive mediators. Tryptase, the most abundant dural mast cell protease, has been demonstrated to stimulate dural mast cells, as well as trigeminal nociceptors by activating the protease activated receptor 2. Mast cell or neuronal protease activated receptors 2 may therefore represent a novel target for migraine treatment. In this study, we characterized and evaluated a novel protease activated receptor 2 monoclonal antibody as a preventive anti-migraine pain therapy in preclinical models.
• Martin, L. F., Patwardhan, A. M., Jain, S. V., Salloum, M. M., Freeman, J., Khanna, R., Gannala, P., Goel, V., Jones-MacFarland, F. N., Killgore, W. D., Porreca, F., & Ibrahim, M. M. (2020). Evaluation of green light exposure on headache frequency and quality of life in migraine patients: A preliminary one-way cross-over clinical trial. Cephalalgia : an international journal of headache, 333102420956711.
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  Pharmacological management of migraine can be ineffective for some patients. We previously demonstrated that exposure to green light resulted in antinociception and reversal of thermal and mechanical hypersensitivity in rodent pain models. Given the safety of green light emitting diodes, we evaluated green light as a potential therapy in patients with episodic or chronic migraine.
• Martin, L., Porreca, F., Mata, E. I., Salloum, M., Goel, V., Gunnala, P., Killgore, W. D., Jain, S., Jones-MacFarland, F. N., Khanna, R., Patwardhan, A., & Ibrahim, M. M. (2020). Green Light Exposure Improves Pain and Quality of Life in Fibromyalgia Patients: A Preliminary One-Way Crossover Clinical Trial. Pain medicine (Malden, Mass.).
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  Fibromyalgia is a functional pain disorder in which patients suffer from widespread pain and poor quality of life. Fibromyalgia pain and its impact on quality of life are not effectively managed with current therapeutics. Previously, in a preclinical rat study, we demonstrated that exposure to green light-emitting diodes (GLED) for 8 hours/day for 5 days resulted in antinociception and reversal of thermal and mechanical hypersensitivity associated with models of injury-related pain. Given the safety of GLED and the ease of its use, our objective is to administer GLED as a potential therapy to patients with fibromyalgia.
• Navratilova, E., Behravesh, S., Oyarzo, J., Dodick, D. W., Banerjee, P., & Porreca, F. (2020). Ubrogepant does not induce latent sensitization in a preclinical model of medication overuse headache. Cephalalgia : an international journal of headache, 40(9), 892-902.
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  Ubrogepant, a small-molecule calcitonin gene-related peptide receptor antagonist, was recently approved as an oral medication for the acute treatment of migraine. This study aimed to determine whether ubrogepant shows efficacy in a preclinical model of migraine-like pain and whether repeated oral administration of ubrogepant induces latent sensitization relevant to medication overuse headache in rats.
• Neugebauer, V., Mazzitelli, M., Cragg, B., Ji, G., Navratilova, E., & Porreca, F. (2020). Amygdala, neuropeptides, and chronic pain-related affective behaviors. Neuropharmacology, 170, 108052.
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  Neuropeptides play important modulatory roles throughout the nervous system, functioning as direct effectors or as interacting partners with other neuropeptide and neurotransmitter systems. Limbic brain areas involved in learning, memory and emotions are particularly rich in neuropeptides. This review will focus on the amygdala, a limbic region that plays a key role in emotional-affective behaviors and pain modulation. The amygdala is comprised of different nuclei; the basolateral (BLA) and central (CeA) nuclei and in between, the intercalated cells (ITC), have been linked to pain-related functions. A wide range of neuropeptides are found in the amygdala, particularly in the CeA, but this review will discuss those neuropeptides that have been explored for their role in pain modulation. Calcitonin gene-related peptide (CGRP) is a key peptide in the afferent nociceptive pathway from the parabrachial area and mediates excitatory drive of CeA neurons. CeA neurons containing corticotropin releasing factor (CRF) and/or somatostatin (SOM) are a source of long-range projections and serve major output functions, but CRF also acts locally to excite neurons in the CeA and BLA. Neuropeptide S (NPS) is associated with inhibitory ITC neurons that gate amygdala output. Oxytocin and vasopressin exert opposite (inhibitory and excitatory, respectively) effects on amygdala output. The opioid system of mu, delta and kappa receptors (MOR, DOR, KOR) and their peptide ligands (β-endorphin, enkephalin, dynorphin) have complex and partially opposing effects on amygdala function. Neuropeptides therefore serve as valuable targets to regulate amygdala function in pain conditions. This article is part of the special issue on Neuropeptides.
• Raghuraman, S., Xie, J. Y., Giacobassi, M. J., Tun, J. O., Chase, K., Lu, D., Teichert, R. W., Porreca, F., & Olivera, B. M. (2020). Chronicling changes in the somatosensory neurons after peripheral nerve injury. Proceedings of the National Academy of Sciences of the United States of America, 117(42), 26414-26421.
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  Current drug discovery efforts focus on identifying lead compounds acting on a molecular target associated with an established pathological state. Concerted molecular changes that occur in specific cell types during disease progression have generally not been identified. Here, we used constellation pharmacology to investigate rat dorsal root ganglion neurons using two models of peripheral nerve injury: chronic constriction injury (CCI) and spinal nerve ligation (SNL). In these well-established models of neuropathic pain, we show that the onset of chronic pain is accompanied by a dramatic, previously unreported increase in the number of bradykinin-responsive neurons, with larger increases observed after SNL relative to CCI. To define the neurons with altered expression, we charted the temporal course of molecular changes following 1, 3, 6, and 14 d after SNL injury and demonstrated that specific molecular changes have different time courses during the progression to a pain state. In particular, ATP receptors up-regulated on day 1 postinjury, whereas the increase in bradykinin receptors was gradual after day 3 postinjury. We specifically tracked changes in two subsets of neurons: peptidergic and nonpeptidergic nociceptors. Significant increases occurred in ATP responses in nAChR-expressing isolectin B4+ nonpeptidergic neurons 1 d postinjury, whereas peptidergic neurons did not display any significant change. We propose that remodeling of ion channels and receptors occurs in a concerted and cell-specific manner, resulting in the appearance of bradykinin-responsive neuronal subclasses that are relevant to chronic pain.
• Rau, J. C., Navratilova, E., Oyarzo, J., Johnson, K. W., Aurora, S. K., Schwedt, T. J., Dodick, D. W., & Porreca, F. (2020). Evaluation of LY573144 (lasmiditan) in a preclinical model of medication overuse headache. Cephalalgia : an international journal of headache, 40(9), 903-912.
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  Medication overuse is a significant issue that complicates the treatment of headache disorders. The most effective medications for the acute treatment of migraine all have the capacity to induce medication overuse headache (MOH). Novel acute migraine-specific treatments are being developed. However, because the mechanism(s) underlying medication overuse headache are not well understood, it is difficult to predict whether any particular acute medication will induce MOH in susceptible individuals. LY573144 (lasmiditan), a 5-HT receptor agonist, has recently been shown to be effective in the acute treatment of migraine in phase 3 trials. The aim of this study is to determine whether frequent administration of lasmiditan induces behaviors consistent with MOH in a pre-clinical rat model.
• Simon, L. S., Taylor, P. C., Choy, E. H., Sebba, A., Quebe, A., Knopp, K. L., & Porreca, F. (2020). The Jak/STAT pathway: A focus on pain in rheumatoid arthritis. Seminars in arthritis and rheumatism, 51(1), 278-284.
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  Pain is a manifestation of rheumatoid arthritis (RA) that is mediated by inflammatory and non-inflammatory mechanisms and negatively affects quality of life. Recent findings from a Phase 3 clinical trial showed that patients with RA who were treated with a Janus kinase 1 (Jak1) and Janus kinase 2 (Jak2) inhibitor achieved significantly greater improvements in pain than those treated with a tumor necrosis factor blocker; both treatments resulted in similar changes in standard clinical measures and markers of inflammation. These findings suggest that Jak1 and Jak2 inhibition may relieve pain in RA caused by inflammatory and non-inflammatory mechanisms and are consistent with the overarching involvement of the Jak-signal transducer and activator of transcription (Jak/STAT) pathway in mediating the action, expression, and regulation of a multitude of pro- and anti-inflammatory cytokines. In this review, we provide an overview of pain in RA, the underlying importance of cytokines regulated directly or indirectly by the Jak/STAT pathway, and therapeutic targeting of the Jak/STAT pathway in RA. As highlighted herein, multiple cytokines directly or indirectly regulated by the Jak/STAT pathway play important roles in mediating various mechanisms underlying pain in RA. Having a better understanding of these mechanisms may help clinicians make treatment decisions that optimize the control of inflammation and pain.
• Ashina, H., Porreca, F., Anderson, T., Amin, F. M., Ashina, M., Schytz, H. W., & Dodick, D. W. (2019). Post-traumatic headache: epidemiology and pathophysiological insights. Nature reviews. Neurology, 15(10), 607-617.
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  Post-traumatic headache (PTH) is a highly disabling secondary headache disorder and one of the most common sequelae of mild traumatic brain injury, also known as concussion. Considerable overlap exists between PTH and common primary headache disorders. The most common PTH phenotypes are migraine-like headache and tension-type-like headache. A better understanding of the pathophysiological similarities and differences between primary headache disorders and PTH could uncover unique treatment targets for PTH. Although possible underlying mechanisms of PTH have been elucidated, a substantial void remains in our understanding, and further research is needed. In this Review, we describe the evidence from animal and human studies that indicates involvement of several potential mechanisms in the development and persistence of PTH. These mechanisms include impaired descending modulation, neurometabolic changes, neuroinflammation and activation of the trigeminal sensory system. Furthermore, we outline future research directions to establish biomarkers involved in progression from acute to persistent PTH, and we identify potential drug targets to prevent and treat persistent PTH.
• Christensen, S. L., Hansen, R. B., Storm, M. A., Olesen, J., Hansen, T. F., Ossipov, M., Izarzugaza, J. M., Porreca, F., & Kristensen, D. M. (2019). Von Frey testing revisited: Provision of an online algorithm for improved accuracy of 50% thresholds. European journal of pain (London, England).
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  In the pain field, it is essential to quantify nociceptive responses. The response to the application of von Frey filaments to the skin measures tactile sensitivity and is a surrogate marker of allodynia in states of peripheral and/or central sensitization. The method is widely used across species within the pain field. However, uncertainties appear to exist regarding the appropriate method for analysing obtained data. Therefore, there is a need for refinement of the calculations for transformation of raw data to quantifiable data.
• Diener, H. C., Dodick, D., Evers, S., Holle, D., Jensen, R. H., Lipton, R. B., Porreca, F., Silberstein, S., & Schwedt, T. (2019). Pathophysiology, prevention, and treatment of medication overuse headache. The Lancet. Neurology, 18(9), 891-902.
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  Regular or frequent use of analgesics and acute antimigraine drugs can increase the frequency of headache, and induce the transition from episodic to chronic headache or medication overuse headache. The 1-year prevalence of this condition in the general population is between 1% and 2%. Medication overuse headache is more common in women and in people with comorbid depression, anxiety, and other chronic pain conditions. Treatment of medication overuse headache has three components. First, patients need education and counselling to reduce the intake of medication for acute headache attacks. Second, some patients benefit from drug withdrawal (discontinuation of the overused medication). Finally, preventive drug therapy and non-medical prevention might be necessary in patients at onset of treatment or in patients who do not respond to the first two steps. The optimal therapeutic approach requires validation in controlled trials.
• Guerrero, M., Urbano, M., Kim, E. K., Gamo, A. M., Riley, S., Abgaryan, L., Leaf, N., Van Orden, L. J., Brown, S. J., Xie, J. Y., Porreca, F., Cameron, M. D., Rosen, H., & Roberts, E. (2019). Design and Synthesis of a Novel and Selective Kappa Opioid Receptor (KOR) Antagonist (BTRX-335140). Journal of medicinal chemistry, 62(4), 1761-1780.
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  κ opioid receptor (KOR) antagonists are potential pharmacotherapies for the treatment of migraine and stress-related mood disorders including depression, anxiety, and drug abuse, thus the development of novel KOR antagonists with an improved potency/selectivity profile and medication-like duration of action has attracted the interest of the medicinal chemistry community. In this paper, we describe the discovery of 1-(6-ethyl-8-fluoro-4-methyl-3-(3-methyl-1,2,4-oxadiazol-5-yl)quinolin-2-yl)- N-(tetrahydro-2 H-pyran-4-yl)piperidin-4 amine (CYM-53093, BTRX-335140) as a potent and selective KOR antagonist, endowed with favorable in vitro ADMET and in vivo pharmacokinetic profiles and medication-like duration of action in rat pharmacodynamic experiments. Orally administered CYM-53093 showed robust efficacy in antagonizing KOR agonist-induced prolactin secretion and in tail-flick analgesia in mice. CYM-53093 exhibited a broad selectivity over a panel of off-target proteins. This compound is in phase 1 clinical trials for the treatment of neuropsychiatric disorders wherein dynorphin is thought to contribute to the underlying pathophysiology.
• Jassar, H., Nascimento, T. D., Kaciroti, N., DosSantos, M. F., Danciu, T., Koeppe, R. A., Smith, Y. R., Bigal, M. E., Porreca, F., Casey, K. L., Zubieta, J. K., & DaSilva, A. F. (2019). Impact of chronic migraine attacks and their severity on the endogenous μ-opioid neurotransmission in the limbic system. NeuroImage. Clinical, 23, 101905.
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  To evaluate, in vivo, the impact of ongoing chronic migraine (CM) attacks on the endogenous μ-opioid neurotransmission.
• Johnstone, T. B., Xie, J. Y., Qu, C., Wasiak, D. J., Hogenkamp, D. J., Porreca, F., & Gee, K. W. (2019). Positive allosteric modulators of nonbenzodiazepine γ-aminobutyric acidA receptor subtypes for the treatment of chronic pain. Pain.
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  Chronic neuropathic pain may be caused, in part, by loss of inhibition in spinal pain processing pathways due to attenuation of local GABAergic tone. Nociception and nocifensive behaviors are reduced after enhancement of tonically activated extrasynaptic GABAAR-mediated currents by agonist ligands for δ subunit-containing GABAARs. However, typical ligands that target δ subunit-containing GABAARs are limited due to sedative effects at higher doses. We used the spinal nerve ligation (SNL) and gp120 models of experimental neuropathic pain to evaluate compound 2-261, a nonbenzodiazepine site positive allosteric modulator of α4β3δ GABAARs optimized to be nonsedative by selective activation of β2/3-subunit-containing GABAARs over receptor subtypes incorporating β1 subunits. Similar levels of 2-261 were detected in the brain and plasma after intraperitoneal administration. Although systemic 2-261 did not alter sensory thresholds in sham-operated animals, it significantly reversed SNL-induced thermal and tactile hypersensitivity in a GABAAR-dependent fashion. Intrathecal 2-261 produced conditioned place preference and elevated dopamine levels in the nucleus accumbens of nerve-injured, but not sham-operated, rats. In addition, systemic pretreatment with 2-261 blocked conditioned place preference from spinal clonidine in SNL rats. Moreover, 2-261 reversed thermal hyperalgesia and partially reversed tactile allodynia in the gp120 model of HIV-related neuropathic pain. The effects of 2-261 likely required interaction with the α4β3δ GABAAR because 2-301, a close structural analog of 2-261 with limited extrasynaptic receptor efficacy, was not active. Thus, 2-261 may produce pain relief with diminished side effects through selective modulation of β2/3-subunit-containing extrasynaptic GABAARs.
• Khanna, R., Patwardhan, A., Yang, X., Li, W., Cai, S., Ji, Y., Chew, L. A., Dorame, A., Bellampalli, S. S., Schmoll, R. W., Gordon, J., Moutal, A., Vanderah, T. W., Porreca, F., & Ibrahim, M. M. (2019). Development and Characterization of An Injury-free Model of Functional Pain in Rats by Exposure to Red Light. The journal of pain : official journal of the American Pain Society, 20(11), 1293-1306.
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  We report the development and characterization of a novel, injury-free rat model in which nociceptive sensitization after red light is observed in multiple body areas reminiscent of widespread pain in functional pain syndromes. Rats were exposed to red light-emitting diodes (RLED) (LEDs, 660 nm) at an intensity of 50 Lux for 8 hours daily for 5 days resulting in time- and dose-dependent thermal hyperalgesia and mechanical allodynia in both male and female rats. Females showed an earlier onset of mechanical allodynia than males. The pronociceptive effects of RLED were mediated through the visual system. RLED-induced thermal hyperalgesia and mechanical allodynia were reversed with medications commonly used for widespread pain, including gabapentin, tricyclic antidepressants, serotonin/norepinephrine reuptake inhibitors, and nonsteroidal anti-inflammatory drugs. Acetaminophen failed to reverse the RLED induced hypersensitivity. The hyperalgesic effects of RLED were blocked when bicuculline, a gamma-aminobutyric acid-A receptor antagonist, was administered into the rostral ventromedial medulla, suggesting a role for increased descending facilitation in the pain pathway. Key experiments were subjected to a replication study with randomization, investigator blinding, inclusion of all data, and high levels of statistical rigor. RLED-induced thermal hyperalgesia and mechanical allodynia without injury offers a novel injury-free rodent model useful for the study of functional pain syndromes with widespread pain. RLED exposure also emphasizes the different biological effects of different colors of light exposure. PERSPECTIVE: This study demonstrates the effect of light exposure on nociceptive thresholds. These biological effects of red LED add evidence to the emerging understanding of the biological effects of light of different colors in animals and humans. Understanding the underlying biology of red light-induced widespread pain may offer insights into functional pain states.
• Khanna, R., Patwardhan, A., Yang, X., Li, W., Cai, S., Ji, Y., Chew, L. A., Dorame, A., Bellampalli, S. S., Schmoll, R. W., Gordon, J., Moutal, A., Vanderah, T. W., Porreca, F., & Ibrahim, M. M. (2019). Erratum to ‛Development and Characterization of An Injury-free Model of Functional Pain in Rats by Exposure to Red Light': The Journal of Pain 20 (2019) 1293-1306. The journal of pain : official journal of the American Pain Society, 20(12), 1509.
• Navratilova, E., & Porreca, F. (2019). Substance P and Inflammatory Pain: Getting It Wrong and Right Simultaneously. Neuron, 101(3), 353-355.
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  How do neuropeptides participate in the classic neuroinflammatory sequelae of tissue injury that includes pain, immune cell infiltration, and swelling? In this issue of Neuron, Green et al. (2019) reveal that MrgprB2/MrgprX2 is a mast cell substance-P-specific receptor that critically links neural and immune systems and offers new approaches for neuroinflammatory therapeutics.
• Navratilova, E., Ji, G., Phelps, C., Qu, C., Hein, M., Yakhnitsa, V., Neugebauer, V., & Porreca, F. (2019). Kappa opioid signaling in the central nucleus of the amygdala promotes disinhibition and aversiveness of chronic neuropathic pain. Pain, 160(4), 824-832.
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  Chronic pain is associated with neuroplastic changes in the amygdala that may promote hyper-responsiveness to mechanical and thermal stimuli (allodynia and hyperalgesia) and/or enhance emotional and affective consequences of pain. Stress promotes dynorphin-mediated signaling at the kappa opioid receptor (KOR) in the amygdala and mechanical hypersensitivity in rodent models of functional pain. Here, we tested the hypothesis that KOR circuits in the central nucleus of the amygdala (CeA) undergo neuroplasticity in chronic neuropathic pain resulting in increased sensory and affective pain responses. After spinal nerve ligation (SNL) injury in rats, pretreatment with a long-acting KOR antagonist, nor-binaltorphimine (nor-BNI), subcutaneously or through microinjection into the right CeA, prevented conditioned place preference (CPP) to intravenous gabapentin, suggesting that nor-BNI eliminated the aversiveness of ongoing pain. By contrast, systemic or intra-CeA administration of nor-BNI had no effect on tactile allodynia in SNL animals. Using whole-cell patch-clamp electrophysiology, we found that nor-BNI decreased synaptically evoked spiking of CeA neurons in brain slices from SNL but not sham rats. This effect was mediated through increased inhibitory postsynaptic currents, suggesting tonic disinhibition of CeA output neurons due to increased KOR activity as a possible mechanism promoting ongoing aversive aspects of neuropathic pain. Interestingly, this mechanism is not involved in SNL-induced mechanical allodynia. Kappa opioid receptor antagonists may therefore represent novel therapies for neuropathic pain by targeting aversive aspects of ongoing pain while preserving protective functions of acute pain.
• Navratilova, E., Nation, K., Remeniuk, B., Neugebauer, V., Bannister, K., Dickenson, A. H., & Porreca, F. (2019). Selective modulation of tonic aversive qualities of neuropathic pain by morphine in the central nucleus of the amygdala requires endogenous opioid signaling in the anterior cingulate cortex. Pain.
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  The amygdala is a key subcortical region believed to contribute to emotional components of pain. As opioid receptors are found in both the central (CeA) and basolateral (BLA) nuclei of the amygdala, we investigated the effects of morphine microinjection on evoked pain responses, pain-motivated behaviors, dopamine release in the nucleus accumbens (NAc), and descending modulation in rats with left-side spinal nerve ligation (SNL). Morphine administered into the right or left CeA had no effect on nerve injury-induced tactile allodynia or mechanical hyperalgesia. Right, but not left, CeA morphine produced conditioned place preference (CPP) and increased extracellular dopamine in the NAc selectively in SNL rats, suggesting relief of aversive qualities of ongoing pain. In SNL rats, CPP and NAc dopamine release following right CeA morphine was abolished by blocking mu opioid receptor signaling in the rostral anterior cingulate cortex (rACC). Right CeA morphine also significantly restored SNL-induced loss of the diffuse noxious inhibitory controls, a spino-bulbo-spinal pain modulatory mechanism, termed conditioned pain modulation in humans. Microinjection of morphine into the BLA had no effects on evoked behaviors and did not produce CPP in nerve-injured rats. These findings demonstrate that the amygdalar action of morphine is specific to the right CeA contralateral to the side of injury and results in enhancement of net descending inhibition. In addition, engagement of mu opioid receptors in the right CeA modulates affective qualities of ongoing pain through endogenous opioid neurotransmission within the rACC, revealing opioid-dependent functional connections from the CeA to the rACC.
• Navratilova, E., Patwardhan, A., & Porreca, F. (2019). Opioid analgesics pass the acid test. Lancet (London, England), 393(10181), 1579-1581.
• Navratilova, E., Rau, J., Oyarzo, J., Tien, J., Mackenzie, K., Stratton, J., Remeniuk, B., Schwedt, T., Anderson, T., Dodick, D., & Porreca, F. (2019). CGRP-dependent and independent mechanisms of acute and persistent post-traumatic headache following mild traumatic brain injury in mice. Cephalalgia : an international journal of headache, 39(14), 1762-1775.
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  Acute and persistent post-traumatic headache are often debilitating consequences of traumatic brain injury. Underlying physiological mechanisms of post-traumatic headache and its persistence remain unknown, and there are currently no approved therapies for these conditions. Post-traumatic headache often presents with a migraine-like phenotype. As calcitonin-gene related peptide promotes migraine headache, we explored the efficacy and timing of intervention with an anti- calcitonin-gene related peptide monoclonal antibody in novel preclinical models of acute post-traumatic headache and persistent post-traumatic headache following a mild traumatic brain injury event in mice.
• Phelps, C. E., Navratilova, E., Dickenson, A. H., Porreca, F., & Bannister, K. (2019). Kappa opioid signaling in the right central amygdala causes hind paw specific loss of diffuse noxious inhibitory controls in experimental neuropathic pain. Pain, 160(7), 1614-1621.
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  Diffuse noxious inhibitory controls (DNICs) is a pain-inhibits-pain phenomenon demonstrated in humans and animals. Diffuse noxious inhibitory control is diminished in many chronic pain states, including neuropathic pain. The efficiency of DNIC has been suggested to prospectively predict both the likelihood of pain chronification and treatment response. Little is known as to why DNIC is dysfunctional in neuropathic pain. Here, we evaluated DNIC in the rat L5/L6 spinal nerve ligation (SNL) model of chronic pain using both behavioral and electrophysiological outcomes. For behavior, nociceptive thresholds were determined using response to noxious paw pressure on both hind paws as the test stimulus before, and after, injection of a conditioning stimulus of capsaicin into the left forepaw. Functionally, the spike firing of spinal wide-dynamic-range neuronal activity was evaluated before and during noxious ear pinch, while stimulating the ipsilateral paw with von Frey hairs of increased bending force. In both assays, the DNIC response was significantly diminished in the ipsilateral (ie, injured) paw of SNL animals. However, behavioral loss of DNIC was not observed on the contralateral (ie, uninjured) paw. Systemic application of nor-binaltorphimine, a kappa opioid antagonist, did not ameliorate SNL-induced hyperalgesia but reversed loss of the behavioral DNIC response. Microinjection of nor-binaltorphimine into the right central amygdala (RCeA) of SNL rats did not affect baseline thresholds but restored DNIC both behaviorally and electrophysiologically. Cumulatively, these data suggest that net enhanced descending facilitations may be mediated by kappa opioid receptor signaling from the right central amygdala to promote diminished DNIC after neuropathy.
• Xie, J. Y., Qu, C., Munro, G., Petersen, K. A., & Porreca, F. (2019). Antihyperalgesic effects of Meteorin in the rat chronic constriction injury model: a replication study. Pain, 160(8), 1847-1855.
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  Data from preclinical research have been suggested to suffer from a lack of inherent reproducibility across laboratories. The goal of our study was to replicate findings from a previous report that demonstrated positive effects of Meteorin, a novel neurotrophic factor, in a rat model of neuropathic pain induced by chronic constriction injury (CCI). Notably, 5 to 6 intermittent subcutaneous (s.c.) injections of Meteorin had been reported to produce reversal of mechanical allodynia/thermal hyperalgesia after injury, wherein maximum efficacy of Meteorin was reached slowly and outlasted the elimination of the compound from the blood by several weeks. Here, we evaluated the efficacy of Meteorin in reversing hindpaw mechanical hyperalgesia and cold allodynia in male, Sprague-Dawley rats with CCI. Nociceptive behavior was monitored before and after CCI, and after drug treatment until day 42 after injury. Systemic administration of recombinant mouse Meteorin (0.5 and 1.8 mg/kg, s.c.) at days 10, 12, 14, 17, and 19 after CCI produced a prolonged reversal of neuropathic hypersensitivity with efficacy comparable with that obtained with gabapentin (100 mg/kg, orally). Despite some protocol deviations (eg, nociceptive endpoint, animal vendor, testing laboratory, investigator, etc.) being incurred, these did not affect study outcome. By paying careful attention to key facets of study design, using bioactive material, and confirming drug exposure, the current data have replicated the salient findings of the previous study, promoting confidence in further advancement of this novel molecule as a potential therapy for neuropathic pain.
• Xie, J. Y., Qu, C., Munro, G., Petersen, K. A., & Porreca, F. (2019). Antihyperalgesic effects of Meteorin in the rat chronic constriction injury model: a replication study. Pain.
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  Data from preclinical research have been suggested to suffer from a lack of inherent reproducibility across laboratories. The goal of our study was to replicate findings from a previous report that demonstrated positive effects of Meteorin, a novel neurotrophic factor, in a rat model of neuropathic pain induced by chronic constriction injury (CCI). Notably, 5 to 6 intermittent subcutaneous (s.c.) injections of Meteorin had been reported to produce reversal of mechanical allodynia/thermal hyperalgesia after injury, wherein maximum efficacy of Meteorin was reached slowly and outlasted the elimination of the compound from the blood by several weeks. Here, we evaluated the efficacy of Meteorin in reversing hindpaw mechanical hyperalgesia and cold allodynia in male, Sprague-Dawley rats with CCI. Nociceptive behavior was monitored before and after CCI, and after drug treatment until day 42 after injury. Systemic administration of recombinant mouse Meteorin (0.5 and 1.8 mg/kg, s.c.) at days 10, 12, 14, 17, and 19 after CCI produced a prolonged reversal of neuropathic hypersensitivity with efficacy comparable with that obtained with gabapentin (100 mg/kg, orally). Despite some protocol deviations (eg, nociceptive endpoint, animal vendor, testing laboratory, investigator, etc.) being incurred, these did not affect study outcome. By paying careful attention to key facets of study design, using bioactive material, and confirming drug exposure, the current data have replicated the salient findings of the previous study, promoting confidence in further advancement of this novel molecule as a potential therapy for neuropathic pain.This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.
• Cowen, S. L., & Porreca, F. (2018). Chronic pain impairs cognitive flexibility and engages novel learning strategies in rats. Pain, 159, 1403-1412.
• Falk, T., Sherman, S. J., Polt, R. L., Porreca, F., Heien, M. L., Parent, K. L., Root, B. K., Bartlett, M. J., & Flores, A. J. (2018). The combination of the opioid glycopeptide MMP-2200 and a NMDA receptor antagonist reduced L-DOPA-induced dyskinesia and MMP-2200 by itself reduced dopamine receptor 2-like agonist-induced dyskinesia. Neuropharmacology, 141, 260-271. doi:10.1016/j.neuropharm.2018.09.005
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  Dopamine (DA)-replacement therapy utilizing l-DOPA is the gold standard symptomatic treatment for Parkinson's disease (PD). A critical complication of this therapy is the development of l-DOPA-induced dyskinesia (LID). The endogenous opioid peptides, including enkephalins and dynorphin, are co-transmitters of dopaminergic, GABAergic, and glutamatergic transmission in the direct and indirect striatal output pathways disrupted in PD, and alterations in expression levels of these peptides and their precursors have been implicated in LID genesis and expression. We have previously shown that the opioid glycopeptide drug MMP-2200 (a.k.a. Lactomorphin), a glycosylated derivative of Leu-enkephalin mediates potent behavioral effects in two rodent models of striatal DA depletion. In this study, the mixed mu-delta agonist MMP-2200 was investigated in standard preclinical rodent models of PD and of LID to evaluate its effects on abnormal involuntary movements (AIMs). MMP-2200 showed antiparkinsonian activity, while increasing l-DOPA-induced limb, axial, and oral (LAO) AIMs by ∼10%, and had no effect on dopamine receptor 1 (D1R)-induced LAO AIMs. In contrast, it markedly reduced dopamine receptor 2 (D2R)-like-induced LAO AIMs. The locomotor AIMs were reduced by MMP-2200 in all three conditions. The N-methyl-d-aspartate receptor (NMDAR) antagonist MK-801 has previously been shown to be anti-dyskinetic, but only at doses that induce parkinsonism. When MMP-2200 was co-administered with MK-801, MK-801-induced pro-parkinsonian activity was suppressed, while a robust anti-dyskinetic effect remained. In summary, the opioid glycopeptide MMP-2200 reduced AIMs induced by a D2R-like agonist, and MMP-2200 modified the effect of MK-801 to result in a potent reduction of l-DOPA-induced AIMs without induction of parkinsonism.
• Flores, A. J., Bartlett, M. J., Root, B. K., Parent, K. L., Heien, M. L., Porreca, F., Polt, R., Sherman, S. J., & Falk, T. (2018). The combination of the opioid glycopeptide MMP-2200 and a NMDA receptor antagonist reduced l-DOPA-induced dyskinesia and MMP-2200 by itself reduced dopamine receptor 2-like agonist-induced dyskinesia. Neuropharmacology, 141, 260-271.
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  Dopamine (DA)-replacement therapy utilizing l-DOPA is the gold standard symptomatic treatment for Parkinson's disease (PD). A critical complication of this therapy is the development of l-DOPA-induced dyskinesia (LID). The endogenous opioid peptides, including enkephalins and dynorphin, are co-transmitters of dopaminergic, GABAergic, and glutamatergic transmission in the direct and indirect striatal output pathways disrupted in PD, and alterations in expression levels of these peptides and their precursors have been implicated in LID genesis and expression. We have previously shown that the opioid glycopeptide drug MMP-2200 (a.k.a. Lactomorphin), a glycosylated derivative of Leu-enkephalin mediates potent behavioral effects in two rodent models of striatal DA depletion. In this study, the mixed mu-delta agonist MMP-2200 was investigated in standard preclinical rodent models of PD and of LID to evaluate its effects on abnormal involuntary movements (AIMs). MMP-2200 showed antiparkinsonian activity, while increasing l-DOPA-induced limb, axial, and oral (LAO) AIMs by ∼10%, and had no effect on dopamine receptor 1 (DR)-induced LAO AIMs. In contrast, it markedly reduced dopamine receptor 2 (DR)-like-induced LAO AIMs. The locomotor AIMs were reduced by MMP-2200 in all three conditions. The N-methyl-d-aspartate receptor (NMDAR) antagonist MK-801 has previously been shown to be anti-dyskinetic, but only at doses that induce parkinsonism. When MMP-2200 was co-administered with MK-801, MK-801-induced pro-parkinsonian activity was suppressed, while a robust anti-dyskinetic effect remained. In summary, the opioid glycopeptide MMP-2200 reduced AIMs induced by a DR-like agonist, and MMP-2200 modified the effect of MK-801 to result in a potent reduction of l-DOPA-induced AIMs without induction of parkinsonism.
• Garami, A., Ibrahim, M., Gilbraith, K., Khanna, R., Pakai, E., Miko, A., Pinter, E., Romanovsky, A. A., Porreca, F., & Patwardhan, A. M. (2018). In Reply. Anesthesiology, 129(2), 378-379.
• Gomtsian, L., Bannister, K., Eyde, N., Robles, D., Dickenson, A. H., Porreca, F., & Navratilova, E. (2018). Morphine effects within the rodent anterior cingulate cortex and rostral ventromedial medulla reveal separable modulation of affective and sensory qualities of acute or chronic pain. Pain, 159(12), 2512-2521.
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  Modulation of pain may result from engagement of opioid receptors in multiple brain regions. Whether sensory and affective qualities of pain are differentially affected by brain opioid receptor circuits remains unclear. We previously reported that opioid actions within the rostral anterior cingulate cortex (ACC) produce selective modulation of affective qualities of neuropathic pain in rodents, but whether such effects may occur in other areas of the ACC is not known. Here, morphine was microinjected into 3 regions of the ACC or into the rostral ventromedial medulla (RVM), and pain behaviors in naive, sham, or spinal nerve ligated (SNL) rats were evaluated. In naive animals, the tail-flick response was inhibited by RVM, but not ACC, morphine. Anterior cingulate cortex morphine did not affect tactile allodynia (the von Frey test) or mechanical (Randall-Selitto) or thermal (Hargreaves) hyperalgesia in spinal nerve ligated rats. In contrary, RVM morphine reduced tactile allodynia and produced both antihyperalgesic and analgesic effects against mechanical and thermal stimuli as well as conditioned place preference selectively in nerve-injured rats. Within the RVM, opioids inhibit nociceptive transmission reflected in both withdrawal thresholds and affective pain behaviors. Activation of mu opioid receptors within specific rostral ACC circuits, however, selectively modulates affective dimensions of ongoing pain without altering withdrawal behaviors. These data suggest that RVM and ACC opioid circuits differentially modulate sensory and affective qualities of pain, allowing for optimal behaviors that promote escape and survival. Targeting specific ACC opioid circuits may allow for treatment of chronic pain while preserving the physiological function of acute pain.
• Nation, K. M., Dodick, D. W., Navratilova, E., & Porreca, F. (2018). Sustained exposure to acute migraine medications combined with repeated noxious stimulation dysregulates descending pain modulatory circuits: Relevance to medication overuse headache. Cephalalgia : an international journal of headache, 333102418804157.
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  Background Loss of conditioned pain modulation/diffuse noxious inhibitory controls has been demonstrated in patients with migraine and medication overuse headache. We hypothesized that exposure to acute migraine medications may lead to dysregulation of central pain modulatory circuits that could be revealed by evaluating diffuse noxious inhibitory controls and that prior noxious stimulus is required for a loss of the diffuse noxious inhibitory control response in rats exposed to these medications. Methods Rats were "primed" by continuous infusion of morphine or one of two doses of sumatriptan. Diffuse noxious inhibitory control was evaluated at the end of drug-priming (day 7) and again after sensory thresholds returned to baseline (day 21). The Randall-Selitto hindpaw pressure test was used as the test stimulus and forepaw capsaicin injection served as the conditioning stimulus. Results Morphine-primed rats showed opioid-induced hyperalgesia accompanied by a loss of diffuse noxious inhibitory controls on day 7. Sumatriptan-primed rats did not develop hyperalgesia or loss of diffuse noxious inhibitory controls on day 7. Morphine-primed and high-dose sumatriptan-primed rats only had a loss of diffuse noxious inhibitory control on day 21 if they received a capsaicin injection on day 7. Conclusions Prolonged exposure to migraine treatments followed by an acute nociceptive stimulation caused long-lasting alterations in descending pain modulation, shown by a loss of diffuse noxious inhibitory controls. Morphine was more detrimental than sumatriptan, consistent with clinical observations of higher medication overuse headache risk with opioids. These data suggest a mechanism of medication overuse headache by which migraine medications combined with repeated episodes of pain may amplify the consequences of nociceptor activation and increase the probability of future migraine attacks as well as risk of medication overuse headache.
• Patel, R., Qu, C., Xie, J. Y., Porreca, F., & Dickenson, A. H. (2018). Selective deficiencies in descending inhibitory modulation in neuropathic rats: implications for enhancing noradrenergic tone. Pain, 159(9), 1887-1899.
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  Pontine noradrenergic neurones form part of a descending inhibitory system that influences spinal nociceptive processing. Weak or absent descending inhibition is a common feature of chronic pain patients. We examined the extent to which the descending noradrenergic system is tonically active, how control of spinal neuronal excitability is integrated into thalamic relays within sensory-discriminative projection pathways, and how this inhibitory control is altered after nerve injury. In vivo electrophysiology was performed in anaesthetised spinal nerve-ligated (SNL) and sham-operated rats to record from wide dynamic range neurones in the ventral posterolateral thalamus (VPL). In sham rats, spinal block of α2-adrenoceptors with atipamezole resulted in enhanced stimulus-evoked and spontaneous firing in the VPL, and produced conditioned place avoidance. However, in SNL rats, these conditioned avoidance behaviours were absent. Furthermore, inhibitory control of evoked neuronal responses was lost, but spinal atipamezole markedly increased spontaneous firing. Augmenting spinal noradrenergic tone in neuropathic rats with reboxetine, a selective noradrenergic reuptake inhibitor, modestly reinstated inhibitory control of evoked responses in the VPL but had no effect on spontaneous firing. By contrast, clonidine, an α2 agonist, inhibited both evoked and spontaneous firing, and exhibited increased potency in SNL rats compared with sham controls. These data suggest descending noradrenergic inhibitory pathways are tonically active in sham rats. Moreover, in neuropathic states, descending inhibitory control is diminished, but not completely absent, and distinguishes between spontaneous and evoked neuronal activity. These observations may have implications for how analgesics targeting the noradrenergic system provide relief.
• Porreca, F. (2018). Activation of ventral tegmental area dopaminergic neurons reverses pathological allodynia resulting from nerve injury or bone cancer. Molecular Pain, 14, 1-11.
• Porreca, F. (2018). Lateralized kappa opioid receptor signaling from the amygdala central nucleus promotes stress-induced functional pain. pain, 159, 919-928.
• Porreca, F. (2018). Nanoparticulate Peptide Delivery Exclusively to the Brain Produces Tolerance Free Analgesia. Journal of Controlled Release, 270, 135-144.
• Porreca, F. (2018). The opioid crisis and… reconsidering the use of drugs that affect body temperature. Temperatures, 5, 1-3.
• Remeniuk, B., King, T., Suktankhar, D., Nippert, A., Li, N., Li, F., Cheng, K., Rice, K. C., & Porreca, F. (2018). Disease modifying actions of IL-6 blockade in a rat model of bone cancer pain. Pain.
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  Metastasis of cancer to the skeleton represents a debilitating turning point in the lives of patients. Skeletal metastasis leads to moderate to severe ongoing pain along with bone remodeling that can result in fracture, events that dramatically diminish quality of life. IL-6 levels are elevated in metastatic breast cancer patients and are associated with a lower survival rate. We therefore determined the consequences of inhibition of IL-6 signaling using a novel small molecule antagonist, TB-2-081, on bone integrity, tumor progression, and pain in a rodent model of breast cancer. Rat MAT B III mammary adenocarcinoma cells were injected and sealed within the tibia of female Fischer rats. Growth of these cells within the rat tibia elicited increased IL-6 levels both within the bone exudate and in the plasma, produced ongoing pain and evoked hypersensitivity, and bone fracture that was observed by approximately day 12. Systemic TB-2-081 delivered by subcutaneous osmotic mini-pumps starting at tumor implantation prevented tumor-induced ongoing bone pain and evoked hypersensitivity without altering tumor growth. Remarkably, TB-2-081 infusion significantly reduced osteolytic and osteoblastic bone remodeling and time to fracture likely by decreasing osteoclastogenesis and associated increase in bone resorption. These findings indicate that blockade of IL-6 signaling may represent a viable, disease-modifying strategy to prevent tumor-induced bone remodeling allowing for stabilization of bone and decreased fractures as well as diminished ongoing pain that may improve quality of life of patients with skeletal metastases. Notably, anti-IL-6 antibodies are clinically available allowing rapid testing of these possibilities in humans.
• Remesic, M., Macedonio, G., Mollica, A., Porreca, F., Hruby, V., & Lee, Y. S. (2018). Cyclic biphalin analogues with a novel linker lead to potent agonist activities at mu, delta, and kappa opioid receptors. Bioorganic & medicinal chemistry, 26(12), 3664-3667.
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  In an effort to improve biphalin's potency and efficacy at the µ-(MOR) and δ-opioid receptors (DOR), a series of cyclic biphalin analogues 1-5 with a cystamine or piperazine linker at the C-terminus were designed and synthesized by solution phase synthesis using Boc-chemistry. Interestingly, all of the analogues showed balanced opioid agonist activities at all opioid receptor subtypes due to enhanced κ-opioid receptor (KOR) activity. Our results indicate that C-terminal flexible linkers play an important role in KOR activity compared to that of the other cyclic biphalin analogues with a hydrazine linker. Among them, analogue 5 is a potent (Ki = 0.27, 0.46, and 0.87 nM; EC = 3.47, 1.45, and 13.5 nM at MOR, DOR, and KOR, respectively) opioid agonist with high efficacy. Based on the high potency and efficacy at the three opioid receptor subtypes, the ligand is expected to have a potential synergistic effect on relieving pain and further studies including in vivo tests are worthwhile.
• Schiene, K., Schröder, W., Linz, K., Frosch, S., Tzschentke, T. M., Christoph, T., Xie, J. Y., & Porreca, F. (2018). Inhibition of experimental visceral pain in rodents by cebranopadol. Behavioural pharmacology.
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  The aim of this study was to investigate the efficacy of cebranopadol in two rodent models of visceral pain. Cebranopadol is a first-in-class analgesic with agonist activity at the nociceptin/orphanin FQ opioid peptide receptor and classical µ-, δ- and κ-opioid peptide receptors. Colitis was induced in Naval Medical Research Institute mice by intra-rectal infusion of mustard oil. The effects of intravenous cebranopadol pretreatment on spontaneous pain behaviours and referred allodynia and hyperalgesia were assessed. Pancreatitis was induced in Sprague-Dawley rats by intravenous administration of dibutyltin dichloride. After 6 days, the effects of intravenous cebranopadol on withdrawal reactions to mechanical abdominal stimulation with von Frey filaments were assessed. In mice with experimental colitis, cebranopadol dose-dependently inhibited spontaneous pain behaviours and allodynic and hyperalgesic withdrawal reactions, with half-maximal effective dose values of 4.6 µg/kg [95% confidence interval (CI): 2.9-7.9] for inhibition of spontaneous pain behaviours, 2.2 µg/kg (95% CI: 1.3-3.4) for inhibition of referred allodynia and 2.4 µg/kg (95% CI: 1.4-3.6) for inhibition of referred hyperalgesia in mice with colitis. In rats with experimental pancreatitis, cebranopadol dose-dependently inhibited abdominal tactile allodynia (half-maximal effective dose, 0.13 µg/kg; 95% CI: 0.03-0.49). Behavioural manifestations of visceral pain were almost completely abolished at the highest doses tested in mice (17.2 µg/kg, intravenous) and rats (2.4 µg/kg, intravenous). We conclude that cebranopadol is a potent and effective antiallodynic and antihyperalgesic agent in rodent models of visceral pain.
• Snyder, L. M., Chiang, M. C., Loeza-Alcocer, E., Omori, Y., Hachisuka, J., Sheahan, T. D., Gale, J. R., Adelman, P. C., Sypek, E. I., Fulton, S. A., Friedman, R. L., Wright, M. C., Duque, M. G., Lee, Y. S., Hu, Z., Huang, H., Cai, X., Meerschaert, K. A., Nagarajan, V., , Hirai, T., et al. (2018). Kappa Opioid Receptor Distribution and Function in Primary Afferents. Neuron, 99(6), 1274-1288.e6.
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  Primary afferents are known to be inhibited by kappa opioid receptor (KOR) signaling. However, the specific types of somatosensory neurons that express KOR remain unclear. Here, using a newly developed KOR-cre knockin allele, viral tracing, single-cell RT-PCR, and ex vivo recordings, we show that KOR is expressed in several populations of primary afferents: a subset of peptidergic sensory neurons, as well as low-threshold mechanoreceptors that form lanceolate or circumferential endings around hair follicles. We find that KOR acts centrally to inhibit excitatory neurotransmission from KOR-cre afferents in laminae I and III, and this effect is likely due to KOR-mediated inhibition of Ca influx, which we observed in sensory neurons from both mouse and human. In the periphery, KOR signaling inhibits neurogenic inflammation and nociceptor sensitization by inflammatory mediators. Finally, peripherally restricted KOR agonists selectively reduce pain and itch behaviors, as well as mechanical hypersensitivity associated with a surgical incision. These experiments provide a rationale for the use of peripherally restricted KOR agonists for therapeutic treatment.
• Watanabe, M., Sugiura, Y., Sugiyama, E., Narita, M., Navratilova, E., Kondo, T., Uchiyama, N., Yamanaka, A., Kuzumaki, N., Porreca, F., & Narita, M. (2018). [EXPRESS] Extracellular N-acetylaspartylglutamate released in the nucleus accumbens modulates the pain sensation: analysis using a microdialysis/mass spectrometry integrated system. Molecular pain, 1744806918754934.
• Bannister, K., Qu, C., Navratilova, E., Oyarzo, J., Xie, J. Y., King, T., Dickenson, A. H., & Porreca, F. (2017). Multiple sites and actions of gabapentin-induced relief of ongoing experimental neuropathic pain. Pain, 158(12), 2386-2395.
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  Gabapentin (GBP) is a first-line therapy for neuropathic pain, but its mechanisms and sites of action remain uncertain. We investigated GBP-induced modulation of neuropathic pain following spinal nerve ligation (SNL) in rats. Intravenous or intrathecal GBP reversed evoked mechanical hypersensitivity and produced conditioned place preference (CPP) and dopamine (DA) release in the nucleus accumbens (NAc) selectively in SNL rats. Spinal GBP also significantly inhibited dorsal horn wide-dynamic-range neuronal responses to a range of evoked stimuli in SNL rats. By contrast, GBP microinjected bilaterally into the rostral anterior cingulate cortex (rACC), produced CPP, and elicited NAc DA release selectively in SNL rats but did not reverse tactile allodynia and had marginal effects on wide-dynamic-range neuronal activity. Moreover, blockade of endogenous opioid signaling in the rACC prevented intravenous GBP-induced CPP and NAc DA release but failed to block its inhibition of tactile allodynia. Gabapentin, therefore, can potentially act to produce its pain relieving effects by (a) inhibition of injury-induced spinal neuronal excitability, evoked hypersensitivity, and ongoing pain and (b) selective supraspinal modulation of affective qualities of pain, without alteration of reflexive behaviors. Consistent with previous findings of pain relief from nonopioid analgesics, GBP requires engagement of rACC endogenous opioid circuits and downstream activation of mesolimbic reward circuits reflected in learned pain-motivated behaviors. These findings support the partial separation of sensory and affective dimensions of pain in this experimental model and suggest that modulation of affective-motivational qualities of pain may be the preferential mechanism of GBP's analgesic effects in patients.
• Chichorro, J. G., Porreca, F., & Sessle, B. (2017). Mechanisms of craniofacial pain. Cephalalgia : an international journal of headache, 37(7), 613-626.
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  Aim To provide an overview of mechanisms underlying craniofacial pain; to highlight peripheral and central adaptations that may promote chronification of pain in craniofacial pain states such as migraine and temporomandibular disorders (TMD). Background Pain is a common symptom associated with disorders involving craniofacial tissues including the teeth and their supporting structures, the temporomandibular joint and the muscles of the head. Most acute painful craniofacial conditions are easily recognized and well managed, but others, especially those that are chronic (e.g., migraine, TMD and trigeminal neuropathies), present clinical challenges. Preclinical studies have provided substantial information about the anatomical and physiological mechanisms related to the initiation and modulation of nociceptive signals in the trigeminal system. While knowledge of the mechanisms underlying chronic craniofacial pain remains limited, both clinical and preclinical investigations suggest that changes in afferent inputs to the brain as well as in brain structure and modulatory pathways occur in chronic pain. Collectively, these changes result in amplification of nociception that promotes and sustains craniofacial chronic pain states. Conclusions The increased understanding gained of the physiological and pathological processing of nociception in the trigeminal system has provided new perspectives for the mechanistic understanding of acute craniofacial pain conditions and the peripheral and central adaptations that are related to pain chronification. Such knowledge may contribute to improvements in currently available treatments as well as to the development of novel analgesic therapies.
• Garami, A., Ibrahim, M., Gilbraith, K., Khanna, R., Pakai, E., Miko, A., Pinter, E., Romanovsky, A. A., Porreca, F., & Patwardhan, A. M. (2017). Transient Receptor Potential Vanilloid 1 Antagonists Prevent Anesthesia-induced Hypothermia and Decrease Postincisional Opioid Dose Requirements in Rodents. Anesthesiology, 127(5), 813-823.
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  Intraoperative hypothermia and postoperative pain control are two important clinical challenges in anesthesiology. Transient receptor potential vanilloid 1 has been implicated both in thermoregulation and pain. Transient receptor potential vanilloid 1 antagonists were not advanced as analgesics in humans in part due to a side effect of hyperthermia. This study tested the hypothesis that a single, preincision injection of a transient receptor potential vanilloid 1 antagonist could prevent anesthesia-induced hypothermia and decrease the opioid requirement for postsurgical hypersensitivity.
• Godfrey, L., Iannitelli, A., Garrett, N. L., Moger, J., Imbert, I., King, T., Porreca, F., Soundararajan, R., Lalatsa, A., Schätzlein, A. G., & Uchegbu, I. F. (2017). Nanoparticulate peptide delivery exclusively to the brain produces tolerance free analgesia. Journal of controlled release : official journal of the Controlled Release Society, 270, 135-144.
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  The delivery of peptide drugs to the brain is challenging, principally due to the blood brain barrier and the low metabolic stability of peptides. Exclusive delivery to the brain with no peripheral exposure has hitherto not been demonstrated with brain quantification data. Here we show that polymer nanoparticles encapsulating leucine5-enkephalin hydrochloride (LENK) are able to transport LENK exclusively to the brain via the intranasal route, with no peripheral exposure and nanoparticle localisation is observed within the brain parenchyma. Animals dosed with LENK nanoparticles (NM0127) showed a strong anti-nociceptive response in multiple assays of evoked and on going pain whereas animals dosed intranasally with LENK alone were unresponsive. Animals did not develop tolerance to the anti-hyperalgesic activity of NM0127 and NM0127 was active in morphine tolerant animals. A microparticulate formulation of clustered nanoparticles was prepared to satisfy regulatory requirements for nasal dosage forms and the polymer nanoparticles alone were found to be biocompatible, via the nasal route, on chronic dosing.
• Havelin, J., Imbert, I., Sukhtankar, D., Remeniuk, B., Pelletier, I., Gentry, J., Okun, A., Tiutan, T., Porreca, F., & King, T. E. (2017). Mediation of Movement-Induced Breakthrough Cancer Pain by IB4-Binding Nociceptors in Rats. The Journal of neuroscience : the official journal of the Society for Neuroscience, 37(20), 5111-5122.
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  Cancer-induced bone pain is characterized by moderate to severe ongoing pain that commonly requires the use of opiates. Even when ongoing pain is well controlled, patients can suffer breakthrough pain (BTP), episodic severe pain that "breaks through" the medication. We developed a novel model of cancer-induced BTP using female rats with mammary adenocarcinoma cells sealed within the tibia. We demonstrated previously that rats with bone cancer learn to prefer a context paired with saphenous nerve block to elicit pain relief (i.e., conditioned place preference, CPP), revealing the presence of ongoing pain. Treatment with systemic morphine abolished CPP to saphenous nerve block, demonstrating control of ongoing pain. Here, we show that pairing BTP induced by experimenter-induced movement of the tumor-bearing hindlimb with a context produces conditioned place avoidance (CPA) in rats treated with morphine to control ongoing pain, consistent with clinical observation of BTP. Preventing movement-induced afferent input by saphenous nerve block before, but not after, hindlimb movement blocked movement-induced BTP. Ablation of isolectin B4 (IB4)-binding, but not TRPV1+, sensory afferents eliminated movement-induced BTP, suggesting that input from IB4-binding fibers mediates BTP. Identification of potential molecular targets specific to this population of fibers may allow for the development of peripherally restricted analgesics that control BTP and improve quality of life in patients with skeletal metastases.SIGNIFICANCE STATEMENT We present a novel preclinical measure of movement-induced breakthrough pain (BTP) that is observed in the presence of morphine controlling ongoing pain. Blockade of sensory input before movement prevented BTP, whereas nerve block after movement failed to reverse BTP. These observations indicate that blocking peripheral sensory input may prevent BTP and targeting central sites may be required for pain relief once BTP has been initiated. Preventing sensory input from TRPV1-expressing fibers failed to alter movement-induced BTP. In contrast, preventing sensory input from isolectin B4 (IB4)-binding fibers blocked movement-induced BTP. Therefore, examining molecular targets on this population of nociceptive fibers may prove useful for developing an improved strategy for preventing BTP in cancer patients with skeletal metastases.
• Ibrahim, M. M., Patwardhan, A., Gilbraith, K. B., Moutal, A., Yang, X., Chew, L. A., Largent-Milnes, T., Malan, T. P., Vanderah, T. W., Porreca, F., & Khanna, R. (2017). Long-lasting antinociceptive effects of green light in acute and chronic pain in rats. Pain, 158(2), 347-360.
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  Treatments for chronic pain are inadequate, and new options are needed. Nonpharmaceutical approaches are especially attractive with many potential advantages including safety. Light therapy has been suggested to be beneficial in certain medical conditions such as depression, but this approach remains to be explored for modulation of pain. We investigated the effects of light-emitting diodes (LEDs), in the visible spectrum, on acute sensory thresholds in naive rats as well as in experimental neuropathic pain. Rats receiving green LED light (wavelength 525 nm, 8 h/d) showed significantly increased paw withdrawal latency to a noxious thermal stimulus; this antinociceptive effect persisted for 4 days after termination of last exposure without development of tolerance. No apparent side effects were noted and motor performance was not impaired. Despite LED exposure, opaque contact lenses prevented antinociception. Rats fitted with green contact lenses exposed to room light exhibited antinociception arguing for a role of the visual system. Antinociception was not due to stress/anxiety but likely due to increased enkephalins expression in the spinal cord. Naloxone reversed the antinociception, suggesting involvement of central opioid circuits. Rostral ventromedial medulla inactivation prevented expression of light-induced antinociception suggesting engagement of descending inhibition. Green LED exposure also reversed thermal and mechanical hyperalgesia in rats with spinal nerve ligation. Pharmacological and proteomic profiling of dorsal root ganglion neurons from green LED-exposed rats identified changes in calcium channel activity, including a decrease in the N-type (CaV2.2) channel, a primary analgesic target. Thus, green LED therapy may represent a novel, nonpharmacological approach for managing pain.
• Moutal, A., Yang, X., Li, W., Gilbraith, K. B., Luo, S., Cai, S., François-Moutal, L., Chew, L. A., Yeon, S. K., Bellampalli, S. S., Qu, C., Xie, J. Y., Ibrahim, M. M., Khanna, M., Park, K. D., Porreca, F., & Khanna, R. (2017). CRISPR/Cas9 editing of Nf1 gene identifies CRMP2 as a therapeutic target in neurofibromatosis type 1-related pain that is reversed by (S)-Lacosamide. Pain, 158(12), 2301-2319.
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  Neurofibromatosis type 1 (NF1) is a rare autosomal dominant disease linked to mutations of the Nf1 gene. Patients with NF1 commonly experience severe pain. Studies on mice with Nf1 haploinsufficiency have been instructive in identifying sensitization of ion channels as a possible cause underlying the heightened pain suffered by patients with NF1. However, behavioral assessments of Nf1 mice have led to uncertain conclusions about the potential causal role of Nf1 in pain. We used the clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (CRISPR/Cas9) genome editing system to create and mechanistically characterize a novel rat model of NF1-related pain. Targeted intrathecal delivery of guide RNA/Cas9 nuclease plasmid in combination with a cationic polymer was used to generate allele-specific C-terminal truncation of neurofibromin, the protein encoded by the Nf1 gene. Rats with truncation of neurofibromin, showed increases in voltage-gated calcium (specifically N-type or CaV2.2) and voltage-gated sodium (particularly tetrodotoxin-sensitive) currents in dorsal root ganglion neurons. These gains-of-function resulted in increased nociceptor excitability and behavioral hyperalgesia. The cytosolic regulatory protein collapsin response mediator protein 2 (CRMP2) regulates activity of these channels, and also binds to the targeted C-terminus of neurofibromin in a tripartite complex, suggesting a possible mechanism underlying NF1 pain. Prevention of CRMP2 phosphorylation with (S)-lacosamide resulted in normalization of channel current densities, excitability, as well as of hyperalgesia following CRISPR/Cas9 truncation of neurofibromin. These studies reveal the protein partners that drive NF1 pain and suggest that CRMP2 is a key target for therapeutic intervention.
• Remesic, M., Hruby, V. J., Porreca, F., & Lee, Y. S. (2017). Recent Advances in the Realm of Allosteric Modulators for Opioid Receptors for Future Therapeutics. ACS chemical neuroscience, 8(6), 1147-1158.
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  Opioids, and more specifically μ-opioid receptor (MOR) agonists such as morphine, have long been clinically used as therapeutics for severe pain states but often come with serious side effects such as addiction and tolerance. Many studies have focused on bringing about analgesia from the MOR with attenuated side effects, but its underlying mechanism is not fully understood. Recently, focus has been geared toward the design and elucidation of the orthosteric site with ligands of various biological profiles and mixed subtype opioid activities and selectivities, but targeting the allosteric site is an area of increasing interest. It has been shown that allosteric modulators play key roles in influencing receptor function such as its tolerance to a ligand and affect downstream pathways. There has been a high variance of chemical structures that provide allosteric modulation at a given receptor, but recent studies and reviews tend to focus on the altered cellular mechanisms instead of providing a more rigorous description of the allosteric ligand's structure-function relationship. In this review, we aim to explore recent developments in the structural motifs that potentiate orthosteric binding and their influences on cellular pathways in an effort to present novel approaches to opioid therapeutic design.
• Vardanyan, R. S., Cain, J. P., Haghighi, S. M., Kumirov, V. K., McIntosh, M. I., Sandweiss, A. J., Porreca, F., & Hruby, V. J. (2017). Synthesis and Investigation of Mixed μ-Opioid and δ-Opioid Agonists as Possible Bivalent Ligands for Treatment of Pain. Journal of heterocyclic chemistry, 54(2), 1228-1235.
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  Several studies have suggested functional association between μ-opioid and δ-opioid receptors and showed that μ-activity could be modulated by δ-ligands. The general conclusion is that agonists for the δ-receptor can enhance the analgesic potency and efficacy of μ-agonists. Our preliminary investigations demonstrate that new bivalent ligands constructed from the μ-agonist fentanyl and the δ-agonist enkephalin-like peptides are promising entities for creation of new analgesics with reduced side effects for treatment of neuropathic pain. A new superposition of the mentioned pharmacophores led to novel μ-bivalent/δ-bivalent compounds that demonstrate both μ-opioid and δ-opioid receptor agonist activity and high efficacy in anti-inflammatory and neuropathic pain models with the potential of reduced unwanted side effects.
• Xie, J. Y., De Felice, M., Kopruszinski, C. M., Eyde, N., LaVigne, J., Remeniuk, B., Hernandez, P., Yue, X., Goshima, N., Ossipov, M., King, T., Streicher, J. M., Navratilova, E., Dodick, D., Rosen, H., Roberts, E., & Porreca, F. (2017). Kappa opioid receptor antagonists: A possible new class of therapeutics for migraine prevention. Cephalalgia : an international journal of headache, 37(8), 780-794.
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  Background Stress is the most commonly reported migraine trigger. Dynorphin, an endogenous opioid peptide acting preferentially at kappa opioid receptors (KORs), is a key mediator of stress responses. The aim of this study was to use an injury-free rat model of functional cephalic pain with features of migraine and medication overuse headache (MOH) to test the possible preventive benefit of KOR blockade on stress-induced cephalic pain. Methods Following sumatriptan priming to model MOH, rats were hyper-responsive to environmental stress, demonstrating delayed cephalic and extracephalic allodynia and increased levels of CGRP in the jugular blood, consistent with commonly observed clinical outcomes during migraine. Nor-binaltorphimine (nor-BNI), a long-acting KOR antagonist or CYM51317, a novel short-acting KOR antagonist, were given systemically either during sumatriptan priming or immediately before environmental stress challenge. The effects of KOR blockade in the amygdala on stress-induced allodynia was determined by administration of nor-BNI into the right or left central nucleus of the amygdala (CeA). Results KOR blockade prevented both stress-induced allodynia and increased plasma CGRP. Stress increased dynorphin content and phosphorylated KOR in both the left and right CeA in sumatriptan-primed rats. However, KOR blockade only in the right CeA prevented stress-induced cephalic allodynia as well as extracephalic allodynia, measured in either the right or left hindpaws. U69,593, a KOR agonist, given into the right, but not the left, CeA, produced allodynia selectively in sumatriptan-primed rats. Both stress and U69,593-induced allodynia were prevented by right CeA U0126, a mitogen-activated protein kinase inhibitor, presumably acting downstream of KOR. Conclusions Our data reveal a novel lateralized KOR circuit that mediated stress-induced cutaneous allodynia and increased plasma CGRP in an injury-free model of functional cephalic pain with features of migraine and medication overuse headache. Selective, small molecule, orally available, and reversible KOR antagonists are currently in development and may represent a novel class of preventive therapeutics for migraine.
• Andrews, N. A., Latrémolière, A., Basbaum, A. I., Mogil, J. S., Porreca, F., Rice, A. S., Woolf, C. J., Currie, G. L., Dworkin, R. H., Eisenach, J. C., Evans, S., Gewandter, J. S., Gover, T. D., Handwerker, H., Huang, W., Iyengar, S., Jensen, M. P., Kennedy, J. D., Lee, N., , Levine, J., et al. (2016). Ensuring transparency and minimization of methodologic bias in preclinical pain research: PPRECISE considerations. Pain, 157(4), 901-9.
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  There is growing concern about lack of scientific rigor and transparent reporting across many preclinical fields of biological research. Poor experimental design and lack of transparent reporting can result in conscious or unconscious experimental bias, producing results that are not replicable. The Analgesic, Anesthetic, and Addiction Clinical Trial Translations, Innovations, Opportunities, and Networks (ACTTION) public-private partnership with the U.S. Food and Drug Administration sponsored a consensus meeting of the Preclinical Pain Research Consortium for Investigating Safety and Efficacy (PPRECISE) Working Group. International participants from universities, funding agencies, government agencies, industry, and a patient advocacy organization attended. Reduction of publication bias, increasing the ability of others to faithfully repeat experimental methods, and increased transparency of data reporting were specifically discussed. Parameters deemed essential to increase confidence in the published literature were clear, specific reporting of an a priori hypothesis and definition of primary outcome measure. Power calculations and whether measurement of minimal meaningful effect size to determine these should be a core component of the preclinical research effort provoked considerable discussion, with many but not all agreeing. Greater transparency of reporting should be driven by scientists, journal editors, reviewers, and grant funders. The conduct of high-quality science that is fully reported should not preclude novelty and innovation in preclinical pain research, and indeed, any efforts that curtail such innovation would be misguided. We believe that to achieve the goal of finding effective new treatments for patients with pain, the pain field needs to deal with these challenging issues.
• Hall, S. M., LeBaron, L., Ramos-Colon, C. N., Qu, C., Xie, J. Y., Porreca, F., Lai, J., Lee, Y. S., & Hruby, V. J. (2016). Discovery of Stable Non-opioid Dynorphin A Analogues Interacting at the Bradykinin Receptors for the Treatment of Neuropathic Pain. ACS chemical neuroscience.
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  Dynorphin A (Dyn A) is a unique endogenous ligand that possesses well-known neuroinhibitory effects via opioid receptors with preference at the kappa receptor but also neuroexcitatory effects, which cause hyperalgesia. We have shown that the neuroexcitatory effects are mediated through bradykinin receptors (BK receptors) and that intrathecal (i.th.) administration of our lead ligand 1, [Des-Arg7]-Dyn A-(4-11), which shows good binding affinity (IC50 = 150 nM) at the BK receptors, blocks Dyn A-induced hyperalgesia in naïve animals and reverses thermal and tactile hypersensitivities in a dose-dependent manner in nerve injured animals. However, 1 has a serious drawback as a potential drug candidate for the treatment of neuropathic pain due to its susceptibility to enzymatic degradation. In an effort to increase the stability, we modified ligand 1 using non-natural amino acids and found that analogues substituted at or near the N-terminus with a D-isomer retain binding at the receptor as well as provide a large increase in stability. In particular when Leu5 was modified, by either the D-isomer or N-methylation, there was a large increase in stability (t1/2= 0.7 h to 160 h in rat plasma) observed. From these studies, we have developed a very stable Dyn A analogue 16, [DLeu5, des-Arg7]-Dyn A-(4-11), that binds to BK receptors (IC50 = 130 nM) in the same range as ligand 1 and shows good anti-hyperalgesic effects both in naïve rats and L5/L6 spinal nerve ligation (SNL) rats.
• Havelin, J., Imbert, I., Cormier, J., Allen, J., Porreca, F., & King, T. (2016). Central Sensitization and Neuropathic Features of Ongoing Pain in a Rat Model of Advanced Osteoarthritis. The journal of pain : official journal of the American Pain Society, 17(3), 374-82.
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  Osteoarthritis (OA) pain is most commonly characterized by movement-triggered joint pain. However, in advanced disease, OA pain becomes persistent, ongoing and resistant to treatment with nonsteroidal anti-inflammatory drugs (NSAIDs). The mechanisms underlying ongoing pain in advanced OA are poorly understood. We recently showed that intra-articular (i.a.) injection of monosodium iodoacetate (MIA) into the rat knee joint produces concentration-dependent outcomes. Thus, a low dose of i.a. MIA produces NSAID-sensitive weight asymmetry without evidence of ongoing pain and a high i.a. MIA dose produces weight asymmetry and NSAID-resistant ongoing pain. In the present study, palpation of the ipsilateral hind limb of rats treated 14 days previously with high, but not low, doses of i.a. MIA produced expression of the early oncogene, FOS, in the spinal dorsal horn. Inactivation of descending pain facilitatory pathways using a microinjection of lidocaine within the rostral ventromedial medulla induced conditioned place preference selectively in rats treated with the high dose of MIA. Conditioned place preference to intra-articular lidocaine was blocked by pretreatment with duloxetine (30 mg/kg, intraperitoneally at -30 minutes). These observations are consistent with the likelihood of a neuropathic component of OA that elicits ongoing, NSAID-resistant pain and central sensitization that is mediated, in part, by descending modulatory mechanisms. This model provides a basis for exploration of underlying mechanisms promoting neuropathic components of OA pain and for the identification of mechanisms that might guide drug discovery for treatment of advanced OA pain without the need for joint replacement.
• Hitomi, S., Kross, K., Kurose, M., Porreca, F., & Meng, I. D. (2016). Activation of dura-sensitive trigeminal neurons and increased c-Fos protein induced by morphine withdrawal in the rostral ventromedial medulla. Cephalalgia : an international journal of headache.
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  Overuse of medications used to treat migraine headache can increase the frequency of headaches. Sudden abstinence from migraine medication can also lead to a period of withdrawal-induced headaches. The aim of this study was to examine the effect of morphine withdrawal localized to the rostral ventromedial medulla (RVM) on the activity of dura-sensitive spinal trigeminal nucleus caudalis (Vc) neurons.
• Kopruszinski, C. M., Xie, J. Y., Eyde, N. M., Remeniuk, B., Walter, S., Stratton, J., Bigal, M., Chichorro, J. G., Dodick, D., & Porreca, F. (2016). Prevention of stress- or nitric oxide donor-induced medication overuse headache by a calcitonin gene-related peptide antibody in rodents. Cephalalgia : an international journal of headache.
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  The objective of this study was the determination of the role of calcitonin gene-related peptide (CGRP) in the induction of medication overuse headache (MOH)-related migraine in an injury-free preclinical model.
• Lee, Y. S., Kupp, R., Remesic, M. V., Ramos-Colon, C., Hall, S. M., Chan, C., Rankin, D., Lai, J., Porreca, F., & Hruby, V. J. (2016). Various modifications of the amphipathic dynorphin A pharmacophore for rat brain bradykinin receptors. Chemical biology & drug design, 88(4), 615-9.
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  As a unique endogenous opioid ligand, dynorphin A shows paradoxical neuroexcitatory effects at bradykinin receptors, and the effects are known to be amplified by the upregulation of dynorphin A under chronic pain and inflammatory conditions. In our earlier structure-activity relationship studies, the amphipathic dynorphin A fragment, [Des-Arg(7) ]-Dyn A-(4-11), was identified as a pharmacophore for the bradykinin receptors along with key structural features. Here, further modifications of the pharmacophore showed that the position of a Pro residue is also an important feature because of its role in making (or disrupting) a β-turn or 310 helix structure which is crucial for receptor recognition.
• Lee, Y. S., Remesic, M., Ramos-Colon, C., Hall, S. M., Kuzmin, A., Rankin, D., Porreca, F., Lai, J., & Hruby, V. J. (2016). Cyclic non-opioid dynorphin A analogues for the bradykinin receptors. Bioorganic & medicinal chemistry letters, 26(22), 5513-5516.
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  Nerve injury and inflammation cause up-regulation of an endogenous opioid ligand, dynorphin A (Dyn A), in the spinal cord resulting in hyperalgesia via the interaction with bradykinin receptors (BRs). This is a non-opioid neuroexcitatory effect that cannot be blocked by opioid antagonists. Our systematic structure-activity relationships study on Dyn A identified lead ligands 1 and 4, along with the key structural feature (i.e. amphipathicity) for the BRs. However, the ligands showed very low metabolic stability in plasma (t1/2 5h) and therefore possesses the potential as a pharmacophoric scaffold to be utilized for drug development.
• Moutal, A., Chew, L. A., Yang, X., Wang, Y., Yeon, S. K., Telemi, E., Meroueh, S., Park, K. D., Shrinivasan, R., Gilbraith, K. B., Qu, C., Xie, J. Y., Patwardhan, A., Vanderah, T. W., Khanna, M., Porreca, F., & Khanna, R. (2016). (S)-lacosamide inhibition of CRMP2 phosphorylation reduces postoperative and neuropathic pain behaviors through distinct classes of sensory neurons identified by constellation pharmacology. Pain, 157(7), 1448-63.
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  Chronic pain affects the life of millions of people. Current treatments have deleterious side effects. We have advanced a strategy for targeting protein interactions which regulate the N-type voltage-gated calcium (CaV2.2) channel as an alternative to direct channel block. Peptides uncoupling CaV2.2 interactions with the axonal collapsin response mediator protein 2 (CRMP2) were antinociceptive without effects on memory, depression, and reward/addiction. A search for small molecules that could recapitulate uncoupling of the CaV2.2-CRMP2 interaction identified (S)-lacosamide [(S)-LCM], the inactive enantiomer of the Food and Drug Administration-approved antiepileptic drug (R)-lacosamide [(R)-LCM, Vimpat]. We show that (S)-LCM, but not (R)-LCM, inhibits CRMP2 phosphorylation by cyclin dependent kinase 5, a step necessary for driving CaV2.2 activity, in sensory neurons. (S)-lacosamide inhibited depolarization-induced Ca influx with a low micromolar IC50. Voltage-clamp electrophysiology experiments demonstrated a commensurate reduction in Ca currents in sensory neurons after an acute application of (S)-LCM. Using constellation pharmacology, a recently described high content phenotypic screening platform for functional fingerprinting of neurons that uses subtype-selective pharmacological agents to elucidate cell-specific combinations (constellations) of key signaling proteins that define specific cell types, we investigated if (S)-LCM preferentially acts on certain types of neurons. (S)-lacosamide decreased the dorsal root ganglion neurons responding to mustard oil, and increased the number of cells responding to menthol. Finally, (S)-LCM reversed thermal hypersensitivity and mechanical allodynia in a model of postoperative pain, and 2 models of neuropathic pain. Thus, using (S)-LCM to inhibit CRMP2 phosphorylation is a novel and efficient strategy to treat pain, which works by targeting specific sensory neuron populations.
• Moutal, A., Eyde, N., Telemi, E., Park, K. D., Xie, J. Y., Dodick, D. W., Porreca, F., & Khanna, R. (2016). Efficacy of (S)-Lacosamide in preclinical models of cephalic pain. Pain reports (Baltimore, Md.), 1(1).
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  Migraine is one of the world's most common neurological disorders. Current acute migraine treatments have sub-optimal efficacy and new therapeutic options are needed. Approaches targeting calcitonin gene related peptide (CGRP) signaling are clinically effective but small molecule antagonists have not been advanced due to toxicity. In this study, we explored the axonal growth/specification collapsin response mediator protein 2 (CRMP2) as a novel "druggable" target for inhibiting CGRP release and for potential relevance for treatment of migraine pain. CRMP2 has been demonstrated to regulate N-type voltage gated Ca(2+) channel (CaV2.2) activity and Ca(2+)-dependent CGRP release in sensory neurons. The co-expression of CRMP2 with CaV2.2 and CGRP in trigeminal ganglia (TG) sensory neurons suggested the possibility of a novel approach to regulate CGRP release in the trigeminal system. Screening protocols surprisingly revealed that (S)-Lacosamide ((S)-LCM), an inactive analog of the clinically-approved small molecule anti-epileptic drug (R)-Lacosamide (Vimpat®), inhibited CRMP2 phosphorylation by cyclin dependent kinase 5 (Cdk5) in rat TG slices and decreased depolarization-evoked Ca(2+) influx in TG cells in culture. (S)-LCM significantly blocked capsaicin-evoked CGRP release from dural nerve terminals in the rat an ex vivo cranial cup preparation. Additionally, cephalic and extracephalic cutaneous allodynia (CA) induced in rats by activation of dural nociceptors with a cocktail of inflammatory mediators, was inhibited by oral administration of (S)-LCM. The confirmation of CRMP2 as an upstream mediator of CGRP release together with the brain penetrance of this molecule suggest (S)-LCM as a potential therapy for acute migraine.
• Navratilova, E., Morimura, K., Xie, J. Y., Atcherley, C. W., Ossipov, M. H., & Porreca, F. (2016). Positive emotions and brain reward circuits in chronic pain. The Journal of comparative neurology, 524(8), 1646-52.
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  Chronic pain is an important public health problem that negatively impacts the quality of life of affected individuals and exacts enormous socioeconomic costs. Chronic pain is often accompanied by comorbid emotional disorders including anxiety, depression, and possibly anhedonia. The neural circuits underlying the intersection of pain and pleasure are not well understood. We summarize recent human and animal investigations and demonstrate that aversive aspects of pain are encoded in brain regions overlapping with areas processing reward and motivation. We highlight findings revealing anatomical and functional alterations of reward/motivation circuits in chronic pain. Finally, we review supporting evidence for the concept that pain relief is rewarding and activates brain reward/motivation circuits. Adaptations in brain reward circuits may be fundamental to the pathology of chronic pain. Knowledge of brain reward processing in the context of pain could lead to the development of new therapeutics for the treatment of emotional aspects of pain and comorbid conditions.
• Okun, A., McKinzie, D. L., Witkin, J. M., Remeniuk, B., Husein, O., Gleason, S. D., Oyarzo, J., Navratilova, E., McElroy, B., Cowen, S., Kennedy, J. D., & Porreca, F. (2016). Hedonic and motivational responses to food reward are unchanged in rats with neuropathic pain. Pain.
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  Rewards influence responses to acute painful stimuli, but the relationship of chronic pain to hedonic or motivational aspects of reward is not well understood. Here, we independently evaluated hedonic qualities of sweet or bitter tastants as well as motivation to seek food reward in rats with experimental neuropathic pain induced by L5/6 spinal nerve ligation (SNL). Hedonic response was measured by implantation of intraoral catheters to allow passive delivery of liquid solutions and "liking/disliking" responses were scored according to a facial reactivity scale. SNL rats did not differ from controls in either "liking" or "disliking" reactions to intraoral sucrose or quinine, respectively, at post-surgery day 21, suggesting no differences in perceived hedonic value of sweet or bitter tastants. To assess possible motivational deficits during acute and chronic pain, we employed fixed- and progressive-ratio response paradigms of sucrose pellet presentation in rats with transient inflammatory or chronic neuropathic pain. Assessment of response acquisition and break points under the progressive ratio schedule revealed no differences between sham and SNL rats for up to 120 days post-injury. However, rats with inflammation showed decrements in lever pressing and break points on post-CFA days 1 and 2 that normalized by day 4, consistent with transient ongoing pain. Thus, while acute, ongoing inflammatory pain may transiently reduce reward motivation, we did not detect influences of chronic neuropathic pain on hedonic or motivational responses to food rewards. Adaptations that allow normal reward responding to food regardless of chronic pain may be of evolutionary benefit to promote survival.
• Ramos-Colon, C. N., Lee, Y. S., Remesic, M., Hall, S. M., LaVigne, J., Davis, P., Sandweiss, A. J., McIntosh, M. I., Hanson, J., Largent-Milnes, T. M., Vanderah, T. W., Streicher, J., Porreca, F., & Hruby, V. J. (2016). Structure-Activity Relationships of [des-Arg(7)]Dynorphin A Analogues at the κ Opioid Receptor. Journal of medicinal chemistry, 59(22), 10291-10298.
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  Dynorphin A (Dyn A) is an endogenous ligand for the opioid receptors with preference for the κ opioid receptor (KOR), and its structure-activity relationship (SAR) has been extensively studied at the KOR to develop selective potent agonists and antagonists. Numerous SAR studies have revealed that the Arg(7) residue is essential for KOR activity. In contrast, our systematic SAR studies on [des-Arg(7)]Dyn A analogues found that Arg(7) is not a key residue and even deletion of the residue does not affect biological activities at the KOR. In addition, it was also found that [des-Arg(7)]Dyn A(1-9)-NH2 is a minimum pharmacophore and its modification at the N-terminus leads to selective KOR antagonists. A lead ligand, 14, with high affinity and antagonist activity showed improved metabolic stability and could block antinociceptive effects of a KOR selective agonist, FE200665, in vivo, indicating high potential to treat KOR mediated disorders such as stress-induced relapse.
• Rice, F. L., Xie, J. Y., Albrecht, P. J., Acker, E., Bourgeois, J., Navratilova, E., Dodick, D. W., & Porreca, F. (2016). Anatomy and immunochemical characterization of the non-arterial peptidergic diffuse dural innervation of the rat and Rhesus monkey: Implications for functional regulation and treatment in migraine. Cephalalgia : an international journal of headache.
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  The interplay between neuronal innervation and other cell types underlies the physiological functions of the dura mater and contributes to pathophysiological conditions such as migraine. We characterized the extensive, but understudied, non-arterial diffuse dural innervation (DDI) of the rat and Rhesus monkey.
• Xie, J. Y., Chew, L. A., Yang, X., Wang, Y., Qu, C., Wang, Y., Federici, L. M., Fitz, S. D., Ripsch, M. S., Due, M. R., Moutal, A., Khanna, M., White, F. A., Vanderah, T. W., Johnson, P. L., Porreca, F., & Khanna, R. (2016). Sustained relief of ongoing experimental neuropathic pain by a CRMP2 peptide aptamer with low abuse potential. Pain, 157(9), 2124-40.
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  Uncoupling the protein-protein interaction between collapsin response mediator protein 2 (CRMP2) and N-type voltage-gated calcium channel (CaV2.2) with an allosteric CRMP2-derived peptide (CBD3) is antinociceptive in rodent models of inflammatory and neuropathic pain. We investigated the efficacy, duration of action, abuse potential, and neurobehavioral toxicity of an improved mutant CRMP2 peptide. A homopolyarginine (R9)-conjugated CBD3-A6K (R9-CBD3-A6K) peptide inhibited the CaV2.2-CRMP2 interaction in a concentration-dependent fashion and diminished surface expression of CaV2.2 and depolarization-evoked Ca influx in rat dorsal root ganglia neurons. In vitro studies demonstrated suppression of excitability of small-to-medium diameter dorsal root ganglion and inhibition of subtypes of voltage-gated Ca channels. Sprague-Dawley rats with tibial nerve injury had profound and long-lasting tactile allodynia and ongoing pain. Immediate administration of R9-CBD3-A6K produced enhanced dopamine release from the nucleus accumbens shell selectively in injured animals, consistent with relief of ongoing pain. R9-CBD3-A6K, when administered repeatedly into the central nervous system ventricles of naive rats, did not result in a positive conditioned place preference demonstrating a lack of abusive liability. Continuous subcutaneous infusion of R9-CBD3-A6K over a 24- to 72-hour period reversed tactile allodynia and ongoing pain, demonstrating a lack of tolerance over this time course. Importantly, continuous infusion of R9-CBD3-A6K did not affect motor activity, anxiety, depression, or memory and learning. Collectively, these results validate the potential therapeutic significance of targeting the CaV-CRMP2 axis for treatment of neuropathic pain.
• Deekonda, S., Rankin, D., Davis, P., Lai, J., Porreca, F., & Hruby, V. J. (2015). Design, synthesis and biological evaluation of multifunctional ligands targeting opioid and bradykinin 2 receptors. Bioorganic & medicinal chemistry letters, 25(19), 4148-52.
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  We report here the design and synthesis of novel multifunctional ligands that act as (μ/δ) opioid agonists and bradykinin 2 receptor antagonists. These multifunctional ligands were designed to interact with the multiple receptors to show an enhanced analgesic effect, with no opioid-induced tolerance. We designed our multifunctional ligands based on the well-known second generation bradykinin 2 receptor antagonist Hoe 140 (DArg-Arg-Pro-Hyp-Gly-Thi-Ser-DTic-Oic-Arg-OH) and the opioid enkephalin analogues Tyr-DAla-Phe, Tyr-DAla-Gly-Phe and Tyr-Pro-Phe. We explored the conjugation of opioid pharmacophore to the Hoe 140 (DArg-Arg-Pro-Hyp-Gly-Thi-Ser-DTic-Oic-Arg-OH) in various positions with and without a linker. These bifunctional ligands showed very good binding affinity towards the both μ and δ opioid receptors. Among these bifunctional ligands 8, 11 and 12 showed excellent and balanced binding affinity at both μ and δ opioid receptors (0.5nM, 2.0nM; 0.3nM, 2nM; 2nM and 3nM), respectively. On the other hand these bifunctional ligands showed very weak and no binding affinity for rat brain bradykinin 2 receptors. Similarly, the Hoe 140 showed very low affinity (>10,000nM and 9000nM) against [(3)H] BK binding in rat brain membranes and in HEK293 cells, respectively. In contrast, the Hoe 140 showed very good binding affinity in guinea pig ileum (0.43nM) similar to that of previously reported. The bradykinin 2 receptors are known to be present in rat brain membrane, guinea pig ileum (GPI) and rabbit jugular vein. Previously the binding affinity of Hoe 140 for bradykinin 2 receptor was reported using guinea pig ileum. The above results suggest that the bradykinin 2 receptors present in rat brain membrane are a different sub type than the bradykinin 2 receptor present in guinea pig ileum (GPI).
• Deekonda, S., Wugalter, L., Kulkarni, V., Rankin, D., Largent-Milnes, T. M., Davis, P., Bassirirad, N. M., Lai, J., Vanderah, T. W., Porreca, F., & Hruby, V. J. (2015). Discovery of 5-substituted tetrahydronaphthalen-2yl-methyl with N-phenyl-N-(piperidin-4-yl)propionamide derivatives as potent opioid receptor ligands. Bioorganic & medicinal chemistry, 23(18), 6185-94.
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  A new series of novel opioid ligands have been designed and synthesized based on the 4-anilidopiperidine scaffold containing a 5-substituted tetrahydronaphthalen-2yl)methyl group with different N-phenyl-N-(piperidin-4-yl)propionamide derivatives to study the biological effects of these substituents on μ and δ opioid receptor interactions. Recently our group reported novel 4-anilidopiperidine analogues, in which several aromatic ring-contained amino acids were conjugated with N-phenyl-N-(piperidin-4-yl)propionamide and examined their biological activities at the μ and δ opioid receptors. In continuation of our efforts in these novel 4-anilidopiperidine analogues, we took a peptidomimetic approach in the present design, in which we substituted aromatic amino acids with tetrahydronaphthalen-2yl methyl moiety with amino, amide and hydroxyl substitutions at the 5th position. In in vitro assays these ligands, showed very good binding affinity and highly selective toward the μ opioid receptor. Among these, the lead ligand 20 showed excellent binding affinity (2nM) and 5000 fold selectivity toward the μ opioid receptor, as well as functional selectivity in GPI assays (55.20±4.30nM) and weak or no agonist activities in MVD assays. Based on the in vitro bioassay results the lead compound 20 was chosen for in vivo assessment for efficacy in naïve rats after intrathecal administration. Compound 20 was not significantly effective in alleviating acute pain. This discrepancy between high in vitro binding affinity, moderate in vitro activity, and low in vivo activity may reflect differences in pharmacodynamics (i.e., engaging signaling pathways) or pharmacokinetics (i.e., metabolic stability). In sum, our data suggest that further optimization of this compound 20 is required to enhance in vivo activity.
• Deekonda, S., Wugalter, L., Rankin, D., Largent-Milnes, T. M., Davis, P., Wang, Y., Bassirirad, N. M., Lai, J., Kulkarni, V., Vanderah, T. W., Porreca, F., & Hruby, V. J. (2015). Design and synthesis of novel bivalent ligands (MOR and DOR) by conjugation of enkephalin analogues with 4-anilidopiperidine derivatives. Bioorganic & medicinal chemistry letters, 25(20), 4683-8.
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  We describe the design and synthesis of novel bivalent ligands based on the conjugation of 4-anilidopiperidine derivatives with enkephalin analogues. The design of non-peptide analogues is explored with 5-amino substituted (tetrahydronaphthalen-2yl) methyl containing 4-anilidopiperidine derivatives, while non-peptide-peptide ligands are explored by conjugating the C-terminus of enkephalin analogues (H-Xxx-DAla-Gly-Phe-OH) to the amino group of 4-anilidopiperidine small molecule derivatives with and without a linker. These novel bivalent ligands are evaluated for biological activities at μ and δ opioid receptors. They exhibit very good affinities at μ and δ opioid receptors, and potent agonist activities in MVD and GPI assays. Among these the lead bivalent ligand 17 showed excellent binding affinities (0.1nM and 0.5nM) at μ and δ opioid receptors respectively, and was found to have very potent agonist activities in MVD (56±5.9nM) and GPI (4.6±1.9nM) assays. In vivo the lead bivalent ligand 17 exhibited a short duration of action (
• Giri, A. K., Apostol, C. R., Wang, Y., Forte, B. L., Largent-Milnes, T. M., Davis, P., Rankin, D., Molnar, G., Olson, K. M., Porreca, F., Vanderah, T. W., & Hruby, V. J. (2015). Discovery of Novel Multifunctional Ligands with μ/δ Opioid Agonist/Neurokinin-1 (NK1) Antagonist Activities for the Treatment of Pain. Journal of medicinal chemistry.
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  Multifunctional ligands with agonist bioactivities at μ/δ opioid receptors (MOR/DOR) and antagonist bioactivity at neurokinin-1 receptor (NK1R) have been designed and synthesized. These peptide-based ligands are anticipated to produce better biological profiles (e.g., higher analgesic effect with significantly less adverse side effects) compared to existing drugs, and to deliver better synergistic effects than co-administration of a mixture of multiple drugs. A systematic Structure-Activity Relationships (SARs) study has been conducted to find multifunctional ligands with desired activities at three receptors. It has been found that introduction of Dmt (2,6-dimethyl-tyrosine) at the 1st position and NMePhe at the 4th position (ligand 3: H-Dmt-D-Ala-Gly-NMePhe-Pro-Leu-Trp-NH-Bn(3',5'-(CF3)2)) displays binding as well as functional selectivity for MOR over DOR while maintaining efficacy, potency and antagonist activity at the NK1R. Dmt at the 1st position and Phe(4-F) at the 4th position (ligand 5: H-Dmt-D-Ala-Gly-Phe(4-F)-Pro-Leu-Trp-NH-Bn(3',5'-(CF3)2)) exhibits balanced binding affinities at MOR and DOR though it has higher agonist activity at DOR over MOR. This study has led to the discovery of several novel ligands including 3 and 5 with excellent in vitro biological activity profiles. Metabolic stability studies in rat plasma with ligands 3, 5 and 7 (H-Tyr-D-Ala-Gly-Phe(4-F)-Pro-Leu-Trp-NH-Bn(3',5'-(CF3)2)) showed that their stability depends on modifications at the 1st and 4th positions (3: T1/2 > 24 h; 5: T1/2 ≈ 6 h; T1/2 > 2 h). Preliminary in vivo studies with these two ligands have shown promising anti-nociceptive activity.
• Guillemyn, K., Kleczkowska, P., Lesniak, A., Dyniewicz, J., Van der Poorten, O., Van den Eynde, I., Keresztes, A., Varga, E., Lai, J., Porreca, F., Chung, N. N., Lemieux, C., Mika, J., Rojewska, E., Makuch, W., Van Duppen, J., Przewlocka, B., Vanden Broeck, J., Lipkowski, A. W., , Schiller, P. W., et al. (2015). Synthesis and biological evaluation of compact, conformationally constrained bifunctional opioid agonist - neurokinin-1 antagonist peptidomimetics. European journal of medicinal chemistry, 92, 64-77.
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  A reported mixed opioid agonist - neurokinin 1 receptor (NK1R) antagonist 4 (Dmt-D-Arg-Aba-Gly-(3',5'-(CF3)2)NMe-benzyl) was modified to identify important features in both pharmacophores. The new dual ligands were tested in vitro and subsequently two compounds (lead structure 4 and one of the new analogues 22, Dmt-D-Arg-Aba-β-Ala-NMe-Bn) were selected for in vivo behavioural assays, which were conducted in acute (tail-flick) and neuropathic pain models (cold plate and von Frey) in rats. Compared to the parent opioid compound 33 (without NK1R pharmacophore), hybrid 22 was more active in the neuropathic pain models. Attenuation of neuropathic pain emerged from NK1R antagonism as demonstrated by the pure NK1R antagonist 6. Surprisingly, despite a lower in vitro activity at NK1R in comparison with 4, compound 22 was more active in the neuropathic pain models. Although potent analgesic effects were observed for 4 and 22, upon chronic administration, both manifested a tolerance profile similar to that of morphine and cross tolerance with morphine in a neuropathic pain model in rat.
• Harasawa, I., Johansen, J. P., Fields, H. L., Porreca, F., & Meng, I. D. (2015). Alterations in the rostral ventromedial medulla after the selective ablation of mu-opioid receptor expressing neurons. Pain.
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  The rostral ventromedial medulla (RVM) exerts both inhibitory and excitatory control over nociceptive neurons in the spinal cord and medullary dorsal horn. Selective ablation of mu-opioid receptor expressing neurons in the RVM using saporin conjugated to the mu-opioid receptor (MOR) agonist dermorphin (derm-sap) attenuates stress and injury induced behavioral hypersensitivity, yet the effect of RVM derm-sap on the functional integrity of the descending inhibitory system and the properties of RVM neurons remains unknown. Three classes of RVM neurons (on-, off-, and neutral cells) have been described with distinct responses to noxious stimuli and MOR agonists. Using single unit recording in lightly anesthetized rats, RVM neurons were characterized following microinjections of derm-sap or saporin. Derm-sap treatment resulted in a reduction in on- and off-cells when compared to saporin controls (p
• Lee, Y. S., Hall, S. M., Ramos-Colon, C., Remesic, M., LeBaron, L., Nguyen, A., Rankin, D., Porreca, F., Lai, J., & Hruby, V. J. (2015). Modification of amphipathic non-opioid dynorphin A analogues for rat brain bradykinin receptors. Bioorganic & medicinal chemistry letters, 25(1), 30-3.
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  It has been shown that under chronic pain or nerve injury conditions, up-regulated dynorphin A (Dyn A) interacts with bradykinin receptors (BRs) to cause hyperalgesia in the spinal cord. Thus BRs antagonist can modulate hyperalgesia by blocking Dyn A's interaction with the BRs in the central nervous system. In our earlier structure-activity relationship (SAR) study, [des-Arg(7)]-Dyn A-(4-11) 13 was discovered as a minimum pharmacophore for rat brain BRs with its antagonist activity (anti-hyperalgesic effect) in in vivo tests using naïve or injured animals. We have pursued further modification on the [des-Arg(7)]-Dyn A analogues and identified a key insight into the pharmacophore of the rat brain BRs: amphipathicity.
• Lee, Y. S., Hall, S. M., Ramos-Colon, C., Remesic, M., Rankin, D., Vanderah, T. W., Porreca, F., Lai, J., & Hruby, V. J. (2015). Blockade of non-opioid excitatory effects of spinal dynorphin A at bradykinin receptors. Receptors & clinical investigation, 2(1).
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  Dynorphin A (Dyn A) is an endogenous opioid ligand that possesses neuroinhibitory (antinociceptive) effects via μ, δ, and κ opioid receptors. However, under chronic pain conditions, up-regulated spinal Dyn A can also interact with bradykinin receptors (BRs) to promote hyperalgesia through a neuroexcitatory(pronociceptive) effect. These excitatory effects cannot be blocked by an opioid antagonist, and thus are non-opioid in nature. On the basis of the structural dissimilarity between Dyn A and endogenous BR ligands, bradykinin(BK) and kallidin (KD), Dyn A's interaction with BRs could not be predicted, and provided an opportunity to identify a novel potential neuroexcitatory target. Systematic structure-activity relationship (SAR) studies discovered a minimum pharmacophore of Dyn A, [des-Arg(7)]-Dyn A-(4-11) LYS1044 for antagonist activity at the BRs, along with insights into the key structural features for BRs recognition, i.e., amphipathicity. The des-Tyr fragment of dynorphin does not bind to opioid receptors. Intrathecal administration of des-Tyr dynorphin produces hyperalgesia reminiscent of behaviors seen in peripheral n europathic pain models and at higher doses, neurotoxicity. Our lead ligand LYS1044 negatively modulated Dyn A-(2-13)-induced neuroexcitatory effects in naïve animals and blocked mechanical hypersensitivity and thermal hyperalgesia in a dose-dependent manner in animals with experimental neuropathic pain. Based on these results, ligand LYS1044 might prevent abnormal pain states by blocking the neuroexcitatory effects of increased levels of Dyn A that are seen in experimental models of neuropathic pain and that likely promote excitation mediated by BRs in the spinal cord.
• Little, J. W., Ford, A., Symons-Liguori, A. M., Chen, Z., Janes, K., Doyle, T., Xie, J., Luongo, L., Tosh, D. K., Maione, S., Bannister, K., Dickenson, A. H., Vanderah, T. W., Porreca, F., Jacobson, K. A., & Salvemini, D. (2015). Endogenous adenosine A3 receptor activation selectively alleviates persistent pain states. Brain : a journal of neurology, 138(Pt 1), 28-35.
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  Chronic pain is a global burden that promotes disability and unnecessary suffering. To date, efficacious treatment of chronic pain has not been achieved. Thus, new therapeutic targets are needed. Here, we demonstrate that increasing endogenous adenosine levels through selective adenosine kinase inhibition produces powerful analgesic effects in rodent models of experimental neuropathic pain through the A3 adenosine receptor (A3AR, now known as ADORA3) signalling pathway. Similar results were obtained by the administration of a novel and highly selective A3AR agonist. These effects were prevented by blockade of spinal and supraspinal A3AR, lost in A3AR knock-out mice, and independent of opioid and endocannabinoid mechanisms. A3AR activation also relieved non-evoked spontaneous pain behaviours without promoting analgesic tolerance or inherent reward. Further examination revealed that A3AR activation reduced spinal cord pain processing by decreasing the excitability of spinal wide dynamic range neurons and producing supraspinal inhibition of spinal nociception through activation of serotonergic and noradrenergic bulbospinal circuits. Critically, engaging the A3AR mechanism did not alter nociceptive thresholds in non-neuropathy animals and therefore produced selective alleviation of persistent neuropathic pain states. These studies reveal A3AR activation by adenosine as an endogenous anti-nociceptive pathway and support the development of A3AR agonists as novel therapeutics to treat chronic pain.
• Nair, P., Yamamoto, T., Cowell, S., Kulkarni, V., Moye, S., Navratilova, E., Davis, P., Ma, S., Vanderah, T. W., Lai, J., Porreca, F., & Hruby, V. J. (2015). Discovery of tripeptide-derived multifunctional ligands possessing delta/mu opioid receptor agonist and neurokinin 1 receptor antagonist activities. Bioorganic & medicinal chemistry letters, 25(17), 3716-20.
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  Several bifunctional peptides were synthesized and characterized based on the pentapeptide-derived ligand NP30 (1: Tyr-DAla-Gly-Phe-Gly-Trp-O-[3',5'-Bzl(CF3)2]). Modification and truncation of amino acid residues were performed, and the tripeptide-derived ligand NP66 (11: Dmt-DAla-Trp-NH-[3',5'-(CF3)2-Bzl]) was obtained based on the overlapping pharmacophore concept. The Trp(3) residue of ligand 11 works as a message residue for both opioid and NK1 activities. The significance lies in the observation that the approach of appropriate truncation of peptide sequence could lead to a tripeptide-derived chimeric ligand with effective binding and functional activities for both mu and delta opioid and NK1 receptors with agonist activities at mu and delta opioid and antagonist activity at NK1 receptors, respectively.
• Navratilova, E., Atcherley, C. W., & Porreca, F. (2015). Brain Circuits Encoding Reward from Pain Relief. Trends in neurosciences, 38(11), 741-50.
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  Relief from pain in humans is rewarding and pleasurable. Primary rewards, or reward-predictive cues, are encoded in brain reward/motivational circuits. While considerable advances have been made in our understanding of reward circuits underlying positive reinforcement, less is known about the circuits underlying the hedonic and reinforcing actions of pain relief. We review findings from electrophysiological, neuroimaging, and behavioral studies supporting the concept that the rewarding effect of pain relief requires opioid signaling in the anterior cingulate cortex (ACC), activation of midbrain dopamine neurons, and the release of dopamine in the nucleus accumbens (NAc). Understanding of circuits that govern the reward of pain relief may allow the discovery of more effective and satisfying therapies for patients with acute or chronic pain.
• Navratilova, E., Xie, J. Y., Meske, D., Qu, C., Morimura, K., Okun, A., Arakawa, N., Ossipov, M., Fields, H. L., & Porreca, F. (2015). Endogenous opioid activity in the anterior cingulate cortex is required for relief of pain. The Journal of neuroscience : the official journal of the Society for Neuroscience, 35(18), 7264-71.
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  Pain is aversive, and its relief elicits reward mediated by dopaminergic signaling in the nucleus accumbens (NAc), a part of the mesolimbic reward motivation pathway. How the reward pathway is engaged by pain-relieving treatments is not known. Endogenous opioid signaling in the anterior cingulate cortex (ACC), an area encoding pain aversiveness, contributes to pain modulation. We examined whether endogenous ACC opioid neurotransmission is required for relief of pain and subsequent downstream activation of NAc dopamine signaling. Conditioned place preference (CPP) and in vivo microdialysis were used to assess negative reinforcement and NAc dopaminergic transmission. In rats with postsurgical or neuropathic pain, blockade of opioid signaling in the rostral ACC (rACC) inhibited CPP and NAc dopamine release resulting from non-opioid pain-relieving treatments, including peripheral nerve block or spinal clonidine, an α2-adrenergic agonist. Conversely, pharmacological activation of rACC opioid receptors of injured, but not pain-free, animals was sufficient to stimulate dopamine release in the NAc and produce CPP. In neuropathic, but not sham-operated, rats, systemic doses of morphine that did not affect withdrawal thresholds elicited CPP and NAc dopamine release, effects that were prevented by blockade of ACC opioid receptors. The data provide a neural explanation for the preferential effects of opioids on pain affect and demonstrate that engagement of NAc dopaminergic transmission by non-opioid pain-relieving treatments depends on upstream ACC opioid circuits. Endogenous opioid signaling in the ACC appears to be both necessary and sufficient for relief of pain aversiveness.
• Porreca, F. (2017). Reward, motivation and emotion of pain and its relief. Pain, e-pub, e-pub. doi:DOI: 10.1097/j.pain.0000000000000798
• Qu, C., Xie, J., Ossipov, M. H., King, T., & Porreca, F. (2015). Gabapentin and duloxetine block spontaneous experimental neuropathic pain in rats. Journal of Pain.
• Remeniuk, B., Sukhtankar, D., Okun, A., Navratilova, E., Xie, J. Y., King, T., & Porreca, F. (2015). Behavioral and neurochemical analysis of ongoing bone cancer pain in rats. Pain, 156(10), 1864-73.
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  Cancer-induced bone pain is described as dull, aching ongoing pain. Ongoing bone cancer pain was characterized after intratibial injection of breast cancer cells in rats. Cancer produced time-dependent bone remodeling and tactile hypersensitivity but no spontaneous flinching. Conditioned place preference (CPP) and enhanced dopamine (DA) release in the nucleus accumbens (NAc) shell was observed after peripheral nerve block (PNB) selectively in tumor-bearing rats revealing nociceptive-driven ongoing pain. Oral diclofenac reversed tumor-induced tactile hypersensitivity but did not block PNB-induced CPP or NAc DA release. Tumor-induced tactile hypersensitivity, and PNB-induced CPP and NAc DA release, was blocked by prior subcutaneous implantation of a morphine pellet. In sham rats, morphine produced a modest but sustained increase in NAc DA release. In contrast, morphine produced a transient 5-fold higher NAc DA release in tumor bearing rats compared with sham morphine rats. The possibility that this increased NAc DA release reflected the reward of pain relief was tested by irreversible blockade of rostral anterior cingulate cortex (rACC) μ-opioid receptors (MORs). The rACC MOR blockade prevented the morphine-induced transient increased NAc DA release in tumor bearing rats but did not affect morphine-induced effects in sham-operated animals. Consistent with clinical experience, ongoing cancer pain was controlled by morphine but not by a dose of diclofenac that reversed evoked hypersensitivity. Additionally, the intrinsic reward of morphine can be dissociated from the reward of relief of cancer pain by blockade of rACC MOR. This approach allows mechanistic and therapeutic assessment of ongoing cancer pain with likely translation relevance.
• Vreeland, R. F., Atcherley, C. W., Russell, W. S., Xie, J. Y., Lu, D., Laude, N. D., Porreca, F., & Heien, M. L. (2015). Biocompatible PEDOT:Nafion composite electrode coatings for selective detection of neurotransmitters in vivo. Analytical chemistry, 87(5), 2600-7.
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  A Nafion and poly(3,4-ethylenedioxythiophene) (PEDOT) containing composite polymer has been electropolymerized on carbon-fiber microelectrodes with the goal of creating a mechanically stable, robust, and controllable electrode coating that increases the selectivity and sensitivity of in vivo electrochemical measurements. The coating is deposited on carbon-fiber microelectrodes by applying a triangle waveform from +1.5 V to -0.8 V and back in a dilute solution of ethylenedioxythiophene (EDOT) and Nafion in acetonitrile. Scanning electron microscopy demonstrated that the coating is uniform and ∼100 nm thick. Energy-dispersive X-ray spectroscopy demonstrated that both sulfur and fluorine are present in the coating, indicating the incorporation of PEDOT (poly(3,4-ethylenedioxythiophene) and Nafion. Two types of PEDOT:Nafion coated electrodes were then analyzed electrochemically. PEDOT:Nafion-coated electrodes made using 200 μM EDOT exhibit a 10-90 response time of 0.46 ± 0.09 s versus 0.45 ± 0.11 s for an uncoated fiber in response to a 1.0 μM bolus of dopamine. The electrodes coated using a higher EDOT concentration (400 μM) are slower with a 10-90 response time of 0.84 ± 0.19 s, but display increased sensitivity to dopamine, at 46 ± 13 nA/μM, compared to 26 ± 6 nA/μM for the electrodes coated in 200 μM EDOT and 13 ± 2 nA/μM for an uncoated fiber. PEDOT:Nafion-coated electrodes were lowered into the nucleus accumbens of a rat, and both spontaneous and electrically evoked dopamine release were measured. In addition to improvements in sensitivity and selectivity, the coating dramatically reduces acute in vivo biofouling.
• Bannister, K., Lee, Y. S., Goncalves, L., Porreca, F., Lai, J., & Dickenson, A. H. (2014). Neuropathic plasticity in the opioid and non-opioid actions of dynorphin A fragments and their interactions with bradykinin B2 receptors on neuronal activity in the rat spinal cord. Neuropharmacology, 85, 375-83.
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  Dynorphin A is an endogenous opioid peptide derived from the precursor prodynorphin. The proteolytic fragment dynorphin A (1-17) exhibits inhibitory effects via opioid receptors. Paradoxically, the activity of the dynorphin system increases with chronic pain and neuropathy is associated with the up-regulation of dynorphin biosynthesis. Dynorphin A (1-17) is cleaved in vivo to produce a non-opioid fragment, dynorphin A (2-17). Previously, a mechanism by which the non-opioid fragment promotes pain through agonist action at bradykinin receptors was revealed. Bradykinin receptor expression is up-regulated after nerve injury and both a truncated version of non-opioid fragment dynorphin A (2-17), referred to as 'Ligand 10', and novel bradykinin receptor antagonist 'Ligand 14', are known to bind to the bradykinin receptor. Here we show that Ligand 10 facilitates the response of wide dynamic range (WDR) neurons to innocuous and noxious mechanical stimuli in neuropathic, but not naïve, animals, while Ligand 14 exhibits inhibitory effects in neuropathic animals only. Furthermore, we reveal an inhibitory effect of Ligand 14 in naïve animals by pre-dosing with either Ligand 10 or a 5-HT3 receptor agonist to reflect activation of descending excitatory drives. Thus remarkably, by mimicking pro-excitatory pharmacological changes that occur after nerve injury in a naïve animal, we induce a state whereby the inhibitory actions of Ligand 14 are now effective. Ultimately our data support an increasing number of studies that suggest that blocking spinal bradykinin receptors may have a therapeutic potential in chronic pain states, here, in particular, in neuropathic pain.
• Borsook, D., Hargreaves, R., Bountra, C., & Porreca, F. (2014). Lost but making progress--Where will new analgesic drugs come from?. Science translational medicine, 6(249), 249sr3.
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  There is a critical need for effective new pharmacotherapies for pain. The paucity of new drugs successfully reaching the clinic calls for a reassessment of current analgesic drug discovery approaches. Many points early in the discovery process present significant hurdles, making it critical to exploit advances in pain neurobiology to increase the probability of success. In this review, we highlight approaches that are being pursued vigorously by the pain community for drug discovery, including innovative preclinical pain models, insights from genetics, mechanistic phenotyping of pain patients, development of biomarkers, and emerging insights into chronic pain as a disorder of both the periphery and the brain. Collaborative efforts between pharmaceutical, academic, and public entities to advance research in these areas promise to de-risk potential targets, stimulate investment, and speed evaluation and development of better pain therapies.
• Dussor, G., Yan, J., Xie, J. Y., Ossipov, M. H., Dodick, D. W., & Porreca, F. (2014). Targeting TRP channels for novel migraine therapeutics. ACS chemical neuroscience, 5(11), 1085-96.
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  Migraine is increasingly understood to be a disorder of the brain. In susceptible individuals, a variety of "triggers" may influence altered central excitability, resulting in the activation and sensitization of trigeminal nociceptive afferents surrounding blood vessels (i.e., the trigeminovascular system), leading to migraine pain. Transient receptor potential (TRP) channels are expressed in a subset of dural afferents, including those containing calcitonin gene related peptide (CGRP). Activation of TRP channels promotes excitation of nociceptive afferent fibers and potentially lead to pain. In addition to pain, allodynia to mechanical and cold stimuli can result from sensitization of both peripheral afferents and of central pain pathways. TRP channels respond to a variety of endogenous conditions including chemical mediators and low pH. These channels can be activated by exogenous stimuli including a wide range of chemical and environmental irritants, some of which have been demonstrated to trigger migraine in humans. Activation of TRP channels can elicit CGRP release, and blocking the effects of CGRP through receptor antagonism or antibody strategies has been demonstrated to be effective in the treatment of migraine. Identification of approaches that can prevent activation of TRP channels provides an additional novel strategy for discovery of migraine therapeutics.
• Fillingim, R. B., Bruehl, S., Dworkin, R. H., Dworkin, S. F., Loeser, J. D., Turk, D. C., Widerstrom-Noga, E., Arnold, L., Bennett, R., Edwards, R. R., Freeman, R., Gewandter, J., Hertz, S., Hochberg, M., Krane, E., Mantyh, P. W., Markman, J., Neogi, T., Ohrbach, R., , Paice, J. A., et al. (2014). The ACTTION-American Pain Society Pain Taxonomy (AAPT): an evidence-based and multidimensional approach to classifying chronic pain conditions. The journal of pain : official journal of the American Pain Society, 15(3), 241-9.
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  Current approaches to classification of chronic pain conditions suffer from the absence of a systematically implemented and evidence-based taxonomy. Moreover, existing diagnostic approaches typically fail to incorporate available knowledge regarding the biopsychosocial mechanisms contributing to pain conditions. To address these gaps, the Analgesic, Anesthetic, and Addiction Clinical Trial Translations Innovations Opportunities and Networks (ACTTION) public-private partnership with the U.S. Food and Drug Administration and the American Pain Society (APS) have joined together to develop an evidence-based chronic pain classification system called the ACTTION-APS Pain Taxonomy. This paper describes the outcome of an ACTTION-APS consensus meeting, at which experts agreed on a structure for this new taxonomy of chronic pain conditions. Several major issues around which discussion revolved are presented and summarized, and the structure of the taxonomy is presented. ACTTION-APS Pain Taxonomy will include the following dimensions: 1) core diagnostic criteria; 2) common features; 3) common medical comorbidities; 4) neurobiological, psychosocial, and functional consequences; and 5) putative neurobiological and psychosocial mechanisms, risk factors, and protective factors. In coming months, expert working groups will apply this taxonomy to clusters of chronic pain conditions, thereby developing a set of diagnostic criteria that have been consistently and systematically implemented across nearly all common chronic pain conditions. It is anticipated that the availability of this evidence-based and mechanistic approach to pain classification will be of substantial benefit to chronic pain research and treatment.
• Green, A. L., Gu, P., De Felice, M., Dodick, D., Ossipov, M. H., & Porreca, F. (2014). Increased susceptibility to cortical spreading depression in an animal model of medication-overuse headache. Cephalalgia : an international journal of headache, 34(8), 594-604.
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  The objective of this article is to evaluate electrically evoked thresholds for cortical spreading depression (CSD) and stress-induced activation of trigeminal afferents in a rat model of medication-overuse headache (MOH).
• King, T., & Porreca, F. (2014). Preclinical assessment of pain: improving models in discovery research. Current topics in behavioral neurosciences, 20, 101-20.
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  To date, animal models have not sufficiently "filtered" targets for new analgesics, increasing the failure rate and cost of drug development. Preclinical assessment of "pain" has historically relied on measures of evoked behavioral responses to sensory stimuli in animals. Such measures can often be observed in decerebrated animals and therefore may not sufficiently capture affective and motivational aspects of pain, potentially diminishing translation from preclinical studies to the clinical setting. Further, evidence indicates that there are important mechanistic differences between evoked behavioral responses of hypersensitivity and ongoing pain, limiting evaluation of mechanisms that could mediate aspects of clinically relevant pain. The mechanisms underlying ongoing pain in preclinical models are currently being explored and may serve to inform decisions towards the transition from drug discovery to drug development for a given target.
• Lee, Y. S., Muthu, D., Hall, S. M., Ramos-Colon, C., Rankin, D., Hu, J., Sandweiss, A. J., De Felice, M., Xie, J. Y., Vanderah, T. W., Porreca, F., Lai, J., & Hruby, V. J. (2014). Discovery of amphipathic dynorphin A analogues to inhibit the neuroexcitatory effects of dynorphin A through bradykinin receptors in the spinal cord. Journal of the American Chemical Society, 136(18), 6608-16.
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  We hypothesized that under chronic pain conditions, up-regulated dynorphin A (Dyn A) interacts with bradykinin receptors (BRs) in the spinal cord to promote hyperalgesia through an excitatory effect, which is opposite to the well-known inhibitory effect of opioid receptors. Considering the structural dissimilarity between Dyn A and endogenous BR ligands, bradykinin (BK) and kallidin (KD), this interaction could not be predicted, but it allowed us to discover a potential neuroexcitatory target. Well-known BR ligands, BK, [des-Arg(10), Leu(9)]-kallidin (DALKD), and HOE140 showed different binding profiles at rat brain BRs than that previously reported. These results suggest that neuronal BRs in the rat central nervous system (CNS) may be pharmacologically distinct from those previously defined in non-neuronal tissues. Systematic structure-activity relationship (SAR) study at the rat brain BRs was performed, and as a result, a new key structural feature of Dyn A for BR recognition was identified: amphipathicity. NMR studies of two lead ligands, Dyn A-(4-11) 7 and [des-Arg(7)]-Dyn A-(4-11) 14, which showed the same high binding affinity, confirmed that the Arg residue in position 7, which is known to be crucial for Dyn A's biological activity, is not necessary, and that a type I β-turn structure at the C-terminal part of both ligands plays an important role in retaining good binding affinities at the BRs. Our lead ligand 14 blocked Dyn A-(2-13) 10-induced hyperalgesic effects and motor impairment in in vivo assays using naïve rats. In a model of peripheral neuropathy, intrathecal (i.th.) administration of ligand 14 reversed thermal hyperalgesia and mechanical hypersensitivity in a dose-dependent manner in nerve-injured rats. Thus, ligand 14 may inhibit abnormal pain states by blocking the neuroexcitatory effects of enhanced levels of Dyn A, which are likely to be mediated by BRs in the spinal cord.
• Lee, Y. S., Rankin, D., Hall, S. M., Ramos-Colon, C., Ortiz, J. J., Kupp, R., Porreca, F., Lai, J., & Hruby, V. J. (2014). Structure-activity relationships of non-opioid [des-Arg(7)]-dynorphin A analogues for bradykinin receptors. Bioorganic & medicinal chemistry letters, 24(21), 4976-9.
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  In our earlier studies, bradykinin receptors (BRs) were identified as a potential target for the neuroexcitatory effects of dynorphin A (Dyn A) in the central nervous system (CNS), and [des-Arg(7)]-Dyn A-(4-11) (6) was discovered as a lead ligand to modulate Dyn A-(2-13) induced neuroexcitatory effects in the CNS as an antagonist. In an effort to gain insights into key structural features of the Dyn A for the BRs, we pursued further structure-activity relationships (SAR) study on the [des-Arg(7)]-Dyn A analogs and confirmed that all of the [des-Arg(7)]-Dyn A analogues showed good binding affinities at the BRs.
• Mamet, J., Klukinov, M., Yaksh, T. L., Malkmus, S. A., Williams, S., Harris, S., Manning, D. C., Taylor, B. K., Donahue, R. R., Porreca, F., Xie, J. Y., Oyarzo, J., Brennan, T. J., Subieta, A., Schmidt, W. K., & Yeomans, D. C. (2014). Single intrathecal administration of the transcription factor decoy AYX1 prevents acute and chronic pain after incisional, inflammatory, or neuropathic injury. Pain, 155(2), 322-33.
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  The persistence of pain after surgery increases the recovery interval from surgery to a normal quality of life. AYX1 is a DNA-decoy drug candidate designed to prevent post-surgical pain following a single intrathecal injection. Tissue injury causes a transient activation of the transcription factor EGR1 in the dorsal root ganglia-dorsal horn network, which then triggers changes in gene expression that induce neuronal hypersensitivity. AYX1 is a potent, specific inhibitor of EGR1 activity that mimics the genomic EGR1-binding sequence. Administered in the peri-operative period, AYX1 dose dependently prevents mechanical hypersensitivity in models of acute incisional (plantar), inflammatory (CFA), and chronic neuropathic pain (SNI) in rats. Furthermore, in a knee surgery model evaluating functional measures of postoperative pain, AYX1 improved weight-bearing incapacitance and spontaneous rearing compared to control. These data illustrate the potential clinical therapeutic benefits of AYX1 for preventing the transition of acute to chronic post-surgical pain.
• Meske, D. S., Xie, J. Y., Oyarzo, J., Badghisi, H., Ossipov, M. H., & Porreca, F. (2014). Opioid and noradrenergic contributions of tapentadol in experimental neuropathic pain. Neuroscience letters, 562, 91-6.
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  Tapentadol is a dual action molecule with mu opioid agonist and norepinephrine (NE) reuptake blocking activity that has recently been introduced for the treatment of moderate to severe pain. The effects of intraperitoneal (i.p.) morphine (10mg/kg), tapentadol (10 or 30 mg/kg) or duloxetine (30 mg/kg), a norepinephrine/serotonin (NE/5HT) reuptake inhibitor, were evaluated in male, Sprague-Dawley rats with spinal nerve ligation (SNL) or sham surgery. Additionally, the effects of these drugs on spinal cerebrospinal fluid (CSF) NE levels were quantified. Response thresholds to von Frey filament stimulation decreased significantly from baseline in SNL, but not sham, operated rats. Duloxetine, tapentadol and morphine produced significant and time-related reversal of tactile hypersensitivity. Duloxetine significantly increased spinal CSF NE levels in both sham and SNL rats and no significant differences were observed in these groups. Tapentadol (10 mg/kg) produced a significant increase in spinal NE levels in SNL, but not in sham, rats. At the higher dose (30 mg/kg), tapentadol produced a significant increase in spinal CSF NE levels in both SNL and sham groups; however, spinal NE levels were elevated for an extended period in the SNL rats. This could be detected 30 min following tapentadol (30 mg/kg) in both sham and SNL groups. Surprisingly, while the dose of morphine studied reversed tactile hypersensitivity in nerve-injured rats, CSF NE levels were significantly reduced in both sham- and SNL rats. The data suggest that tapentadol elicits enhanced elevation in spinal NE levels in a model of experimental neuropathic pain offering a mechanistic correlate to observed clinical efficacy in this pain state.
• Mollica, A., Carotenuto, A., Novellino, E., Limatola, A., Costante, R., Pinnen, F., Stefanucci, A., Pieretti, S., Borsodi, A., Samavati, R., Zador, F., Benyhe, S., Davis, P., Porreca, F., & Hruby, V. J. (2014). Novel cyclic biphalin analogue with improved antinociceptive properties. ACS medicinal chemistry letters, 5(9), 1032-6.
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  Two novel opioid analogues have been designed by substituting the native d-Ala residues in position 2,2' of biphalin with two residues of d-penicillamine or l-penicillamine and by forming a disulfide bond between the thiol groups. The so-obtained compound 9 containing d-penicillamines showed excellent μ/δ mixed receptor affinities (K i (δ) = 5.2 nM; K i (μ) = 1.9 nM), together with an efficacious capacity to trigger the second messenger and a very good in vivo antinociceptive activity, whereas product 10 was scarcely active. An explanation of the two different pharmacological behaviors of products 9 and 10 was found by studying their conformational properties.
• Navratilova, E., & Porreca, F. (2014). Reward and motivation in pain and pain relief. Nature neuroscience, 17(10), 1304-12.
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  Pain is fundamentally unpleasant, a feature that protects the organism by promoting motivation and learning. Relief of aversive states, including pain, is rewarding. The aversiveness of pain, as well as the reward from relief of pain, is encoded by brain reward/motivational mesocorticolimbic circuitry. In this Review, we describe current knowledge of the impact of acute and chronic pain on reward/motivation circuits gained from preclinical models and from human neuroimaging. We highlight emerging clinical evidence suggesting that anatomical and functional changes in these circuits contribute to the transition from acute to chronic pain. We propose that assessing activity in these conserved circuits can offer new outcome measures for preclinical evaluation of analgesic efficacy to improve translation and speed drug discovery. We further suggest that targeting reward/motivation circuits may provide a path for normalizing the consequences of chronic pain to the brain, surpassing symptomatic management to promote recovery from chronic pain.
• Ossipov, M. H., Morimura, K., & Porreca, F. (2014). Descending pain modulation and chronification of pain. Current opinion in supportive and palliative care, 8(2), 143-51.
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  Chronic pain is an important public health problem that negatively impacts quality of life of affected individuals and exacts an enormous socio-economic cost. Currently available therapeutics provide inadequate management of pain in many patients. Acute pain states generally resolve in most patients. However, for reasons that are poorly understood, in some individuals, acute pain can transform to a chronic state. Our understanding of the risk factors that underlie the development of chronic pain is limited. Recent studies have suggested an important contribution of dysfunction in descending pain modulatory circuits to pain 'chronification'. Human studies provide insights into possible endogenous and exogenous factors that may promote the conversion of pain into a chronic condition.
• Wang, R., Rossomando, A., Sah, D. W., Ossipov, M. H., King, T., & Porreca, F. (2014). Artemin induced functional recovery and reinnervation after partial nerve injury. Pain, 155(3), 476-84.
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  Systemic artemin promotes regeneration of dorsal roots to the spinal cord after crush injury. However, it is unclear whether systemic artemin can also promote peripheral nerve regeneration, and functional recovery after partial lesions distal to the dorsal root ganglion (DRG) remains unknown. In the present investigation, male Sprague Dawley rats received axotomy, ligation, or crush of the L5 spinal nerve or sham surgery. Starting the day of injury, animals received intermittent subcutaneous artemin or vehicle across 2weeks. Sensory thresholds to tactile or thermal stimuli were monitored for 6weeks after injury. Immunohistochemical analyses of the DRG and nerve regeneration were performed at the 6-week time point. Artemin transiently reversed tactile and thermal hypersensitivity after axotomy, ligation, or crush injury. Thermal and tactile hypersensitivity reemerged within 1week of treatment termination. However, artemin-treated rats with nerve crush, but not axotomy or ligation, subsequently showed gradual return of sensory thresholds to preinjury baseline levels by 6weeks after injury. Artemin normalized labeling for NF200, IB4, and CGRP in nerve fibers distal to the crush injury, suggesting persistent normalization of nerve crush-induced neurochemical changes. Sciatic and intradermal administration of dextran or cholera toxin B distal to the crush injury site resulted in labeling of neuronal profiles in the L5 DRG, suggesting regeneration functional restoration of nonmyelinated and myelinated fibers across the injury site into cutaneous tissue. Artemin also diminished ATF3 and caspase 3 expression in the L5 DRG, suggesting persistent neuroprotective actions. A limited period of artemin treatment elicits disease modification by promoting sensory reinnervation of distal territories and restoring preinjury sensory thresholds.
• Xie, J. Y., Qu, C., Patwardhan, A., Ossipov, M. H., Navratilova, E., Becerra, L., Borsook, D., & Porreca, F. (2014). Activation of mesocorticolimbic reward circuits for assessment of relief of ongoing pain: a potential biomarker of efficacy. Pain, 155(8), 1659-66.
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  Preclinical assessment of pain has increasingly explored operant methods that may allow behavioral assessment of ongoing pain. In animals with incisional injury, peripheral nerve block produces conditioned place preference (CPP) and activates the mesolimbic dopaminergic reward pathway. We hypothesized that activation of this circuit could serve as a neurochemical output measure of relief of ongoing pain. Medications commonly used clinically, including gabapentin and nonsteroidal anti-inflammatory drugs (NSAIDs), were evaluated in models of post-surgical (1 day after incision) or neuropathic (14 days after spinal nerve ligation [SNL]) pain to determine whether the clinical efficacy profile of these drugs in these pain conditions was reflected by extracellular dopamine (DA) release in the nucleus accumbens (NAc) shell. Microdialysis was performed in awake rats. Basal DA levels were not significantly different between experimental groups, and no significant treatment effects were seen in sham-operated animals. Consistent with clinical observation, spinal clonidine produced CPP and produced a dose-related increase in net NAc DA release in SNL rats. Gabapentin, commonly used to treat neuropathic pain, produced increased NAc DA in rats with SNL but not in animals with incisional, injury. In contrast, ketorolac or naproxen produced increased NAc DA in animals with incisional but not neuropathic pain. Increased extracellular NAc DA release was consistent with CPP and was observed selectively with treatments commonly used clinically for post-surgical or neuropathic pain. Evaluation of NAc DA efflux in animal pain models may represent an objective neurochemical assay that may serve as a biomarker of efficacy for novel pain-relieving mechanisms.
• Yoshimura, R. F., Tran, M. B., Hogenkamp, D. J., Johnstone, T. B., Xie, J. Y., Porreca, F., & Gee, K. W. (2014). Limited central side effects of a β-subunit subtype-selective GABAA receptor allosteric modulator. Journal of psychopharmacology (Oxford, England), 28(5), 472-8.
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  GABAergic anxiolytics have well-documented centrally mediated side effects including sedation, potentiation of ethanol, tolerance, abuse liability and memory impairment. Most research directed towards identifying an anxioselective GABAergic therapeutic has been based upon the theory that these side effects could be mitigated by avoiding α1/5-subunit GABAA receptors while specifically targeting those with the α2/3-subunit. Unfortunately, there are prominent exceptions to this theory and it has yet to be translated into clinical success. We previously demonstrated that β2/3-subunit-selective GABAA receptor-positive allosteric modulators act as anxiolytics with reduced sedation and ethanol potentiation regardless of their activity at α1-subunit GABAA receptors. The prototypical β2/3-subunit-selective positive allosteric modulator, 2-261, is further characterized here for additional side effects commonly associated with central GABAA receptor activation. In mice, 10 times the anxiolytic dose (10 mg/kg) of 2-261 does not induce behavioral tolerance in the elevated plus maze following a 2 week subchronic treatment. In rats, an anxiolytic dose (10 mg/kg) of 2-261 is inactive in conditioned place preference, suggesting a reduced abuse liability. In rats, 10 times the anxiolytic dose (100 mg/kg) of 2-261 does not have a significant amnestic effect in the radial arm maze, suggesting a greater therapeutic index for memory impairment. These results suggest that β2/3-subunit subtype-selective GABAA receptor-positive allosteric modulators not only have reduced sedative liability, but also a reduction in other central side effects commonly associated with broader GABAA receptor activation. β2/3-subunit-selective compounds may represent a novel design template for anxiolytics with benzodiazepine-like efficacy and mitigated side effects.
• De Felice, M., Eyde, N., Dodick, D., Dussor, G. O., Ossipov, M. H., Fields, H. L., & Porreca, F. (2013). Capturing the aversive state of cephalic pain preclinically. Annals of neurology, 74(2), 257-65.
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  Preclinical evaluation of headache by behavioral assessment of reward from pain relief.
• Lee, Y. S., Qu, H. C., Davis, P., Ma, S. W., Vardanyan, R., Lai, J., Porreca, F., & Hruby, V. J. (2013). Chiral Effect of a Phe Residue in Position 3 of the Dmt1-L(or D)-Tic2 Analogues on Opioid Functional Activities. ACS medicinal chemistry letters, 4(7), 656-659.
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  In this letter, we describe a structure-activity relationships study, specifically related to the chirality of third amino acid residue in our H-Dmt-L(or D)-Tic analogues, of which C-terminus is attached to a piperidinyl moiety. Observed selectivities and functional activities of these analogues demonstrated that the chiralities of the second and third position residues are crucial for determining whether these ligands act as antagonists or agonists at the δ opioid receptor, but not at the μ opioid receptor.
• Lozano-Ondoua, A. N., Hanlon, K. E., Symons-Liguori, A. M., Largent-Milnes, T. M., Havelin, J. J., Ferland, H. L., Chandramouli, A., Owusu-Ankomah, M., Nikolich-Zugich, T., Bloom, A. P., Jimenez-Andrade, J. M., King, T., Porreca, F., Nelson, M. A., Mantyh, P. W., & Vanderah, T. W. (2013). Disease modification of breast cancer-induced bone remodeling by cannabinoid 2 receptor agonists. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 28(1), 92-107.
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  Most commonly originating from breast malignancies, metastatic bone cancer causes bone destruction and severe pain. Although novel chemotherapeutic agents have increased life expectancy, patients are experiencing higher incidences of fracture, pain, and drug-induced side effects; furthermore, recent findings suggest that patients are severely undertreated for their cancer pain. Strong analgesics, namely opiates, are first-line therapy in alleviating cancer-related pain despite the severe side effects, including enhanced bone destruction with sustained administration. Bone resorption is primarily treated with bisphosphonates, which are associated with highly undesirable side effects, including nephrotoxicity and osteonecrosis of the jaw. In contrast, cannabinoid receptor 2 (CB(2) ) receptor-specific agonists have been shown to reduce bone loss and stimulate bone formation in a model of osteoporosis. CB(2) agonists produce analgesia in both inflammatory and neuropathic pain models. Notably, mixed CB(1) /CB(2) agonists also demonstrate a reduction in ErbB2-driven breast cancer progression. Here we demonstrate for the first time that CB(2) agonists reduce breast cancer-induced bone pain, bone loss, and breast cancer proliferation via cytokine/chemokine suppression. Studies used the spontaneously-occurring murine mammary cell line (66.1) implanted into the femur intramedullary space; measurements of spontaneous pain, bone loss, and cancer proliferation were made. The systemic administration of a CB(2) agonist, JWH015, for 7 days significantly attenuated bone remodeling, assuaged spontaneous pain, and decreased primary tumor burden. CB(2) -mediated effects in vivo were reversed by concurrent treatment with a CB(2) antagonist/inverse agonist but not with a CB(1) antagonist/inverse agonist. In vitro, JWH015 reduced cancer cell proliferation and inflammatory mediators that have been shown to promote pain, bone loss, and proliferation. Taken together, these results suggest CB(2) agonists as a novel treatment for breast cancer-induced bone pain, in which disease modifications include a reduction in bone loss, suppression of cancer growth, attenuation of severe bone pain, and increased survival without the major side effects of current therapeutic options.
• Mollica, A., Pinnen, F., Costante, R., Locatelli, M., Stefanucci, A., Pieretti, S., Davis, P., Lai, J., Rankin, D., Porreca, F., & Hruby, V. J. (2013). Biological active analogues of the opioid peptide biphalin: mixed α/β(3)-peptides. Journal of medicinal chemistry, 56(8), 3419-23.
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  Natural residues of the dimeric opioid peptide Biphalin were replaced by the corresponding homo-β(3) amino acids. The derivative 1 containing hβ(3) Phe in place of Phe showed good μ- and δ-receptor affinities (Ki(δ) = 0.72 nM; Ki(μ) = 1.1 nM) and antinociceptive activity in vivo together with an increased enzymatic stability in human plasma.
• Nair, P., Yamamoto, T., Largent-Milnes, T. M., Cowell, S., Kulkarni, V., Moye, S., Navratilova, E., Davis, P., Ma, S. W., Vanderah, T. W., Lai, J., Porreca, F., & Hruby, V. J. (2013). Truncation of the peptide sequence in bifunctional ligands with mu and delta opioid receptor agonist and neurokinin 1 receptor antagonist activities. Bioorganic & medicinal chemistry letters, 23(17), 4975-8.
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  The optimization and truncation of our lead peptide-derived ligand TY005 possessing eight amino-acid residues was performed. Among the synthesized derivatives, NP30 (Tyr(1)-DAla(2)-Gly(3)-Phe(4)-Gly(5)-Trp(6)-O-[3',5'-Bzl(CF3)2]) showed balanced and potent opioid agonist as well as substance P antagonist activities in isolated tissue-based assays, together with significant antinociceptive and antiallodynic activities in vivo.
• Navratilova, E., Xie, J. Y., King, T., & Porreca, F. (2013). Evaluation of reward from pain relief. Annals of the New York Academy of Sciences, 1282, 1-11.
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  The human experience of pain is multidimensional and comprises sensory, affective, and cognitive dimensions. Preclinical assessment of pain has been largely focused on the sensory features that contribute to nociception. The affective (aversive) qualities of pain are clinically significant but have received relatively less mechanistic investigation in preclinical models. Recently, operant behaviors such as conditioned place preference, avoidance, escape from noxious stimulus, and analgesic drug self-administration have been used in rodents to evaluate affective aspects of pain. An important advance of such operant behaviors is that these approaches may allow the detection and mechanistic investigation of spontaneous neuropathic or ongoing inflammatory/nociceptive (i.e., nonevoked) pain that is otherwise difficult to assess in nonverbal animals. Operant measures may allow the identification of mechanisms that contribute differentially to reflexive hypersensitivity or to pain affect and may inform the decision to progress novel mechanisms to clinical trials for pain therapy. Additionally, operant behaviors may allow investigation of the poorly understood mechanisms and neural circuits underlying motivational aspects of pain and the reward of pain relief.
• Petrov, R. R., Lee, Y. S., Vardanyan, R. S., Liu, L., Ma, S. W., Davis, P., Lai, J., Porreca, F., Vanderah, T. W., & Hruby, V. J. (2013). Effect of anchoring 4-anilidopiperidines to opioid peptides. Bioorganic & medicinal chemistry letters, 23(11), 3434-7.
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  We report here the design, synthesis, and in vitro characterization of new opioid peptides featuring a 4-anilidopiperidine moiety. Despite the fact that the chemical structures of fentanyl surrogates have been found suboptimal per se for the opioid activity, the corresponding conjugates with opioid peptides displayed potent opioid activity. These studies shed an instructive light on the strategies and potential therapeutic values of anchoring the 4-anilidopiperidine scaffold to different classes of opioid peptides.
• Stahl, S. M., Porreca, F., Taylor, C. P., Cheung, R., Thorpe, A. J., & Clair, A. (2013). The diverse therapeutic actions of pregabalin: is a single mechanism responsible for several pharmacological activities?. Trends in pharmacological sciences, 34(6), 332-9.
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  Pregabalin is a specific ligand of the alpha2-delta (α2-δ) auxiliary subunit of voltage-gated calcium channels. A growing body of evidence from studies of anxiety and pain indicate that the observed responses with pregabalin may result from activity at the α2-δ auxiliary protein expressed presynaptically, in several different circuits of the central nervous system (CNS). The disorders that appear to be effectively treated with pregabalin are thematically linked by neuronal dysregulation or hyperexcitation within the CNS. This review proposes how binding to the α2-δ protein target in different regions of the CNS may contribute to the observed clinical activity of pregabalin, as well as to the adverse event profile of the compound. Whether this compound regulates synaptic function via α2-δ in additional conditions is yet to be discovered. The potential of pregabalin to regulate neuronal hyperactivity involving other CNS circuits will require further exploration.
• Sui, P., Watanabe, H., Ossipov, M. H., Porreca, F., Bakalkin, G., Bergquist, J., & Artemenko, K. (2013). Dimethyl-labeling-based protein quantification and pathway search: a novel method of spinal cord analysis applicable for neurological studies. Journal of proteome research, 12(5), 2245-52.
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  In this paper we describe a simple, fast, and inexpensive approach for quantitative analysis of proteins originated from small central nervous system (CNS) samples, i.e., rat spinal cord. The presented sample preparation protocol and quantification results from isotope dimethyl labeling were statistically evaluated and approved as a reliable and robust method for animal model studies of neurological disorders. Combined with the biopathway analysis tool IPA, the method was applied for comparative analysis of proteins in the dorsal and ventral segments of the rat spinal cord. The results are in agreement with the previously published protein patterns in these tissues. A majority (73%) of proteins identified as "related with CNS development and functions" were found to be overexpressed in the dorsal section compared to the ventral segment. The pathway related to neuropathic pain was overrepresented in the dorsal tissue samples. The developed novel approach may be applied for analyses of the spinal cord mediated neurological dysfunctions and pathological pain.
• Taylor, R., Pergolizzi, J. V., Porreca, F., & Raffa, R. B. (2013). Opioid antagonists for pain. Expert opinion on investigational drugs, 22(4), 517-25.
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  Opioid receptor antagonists are well known for their ability to attenuate or reverse the effects of opioid agonists. This property has made them useful in mitigating opioid side effects, overdose and abuse. Paradoxically, opioid antagonists have been reported to produce analgesia or enhance analgesia of opioid agonists. The authors review the current state of the clinical use of opioid antagonists as analgesics.
• Wang, R., King, T., De Felice, M., Guo, W., Ossipov, M. H., & Porreca, F. (2013). Descending facilitation maintains long-term spontaneous neuropathic pain. The journal of pain : official journal of the American Pain Society, 14(8), 845-53.
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  Neuropathic pain is frequently characterized by spontaneous pain (ie, pain at rest) and, in some cases, by cold- and touch-induced allodynia. Mechanisms underlying the chronicity of neuropathic pain are not well understood. Rats received spinal nerve ligation (SNL) and were monitored for tactile and thermal thresholds. While heat hypersensitivity returned to baseline levels within approximately 35 to 40 days, tactile hypersensitivity was still present at 580 days after SNL. Tactile hypersensitivity at post-SNL day 60 (D60) was reversed by microinjection of 1) lidocaine; 2) a cholecystokinin 2 receptor antagonist into the rostral ventromedial medulla; or 3) dorsolateral funiculus lesion. Rostral ventromedial medulla lidocaine at D60 or spinal ondansetron, a 5-hydroxytryptamine 3 antagonist, at post-SNL D42 produced conditioned place preference selectively in SNL-treated rats, suggesting long-lasting spontaneous pain. Touch-induced FOS was increased in the spinal dorsal horn of SNL rats at D60 and prevented by prior dorsolateral funiculus lesion, suggesting that long-lasting tactile hypersensitivity depends upon spinal sensitization, which is mediated in part by descending facilitation, in spite of resolution of heat hypersensitivity.
• Annedi, S. C., Maddaford, S. P., Ramnauth, J., Renton, P., Rybak, T., Silverman, S., Rakhit, S., Mladenova, G., Dove, P., Andrews, J. S., Zhang, D., & Porreca, F. (2012). Discovery of a potent, orally bioavailable and highly selective human neuronal nitric oxide synthase (nNOS) inhibitor, N-(1-(piperidin-4-yl)indolin-5-yl)thiophene-2-carboximidamide as a pre-clinical development candidate for the treatment of migraine. European journal of medicinal chemistry, 55, 94-107.
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  We recently reported a series of 1,6-disubstituted indoline-based thiophene amidine compounds (5) as selective neuronal nitric oxide synthase (nNOS) inhibitors to mitigate the cardiovascular liabilities associated with hERG K(+) channel inhibition (IC(50) = 4.7 μM) with previously reported tetrahydroquinoline-based selective nNOS inhibitors (4). The extended structure-activity relationship studies within the indoline core led to the identification of 43 as a selection candidate for further evaluations. The in vivo activity in two different pain (spinal nerve ligation and migraine pain) models, the excellent physicochemical and pharmacokinetic properties, oral bioavailability (F(po) = 91%), and the in vitro safety profile disclosed in this report make 43 an ideal candidate for further evaluation in clinical applications related to migraine pain.
• De Felice, M., Melchiorri, P., Ossipov, M. H., Vanderah, T. W., Porreca, F., & Negri, L. (2012). Mechanisms of Bv8-induced biphasic hyperalgesia: increased excitatory transmitter release and expression. Neuroscience letters, 521(1), 40-5.
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  Bv8 is a pronociceptive peptide that binds to two G-protein coupled prokineticin receptors, PK-R1 and PK-R2. These receptors are localized in the dorsal horn of the spinal cord and dorsal root ganglia (DRG) of nociceptive neurons in rodents. Systemic administration of Bv8 elicits a biphasic reduction in nociceptive thresholds to thermal and mechanical stimuli. Here, the possibility that Bv8 might directly modulate the expression and release of excitatory transmitters within the early and late phases of hyperalgesia was evaluated. Administration of Bv8 to mouse lumbar spinal cord sections produced a direct, significant and concentration-related release of CGRP. Bv8- or capsaicin-stimulated CGRP release was markedly enhanced in tissues taken from Bv8-pretreated mice during the late, but not the early, phase of hyperalgesia. Pretreatment of rats with protein synthesis inhibitors blocked the expression of the late, but not early, phase of Bv8-induced hyperalgesia. Finally, during the late-phase of hyperalgesia, there was an upregulation of CGRP and substance P immunoreactivity in the rat lumbar dorsal horn and DRG. Such upregulation was prevented by pretreatment with protein synthesis inhibitors. These data suggest that Bv8 induces hyperalgesia by direct release of excitatory transmitters in the spinal cord, consistent with the first phase of hyperalgesia. Additionally, Bv8 elicits a subsequent, protein-synthesis dependent increase in expression and release of excitatory transmitters that may underlie the long-lasting second phase of hyperalgesia. Activation of prokineticin receptors may therefore contribute to persistent hyperalgesia occurring as a consequence of tissue injury further suggesting that these receptors are attractive targets for development of therapeutics for pain treatment.
• Edelmayer, R. M., Le, L. N., Yan, J., Wei, X., Nassini, R., Materazzi, S., Preti, D., Appendino, G., Geppetti, P., Dodick, D. W., Vanderah, T. W., Porreca, F., & Dussor, G. (2012). Activation of TRPA1 on dural afferents: a potential mechanism of headache pain. Pain, 153(9), 1949-58.
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  Activation of transient receptor potential ankyrin-1 (TRPA1) on meningeal nerve endings has been suggested to contribute to environmental irritant-induced headache, but this channel may also contribute to other forms of headache, such as migraine. The preclinical studies described here examined functional expression of TRPA1 on dural afferents and investigated whether activation of TRPA1 contributes to headache-like behaviors. Whole-cell patch-clamp recordings were performed in vitro with 2 TRPA1 agonists, mustard oil (MO), and the environmental irritant umbellulone (UMB) on dural-projecting trigeminal ganglion neurons. Application of MO and UMB to dural afferents produced TRPA1-like currents in approximately 42% and 38% of cells, respectively. By means of an established in vivo behavioral model of migraine-related allodynia, dural application of MO and UMB produced robust time-related tactile facial and hind paw allodynia that was attenuated by pretreatment with the TRPA1 antagonist HC-030031. Additionally, MO or UMB were applied to the dura, and exploratory activity was monitored for 30min with an automated open-field activity chamber. Dural MO and UMB decreased the number of vertical rearing episodes and the time spent rearing in comparison to vehicle-treated animals. This change in activity was prevented in rats pretreated with HC-030031 as well as sumatriptan, a clinically effective antimigraine agent. These data indicate that TRPA1 is expressed on a substantial fraction of dural afferents, and activation of meningeal TRPA1 produces behaviors consistent with those observed in patients during migraine attacks. Further, they suggest that activation of meningeal TRPA1 via endogenous or exogenous mechanisms can lead to afferent signaling and headache.
• He, Y., Tian, X., Hu, X., Porreca, F., & Wang, Z. J. (2012). Negative reinforcement reveals non-evoked ongoing pain in mice with tissue or nerve injury. The journal of pain : official journal of the American Pain Society, 13(6), 598-607.
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  Patients with chronic pain experience spontaneous or ongoing pain as well as enhanced sensitivity to evoked stimuli. Spontaneous or ongoing pain is rarely evaluated in preclinical studies. In fact, it remains controversial whether ongoing or spontaneous pain even develops in mice after tissue or nerve injury. This study tested a hypothesis that negative reinforcement can be used to unmask the presence of pain in mice with tissue or nerve injury. We found that spinal administration of clonidine or lidocaine did not elicit conditioned place preference (CPP) in uninjured or sham-operated mice. However, these agents produced CPP in mice with chronic inflammation induced by complete Freund's adjuvant (CFA) or following L5/L6 spinal nerve ligation (SNL). These data indicate the presence of non-evoked (ie, stimulus-independent) ongoing pain in mice with chronic inflammation (CFA) or following nerve injury (SNL). In addition, this study validates the use of negative reinforcement to unmask non-evoked ongoing pain in mice. Given the existence of a large collection of transgenic and knockout mice, our data show the application of this approach to elucidate molecular mechanisms underlying non-evoked pain and to contribute to drug discovery for pain.
• Hughes, J. P., Chessell, I., Malamut, R., Perkins, M., Bačkonja, M., Baron, R., Farrar, J. T., Field, M. J., Gereau, R. W., Gilron, I., McMahon, S. B., Porreca, F., Rappaport, B. A., Rice, F., Richman, L. K., Segerdahl, M., Seminowicz, D. A., Watkins, L. R., Waxman, S. G., , Wiech, K., et al. (2012). Understanding chronic inflammatory and neuropathic pain. Annals of the New York Academy of Sciences, 1255, 30-44.
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  This meeting report highlights the main topics presented at the conference "Chronic Inflammatory and Neuropathic Pain," convened jointly by the New York Academy of Sciences, MedImmune, and Grünenthal GmbH, on June 2-3, 2011, with the goal of providing a conducive environment for lively, informed, and synergistic conversation among participants from academia, industry, clinical practice, and government to explore new frontiers in our understanding and treatment of chronic and neuropathic pain. The program included leading and emerging investigators studying the pathophysiological mechanisms underlying neuropathic and chronic pain, and experts in the clinical development of pain therapies. Discussion included novel issues, current challenges, and future directions of basic research in pain and preclinical and clinical development of new therapies for chronic pain.
• King, T., Qu, C., Okun, A., Melemedjian, O. K., Mandell, E. K., Maskaykina, I. Y., Navratilova, E., Dussor, G. O., Ghosh, S., Price, T. J., & Porreca, F. (2012). Contribution of PKMζ-dependent and independent amplification to components of experimental neuropathic pain. Pain, 153(6), 1263-73.
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  Injuries can induce adaptations in pain processing that result in amplification of signaling. One mechanism may be analogous to long-term potentiation and involve the atypical protein kinase C, PKMζ. The possible contribution of PKMζ-dependent and independent amplification mechanisms to experimental neuropathic pain was explored in rats with spinal nerve ligation (SNL) injury. SNL increased p-PKMζ in the rostral anterior cingulate cortex (rACC), a site that mediates, in part, the unpleasant aspects of pain. Inhibition of PKMζ within the rACC by a single administration of ζ-pseudosubstrate inhibitory peptide (ZIP) reversed SNL-induced aversiveness within 24 hours, whereas N-methyl-d-aspartate receptor blockade with MK-801 had no effects. The SNL-induced aversive state (reflecting "spontaneous" pain), was re-established in a time-dependent manner, with full recovery observed 7 days post-ZIP administration. Neither rACC ZIP nor MK-801 altered evoked responses. In contrast, spinal ZIP or MK-801, but not scrambled peptide, transiently reversed evoked hypersensitivity, but had no effect on nerve injury-induced spontaneous pain. PKMζ phosphorylation was not altered by SNL in the spinal dorsal horn. These data suggest that amplification mechanisms contribute to different aspects of neuropathic pain at different levels of the neuraxis. Thus, PKMζ-dependent amplification contributes to nerve injury-induced aversiveness within the rACC. Moreover, unlike mechanisms maintaining memory, the consequences of PKMζ inhibition within the rACC are not permanent in neuropathic pain, possibly reflecting the re-establishment of amplification mechanisms by ongoing activity of injured nerves. In the spinal cord, however, both PKMζ-dependent and independent mechanisms contribute to amplification of evoked responses, but apparently not spontaneous pain.
• Little, J. W., Chen, Z., Doyle, T., Porreca, F., Ghaffari, M., Bryant, L., Neumann, W. L., & Salvemini, D. (2012). Supraspinal peroxynitrite modulates pain signaling by suppressing the endogenous opioid pathway. The Journal of neuroscience : the official journal of the Society for Neuroscience, 32(32), 10797-808.
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  Peroxynitrite (PN, ONOO(-)) is a potent oxidant and nitrating agent that contributes to pain through peripheral and spinal mechanisms, but its supraspinal role is unknown. We present evidence here that PN in the rostral ventromedial medulla (RVM) is essential for descending nociceptive modulation in rats during inflammatory and neuropathic pain through PN-mediated suppression of opioid signaling. Carrageenan-induced thermal hyperalgesia was associated with increased 3-nitrotyrosine (NT), a PN biomarker, in the RVM. Furthermore, intra-RVM microinjections of the PN decomposition catalyst Fe(III)-5,10,15,20-tetrakis(N-methyl-pyridinium-4-yl)porphyrin (FeTMPyP(5+)) dose-dependently reversed this thermal hyperalgesia. These effects of FeTMPyP(5+) were abrogated by intra-RVM naloxone, implicating potential interplay between PN and opioids. In support, we identified NT colocalization with the endogenous opioid enkephalin (ENK) in the RVM during thermal hyperalgesia, suggesting potential in situ interactions. To address the functional significance of such interactions, we exposed methionine-enkephalin (MENK) to PN and identified the major metabolite, 3-nitrotyrosine-methionine-sulfoxide (NSO)-MENK, using liquid chromatography-mass spectrometry. Next, we isolated, purified, and tested NSO-MENK for opioid receptor binding affinity and analgesic effects. Compared to MENK, this NSO-MENK metabolite lacked appreciable binding affinity for δ, μ, and κ opioid receptors. Intrathecal injection of NSO-MENK in rats did not evoke antinociception, suggesting that PN-mediated chemical modifications of ENK suppress opioid signaling. When extended to chronic pain, intra-RVM FeTMPyP(5+) produced naloxone-sensitive reversal of mechanical allodynia in rats following chronic constriction injury of the sciatic nerve. Collectively, our data reveal the central role of PN in RVM descending facilitation during inflammatory and neuropathic pain potentially through anti-opioid activity.
• Mladenova, G., Annedi, S. C., Ramnauth, J., Maddaford, S. P., Rakhit, S., Andrews, J. S., Zhang, D., & Porreca, F. (2012). First-in-class, dual-action, 3,5-disubstituted indole derivatives having human nitric oxide synthase (nNOS) and norepinephrine reuptake inhibitory (NERI) activity for the treatment of neuropathic pain. Journal of medicinal chemistry, 55(7), 3488-501.
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  A family of different 3,5-disubstituted indole derivatives having 6-membered rings were designed, synthesized, and demonstrated inhibition of human nitric oxide synthase (NOS) with norepinephrine reuptake inhibitory activity (NERI). The structure-activity relationship (SAR) within the cyclohexane ring showed the cis-isomers to be more potent for neuronal NOS and selective over endothelial NOS compared to their trans-counterparts. Compounds, such as cis-(+)-37, exhibited dual nNOS and NET inhibition (IC(50) of 0.56 and 1.0 μM, respectively) and excellent selectivity (88-fold and 12-fold) over eNOS and iNOS, respectively. The lead compound (cis-(+)-37) showed lack of any direct vasoconstriction or inhibition of ACh-mediated vasorelaxation in isolated human coronary arteries. Additionally, cis-(+)-37 was effective at reversing both allodynia and thermal hyperalgesia in a standard Chung (spinal nerve ligation) rat neuropathic pain model. Overall, the data suggest that cis-(+)-37 is a promising dual action development candidate having therapeutic potential for the treatment of neuropathic pain.
• Mollica, A., Pinnen, F., Stefanucci, A., Feliciani, F., Campestre, C., Mannina, L., Sobolev, A. P., Lucente, G., Davis, P., Lai, J., Ma, S. W., Porreca, F., & Hruby, V. J. (2012). The cis-4-amino-L-proline residue as a scaffold for the synthesis of cyclic and linear endomorphin-2 analogues. Journal of medicinal chemistry, 55(7), 3027-35.
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  Endomorphin-2 (EM-2: Tyr-Pro-Phe-Phe-NH(2)) is an endogenous tetrapeptide that combines potency and efficacy with high affinity and selectivity toward the μ opioid receptor, the most responsible for analgesic effects in the central nervous system. The presence of the Pro(2) represents a crucial factor for the ligand structural and conformational properties. Proline is in fact an efficient stereochemical spacer, capable of inducing favorable spatial orientation of aromatic rings, a key factor for ligand recognition and interaction with receptors. Here the Pro(2) has been replaced by 4(S)-NH(2)-2(S)-proline (cAmp), a proline/GABA cis-chimera residue. This bivalent amino acid maintains the capacity to influenc the tetrapeptide conformation and offers the opportunity to generate new linear models and unusually constrained cyclic analogues characterized by an N-terminal Tyr bearing a free α-amino group. The results indicate that the new analogues do not show affinity for both δ and κ opioid receptors and bind only poorly to the μ receptors (for cyclopeptide 9: K(i)(μ) = 660 nM; GPI (IC(50)) = 1.4% at 1 μM; for linear tetrapeptide acid 13: K(i)(μ) = 2000 nM; GPI (IC(50)) = 0% at 1 μM; for linear tetrapeptide amide 15: K(i)(μ) = 310 nM; GPI (IC(50)) = 894 nM).
• Mollica, A., Pinnen, F., Stefanucci, A., Mannina, L., Sobolev, A. P., Lucente, G., Davis, P., Lai, J., Ma, S. W., Porreca, F., & Hruby, V. J. (2012). cis-4-amino-L-proline residue as a scaffold for the synthesis of cyclic and linear endomorphin-2 analogues: part 2. Journal of medicinal chemistry, 55(19), 8477-82.
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  Recently, we reported synthesis and activity of a constrained cyclic analogue of endomorphin-2 (EM-2: Tyr-Pro-Phe-Phe-NH(2)) and related linear models containing the cis-4-amino-L-proline (cAmp) in place of native Pro(2). In the present article, the adopted rationale is the possible modulation of the receptor affinity of the cAmp containing EM-2 analogues by assigning a different stereochemistry to the Phe(3) and Phe(4) residues present in the ring. Thus, eight more analogues with different absolute configuration at the chiral center of the aromatic residues in positions 3 and 4 have been synthesized and their opioid activity examined. The stereochemical change at the α-carbon atoms leads to a meaningful enhancement of the affinity and activity toward μ opioid receptors with respect to the prototype compound 9: e.g., 9a, K(i)(μ) = 63 nM, GPI (IC(50)) = 480 nM; 9b, K(i)(μ) = 38 nM, GPI (IC(50)) = 330 nM.
• Navratilova, E., Xie, J. Y., Okun, A., Qu, C., Eyde, N., Ci, S., Ossipov, M. H., King, T., Fields, H. L., & Porreca, F. (2012). Pain relief produces negative reinforcement through activation of mesolimbic reward-valuation circuitry. Proceedings of the National Academy of Sciences of the United States of America, 109(50), 20709-13.
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  Relief of pain is rewarding. Using a model of experimental postsurgical pain we show that blockade of afferent input from the injury with local anesthetic elicits conditioned place preference, activates ventral tegmental dopaminergic cells, and increases dopamine release in the nucleus accumbens. Importantly, place preference is associated with increased activity in midbrain dopaminergic neurons and blocked by dopamine antagonists injected into the nucleus accumbens. The data directly support the hypothesis that relief of pain produces negative reinforcement through activation of the mesolimbic reward-valuation circuitry.
• Pajouhesh, H., Feng, Z. P., Zhang, L., Pajouhesh, H., Jiang, X., Hendricson, A., Dong, H., Tringham, E., Ding, Y., Vanderah, T. W., Porreca, F., Belardetti, F., Zamponi, G. W., Mitscher, L. A., & Snutch, T. P. (2012). Structure-activity relationships of trimethoxybenzyl piperazine N-type calcium channel inhibitors. Bioorganic & medicinal chemistry letters, 22(12), 4153-8.
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  We previously reported the small organic N-type calcium channel blocker NP078585 that while efficacious in animal models for pain, exhibited modest L-type calcium channel selectivity and substantial off-target inhibition against the hERG potassium channel. Structure-activity studies to optimize NP078585 preclinical properties resulted in compound 16, which maintained high potency for N-type calcium channel blockade, and possessed excellent selectivity over the hERG (~120-fold) and L-type (~3600-fold) channels. Compound 16 shows significant anti-hyperalgesic activity in the spinal nerve ligation model of neuropathic pain and is also efficacious in the rat formalin model of inflammatory pain.
• Sherman, S. J., Polt, R., Porreca, F., Szabo, L., Zuniga, L. A., Falk, T., & Yue, X. (2011). Effects of the novel glycopeptide opioid agonist MMP-2200 in preclinical models of Parkinson's disease.. Brain research, 1413, 72-83. doi:10.1016/j.brainres.2011.07.038
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  In Parkinson's disease (PD), the consequence of dopaminergic denervation is an imbalance in the activity of the direct and indirect striatofugal pathways, which include potentially important changes in opioid peptide expression and/or activity. The systemic administration of a novel glycosylated opioid peptide MMP-2200 (a.k.a. lactomorphin) was shown to have potent effects in two standard models of PD: 1) amphetamine-induced rotations in the hemi-Parkinsonian 6-hydroxydopamine (6-OHDA)-treated rat and 2) locomotion in the reserpine-treated rat. MMP-2200, an opioid mu and delta receptor agonist, reduced amphetamine-induced rotations in severely-lesioned hemi-Parkinsonian rats; this effect was fully blocked by naloxone, an opioid receptor antagonist. The selective δ-opioid receptor antagonist naltrindole only partially blocked the effect of MMP-2200. MMP-2200 alone did not induce rotations. This effect was also observed in a mild progressive rat 6-OHDA-lesion model. In animals treated with reserpine, profound akinesia was induced that was reversed with apomorphine. There was a prominent overshoot in animals that received apomorphine compared to non-reserpine treated animals, reflecting the well described phenomenon of dopamine supersensitivity indicating that apomorphine not only reversed akinesia but also induced hyper-kinesia. The opioid peptide MMP-2200 blocked the apomorphine-induced hyper-kinesia. This effect of MMP-2200 was prevented by pre-administration of naloxone. MMP-2200 had no effect in preventing the reserpine-induced akinesia, nor did it affect locomotion in control animals. Taken together, the results from these two models are consistent with the glycopeptide opioid agonist MMP-2200 having a potent effect on movements related to dopaminergic hyper-stimulation following striatal dopamine depletion that are best explained by a reduction in the downstream effects of dopamine agonists in these models.

Poster Presentations

• Falk, T., Porreca, F., Sherman, S. J., Heien, M. L., Siegenthaler, J., Sexauer, M. R., Bartlett, M. J., Flores, A. J., & Nava, R. (2018, Fall). The kappa opioid receptor antagonist nor-BNI accelerates development of L-DOPA-induced dyskinesia in a model of mild Parkinson’s disease. Society for Neuroscience Meeting.
• Falk, T., Porreca, F., Sherman, S. J., Heien, M. L., Siegenthaler, J., Sexauer, M. R., Bartlett, M. J., Flores, A. J., & Nava, R. (2018, Fall). The kappa opioid receptor antagonist nor-BNI accelerates development of L-DOPA-induced dyskinesia in a model of mild Parkinson’s disease. Society for Neuroscience.