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 (PNDC), Fe(III)-5,10,15,20-tetrakis(N-methyl-pyridinium-4-yl)porphyrin (FeTMPyP5+) dose-dependently reversed this thermal hyperalgesia. These effects of FeTMPyP5+ were abrogated by intra-RVM naloxone, implicating potential interplay between PN and opioids. In support, we identified NT co-localization 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 (LCMS). 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 FeTMPyP5+ produced naloxone-sensitive reversal of mechanical allodynia in rats following chronic constriction injury (CCI) 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.

Cholecystokinin (CCK) has been suggested to be both pro-nociceptive and anti-opioid by actions on pain modulatory cells within the RVM. One consequence of activation of RVM CCK2 receptors may be enhanced spinal nociceptive transmission but how this might occur, especially in states of pathological pain is unknown. Here, in vivo microdialysis was used to demonstrate that levels of RVM CCK increased by approximately 2-fold following ligation of L5/L6 spinal nerves (SNL). Microinjection of CCK into the RVM of naïve rats elicited hypersensitivity to tactile stimulation of the hindpaw. Additionally, RVM CCK elicited a time-related increase in PGE2 measured in cerebrospinal fluid from the lumbar spinal cord. The peak increase in spinal PGE2 was approximately 5-fold and was observed at approximately 80-min post-RVM CCK, a time coincident with maximal RVM CCK-induced mechanical hypersensitivity. Spinal administration of naproxen, a non-selective COX-inhibitor, significantly attenuated RVM CCK-induced hindpaw tactile hypersensitivity. RVM-CCK also resulted in a 2-fold increase in spinal 5-HIAA, a 5-HT metabolite, as compared to controls, and mechanical hypersensitivity that was attenuated by spinal application of ondansetron, a 5-HT3 antagonist. The present studies suggest that chronic nerve injury can result in activation of descending facilitatory mechanisms that may promote hyperalgesia via ultimate release of PGE2 and 5-HT in the spinal cord.

In order to obtain a metabolically more stable analgesic peptide derivative, O-β-glycosylated serine (Ser(Glc)) was introduced into TY027 (Tyr-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-NH-3’,5’-Bzl(CF3)2) which was a previously reported bifunctional compound with delta/mu opioid agonist and neurokinin-1 receptor antagonist activities, and with a half life of 4.8 h in rat plasma. Incorporation of Ser(Glc) into various positions of TY027 gave analogues with variable bioactivities. Analogue 6 (Tyr-D-Ala-Gly-Phe-Nle-Pro-Leu-Ser(Glc)-Trp-NH-3’,5’-Bzl(CF3)2) was found to have effective bifunctional activities with a well-defined conformation with two β-turns based on the NMR conformational analysis in the presence of DPC micelles. In addition, 6 showed significant improvement in its metabolic stability (70 ± 9 % of 6 was intact after 24 h incubation in rat plasma). This improved metabolic stability, along with its effective and delta selective bifunctional activities, suggests that 6 could be an interesting research tool and possibly a promising candidate as a novel analgesic drug.

A puzzling observation is why peripheral nerve injury results in chronic pain in some, but not all, patients. We explored potential mechanisms that may prevent the expression of chronic pain. Sprague-Dawley (SD) or Holtzman (HZ) rats showed no differences in baseline sensory thresholds or responses to inflammatory stimuli. However, spinal nerve ligation (SNL)-induced tactile allodynia occurred in approximately 85% of SD and 50% of HZ rats, respectively. No apparent differences were observed in a survey of DRG or spinal “neuropathic markers” following SNL regardless of allodynic phenotype. SNL-induced allodynia was reversed by administration of lidocaine within the rostral ventromedial medulla (RVM), a site that integrates descending pain modulation via pain inhibitory (i.e., OFF) and excitatory (i.e., ON) cells. However, in SD or HZ rats with SNL but without allodynia, RVM lidocaine precipitated allodynia. Additionally, RVM lidocaine produced conditioned place preference in allodynic SD or HZ rats but conditioned place aversion in non-allodynic HZ rats. Similarly, RVM U69,593 (kappa opioid agonist) or blockade of spinal α2 adrenergic receptors precipitated allodynia in previously non-allodynic HZ rats with SNL. All rats showed an equivalent first phase formalin responses. However, HZ rats had reduced second phase formalin behaviors along with fewer RVM OFF cell pauses and RVM ON cell bursts. Thus, expression of nerve-injury induced pain may ultimately depend on descending modulation. Engagement of descending inhibition protects in the transition from acute to chronic pain. These unexpected findings might provide a mechanistic explanation for medications that engage descending inhibition or mimic its consequences.

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 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.

A predominant complaint in patients with neuropathic pain is spontaneous pain, often described as “burning”. Recent studies have demonstrated that negative reinforcement can be used to unmask spontaneous neuropathic pain allowing for mechanistic investigations. Here, ascending pathways that might contribute to evoked and spontaneous components of experimental neuropathic pain model were explored. Desensitization of TRPV1 positive fibers with systemic resiniferatoxin (RTX) abolished spinal nerve ligation (SNL) injury-induced thermal hypersensitivity and spontaneous pain, but had no effect on tactile hypersensitivity. Ablation of spinal NK-1 receptor expressing neurons blocked SNL-induced thermal and tactile hypersensitivity as well as spontaneous pain. Following nerve injury, upregulation of neuropeptide Y (NPY) is observed almost exclusively in large diameter fibers and inactivation of the brainstem target of these fibers in the n. gracilis prevents tactile, but not thermal, hypersensitivity. Blockade of NPY signaling within the n. gracilis failed to block SNL-induced spontaneous pain or thermal hyperalgesia while fully reversing tactile hypersensitivity. Moreover, microinjection of NPY into n. gracilis produced robust tactile hypersensitivity, but failed to induce conditioned place aversion. These data suggest that spontaneous neuropathic pain and thermal hyperalgesia are mediated by TRPV1 positive fibers and spinal NK-1 positive ascending projections. In contrast, the large diameter dorsal column projection can mediate nerve injury-induced tactile hypersensitivity, but does not contribute to spontaneous pain. As inhibition of tactile hypersensitivity can be achieved either by spinal manipulations or by inactivation of signaling within the n. gracilis, the enhanced paw withdrawal response evoked by tactile stimulation does not necessarily reflect “allodynia”.

Tonic pain has been difficult to demonstrate in animals. Because relief of pain is rewarding, analgesic agents that are not rewarding in the absence of pain should become rewarding only when there is ongoing pain. We used conditioned place preference to concomitantly determine the presence of tonic pain in rats and the efficacy of agents that relieve it. This provides a new approach for investigating tonic pain in animals and for evaluating the analgesic effects of drugs.

Background

Opioid induced hyperalgesia is recognised in the laboratory and the clinic, generating central hyperexcitability in the absence of peripheral pathology. We investigated pregabalin, indicated for neuropathic pain, and ondansetron, a drug that disrupts descending serotonergic processing in the central nervous system, on spinal neuronal hyperexcitability and visceral hypersensitivity in a rat model of opioid induced hyperalgesia.

Methods

Sprague-Dawley rats (180-200 g) were implanted with morphine (90μg · μl−1 · hr−1) or saline (0.9% w/v) filled osmotic mini-pumps. On days 7-10 in isoflurane anaesthetized animals we evaluated the effects of (a) systemic pregabalin on spinal neuronal and visceromotor responses and (b) spinal ondansetron on dorsal horn neuronal responses. The messenger RNA levels of α2δ-1, 5HT3A and mu-opioid receptor in the dorsal root ganglia of all animals were analysed.

Results

In morphine-treated animals the evoked spinal neuronal responses were enhanced to a sub-set of thermal and mechanical stimuli. This activity was attenuated by pregabalin (by at least 71%) and ondansetron (37%), and the visceromotor response to a sub-set of colorectal distension pressures was attenuated by pregabalin (52.8%) (n = 8 for all measures, P < 0.05). Messenger RNA levels were unchanged.

Conclusions

The inhibitory action of pregabalin in opioid induced hyperalgesia animals is not neuropathy-dependent nor reliant on up-regulation of the α2δ-1 subunit of voltage gated calcium channels, mechanisms proposed essential for pregabalin’s efficacy in neuropathy. In opioid induced hyperalgesia, which extends to colonic distension, a serotonergic facilitatory system may be upregulated creating an environment that’s permissive for pregabalin-mediated analgesia without peripheral pathology.

Dorsal root injury results in substantial and often irreversible loss of sensory functions as a result of the limited regenerative capacity of sensory axons and the inhibitory barriers that prevent both axonal entry into and regeneration in the spinal cord. Here, we describe previously unknown effects of the growth factor artemin after crush injury of the dorsal spinal nerve roots in rats. Artemin not only promoted re-entry of multiple classes of sensory fibers into the spinal cord and re-establishment of synaptic function and simple behavior, but it also, surprisingly, promoted the recovery of complex behavior. These effects occurred after a 2-week schedule of intermittent, systemic administration of artemin and persisted for at least 6 months following treatment, suggesting a substantial translational advantage. Systemic artemin administration produced essentially complete and persistent restoration of nociceptive and sensorimotor functions, and could represent a promising therapy that may effectively promote sensory neuronal regeneration and functional recovery after injury.

Osteoarthritis (OA) isa chronic pain condition characterized by pain during joint useas well aspain at rest (i.e., ongoing pain). Although injection of monosodium iodoacetate (MIA)into the intra-articular space of the rodent knee is a well established model of OA pain that is characterized by changes in weight bearing and hypersensitivity to tactile and thermal stimuli, it is not known if this procedure elicits ongoing pain. Further, the time-course and possible underlying mechanisms of these components of pain remain poorly understood. In these studies, we demonstrated the presence ofongoing painin addition to changes in weight bearing and evoked hypersensitivity. Twenty-eight days following MIA injection, spinal clonidine blockedchanges in weight bearing and thermal hypersensitivityand produced place preference indicating that MIA induces ongoing and evoked pain.These findings demonstrate the presence of ongoing pain in this model that is present at a late-time point after MIA allowing for mechanistic investigation.

Neuropathic pain is often “spontaneous” or “stimulus-independent.” Such pain may result from spontaneous discharge in primary afferent nociceptors in injured peripheral nerves. However, whether axotomized primary afferent nociceptors give rise to pain is unclear. The rostral anterior cingulate cortex (rACC) mediates the negative affective component of inflammatory pain. Whether the rACC integrates the aversive component of chronic spontaneous pain arising from nerve injury is not known. Here, we used the principle of negative reinforcement to show that axotomy produces an aversive state reflecting spontaneous pain driven from injured nerves. Additionally, we investigated whether the rACC contributes to the aversiveness of nerve injury induced spontaneous pain. Partial or complete hindpaw denervation was produced by sciatic or sciatic/saphenous axotomy, respectively. Conditioned place preference resulting from presumed pain relief was observed following spinal clonidinein animals with sciatic axotomy but not in sham-operated controls. Similarly, lidocaine administration into the rostral ventromedial medulla (RVM) produced place preference selectively in animals with sciatic/saphenous axotomy. In rats with spinal nerve ligation (SNL) injury lesion of the rACC blocked the reward elicited by RVM lidocaine but did not alter acute stimulus evoked hypersensitivity. Lesion of the rACC did not block cocaine-induced reward indicating that rACC blockade did not impair memory encoding or retrieval but did impair spontaneous aversiveness. These data indicate that spontaneous pain arising from injured nerve fibers produces a tonic aversive state that is mediated by the rACC. Identification of the circuits mediating aversiveness of chronic pain should facilitate the development of improved therapies.

Opiates are currently the mainstay for treatment of moderate to severe pain. However, prolonged administration of opiates has been reported to elicit hyperalgesia in animals and examples of opiate-induced hyperalgesia have been reported in humans as well. In spite of the potential clinical significance of such opiate-induced actions, the mechanisms of opiate-induced hypersensitivity remain unknown. The TRPV1 receptor, a molecular sensor of noxious heat, acts as an integrator of multiple forms of noxious stimuli and plays an important role in the development of inflammation-induced hyperalgesia. As animals treated with opiates show thermal hyperalgesia, we examined the possible role of TRPV1 receptors in the development of morphine-induced hyperalgesia using TRPV1 wild-type (WT) and knock-out (KO) mice and with administration of a TRPV1 antagonist in mice and rats. Administration of morphine by subcutaneous implantation of morphine pellets elicited both thermal and tactile hypersensitivity in TRPV1 WT mice, but not in TRPV1 KO mice. Moreover, oral administration of a TRPV1 antagonist reversed both thermal and tactile hypersensitivity induced by sustained morphine administration in mice and rats. Immunohistochemical analyses indicate that sustained morphine administration modestly increases TRPV1 labeling in the dorsal root ganglia (DRG). In addition, sustained morphine increased flinching and plasma extravasation after peripheral stimulation with capsaicin, suggesting an increase in TRPV1 receptor function in the periphery in morphine treated animals. Collectively our data indicate that the TRPV1 receptor is an essential peripheral mechanism in expression of morphine-induced hyperalgesia.

PERSPECTIVE

Opioid-induced hyperalgesia possibly limits the usefulness of opioids, emphasizing the value of alternative methods of pain control. We demonstrate that TRPV1 channels play an important role in peripheral mechanisms of opioid-induced hyperalgesia. Such information may lead to the discovery of analgesics lacking such adaptations and improving treatment of chronic pain.

The conventional design of high affinity drugs targeted to a single molecule has not resulted in clinically useful therapies for pain relief. Recent reviews have suggested that newly designed analgesic drugs should incorporate multiple targets. The distributions of cholecystokinin (CCK) and CCK receptors in the central nervous system (CNS) overlap significantly with endogenous opioid systems and can be dually targeted. CCK has been shown to act as an endogenous “anti-analgesic” peptide and neuropathic pain conditions promote endogenous CCK release in CNS regions of pain modulation. Administration of CCK into nuclei of the rostral ventromedial medulla induces pronociceptive behaviors in rats. RSA 504 and RSA 601 are novel bifunctional compounds developed to target neuropathic pain by simultaneously acting as agonists at two distinct opioid receptors and antagonizing CCK receptors in the CNS. RSA 504 and RSA 601 demonstrate agonist activity in vitro and antihypersensitivity to mechanical and thermal stimuli in vivo using the spinal nerve ligation model of neuropathic pain. Intrathecal administration of RSA 504 and RSA 601 did not demonstrate antinociceptive tolerance over 7 days of administration and did not display motor impairment or sedation using a rotarod. These are the first behavioral studies that demonstrate how multi-targeted molecule design can address the pathology of neuropathic pain. These compounds with δ and μ opioid agonist activity and CCK antagonist activity within one molecule offer a novel approach with efficacy for neuropathic pain while lacking the side effects typically caused by conventional opioid therapies.

Multivalent ligands with delta/mu opioid agonist and NK1 antagonist activities have shown promising analgesic potency without detectable sign of toxicities, including motor skill impairment and opioid-induced tolerance. To improve their biological activities and metabolic stability, structural optimization was performed on our peptide-derived lead compounds by introducing 2′, 6′-Dimethyl-L-Tyrosine (Dmt) instead of Tyr at the first position. The compound 7 (Dmt-D-Ala-Gly-Phe-MetPro-Leu-Trp-NH-[3′,5′-(CF3)2-Bzl]) showed improved multivalent bioactivities compared to those of the lead compounds, had more than 6 h half-life in rat plasma, and significant antinociceptive efficacy in vivo. The NMR structural analysis suggested that Dmt1 incorporation in compound 7 induces the structured conformation in the opioid pharmacophore (N-terminus), and simultaneously shifts the orientation of the NK1 pharmacophore (C-terminus), consistent with its affinities and activities at both opioid and NK1 receptors. These results indicate that compound 7 is a valuable research tool to seek a novel analgesic drug.

A screening of conformationally constrained aromatic amino acids as base cores for the preparation of new NK1 receptor antagonists resulted in the discovery of three new NK1 receptor antagonists, 19 [Ac-Aba-Gly-NH-3′,5′-(CF3)2-Bn], 20 [Ac-Aba-Gly-NMe-3′,5′-(CF3)2-Bn] and 23 [Ac-Tic-NMe-3′,5′-(CF3)2-Bn], which were able to counteract the agonist effect of substance P, the endogenous ligand of NK1R. The most active NK1 antagonist of the series, 20 [Ac-Aba-Gly-NMe-3′,5′-(CF3)2-Bn], was then used in the design of a novel, potent chimeric opioid agonist-NK1 receptor antagonist, 35 [Dmt-D-Arg-Aba-Gly-NMe-3′,5′-(CF3)2-Bn], which combines the N-terminus of the established Dmt1-DALDA agonist opioid pharmacophore (H-Dmt-D-Arg-Phe-Lys-NH2) and 20, the NK1R ligand. The opioid component of the chimeric compound 35, i.e. Dmt-D-Arg-Aba-Gly-NH2 36, also proved to be an extremely potent and balanced μ- and δ opioid receptor agonist with subnanomolar binding and in vitro functional activity.

Pancreatic pain resulting from chronic inflammation of the pancreas is often intractable and clinically difficult to manage with available analgesics reflecting the need for more effective therapies. Mechanisms underlying pancreatitis pain are not well understood. Here, the possibility that interleukin-6 (IL-6) may promote pancreatitis pain was investigated with TB-2-081 (3-O-formyl-20R,21-epoxyresibufogenin, EBRF), a small molecule IL-6 receptor antagonist that was semi-synthetically derived from natural sources. The potential activity and mechanism of TB-2-081 was investigated following induction of persistent pancreatitis using dibutyltin dichloride (DBTC) in rats. TB-2-081 displaces binding of IL-6 to the human recombinant soluble IL-6 receptor with apparent high affinity and inhibits IL-6 mediated cell growth. Systemic or oral, but not intrathecal, administration of TB-2-081 reversed DBTC-induced abdominal hypersensitivity in a dose- and time-dependent manner. IL-6 levels were significantly upregulated in the dorsal root ganglia (DRG) of rats with pancreatitis on day 6 after DBTC injection. IL-6 enhanced capsaicin-evoked release of calcitonin gene related peptide from cultured DRG neurons was blocked by TB-2-081. Our data demonstrate that TB-2-081 acts as a systemically available and orally active small molecule IL-6 receptor antagonist. TB-2-081 effectively reduces pancreatitis-induced pain through peripheral mechanisms that are likely due to (a) increased expression of IL-6 in the DRG and (b) IL-6-mediated sensitization of nociceptive neurons. The activity of TB-2-081 implicates an important role for IL-6 in sustaining pancreatitis pain. Strategies targeting IL-6 actions through small molecule antagonists may offer novel approaches to improve therapy of chronic pancreatitis and other chronic pain states.

An SAR study on the Dmt-substituted enkephalin-like tetrapeptide with a N-phenyl-N-piperidin-4-yl propionamide moiety at C-terminal was performed, and has resulted in highly potent ligands at μ and δ opioid receptors. In general, ligands with the substitution of D-Nle2 and halogenation of the aromatic ring of Phe4 showed highly increased opioid activities. Ligand 6 with good biological activities in vitro demonstrated potent in vivo antihyperalgesic and antiallodynic effects in the tail-flick assay.

Migraine headache is one of the most common neurological disorders. The pathological conditions that directly initiate afferent pain signaling are poorly understood. In trigeminal neurons retrogradely labeled from the cranial meninges, we have recorded pH-evoked currents using whole-cell patch-clamp electrophysiology. Approximately 80% of dural afferent neurons responded to a pH 6.0 application with a rapidly activating and rapidly desensitizing ASIC-like current that often exceeded 20 nA in amplitude. Inward currents were observed in response to a wide range of pH values and 30% of the neurons exhibited inward currents at pH 7.1. These currents led to action potentials in 53%, 30% and 7% of the dural afferents at pH 6.8, 6.9 and 7.0, respectively. Small decreases in extracellular pH were also able to generate sustained window currents and sustained membrane depolarizations. Amiloride, a non specific blocker of ASIC channels, inhibited the peak currents evoked upon application of decreased pH while no inhibition was observed upon application of TRPV1 antagonists. The desensitization time constants of pH 6.0-evoked currents in the majority of dural afferents was less than 500 ms which is consistent with that reported for ASIC3 homomeric or heteromeric channels. Finally, application of pH 5.0 synthetic-interstitial fluid to the dura produced significant decreases in facial and hind-paw withdrawal threshold, an effect blocked by amiloride but not TRPV1 antagonists, suggesting that ASIC activation produces migraine-related behavior in vivo. These data provide a cellular mechanism by which decreased pH in the meninges following ischemic or inflammatory events directly excites afferent pain-sensing neurons potentially contributing to migraine headache.

Existing clinical imaging procedures lack sensitivity and specificity in detecting early prostate cancer bone metastatic lesions. In this study, we developed a highly reproducible bone metastasis xenograft model and identified possible molecular imaging candidates for detecting early bone metastatic lesions. Bone trophic human prostate cells (PC-3B1) were isolated and characterized for their ability to reach bone after intracardiac injection into SCID mice. The appearances of skeletal metastases were evaluated using digital radiographic imaging and confirmed by necropsy and histology. The PC-3B1 cells retain a bone homing phenotype after long term propagation in tissue culture and exhibit progressive bone lesions within 3 weeks following intracardiac injection. Comparative transcription signatures of PC-3 and PC-3B1 cells were determined using a cancer specific microarray and confirmed by RT-PCR analysis. The analysis identified increased expression of four cell surface molecules in PC-3B1 cells that may be suitable as molecular imaging candidates to detect bone micro metastases.

Sensitization of the pain pathway is believed to promote clinical pain disorders. We hypothesized that the persistence of a sensitized state in the spinal dorsal horn might depend on the activity of protein kinase M zeta (PKMζ), an essential mechanism of late long-term potentiation (LTP). To test this hypothesis we utilized intraplantar injections of interleukin-6 (IL-6) in mice to elicit a transient allodynic state that endured approximately 3 days. After the resolution of IL-6-induced allodynia, a subsequent intraplantar injection of prostaglandin E2 (PGE2) or intrathecal (i.t.) injection of the mGluR1/5 agonist, DHPG, precipitated allodynia and/or nocifensive responses. Intraplantar injection of IL-6 followed immediately by intrathecal (i.t.) injection of a PKMζ inhibitor prevented the expression of subsequent PGE2-induced allodynia. Inhibitors of protein translation were effective in preventing PGE-2-induced allodynia when given immediately after IL-6, but not after the initial allodynia had resolved. In contrast, spinal PKMζ inhibition completely abolished both prolonged allodynia to hindpaw PGE2 and enhanced nocifensive behaviors evoked by i.t. mGluR1/5 agonist injection after the resolution of IL-6-induced allodynia. Moreover, spinal PKMζ inhibition prevented the enhanced response to subsequent stimuli following resolution of hypersensitivity induced by plantar incision. The present findings demonstrate that the spinal cord encodes an engram for persistent nociceptive sensitization that is analogous to molecular mechanisms of late-LTP and suggest that spinally-directed PKMζ inhibitors may offer therapeutic benefit for injury-induced pain states.

Background

Mechanisms driving cancer-induced bone pain are poorly understood. A central factor implicated to be a key player in the process of tumorigenesis, osteoclastogenesis and nociception is p38 MAPK. We determined the role of p38 MAPK in a mouse model of breast cancer induced bone pain in which mixed osteolytic and osteoblastic remodeling occurs.

Results

In cancer-treated mice, acute as well as chronic inhibition of p38 MAPK with SB203580 blocked flinching and guarding behaviors in a dose-dependent manner whereas no effect on thresholds to tactile stimuli was observed. Radiographic analyses of bones demonstrated that chronic inhibition of p38 MAPK reduced bone loss and incidence of spontaneous fracture in cancer-treated mice. Histological analysis of bones collected from mice treated with the p38 MAPK inhibitor showed complete absence of osteoblastic growth in the intramedullary space as well as significantly reduced tumor burden.

Conclusions

Blockade of non-evoked pain behaviors but not hypersensitivity suggests differences in the underlying mechanisms of specific components of the pain syndrome and a possibility to individualize aspects of pain management. While it is not known whether the role of p38 MAPK signaling can be expanded to other cancers, the data suggest a need for understanding molecular mechanisms and cellular events that initiate and maintain cancer-induced bone pain for effective management for both ongoing pain as well as breakthrough pain.

Overuse of medications used to treat migraine headache can produce a chronic daily headache, termed medication overuse headache (MOH). Although “overuse” of opioids, triptans, and over-the-counter analgesics can all produce MOH, the neuronal mechanisms remain unknown. Headache pain is likely to be produced by stimulation of primary afferent neurons that innervate the intracranial vasculature and the resulting activation of medullary dorsal horn (MDH) neurons. The present study compared the receptive field properties of MDH dura sensitive neurons in rats treated with morphine to those given vehicle. Animals were implanted with osmotic mini-pumps or pellets for sustained subcutaneous administration of morphine or vehicle 6–7 days prior to recording from dura-sensitive neurons. Electrical and mechanical activation thresholds from the dura were significantly lower in chronic morphine treated animals when compared to vehicle controls. In addition, sustained morphine increased the cutaneous receptive field sizes. The presence of diffuse noxious inhibitory controls (DNIC) was examined by placing the tail in 55°C water during concomitant noxious thermal stimulation of the cutaneous receptive field, usually located in the ophthalmic region. The DNIC stimulus produced significant inhibition of heat-evoked activity in vehicle, but not chronic morphine treated animals. Inactivation of the rostral ventromedial medulla (RVM) with 4% lidocaine reinstated DNIC in chronic morphine treated animals. These results are consistent with studies demonstrating a loss of DNIC in patients that suffer from chronic daily headache and may partially explain why overuse of medication used to treat migraine can induce headaches.

Neuropathic pain states and tolerance to opioids can result from system changes in the CNS, such as up-regulation of the NK1 receptor and substance P, which have anti-opioid effects in ascending or descending pain-signaling pathways. Bifunctional compounds, possessing both the NK1 antagonist pharmacophore and the opioid agonist pharmacophore with delta-selectivity, could counteract these system changes to have significant analgesic efficacy without undesirable side effects. As a result of the introduction of cyclic and topological constraints with penicillamines, 2 (Tyr-cyclo[D-Pen-Gly-Phe-Pen]-Pro-Leu-Trp-NH-[3′,5′-(CF3)2-Bzl]) was found as the best bifunctional compound with effective NK1 antagonist and potent opioid agonist activities, and 1400-fold delta-selectivity over the mu-receptor. The NMR structural analysis of 2 revealed that the relative positioning of the two connected pharmacophores as well as its cyclic and topological constraints might be responsible for its excellent bifunctional activities as well as its significant delta-opioid selectivity. Together with the observed high metabolic stability, 2 could be considered as a valuable research tool and possibly a promising candidate for a novel analgesic drug.

Migraine is a common neurological disorder often treated with triptans. Triptan overuse can lead to increased frequency of headache in some patients, a phenomenon termed medication overuse headache. Previous preclinical studies have demonstrated that repeated or sustained triptan administration for several days can elicit persistent neural adaptations in trigeminal ganglion cells innervating the dura, prominently characterized by increased labelling of neuronal profiles for calcitonin gene related peptide. Additionally, triptan administration elicited a behavioural syndrome of enhanced sensitivity to surrogate triggers of migraine that was maintained for weeks following discontinuation of drug, a phenomenon termed ‘triptan-induced latent sensitization’. Here, we demonstrate that triptan administration elicits a long-lasting increase in identified rat trigeminal dural afferents labelled for neuronal nitric oxide synthase in the trigeminal ganglion. Cutaneous allodynia observed during the period of triptan administration was reversed by NXN-323, a selective inhibitor of neuronal nitric oxide synthase. Additionally, neuronal nitric oxide synthase inhibition prevented environmental stress-induced hypersensitivity in the post-triptan administration period. Co-administration of NXN-323 with sumatriptan over several days prevented the expression of allodynia and enhanced sensitivity to stress observed following latent sensitization, but not the triptan-induced increased labelling of neuronal nitric oxide synthase in dural afferents. Triptan administration thus promotes increased expression of neuronal nitric oxide synthase in dural afferents, which is critical for enhanced sensitivity to environmental stress. These data provide a biological basis for increased frequency of headache following triptans and highlight the potential clinical utility of neuronal nitric oxide synthase inhibition in preventing or treating medication overuse headache.