Targeting MOR-mGluR5 heteromers reduces bone cancer pain by activating MOR and inhibiting mGluR5.

Pain is among the most common symptoms in cancer and approximately 90% of patients experience end-stage cancer pain. The management of cancer pain is challenging due to the significant side effects associated with opioids, and novel therapeutic approaches are needed. MMG22 is a bivalent ligand containing MOR agonist and mGluR5 antagonist pharmacophores joined by a 22-atom spacer. MMG22 exhibited extraordinary analgesia following intrathecal administration in a mouse model of bone cancer pain. Here, we assessed the effectiveness of systemic administration of MMG22 in reducing cancer pain and evaluated whether MMG22 displays side effects associated with opioids. Fibrosarcoma cells were injected into and around the calcaneus bone in C3H mice. Mechanical hyperalgesia was defined as an increase in the paw withdrawal frequencies (PWFs) evoked by application of a von Frey monofilament (3.9 mN bending force) applied to the plantar surface of the hind paw Subcutaneous (s.c.), intramuscular (i.m.), and oral (p.o.) administration of MMG22 produced robust dose-dependent antihyperalgesia, whose ED50 was orders of magnitude lower than morphine. Moreover, the ED50 for MMG22 decreased with disease progression. Importantly, s.c. administration of MMG22 did not produce acute (24 h) or long-term (9 days) tolerance, was not rewarding (conditioned place preference test), and did not produce naloxone-induced precipitated withdrawal or alter motor function. A possible mechanism of action of MMG22 is discussed in terms of inhibition of spinal NMDAR via antagonism of its co-receptor, mGluR5, and concomitant activation of neuronal MOR. We suggest that MMG22 may be a powerful alternative to traditional opioids for managing cancer pain. This article is part of the Special Issue entitled 'New Vistas in Opioid Pharmacology'.

Increased anandamide uptake by sensory neurons contributes to hyperalgesia in a model of cancer pain.

Opioids do not effectively manage pain in many patients with advanced cancer. Because anandamide (AEA) activation of cannabinoid type-1 receptors (CB1R) on nociceptors reduces nociception, manipulation of AEA metabolism in the periphery may be an effective alternative or adjuvant therapy in the management of cancer pain. AEA is hydrolyzed by the intracellular enzyme fatty acid amide hydrolase (FAAH), and this enzyme activity contributes to uptake of AEA into neurons and to reduction of AEA available to activate CB1R. We used an in vitro preparation of adult murine dorsal root ganglion (DRG) neurons co-cultured with fibrosarcoma cells to investigate how tumors alter the uptake of AEA into neurons. Evidence that the uptake of [(3)H]AEA into dissociated DRG cells in the co-culture model mimicked the increase in uptake that occurred in DRG cells from tumor-bearing mice supported the utility of the in vitro model to study AEA uptake. Results with the fluorescent AEA analog CAY10455 confirmed that an increase in uptake in the co-culture model occurred in neurons. One factor that contributed to the increase in [(3)H]AEA uptake was an increase in total cellular cholesterol in the cancer condition. Treatment with the FAAH inhibitor URB597 reduced CAY10455 uptake in the co-culture model to the level observed in DRG neurons maintained in the control condition (i.e., in the absence of fibrosarcoma cells), and this effect was paralleled by OMDM-1, an inhibitor of AEA uptake, at a concentration that had no effect on FAAH activity. Maximally effective concentrations of the two drugs together produced a greater reduction than was observed with each drug alone. Treatment with BMS309403, which competes for AEA binding to fatty acid binding protein-5, mimicked the effect of OMDM-1 in vitro. Local injection of OMDM-1 reduced hyperalgesia in vivo in mice with unilateral tumors in and around the calcaneous bone. Intraplantar injection of OMDM-1 (5µg) into the tumor-bearing paw reduced mechanical hyperalgesia through a CB1R-dependent mechanism and also reduced a spontaneous nocifensive behavior. The same dose reduced withdrawal responses evoked by suprathreshold mechanical stimuli in naive mice. These data support the conclusion that OMDM-1 inhibits AEA uptake by a mechanism that is independent of inhibition of FAAH and provide a rationale for the development of peripherally restricted drugs that decrease AEA uptake for the management of cancer pain.

Cannabinoid type-1 receptor reduces pain and neurotoxicity produced by chemotherapy.

Painful peripheral neuropathy is a dose-limiting complication of chemotherapy. Cisplatin produces a cumulative toxic effect on peripheral nerves, and 30-40% of cancer patients receiving this agent experience pain. By modeling cisplatin-induced hyperalgesia in mice with daily injections of cisplatin (1 mg/kg, i.p.) for 7 d, we investigated the anti-hyperalgesic effects of anandamide (AEA) and cyclohexylcarbamic acid 3'-carbamoyl-biphenyl-3-yl ester (URB597), an inhibitor of AEA hydrolysis. Cisplatin-induced mechanical and heat hyperalgesia were accompanied by a decrease in the level of AEA in plantar paw skin. No changes in motor activity were observed after seven injections of cisplatin. Intraplantar injection of AEA (10 µg/10 µl) or URB597 (9 µg/10 µl) transiently attenuated hyperalgesia through activation of peripheral CB1 receptors. Co-injections of URB597 (0.3 mg/kg daily, i.p.) with cisplatin decreased and delayed the development of mechanical and heat hyperalgesia. The effect of URB597 was mediated by CB1 receptors since AM281 (0.33 mg/kg daily, i.p.) blocked the effect of URB597. Co-injection of URB597 also normalized the cisplatin-induced decrease in conduction velocity of Aα/Aß-fibers and reduced the increase of ATF-3 and TRPV1 immunoreactivity in dorsal root ganglion (DRG) neurons. Since DRGs are a primary site of toxicity by cisplatin, effects of cisplatin were studied on cultured DRG neurons. Incubation of DRG neurons with cisplatin (4 µg/ml) for 24 h decreased the total length of neurites. URB597 (100 nM) attenuated these changes through activation of CB1 receptors. Collectively, these results suggest that pharmacological facilitation of AEA signaling is a promising strategy for attenuating cisplatin-associated sensory neuropathy.

Sex differences in neuropeptide content and release from rat dental pulp.

INTRODUCTION: Studies to examine sex differences in response to pain have suggested that females exhibit lower threshold responses to painful stimuli and that threshold response varies greatly at different stages of the menstrual cycle. Additional studies suggest that sex differences may be caused by societal sex roles or differences in anxiety responses by men and women. OBJECTIVE: The purpose of this study was to evaluate biologically evident sex differences in male and female rats chronically treated with a systemic algogen, the nerve growth factor (NGF), by measuring neuropeptides (calcitonin gene-related peptide) content and release from isolated dental pulp. METHODS: Rats were injected subcutaneously every other day with either murine NGF (1 mg/kg) or vehicle for 7 or 13 days. Isolated incisor pulp tissue was evaluated from these male and female rats (n = 96). Capsaicin-evoked neurosecretion of CGRP and tissue content were measured using a previously validated radioimmunoassay. RESULTS: Dental pulp from female rats at 7 days showed significantly increased capsaicin-evoked immunoreactive CGRP release (>50% increase) compared with tissue from male rats. After 13 days, this release was significantly increased only in NGF-treated female rats (3-fold increase) when compared with control females or both male groups. The CGRP content in tissue from both female groups was also significantly increased after 7 days of treatment (>3 fold), but after 13 days this content was only significantly increased in tissue from NGF-treated female rats (P = .0001). CONCLUSIONS: These data suggest that sex differences affect the role of NGF in the modulation of inflammation through the regulation of peripheral neuropeptide release and content.

CB1 and CB2 receptor agonists promote analgesia through synergy in a murine model of tumor pain.

In light of the adverse side-effects of opioids, cannabinoid receptor agonists may provide an effective alternative for the treatment of cancer pain. This study examined the potency and efficacy of synthetic CB1 and CB2 receptor agonists in a murine model of tumor pain. Intraplantar injection of the CB1 receptor agonist arachidonylcyclopropylamide (ED(50) of 18.4 µg) reduced tumor-related mechanical hyperalgesia by activation of peripheral CB1 but not CB2 receptors. Similar injection of the CB2 receptor agonist AM1241 (ED50 of 19.5 µg) reduced mechanical hyperalgesia by activation of peripheral CB2 but not CB1 receptors. Both agonists had an efficacy comparable with that of morphine (intraplantar), but their analgesic effects were independent of opioid receptors. Isobolographic analysis of the coinjection of arachidonylcyclopropylamide and AM1241 determined that the CB1 and CB2 receptor agonists interacted synergistically to reduce mechanical hyperalgesia in the tumor-bearing paw. These data extend our previous findings that the peripheral cannabinoid receptors are a promising target for the management of cancer pain and mixed cannabinoid receptor agonists may have a therapeutic advantage over selective agonists.

Increasing 2-arachidonoyl glycerol signaling in the periphery attenuates mechanical hyperalgesia in a model of bone cancer pain.

Metastatic and primary bone cancers are usually accompanied by severe pain that is difficult to manage. In light of the adverse side effects of opioids, manipulation of the endocannabinoid system may provide an effective alternative for the treatment of cancer pain. The present study determined that a local, peripheral increase in the endocannabinoid 2-arachidonoyl glycerol (2-AG) reduced mechanical hyperalgesia evoked by the growth of a fibrosarcoma tumor in and around the calcaneous bone. Intraplantar (ipl) injection of 2-AG attenuated hyperalgesia (ED(50) of 8.2 µg) by activation of peripheral CB2 but not CB1 receptors and had an efficacy comparable to that of morphine. JZL184 (10 µg, ipl), an inhibitor of 2-AG degradation, increased the local level of 2-AG and mimicked the anti-hyperalgesic effect of 2-AG, also through a CB2 receptor-dependent mechanism. These effects were accompanied by an increase in CB2 receptor protein in plantar skin of the tumor-bearing paw as well as an increase in the level of 2-AG. In naïve mice, intraplantar administration of the CB2 receptor antagonist AM630 did not alter responses to mechanical stimuli demonstrating that peripheral CB2 receptor tone does not modulate mechanical sensitivity. These data extend our previous findings with anandamide in the same model and suggest that the peripheral endocannabinoid system is a promising target for the management of cancer pain.

A decrease in anandamide signaling contributes to the maintenance of cutaneous mechanical hyperalgesia in a model of bone cancer pain.

Tumors in bone are associated with pain in humans. Data generated in a murine model of bone cancer pain suggest that a disturbance of local endocannabinoid signaling contributes to the pain. When tumors formed after injection of osteolytic fibrosarcoma cells into the calcaneus bone of mice, cutaneous mechanical hyperalgesia was associated with a decrease in the level of anandamide (AEA) in plantar paw skin ipsilateral to tumors. The decrease in AEA occurred in conjunction with increased degradation of AEA by fatty acid amide hydrolase (FAAH). Intraplantar injection of AEA reduced the hyperalgesia, and intraplantar injection of URB597, an inhibitor of FAAH, increased the local level of AEA and also reduced hyperalgesia. An increase in FAAH mRNA and enzyme activity in dorsal root ganglia (DRG) L3-L5 ipsilateral to the affected paw suggests DRG neurons contribute to the increased FAAH activity in skin in tumor-bearing mice. Importantly, the anti-hyperalgesic effects of AEA and URB597 were blocked by a CB1 receptor antagonist. Increased expression of CB1 receptors by DRG neurons ipsilateral to tumor-bearing limbs may contribute to the anti-hyperalgesic effect of elevated AEA levels. Furthermore, CB1 receptor protein-immunoreactivity as well as inhibitory effects of AEA and URB597 on the depolarization-evoked Ca(2+) transient were increased in small DRG neurons cocultured with fibrosarcoma cells indicating that fibrosarcoma cells are sufficient to evoke phenotypic changes in AEA signaling in DRG neurons. Together, the data provide evidence that manipulation of peripheral endocannabinoid signaling is a promising strategy for the management of bone cancer pain.

The cannabinoid receptor agonist, WIN 55, 212-2, attenuates tumor-evoked hyperalgesia through peripheral mechanisms.

Several lines of evidence suggest that cannabinoids can attenuate various types of pain and hyperalgesia through peripheral mechanisms. The development of rodent cancer pain models has provided the opportunity to investigate novel approaches to treat this common form of pain. In the present study, we examined the ability of peripherally administered cannabinoids to attenuate tumor-evoked mechanical hyperalgesia in a murine model of cancer pain. Unilateral injection of osteolytic fibrosarcoma cells into and around the calcaneus bone resulted in tumor formation and mechanical hyperalgesia in the injected hindpaw. Mechanical hyperalgesia was defined as an increase in the frequency of paw withdrawals to a suprathreshold von Frey filament (3.4 mN) applied to the plantar surface of the hindpaw. WIN 55, 212-2 (1.5 to 10 microg) injected subcutaneously into the tumor-bearing hindpaw produced a dose-dependent decrease in paw withdrawal frequencies to suprathreshold von Frey filament stimulation. Injection of WIN 55,212-2 (10 microg) into the contralateral hindpaw did not decrease paw withdrawal frequencies in the tumor-bearing hindpaw. Injection of the highest antihyperalgesic dose of WIN 55,212-2 (10 microg) did not produce catalepsy as determined by the bar test. Co-administration of WIN 55,212-2 with either cannabinoid 1 (AM251) or cannabinoid 2 (AM630) receptor antagonists attenuated the antihyperalgesic effects of WIN 55, 212-2. In conclusion, peripherally administered WIN 55,212-2 attenuated tumor-evoked mechanical hyperalgesia by activation of both peripheral cannabinoid 1 and cannabinoid 2 receptors. These results suggest that peripherally-administered cannabinoids may be effective in attenuating cancer pain.

Tumor-evoked hyperalgesia and sensitization of nociceptive dorsal horn neurons in a murine model of cancer pain.

Pain associated with cancer, particularly when tumors metastasize to bone, is often severe and debilitating. Better understanding of the neurobiological mechanisms underlying cancer pain will likely lead to the development of more effective treatments. The aim of this study was to characterize changes in response properties of nociceptive dorsal horn neurons following implantation of fibrosarcoma cells into and around the calcaneus bone, an established model of cancer pain. Extracellular electrophysiological recordings were made from wide dynamic range (WDR) and high threshold (HT) dorsal horn neurons in mice with tumor-evoked hyperalgesia and control mice. WDR and HT neurons were examined for ongoing activity and responses to mechanical, heat, and cold stimuli applied to the plantar surface of the hind paw. Behavioral experiments showed that mice exhibited hyperalgesia to mechanical and heat stimuli applied to their tumor-bearing hind paw. WDR, but not HT, nociceptive dorsal horn neurons in tumor-bearing mice exhibited sensitization to mechanical, heat, and cold stimuli and may contribute to tumor-evoked hyperalgesia. Specifically, the proportion of WDR neurons that exhibited ongoing activity and their evoked discharge rates were greater in tumor-bearing than in control mice. In addition, WDR neurons exhibited lower response thresholds for mechanical and heat stimuli, and increased responses to suprathreshold mechanical, heat, and cold stimuli. Our findings show that sensitization of WDR neurons contributes to cancer pain and supports the notion that the mechanisms underlying cancer pain differ from those that contribute to inflammatory and neuropathic pain.

Chemical interactions between fibrosarcoma cancer cells and sensory neurons contribute to cancer pain.

In an experimental model of cancer pain, the hyperalgesia that occurs with osteolytic tumor growth is associated with the sensitization of nociceptors. We examined functional and molecular changes in small-diameter dorsal root ganglion (DRG) neurons to determine cellular mechanisms underlying this sensitization. The occurrence of a Ca2+ transient in response to either KCl (25 mM) or capsaicin (500 nM) increased in small neurons isolated from murine L3-L6 DRGs ipsilateral to fibrosarcoma cell tumors. The increased responses were associated with increased mRNA levels for the Ca2+ channel subunit alpha2delta1 and TRPV1 receptor. Pretreatment with gabapentin, an inhibitor of the alpha2delta1 subunit, blocked the increased response to KCl in vitro and the mechanical hyperalgesia in tumor-bearing mice in vivo. Similar increases in neuronal responsiveness occurred when DRG neurons from naive mice and fibrosarcoma cells were cocultured for 48 h. The CC chemokine ligand 2 (CCL2) may contribute to the tumor cell-induced sensitization because CCL2 immunoreactivity was present in tumors, high levels of CCL2 peptide were present in microperfusates from tumors, and treatment of DRG neurons in vitro with CCL2 increased the amount of mRNA for the alpha2delta1 subunit. Together, our data provide strong evidence that the chemical mediator CCL2 is released from tumor cells and evokes phenotypic changes in sensory neurons, including increases in voltage-gated Ca2+ channels that likely underlie the mechanical hyperalgesia in the fibrosarcoma cancer model. More broadly, this study provides a novel in vitro model to resolve the cellular and molecular mechanisms by which tumor cells drive functional changes in nociceptors.

Chronic nerve growth factor administration increases the peripheral exocytotic activity of capsaicin-sensitive cutaneous neurons.

Nerve growth factor (NGF) plays an important role in inflammation and pain and has been suggested to regulate the responsiveness and sensitivity of nociceptive fibers. However, no study has evaluated whether chronic NGF alters the exocytotic capacity of peripheral terminals of peptidergic fibers. To test this hypothesis, rats were injected subcutaneously every other day with either murine recombinant NGF (mNGF; 1.0 mg/kg) or vehicle for 7 days; or mNGF (0.1 mg/kg), mNGF (1 mg/kg) or vehicle every other day for 13 days. Treatment of rats with NGF over a 13-day period produced a significant increase in capsaicin-evoked iCGRP release from isolated biopsies of hindpaw skin, as assessed by in vitro superfusion and RIA. This effect was dose-dependent and exhibited a temporal requirement, because the enhancement was only observed after 13 days of treatment and was not evident after 7 days of treatment. This NGF enhancement of capsaicin-evoked iCGRP release was not due solely to increases in peripheral iCGRP content since only the 1mg/kg dose of NGF elevated cutaneous pools of iCGRP, whereas both doses significantly increased capsaicin-evoked peptide release. Moreover, NGF also enhanced capsaicin-evoked thermal hyperalgesia under similar dose- and time-related conditions. Collectively, the chronic administration of NGF not only increases capsaicin-evoked hyperalgesia, but also significantly primes peripheral fibers to enhanced peptidergic exocytosis following activation of the capsaicin receptor. Collectively, these data are consistent with the hypothesis that persistently elevated NGF levels may contribute to enhanced neurogenic regulation of inflammatory and wound healing processes in injured tissue.

Re-organization of P2X3 receptor localization on epidermal nerve fibers in a murine model of cancer pain.

To determine whether ATP and P2X3 receptors contribute to bone-cancer pain in a mouse model, immunohistochemical techniques were used to identify whether changes in the labeling of P2X3 receptors on epidermal nerve fibers (ENFs) occurred during tumor development. C3H mice were injected with osteolytic fibrosarcoma cells in and around the calcaneus bone. These mice exhibited mechanical hyperalgesia by day 10 post-implantation as assessed using von Frey monofilaments. Biopsies of the plantar skin overlying the tumor were obtained at days 10, 14, and 18 post-implantation. Confocal images were analyzed for the number of PGP 9.5, P2X3, and CGRP immunoreactive (ir) ENFs. The overall ENF population (PGP-ir) decreased progressively over time, whereas the subsets of P2X3-ir fibers demonstrated a modest increase and CGRP-ir nerve fibers remained fairly constant. Importantly, the proportion of CGRP-ir fibers that labeled for P2X3 increased from approximately 6% in control animals to nearly 30% at day 14 following tumor cell implantation. These studies demonstrate increased expression of P2X3 receptors on CGRP-ir ENFs during tumor growth and suggest a role for ATP in cancer-related pain.

Nerve growth factor treatment enhances release of immunoreactive calcitonin gene-related peptide but not substance P from spinal dorsal horn slices in rats.

The effect of systemic nerve growth factor (NGF) on neuropeptide content and capsaicin-evoked release of neuropeptide from in vitro spinal cord dorsal horn slices was examined. Rats were injected subcutaneously every other day with murine NGF (mNGF) 1 mg/kg or saline for 7 days, or mNGF 0.1/kg, mNGF 1 mg/kg or saline for 13 days. Lumbar dorsal horn slices of the rat spinal cord from all groups showed a significant increase in immunoreactive calcitonin gene-related peptide (CGRP) release upon exposure to capsaicin. This release was enhanced in rats pretreated with mNGF 1 mg/kg for 7 days, but not after 13 days. No enhancement was seen after 7 or 13 days in any treatment group for immunoreactive substance P release. Upon examination of neuropeptide content in dorsal horn, no significant differences were noted between treatment groups. The increased iCGRP release from dorsal horn slices suggests a preferential release of CGRP and provides further evidence that NGF indirectly plays a role in the modulation of inflammation through the regulation of neuropeptide release.

Capsaicin-evoked release of immunoreactive calcitonin gene-related peptide from rat trigeminal ganglion: evidence for intraganglionic neurotransmission.

Chemically-mediated cross-excitation has been described between neurons within sensory ganglia. However, the identity and source of the chemical mediators is not known. Ca(2+)-dependent release of neurotransmitters from cultured sensory neurons in vitro has been observed, although neurite outgrowth may confound the ability to extrapolate findings from culture systems to in vivo conditions. Thus, the present studies evaluate the hypothesis of capsaicin-sensitive intraganglionic neuropeptide release from freshly prepared slices of rat sensory ganglia. The ganglionic slice preparation provides an advantage over neuronal cultures, because release may be assessed within minutes after tissue collection (minimizing phenotypic changes) and while maintaining gross anatomical relationships. Trigeminal ganglia (TGG) were quickly removed from male, Sprague--Dawley rats (175--200 g), chopped into 200 microm slices and placed into chambers within 3 min of collection. Chambers were perfused with buffer, and superfusates were collected and assayed for immunoreactive calcitonin gene-related peptide (iCGRP) release via radioimmunoassay. After about 90 min of baseline collection, tissue was treated with capsaicin followed by a washout period. Capsaicin (1--100 microM) evoked concentration-dependent increases in iCGRP release. A competitive capsaicin receptor antagonist, capsazepine, significantly inhibited capsaicin-evoked release of iCGRP. In addition, capsaicin-evoked release of iCGRP was dependent on the presence of extracellular calcium. Furthermore, capsaicin-evoked release from TGG slices was significantly greater than that from slices of equivalent weights of adjacent trigeminal nerve shown histologically to be free of neuronal somata. These data support the hypothesis that Ca(2+)-dependent exocytosis of neuropeptides may occur within the TGG in vivo and that the majority of this release derives from neuronal somata.

Peripheral CGRP release as a marker for neurogenic inflammation: a model system for the study of neuropeptide secretion in rat paw skin.

The local release of pro-inflammatory neuropeptides in the periphery has been associated with the development of neurogenic inflammation. However, there is an increasing number of reports demonstrating tissue-dependent differences regarding the mechanisms engaged by these neuropeptides to initiate and maintain the inflammatory response in the target tissue. Since skin is often involved in tissue injury, the present studies were designed to develop a model for assessing cutaneous peptide secretion as a marker for neurogenic inflammation in skin tissue. Calcitonin gene-related peptide (CGRP), as one of several neuropeptides known to be involved in neurogenic inflammation, was chosen to study capsaicin-induced effects on peripheral neurosecretion. The corial surface of the hairy skin of a rat hindlimb was superfused in vitro, and the basal and capsaicin-evoked peripheral release of immunoreactive CGRP (iCGRP) was measured using a radioimmunoassay. The main objectives of these studies were to characterize the various properties of this release including dose-dependency, exocytosis and receptor-mediation as well as the effects of acute and long-term capsaicin desensitization. Capsaicin significantly and dose-dependently increased the release of iCGRP at concentrations ranging from 3 to 300 microM. Omission of calcium ions or treatment with the competitive capsaicin receptor antagonist capsazepine completely inhibited the capsaicin-induced iCGRP release. Superfusion of the skin with 100 microM capsaicin following a conditioning stimulation with capsaicin at concentrations ranging from 0.3 to 100 microM led to an acute, dose-dependent desensitization of the CGRP response. In addition, chronic desensitization following the neonatal injection of capsaicin completely abolished the acute iCGRP response to capsaicin. The method described here should prove to be a valuable tool for the evaluation of the processes regulating the peripheral, cutaneous release of pro-inflammatory neuropeptides. This strategy, therefore, may lead to a better understanding of the mechanisms involved in the development and maintenance of neurogenic inflammation, particularly in the skin.