Tally M. Largent-Milnes

Novel d-amino acid tetrapeptides produce potent antinociception by selectively acting at peripheral κ-opioid receptors,

Kappa-(kappa) opioid receptors are widely distributed in the periphery and activation results in antinociception; however supraspinal acting kappa-agonists result in unwanted side effects. Two novel, all d-amino acid, tetrapeptide kappa-opioid receptor agonists, FE 200665 and FE 200666, were identified and compared to brain penetrating (enadoline) and peripherally selective (asimadoline) kappa-agonists as potential analgesics lacking unwanted central nervous system (CNS) side effects. In vitro characterization was performed using radioligand binding and GTP gamma S binding. Antinociception was evaluated in both mice and rats. Rotarod tests were performed to determine motor impairment effects of the kappa-agonists. FE 200665 and FE 200666 showed high affinity for human kappa-opioid receptor 1 (Ki of 0.24 nM and 0.08 nM, respectively) and selectivity for human kappa-opioid receptor 1 (human kappa-opioid receptor 1/human mu-opioid receptor/human delta-opioid receptor selectivity ratios of 1/16,900/84,600 and 1/88,600/>1,250,000, respectively). Both compounds demonstrated agonist activity in the human kappa-opioid receptor 1 [35S]GTP gamma S binding assay (EC50 of 0.08 nM and 0.03 nM) and resulted in dose-related antinociception in the mouse writhing test (A50: 0.007 and 0.013 mg/kg, i.v., respectively). Markedly higher doses of FE 200665 and FE 200666 were required to induce centrally-mediated effects in the rotarod assay (548- and 182-fold higher doses, respectively), and antinociception determined in the mouse tail-flick assay (>1429- and 430-fold fold higher doses, respectively) after peripheral administration supporting a peripheral site of action. The potency ratios between central and peripheral activity suggest a therapeutic window significantly higher than previous kappa-agonists. Furthermore, FE 200665 has entered into clinical trials with great promise as a novel analgesic lacking unwanted side effects seen with current therapeutics.

A cannabinoid 2 receptor agonist attenuates bone cancer-induced pain and bone loss

AIMS Cannabinoid CB(2) agonists have been shown to alleviate behavioral signs of inflammatory and neuropathic pain in animal models. AM1241, a CB(2) agonist, does not demonstrate central nervous system side effects seen with CB(1) agonists such as hypothermia and catalepsy. Metastatic bone cancer causes severe pain in patients and is treated with analgesics such as opiates. Recent reports suggest that sustained opiates can produce paradoxical hyperalgesic actions and enhance bone destruction in a murine model of bone cancer. In contrast, CB(2) selective agonists have been shown to reduce bone loss associated with a model of osteoporosis. Here we tested whether a CB(2) agonist administered over a 7day period inhibits bone cancer-induced pain as well as attenuates cancer-induced bone degradation. MAIN METHODS A murine bone cancer model was used in which osteolytic sarcoma cells were injected into the intramedullary space of the distal end of the femur. Behavioral and radiographic image analysis was performed at days 7, 10 and 14 after injection of tumor cells into the femur. KEY FINDINGS Osteolytic sarcoma within the femur produced spontaneous and touch evoked behavioral signs of pain within the tumor-bearing limb. The systemic administration of AM1241 acutely or for 7days significantly attenuated spontaneous and evoked pain in the inoculated limb. Sustained AM1241 significantly reduced bone loss and decreased the incidence of cancer-induced bone fractures. SIGNIFICANCE These findings suggest a novel therapy for cancer-induced bone pain, bone loss and bone fracture while lacking many unwanted side effects seen with current treatments for bone cancer pain.

Oxycodone Plus Ultra-Low-Dose Naltrexone Attenuates Neuropathic Pain and Associated μ-Opioid Receptor–Gs Coupling

UNLABELLED Both peripheral nerve injury and chronic opioid treatment can result in hyperalgesia associated with enhanced excitatory neurotransmission at the level of the spinal cord. Chronic opioid administration leads to a shift in mu-opioid receptor (MOR)-G protein coupling from G(i/o) to G(s) that can be prevented by cotreatment with an ultra-low-dose opioid antagonist. In this study, using lumbar spinal cord tissue from rats with L(5)/L(6) spinal nerve ligation (SNL), we demonstrated that SNL injury induces MOR linkage to G(s) in the damaged (ipsilateral) spinal dorsal horn. This MOR-G(s) coupling occurred without changing G(i/o) coupling levels and without changing the expression of MOR or Galpha proteins. Repeated administration of oxycodone alone or in combination with ultra-low-dose naltrexone (NTX) was assessed on the SNL-induced MOR-G(s) coupling as well as on neuropathic pain behavior. Repeated spinal oxycodone exacerbated the SNL-induced MOR-G(s) coupling, whereas ultra-low-dose NTX cotreatment slightly but significantly attenuated this G(s) coupling. Either spinal or oral administration of oxycodone plus ultra-low-dose NTX markedly enhanced the reductions in allodynia and thermal hyperalgesia produced by oxycodone alone and minimized tolerance to these effects. The MOR-G(s) coupling observed in response to SNL may in part contribute to the excitatory neurotransmission in spinal dorsal horn in neuropathic pain states. The antihyperalgesic and antiallodynic effects of oxycodone plus ultra-low-dose NTX (Oxytrex, Pain Therapeutics, Inc., San Mateo, CA) suggest a promising new treatment for neuropathic pain. PERSPECTIVE The current study investigates whether Oxytrex (oxycodone with an ultra-low dose of naltrexone) alleviates mechanical and thermal hypersensitivities in an animal model of neuropathic pain over a period of 7 days, given locally or systemically. In this report, we first describe an injury-induced shift in mu-opioid receptor coupling from G(i/o) to G(s), suggesting why a mu-opioid agonist may have reduced efficacy in the nerve-injured state. These data present a novel approach to neuropathic pain therapy.

A Structure–Activity Relationship Study and Combinatorial Synthetic Approach of C-Terminal Modified Bifunctional Peptides That Are δ/μ Opioid Receptor Agonists and Neurokinin 1 Receptor Antagonists

A series of bifunctional peptides with opioid agonist and substance P antagonist bioactivities were designed with the concept of overlapping pharmacophores. In this concept, the bifunctional peptides were expected to interact with each receptor separately in the spinal dorsal horn where both the opioid receptors and the NK1 receptors were found to be expressed, to show an enhanced analgesic effect, no opioid-induced tolerance, and to provide better compliance than coadministration of two drugs. Compounds were synthesized using a two-step combinatorial method for C-terminal modification. In the method, the protected C-terminal-free carboxyl peptide, Boc-Tyr( tBu)- d-Ala-Gly Phe-Pro-Leu-Trp(Boc)-OH, was synthesized as a shared intermediate using Fmoc solid phase chemistry on a 2-chlorotrityl resin. This intermediate was esterified or amidated in solution phase. The structure-activity relationships (SAR) showed that the C-terminus acted as not only a critical pharmacophore for the substance P antagonist activities, but as an address region for the opioid agonist pharmacophore that is structurally distant from the C-terminal. Among the peptides, H-Tyr- d -Ala-Gly-Phe-Pro-Leu-Trp-NH-Bzl ( 3) demonstrated high binding affinities at both delta and mu receptors ( K i = 10 and 0.65 nM, respectively) with efficient agonist functional activity in the mouse isolated vas deferens (MVD) and guinea pig isolated ileum (GPI) assays (IC 50 = 50 and 13 nM, respectively). Compound 3 also showed a good antagonist activity in the GPI assay with substance P stimulation ( K e = 26 nM) and good affinity for the hNK1 receptor ( K i = 14 nM). Consequently, compound 3 is expected to be a promising and novel type of analgesic with bifunctional activities.

Spinal or systemic TY005, a peptidic opioid agonist/neurokinin 1 antagonist, attenuates pain with reduced tolerance.

BACKGROUND AND PURPOSE The use of opioids in treating pain is limited due to significant side effects including somnolence, constipation, analgesic tolerance, addiction and respiratory depression. Pre‐clinical studies have shown that neurokinin 1 (NK1) receptor antagonists block opioid‐induced antinociceptive tolerance and may inhibit opioid‐induced rewarding behaviours. Here, we have characterized a bifunctional peptide with both opioid agonist and NK1 antagonist pharmacophores in a rodent model of neuropathic pain.

Disease modification of breast cancer-induced bone remodeling by cannabinoid 2 receptor agonists.

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 (CB2) receptor‐specific agonists have been shown to reduce bone loss and stimulate bone formation in a model of osteoporosis. CB2 agonists produce analgesia in both inflammatory and neuropathic pain models. Notably, mixed CB1/CB2 agonists also demonstrate a reduction in ErbB2‐driven breast cancer progression. Here we demonstrate for the first time that CB2 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 CB2 agonist, JWH015, for 7 days significantly attenuated bone remodeling, assuaged spontaneous pain, and decreased primary tumor burden. CB2‐mediated effects in vivo were reversed by concurrent treatment with a CB2 antagonist/inverse agonist but not with a CB1 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 CB2 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.

Recently patented and promising ORL-1 ligands: where have we been and where are we going?

Importance of the field: The interactions of nociceptin/orphanin FQ (N/OFQ) and the opioid receptor-like receptor 1 (nociceptin opioid peptide – NOP) have been implicated in a variety of systems including cardiovascular, respiratory, immune, and the central and peripheral nervous systems. Areas covered in this review: To elucidate the endogenous role of the N/OFQ–NOP system through the use of knockout and knockdown animal preparations, though most advances have been made using a host of synthetic agonists and antagonists. This review gives a brief history of the receptor–ligand discovery, the development of these agonists and antagonists within the last 10 years as published, and the therapeutic indications thereof focusing on pain. What the reader will gain: The use of NOP ligands in pain has been controversial at best; however, there are indications that both agonists and antagonists have a place in the clinical setting for acute and chronic pain. NOP ligands have potential as novel therapeutics, interestingly, when incorporated into a rationally-designed multi-target agent. Take home message: The discovery of N/OFQ and NOP opened a new option for the treatment of pain with the potential for a decreased side effect profile. Numerous compounds have been designed to target this system, the most promising of which have mixed profiles.

Activation of descending pain-facilitatory pathways from the rostral ventromedial medulla by cholecystokinin elicits release of prostaglandin-E2 in the spinal cord

Summary Nerve injury significantly increases endogenous cholecystokinin (CCK) in the rostral ventromedial medulla (RVM), and CCK drives descending facilitatory pathways from the RVM in naïve animals to increase spinal prostaglandin‐E2 and serotonin (5‐hydoxytryptophan/5‐hydroxyindoleacetic acid). ABSTRACT Cholecystokinin (CCK) has been suggested to be both pro‐nociceptive and “anti‐opioid” by actions on pain‐modulatory cells within the rostral ventromedial medulla (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 after ligation of L5/L6 spinal nerves (SNL). Microinjection of CCK into the RVM of naïve rats elicited hypersensitivity to tactile stimulation of the hindpaw. In addition, RVM CCK elicited a time‐related increase in (prostaglandin‐E2) 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 minutes post‐RVM CCK, a time coincident with maximal RVM CCK‐induced mechanical hypersensitivity. Spinal administration of naproxen, a nonselective COX‐inhibitor, significantly attenuated RVM CCK‐induced hindpaw tactile hypersensitivity. RVM‐CCK also resulted in a 2‐fold increase in spinal 5‐hydroxyindoleacetic acid (5‐HIAA), a 5‐hydoxytryptophan (5‐HT) metabolite, as compared with 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.

Repeated morphine treatment-mediated hyperalgesia, allodynia and spinal glial activation are blocked by co-administration of a selective cannabinoid receptor type-2 agonist

Spinal glial activation has been implicated in sustained morphine-mediated paradoxical pain sensitization. Since activation of glial CB2 cannabinoid receptors attenuates spinal glial activation in neuropathies, we hypothesized that CB2 agonists may also attenuate sustained morphine-mediated spinal glial activation and pain sensitization. Our data indicate that co-administration of a CB2-selective agonist (AM 1241) attenuates morphine (intraperitoneal; twice daily; 6 days)-mediated thermal hyperalgesia and tactile allodynia in rats. A CB2 (AM 630) but not a CB1 (AM 251) antagonist mitigated this effect. AM 1241 co-treatment also attenuated spinal astrocyte and microglial marker and pro-inflammatory mediator (IL-1β, TNFα) immunoreactivities in morphine-treated rats, suggesting that CB2 agonists may be useful to prevent the neuroinflammatory consequences of sustained morphine treatment.

A membrane-delimited N-myristoylated CRMP2 peptide aptamer inhibits CaV2.2 trafficking and reverses inflammatory and postoperative pain behaviors.

Abstract Targeting proteins within the N-type voltage-gated calcium channel (CaV2.2) complex has proven to be an effective strategy for developing novel pain therapeutics. We describe a novel peptide aptamer derived from the collapsin response mediator protein 2 (CRMP2), a CaV2.2-regulatory protein. Addition of a 14-carbon myristate group to the peptide (myr-tat-CBD3) tethered it to the membrane of primary sensory neurons near surface CaV2.2. Pull-down studies demonstrated that myr-tat-CBD3 peptide interfered with the CRMP2–CaV2.2 interaction. Quantitative confocal immunofluorescence revealed a pronounced reduction of CaV2.2 trafficking after myr-tat-CBD3 treatment and increased efficiency in disrupting CRMP2-CaV2.2 colocalization compared with peptide tat-CBD3. Consequently, myr-tat-CBD3 inhibited depolarization-induced calcium influx in sensory neurons. Voltage clamp electrophysiology experiments revealed a reduction of Ca2+, but not Na+, currents in sensory neurons after myr-tat-CBD3 exposure. Current clamp electrophysiology experiments demonstrated a reduction in excitability of small-diameter dorsal root ganglion neurons after exposure to myr-tat-CBD3. Myr-tat-CBD3 was effective in significantly attenuating carrageenan-induced thermal hypersensitivity and reversing thermal hypersensitivity induced by a surgical incision of the plantar surface of the rat hind paw, a model of postoperative pain. These effects are compared with those of tat-CBD3—the nonmyristoylated tat-conjugated CRMP2 peptide as well as scrambled versions of CBD3 and CBD3-lacking control peptides. Our results demonstrate that the myristoyl tag enhances intracellular delivery and local concentration of the CRMP2 peptide aptamer near membrane-delimited calcium channels resulting in pronounced interference with the calcium channel complex, superior suppression of calcium influx, and better antinociceptive potential.