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We have determined the effect of naltrexone, naloxone, [d-Ala2,d-Leu5]enkephalin (DADLE), and morphine on the μ-S196A opioid receptor knock-in and μ-opioid receptor knockout mouse vas deferens preparations. The antagonists, naltrexone and naloxone, exhibited agonist activity and possessed IC50 values that were 14- and 37-fold greater than morphine on the S196A preparation. Morphine was found to be threefold more potent at S196A relative to wild-type μ-opioid receptor. The mouse vas deferens data suggest that S196 in transmembrane helix 4 of the μ-opioid receptor modulates efficacy. It is proposed that this may be due to decreased dimerization of the receptor. Identical IC50 values of DADLE obtained on the wild-type, S196A knock-in, and μ-opioid receptor knockout preparations support the absence of μ–δ heterodimers in the mouse vas deferens. [Copyright &y& Elsevier]

In view of the co-localization of spinal δ- and κ-opioid receptors, we have investigated the interaction of selective opioid receptor agonists and antagonists in the spinal cord of mice in order to determine if these receptors are organized as heteromers. The finding that norbinaltorphimine (κ) antagonized [d-Pen2,5]enkephalin (δ1), but not deltorphin II (δ2), strongly suggests that the putative δ1-subtype is a δ–κ heteromer. Studies with selective opioid receptor (ant)agonists support this conclusion. [Copyright &y& Elsevier]

• The bivalent ligand MMG22 consists of a MOR agonist and mGluR5 antagonist. • MMG22 reduced mechanical hyperalgesia produced by cisplatin in mice. • MMG22 reduced hyperalgesia without tolerance. • MMG22 may produce antinociception through the formation of a MOR-mGluR5 heteromer. MMG22 is a bivalent ligand containing MOR agonist and mGluR5 antagonist pharmacophores connected by a 22-atom linker. Intrathecal (i.t.) administration of MMG22 to inflamed mice has been reported to produce fmol-range antinociception in the reversal of LPS-induced hyperalgesia. MMG22 reduced hyperalgesia in the spared nerve injury (SNI) model of neuropathic pain at 10 days after injury but not at 30 days after injury, perhaps related to the inflammation that occurs early after injury but subsequently subsides. The present study determined the efficacy of MMG22 in cisplatin-treated male mice in order to provide data relating to the efficacy of MMG22 in the treatment of neuropathic pain that is associated with inflammation. Groups of eight mice each received daily intraperitoneal (i.p.) injections of cisplatin for seven days to produce robust mechanical allodynia defined by the decrease in withdrawal threshold using an electronic von Frey applied to the plantar surface of the hind paw. Intrathecal administration of MMG22 potently reduced mechanical hyperalgesia (ED 50 0.04 fmol/mouse) without tolerance, whereas MMG10 was essentially inactive. Morphine was less potent than MMG22 by >5-orders of magnitude and displayed tolerance. Subcutaneous MMG22 was effective (ED 50 = 2.41 mg/kg) and devoid of chronic tolerance. We propose that MMG22 induces the formation of a MOR-mGluR5 heteromer through selective interaction with the upregulated NR2B subunit of activated NMDAR, in view of the 4600-fold reduction of i.t. MMG22 antinociception by the selective NR2B antagonist, Ro25-6981. A possible explanation for the substantially reduced potency for MMG22 in the SNI model is discussed. [ABSTRACT FROM AUTHOR]

The therapeutic management of chronic pain associated with many cancers is problematic due to the development of tolerance and other adverse effects during the disease progression. Recently we reported on a bivalent ligand (MMG22) containing both mu agonist and mGluR 5 antagonist pharmacophores that produced potent antinociception in mice with LPS-induced acute inflammatory pain via a putative MOR-mGluR 5 heteromer. In the present study we have investigated the antinociception of MMG22 in a mouse model of bone cancer pain to determine its effectiveness in reducing this type of chronic nociception. There was a 572-fold increase in the potency of MMG22 over a period of 3–21 days that correlated with the progressive increase in hyperalgesia induced by bone tumor growth following implantation of fibrosarcoma cells in mice. The enhancement of antinociception with the progression of the cancer is possibly due to inhibition of NMDA receptor-mediated hyperalgesia via antagonism of mGluR 5 and concomitant activation of MOR by the MMG22-occupied heteromer. Notably, MMG22 was 3.6-million-fold more potent than morphine at PID 21. Since MMG22 exhibited a 250,000-times greater potency than that of a mixture of the mu opioid (M19) agonist and mGluR 5 antagonist (MG20) monovalent ligands, the data suggest that targeting the putative MOR-mGluR 5 heteromer is far superior to univalent interaction with receptors in reducing tumor-induced nociception. In view of the high potency, long duration (>24 h) of action and minimal side effects, MMG22 has the potential to be a superior pharmacological agent than morphine and other opiates in the treatment of chronic cancer pain and to serve as a novel pharmacologic tool. [ABSTRACT FROM AUTHOR]

Allosteric binding pockets in peptide-binding G protein-coupled receptors create opportunities for the development of small molecule drugs with substantial benefits over orthosteric ligands. To gain insights into molecular determinants for this pocket within type 1 and 2 cholecystokinin receptors (CCK1R and CCK2R), we prepared a series of receptor constructs in which six distinct residues in TM2, -3, -6, and -7 were reversed. Two novel iodinated CCK1R- and CCK2R-selective 1,4-benzodiazepine antagonists, differing only in stereochemistry at C3, were used. When all six residues within CCK1R were mutated to corresponding CCK2R residues, benzodiazepine selectivity was reversed, yet peptide binding selectivity was unaffected. Detailed analysis, including observations of gain of function, demonstrated that residues 6.51, 6.52, and 7.39 were most important for binding the CCK1R-selective ligand, whereas residues 2.61 and 7.39 were most important for binding CCK2Rselective ligand, although the effect of substitution of residue 2.61 was likely indirect. Ligand-guided homology modeling was applied to wild type receptors and those reversing benzodiazepine binding selectivity. The models had high predictive power in enriching known receptor-selective ligands from related decoys, indicating a high degree of precision in pocket definition. The benzodiazepines docked in similar poses in both receptors, with C3 urea substituents pointing upward, whereas different stereochemistry at C3 directed the C5 phenyl rings and N1 methyl groups into opposite orientations. The geometry of the binding pockets and specific interactions predicted for ligand docking in these models provide a molecular framework for understanding ligand selectivity at these receptor subtypes. Furthermore, the strong predictive power of these models suggests their usefulness in the discovery of lead compounds and in drug development programs. [ABSTRACT FROM AUTHOR]

Abstract: The current status of the GRAS evaluation program of flavoring substances operated by the Expert Panel of FEMA is discussed. The Panel maintains a rigorous rotating 10-year program of continuous review of scientific data related to the safety evaluation of flavoring substances. The Panel concluded a comprehensive review of the GRAS (GRASa) status of flavors in 1985 and began a second comprehensive review of the same substances and any recently GRAS materials in 1994. This second re-evaluation program of chemical groups of flavor ingredients, recognized as the GRAS reaffirmation (GRASr) program, is scheduled to be completed in 2005. The evaluation criteria used by the Panel during the GRASr program reflects the significant impact of advances in biochemistry, molecular biology and toxicology that have allowed for a more complete understanding of the molecular events associated with toxicity. The interpretation of novel data on the relationship of dose to metabolic fate, formation of protein and DNA adducts, enzyme induction, and the cascade of cellular events leading to toxicity provides a more comprehensive basis upon which to evaluate the safety of the intake of flavor ingredients under conditions of intended use. The interpretation of genotoxicity data is evaluated in the context of other data such as in vivo animal metabolism and lifetime animal feeding studies that are more closely related to actual human experience. Data are not viewed in isolation, but comprise one component that is factored into the Panel’s overall safety assessment. The convergence of different methodologies that assess intake of flavoring substances provides a greater degree of confidence in the estimated intake of flavor ingredients. When these intakes are compared to dose levels that in some cases result in related chemical and biological effects and the subsequent toxicity, it is clear that exposure to these substances through flavor use presents no significant human health risk. [Copyright &y& Elsevier]

This publication is the seventh in a series of safety evaluations performed by the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA). In 1993, the Panel initiated a comprehensive program to re-evaluate the safety of more than 1700 GRAS flavoring substances under conditions of intended use. Elements that are fundamental to the safety evaluation of flavor ingredients include exposure, structural analogy, metabolism, pharmacokinetics and toxicology. Flavor ingredients are evaluated individually and in the context of the available scientific information on the group of structurally related substances. Scientific data relevant to the safety evaluation of the use of cinnamyl derivatives as flavoring ingredients is evaluated. [Copyright &y& Elsevier]

Abstract: In an effort to develop antagonists for κ–μ opioid receptor heterodimers, a series of bivalent ligands 3–6 containing κ- and μ-antagonist pharmacophores were designed and synthesized. Evaluation of the series in HEK-293 cells revealed 4 (KMN-21) to selectively antagonize the activation of κ–μ heterodimers, suggesting possible bridging of receptors when the bivalent ligand spacer contains 21 atoms. [Copyright &y& Elsevier]

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 mGluR 5 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 ED 50 was orders of magnitude lower than morphine. Moreover, the ED 50 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, mGluR 5 , 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'. • Systemic administration OF MMG22 targets a MOR-mGluR5 heteromer and potently reduces bone cancer pain in mice. • MMG22 does not possess adverse effects associated with traditional opioids. • MMG22 activates MOR and antagonizes mGluR5. [ABSTRACT FROM AUTHOR]

Cisplatin and other widely employed platinum-based anticancer agents produce chemotherapy-induced peripheral neuropathy (CIPN) that often results in pain and hyperalgesia that are difficult to manage. We investigated the efficacy of a novel bivalent ligand, MCC22, for the treatment of pain arising from CIPN. MCC22 consists of mu opioid receptor (MOR) agonist and chemokine receptor 5 (CCR5) antagonist pharmacophores connected through a 22-atom spacer and was designed to target a putative MOR-CCR5 heteromer localized in pain processing areas. Mice received once daily intraperitoneal (i.p.) injections of cisplatin (1 mg/kg) for seven days and behavior testing began 7 days later. Cisplatin produced mechanical hyperalgesia that was decreased dose-dependently by MCC22 given by intrathecal (ED 50 = 0.004 pmol) or i.p. (3.07 mg/kg) routes. The decrease in hyperalgesia was associated with decreased inflammatory response by microglia in the spinal cord. Unlike morphine, MCC22 given daily for nine days did not exhibit tolerance to its analgesic effect and its characteristic antihyperalgesic activity was fully retained in morphine-tolerant mice. Furthermore, MCC22 did not alter motor function and did not exhibit rewarding properties. Given the exceptional potency of MCC22 without tolerance or reward, MCC22 has the potential to vastly improve management of chronic pain due to CIPN. This article is part of the Special Issue entitled 'New Vistas in Opioid Pharmacology'. • Chemotherapy often causes peripheral neuropathy that can be chronic and painful. • The bivalent ligand, MCC22, consists of a mµ opioid receptor agonist and a CCR5 receptor antagonist. • MCC22 potently reduced chemotherapy-induced hyperalgesia produced by cisplatin in mice. • MCC22 did not produce tolerance or reward. [ABSTRACT FROM AUTHOR]