Current Pharmaceutical Design - Volume 19, Issue 42, 2013

Opioid receptors are seven-transmembrane domain receptors that couple to intracellular signaling molecules by activating heterotrimeric G proteins. However, the receptor and G protein do not function in isolation but their activities are modulated by several accessory and scaffolding proteins. Examples include arrestins, kinases, and regulators of G protein signaling proteins. Accessory proteins contribute to the observed potency and efficacy of agonists, but also to the direction of signaling and the phenomenon of biased agonism. This review will present current knowledge of such proteins and how they may provide targets for future drug design.

Here we have studied regulatory changes of μ-opioid receptors accompanying in vivo 14-methoxymetopon treatments of rats. Previously, this ligand has been shown to be an extremely potent, centrally acting μ-opioid specific analgesic with low physical dependence, tolerance, respiratory depression, constipation and other side effects. Our work shows that it is a highly potent full agonist of μ-opioid receptor coupled G-protein signaling in vitro, alike the well-known opioid agonist, etorphine. However, unlike etorphine, which desensitized and down-regulated the endogenous μ-opioid receptors, 14-methoxymetopon, given to rats intraperitoneally (i.p.) either acutely or chronically, did not change the binding or G-protein signaling of μ-opioid receptors in rat brain subcellular membranes. Thereby, these data provide further evidence that there is no direct relationship between the efficacy of the ligand in signaling and its ability to internalize or desensitize the receptor. Viewed collectively with published work, it is discussed that μ-opioid receptors display functional selectivity, also called ‘biased agonism’. This concept implies that each ligand may induce unique, ligand-specific receptor conformation that can result in distinct agonist- directed trafficking and/or signal transduction pathways associated with the receptor. Ligand-specific signaling may open up new directions for designing potent analgesics that do not interact with unwanted signaling pathways, which mediate undesired side-effects, such as tolerance and dependence.

Herein, we investigated the role of periaqueductal gray (PAG)-resident microglia in the development of morphine tolerance and its underlying mechanisms. We showed that clodronate and minocycline known as microglia inhibitors reversed morphine tolerance, providing proof that microglia activation has key role in the development of morphine tolerance. The microglia-mediated anti-opioid mechanism occurs via sequential BDNF release and NMDA expression. Experimental evidence is provided here as conditional bdnf knockout mice (bdnf-/-) failed to develop tolerance following Cre-recombinase adenovirus treatment. Increased BDNF expression followed microglia activation in acute minocycline treatment reversible manner. Following BDNF release, NR2A subunit of NMDA receptor was upregulated in anti-BDNF reversible manner showing the contribution of BDNF signaling in the control of NMDA receptor expression following chronic morphine treatment. Our data provide compelling evidence that microglia activation and BDNF release are key regulators in opioid tolerance mechanism via glutaminergic synapse plasticity.

The κ opioid receptor (KOR) plays a significant role in many physiological functions, including pain relief, stress, depression, drug abuse, anxiety and psychotic behaviors. KORs are widely distributed in the central and peripheral nervous systems, and are specifically activated by endogenous opioids derived from prodynorphin. They are members of the G protein-coupled receptor superfamily, and the crystal structure of the human KOR was recently elucidated. KORs were initially studied for their involvement in mediation of pain as stimulation of KOR produces analgesia and minimizes abuse liability and other side effects. Nowadays, the KOR is rapidly emerging as an important target for the treatment of a variety of other human disorders. Specifically, the KOR system has become increasingly implicated as a modulator of stress-related and addictive behaviors. Several selective KOR partial agonists and antagonists have been developed as potential antidepressants, anxiolytic and anti-addiction medications. Although many KOR ligands have not demonstrated desirable pharmacological properties, some have been shown to be viable drug candidates. Herein, we describe chemical and pharmacological developments on KOR ligands, advantages and challenges, and potential therapeutic applications of ligands for KORs. In the second part, recent advances in the KOR drug design utilizing computational approaches are presented, with focus on the discovery of a new naturally derived scaffold, sewarine, as a novel class of selective KOR ligands with antagonist properties, using a pharmacophore-based virtual screening strategy.

The three opioid receptors, mu, delta and kappa all mediate analgesia, and knockout mice with opioid receptor deletion have proven unique tools to investigate in vivo opioid pharmacology. Since a few years, a number of new mouse lines have been engineered, with several distinct mutations of the mu receptor, to assess the role of specific amino acid residues or peptidic sequences of this receptor in analgesia and tolerance. The analysis of analgesia in mice deleted in kappa receptor and triple mu/delta/kappa receptor knockout mice have provided advances in opioid-induced analgesia. Also, the global and conditional deletion of the delta receptor in peripheral nociceptive neurons has revealed the participation of the targeted receptors in opioid-induced analgesia. Another approach for the study of opioid receptors is the visualization of these receptors in vivo, by engineering of knock-in mice with fluorescently tagged receptors. A mouse line with a fluorescent delta receptor has allowed live imaging of this receptor in behavioral paradigms and first studies on ligand-biased agonism at this receptor in vivo. The studies with mutant mouse lines for opioid receptors, combined with novel molecular and pharmacological approaches, will allow to develop novel strategies for opioid-based analgesia. This review summarizes the different genetically modified mouse lines for opioid receptors as well as the data and concepts inferred from analgesia results on these mutant mouse lines.

Interest in opioid drugs like morphine, as the oldest and most potent pain-killing agents known, has been maintained through the years. One of the most frequent chronic pain sensations people experience is associated with pathological conditions of the musculoskeletal system. Chronic musculoskeletal pain is a major health problem, and an adequate management requires understanding of both peripheral and central components, with more attention drawn to the former. Intense experimental and clinical research activities resulted in important knowledge on the mechanisms and functions of the endogenous opioid system located in the periphery. This review describes the occurrence and distribution of endogenous opioids and their receptors in the musculoskeletal system, and their role in pain control in musculoskeletal disorders, such as rheumatoid arthritis and osteoarthritis. Using different techniques, including immunohistochemistry, electron microscopy or radioimmunoassay, expression of enkephalins, dynorphin, β-endorphin, and endomorphins was demonstrated in musculoskeletal tissues of animals and humans. Localization of opioid peptides was found in synovial membrane, periosteum, bone and bone marrow, loose connective tissue, the paratenon and musculotendinous junction of the achilles tendon. Animal and human studies have also demonstrated expression of μ, δ and κ opioid receptor proteins in musculoskeletal tissues using radioligand binding assays, autoradiography, electrophysiology, immunohistochemistry and Western blotting. Opioid receptor gene expression was reported based on polymerase chain reaction and in situ hybridization techniques. Combining morphological and quantitative approaches, important evidence that the musculoskeletal apparatus is equipped with a peripheral opioid system is provided. Demonstration of the occurrence of an endogenous opioid system in bone and joint tissues represents an essential step for defining novel pharmacological strategies to attain peripheral control of pain in musculoskeletal disorders.

The well-known opioid agonists, oxycodone and oxymorphone, and the opioid antagonists, naloxone and naltrexone, are commonly used clinical agents and research tools in the opioid field. They belong to the class of morphinan-6-ones, and produce their pharmacological effects by interacting with opioid receptors, i.e. mu (MOR), delta (DOR) and kappa (KOR). The search for potent agonists and antagonists has continuously engaged the interest of pharmaceutical research, aiming for the identification of safer therapeutic agents or discovery of opioids with novel therapeutic properties and with lesser unwanted side effects. The chemically highly versatile carbonyl group in position 6 of mophinan-6-ones permits functionalization and modification leading to numerous opioid ligands. We have focused on representative examples of various derivatives and interesting approaches for the development of structurally distinct molecules with substitution at C6 (e.g. 6-methylene, 6-hydroxy, 6-amido, bifunctional ligands), as preclinically and clinically valuable opioids. In this work, the development of 6-amino and 6-guanidino substituted 14-alkoxymorphinans, including the synthesis and pharmacological investigations is presented. The new approach represented by the introduction of amino and guanidino groups into position 6 of the morphinan skeleton of 14-O-methyloxymorphone, led to compounds with high efficacy, MOR affinity and selectivity, which act as potent antinociceptive agents. Altogether, as a consequence of target drug design and synthetic efforts in the field of morphinan-6-ones, we achieve a better understanding of the function of the opioid system, and such efforts may open new avenues for further investigations.

The selective κ opioid receptor agonist nalfurafine was launched in 2009 as an antipruritic drug for patients undergoing hemodialysis. It is the first clinically used compound with high selectivity for the κ opioid receptor. Nalfurafine had a different pharmacological feature from other κ opioid agonists. Nalfurafine induced neither addictive nor aversive effects, whereas other κ agonists such as U- 50,488H or salvinorin A produced psychotomimetic effects like dysphoria. Therefore, identification of the essential structural moieties of nalfurafine for binding to the κ opioid receptor was important for elucidation of the pharmacological discrepancies observed with these κ opioid agonists. Based on the investigations of various nalfurafine derivatives, the essential structural moieties of nalfurafine were unveiled. Both the nitrogen substituted by a cyclopropylmethyl group and the 6-amide side chain were indispensable. The phenol ring was important for obtaining strong binding affinities for the opioid receptors, but not indispensable for exerting selectivity for the κ receptor. This structure-activity information is expected to lead to the development of novel κ opioid receptor selective agonists.

Although the μ opioid receptor (MOR) was pharmacologically and biochemically identified in binding studies forty years ago, its structure, function, and true complexity only have emerged after its cloning in 1993. Continuous efforts from many laboratories have greatly advanced our understanding of MORs, ranging from their anatomic distribution to cellular and molecular mechanisms, and from cell lines to in vivo systems. The MOR is recognized as the main target for effective pain relief, but its involvement in many other physiological functions has also been recognized. This review provides a synopsis on the history of research on MORs and ligands acting at the MOR with the focus on their clinical and potential use as therapeutic drugs, or as valuable research tools. Since the elucidation of the chemical structure of morphine and the characterization of endogenous opioid peptides, research has stimulated the development of new generations of MOR ligands with distinct pharmacological profiles (agonist, antagonist, mixed agonist/antagonist and partial agonist) or site of action (central/peripheral). Discovery of therapeutically useful morphine-like drugs and innovative drugs with new scaffolds, with several outstanding representatives, is discussed. Extensive efforts on modifications of endogenous peptides to attain stable and MOR selective analogs are overviewed with stimulating results for the development of peptide-based pharmaceuticals. With pharmacophore modeling as an important tool in drug discovery, application of modern computational methodologies for the development of morphinans as new MOR ligands is described. Moreover, the crystal structure of the MOR available today will enable the application of structurebased approaches to design better drugs for the management of pain, addiction and other human diseases, where MORs play a key role.

To address the different types of pain (e.g. acute, chronic, neuropathic) different classes of medications, mainly non-steroidal anti-inflammatory drugs and narcotics (opioids), are used. More specifically, the alleviation or treatment of moderate to severe pain states commonly invokes the use of opioids. Unfortunately, their chronic administration induces various undesirable side effects, such as for example physical dependence and tolerance. One strategy to overcome these major side effects and to prolong the antinociceptive efficiency of the applied drugs involves the creation of multifunctional compounds which contain hybridized structures. Combination of opioid agonist and antagonist pharmacophores in a single chemical entity has been considered and extensively investigated, but opioids have also been combined with other bioactive neurotransmitters and peptide hormones that are involved in pain perception (e.g. substance P, neurotensin, cholecystokinin, cannabinoids, melanocortin ligands, etc.). Such novel chimeras (also called designed multiple ligands or twin/triplet drugs), may interact independently with their respective receptors and potentially result in more effective antinociceptive properties. The designed multiple ligands presented in this work include opioid-non-opioid peptide dimer analogs, mixed peptidic- non-peptidic bifunctional ligands and dual non-peptidic dimers. The main focus herein is placed on the design and biological evaluation of these multiple opioid compounds, rather than the synthetic approach and preparation.

The NOP receptor, the fourth receptor in the opioid receptor family, is found throughout the brain and is involved in a variety of CNS systems and pathways. The endogenous ligand for NOP receptors, nociceptin/orphanin FQ (now called N/OFQ), was originally thought to increase a painful stimulus since intracerebroventricular (i.c.v.) administration of this heptadecapeptide led to a decrease in tail-flick and hot-plate latency in mice. Further studies suggested that N/OFQ blocks opiate analgesia when administered i.c.v. but potentiates opiate analgesia and has antinociceptive activity when administered intrathecally. I.c.v. administration of N/OFQ has other beneficial actions including inhibition of reward induced by several different abused drugs, as well as anti-anxiety activity. Recent work has demonstrated that individual small molecules that activate both NOP and mu receptors possess mu-mediated antinociceptive activity with reduced reward, as determined by conditioned place preference tests. Furthermore, selective NOP receptor agonists appear to be active in certain chronic pain models and reduce both drug craving and anxiety. NOP receptor antagonists may also have therapeutic benefits since both peptide and small molecule antagonists have anti-depressant activity in two different animal models. Therefore, both selective NOP receptor compounds and non-selective compounds, with both NOP receptor and mu opioid receptor activity, appear to have potential for clinical use for several neurological and psychiatric disorders including acute and chronic pain, drug abuse, anxiety and depression.

Tritiated opioid ligands are essential tools for the identification of opioid receptors. This review deals with the syntheses of tritiated opioid peptide derivatives, including enkephalins, dynorphins, dermorphins, deltorphins and endomorphins, and also discusses tritium-labeled nonpeptide opioids. It additionally focuses on the relevance of tritium-labeled opioid compounds as research tools for investigating opioid receptor pharmacology. Agonists and antagonists are used for the characterization of new opioid ligands by means of radioreceptor binding assays. Further topics covered in this review are the distribution of the endogenous peptides in the central nervous system and peripheral tissues, and degradation studies of opioids in brain membrane preparations and the blood.

Water caltrop is a popular traditional vegetable in China, and its pericarps are always wasted. In the present work reported here, pericarps from three different Chinese water caltrop cultivars were collected and extracted using 70% methanol and hot water. All the extracts contained significant amounts of polyphenols (183.7-201.7 mg GAE/g), flavonoids (34.3-54.6 mg RE/g) and saponins (23.2- 36.3 mg GRE/g). These extracts exhibited strong antioxidant capacity as assessed by DPPH, ABTS and FRAP methods. High correlations were found in DPPH, ABTS and polyphenols, FRAP and saponins. All the three extracts inhibited proliferation of SGC7901 human gastric cancer cells and HepG2 human hepatocarcinoma cells in a dose dependent manner without detectable cytotoxicity on HUVEC normal cells. Flow cytometry showed that apoptosis of SGC7901 and HepG2 cells was induced by water caltrop extracts while HUVEC cells were relatively resistant to apoptosis. Hot water extracts showed similar bioactivities as methanol extracts, which indicated that hot water could be used to extract bioactive compounds instead of organic solvents. These results suggest that water caltrop pericarps could be explored for their potential as anti-cancer drugs in future studies.

Background and Purpose. Activated Protein C (APC) stimulates multiple cytoprotective pathways via the protease activated receptor-1 (PAR-1) and promotes anticoagulation. 3K3A-APC was designed for preserved activity at PAR-1 with reduced anticoagulation. This Phase 1 trial characterized pharmacokinetics and anticoagulation effects of 3K3A-APC. Methods. Subjects (n=64) were randomly assigned to receive 3K3A-APC (n=4) at 6, 30, 90, 180, 360, 540 or 720 μg/kg or placebo (n=6) and were observed for 24 hr. After safety review additional subjects received drug every 12 hr for 5 doses (n=6 per group) at 90, 180, 360, or 540 μg/kg or placebo (n=8) and were observed for 24 hr. Results. All subjects returned for safety assessments at 72 hours and 15 days. We found few adverse events in all groups. Systolic blood pressure increased in both active and placebo groups. Moderately severe headache, nausea and vomiting were reported in one of two subjects treated with 720 g/kg so 540 μg/kg was considered the highest tolerated dose. Mean plasma concentrations increased in proportion to dose. Clearance ranged from 11,693 ± 807 to 18,701 ± 4,797 mL/hr, volume of distribution ranged from 4,873±828 to 6,971 ± 1,169 mL, and elimination half-life ranged from 0.211 ± 0.097 to 0.294 ± 0.054 hours. Elevations in aPTT were minimal. Conclusions. 3K3A-APC was well tolerated at multiple doses as high as 540 μg/kg. These results should be confirmed in stroke patients with relevant co-morbidities. Clinical Trial Registration-URL: http://www.clinicaltrials.gov. Unique identifier: NCT01660230