FBNTI, a DOR-Selective Antagonist That Allosterically Activates MOR within a MOR-DOR Heteromer.

This report describes the unique pharmacological profile of FBNTI, a potent DOR antagonist that acts as a MOR agonist via an allosteric mechanism. Binding of FBNTI to opioid receptors expressed in HEK 293 cells revealed a 190-fold greater affinity for DOR (Ki = 0.84 nM) over MOR (Ki = 160 nM). In mice, intrathecal FBNTI produced potent antinociception (ED50 = 46.9 pmol/mouse), which was antagonized by selective MOR antagonists (CTOP, ß-FNA). Autoantagonism of the MOR agonism by FBNTI was observed above the ED75 dose, suggesting antagonism of activated MOR. That FBNTI is devoid of agonism in DOR knockout mice is consistent with allosteric activation of the MOR protomer via FBNTI bound to within a MOR-DOR heteromer. This proposed mechanism is supported by calcium mobilization assays, which indicate that FBNTI selectively activates the MOR-DOR heteromer and functionally antagonizes the MOR protomer at >ED75. The unprecedented mode of MOR activation by FBNTI may be responsible for the lack of tolerance after intrathecal (i.t.) administration. FBNTI was highly effective upon topical administration to the ipsolateral hind paw in the Hargreaves assay (EC50 = 0.17 ± 0.08 µM) and without significant contralateral activity, suggesting a lack of systemic exposure.

Combination of a δ-opioid Receptor Agonist and Loperamide Produces Peripherally-mediated Analgesic Synergy in Mice.

BACKGROUND: The long-term use of opioids for analgesia carries significant risk for tolerance, addiction, and diversion. These adverse effects are largely mediated by µ-opioid receptors in the central nervous system. Based on the authors' previous observation that morphine and δ-opioid receptor agonists synergize in spinal cord in a protein kinase Cε-dependent manner, they predicted that this µ-opioid receptor-δ-opioid receptor synergy would take place in the central terminals of nociceptive afferent fibers and generalize to their peripheral terminals. Therefore, the authors hypothesized that loperamide, a highly efficacious µ-opioid receptor agonist that is excluded from the central nervous system, and oxymorphindole, a δ-opioid receptor agonist that was shown to synergize with morphine spinally, would synergistically reverse complete Freund's adjuvant-induced hyperalgesia. METHODS: Using the Hargreaves assay for thermal nociception, the von Frey assay for mechanical nociception and the complete Freund's adjuvant-induced model of inflammatory pain, we tested the antinociceptive and antihyperalgesic effect of loperamide, oxymorphindole, or the loperamide-oxymorphindole combination. Animals (Institute for Cancer Research [ICR] CD1 strain mice; n = 511) received drug by systemic injection, intraplantar injection to the injured paw, or a transdermal solution on the injured paw. Dose-response curves for each route of administration and each nociceptive test were generated, and analgesic synergy was assessed by isobolographic analysis. RESULTS: In naïve animals, the loperamide-oxymorphindole combination ED50 value was 10 times lower than the theoretical additive ED50 value whether given systemically or locally. In inflamed animals, the combination was 150 times more potent systemically, and 84 times more potent locally. All combinations showed statistically significant synergy when compared to the theoretical additive values, as verified by isobolographic analysis. The antihyperalgesia was ablated by a peripherally-restricted opioid antagonist. CONCLUSIONS: From these data we conclude that the loperamide-oxymorphindole combination synergistically reverses complete Freund's adjuvant-induced inflammatory hyperalgesia. The authors also conclude that this interaction is mediated by opioid receptors located in the peripheral nervous system.

Ligands that interact with putative MOR-mGluR5 heteromer in mice with inflammatory pain produce potent antinociception.

The low effectiveness of morphine and related mu opioid analgesics for the treatment of chronic inflammatory pain is a result of opioid-induced release of proinflammatory cytokines and glutamate that lower the pain threshold. In this regard, the use of opioids with metabotropic glutamate-5 receptor (mGluR5) antagonist has been reported to increase the efficacy of morphine and prevent the establishment of adverse effects during chronic use. Given the presence of opioid receptors (MORs) and mGluR5 in glia and neurons, together with reports that suggest coexpressed MOR/mGluR5 receptors in cultured cells associate as a heteromer, the possibility that such a heteromer could be a target in vivo was addressed by the design and synthesis of a series of bivalent ligands that contain mu opioid agonist and mGluR5 antagonist pharmacophores linked through spacers of varying length (10-24 atoms). The series was evaluated for antinociception using the tail-flick and von Frey assays in mice pretreated with lipopolysaccharide (LPS) or in mice with bone cancer. In LPS-pretreated mice, MMG22 (4c, 22-atom spacer) was the most potent member of the series (intrathecal ED50 ∼9 fmol per mouse), whereas in untreated mice its ED50 was more than three orders of magnitude higher. As members of the series with shorter or longer spacers have ≥500-fold higher ED50s in LPS-treated mice, the exceptional potency of MMG22 may be a result of the optimal bridging of protomers in a putative MOR-mGluR5 heteromer. The finding that MMG22 possesses a >10(6) therapeutic ratio suggests that it may be an excellent candidate for treatment of chronic, intractable pain via spinal administration.

Molecular basis for binding and subtype selectivity of 1,4-benzodiazepine antagonist ligands of the cholecystokinin receptor.

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 CCK2R-selective 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.

MMG22 Potently Blocks Hyperalgesia in Cisplatin-treated Mice.

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 (ED50 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 (ED50 = 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.

The bivalent ligand, MMG22, reduces neuropathic pain after nerve injury without the side effects of traditional opioids.

ABSTRACT: Functional interactions between the mu opioid receptor (MOR) and the metabotropic glutamate receptor 5 (mGluR5) in pain and analgesia have been well established. MMG22 is a bivalent ligand containing MOR agonist (oxymorphamine) and mGluR5 antagonist (MPEP) pharmacophores tethered by a 22-atom linker. MMG22 has been shown to produce potent analgesia in several models of chronic inflammatory and neuropathic pain (NP). This study assessed the efficacy of systemic administration of MMG22 at reducing pain behavior in the spared nerve injury (SNI) model of NP in mice, as well as its side-effect profile and abuse potential. MMG22 reduced mechanical hyperalgesia and spontaneous ongoing pain after SNI, with greater potency early (10 days) as compared to late (30 days) after injury. Systemic administration of MMG22 did not induce place preference in naive animals, suggesting absence of abuse liability when compared to traditional opioids. MMG22 also lacked the central locomotor, respiratory, and anxiolytic side effects of its monomeric pharmacophores. Evaluation of mRNA expression showed the transcripts for both receptors were colocalized in cells in the dorsal horn of the lumbar spinal cord and dorsal root ganglia. Thus, MMG22 reduces hyperalgesia after injury in the SNI model of NP without the typical centrally mediated side effects associated with traditional opioids.

Combined Glia Inhibition and Opioid Receptor Agonism Afford Highly Potent Analgesics without Tolerance.

Commonly prescribed opioid analgesics produce tolerance upon chronic use due in part to induction of hyperalgesia. Given that two reported bivalent ligands (MMG22 and MCC22) produce potent antinociception without tolerance only in inflamed mice, we have investigated the possible cellular and receptor targets of these ligands. The selective microglia inhibitors, minocycline and SB290157, antagonized intrathecal (i.t.) MCC22 antinociception orders of magnitude more potently than MMG22, suggesting that MCC22 selectively targets activated microglia. The astrocyte toxin, l-α-aminoadipic acid antagonized MMG22 antinociception 126-fold without reducing the potency of MCC22, indicating that activated astrocytes are targets of MMG22. MK-801 and Ro25-6981 antagonism of MMG22 antinociception, but not MCC22, is consistent with selective inhibition of activated NMDAR in astrocytes. The antinociception produced by i.t. MMG22 or MCC22 were both antagonized by the selective mu opioid receptor antagonist, ß-FNA, implicating interaction of these ligands with MOR in spinal afferent neurons. MCC22 antinociception was potently blocked by kainate or AMPA ion channel antagonists (LY382884; NBQX), in contrast to MMG22. It is concluded that i.t. MMG22 and MCC22 produce exceptional antinociception via potent inhibition of activated spinal glia, thereby leading to desensitization of spinal neurons and enhanced activation of neuronal MOR. Thus, the present study suggests a new approach to treatment of chronic inflammatory pain without tolerance through a single molecular entity that simultaneously inhibits activated glia and stimulates MOR in spinal neurons.

The bivalent ligand MCC22 potently attenuates hyperalgesia in a mouse model of cisplatin-evoked neuropathic pain without tolerance or reward.

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 (ED50 = 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'.

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'.

A bivalent compound targeting CCR5 and the mu opioid receptor treats inflammatory arthritis pain in mice without inducing pharmacologic tolerance.

BACKGROUND: Pain accompanies rheumatoid arthritis and other chronic inflammatory conditions and is difficult to manage. Although opioids provide potent analgesia, chronic opioid use can cause tolerance and addiction. Recent studies have demonstrated functional interactions between chemokine and opioid receptor signaling pathways. Reported heterodimerization of chemokine and opioid receptors led our group to develop bivalent compounds that bind both types of receptors, with the goal of targeting opioids to sites of inflammation. MCC22 is a novel bivalent compound containing a CCR5 antagonist and mu opioid receptor (MOR) agonist pharmacophores linked through a 22-atom spacer. We evaluated the efficacy of MCC22 in the K/B.g7 T-cell receptor transgenic mouse model of spontaneous inflammatory arthritis. METHODS: MCC22 or morphine was administered intraperitoneally at varying doses to arthritic K/B.g7 mice or nonarthritic control mice. Mechanical pain hypersensitivity was measured each day before and after drug administration, using the electronic von Frey test. The potency of MCC22 relative to that of morphine was calculated. Functional readouts of pain included grip strength and nesting behavior. A separate dosing regimen was used to determine whether the drugs induced pharmacologic tolerance. RESULTS: MCC22 provided ~ 3000-fold more potent analgesia than morphine in this model. Daily treatment with MCC22 also led to a cumulative analgesic effect, reducing the daily baseline pain level. MCC22 produced no observable analgesic effect in nonarthritic control mice. Importantly, repeated administration of MCC22 did not induce pharmacologic tolerance, whereas a similar regimen of morphine did. Both grip strength and nesting behaviors improved among arthritic mice treated with MCC22. Ankle thickness and arthritis scores were not affected by MCC22. The analgesic effect of MCC22 was abolished in K/B.g7 mice genetically lacking CCR5, demonstrating the receptor specificity of the antagonist pharmacophore. CONCLUSIONS: MCC22 is a novel bivalent ligand that targets CCR5 and MOR. Our findings demonstrate that MCC22 provides highly potent analgesia and improved functional outcomes in a model of inflammatory arthritis, without inducing typical opioid tolerance. These findings suggest that MCC22 or similar compounds could be used to treat the pain associated with inflammatory arthritis and related conditions, while minimizing the risks typically associated with chronic opioid use.

Bivalent ligand MCC22 potently attenuates nociception in a murine model of sickle cell disease.

Sickle cell disease (SCD) is a chronic inflammatory disorder accompanied by chronic pain. In addition to ongoing pain and hyperalgesia, vaso-occlusive crises-induced pain can be chronic or episodic. Because analgesics typically used to treat pain are not very effective in SCD, opioids, including morphine, are a primary treatment for managing pain in SCD but are associated with many serious side effects, including constipation, tolerance, addiction, and respiratory depression. Thus, there is a need for the development of novel treatments for pain in SCD. In this study, we used the Townes transgenic mouse model of SCD to investigate the antinociceptive efficacy of the bivalent ligand, MCC22, and compared its effectiveness with morphine. MCC22 consists of a mu-opioid receptor agonist and a chemokine receptor-5 (CCR5) antagonist that are linked through a 22-atom spacer. Our results show that intraperitoneal administration of MCC22 produced exceptionally potent dose-dependent antihyperalgesia as compared to morphine, dramatically decreased evoked responses of nociceptive dorsal horn neurons, and decreased expression of proinflammatory cytokines in the spinal cord. Moreover, tolerance did not develop to its analgesic effects after repeated administration. In view of the extraordinary potency of MCC22 without tolerance, MCC22 and similar compounds may vastly improve the management of pain associated with SCD.

Heteromer Induction: An Approach to Unique Pharmacology?

It is proposed that two types of opioid receptor heteromers exist: a) those that are constitutive and b) those that are induced by bivalent ligands. Mu opioid agonists interact with constitutive MOR-DOR heteromer to mediate tolerance and dependence. Bivalent ligand, MDAN21, is devoid of these adverse effects by virtue of its DOR antagonist pharmacophore. We propose that bivalent ligands MMG22 and MCC22 induce colocalized receptors to form heteromers (MOR-mGluR5 and MOR-CCR5, respectively) that do not occur naturally, thereby promoting unique pharmacology. Heteromer induction with bivalent ligands offers a general approach to unique pharmacology that complements traditional SAR.

Bivalent ligand that activates mu opioid receptor and antagonizes mGluR5 receptor reduces neuropathic pain in mice.

The mu opioid receptor (MOR) and metabotropic glutamate receptor 5 (mGluR5) are well-established pharmacological targets in the management of chronic pain. Both receptors are expressed in the spinal cord. MMG22, a bivalent ligand containing 2 pharmacophores separated by 22 atoms, which simultaneously activates MOR and antagonizes mGluR5, has been shown to produce potent reversal of tactile hypersensitivity in rodent models of lipopolysaccharide (LPS)-and bone cancer-induced chronic pain. This study assessed whether intrathecal MMG22 also is effective in reducing pain of neuropathic origin. Furthermore, we theorized that MMG22 should reduce hyperalgesia in nerve-injured mice in a manner consistent with a synergistic interaction between MOR and mGluR5. Several weeks after spared nerve injury, tactile hypersensitivity was reversed in mice by the intrathecal injection of MMG22 (0.01-10 nmol) but also by its shorter spacer analog, MMG10, with similar potency. The potencies of the bivalent ligands were 10- to 14-fold higher than those of the compounds upon which the bivalent structure was based, the MOR agonist oxymorphone and the mGluR5 antagonist MPEP. Coadministration of oxymorphone and MPEP demonstrated analgesic synergism, an interaction confirmed by isobolographic analysis. This study indicates that in the spared nerve injury-induced model of neuropathic pain, the 2 pharmacophores of the bivalent ligands MMG22 and MMG10 target MOR and mGluR5 as separate receptor monomers. The observed increase in the potency of MMG22 and MMG10, compared with oxymorphone and MPEP, may reflect the synergistic interaction of the 2 pharmacophores of the bivalent ligand acting at their respective separate receptor monomers.

A novel Schiff base: Synthesis, structural characterisation and comparative sensor studies for metal ion detections.

A novel Schiff base ligand was synthesized by the condensation reaction of 2,6-diformylpyridine and 4-aminoantipyrine in MeOH and characterised by its melting point, elemental analysis, FT-IR, (1)H, (13)C NMR and mass spectroscopic studies. Molecular structure of the ligand was determined by single crystal X-ray diffraction technique. The electrochemical properties of the Schiff base ligand were studied in different solvents at various scan rates. Sensor ability of the Schiff base ligand was investigated by colorimetric and fluorometric methods. Visual colour change of the ligand was investigated in MeOH solvent in presence of various metal ions Na(+), Mg(2+), Al(3+), K(+), Cr(3+), Mn(2+), Fe(3+), Co(2+), Ni(2+), Cu(2+), Zn(2+), Cd(2+), Hg(2+) and Pb(2+). Upon addition of Al(3+) ion into a MeOH solution of the ligand, an orange colour developed which is detectable by naked eye. Fluorescence emission studies showed that the ligand showed single emission band at 630-665nm upon excitation at 560nm. Addition of metal ions Na(+), Mg(2+), K(+), Cr(3+), Mn(2+), Fe(3+), Co(2+), Ni(2+), Cu(2+), Zn(2+), Cd(2+), Hg(2+) and Pb(2+) (1:1M ratio) cause fluorescence quenching, however addition of Al(+3) resulted in an increase in fluorescence intensity. No significant variation was observed in the fluorescence intensity caused by Al(3+) in presence of other metal ions. Therefore, the Schiff base ligand can be used for selective detection of Al(3+) ions in the presence of the other metal ions studied.

Inhibition of Inflammatory and Neuropathic Pain by Targeting a Mu Opioid Receptor/Chemokine Receptor5 Heteromer (MOR-CCR5).

Chemokine release promotes cross-talk between opioid and chemokine receptors that in part leads to reduced efficacy of morphine in the treatment of chronic pain. On the basis of the possibility that a MOR-CCR5 heteromer is involved in such cross-talk, we have synthesized bivalent ligands (MCC series) that contain mu opioid agonist and CCR5 antagonist pharmacophores linked through homologous spacers (14-24 atoms). When tested on lipopolysaccharide-inflamed mice, a member of the series (MCC22; 3e) with a 22-atom spacer exhibited profound antinociception (i.t. ED50 = 0.0146 pmol/mouse) that was 2000× greater than morphine. Moreover, MCC22 was ~3500× more potent than a mixture of mu agonist and CCR5 antagonist monovalent ligands. These data strongly suggest that MCC22 acts by bridging the protomers of a MOR-CCR5 heteromer having a TM5,6 interface. Molecular simulation studies are consistent with such bridging. This study supports the MOR-CCR5 heteromer as a novel target for the treatment of chronic pain.

Targeting putative mu opioid/metabotropic glutamate receptor-5 heteromers produces potent antinociception in a chronic murine bone cancer model.

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 mGluR5 antagonist pharmacophores that produced potent antinociception in mice with LPS-induced acute inflammatory pain via a putative MOR-mGluR5 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 mGluR5 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 mGluR5 antagonist (MG20) monovalent ligands, the data suggest that targeting the putative MOR-mGluR5 heteromer is far superior to univalent interaction with receptors in reducing tumor-induced nociception. In view of the high potency, long duration (>24h) 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.

Bivalent ligands that target µ opioid (MOP) and cannabinoid1 (CB1) receptors are potent analgesics devoid of tolerance.

Given that µ opioid (MOP) and canabinoid (CB1) receptors are colocalized in various regions of the central nervous system and have been reported to associate as heteromer (MOP-CB1) in cultured cells, the possibility of functional, endogenous MOP-CB1 in nociception and other pharmacologic effects has been raised. As a first step in investigating this possibility, we have synthesized a series of bivalent ligands 1-5 that contain both µ agonist and CB1 antagonist pharmacophores for use as tools to study the functional interaction between MOP and CB1 receptors in vivo. Immunofluorescent studies on HEK293 cells coexpressing both receptors suggested 5 (20-atom spacer) to be the only member of the series that bridges the protomers of the heteromer. Antinociceptive testing in mice revealed 5 to be the most potent member of the series. As neither a mixture of monovalent ligands 9 + 10 nor bivalents 2-5 produced tolerance in mice, MOR-CB1 apparently is not an important target for reducing tolerance.

MDAN-21: A Bivalent Opioid Ligand Containing mu-Agonist and Delta-Antagonist Pharmacophores and Its Effects in Rhesus Monkeys.

MDAN-21, 7'-{2-[(7-{2-[({(5α, 6α)-4,5-Epoxy-3,14-dihydroxy-17-methylmorphin-6-yl}-aminocarbonyl)metoxy]-acetylamino}-heptylaminocarbonyl)-methoxy]-acetylamino}-naltrindole, a bivalent opioid ligand containing a mu-opioid receptor agonist (derived from oxymorphone) linked to the delta-opioid receptor antagonist (related to naltrindole) by a spacer of 21 atoms, was reported to have potent analgesic properties in mice. Tolerance, physical dependence, and conditioned place preference were not evident in that species. The finding that bivalent ligands in this series, with spacers 19 atoms or greater, were devoid of tolerance and dependence led to the proposal that MDAN-21 targets heteromeric mu-delta-opioid receptors. The present study focused on its effects in nonhuman primates (Macaca mulatta), a species with a physiology and behavioral repertoire not unlike humans. With regard to opioids, this species usually better predicts clinical outcomes. MDAN-21 substituted for morphine in morphine-dependent monkeys in the remarkably low dose range 0.006-0.032 mg/kg, subcutaneously. Although MDAN-21 failed to produce reliable thermal analgesia in the dose range 0.0032-0.032 mg/kg, intramuscularly, it was active in the same dose range and by the same route of administration, in the capsaicin-induced thermal allodynia assay. The results suggest that MDAN-21 may be useful in the treatment of opioid dependence and allodynia. The data provide additional evidence that opioid withdrawal is associated with sensitized pain.

Modulation of cell surface expression of nonactivated cholecystokinin receptors using bivalent ligand-induced internalization.

CCK(2) receptor antagonists potentiate pain relief by MOP receptor agonists. In an attempt to enhance this effect, we prepared bivalent ligands incorporating CCK(2) receptor antagonist and MOP receptor agonist pharmacophores. (9) Ligands with 16- to 22-atom spacers could simultaneously bind both receptors but provided no advantage in activity over individual ligands. We now examine the effect of these ligands on receptor internalization as a mechanism of receptor regulation. We prepared CHO cell lines expressing nonfluorescent halves (YN and YC) of yellow fluorescent protein attached to each receptor. Spatial approximation of constructs was needed to yield fluorescence. Monovalent MOP agonist 1 signaled normally and internalized the MOP receptor. Monovalent CCK(2) antagonist 2 did not stimulate receptor internalization. In the dual receptor-bearing cells, bivalent ligands 3a-c capable of simultaneously binding both receptors resulted in cell surface fluorescence and internalization of the fluorescent complex in a time- and temperature-dependent manner. Bivalent ligand 4 with spacer too short to occupy both receptors simultaneously yielded no signal. Receptor tethering with appropriate bivalent ligands can down-regulate signaling by moving a nonactivated receptor into the endocytic pathway.

Induced association of mu opioid (MOP) and type 2 cholecystokinin (CCK2) receptors by novel bivalent ligands.

Both mu-opioid (MOP) and type 2 cholecystokinin (CCK2) receptors are present in areas of the central nervous system that are involved in modulation of pain processing. We conducted bioluminescence resonance energy transfer (BRET) studies on COS cells coexpressing MOP and CCK2 receptors to determine whether receptor heterodimerization is involved in such modulation. These studies revealed the absence of constitutive or monovalent ligand-induced heterodimerization. Heterodimerization of MOP and CCK2 receptors therefore is unlikely to be responsible for the opposing effects between morphine and CCK in the CNS. However, association was induced, as indicated by a positive BRET signal, on exposure of the cells to bivalent ligands containing mu-opioid agonist and CCK2 receptor antagonist pharmacophores linked through spacers containing 16-22 atoms but not with a shorter (9-atom) spacer. These studies demonstrate for the first time that an appropriately designed bivalent ligand is capable of inducing association of G-protein-coupled receptors. The finding that opioid tolerance studies with these ligands in mice showed no correlation with the BRET data is consistent with the absence of association of MOP and CCK2 receptors in vivo.