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.

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.

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.

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'.

Stereochemistry and innate immune recognition: (+)-norbinaltorphimine targets myeloid differentiation protein 2 and inhibits toll-like receptor 4 signaling.

Deregulation of innate immune TLR4 signaling contributes to various diseases including neuropathic pain and drug addiction. Naltrexone is one of the rare TLR4 antagonists with good blood-brain barrier permeability and showing no stereoselectivity for TLR4. By linking 2 naltrexone units through a rigid pyrrole spacer, the bivalent ligand norbinaltorphimine was formed. Interestingly, (+)-norbinaltorphimine [(+)-1] showed ∼25 times better TLR4 antagonist activity than naltrexone in microglial BV-2 cell line, whereas (-)-norbinaltorphimine [(-)-1] lost TLR4 activity. The enantioselectivity of norbinaltorphimine was further confirmed in primary microglia, astrocytes, and macrophages. The activities of meso isomer of norbinaltorphimine and the molecular dynamic simulation results demonstrate that the stereochemistry of (+)-1 is derived from the (+)-naltrexone pharmacophore. Moreover, (+)-1 significantly increased and prolonged morphine analgesia in vivo. The efficacy of (+)-1 is long lasting. This is the first report showing enantioselective modulation of the innate immune TLR signaling.-Zhang, X., Peng, Y., Grace, P. M., Metcalf, M. D., Kwilasz, A. J., Wang, Y., Zhang, T., Wu, S., Selfridge, B. R., Portoghese, P. S., Rice, K. C., Watkins, L. R., Hutchinson, M. R., Wang, X. Stereochemistry and innate immune recognition: (+)-norbinaltorphimine targets myeloid differentiation protein 2 and inhibits toll-like receptor 4 signaling.

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'.

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.

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.

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.

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.

Modulation of musculoskeletal hyperalgesia by brown adipose tissue activity in mice.

Cold exposure and a variety of types of mild stress increase pain in patients with painful disorders such as fibromyalgia syndrome. Acutely, stress induces thermogenesis by increasing sympathetic activation of beta-3 (ß3) adrenergic receptors in brown adipose tissue. Chronic stress leads to the hypertrophy of brown adipose, a phenomenon termed adaptive thermogenesis. Based on the innervation of skeletal muscle by collaterals of nerves projecting to brown adipose, we theorized an association between brown adipose tissue activity and musculoskeletal hyperalgesia and tested this hypothesis in mice. Exposure to a cold swim or injection of BRL37344 (ß3 adrenergic agonist) each enhanced musculoskeletal hyperalgesia, as indicated by morphine-sensitive decreases in grip force responses, whereas SR59230A (ß3 adrenergic antagonist) attenuated swim-induced hyperalgesia. Chemical ablation of interscapular brown adipose, using Rose Bengal, attenuated the development of hyperalgesia in response to either swim stress or BRL37344. In addition, elimination of the gene expressing uncoupling protein-1 (UCP1), the enzyme responsible for thermogenesis, prevented musculoskeletal hyperalgesia in response to either a swim or BRL37344, as documented in UCP1-knockout (UCP1-KO) mice compared with wild-type controls. Together, these data provide a convergence of evidence suggesting that activation of brown adipose contributes to stress-induced musculoskeletal hyperalgesia.

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.

Putative kappa opioid heteromers as targets for developing analgesics free of adverse effects.

It is now generally recognized that upon activation by an agonist, ß-arrestin associates with G protein-coupled receptors and acts as a scaffold in creating a diverse signaling network that could lead to adverse effects. As an approach to reducing side effects associated with κ opioid agonists, a series of ß-naltrexamides 3-10 was synthesized in an effort to selectively target putative κ opioid heteromers without recruiting ß-arrestin upon activation. The most potent derivative 3 (INTA) strongly activated KOR-DOR and KOR-MOR heteromers in HEK293 cells. In vivo studies revealed 3 to produce potent antinociception, which, when taken together with antagonism data, was consistent with the activation of both heteromers. 3 was devoid of tolerance, dependence, and showed no aversive effect in the conditioned place preference assay. As immunofluorescence studies indicated no recruitment of ß-arrestin2 to membranes in coexpressed KOR-DOR cells, this study suggests that targeting of specific putative heteromers has the potential to identify leads for analgesics devoid of adverse effects.

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.

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.

An immunocytochemical-derived correlate for evaluating the bridging of heteromeric mu-delta opioid protomers by bivalent ligands.

Bivalent ligands that contain two pharmacophores linked by a spacer are promising tools to investigate the pharmacology of opioid receptor heteromers. Evidence for occupation of neighboring protomers by two phamacophores of a single bivalent ligand (bridging) has relied mainly on pharmacological data. In the present study, we have employed an immunocytochemical correlate to support in vivo biological studies that are consistent with bridging. We show that a bivalent mu agonist/delta antagonist (MDAN-21) that is devoid of tolerance due to possible bridging of mu and delta protomers prevents endocytosis of the heteromeric receptors in HEK-293 cells. Conversely, a bivalent ligand (MDAN-16) with a short spacer or monovalent mu agonist give rise to robust internalization. The data suggest that the immobilization of proximal mu and delta protomers is due to bridging by MDAN-21. The finding that MDAN-21 and its shorter spacer homologue MDAN-16 possess equivalent activity in HEK-293 cells, but produce dramatically divergent internalization of mu-delta heteromer, is relevant to the role of internalization and tolerance.

Reduced antinociception of opioids in rats and mice by vaccination with immunogens containing oxycodone and hydrocodone haptens.

Prescription opioids abuse and associated deaths are an emerging concern in the USA. Vaccination against prescription opioids may provide an alternative to pharmacotherapy. An oxycodone hapten containing a tetraglycine linker at the C6 position (6OXY(Gly)(4)OH) conjugated to keyhole limpet hemocyanin (KLH) has shown early proof-of-efficacy in rodents as a candidate immunogen (6OXY(Gly)(4)-KLH) for the treatment of oxycodone abuse. In this study, oxycodone-based and hydrocodone-based haptens were conjugated to KLH to generate immunogens that would recognize both oxycodone and hydrocodone. Vaccination with 6OXY(Gly)(4)-KLH increased drug binding in serum, reduced drug distribution to brain, and blunted analgesia for both oxycodone and hydrocodone. An analogous C6-linked hydrocodone vaccine blocked hydrocodone effects but less so than 6OXY(Gly)(4)-KLH. C8-Linked hydrocodone immunogens had only limited efficacy. Amide conjugation showed higher haptenation ratios and greater efficacy than thioether conjugation to maleimide activated KLH (mKLH). The 6OXY(Gly)(4)-KLH vaccine may be used for treatment of prescription opioid abuse.