δ-opioid receptor ( IC50 = 2.73 nM ); μ-opioid receptor ( IC50 = 5457 nM ); δ-opioid receptor) ( Ki = 1.78 nM ); μ-opioid receptor ( IC50 = 881.5 nM ); κ-opioid receptor ( IC50 = 441.8 nM )

SNC80 selectively activates the μ-δ heteromer in HEK293 cells with an EC50 of 52.8 nM. SNC80 shows noticeably more activity in cells that coexpress μ- and δ-opioid receptors than in those that either express δ-opioid receptors alone or in combination with ν- and κ-opioid receptors[4].

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The present study has investigated the pharmacology of SNC80, a nonpeptidic ligand proposed to be a selective delta agonist in vitro and in vivo. SNC 80 was potent in producing inhibition of electrically induced contractions of mouse vas deferens, but not in inhibiting contractions of the guinea pig isolated ileum (IC50 values of 2.73 nM and 5457 nM, respectively). The delta selective antagonist ICI 174,864 (1 microM) and the mu selective antagonist CTAP (1 microM) produced 236- and 1.9-fold increases, respectively, in the SNC 80 IC50 value in the mouse vas deferens. SNC 80 preferentially competed against sites labeled by [3H]naltrindole (delta receptors) rather than against those labeled by [3H]DAMGO (mu receptors) or [3H]U69, 593 kappa receptors) in mouse whole-brain assays. The ratios of the calculated Ki values for SNC80 at mu/delta and kappa/delta sites were 495- and 248-fold, respectively, which indicates a significant degree of delta selectivity for this compound in radioligand binding assays. [2]

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SNC80 was assayed in heterologous HEK293 cells singly or coexpressing opioid receptors, the same coexpressing cell lines where functional heteromer was established by coimmunoprecipitation. The internal calcium release [Ca2+]i assay, previously established to determine opioid receptor activation, was used to determine the selective efficacy of SNC80-induced receptor activation. In this assay of opioid efficacy, the opioid receptor activation is shifted by the chimeric Δ6-Gqi4-myr protein to a Gq response (release of intracellular calcium), which was measured fluorescently. Importantly, opioid efficacy in the [Ca2+]i assay has produced similar and consistent results when compared with opioid efficacy in the [35S]GTPγS assay providing a convenient, nonradiological measure of whole cell efficacy. SNC80 selectively activated μ–δ heteromer in HEK293 cells with an EC50 = 52.8 ± 27.8 nM (SEM) (Figure 3), with a mean peak ΔRFU effect of 746 ± 83 (SEM), n = 3 (12). Cells expressing other opioid receptors were substantially less potently activated. In this regard, SNC80 potency in cells expressing only δ-opioid receptors was at least 100-fold less than in cells that coexpressed μ–δ heteromers.[4]

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The in vitro efficacy data derived from intact HEK-293 cells in the present study support the concept that the principal targets of SNC80 are heteromeric μ–δ receptors. As illustrated in Figure 3 and Figure S1, Supporting Information, SNC80 exhibits substantially greater activity in cells coexpressing μ- and δ-opioid receptors than in cells either singly expressing δ-opioid receptors or coexpressing δ- and κ-opioid receptors. Given the evidence for physical association of these receptors, the implication is that μ–δ heteromers are selectively activated by SNC80, particularly since the singly expressed δ-receptor does not produce potent activation. These findings, taken together with the reported low binding affinity of SNC80 for μ-receptors,4 suggest that it targets the δ-protomer of the μ–δ heteromer, thereby leading to activation of the complex [4].

SNC80 (10 mg/kg; intraperitoneal injection; once; C57BL6/J mice) treatment significantly reduced the allodynia brought on by excessive sumatriptan usage[1].

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Headaches are highly disabling and are among the most common neurological disorders worldwide. Despite the high prevalence of headache, therapeutic options are limited. We recently identified the delta opioid receptor (DOR) as an emerging therapeutic target for migraine. In this study, we examined the effectiveness of a hallmark DOR agonist, SNC80, in disease models reflecting diverse headache disorders including: chronic migraine, post-traumatic headache (PTH), medication overuse headache by triptans (MOH), and opioid-induced hyperalgesia (OIH). To model chronic migraine C57BL/6J mice received chronic intermittent treatment with the known human migraine trigger, nitroglycerin. PTH was modeled by combining the closed head weight drop model with the nitroglycerin model of chronic migraine. For MOH and OIH, mice were chronically treated with sumatriptan or morphine, respectively. The development of periorbital and peripheral allodynia was observed in all four models; and SNC80 significantly inhibited allodynia in all cases. In addition, we also determined if chronic daily treatment with SNC80 would induce MOH/OIH, and we observed limited hyperalgesia relative to sumatriptan or morphine. Together, our results indicate that DOR agonists could be effective in multiple headache disorders, despite their distinct etiology, thus presenting a novel therapeutic target for headache. [1]

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SNC80 produced dose- and time-related antinociception in the mouse warm-water tail-flick test after i.c.v., i.th. and i.p. administration. The calculated A50 values (and 95% C.I.) for SNC 80 administered i.c.v., i.th. and i.p. were 104.9 (63.7-172.7) nmol, 69 (51.8-92.1) nmol and 57 (44.5-73.1) mg/kg, respectively. The i.c.v. administration of SNC 80 also produced dose- and time-related antinociception in the hot-plate test, with a calculated A50 value (and 95% C.I.) of 91.9 (60.3-140.0) nmol.[2]

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The antinociceptive activity of SNC80 was determined in mice to investigate the identity of its receptor target in vivo. In this regard, we hypothesized that SNC80 would have reduced activity in knockout animals lacking one of the receptors due to inability to form the μ–δ heteromer. Wild-type, μ-KO, and δ-KO mice were employed to determine the contribution of both the μ- and δ-opioid receptors on SNC80-induced antinociception. SNC80 was administered via the intrathecal route of injection (i.t.) to mice and assessed in the warm water (52.2 °C) tail withdrawal assay (Figure 2), focusing attention on the spinal cord where in vivo data support receptor colocalization. A cumulative dosing schedule was chosen partly to address the limited solubility of SNC-80, given that the three highest doses totaling 300 nmol greatly exceeded the amount of the compound that could be dissolved in a single 5 μL intrathecal injection volume. In addition, multiple comparisons with previous noncumulative dosing experiments revealed no significant differences in outcomes, validating the approach. The graph (Figure 2) shows two different dose–response curves to illustrate the lack of effect of SNC80 at low doses in μ-KO mice. In WT (C57/129) mice, SNC80 has an ED50 = 49 nmol, 95% CI (43–56). The dose–response curve (calculated from the second, higher dosage curve) is right-shifted in a parallel manner 2.7-fold in μ-KO mice (ED50 = 131 nmol, 95% CI (111–153), Figure 2A). In the WT (C57BL/6) mice, SNC80 displayed an ED50 = 53.6 nmol, 95% CI (47.0–61.1), which was right-shifted 6.1-fold in δ-KO mice, ED50 = 327 nmol, 95% CI (216–494) (∼50% MPE, Figure 2B). The results indicate that the antinociceptive activity of SNC80 is reduced in either μ- or δ-opioid receptor knockout mice. [4]

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Our in vivo data also suggest that SNC80-induced antinociception is produced via selective activation of μ–δ heteromer in the spinal cord. The rightward shift in the SNC80 dose–response curve in both μ-KO and δ-KO mice demonstrates that both μ- and δ-opioid receptor protomers in the heteromeric complex contribute to the antinociceptive activity of SNC80 in wild-type mice. This result is consistent with prior studies showing that SNC80-induced antinociception possesses both δ- and μ-opioid receptor-mediated components. The in vivo contribution of both receptors, as evidenced by reduced potency of SNC80 in both δ- and μ-knockout animals, is also one of three specific criteria for the establishment of the heteromeric complex established by IUPHAR guidelines. [4]

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In Gαo RGS-insensitive heterozygous knock-in mice, the potency of SNC80 to produce antihyperalgesia and antidepressant-like effects was enhanced with no change in SNC80-induced convulsions. Conversely, in Gαo heterozygous knockout mice, SNC80-induced antihyperalgesia was abolished while antidepressant-like effects and convulsions were unaltered. No changes in SNC80-induced behaviours were observed in arrestin 3 knockout mice. SNC80-induced convulsions were potentiated in arrestin 2 knockout mice. Conclusions and implications: Taken together, these findings suggest that different signalling molecules may underlie the convulsive effects of the δ-receptor relative to its antihyperalgesic and antidepressant-like effects [6].

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In the present study, δ‐receptor‐mediated convulsions were not altered in Gαo RGSi and Gαo knockout mice. In addition, we previously observed that SNC80‐induced convulsions were unaltered in RGS4 knockout mice (Dripps et al., 2017). Overall, these data may suggest that signalling mechanisms mediating δ‐receptor agonist‐induced convulsions are distinct from those mediating antihyperalgesia and antidepressant‐like effects. These behavioural measures could be regulated differentially by specific G protein subunits, G protein‐independent signalling and/or the selective expression of signalling molecules within specific brain circuits or regions.

To address this question, we explored the hypothesis that SNC80‐induced convulsions are produced by a G protein‐independent, arrestin‐mediated mechanism. As first shown by Bohn et al. (1999), we observed potentiation of morphine‐induced antinociception in arrestin 3 knockout mice. Although class A GPCRs are thought to preferentially interact with arrestin 3 (Oakley et al., 2000), no significant changes in δ‐receptor‐mediated behaviours, including convulsions, were observed in arrestin 3 knockout mice. It should be noted that these data are the result of acute administration of SNC80 and it is possible that arrestin 3 could play a role in regulating the effects of repeated doses of SNC80 or other δ‐receptor agonists. This observation with SNC80 is consistent with previous reports that found that loss of arrestin 3 in mice did not alter the analgesic profile of δ‐receptor agonists and had no effect on the enhanced coupling of δ‐receptors to voltage‐dependent calcium channels observed in the complete Freund's adjuvant (CFA) model of chronic inflammatory pain (Pradhan et al., 2013; Pradhan et al., 2016). Overall, our findings indicate that arrestin 3 is not required for δ‐receptor‐mediated antihyperalgesia, antidepressant‐like effects or convulsions.

In arrestin 2 knockout mice, we observed no changes in the effects of SNC80 in response to NTG‐induced thermal hyperalgesia. However, it was previously demonstrated that the effects of SNC80 on CFA‐induced mechanical hyperalgesia were potentiated in arrestin 2 knockout mice (Pradhan et al., 2016). It is possible that the δ‐receptor‐mediated responses to these distinct pain modalities (CFA vs. NTG; mechanical vs. thermal) are differentially regulated by arrestin 2. Further studies should investigate differences in the signalling molecules and pathways mediating different types of δ‐receptor‐mediated antihyperalgesia. The convulsive effects of SNC80 were strongly enhanced in arrestin 2 knockout mice. The potency of SNC80 to induce convulsions was enhanced in arrestin 2 knockout mice, suggesting that arrestin 2 acts as a negative regulator of δ‐receptor‐mediated convulsions. Second, arrestin 2 knockout mice convulsed multiple times in response to a single dose of SNC80.

Tolerance to δ‐receptor‐mediated convulsions is typically acute and long lasting (Comer et al., 1993; Hong et al., 1998). In addition, the changes in the electroencephalographic waveform produced by SNC80 return to normal baseline activity following the end of catalepsy (Jutkiewicz et al., 2006). To our knowledge, this is the first report of multiple convulsive events in response to a δ‐receptor agonist in rodents. One possible explanation for this observation is that loss of arrestin 2 produces these behavioural changes by preventing δ‐receptor desensitization and/or up‐regulating δ‐receptor trafficking to the cell membrane resulting in enhanced δ‐receptor signalling (Mittal et al., 2013). However, in the current study, δ‐receptor‐mediated antidepressant‐like effects and thermal antihyperalgesia were not significantly altered in arrestin 2 knockout mice. Therefore, it is possible that the behavioural effects of SNC80 are differentially regulated by arrestin 2 due to differences in regional expression, behavioural mechanisms and/or signalling down‐regulation and/or tolerance to the convulsive effects of SNC80. Thus, loss of arrestin 2 could allow signalling pathways that would normally be terminated to persist and produce multiple convulsive events. Future work will examine whether arrestin 2 also regulates tolerance to other behavioural effects of δ‐receptor agonists[6].

δ‐receptor saturation binding [6]
Mice were decapitated following cervical dislocation, the forebrain was removed immediately, and membranes were freshly prepared as previously described (Broom et al., 2002a). Tissue collection without anaesthesia was used to limit modification to δ‐receptor number, conformation and/or localization and is conditionally acceptable with justification under the American Veterinary Medical Association Guidelines for the Euthanasia of Animals. Protein concentrations were determined with a BCA assay kit. Specific binding of the δ‐receptor agonist [3H]DPDPE was determined as described using 10 μM of the opioid antagonist naloxone to define non‐specific binding as described by Broom et al., (2002a). Reactions were incubated for 60 min at 26°C and stopped by rapid filtration through GF/C filter mats soaked in 0.1% PEI using an MLR‐24 harvester. Bound [3H]DPDPE was determined by scintillation counting, and B max and K d values were calculated using nonlinear regression analysis with GraphPad Prism version 6.02. To ensure the reliability of single values, membranes from each mouse (n = 5 per group) were assayed in triplicate.

Intracellular Calcium Release Assay [4]
The intracellular calcium release assay as described previously was used to determine the selectivity of SNC80 in activating opioid receptors with minor modifications. Briefly, HEK293 cells stably expressing opioid receptors were grown in DMEM (10% FBS, 1% P/S) in 10% CO2 atmosphere, transiently transfected with a chimeric G-protein Δ6-Gqi4-myr using lipofectamine 2000, plated into 96 well half area plates 24 h later, and assayed for intracellular calcium release 48 h after transfection. In the experiments involving HEK293 cells stably expressing the chimeric Δ6-Gqi4-myr protein, the procedure was identical except opioid receptor DNA was transiently transfected, and the amount of lipofectamine was doubled in the cotransfected cells. Assays employed the standard explorer FLIPR calcium dye kit in a Flexstation 3 apparatus. SNC80 freebase was initially dissolved in DMSO. DMSO was employed such that the final DMSO concentration did not exceed 0.1% v/v at maximum, a concentration that did not significantly alter calcium flux from basal responses. To minimize experimental variability, all experiments were conducted at least three times with four internal repetitions, n ≥ 3 (12), except cells expressing κ- or μ–κ receptors where n ≥ 2 (8). Assay controls (blanks and standard ligands) were performed on every plate to eliminate technical variability and ensure uniform response.

Male and female C57BL6/J mice (20-30g) injected with Sumatriptan
10 mg/kg
Intraperitoneal injection; once

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Acute treatment with DOR agonist [1]
Eighteen to twenty-four hours after the last drug administration day we determined the effect of SNC80. On this challenge test day, basal hind paw and cephalic mechanical thresholds were determined, after which mice received either vehicle (VEH) or SNC80 (10 mg/kg, ip). SNC80 was diluted to 1 mg/mL in 0.33% 1N HCl/0.9% saline. Post-SNC80 thresholds were assessed 2 hours after basal testing, and 45 minutes after SNC80 injection.

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Chronic treatment with DOR agonist [1]
To determine whether chronic DOR activation caused hypersensitivity similar to MOH, Mice were treated once daily with vehicle, SUMA (0.6 mg/kg, ip), or SNC80 (10 mg/kg, ip) over 11 days. For hind paw experiments, mice were tested on days 1, 3, 5, 7, 9, and 11, and another cohort of mice were tested on days 1 and 11. For cephalic experiments, mice were tested on days 1 and 11, and days 1, 5 and 11 depending on the experiment.

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Drug stock solutions were dissolved in sterile water and diluted in sterile saline solution with the exception of SNC80, which was initially mixed with 1 equiv of tartaric acid before dissolution in sterile water. Antinociceptive effects of SNC80 were assessed utilizing the warm water (52.5 °C) tail immersion assay. Animals employed were 129sv/C57BL6 mice (μ-opioid receptor WT), μ-KO (−/−) mice on a 129sv/C57BL6 background, C57BL/6 mice (δ-opioid receptor WT), and δ-KO (−/−) mice on a C57BL/6 background, all with ad libitum food access and a 12 hour light/dark cycle. Intrathecal (i.t.) drug administration was accomplished by direct lumbar puncture as modified. A minimum of six mice were assayed at each dose tested, each mouse was used twice, and a total of 48 mice were used in the study. No antinociceptive differences were observed between male and female mice in any of the experiments. Tail flick (TF) latencies were obtained before drug administration to establish a baseline prior to drug treatment; shortly after baseline testing, the lowest dose of drug was injected intrathecally in 5 μL of vehicle, and TF latency was determined 7 min later. Immediately after testing, the subsequent dose of a cumulative dose–response curve was administered, and TF latency was determined 5 min later; three to four doses were thereby administered in rapid succession, and a cumulative dose–response curve was determined. Antagonists were administered 7 min prior to the initial agonist injection. A 12 s cutoff was employed in cases of no detectable response to avoid tissue damage. [4]

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Forced swim test [6]
The forced swim test (FST) is an assay that is widely used to evaluate the antidepressant‐like effects of drugs in rodents (Barkus, 2013). Our experiments were adapted from Porsolt et al. (1977) and performed as previously described (Dripps et al., 2017). Briefly, 60 min after SNC80 (0.1, 0.32, 1, 3.2, 10 or 32 mg·kg−1) or vehicle injection, each mouse was placed in a 4 L beaker filled with 15 cm of 25 ± 1°C water, and its behaviour was recorded for 6 min using a Sony HDR‐CX220 digital camcorder. Videos were analysed by individuals blind to the experimental conditions, and the amount of time the animals spent immobile was quantified. Immobility was defined as the mouse not actively traveling through the water and making only movements necessary to stay afloat. The time the mouse spends immobile after the first 30 s of the assay was recorded.

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Nitroglycerin‐induced hyperalgesia [6]
The NTG‐induced hyperalgesia assay was adapted from Bates et al. (2010) using modifications described in Pradhan et al. (2014) and performed as previously described (Dripps et al., 2017). In brief, male and female mice were used to evaluate NTG‐induced hyperalgesia. Hyperalgesia was assessed by immersing the tail (~5 cm from the tip) in a 46°C water bath and determining the latency for the animal to withdraw its tail with a cut‐off time of 60 s. After determining baseline withdrawal latencies, 10 mg·kg−1 NTG (i.p.) was administered to each animal. Tail withdrawal latency was assessed again 1 h after NTG administration. At 90 min post‐NTG, animals received an injection of SNC80 (0.32, 1, 3.2, 10 or 32 mg·kg−1) or vehicle, and mice were observed continuously in individual cages for 30 min to observe for convulsions (see section below). Tail withdrawal latencies were assessed again 30 min after SNC80 administration.

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SNC80‐induced convulsions [6]
Mice were observed continuously in individual cages for convulsions. Unless otherwise noted, NTG treatment had no significant effect on the frequency or nature of SNC80‐induced convulsions (see Supporting Information). Convulsions were typically composed of a single tonic phase characterized by sudden tensing of the musculature and extension of the forepaws followed by clonic contractions that extended the length of the body. Mice would frequently lose balance and fall on their side, although the so‐called barrel rolling was rarely observed. Convulsions were followed by a period of catalepsy that lasted 2–5 min after which the animals were hyperlocomotive but otherwise indistinguishable from untreated controls. The severity of each convulsion was quantified using the following modified Racine (1972) scale adapted from Jutkiewicz et al. (2006): 1 – teeth chattering or face twitching; 2 – head bobbing or twitching; 3 – tonic extension or clonic convulsion lasting less than 3 s; 4 – tonic extension or clonic convulsion lasting longer than 3 s; and 5 – tonic extension or clonic convulsion lasting more than 3 s with loss of balance. Post‐convulsion catalepsy‐like behaviour was assessed by placing a horizontal rod under the forepaws of the mouse, and a positive catalepsy score was assigned if the mouse did not remove its forepaws after 30 s. Two arrestin 2 knockout mice that received 32 mg·kg−1 SNC80 exhibited sustained convulsions after the observation period and were killed by pentobarbital overdose.
SNC80 was dissolved in 1 M HCl and diluted in sterile water to a concentration of 3% HCl.

[1]. Delta opioid receptor agonists are effective for multiple types of headache disorders. Neuropharmacology. 2019 Apr;148:77-86.

[2]. SNC 80, a selective, nonpeptidic and systemically active opioid delta agonist. J Pharmacol Exp Ther. 1995 Apr;273(1):359-66.

[3]. Probes for narcotic receptor mediated phenomena. 19. Synthesis of (+)-4-[(alpha R)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3- methoxybenzyl]-N,N-diethylbenzamide (SNC 80): a highly selective, nonpeptide delta opioid receptor. J Med Chem. 1994 Jul 8;37(14):2125-8.

[4]. The δ opioid receptor agonist SNC80 selectively activates heteromeric μ-δ opioid receptors. ACS Chem Neurosci. 2012 Jul 18;3(7):505-9.

[5]. The delta opioid receptor tool box. Neuroscience. 2016 Dec 3;338:145-159.

[6]. Role of signalling molecules in behaviours mediated by the δ opioid receptor agonist SNC80. Br J Pharmacol. 2018 Mar;175(6):891-901.

4-[(R)-[(2S,5R)-2,5-dimethyl-4-prop-2-enyl-1-piperazinyl]-(3-methoxyphenyl)methyl]-N,N-diethylbenzamide is a diarylmethane.

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Coexpressed and colocalized μ- and δ-opioid receptors have been established to exist as heteromers in cultured cells and in vivo. However the biological significance of opioid receptor heteromer activation is less clear. To explore this significance, the efficacy of selective activation of opioid receptors by SNC80 was assessed in vitro in cells singly and coexpressing opioid receptors using a chimeric G-protein-mediated calcium fluorescence assay, SNC80 produced a substantially more robust response in cells expressing μ-δ heteromers than in all other cell lines. Intrathecal SNC80 administration in μ- and δ-opioid receptor knockout mice produced diminished antinociceptive activity compared with wild type. The combined in vivo and in vitro results suggest that SNC80 selectively activates μ-δ heteromers to produce maximal antinociception. These data contrast with the current view that SNC80 selectively activates δ-opioid receptor homomers to produce antinociception. Thus, the data suggest that heteromeric μ-δ receptors should be considered as a target when SNC80 is employed as a pharmacological tool in vivo.[4]

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In recent years, the delta opioid receptor has attracted increasing interest as a target for the treatment of chronic pain and emotional disorders. Due to their therapeutic potential, numerous tools have been developed to study the delta opioid receptor from both a molecular and a functional perspective. This review summarizes the most commonly available tools, with an emphasis on their use and limitations. Here, we describe (1) the cell-based assays used to study the delta opioid receptor. (2) The features of several delta opioid receptor ligands, including peptide and non-peptide drugs. (3) The existing approaches to detect delta opioid receptors in fixed tissue, and debates that surround these techniques. (4) Behavioral assays used to study the in vivo effects of delta opioid receptor agonists; including locomotor stimulation and convulsions that are induced by some ligands, but not others. (5) The characterization of genetically modified mice used specifically to study the delta opioid receptor. Overall, this review aims to provide a guideline for the use of these tools with the final goal of increasing our understanding of delta opioid receptor physiology. [5]

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Background and purpose: GPCRs exist in multiple conformations that can engage distinct signalling mechanisms which in turn may lead to diverse behavioural outputs. In rodent models, activation of the δ opioid receptor (δ-receptor) has been shown to elicit antihyperalgesia, antidepressant-like effects and convulsions. We recently showed that these δ-receptor-mediated behaviours are differentially regulated by the GTPase-activating protein regulator of G protein signalling 4 (RGS4), which facilitates termination of G protein signalling. To further evaluate the signalling mechanisms underlying δ-receptor-mediated antihyperalgesia, antidepressant-like effects and convulsions, we observed how changes in Gαo or arrestin proteins in vivo affected behaviours elicited by the δ-receptor agonist SNC80 in mice. Experimental approach: Transgenic mice with altered expression of various signalling molecules were used in the current studies. Antihyperalgesia was measured in a nitroglycerin-induced thermal hyperalgesia assay. Antidepressant-like effects were evaluated in the forced swim test. Mice were also observed for convulsive activity following SNC80 treatment. [6]

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One of our aims was to determine if chronic treatment with a DOR agonist would result in OIH/MOH. Interestingly, we found that daily treatment and testing with SNC80 resulted in subsequent hyperalgesia, but that daily treatment alone did not result in increased pain sensitivity, unlike sumatriptan. These results suggest that pharmacological activation of DOR does not produce OIH/MOH, however, if chronic SNC80 is paired with repeated testing then DOR activation might facilitate associative learning resulting in behavioral sensitization. The DOR is expressed in a number of brain regions that can regulate different kinds of learning; including the hippocampus, amygdala, and striatum (Le Merrer et al., 2009; Pellissier et al., 2016; Pradhan et al., 2011). Knockout of DOR results in impairment in object recognition tasks (Le Merrer et al., 2013), as well as deficiencies in place conditioning tasks (Le Merrer et al., 2011). In addition, DORs in the nucleus accumbens shell have been shown to modulate predictive learning (Bertran-Gonzalez et al., 2013; Laurent et al., 2014; Laurent et al., 2015a; Laurent et al., 2015b). We have also previously demonstrated that tolerance to SNC80 is significantly dependent on associative learning (Pradhan et al., 2010; Vicente-Sanchez et al., 2018), and environmental cues related to memory and learning can modulate behavioral outcomes to repeated exposure to opioids (Gamble et al., 1989; Mitchell et al., 2000). Our results should be considered during the development of DOR agonists for headache, as chronic DOR activation could facilitate associated learned behavior in migraineurs. [1]

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Additionally, our in vitro efficacy data demonstrating selective activation of μ–δ heteromers by SNC80 is further supported by trafficking and binding studies. That these trafficking studies revealed SNC80-induced cointernalization of μ- and δ-opioid receptors is consistent with activation of a μ–δ heteromer as the signaling unit. The present study demonstrates that potent SNC80 activity requires the presence of μ- and δ-opioid receptors both in vitro and in vivo. Considered together, and in light of prior studies, the combined results presented here suggest that SNC80, in vivo, interacts selectively with the δ-protomer of the μ–δ heteromer, thereby leading to the activation of the heteromeric complex. While this conclusion does not invalidate previous research demonstrating that SNC80 is a δ-selective ligand in receptor homomer models, it strongly suggests the involvement of the μ–δ heteromer as a selective target in the signaling pathway. The implications of μ–δ receptor heteromers as a target for SNC80 suggest a more complex role for this ligand in vivo, given that μ-selective ligands (based on binding) also target μ–δ heteromers but via the μ-protomer. It therefore appears that the output of the agonist depends on which protomer in the μ–δ heteromer is targeted for activation. The fact that, unlike μ-agonists, SNC80 does not produce physical dependence illustrates the complexity of the heteromeric system and suggests that different signaling pathways are dependent on the protomer that is activated in the μ–δ heteromeric complex.[4]

C28H39N3O2

449.64

449.304

C, 74.80; H, 8.74; N, 9.35; O, 7.12

156727-74-1

123924

Off-white to light yellow solid powder

1.0±0.1 g/cm3

564.8±50.0 °C at 760 mmHg

122-123ºC

295.4±30.1 °C

0.0±1.5 mmHg at 25°C

1.545

3.37

0

4

9

33

614

3

C=CCN1C[C@H](C)N(C[C@H]1C)[C@H](C2=CC=C(C=C2)C(=O)N(CC)CC)C3=CC(=CC=C3)OC

KQWVAUSXZDRQPZ-UMTXDNHDSA-N

InChI=1S/C28H39N3O2/c1-7-17-30-19-22(5)31(20-21(30)4)27(25-11-10-12-26(18-25)33-6)23-13-15-24(16-14-23)28(32)29(8-2)9-3/h7,10-16,18,21-22,27H,1,8-9,17,19-20H2,2-6H3/t21-,22+,27-/m1/s1

4-[(R)-[(2S,5R)-2,5-dimethyl-4-prop-2-enylpiperazin-1-yl]-(3-methoxyphenyl)methyl]-N,N-diethylbenzamide

SNC80; SNC-80; 156727-74-1; 4-(alpha-(4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl)-N,N-diethylbenzamide; 4-[(R)-[(2S,5R)-2,5-dimethyl-4-prop-2-enylpiperazin-1-yl]-(3-methoxyphenyl)methyl]-N,N-diethylbenzamide; Benzamide, 4-[(R)-[(2S,5R)-2,5-dimethyl-4-(2-propen-1-yl)-1-piperazinyl](3-methoxyphenyl)methyl]-N,N-diethyl-; CHEMBL13470; SNC80

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~33.33 mg/mL (~74.1 mM)

Solubility in Formulation 1: ≥ 3.33 mg/mL (7.41 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 33.3 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 3.33 mg/mL (7.41 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 33.3 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 3.33 mg/mL (7.41 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 33.3 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)