mGluR2 ( EC50 = 285 nM )

AZD-8529 increases the effects of glutamate at mGluR2 with an EC50 of 195 nM [1].
AZD-8529 does not cause antagonist reactions on mGluRs at 25 μM[1].

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AZD8529 potentiation of mGluR2 receptor function[1]
We assessed the effect of AZD8529 at the human mGluR2 receptor by measuring the potentiation of [35S]GTPγS binding in the presence of increasing concentrations of exogenously applied agonist (L-glutamate). AZD8529 potentiated the effects of glutamate at mGluR2 with an EC50 of 195±62 nM and an Emax of 110%±11% (Figure 1A). In order to assess the selectivity of AZD8529 against the family of mGluRs, we used fluorescence-based assays. AZD8529 potentiated mGluR2 activity with an EC50 of 285±20 nM and did not produce any positive allosteric modulator responses at 20-25 M on the mGluR1, 3, 4, 5, 6, 7, and 8 subtypes (Table 1). In addition, at 25 μM AZD8529 did not elicit antagonist responses on mGluRs. When AZD8529 (10 μM) was studied in a broad receptor screen (Table 2), we observed >50% inhibition of ligand biding at adenosine A3 receptors (51% inhibition) and the norepinephrine transporter (NET, IC50=4.73 μM).

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In vitro electrophysiology tests [2]
The mGlu2/3 receptor agonist DCG-IV induced inhibitory effects on fEPSPs in the pyramidal cells in the CA1 region of the hippocampus. The inhibitory effects of DCG-IV on synaptic transmission were potentiated by AZD8418 and AZD8529, with EC50 values of 0.86 and 1.4 μM, respectively (Fig. 2a). AZD8418 produced a greater maximal potentiation (79.9 ± 4.4) than AZD8529 (61.4 ± 4.3, p < 0.05). The mGlu2/3 receptor antagonist LY341495 (1 μM) effectively blocked the inhibition of fEPSPs that was induced by DCG-IV alone (10 nM) or the combination of DCG-IV with either AZD8529 (10 μM) or AZD8418 (10 μM; Fig. 2b).

AZD-8529 (0.3-mg/kg, i.m.) AZD-8529 mesylate (0.3 mg/kg, i.m.) lessens cue- and priming-induced reinstatement in squirrel monkeys[1].
AZD-8529 (30 mg/kg; i.p.) reduces the increased extracellular dopamine induced by nicotine in accumbens shell of freely-moving rats[1].

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In monkeys, AZD8529 decreased nicotine self-administration at doses (.3-3 mg/kg) that did not affect food self-administration. AZD8529 also reduced nicotine priming- and cue-induced reinstatement of nicotine seeking after extinction of the drug-reinforced responding. In rats, AZD8529 decreased nicotine-induced accumbens dopamine release. Conclusions: These results provide evidence for efficacy of positive allosteric modulators of mGluR2 in nonhuman primate models of nicotine reinforcement and relapse. This drug class should be considered for nicotine addiction treatment.

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Acute treatment with AZD8418 (0.37, 1.12, 3.73, 7.46, and 14.92 mg/kg) and AZD8529 (1.75, 5.83, 17.5, and 58.3 mg/kg) deceased nicotine self-administration and had no effect on food-maintained responding. Chronic treatment with AZD8418 attenuated nicotine self-administration, but tolerance to this effect developed quickly. The inhibition of nicotine self-administration by chronic AZD8529 administration persisted throughout the 14 days of treatment. Chronic treatment with either PAMs inhibited food self-administration. AZD8418 (acute) and AZD8529 (acute and subchronic) blocked cue-induced reinstatement of nicotine- and food-seeking behavior. Conclusions: These findings indicate an important role for mGlu2 receptors in the reinforcing properties of self-administered nicotine and the motivational impact of cues that were previously associated with nicotine administration (i.e., cue-induced reinstatement of nicotine-seeking behavior). Thus, mGlu2 PAMs may be useful medications to assist people to quit tobacco smoking and prevent relapse [2].

Functional mGluR2 assays [1]
Receptor selectivity assay [1]
To determine the selectivity of AZD8529 within the mGluR family, we used fluorescence-based assays and HEK 293 cell-lines expressing human mGluR constructs. The cell lines expressed chimeric fusion constructs hmGluR2/hCaR*, hmGluR1/hCaR*, hmGluR3/hCaR*, hmGluR4/hCaR*, hmGluR5/hCaR*, hmGluR6/hCaR*, hmGluR7/hCaR*, hmGluR8/hCaR*, each comprising the extracellular domain and transmembrane domain of human mGluR, and the intracellular domain of the human calcium receptor fused to the promiscuous chimeric protein Gqi5 as described previously.

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Receptor screening [1]
We evaluated AZD8529 at 10 μM for off-target effects using radioligand binding assays (MDS Pharma) based on published methods. We ran reference standards for each assay. We determined IC50 values using non-linear, least squares regression analysis of the Data Analysis Toolbox (MDL Information Systems).

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[35S]GTPγS binding human mGlu2-CHO membranes [1]
We used membranes prepared from a CHO cell line expressing the human mGluR2 and performed the assay in a scintillation proximity assay (SPA) format. We grew Chinese hamster ovary (CHO) cells expressing the human mGluR2 to approximately 80% confluence, washed the cells in ice-cold phosphate-buffered saline, and stored them frozen until membrane preparation. Assay buffer contained 0.05 M HEPES, 0.10 M NaCl, 0.01 M MgCl2, pH 7.4 plus 100 M dithiothreitol and 3 M guanosine diphosphate. We started the assay by adding a mixture of wheat germ agglutinin SPA beads (0.75 mg/ml) and membranes (6 g/ml) in assay buffer containing AZD8529 or vehicle. After 15-min incubation, we added a solution containing the [35S]GTPγS and L- glutamate (final concentrations 100 pM [35S]GTPγS and 0-100 M glutamate). Following incubation at room temperature (60 min), we centrifuged the assay plates and read them on the TopCount™ scintillation counter. We determined [35S]GTPγS binding in the absence of glutamate and in the presence of 100- M glutamate as 0% and 100% levels, respectively. We estimated the modulator activity of AZD8529 on mGluR2 activation from the concentration response curves of AZD8529 fitted with a 4-parameter logistic equation to calculate the apparent potency (EC50) and maximal efficacy (Emax).

[35S]GTPγS autoradiography in cynomolgus monkey brain slices [1]
We anaesthetized the monkey with sodium pentobarbital (100 mg/kg), perfused it with saline, and then removed the brain and froze it in cooled isopentane. We cut 20-μm striatum and hippocampus sections on a cryostat, mounted the sections on glass slides and stored them at 80°C until use. We warmed the sections to room temperature in a vacuum chamber over 3 hr on the day of the experiment. We incubated the sections in 50 mM Tris HCl, 3 mM MgCl2, 0.2 mM EGTA, 100 mM NaCl, and 0.2 mM DTT (Tris Assay Buffer, TAB); pH 7.4 at 25°C for 10 min. We then incubated the slides in TAB containing 2 mM guanosine diphosphate (GDP) for 15 min at 25°C. We placed the slides in one of the following four conditions for 2 hr at 25°C: Basal: TAB + 2 mM GDP + 0.04 nM [35S]GTPγ S; Agonist alone: TAB + 2 mM GDP + 0.04 nM [35S]GTPγS + 1 μM LY379268; Modulator alone: TAB + 2 mM GDP + 0.04 nM [35S]GTPγS + 3 μM AZD8529; Modulator + Agonist: TAB + 2 mM GDP + 0.04 nM [35S]GTPγS + 1 μM LY379268 + 3 μM AZD8529; Modulator + Agonist + Antagonist: TAB+2 mM GDP+0.04 nM [35S]GTPγS+1 μM LY379268+ 3 μM AZD8529+1 μM LY341495. We then washed the sections 2 times in 4°C 50 mM Tris HCl, pH 7.4, 5 min each, rinsed them in ice cold H2O and air dried the slides. We then exposed the slides to Biomax MR film for 2 days and developed using standard techniques, digitized, and analyzed.

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Slice recording [2]
Slices were submerged in a slice chamber and bathed in 32 °C aCSF at a flow rate of 1–2 ml/min. The slices were held in place with a weight made of platinum wire. Schaeffer collateral fibers in the stratum radiatum were stimulated with a monopolar tungsten electrode (model 575300, 0.5–1 mΩ) connected to an isolated pulse stimulator (model 2100). Recordings of the extracellular population spike from pyramidal cells in layers of the CA1 were made with electrodes that were pulled from borosilicate glass (model TW150-4) and filled with 2 mM NaCl. The slices were stimulated with single 10 ms pulses that were delivered every 30 s. A baseline response was established (50–70 % of maximum), and then an approximately 15-min control period was recorded. Following the control period, the compounds were bath applied for 40 min or until a steady-state response was reached.

Sprague-Dawley rats
10 mg/kg, 30 mg/kg
Intraperitoneal injection; 2 hours before nicotine injections

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Nicotine self-administration [1]
We performed this phase over a period of 14 weeks and it included 1-hr sessions from Monday through Friday. Before the start of each session, we placed the monkeys into the Plexiglas chairs and restrained them in the seated position by waist locks. We first trained the monkeys to lever-press under a fixed-ratio schedule (FR10, timeout 60 s) of intravenous nicotine (30 μg/kg/injection) reinforcement. After flushing the catheters with 1 ml physiological saline, we connected them to a motor-driven syringe. At the start of each session, the white house-light was turned off and the green stimulus light was turned on; 10 lever-presses turned off the green light and produced 2-s amber light paired with nicotine injection (0.2 ml). During the 60-s timeout period the chamber was dark and lever-presses had no programmed consequences. When responses showed <15% variability for at least 5 consecutive sessions, we tested the effect of AZD8529 pretreatment (0.03, 0.3, 1, 3, and 10 mg/kg, i.m., 3 hr before the session) on nicotine self-administration for 3 sessions; we compared these data to three consecutive session of vehicle pretreatment immediately preceding each test session. The 3-hr pretreatment time is based on AstraZeneca Tmax pharmacokinetic studies (data not shown).

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Reinstatement of nicotine seeking [1]
We performed this phase of the study over a period of 9 weeks. We first tested the monkeys for nicotine priming-induced reinstatement after extinction of the drug-reinforced responding. We then retrained them to self-administer nicotine over 5 days and then tested them for cue-induced reinstatement after extinction of the drug-reinforced responding. We tested AZD8529 doses of 3 mg/kg or lower, because 3 mg/kg was the highest effective dose that reduced nicotine but not food self-administration.

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Nicotine priming-induced reinstatement [1]
We performed tests for nicotine priming-induced reinstatement after the monkeys underwent daily extinction sessions during which lever-presses led to saline infusions plus the visual cues previously paired with nicotine infusions, but not nicotine. We gave a non-contingent saline injection before each extinction session as a vehicle control for the nicotine-priming injections. After at least two extinction sessions, when responding had reached a low, stable level, we determined the effect of pretreatment with AZD8529 (0.3, 1 or 3 mg/kg, i.m.) or its vehicle on nicotine (0.1 mg/kg i.v.)-induced reinstatement. We gave the nicotine priming injections immediately before the start of the test sessions. During testing, lever-presses (FR10) continued to produce only saline injections and the discrete cues. We also tested the effect of 3 mg/kg of AZD8529 on saline priming to determine whether AZD8529 alone would affect nicotine seeking after extinction.

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Cue-induced reinstatement [1]
After the completion of nicotine priming tests, we retrained the monkeys to self-administer nicotine for ~10 sessions. We then gave them 3 extinction sessions during which lever-presses had no reinforced consequences (neither nicotine nor cue were available); additionally, we did not inject monkeys with saline priming before these sessions. After extinction, we determined the effect of pretreatment with AZD8529 (0.3, 1 or 3 mg/kg, i.m.) or its vehicle on cue-induced reinstatement. During testing, lever-presses (FR10) produced the i.v. saline infusions and visual cue presentations. We also determined the effect of 3 mg/kg of AZD8529 on extinction responding in the absence of the cue. Each cue-induced reinstatement test was followed by one or two extinction sessions.

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Food self-administration [1]
We determined the effect of AZD8529 in a separate group of monkeys that self-administered 190-mg food pellets under reinforcement schedule conditions identical to those we used with nicotine (FR 10, TO 60 s). We restricted food intake to maintain monkeys’ weights at a level that facilitates food-reinforced responding (no more than 10% below free-feeding weight). The number of reinforcers delivered per session, as well as rates of responding, in this group were very similar to the nicotine group (Figure 2). We injected each dose of AZD8529 (3, 10 and 30 mg/kg, i.m.) for three consecutive sessions, which was preceded by three sessions with vehicle injections before the sessions.

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AZD8529 plasma levels in squirrel monkeys [1]
To determine whether plasma levels during the behavioral experiments reach levels that are well tolerated in humans (per AstraZeneca company information), we injected 3 squirrel monkeys with AZD8529 (1 mg/kg, i.m.) and 3 hr later we collected venous blood samples (approximately 1.5 ml) from the femoral vein under light ketamine (10 mg/kg, i.m.) anesthesia. We rapidly mixed the blood samples and immediately cooled them on ice until centrifugation. Plasma was prepared by centrifugation at 4°C for 10 min at 1500 x g within 30 min of blood sampling. We separated the plasma and transferred it to two 2-ml micro-centrifuge tubes. We stored the plasma samples at −80°C. We shipped the samples on dry ice to AstraZeneca where AZD8529 levels were measured using a standardized LC/MS/MS method.

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In vivo microdialysis in rats [1]
The general procedure was described previously (36). We performed microdialysis in Sprague-Dawley rats 20-24 hr after implantation of probes aimed at the accumbens shell (2.0 mm anterior,1.1 mm lateral from bregma, and 8.0 mm below the dura) (37). We collected samples (20 μl) every 20 min (perfusion rate: 1ul per min) and immediately analyzed dopamine levels by HPLC coupled to electrochemical detection. We injected the test drugs or their vehicle after observing stable dopamine levels (<15% variation) in 3 consecutive samples. We injected vehicle or AZD8529 (10 or 30 mg/kg i.p.) 2 hr before vehicle or nicotine (0.4 mg/kg s.c.) injections. We collected dialysate samples for 2 hr after nicotine injections. We based the AZD8529 doses on previous unpublished work of AstraZeneca in rat behavioral models and a recent study on the effect of the drug on ‘incubation’ of methamphetamine craving in rats. [1]
AZD8529 free-base was dissolved in sterile water.[1]

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Experimental design [2]
Experiment 1: effects of acute AZD8418 and AZD8529 treatment on nicotine and food self-administration [2]
After establishing stable nicotine or food self-administration (<20 % variability in responding over three consecutive days), the effects of acute AZD8418 (0, 0.37, 1.12, 3.73, 7.46, and 14.92 mg/kg) and AZD8529 (0, 1.75, 5.83, 17.5, and 58.3 mg/kg) treatment on nicotine and food self-administration were assessed using a within-subjects Latin square design. Four groups of naive rats were used to examine the effects of acute treatment with (i) AZD8418 on nicotine self-administration (n = 12) and food self-administration (n = 10) and (ii) AZD8529 on nicotine self-administration (n = 12) and food self-administration (n = 7). At least 5 days elapsed between drug administrations.

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Experiment 2: effects of chronic AZD8418 and AZD8529 treatment on nicotine and food self-administration [2]
The effects of 14-day repeated AZD8418 and AZD8529 treatment regimens on nicotine and food self-administration were assessed using a between-subjects design. Four groups of naive rats were used to examine the effects of chronic treatment with (i) AZD8418 (0, 3.73, 7.46, and 14.92 mg/kg/day) on nicotine self-administration (n = 10–11/subgroup), (ii) AZD8418 (0 and 14.92 mg/kg/day) on food-self-administration (n = 10–11/subgroup), (iii) AZD8529 (0 and 58.3 mg/kg/day) on nicotine self-administration (n = 10–12/subgroup), and (iv) AZD8529 (0 and 58.3 mg/kg/day) on food self-administration (n = 8–13/subgroup). The subgroups for each tested compound were balanced for weight and nicotine/food intake before initiating the chronic treatments.

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Experiment 3: effects of acute AZD8418 and AZD8529 treatment on cue-induced reinstatement of nicotine- and food-seeking behavior [2]
After completing experiment 1, nicotine self-administering rats were tested under extinction conditions. All of the rats reached the predetermined criterion of extinction (see above) by the end of the 10th extinction session. The first reinstatement session was conducted after vehicle administration to ensure that each subject exhibited robust reinstatement as defined above (>50 % increase in responding compared to the mean of the last three extinction sessions) before initiating the drug treatments. Only rats that exhibited robust nicotine-seeking behavior during this first reinstatement session were included in the remainder of the experiments. Each reinstatement session was preceded by three daily extinction sessions to re-extinguish responding. AZD8418 (0, 1.12, 3.73, 7.46, and 14.92 mg/kg) and AZD8529 (0, 1.75, 5.83, 17.5, and 58.3 mg/kg) were administered prior to each reinstatement session using a within-subjects Latin square design. Independent naive rats (n = 9–13/group) that were trained to self-administer food were used to assess the effects of AZD8418 (0, 3.73, 7.46, and 14.92 mg/kg) and AZD8529 (0, 1.75, 5.83, 17.5, and 58.3 mg/kg) on cue-induced reinstatement of food-seeking behavior using a between-subjects design because food-seeking behavior exhibits rapid extinction with repeated reinstatement testing (Bespalov et al. 2005). The groups were balanced for weight and food responding before treatment.

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Pharmacokinetic studies [2]
AZD8529 (4.7 mg/kg) and AZD8418 (5 mg/kg) were administered orally by gavage to groups of male Wistar rats (n = 3–4), either as a single dose or as daily doses for 7 days. Tail vein blood samples (0.25 ml) were collected from all rats at 0.5, 1, 3, 6, 12, and 24 h after drug administration on day 1 or 7 of dosing. The plasma was prepared by centrifugation at 4 °C for 10 min at 1500×g within 30 min of blood sampling and analyzed for concentrations of AZD8529 or AZD8418 by a standard reverse-phase liquid chromatography and electrospray ionization tandem mass spectrometry (LC/MS/MS) method.

PK profiles of AZD8529 [2] Peak plasma concentrations (T max) of AZD8418 were reached at 1 h post-administration after a single dose of 5 mg/kg. Peak plasma concentrations of AZD8529 were reached at 3 h post-administration after a single dose of 4.7 mg/kg. The peak plasma concentration (C max) for AZD8418 (690 ± 108 nM) was considerably higher than that for AZD8529 (158 ± 30 nM). Similarly, the area under the curve (AUC) for AZD8418 was also higher than that for AZD8529, indicating higher bioavailability of AZD8418 than that of AZD8529. Repeated daily administration of AZD8418 or AZD8529 for 7 days did not alter the T max or C max of plasma exposure. Based on findings of the PK studies, doses and pretreatment time were determined for AZD8418 (0, 0.37, 1.12, 3.73, 7.46, and 14.92 mg/kg; 1 h pretreatment) and AZD8529 (0, 1.75, 5.83, 17.5, and 58.3 mg/kg; 3 h pretreatment) administration to reflect differences in C max and T max.

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Plasma concentrations of AZD8529 [2]
In a group of squirrel monkeys (n=3), the plasma concentration of AZD8529 3 hr (the pretreatment time in the self-administration and reinstatement experiments) after drug (1 mg/kg) injections was 112±17 nM. Effect of AZD8529 on nicotine-induced dopamine release in the rat accumbens shell We determined the effect of systemic AZD8529 injections on nicotine-induced elevations of extracellular dopamine levels in accumbens shell of freely-moving rats. Nicotine (0.4 mg/kg, s.c.) increased extracellular dopamine and this effect was decreased by 30 mg/kg but not 10 mg/kg AZD8529 (Supplementary Figure S1; AZD8529 Dose x Time interaction: F(34,170)=2.24; p<0.001). When given alone, AZD8529 (10 or 30 mg/kg) had no effect on dopamine levels (Supplementary Figure S1).

[1]. The Novel Metabotropic Glutamate Receptor 2 Positive Allosteric Modulator, AZD8529, Decreases Nicotine Self-Administration and Relapse in Squirrel Monkeys. Biol Psychiatry. 2015 Oct 1;78(7):452-62.

[2]. Attenuation of nicotine-taking and nicotine-seeking behavior by the mGlu2 receptor positive allosteric modulators AZD8418 and AZD8529 in rats. Psychopharmacology (Berl). 2016 May;233(10):1801-14.

AZD8529 is under investigation in clinical trial NCT00921804 (Study to Assess the Efficacy, Safety, and Tolerability of AZD8529 in Adult Schizophrenia Patients).

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Background: Based on rodent studies, group II metabotropic glutamate receptors (mGluR2 and mGluR3) were suggested as targets for addiction treatment. However, LY379268 and other group II agonists do not discriminate between the mainly presynaptic inhibitory mGluR2 (the proposed treatment target) and mGluR3. These agonists also produce tolerance over repeated administration and are no longer considered for addiction treatment. Here, we determined the effects of AZD8529, a selective positive allosteric modulator of mGluR2, on abuse-related effects of nicotine in squirrel monkeys and rats. Methods: We first assessed modulation of mGluR2 function by AZD8529 using functional in vitro assays in membranes prepared from a cell line expressing human mGluR2 and in primate brain slices. We then determined AZD8529 (.03-10 mg/kg, intramuscular injection) effects on intravenous nicotine self-administration and reinstatement of nicotine seeking induced by nicotine priming or nicotine-associated cues. We also determined AZD8529 effects on food self-administration in monkeys and nicotine-induced dopamine release in accumbens shell in rats.[1]

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Rationale: Numerous medication development strategies seek to decrease nicotine consumption and prevent relapse to tobacco smoking by blocking glutamate transmission. Decreasing glutamate release by activating presynaptic inhibitory metabotropic glutamate (mGlu)2/3 receptors inhibits the reinforcing effects of nicotine and blocks cue-induced reinstatement of nicotine-seeking behavior in rats. However, the relative contribution of mGlu2 receptors in nicotine dependence is still unknown. Objectives: The present study evaluated the role of mGlu2 receptors in nicotine-taking and nicotine-seeking behavior using the novel, relatively selective mGlu2 positive allosteric modulators (PAMs) AZD8418 and AZD8529. Results: Acute treatment with AZD8418 (0.37, 1.12, 3.73, 7.46, and 14.92 mg/kg) and AZD8529 (1.75, 5.83, 17.5, and 58.3 mg/kg) deceased nicotine self-administration and had no effect on food-maintained responding. Chronic treatment with AZD8418 attenuated nicotine self-administration, but tolerance to this effect developed quickly. The inhibition of nicotine self-administration by chronic AZD8529 administration persisted throughout the 14 days of treatment. Chronic treatment with either PAMs inhibited food self-administration. AZD8418 (acute) and AZD8529 (acute and subchronic) blocked cue-induced reinstatement of nicotine- and food-seeking behavior. Conclusions: These findings indicate an important role for mGlu2 receptors in the reinforcing properties of self-administered nicotine and the motivational impact of cues that were previously associated with nicotine administration (i.e., cue-induced reinstatement of nicotine-seeking behavior). Thus, mGlu2 PAMs may be useful medications to assist people to quit tobacco smoking and prevent relapse.[2]

C24H24F3N5O3

487.474275588989

487.183

C, 59.13; H, 4.96; F, 11.69; N, 14.37; O, 9.85

1092453-15-0

AZD-8529 mesylate; 1314217-69-0

25125217

Off-white to light yellow solid powder

1.4±0.1 g/cm3

650.9±65.0 °C at 760 mmHg

347.4±34.3 °C

0.0±1.9 mmHg at 25°C

1.585

3.17

1

10

6

35

730

0

FC(OC1C=CC(=CC=1)CN1C(C2C(C)=CC(C3=NC(CN4CCNCC4)=NO3)=CC=2C1)=O)(F)F

IPCYZQQFECEHLI-UHFFFAOYSA-N

InChI=1S/C24H24F3N5O3/c1-15-10-17(22-29-20(30-35-22)14-31-8-6-28-7-9-31)11-18-13-32(23(33)21(15)18)12-16-2-4-19(5-3-16)34-24(25,26)27/h2-5,10-11,28H,6-9,12-14H2,1H3

7-methyl-5-[3-(piperazin-1-ylmethyl)-1,2,4-oxadiazol-5-yl]-2-[[4-(trifluoromethoxy)phenyl]methyl]-3H-isoindol-1-one

AZD8529; AZD-8529; AZD-8529; 1092453-15-0; AZD-8529 free base; AZD8529; 7-methyl-5-[3-(piperazin-1-ylmethyl)-1,2,4-oxadiazol-5-yl]-2-[[4-(trifluoromethoxy)phenyl]methyl]-3H-isoindol-1-one; CHEMBL3937907; UNII-6H81G454I7; 1092453-15-0 (free base); AZD 8529

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: ~128.2 mg/mL (~71.4 mM)

Solubility in Formulation 1: ≥ 6.25 mg/mL (12.82 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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 62.5 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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 (Please use freshly prepared in vivo formulations for optimal results.)