mGlu7/metabotropic glutamate receptor 7

The internal cell lines of ADX71743 have an IC50 of 300 nM. When ADX71743 (3 μM; 20 min) is pretreated prior to high-frequency stimulation (HFS), LTP induction is virtually entirely blocked [1]. ADX71743 (0.1, 10 μM) promotes a concentration-dependent reversal of L-AP4-induced depression and reverses the depression of synaptic transmission caused by L-AP4. 10% and 11% of the effects of L-AP4 can be reversed by 10 μM and 0.1 μM ADX71743, respectively [2]. EC80 (IC50 of 22 nM) of ADX71743 antagonizes glutamate, while EC80 (IC50 of 125 nM) of L-AP4 does the same [2].

At lower dosages (50 and 100 mg/kg), ADX71743 (50, 100, and 150 mg/kg; SC) dramatically decreased the amount of buried marble to near-maximum values [2]. In mice, ADX71743 (mouse 12.5, 100 mg/kg, rat 100 mg/kg; SC) has a Cmax of 1380, 12766 ng/ml (12.5 mg/kg and 100 mg/kg) and a T1/2 of 0.68, 0.40 hours [2].

Of the eight metabotropic glutamate (mGlu) receptor subtypes, only mGlu7 is expressed presynaptically at the Schaffer collateral (SC)-CA1 synapse in the hippocampus in adult animals. Coupled with the inhibitory effects of Group III mGlu receptor agonists on transmission at this synapse, mGlu7 is thought to be the predominant autoreceptor responsible for regulating glutamate release at SC terminals. However, the lack of mGlu7-selective pharmacological tools has hampered direct testing of this hypothesis. We used a novel, selective mGlu7-negative allosteric modulator (NAM), ADX71743, and a newly described Group III mGlu receptor agonist, LSP4-2022, to elucidate the role of mGlu7 in modulating transmission in hippocampal area CA1 in adult C57BL/6J male mice. Interestingly, although mGlu7 agonists inhibit SC-CA1 EPSPs, we found no evidence for activation of mGlu7 by stimulation of SC-CA1 afferents. However, LSP4-2022 also reduced evoked monosynaptic IPSCs in CA1 pyramidal cells and, in contrast to its effect on SC-CA1 EPSPs, ADX71743 reversed the ability of high-frequency stimulation of SC afferents to reduce IPSC amplitudes. Furthermore, blockade of mGlu7 prevented induction of LTP at the SC-CA1 synapse and activation of mGlu7 potentiated submaximal LTP. Together, these data suggest that mGlu7 serves as a heteroreceptor at inhibitory synapses in area CA1 and that the predominant effect of activation of mGlu7 by stimulation of glutamatergic afferents is disinhibition, rather than reduced excitatory transmission. Furthermore, this mGlu7-mediated disinhibition is required for induction of LTP at the SC-CA1 synapse, suggesting that mGlu7 could serve as a novel therapeutic target for treatment of cognitive disorders[1].

cAMP[2]
A homogeneous time-resolved fluorescence (HTRF) cAMP dynamic 2 assay was performed as previously described (Chruścicka et al., 2015) with recombinant cell lines. Briefly, HEK 293 T-REx cells stably expressing mGlu7 receptor, were collected and suspended in Hanks-HEPES buffer. The cell suspension was added to compounds solution with 5 μM of forskolin (final concentration). After 5 min incubation in 37°C, 5 μl of cAMP-d2 conjugate in lysis buffer was added and mixed with the 10 μl cell suspension by means of an automated pipetting system. Next, 5 μl anti-cAMP cryptate conjugate was added and the fluorescence at 620 and 665 nm was read after 1 h . The results are shown as the 665 nm/620 nm ratio multiplied by 104. The detected signal was inversely proportional to the concentration of cAMP in the sample. Antagonist activity of ADX71743 or MMPIP are shown as a percentage of the inhibition of L-Glu activity at its EC80 concentration. Dose response data from ADX71743 or MMPIP were analyzed with Prism Version 7.03. Each experiment was performed three times (n = 3), and each data point was in triplicate.

Animal/Disease Models: Adult male C57Bl6/J mice (24-30 g) and SD (SD (Sprague-Dawley)) rats (250-350 g) [2]
Doses: mice 12.5, 100 mg/kg, rats 100 mg/kg (drug Metabokinetic analysis)
Route of Administration: SC
Experimental Results: T1/2 in mice were 0.68, 0.40 hrs (hrs (hours)), Cmax were 1380, 12766ng/ml, 12.5mg/kg and 100mg/kg. The T1/2 of 100 mg/kg in rats is 1.5 hrs (hrs (hours)) and the Cmax is 16800 ng/ml.

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ADX71743 was dissolved in small amount of DMSO and then titrated in 20% captisol [Front Mol Neurosci. 2018 Sep 20;11:316.]

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Pharmacokinetic Studies[Front Mol Neurosci. 2018 Sep 20;11:316.]
The method described below was successfully applied to a pharmacokinetic study of ADX71743 and MMPIP in mouse (Albino Swiss) after i.p. injection. Compound ADX71743 and MMPIP were administered to mice at 10 mg/kg i.p. At 0.25, 0.50, 1.0, 2.0, 4.0, 6.0 h, the mice were anesthetized, and the blood was collected from the portal vein to the tubes containing 5% EDTA. The mice were then perfused with 0.1M PBS to remove remaining blood from the body, and the brains were taken out for the analysis. Blood was centrifuged at 2000 rpm for 10 min at 4°C, and the plasma was collected and frozen at -80°C for further analysis. [2]
Plasma and tissue samples from all drug-treated animals were thawed at room temperature prior to use. Standard protocol of sample preparation: 200 μl acetonitrile was added to the eppendorfs with 50 μl of studied plasma samples or tissue homogenate. Samples were mixed for 5 min on a mixer at 25°C and 1400 rpm. Tubes were then centrifuged at 2000 × g for 15 min at 4°C. About 180 μl of each supernatant was transferred into a plate well. Finally, each sample was injected into the column.

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MK-801-Induced Hyperactivity[Front Mol Neurosci. 2018 Sep 20;11:316.]
The locomotor activity was recorded individually for each animal in OPTO-M3 locomotor activity cages linked online to a compatible PC activity, as described previously by Woźniak et al., 2016b. Each cage (13 cm × 23 cm × 15 cm) was surrounded with an array of photocell beams. Interruptions of these photobeams resulted in horizontal activity defined as ambulation counts. The mice were placed in the locomotor activity cages for acclimatization for 30 min Then, MMPIP (10, 15 mg/kg) or ADX71743 (5, 10 mg/kg) were administered i.p. Both drugs were given 30 min prior to MK-801 injection (0.35 mg/kg, i.p.). The locomotor activity was measured for 60 min immediately after MK-801 administration.

The pharmacokinetic analysis of ADX71743 in mice and rats revealed that it is bioavailable after s.c. administration and is brain penetrant (cerebrospinal fluid concentration/total plasma concentration ratio at C(max) = 5.3%). [2]

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The concentration of ADX71743 and MMPIP in mouse plasma and brain are shown in Table ​Table11. Cmax was evident in brain and plasma 0.25 h after injection of ADX71743, and 0.5 h after MMPIP administration. Figure ​Figure33 represents comparison between ADX71743 and MMPIP concentrations in the brain in selected time points after administration.[Front Mol Neurosci. 2018 Sep 20;11:316.]
Data presented in Table ​Table22 showed that ADX71743 and MMPIP had different cytochrome P450 inhibition profile. Weak inhibition (IC50 > 10μM) of cytochrome P450 was observed in case of 1A2, 2B6, 2C9, 2D6 isoforms for both NAM mGluR7 standards. Mild inhibition (3.3 < IC50 < 10) of isoform 2C19 was determined for ADX71743 standard, while strong inhibition (IC50 < 1.1) was observed only for MMPIP in case of isoform 3A4 as well as 2C19.[Front Mol Neurosci. 2018 Sep 20;11:316.]

[1]. Activation of Metabotropic Glutamate Receptor 7 Is Required for Induction of Long-Term Potentiation at SC-CA1 Synapses in the Hippocampus. J Neurosci. 2015 May 13;35(19):7600-15.

[2]. ADX71743, a Potent and Selective Negative Allosteric Modulator of Metabotropic Glutamate Receptor 7: In Vitro and in Vivo Characterization. Pharmacol Exp Ther. 2013 Mar;344(3):624-36.

Metabotropic glutamate receptor 7 (mGlu(7)) has been suggested to be a promising novel target for treatment of a range of disorders, including anxiety, post-traumatic stress disorder, depression, drug abuse, and schizophrenia. Here we characterized a potent and selective mGlu(7) negative allosteric modulator (NAM) (+)-6-(2,4-dimethylphenyl)-2-ethyl-6,7-dihydrobenzo[d]oxazol-4(5H)-one (ADX71743). In vitro, Schild plot analysis and reversibility tests at the target confirmed the NAM properties of the compound and attenuation of L-(+)-2-amino-4-phosphonobutyric acid-induced synaptic depression confirmed activity at the native receptor. The pharmacokinetic analysis of ADX71743 in mice and rats revealed that it is bioavailable after s.c. administration and is brain penetrant (cerebrospinal fluid concentration/total plasma concentration ratio at C(max) = 5.3%). In vivo, ADX71743 (50, 100, 150 mg/kg, s.c.) caused no impairment of locomotor activity in rats and mice or activity on rotarod in mice. ADX71743 had an anxiolytic-like profile in the marble burying and elevated plus maze tests, dose-dependently reducing the number of buried marbles and increasing open arm exploration, respectively. Whereas ADX71743 caused a small reduction in amphetamine-induced hyperactivity in mice, it was inactive in the mouse 2,5-dimethoxy-4-iodoamphetamine-induced head twitch and the rat conditioned avoidance response tests. In addition, the compound was inactive in the mouse forced swim test. These data suggest that ADX71743 is a suitable compound to help unravel the physiologic role of mGlu(7) and to better understand its implication in central nervous system diseases. Our in vivo tests using ADX71743, reported here, suggest that pharmacological inhibition of mGlu(7) is a valid approach for developing novel pharmacotherapies to treat anxiety disorders, but may not be suitable for treatment of depression or psychosis.[2]

C17H19NO2

269.338264703751

269.141578

C, 75.81; H, 7.11; N, 5.20; O, 11.88

1431641-29-0

53391766

White to off-white solid powder

3.8

0

3

2

20

370

0

O1C(CC)=NC2C(CC(C3C=CC(C)=CC=3C)CC1=2)=O

CPKZCQHJDFSOJT-UHFFFAOYSA-N

InChI=1S/C17H19NO2/c1-4-16-18-17-14(19)8-12(9-15(17)20-16)13-6-5-10(2)7-11(13)3/h5-7,12H,4,8-9H2,1-3H3

6-(2,4-dimethylphenyl)-2-ethyl-6,7-dihydro-5H-1,3-benzoxazol-4-one

(+/-)-ADX71743; CHEMBL4174742; 6-(2,4-Dimethylphenyl)-2-ethyl-6,7-dihydrobenzo[d]oxazol-4(5H)-one; 6-(2,4-dimethylphenyl)-2-ethyl-4,5,6,7-tetrahydro-1,3-benzoxazol-4-one; 6-(2,4-dimethylphenyl)-2-ethyl-6,7-dihydro-5H-1,3-benzoxazol-4-one; (+)-6-(2,4-Dimethylphenyl)-2-ethyl-6,7-dihydro-4(5H)-benzoxazolone;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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