CB2 receptor ( Ki = 31.2 nM )

AM630 exhibits different behaviors in AM630-pre-incubated cells, such as a low-potency neutral competitive antagonist, a low-potency agonist, and a much higher-potency inverse agonist/antagonist in vehicle-pre-incubated cells. The hCB2 receptor has two forms: constitutively active, which has a low affinity for producing agonism or neutral antagonism, and constitutively inactive, which has a much higher affinity for producing inverse agonism. AM630 is a protean ligand that can target both of these forms of the receptor.[2]

Persistent AM630 therapy reduces anxiety in elevated plus maze (EPM) and light-dark box (LDB) tests. Long-term AM630 treatment raises the gene and decreases the protein expression of GABAAγ2 and GABAAγ2 (CB2 receptors) in the cortex and amygdala. Furthermore, chronic administration of AM630 reduces DBA/2 mice's anxiety in the LDB test. The ability of AM630 to effectively lessen anxiety in the spontaneously anxious DBA/2 strain of mice emphasizes the CB2 receptor's potential as a novel target for the treatment of anxiety-related disorders. [1]

Analysis of gene expression of CB2 receptors, GABAAα2 and GABAAγ2 receptor subunits by RT-PCR [1]
The gene expression of CB2 receptors and the main anxiolytic subunits of GABAA receptor, GABAAα2 and GABAAγ2, were examined by RT-PCR in the cortex and amygdala of Swiss ICR mice, chronically treated with AM630 and JWH133. Briefly, 18 h after the last administration of AM630, JWH133 or its corresponding vehicle, mice were killed, and the brains were removed from the skull and frozen over dry ice. Brain sections (500 µm) were cut at different levels containing the cingulated cortex (figure 20 and 1.34 mm from the bregma) and amygdala (basolateral and central amygdaloid nuclei; figure 40, −1.6 mm from the bregma) according to Paxinos and Franklin (2001), mounted onto slides and stored at −80°C. Sections were dissected following the method described by Palkovits (1983). Total RNA was obtained from brain punches using Biozol® Total RNA extraction reagent. After DNAse digestion, the reverse transcription was carried out following the instructions of the manufacturer. CB2 receptor, GABAAα2 and GABAAγ2 gene expression was measured by using Taqman® Gene Expression assays (Mm 00438286_m1, Mm00433435_m1 and Mm00433489_m1 respectively) as a double-stranded DNA-specific fluorescent dye and performed on the AbbiPrism 7700 Real Time Cycler. The reference gene used was 18S rRNA, detected using Taqman ribosomal RNA control reagents. All primer-probe combinations were optimized and validated for relative quantification of gene expression. Briefly, data for each target gene were normalized to the endogenous reference gene, and the fold change in target gene abundance was determined using the 2-ΔΔCt method.[1]

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Analysis of expression of CB2 receptors, GABAAα2 and GABAAγ2 receptor subunits protein by Western blot[1]
After determination and adjusting protein levels, homogenates of cortex and amygdala tissues were centrifuged (12 000×g, 20 min at 4°C) and mixed with Laemmli sample buffer (SDS 10%, distilled H2O, glycerol 50%, Tris HCl 1 M pH 6,8, dithiotreitol and blue bromophenol) containing β-mercapthoethanol (50 µL per mL of Laemmli). Once separated by molecular weight, proteins from the electrophoresis gels were blotted onto a nitrocellulose membrane by semidry transfer system and incubated with a specific primary antibodies against CB2 receptors (1:1000 dilution), GABAAα2 (1:1000 dilution) and GABAAγ2 antibody (Abcam; 1:1000 dilution). Proteins recognized by the respective horseradish peroxidase-linked secondary antibodies were revealed by ECL™-kit following the manufacturer's instructions (Amersham) and visualised on X-ray film (Amersham). Autoradiographs were quantified by densitometry, and several time exposures were analysed to ensure the linearity of the band intensities. All densitometries are expressed in arbitrary units (AU). In all the Western blot analyses, the housekeeping gene β-actin was used as loading control.

The hCB2 CHO cell membrane is used in binding assays with [3H]-CP55940 or [35S]-GTPγS. After undergoing a full 24-hour incubation period in complete medium containing 10 μM AM630 or dimethyl sulphoxide as a vehicle, the hCB2 CHO cells are rigorously washed. The process of preparing membranes involves scraping the cells out of the flasks, centrifuging them at 489 x g, and freezing them as a pellet at -20°C until needed. Prior to being used in an experiment involving radioligand binding, cells are frozen and diluted in either GTPγS binding buffer ([35S]-GTPγS binding assay) or Tris binding buffer (radioligand displacement assay).

male Swiss ICR mice, DBA/2 Ola Hs mice
1 mg/kg, 2 mg/kg, 3 mg/kg
IP

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Effects of acute and chronic treatment with the CB(2) receptor agonist JWH133 and CB(2) receptor antagonist AM630 were evaluated in the light-dark box (LDB) and elevated plus maze (EPM) tests in Swiss ICR mice. CB(2) receptor, GABA(A) α(2) and GABA(A) γ(2) gene and protein expression in the cortex and amygdala of mice chronically treated with JWH133 or AM630 were examined by RT-PCR and Western blot. Effects of chronic AM630 treatment were evaluated in spontaneously anxious DBA/2 mice in LDB.[1]

[1]. Br J Pharmacol . 2012 Feb;165(4):951-64.

[2]. Br J Pharmacol . 2012 Apr;165(8):2561-74.

[6-Iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-3-indolyl]-(4-methoxyphenyl)methyl ketone is an N-acylindole.

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Background and Objectives: This study aimed to investigate the roles of CB(2) receptor agonists and antagonists in regulating anxiety-like behavior. Experimental Methods: The acute and chronic therapeutic effects of the CB(2) receptor agonist JWH133 and the CB(2) receptor antagonist AM630 were evaluated in Swiss ICR mice using the light-dark box (LDB) and elevated cross maze (EPM) experiments. The expression of CB(2) receptor, GABA(A)α(2), and GABA(A)γ(2) genes and proteins in the cortex and amygdala of mice treated with JWH133 or AM630 long-term was detected by RT-PCR and Western blot. This study evaluated the effects of long-term AM630 treatment on spontaneous anxiety in DBA/2 mice in the LDB model. Main Results: Acute JWH133 treatment had no effect. Acute AM630 treatment increased anxiety in mice, while JWH133 pretreatment blocked this effect. Chronic JWH133 treatment increased anxiety-like behavior in mice in the LDB and EPM tests, while chronic AM630 treatment showed an anti-anxiety effect. Chronic AM630 treatment increased gene expression of CB(2) receptors, GABA(A)α(2), and GABA(A)γ(2) in the cortex and amygdala, and decreased their protein expression. Chronic JWH133 treatment resulted in the opposite gene and protein changes. In addition, chronic AM630 administration reduced anxiety levels in DBA/2 mice in the LDB test. Conclusions and Implications: The opposite behavioral and molecular changes observed after long-term AM630 or JWH133 treatment support the key role of CB(2) receptors in anxiety regulation. In fact, AM630 effectively reduced the anxiety level in spontaneously anxious DBA/2 strain mice, which further confirms the potential of CB(2) receptors as a novel target for treating anxiety-related diseases. [1]

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Background and Objectives: We investigated how pre-incubation of hCB(2) CHO cells with the CB(2) receptor antagonists/inverse agonists AM630 and SR144528 affected the way these ligands and other ligands targeted these cells or the hCB(2) receptors on their cell membranes. Experimental Methods: We examined the ability of AM630, SR144528, and the CB(1)/CB(2) receptor agonists CP55940 and R-(+)-WIN55212 to regulate fossiclin-stimulated cAMP production in hCB(2) CHO cells, the binding of [(35)S]-GTPγS to these cell membranes, and the ability of [(3)H]-CP55940 to displace [(3)H]-CP55940 from intact cells and cell membranes. We also conducted experiments using the CB(2) receptor partial agonist Δ(9)-tetrahydrocannabinol. Cells were pre-incubated with AM630 or SR144528 and then thoroughly washed. Main results: AM630 acted as a low-potency neutral competitive antagonist in cells pre-incubated with AM630; as a low-potency agonist in cells pre-incubated with SR144528; and as a high-potency inverse agonist/antagonist in cells pre-incubated with the vector. AM630 pre-incubation (i) reduced the inverse potency of SR144528, but did not completely eliminate its effect; (ii) increased the potency of Δ(9)-tetrahydrocannabinol; and (iii) did not affect the potency of AM630 in displacing [(3)H]-CP55940 from intact cells, nor did it affect its inverse agonist potency and potency in the [(35)S]-GTPγS membrane assay. Conclusions and significance: These results indicate that AM630 is a multifunctional ligand that can exert agonistic or neutral antagonistic effects by targeting the constitutive active form (R) of the hCB(2) receptor with low affinity and reverse agonistic effects by targeting the constitutive inactive form (R) of the receptor with higher affinity. Furthermore, pre-incubation with AM630 resulted in less reduction of constitutive activity in the whole cell compared to the highly potent reverse agonist SR144528. [2]

C23H25IN2O3

504.3685

504.09

C, 54.77; H, 5.00; I, 25.16; N, 5.55; O, 9.52

164178-33-0

164178-33-0

4302963

Solid powder

1.5±0.1 g/cm3

605.9±55.0 °C at 760 mmHg

320.3±31.5 °C

0.0±1.7 mmHg at 25°C

1.647

4.65

0

4

6

29

548

0

CC1=C(C2=C(C=C(C=C2)I)N1CCN3CCOCC3)C(=O)C4=CC=C(C=C4)OC

JHOTYHDSLIUKCJ-UHFFFAOYSA-N

InChI=1S/C23H25IN2O3/c1-16-22(23(27)17-3-6-19(28-2)7-4-17)20-8-5-18(24)15-21(20)26(16)10-9-25-11-13-29-14-12-25/h3-8,15H,9-14H2,1-2H3

[6-iodo-2-methyl-1-(2-morpholin-4-ylethyl)indol-3-yl]-(4-methoxyphenyl)methanone

iodopravadoline; AM630; AM-630; (6-iodo-2-methyl-1-(2-morpholinoethyl)-1H-indol-3-yl)(4-methoxyphenyl)methanone; AM-630; 6-iodopravadoline; iodopravadoline; AM 630; Iodopravadoline (AM630); AM 630

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: ~50 mg/mL (~99.1 mM)

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.)