nAChR/nicotinic acetylcholine receptors
MG624 targets neuronal nicotinic acetylcholine receptors, particularly those containing the α7 subunit. The compound inhibits α-bungarotoxin binding to chick α7 nAChRs with a Ki of 106 nM. It is selective for neuronal nAChRs over muscle-type AChRs (Ki = 70 µM in TE671 cells that express muscle-type AChRs). MG624 inhibits α-bungarotoxin binding to chick α4β2 subtypes with a Ki of 84 μM, demonstrating significant selectivity for α7-containing receptors over α4β2 receptors. By blocking α7 nAChRs, MG624 modulates cholinergic signaling in the nervous system. The α7 nAChR is involved in various physiological processes, including synaptic transmission, inflammation, and cancer progression. MG624's selectivity for neuronal nAChRs makes it a valuable tool for studying the role of these receptors in health and disease.

In this study, researchers assessed the pharmacological activity of triethyl-(β-4-stilbenoxy-ethyl) ammonium (MG624), a drug that is active on neuronal nicotinic receptors (nicotinic AChR). Experiments on the major nicotinic AChR subtypes present in chick brain, showed that it inhibits the binding of [125I]-αBungarotoxin (αBgtx) to the α7 subtype, and that of [3H]-epibatidine (Epi) to the α4β2 subtype, with Ki values of respectively 106 nm and 84 μm.[1]
MG624 also inhibited ACh elicited currents (IACh) in the oocyte-expressed α7 and α4β2 chick subtypes with half-inhibitory concentrations (IC50) of respectively 109 nm and 3.2 μm.[1]
When tested on muscle-type AChR, it inhibited [125I]-αBgtx binding with a Ki of 32 μm and ACh elicited currents (IACh) in the oocyte-expressed α1β1γδ chick subtype with an IC50 of 2.9 μm.[1]
The interaction of MG624 with the α7 subtype was investigated using an α7 homomeric mutant receptor with a threonine-for-leucine 247 substitution (L247T α7). MG624 did not induce any current in oocytes expressing the wild type α7 receptor, but did induce large currents in the oocyte-expressed L247T α7 receptor. The MG624 elicited current (IMG624) has an EC50 of 0.2 nm and a Hill coefficient nH of 1.9, and is blocked by the nicotinic receptor antagonist methyllycaconitine (MLA).[1]
These binding and electrophysiological studies show that MG624 is a potent antagonist of neuronal chick α7 nicotinic AChR, and becomes a competitive agonist following the mutation of the highly conserved leucine residue 247 located in the M2 channel domain.[1]
In vitro, MG624 demonstrates potent antagonism of neuronal nicotinic acetylcholine receptors. The compound inhibits α-bungarotoxin binding to chick α7 nAChRs with a Ki of 106 nM. It inhibits α-bungarotoxin binding to chick α4β2 subtypes with a Ki of 84 μM. MG624 is selective for neuronal nAChRs over muscle-type AChRs (Ki = 70 µM in TE671 cells that express muscle-type AChRs). The compound can decrease vagus nerve stimulation-induced contractions of isolated guinea pig vagus nerve-stomach preparations. In cellular studies, MG624 potently suppresses proliferation of primary human microvascular endothelial cells of the lung. It decreases nicotine-induced early growth response gene 1 (Egr-1) levels in HMEC-Ls, and reduces the levels of Egr-1 on the FGF2 promoter. MG624's ability to inhibit α7 nAChR signaling suggests potential applications in cancer research.

The administration of 20 μM MG624 attenuated the nicotine-induced angiogenesis of H69 human SCLC tumors (implanted on CAM) by approximately 50%, as compared to control H69 tumors treated with nicotine only.[2]
MG624 inhibited the growth of human SCLC tumors implanted in nude mice (Fig. 4b). One of our concerns was whether MG624 would cause any side effects in these mice. We observed that the administration of 50 mg MG624/kg food in the diet was well tolerated and there was no sign of discomfort or change in food intake (mean = 5.6 g/day/mouse for both diets), water consumption (vehicle treated mean = 29.0 mL/week/mouse; MG624 treated mean = 29.2 mL/week/mouse) or body weights (vehicle treated mean = 28.1 g/mouse; MG624 treated mean = 27.9 g/mouse) in mice administered nicotine and/or MG624. Immunohistochemical staining of tumor sections showed decreased staining of angiogenic markers, namely CD31 and von Willebrand Factor (vWF), in the MG624-treated tumors as compared to the tumors isolated from nicotine-treated mice[2].
In vivo, MG624 demonstrates anti-angiogenic activity in various experimental models. The anti-angiogenic activity of MG624 was assessed by two in vivo models, namely the chicken chorioallantoic membrane model and the nude mice model. In both of these experimental models, MG624 inhibited angiogenesis of human small cell lung cancer (SCLC) tumors. MG624, an α7-nAChR antagonist, inhibits angiogenesis via the Egr-1/FGF2 pathway. The compound decreased nicotine-induced early growth response gene 1 (Egr-1) levels in HMEC-Ls, and reduced the levels of Egr-1 on the FGF2 promoter. MG624 inhibits growth of human small cell lung cancer by induction of p73. The compound's in vivo efficacy in inhibiting angiogenesis and tumor growth suggests potential applications in cancer therapy. However, MG624 is primarily a research compound with no approved clinical indications.

Binding of nicotinic ligands to immobilized subtypes[1]
In order to assess the pharmacological profiles of the immuno-immobilized subtypes we performed competition experiments using MG624, d-Tubocurarine (d-Tub) and MLA.
The drugs were dissolved in buffer C just before use and serial dilutions were preincubated for 30 min at RT. Subsequently a final concentration of 0.1 nm [3H]-Epi was added to the α4β2 subtype, and a final concentration of 0.5 nm or 0.3 nm of [125I]-αBgtx was added to the α7 or α1β1γδ subtypes. The α4β2 and α7 subtypes were incubated overnight at 20°C, the α1β1γδ subtype was incubated for 48 h at 20°C.
After incubation, the wells were washed seven times with ice-cold PBS containing 0.05% Tween 20, and the bound radioactivity was recovered by means of incubation with 200 μl of 2N NaOH for 2 h. The bound radioactivity was then determined by means of liquid scintillation counting in a β counter in the case of [3H]-Epi, or direct counting in a γ counter in the case of [125I]-αBgtx.
In vitro receptor binding assays for MG624 employ radioligand displacement techniques using membrane preparations from tissues or cells expressing nAChR subtypes. For α7 receptor binding, membranes from chick optic lobe or cells expressing the α7 receptor are used. The assay involves incubating MG624 at varying concentrations with the membrane preparation and a fixed concentration of 125I-labeled α-bungarotoxin. Following incubation, bound and free radioligand are separated by rapid filtration, and radioactivity is measured by gamma counting. Non-specific binding is determined in the presence of a high concentration of unlabeled α-bungarotoxin. Binding affinity (Ki) values are calculated from competition curves using nonlinear regression analysis. For α4β2 receptor binding, similar methodologies are employed using [³H]-cytisine or other radioligands. For muscle-type AChR binding, TE671 cells expressing muscle-type AChRs are used.

BrdU proliferation assays[2]
HMEC-Ls were plated in 96-well plates at a density of 10,000 cells/well and rendered quiescent in EGM-R media for 24 h. After 24 h, these cells were treated with 100 nM nicotine for 18 h (which is the time required for S-phase entry) in the presence or absence of varying concentrations of MG624. The rate of BrdU incorporation was measured by the BrdU ELISA kit, according to manufacturer’s instructions. The absorbance of nicotine-treated cells was assumed to be 100%. MG624-induced decreases in the number of cells in S-phase were calculated as a percentage of the nicotine-treated cells. Each sample was tested in duplicate, and the BrdU assay was performed 2 independent times.

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PCNA cell proliferation assays[2]
The effect of MG624 on HMEC-L proliferation was also examined by measuring the levels of proliferating cell nuclear antigen (PCNA). HMEC-Ls were plated in 6-well plates at a density of 250,000 cells/well and treated in an identical manner as in the BrdU assay. The level of PCNA in the cell lysate was quantified by using a PCNA ELISA kit, according to manufacturer’s protocols. Each sample was tested in duplicate, and the assay was performed 2 independent times.
In vitro cellular assays for MG624 typically employ cell lines expressing nAChRs or primary cells that respond to nicotinic stimulation. For studies investigating the compound's effects on cancer, human small cell lung cancer cells or primary human microvascular endothelial cells of the lung are cultured in appropriate media and treated with MG624 at various concentrations. Cell proliferation is assessed using standard assays such as MTT or resazurin reduction. For studies investigating the mechanism of action, the expression of Egr-1, FGF2, and p73 is measured by Western blotting or qPCR. For studies investigating the effects on vagus nerve stimulation, isolated guinea pig vagus nerve-stomach preparations are used, and contractions are measured. The compound's effects on cell viability, proliferation, and signaling pathways can be assessed in various cell types.

Antitumor studies in nude mice[2]
Fourteen 4-week-old male nude mice were acclimatized for 1 week. H69 cells were harvested and re-suspended in a 1:1 (v/v) solution of serum-free RPMI and Matrigel matrix. Two million cells in 100 μL were injected subcutaneously between the scapulae of each mouse. After the tumors reached 100 mm3, the mice were randomized into 2 groups. The control group (n = 7) was fed AIN-76A diet containing vehicle [0.01% DMSO and 10% corn oil]. The treatment group (n = 7) was changed to an AIN76A diet containing 50 mg MG624/kg food (approximately 10 mg MG624/kg body weight of mouse per day). Both of the groups were administered 200 μg/mL nicotine in the drinking water containing 2% saccharin sodium. The food was changed and monitored daily. Nicotine-containing water was changed weekly and monitored daily. Mice were weighed once per week. The administration of MG624 caused no discomfort, lethargy or weight loss in mice. Additionally, food intake and water consumption was similar between control and MG624-treated mice.
In vivo animal experiments for MG624 employ models of angiogenesis and tumor growth. The chicken chorioallantoic membrane (CAM) model is used to assess the compound's anti-angiogenic activity. Fertilized chicken eggs are incubated, and MG624 is applied to the CAM. Angiogenesis is assessed by measuring the number and length of blood vessels. The nude mice model is used to assess the compound's effects on tumor growth. Human small cell lung cancer cells are injected subcutaneously into nude mice, and MG624 is administered by various routes. Tumor growth is measured using calipers, and tumor volume is calculated. The compound's effects on angiogenesis are assessed by immunohistochemistry or other methods. Dosing regimens vary depending on the experimental objectives, with local or systemic administration being used.

The pharmacokinetic properties of MG624 have not been extensively characterized in the literature, as the compound is primarily used as a research tool. MG624 has a molecular weight of 451.38 and a molecular formula of C₂₂H₃₀INO. The compound is a quaternary ammonium salt, which may limit its oral bioavailability and ability to cross the blood-brain barrier. However, the compound has been shown to be active in vivo in the CAM and nude mice models, suggesting adequate bioavailability for these applications. The compound's metabolism and elimination pathways have not been fully elucidated. As with any research compound, appropriate handling and storage procedures should be followed. The compound's pharmacokinetic properties may be influenced by its quaternary ammonium structure.

The intraperitoneal LD50 in mice was 28 mg/kg.
The toxicity profile of MG624 has not been extensively characterized in the literature, as the compound is primarily used as a research tool. In cellular assays, MG624 potently suppresses proliferation of primary human microvascular endothelial cells of the lung. The compound's effects on cell viability in other cell types have not been extensively reported. In animal studies, MG624 has been administered in the CAM and nude mice models without reports of significant toxicity. The compound's safety in humans has not been established, as it has not been developed for clinical use. As with any research compound, appropriate safety precautions should be taken when handling MG624. The compound's potential for off-target effects or drug-drug interactions has not been extensively studied. The compound is intended for research use only and is not approved for human therapeutic use.

[1]. Selective effects of a 4-oxystilbene derivative on wild and mutant neuronal chick alpha7 nicotinic receptor. Br J Pharmacol. 1999 Jan;126(1):285-95.

Small cell lung cancer (SCLC) is closely etiologically linked to smoking. Although cigarette smoke is composed of approximately 4,000 compounds, nicotine is the main addictive component. Multiple studies have shown that nicotine promotes angiogenesis in lung cancer through the α7-nicotine acetylcholine receptor (α7-nAChR) on endothelial cells. Therefore, we hypothesized that α7-nAChR antagonists might attenuate nicotine-induced angiogenesis and hold promise for treating human SCLC. This study is the first to explore the anti-angiogenic activity of the small molecule α7-nAChR antagonist MG624 in multiple angiogenesis experimental models. We observed that MG624 effectively inhibited the proliferation of primary human lung microvascular endothelial cells (HMEC-Ls). Furthermore, MG624 exhibited significant anti-angiogenic activity in Matrigel matrix, rat aortic ring, and rat retinal explant experiments. The anti-angiogenic activity of MG624 was evaluated using two in vivo models: the chicken chorioallantoic membrane model and the nude mouse model. In both experimental models, MG624 inhibited angiogenesis in human small cell lung cancer (SCLC) tumors. More importantly, MG624 administration did not cause any toxic side effects, drowsiness, or discomfort in mice. The anti-angiogenic activity of MG624 is achieved by inhibiting the expression level of FGF2 in nicotine-induced HMEC-L cells. MG624 reduced the expression level of nicotine-induced early growth response gene 1 (Egr-1) in HMEC-L cells and reduced the Egr-1 level on the FGF2 promoter. Therefore, this process reduced the expression level of FGF2, thereby inhibiting angiogenesis. Our results suggest that the anti-angiogenic effect of MG624 may be useful for anti-angiogenic therapy in human small cell lung cancer. [2]
MG624 is a selective antagonist of neuronal nicotinic acetylcholine receptors with high affinity for α7 subunit-containing nAChRs. It inhibits α-bungarotoxin binding to chick α7 with a Ki of 106 nM and to chick α4β2 subtypes with a Ki of 84 μM. MG624 is selective for neuronal nAChRs over muscle-type AChRs (Ki = 70 µM in TE671 cells). It has a molecular weight of 451.38 and a molecular formula of C₂₂H₃₀INO. MG624 can decrease vagus nerve stimulation-induced contractions of isolated guinea pig vagus nerve-stomach preparations. In vivo, MG624 inhibits angiogenesis of human small cell lung cancer tumors in the chicken chorioallantoic membrane model and the nude mice model. MG624 inhibits growth of human small cell lung cancer by induction of p73. It is a research compound for studying nAChR function and cancer biology with no approved clinical indications.

C22H30INO

451.3925

324.233

C, 58.54; H, 6.70; I, 28.11; N, 3.10; O, 3.54

77257-42-2

6433339

White to off-white solid powder

5.112

0

2

9

25

339

0

CC[N+](CC)(CC)CCOC1=CC=C(C=C1)/C=C/C2=CC=CC=C2.[I-]

RDTKUZXIHMTSJO-UEIGIMKUSA-M

InChI=1S/C22H30NO.HI/c1-4-23(5-2,6-3)18-19-24-22-16-14-21(15-17-22)13-12-20-10-8-7-9-11-20;/h7-17H,4-6,18-19H2,1-3H3;1H/q+1;/p-1/b13-12+;

triethyl-[2-[4-[(E)-2-phenylethenyl]phenoxy]ethyl]azanium;iodide

MG-624; MG 624; Stilonium iodide; MG 624; 77257-42-2; MG624; MG-624; 2551-76-0; M.G. 624; DTXSID2045781; MG624

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

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.)