TCF-dependent transcriptional activity (EC50 = 700 nM)

In comparison to the NC group, BML-284 (10 µM; 24 hours) dramatically increased the expression of β-catenin. Additionally, as compared to the pizotifen treatment group, it partially counteracted the effects of pizotifen on the expression of N- and E-cadherin in MNK45 and AGS cells [1]. The migration and invasion capacities of MNK45 and AGS cells are markedly enhanced by BML-284 (10 µM; 24 hours), while the migration and invasion capacities of cells inhibited by bentiftine are partially restored [1].

BML-284 hydrochloride (10 ng) coupled with pyrimethanil (4 mg/L) can partially repair the teratogenic phenotype and heart abnormalities induced by pyrimethanil in Tg (myl7:EGFP) transgenic embryos at 5.5 hpf. Transfer to a plate containing 20 embryos [1].

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AMBMP Targets CaMKIIβ In Vivo, https://pmc.ncbi.nlm.nih.gov/articles/PMC7659555/ Researchers next asked whether AMBMP acts by enhancing CaMKIIβ activity in vivo. C3KO and C57BL/6 WT mice were treated with AMBMP and then their muscles were evaluated for CaMKIIβ and other signaling pathways. The activation of signaling was carried out by western blotting with antibodies specific for the active forms of these signaling pathways. Treatment with AMBMP (daily i.p. injection 7.5 mg/kg) led to CaMKIIβ activation in both WT and C3KO mice (Figures 4C and 4D). The drug appears to engage CaMKIIβ specifically as it does not activate AKT nor AMPK (nor other pathways that control muscle remodeling and oxidative metabolism) (Figures 4E and 4F). Furthermore, the effect of AMBMP on CaMKIIβ was likely post-transcriptional, and there was no significant change in the expression level of the Camk2b gene (Figure 4G). Thus, these studies establish proof of concept for the ability of AMBMP to activate CaMKII and subsequently to promote oxidative metabolism and benefit the LGMDR1 phenotype.

Western Blot Analysis [1]
Cell Types: Human gastric cancer cell line MNK45 and AGS
Tested Concentrations: 10 µM
Incubation Duration: 24 hrs (hours)
Experimental Results: β-catenin expression was induced and E-cadherin and N-cadherin expression were retained in MNK45 and AGS cells.

Compound pharmacokinetics assay
For pharmacokinetics, AMBMP was administered by different routes of delivery (subcutaneous, intraperitoneal, and oral, in food or by gavage) at two different dosages (10 mg/kg and 30 mg/kg). The blood was collected at 0.5 h, 1 h, 2 h, 4 h and 6 h post treatment by heart puncture. The concentrations of compounds in plasma were analyzed by Integrated Analytical Solutions, Inc.https://pmc.ncbi.nlm.nih.gov/articles/PMC7659555/

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Seahorse analysis of Extracts from Frozen Muscle
For Seahorse analysis, frozen soleus muscles from DMSO or AMBMP-treated mice (daily IP injections at 7.5 mg/kg) were homogenized by hand in a Dounce homogenizer in 200 mL of mitochondrial buffer (70 mM sucrose, 220 mM mannitol, 5 mM KH2PO4, 5 mM MgCl2, 1 mM EGTA, 2 mM HEPES, adjusted to pH 7.4 with KOH) on ice. Muscle homogenates were centrifuged at 900xg for 5 min at 4°C. Supernatants were transferred to new tubes; protein concentrations were measured using BCA protein Assay Kit. The samples (4 μg/well) were analyzed in the UCLA Mitochondrial and Metabolism Core using a Seahorse XF96 Analyzer. Data were normalized to total protein. Seahorse analysis was carried out according to Acin-Perez et al.https://pmc.ncbi.nlm.nih.gov/articles/PMC7659555/

[1]. A small-molecule agonist of the Wnt signaling pathway. Angew Chem Int Ed Engl. 2005 Mar 18;44(13):1987-90.

[2]. Pizotifen inhibits the proliferation and invasion of gastric cancer cells. Exp Ther Med. 2020 Feb;19(2):817-824.

[3]. Exposure to pyrimethanil induces developmental toxicity and cardiotoxicity in zebrafish. Chemosphere. 2020 Sep;255:126889.

N4-(1,3-benzodioxol-5-ylmethyl)-6-(3-methoxyphenyl)pyrimidine-2,4-diamine is a member of pyrimidines.

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Gastric cancer is the fifth most common malignancy and the third highest cause of cancer-associated mortality worldwide. Therefore, research on the pathogenesis of gastric cancer is of utmost importance. It has been reported that aberrant activation of the Wnt/β-catenin signaling pathway is involved in the occurrence and development of gastric cancer. In the present study, it was found that pizotifen could inhibit the viability of gastric cancer cell lines MNK45 and AGS cells in a dose-dependent manner. Pizotifen treatment suppressed cell migration and invasion in MNK45 and AGS cells, whilst also inducing apoptosis. Western blot analysis demonstrated that pizotifen blocked the expression of Wnt3a, β-catenin and N-cadherin, whilst increasing E-cadherin expression. In addition, BML-284, a pharmacological Wnt signaling activator, partially reversed the changes in the expression levels of β-catenin, N-cadherin and E-cadherin in MNK45 and AGS cells induced by pizotifen. Collectively, these findings suggested that pizotifen demonstrates potential as a novel anti-cancer drug for the treatment of gastric cancer by inhibiting the Wnt/β-catenin pathway.[2]

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Pyrimethanil is a broad-spectrum fungicide commonly used in the prevention and treatment of Botrytis cinerea. However, little information is available in the literature to show the toxicity of Pyrimethanil to cardiac development. In this study, we used an experimental animal model to explore the developmental and cardiac toxicity of Pyrimethanil in aquatic vertebrates; we exposed zebrafish embryos to Pyrimethanil at concentrations of 2, 4, and 6 mg/L from 5.5 to 72 h post fertilisation. We found that Pyrimethanil caused a decrease in the hatching rate, heart rate, and survival rate of zebrafish embryos. Pyrimethanil exposure also resulted in pericardial and yolk sac edema, spinal deformity, and heart loop failure. Moreover, Pyrimethanil increased reactive oxygen stress levels and heightened the activity of superoxide dismutase and catalase. Alterations were induced in the transcription of apoptosis-related genes (p53, Bax, Bcl2, Casp 9, and Casp6l1) and heart development-related genes (Tbx2b, Gata4, Myh6, Vmhc, Nppa, Bmp2b, Bpm 4, and Bpm 10). Our data showed that the activation of Wnt signalling by BML-284 could partially rescue the malformed phenotype caused by Pyrimethanil. Our results provide new evidence for Pyrimethanil's toxicity and the danger of its residues in the environment and agricultural products.[3]

C19H19CLN4O3

386.84

386.114

C, 58.99; H, 4.95; Cl, 9.16; N, 14.48; O, 12.41

2095432-75-8

BML-284;853220-52-7

122705993

Solid powder

3

7

5

27

455

0

C1(OC)C=C(C=CC=1)C1=NC(=NC(=C1)NCC1=CC2OCOC=2C=C1)N.Cl

XZOFNDFDGVAIEH-UHFFFAOYSA-N

InChI=1S/C19H18N4O3.ClH/c1-24-14-4-2-3-13(8-14)15-9-18(23-19(20)22-15)21-10-12-5-6-16-17(7-12)26-11-25-16;/h2-9H,10-11H2,1H3,(H3,20,21,22,23);1H

4-N-(1,3-benzodioxol-5-ylmethyl)-6-(3-methoxyphenyl)pyrimidine-2,4-diamine;hydrochloride

AMBMP Hydrochloride BML284 HCl Wnt Agonist BML284 HCl

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.)