TCF-dependent transcriptional activity (EC50 = 700 nM)

与 NC 组相比，BML-284（10 µM；24 小时）显着增加了 β-catenin 的表达。此外，与匹佐替芬治疗组相比，它部分抵消了匹佐替芬对MNK45和AGS细胞中N-和E-钙粘蛋白表达的影响[1]。 BML-284（10 µM；24小时）显着增强MNK45和AGS细胞的迁移和侵袭能力，而苯替夫汀抑制的细胞的迁移和侵袭能力部分恢复[1]。

BML-284 盐酸盐 (10 ng) 与嘧霉胺 (4 mg/L) 联用可部分修复 5.5 hpf 时 Tg (myl7:EGFP) 转基因胚胎中由嘧霉胺诱导的致畸表型和心脏异常。转移到含有 20 个胚胎的平板上 [1]。

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AMBMP在体内靶向CaMKIIβ，https://pmc.ncbi.nlm.nih.gov/articles/PMC7659555/ 研究人员接下来询问AMBMP是否通过增强体内CaMKIIβ活性起作用。用AMBMP处理C3KO和C57BL/6 WT小鼠，然后评估其肌肉中的CaMKIIβ和其他信号通路。通过用对这些信号通路的活性形式特异的抗体进行蛋白质印迹来激活信号传导。AMBMP治疗（每日腹腔注射7.5mg/kg）导致WT和C3KO小鼠的CaMKIIβ激活（图4C和4D）。该药物似乎专门与CaMKIIβ结合，因为它不会激活AKT或AMPK（也不会激活控制肌肉重塑和氧化代谢的其他途径）（图4E和4F）。此外，AMBMP对CaMKIIβ的影响可能是转录后的，Camk2b基因的表达水平没有显著变化（图4G）。因此，这些研究为AMBMP激活CaMKII并随后促进氧化代谢和有益于LGMDR1表型的能力提供了概念证明。

蛋白质印迹分析 [1]
细胞类型：人胃癌细胞系 MNK45 和 AGS
测试浓度： 10 µM
孵育时间：24小时
实验结果：MNK45和AGS细胞中β-catenin表达被诱导，E-cadherin和N-cadherin表达得以保留。

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

91.5

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

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。