甲氨蝶呤（Methotrexate）是一种抗叶酸类抗代谢药，有效抑制二氢叶酸还原酶（DHFR）活性，IC50约为7nM。
Cas No.:59-05-2
Sample solution is provided at 25 µL, 10mM.
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Methotrexate is an antifolate antimetabolite that effectively inhibits the activity of dihydrofolate reductase (DHFR), with an IC50 of approximately 7nM [1]. Folic acid is essential for the biosynthesis of purine and pyrimidine bases, so methotrexate interferes with the synthesis of purines and pyrimidines required for DNA replication and cell proliferation [2]. Methotrexate also inhibits adenosine deaminase (ADA), resulting in increased extracellular adenine nucleotide levels [3]. Methotrexate has anticancer, antirheumatic, anti-inflammatory and immunomodulatory activities [4].
In vitro, methotrexate (5 μg/ml) has a wide range of IC50s against various cancer cell lines, from 6.05±0.81 nM to >1,000 nM. Methotrexate resistance in Saos-2 and MCF-7 cells The most potent, AGS and HCT-116 cells are highly sensitive to methotrexate, with IC50 of 6.05±0.81nM and 13.56±3.76nM respectively. NCI-H23 and A549 cells show similar sensitivity to methotrexate [5]. Methotrexate (3ng/ml-1μg/ml) measured the degree of drug resistance of the U-2OS cell line and found that the cell resistance was related to the increase in intracellular DHFR levels [6].
In vivo, treatment of arthritic DBA/1J mice with methotrexate (2-50 mg/kg; s.c.) dose-dependently reduced disease activity and mean paw volume and increased mean body weight percentage [7]. Methotrexate (2 mg/kg; i.p.) treats arthritis in rats and has significant anti-arthritic effects and prevents the occurrence of hematological toxicity [8].
References:
[1]Gurdag S, Khandare J, Stapels S, et al. Activity of dendrimer−methotrexate conjugates on methotrexate-sensitive and-resistant cell lines[J]. Bioconjugate chemistry, 2006, 17(2): 275-283.
[2] Rajagopalan P T R , Zhang Z , Mccourt L ,et al.Interaction of dihydrofolate reductase with methotrexate: Ensemble and single-molecule kinetics[J].Proceedings of the National Academy of Sciences, 2002, 99(21):13481-13486.
[3] Sramek M, Neradil J, Veselska R. Much more than you expected: the non-DHFR-mediated effects of methotrexate[J]. Biochimica et Biophysica Acta (BBA)-General Subjects, 2017, 1861(3): 499-503.
[4] Bedoui Y, Guillot X, Sélambarom J, et al. Methotrexate an old drug with new tricks[J]. International journal of molecular sciences, 2019, 20(20): 5023.
[5] Yoon S A , Choi J R , Kim J O ,et al. Influence of Reduced Folate Carrier and Dihydrofolate Reductase Genes on Methotrexate-Induced Cytotoxicity[J].Cancer Research & Treatment, 2010, 42(3):163-171.
[6]Serra M , Reverter B G , Maurici D ,et al.Analysis of dihydrofolate reductase and reduced folate carrier gene status in relation to methotrexate resistance in osteosarcoma cells.[J].Annals of oncology, 2004.
[7] Singh, Rakesh K.van Haandel, LeonKiptoo, et al . Methotrexate disposition, anti-folate activity and efficacy in the collagen-induced arthritis mouse model[J].European Journal of Pharmacology: An International Journal, 2019, 853.
[8] Banji D , Banji O F , Reddy K , et al. Evaluation of the concomitant use of methotrexate and curcumin on Freund's complete adjuvant-induced arthritis and hematological indices in rats[J]. Indian Journal of Pharmacology, 2011, 43(5):546-550.
甲氨蝶呤（Methotrexate）是一种抗叶酸类抗代谢药，有效抑制二氢叶酸还原酶（DHFR）活性，IC50约为7nM[1]。叶酸对于嘌呤和嘧啶碱的生物合成至关重要，因此甲氨蝶呤会干扰DNA复制和细胞增殖所需的嘌呤和嘧啶合成[2]。甲氨蝶呤还会抑制腺苷脱氨酶（ADA），导致细胞外腺嘌呤核苷酸水平升高[3]。甲氨蝶呤具有抗癌、抗风湿、抗炎和免疫调节活性[4]。
在体外，甲氨蝶呤（5μg/ml）对各种癌细胞系的IC50范围很广，从6.05±0.81nM 到>1,000nM， Saos-2和MCF-7细胞对甲氨蝶呤的耐药性最强，AGS和 HCT-116细胞对甲氨蝶呤高度敏感，IC50分别为6.05±0.81nM和13.56±3.76nM。NCI-H23和A549细胞对甲氨蝶呤表现出相似的敏感性[5]。甲氨蝶呤（3ng/ml-1μg/ml）测量U-2OS细胞系的耐药程度，发现该细胞耐药性与胞内DHFR水平的增加有关[6]。
在体内，甲氨蝶呤（2-50mg/kg; s.c.）治疗关节炎DBA/1J小鼠后，剂量依赖性地降低了疾病活动性和平均爪体积，增加了平均体重百分比[7]。甲氨蝶呤（2 mg/kg;i.p.）治疗大鼠关节炎，具有显著的抗关节炎作用，并且预防了血液毒性的发生[8]。
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Cell experiment [1]: |
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Cell lines |
MCF-7, AGS, A549, NCI-H23, HCT-116 and Saos-2 cell lines |
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Preparation method |
The cytotoxicity of Methotrexate was repeatedly tested at 8 different concentrations for control: 0.03, 0.1, 0.3, 1, 3, 10, 30 and 100µg/mL. The cells were incubated for 24h to allow sufficient cell adhesion, and all the microplates were incubated for a total of 72h after Methotrexate administration. |
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Reaction Conditions |
0.03, 0.1, 0.3, 1, 3, 10, 30 and 100µg/mL; 72h |
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Applications |
The IC50s of Methotrexate was in an extensively broad range from 6.05±0.81nM to>1,000nM in the cell lines. The Saos-2 and MCF-7 cells were most resistant to Methotrexate; in contrast, the AGS and HCT-116 cells were highly sensitive to Methotrexate with an IC50 of 6.05±0.81nM and 13.56±3.76nM, respectively. The NCI-H23 and A549 cells demonstrated similar sensitivity to Methotrexate. |
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Animal experiment [2]: |
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Animal models |
Male DBA/1J mice |
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Preparation method |
The Methotrexate treatment group were further stratified into 4 subgroups and administered subcutaneous Methotrexate at doses of 2, 10, 20, or 50mg/kg weekly starting on day 14 for a total of 6 doses. Disease activity scores, paw volume measurements, and animal weight were collected. |
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Dosage form |
2, 10, 20, or 50mg/kg; s.c. |
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Applications |
Methotrexate treatment resulted in a reduction in disease activity that was variable and dose-dependent. Mean paw volume measurements were significantly reduced in mice treated with Methotrexate. Mean percent body weight increased significantly in mice treated with Methotrexate. |
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References: [1] Yoon S A , Choi J R , Kim J O ,et al. Influence of Reduced Folate Carrier and Dihydrofolate Reductase Genes on Methotrexate-Induced Cytotoxicity[J].Cancer Research & Treatment, 2010, 42(3):163-171. [2] Singh, Rakesh K.van Haandel, LeonKiptoo, et al . Methotrexate disposition, anti-folate activity and efficacy in the collagen-induced arthritis mouse model[J].European Journal of Pharmacology: An International Journal, 2019, 853. |
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| Cas No. | 59-05-2 | SDF | |
| 别名 | 甲氨蝶呤; Amethopterin; CL14377; WR19039 | ||
| 化学名 | (2S)-2-[[4-[(2,4-diaminopteridin-6-yl)methyl-methylamino]benzoyl]amino]pentanedioic acid | ||
| Canonical SMILES | CN(CC1=CN=C2C(=N1)C(=NC(=N2)N)N)C3=CC=C(C=C3)C(=O)NC(CCC(=O)O)C(=O)O | ||
| 分子式 | C20H22N8O5 | 分子量 | 454.44 |
| 溶解度 | ≥ 21.55 mg/mL in DMSO | 储存条件 | 4°C, protect from light |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
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| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
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1 mg | 5 mg | 10 mg |
| 1 mM | 2.2005 mL | 11.0026 mL | 22.0051 mL |
| 5 mM | 440.1 μL | 2.2005 mL | 4.401 mL |
| 10 mM | 220.1 μL | 1.1003 mL | 2.2005 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。
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- Purity: >98.00% Appearance: A solid
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