DHFR/dihydrofolate reductase; DNA synthesis; antimetabolite; antifolate

体外活性：甲氨蝶呤 (0.1-10 mM) 诱导人外周血体外活化 T 细胞凋亡。甲氨蝶呤在混合淋巴细胞反应中实现活化 T 细胞的克隆删除。甲氨蝶呤可以通过 CD95 独立途径选择性地删除活化的外周血 T 细胞。甲氨蝶呤通过还原叶酸载体被细胞吸收，然后在细胞内转化为聚谷氨酸盐。甲氨蝶呤会导致离体刺激的中性粒细胞产生的白三烯 B4 减少。甲氨蝶呤聚谷氨酸盐比其他参与嘌呤生物合成的酶更有效地抑制氨基咪唑羧酰胺腺苷核糖核苷酸 (AICAR) 转化酶。甲氨蝶呤还已知通过在体外抑制 TNF 诱导的核因子 -κB 激活来抑制 TNF 活性，部分与减少该因子抑制剂 IκBα 的降解和失活有关，并且可能与腺苷的释放有关。甲氨蝶呤抑制来自健康人类供体和 RA 患者的 T 细胞受体引发的 T 淋巴细胞产生 TNF 和 IFN-γ。甲氨蝶呤治疗与 TNF-α 阳性 CD4+ T 细胞显着减少有关，而表达抗炎细胞因子 IL-10 的 T 细胞数量增加。细胞测定：使用 96 孔微量滴定板在生长抑制实验中研究每种细胞系。由于 antifols 具有时间表依赖性，初步实验旨在确定最长的暴露持续时间，从而允许细胞连续对数期生长而不改变培养基，同时保持 SRB 光密度和细胞数量之间的线性关系。细胞铺板后 24 小时，将细胞系暴露于 antifol 中 120 小时（每个实验重复 3 次）。为了确保可以观察到完整的 S 形生存浓度曲线，研究了以下药物浓度：甲氨蝶呤 (0.002-5 μM)、AMT (0.0001-1 μM)、PXD (0.0003-10 μM)、TLX (0.0002-0.5)微米）。实验至少重复两次。

氨甲喋呤或甲氨蝶呤可降低小鼠的胸腺和脾脏指数。剂量≥5 mg/kg时，甲氨蝶呤显着减少脾脏、胸腺和白细胞。然而，模型组和治疗加对照组差异显着（p <0.01）。很明显，葡萄籽原花青素与西伯利亚人参刺五加苷一起给药可减少甲氨蝶呤对小鼠胸腺和脾脏指数的负面影响[2]。甲氨蝶呤 (MTX)（2 毫克/公斤；腹膜内注射；每周一次）持续五周，可有效治疗弗氏完全佐剂诱发的关节炎。姜黄素（30 mg/kg 和 100 mg/kg，每周 3 次，持续五周；腹腔注射）和甲氨蝶呤（1 mg/kg；腹腔注射；每周一次，持续五周）一起具有很强的抗关节炎作用和保护作用。抗血液毒性[4]。

甲氨蝶呤进入组织，被叶酸聚谷氨酸转化为甲氨蝶呤聚谷氨酸盐。甲氨蝶呤的作用机制是由于它抑制了负责核苷酸合成的酶，包括二氢叶酸还原酶、胸苷酸合酶、氨基咪唑卡巴酰胺核糖核苷酸转化酶（AICART）和氨基磷酸核糖基转移酶。核苷酸合成的抑制会阻止细胞分裂。在类风湿性关节炎中，甲氨蝶呤聚谷氨酸盐比甲氨蝶呤更能抑制AICART。这种抑制导致AICART核糖核苷酸的积累，从而抑制腺苷脱氨酶，导致三磷酸腺苷和腺苷在细胞外空间的积累，刺激腺苷受体，从而产生抗炎作用。

Arthritis was induced in rats following a single subplantar injection of Freund's complete adjuvant (0.1 ml). Rats were divided into six groups of six animals each. Group I and II were control injected with saline and Freund's complete adjuvant (0.1 ml), respectively. Group III arthritic rats were treated with curcumin (100 mg/kg, i.p.) on alternate days. Group IV received methotrexate (MTX) (2 mg/kg, i.p.) once in a week. Group-V and VI were treated with MTX (1 mg/kg, i.p.) once in a week and after 30 min received curcumin (30 mg/kg and 100 mg/kg, thrice a week, i.p.) from 10(th) to 45(th) days, respectively. Body weight and the paw volume was measured on 9(th), 16(th), 23(rd), 30(th), 37(th), and 45(th) days. Determination of complete blood cell counts, hemoglobin concentration, hematocrit, mean corpuscular volume, and mean corpuscular hemoglobin concentration was determined on the 46(th) day. [4]

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The combination of bioactive phytochemicals is administered one week prior to the Methotrexate exposure. Treatment group I: mice are given a combination of green tea polyphenols and eleutherosides from Siberian ginseng (0.2 mL/10 g, i.g. once daily) for 15 days, and a single dose of Methotrexate (2 mg/kg, i.p. once daily) is added on the 8th day. Treatment group II: mice are given a combination of grape seed proanthocyanidins and eleutherosides from Siberian ginseng for 15 days, and Methotrexate is administered on the 8th day in a similar manner. Model group: animals received distilled water instead of bioactive phytochemicals combinations for 15 days and the same Methotrexate protocol applied to this group on the 8th day. Control group: mice are given distilled water through 15 days and physiological saline instead of Methotrexate is administered on the 8th day in a similar manner. Twelve hours after the final doses, the animals are euthanized by cervical dislocation.

Mice

Absorption, Distribution and Excretion
Methotrexate has a bioavailability of 64-90%, though this decreases at oral doses above 25mg due to saturation of the carrier mediated transport of methotrexate.. Methotrexate has a Tmax of 1 to 2 hours. oral doses of 10-15µg reach serum levels of 0.01-0.1µM.
Methotrexate is >80% excreted as the unchanged drug and approximately 3% as the 7-hydroxylated metabolite. Methotrexate is primarily excreted in the urine with 8.7-26% of an intravenous dose appearing in the bile.
The volume of distribution of methotrexate at steady state is approximately 1L/kg.
Methotrexate clearance varies widely between patients and decreases with increasing doses. Currently, predicting clearance of methotrexate is difficult and exceedingly high serum levels of methotrexate can still occur when all precautions are taken.
In adults, oral absorption of methotrexate appears to be dose dependent. Peak serum levels are reached within one to two hours. At doses of 30 mg/sq m or less, methotrexate is generally well absorbed with a mean bioavailability of about 60%. The absorption of doses greater than 80 mg/sq m is significantly less, possibly due to a saturation effect.
After intravenous administration, the initial volume of distribution is approximately 0.18 L/kg (18% of body weight) and steady-state volume of distribution is approximately 0.4 to 0.8 L/kg (40% to 80% of body weight).
Protein binding: Moderate (approximately 50%), primarily to albumin.
At serum methotrexate concentrations exceeding 0.1 umol/mL passive diffusion becomes a major means of intracellular transport of the drug. The drug is widely distributed into body tissues with highest concn in the kidneys, gallbladder, spleen, liver, and skin.
For more Absorption, Distribution and Excretion (Complete) data for METHOTREXATE (10 total), please visit the HSDB record page.

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Metabolism / Metabolites
Methotrexate is metabolized by folylpolyglutamate synthase to methotrexate polyglutamate in the liver as well as in tissues. Gamma-glutamyl hydrolase hydrolyzes the glutamyl chains of methotrexate polyglutamates converting them back to methotrexate. A small amount of methotrexate is also converted to 7-hydroxymethotrexate.
After absorption, methotrexate undergoes hepatic and intracellular metabolism to form methotrexate polyglutamate, metabolites which by hydrolysis may be converted back to methotrexate. Methotrexate polyglutamates inhibit dihydrofolate reductase and thymidylate synthetase. Small amounts of these polyglutamate metabolites may remain in tissues for extended periods; the retention and prolonged action of these active metabolites vary among different cells, tissues, and tumors. In addition, small amounts of methotrexate polyglutamate may be converted to 7-hydroxymethotrexate; accumulation of this metabolite may become substantial following administration of high doses of methotrexate, since the aqueous solubility of 7-hydroxymethotrexate is threefold to fivefold lower than that of the parent compound. Following oral administration of methotrexate, the drug also is partially metabolized by the intestinal flora.
After absorption, methotrexate undergoes hepatic and intracellular metabolism to form methotrexate polyglutamate, metabolites which by hydrolysis may be converted back to methotrexate. Methotrexate polyglutamates inhibit dihydrofolate reductase and thymidylate synthetase. Small amounts of these polyglutamate metabolites may remain in tissues for extended periods; the retention and prolonged action of these active metabolites vary among different cells, tissues, and tumors. In addition, small amounts of methotrexate polyglutamate may be converted to 7-hydroxymethotrexate; accumulation of this metabolite may become substantial following administration of high doses of methotrexate, since the aqueous solubility of 7-hydroxymethotrexate is threefold to fivefold lower than that of the parent compound. Following oral administration of methotrexate, the drug also is partially metabolized by the intestinal flora. Renal excretion is the primary route of elimination, and is dependent upon dosage and route of administration (A620).
Route of Elimination: Renal excretion is the primary route of elimination and is dependent upon dosage and route of administration. IV administration, 80% to 90% of the administered dose is excreted unchanged in the urine within 24 hours. There is limited biliary excretion amounting to 10% or less of the administered dose.
Half Life: Low doses (less than 30 mg/m^2): 3 to 10 hours; High doses: 8 to 15 hours.

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Biological Half-Life
The half life of low dose methotrexate is 3 to 10 hours in adults. The half life for high dose methotrexate is 8 to 15 hours. Pediatric patients taking methotrexate for acute lymphoblastic anemia experience a terminal half life of 0.7 to 5.8 hours. Pediatric patients taking methotrexate for juvenile idiopathic arthritis experience a half life of 0.9 to 2.3 hours.
Terminal: Low doses: 3 to 10 hours. High doses: 8 to 15 hours. Note: There is wide interindividual variation in clearance rates. Small amounts of methotrexate and its metabolites are protein-bound and may remain in tissues (kidneys, liver) for weeks to months; the presence of fluid loads, such as ascites or pleural effusion, and renal function impairment will also delay clearance.

Toxicity Summary
Methotrexate anti-tumor activity is a result of the inhibition of folic acid reductase, leading to inhibition of DNA synthesis and inhibition of cellular replication. The mechanism involved in its activity against rheumatoid arthritis is not known.

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Toxicity Data
Man(iv): TD: 740 mg/kg
Mouse(ip): LD50 mg/kg
Rat(po): LD50 135 mg/kg
Rat(ip): LD50 6 mg/kg
LD50: 43 mg/kg (Oral, Rat) (A308)

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Interactions
Oral neomycin may decreases absorption of oral methotrexate.
Severe, sometimes fatal, toxicity (including hematologic and GI toxicity) has occurred following administration of a non-steroidal anti-inflammatory agent (eg, indomethacin, ketoprofen) concomitantly with methotrexate (particularly with high dose therapy) in patients with various malignant neoplasms, psoriasis, or rheumatoid arthritis.
Concomitant use of penicillins (e.g., amoxicillin, carbenicillin, mezlocillin) may decrease renal clearance of methotrexate, presumably by inhibiting renal tubular secretion of the drug. Increased serum concentrations of methotrexate, resulting in GI or hematologic toxicity, have been reported in patients receiving low- or high-dose methotrexate therapy concomitantly with penicillins, and patients receiving the drugs concomitantly should be carefully monitored.
Concurrent adminstration of intrathecal methotrexate and acyclovir may result in neurological abnormalities; use with caution.
For more Interactions (Complete) data for METHOTREXATE (16 total), please visit the HSDB record page.

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Non-Human Toxicity Values
LD50 Rat oral 180 +/- 45 mg/kg body weight
LD50 Rat ip 6-25 mg/kg body weight
LD50 Mice ip 94 +/- 9 mg/kg body weight

[1]. Understanding the mechanisms of action of methotrexate: implications for the treatment of rheumatoid arthritis. Bull NYU Hosp Jt Dis. 2007;65(3):168-73.

[2]. Methotrexate in rheumatoid arthritis. Pharmacol Rep. 2006 Jul-Aug;58(4):473-92.

[3]. The Effect of L-carnitine on Amethopterin-induced Toxicity in Rat Large Intestine.

[4]. Evaluation of the concomitant use of methotrexate and curcumin on Freund's complete adjuvant-induced arthritis and hematological indices in rats. Indian J Pharmacol. 2011;43(5):546-550.

Therapeutic Uses
Abortifacient Agents, Nonsteroidal; Antimetabolites, Antineoplastic; Antirheumatic Agents; Dermatologic Agents; Enzyme Inhibitors; Folic Acid Antagonists; Immunosuppressive Agents; Nucleic Acid Synthesis Inhibitors
Methotrexate is indicated for treatment of breast carcinoma, head and neck cancers (epidermoid), non-small cell lung carcinoma (especially squamous cell types), small cell lung carcinoma, and gestational trophoblastic tumors (gestational choriocarcinoma, chorioadenoma destruens, hydatidiform mole). /Included in US product labeling/
Methotrexate is indicated for treatment of cervical carcinoma, ovarian carcinoma, bladder carcinoma, colorectal carcinoma, esophageal carcinoma, gastric carcinoma, pancreatic carcinoma, and penile carcinoma. /NOT included in US product labeling/
Methotrexate is indicated for treatment of acute lymphocytic leukemia and prophylaxis and treatment of meningeal leukemia. /Included in US product labeling/
For more Therapeutic Uses (Complete) data for METHOTREXATE (17 total), please visit the HSDB record page.

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Drug Warnings
Methotrexate is a highly toxic drug with a very low therapeutic index and a therapeutic response is not likely to occur without some evidence of toxicity. ... When methotrexate is used in combination with other antineoplastic agents and/or radiation therapy, toxic reactions may be more severe than would occur with methotrexate therapy alone. Although doses of methotrexate used in the management of psoriasis and rheumatoid arthritis are usually lower than those used in antineoplastic chemotherapy, severe toxicity may occur in any patient receiving the drug and deaths have been reported with the use of methotrexate in the management of psoriasis and rheumatoid arthritis.
Methotrexate should be used with extreme caution in patients with infection, peptic ulcer, ulcerative colitis, or debility, and in very young or geriatric patients. Methotrexate should be used with extreme caution, if at all, in patients with malignant disease who have preexisting liver damage or impaired hepatic function, preexisting bone marrow depression, aplasia, leukopenia, thrombocytopenia, or anemia; the drug is usually contraindicated in patients with impaired renal function. In the management of psoriasis, methotrexate is contraindicated in patients with poor nutritional status or severe renal or hepatic disorders, those with overt or laboratory evidence of an immunodeficiency syndrome, and in those with preexisting blood dyscrasias such as bone marrow hypoplasia, leukopenia, thrombocytopenia, or clinically important anemia; relative contraindications also include cirrhosis, active or recent hepatitis, or excessive alcohol consumption. In the management of rheumatoid arthritis, methotrexate is contraindicated in patients with preexisting blood dyscrasias such as bone marrow hypoplasia, leukopenia, thrombocytopenia, or clinically important anemia; those with overt or laboratory evidence of immunodeficiency syndromes; and those with excessive alcohol consumption, alcoholic liver disease, or chronic liver disease.
Elevations in serum uric acid concentrations may occur in patients receiving methotrexate as a result of cell destruction and hepatic and renal damage. In some patients, uric acid nephropathy and acute renal failure may result. Tumor lysis syndrome associated with other cytotoxic drugs (e.g., fludarabine, cladribine), also has been reported in patients with rapidly growing tumors who were receiving methotrexate. Pharmacologic and appropriate supportive treatment may prevent or alleviate this complication. Methotrexate also was reported to precipitate acute gouty arthritis in two patients being treated for psoriasis. Administration of large volumes of fluids, alkalinization of the urine, and/or administration of allopurinol may be useful in preventing acute attacks of hyperuricemia and uric acid nephropathy.
Severe nephropathy manifested by azotemia, hematuria, and renal failure may occur in patients receiving methotrexate; fatalities have been reported. In one study, postmortem examination revealed extensive necrosis of the epithelium of the convoluted tubules. In patients with renal impairment, methotrexate accumulation and increased toxicity or additional renal damage may occur.
For more Drug Warnings (Complete) data for METHOTREXATE (22 total), please visit the HSDB record page.

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Pharmacodynamics
Methotrexate inhibits enzymes responsible for nucleotide synthesis which prevents cell division and leads to anti-inflammatory actions. It has a long duration of action and is generally given to patients once weekly. Methotrexate has a narrow therapeutic index. Do not take methotrexate daily.

C20H22N8O5

454.44

454.171

C, 52.86; H, 4.88; N, 24.66; O, 17.60

59-05-2

Methotrexate disodium;7413-34-5;Methotrexate hydrate;133073-73-1;Methotrexate monohydrate;6745-93-3; Methotrexate-d3; 432545-63-6; 7413-34-5 (disodium); 7532-09-4 (monosodium); 15475-56-6 (sodium); 59-05-2 (free acid)

126941

Light yellow to yellow solid powder

1.4080

561.26°C

195°C

11℃

1.6910

-0.24

210.54

5

12

9

33

704

1

CN(CC1=CN=C2C(=N1)C(=NC(=N2)N)N)C3=CC=C(C=C3)C(=O)N[C@@H](CCC(=O)O)C(=O)O

FBOZXECLQNJBKD-ZDUSSCGKSA-N

InChI=1S/C20H22N8O5/c1-28(9-11-8-23-17-15(24-11)16(21)26-20(22)27-17)12-4-2-10(3-5-12)18(31)25-13(19(32)33)6-7-14(29)30/h2-5,8,13H,6-7,9H2,1H3,(H,25,31)(H,29,30)(H,32,33)(H4,21,22,23,26,27)/t13-/m0/s1

(S)-2-(4-(((2,4-diaminopteridin-6-yl)methyl)(methyl)amino)benzamido)pentanedioic acid.

alphamethopterin; amethopterin; methylaminopterin; CL 14377; NCIC04671; WR19039; WR-19039; Rheumatrex; Metatrexan; Hdmtx; Abitrexate; WR 19039; MTX; NCI-C04671; NCI C04671; CL14377; CL-14377; Methotrexate.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 本产品在运输和储存过程中需避光。

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

配方 1 中的溶解度: ≥ 2.5 mg/mL (5.50 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (5.50 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (5.50 mM) (饱和度未知) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 4 中的溶解度: ≥ 2.5 mg/mL (5.50 mM) (饱和度未知) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 5 中的溶解度: 2% DMSO+30% PEG 300+5% Tween 80+ddH2O:5 mg/mL

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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。