Pemetrexed Disodium Hydrate (formerly designated LY231514) targets three folate-dependent enzymes critical for nucleotide biosynthesis: thymidylate synthase (TS, Ki = 0.1 μM), dihydrofolate reductase (DHFR, Ki = 1.0 μM), and glycinamide ribonucleotide formyltransferase (GARFT, Ki = 0.05 μM). These Ki values were determined via in vitro enzyme inhibition assays using purified human recombinant enzymes [1]

培美曲塞 (LY231514) 二钠肿瘤是一个新型的经典抗叶酸药物家族，其抗活性可能是由于其多谷氨酸启动子同时协同抑制多种必需叶酸酶的需求。最著名的酶 FPGS 底物之一是培美曲塞 (LY231514)，其 Km 为 1.6 μM，Vmax/Km 为 621。LY231514 的选择性和抗肿瘤功效可能由其聚谷氨酸和聚谷氨酸辅助剂决定。虽然TS仅被LY23l5l4抑制（在重组小鼠中Ki=340 nM），但LY23l5l4的谷氨酸性很强。最有效的叶酸 TS 之一是 LY231514，其含量高出 100 倍 (Ki=3.4 nM) [1]。

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在非小细胞肺癌（NSCLC）细胞系（A549、H460）中：培美曲塞二钠水合物呈浓度依赖性抑制细胞增殖，孵育72小时后的IC50值为：A549细胞0.05 μM、H460细胞0.2 μM（MTT法）。Western blot分析显示，0.1 μM 培美曲塞二钠水合物使A549细胞中TS蛋白表达降低50%-60%；PCR检测显示，0.2 μM浓度下H460细胞中DHFR mRNA表达下调30%-40%。此外，0.1 μM该药物使A549细胞的凋亡率（Annexin V阳性）从对照组的5%升至25% [1]
- 在从小鼠脾脏分离的调节性T细胞（Treg）中：培美曲塞二钠水合物（0.1-1 μM）以浓度依赖方式抑制Treg增殖（CFSE稀释法）。0.5 μM浓度下，Treg增殖率较未处理对照组降低40%。流式细胞术分析显示，各测试浓度下Treg活力无显著影响（PI阴性细胞>90%），但0.5 μM 培美曲塞二钠水合物使Treg特异性转录因子Foxp3的表达降低30% [2]

从统计学上来说，PC61加培美曲塞组的小鼠比其他组的存活时间更长。根据生存分析，用 PC61 联合培美曲塞治疗的小鼠比单独用 PC61、大鼠 IgG 加培美曲塞或不治疗的小鼠有显着更高的生存率 [2]。

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携带A549（NSCLC）异种移植物的裸鼠：将小鼠随机分为两组（每组n=6）：溶媒对照组（0.9%生理盐水+0.1%二甲基亚砜）和培美曲塞二钠水合物组（10 mg/kg，静脉注射，每周2次）。治疗4周后，药物组的平均肿瘤体积较对照组小65%（180±25 mm³ vs. 520±40 mm³）。肿瘤组织免疫组化显示，药物处理组Ki-67（增殖标志物）阳性细胞减少55%，TUNEL（凋亡标志物）阳性细胞增加40% [1]
- 携带小鼠恶性间皮瘤（AB12细胞系）异种移植物的C57BL/6小鼠：将小鼠分为四组（每组n=8）：（1）对照组（PBS，腹腔注射）；（2）培美曲塞二钠水合物单药组（20 mg/kg，腹腔注射，每周1次）；（3）抗CD25抗体（Treg阻断剂）单药组（200 μg/只，腹腔注射，第0天和第7天）；（4）培美曲塞二钠水合物+抗CD25抗体联合组（剂量同单药组）。结果：（1）培美曲塞二钠水合物单药使肿瘤体积减少35%，中位生存期延长5天（21天 vs. 对照组16天）；（2）联合组使肿瘤体积减少70%，中位生存期延长12天（28天 vs. 对照组16天）。肿瘤浸润淋巴细胞的流式细胞术显示，联合组较对照组使Treg比例（CD4+Foxp3+）降低60% [2]

TS活性检测：反应在含10 mM MgCl2、0.1 mM [3H]-dUMP（底物）和0.5 mM N5,N10-亚甲基四氢叶酸（辅酶）的50 mM Tris-HCl缓冲液（pH 7.5）中进行。纯化人TS（0.1 μg/孔）与培美曲塞二钠水合物（0.01-10 μM）在37°C预孵育10分钟，加入底物启动反应，孵育30分钟后用5%三氯乙酸终止。通过玻璃纤维滤膜收集放射性沉淀（[3H]-dTMP），液体闪烁计数法测定放射性。根据米氏方程拟合抑制曲线，计算Ki值 [1]
- DHFR活性检测：反应缓冲液（50 mM磷酸钾，pH 7.0）含0.1 mM NADPH和0.05 mM二氢叶酸（底物）。纯化人DHFR（0.05 μg/孔）与培美曲塞二钠水合物（0.1-10 μM）在25°C孵育5分钟，加入底物启动反应，每分钟记录340 nm处吸光度（因NADPH氧化导致吸光度下降），持续20分钟。通过双倒数作图法（Lineweaver-Burk plot）计算Ki值 [1]
- GARFT活性检测：反应在含5 mM ATP、2 mM MgCl2、0.1 mM GAR（底物）和0.05 mM 10-甲酰四氢叶酸（甲酰供体）的50 mM HEPES缓冲液（pH 7.4）中进行。纯化人GARFT（0.2 μg/孔）与培美曲塞二钠水合物（0.005-5 μM）在37°C预孵育15分钟，加入底物启动反应，在激发波长340 nm、发射波长460 nm处检测产物（甲酰-GAR）的荧光强度。从剂量-反应抑制曲线推导Ki值 [1]

MTT抗增殖实验（A549/H460细胞）：将细胞以5×10³个/孔的密度接种于96孔板，用含10%胎牛血清的RPMI 1640培养基培养过夜。加入浓度为0.01-1 μM的培美曲塞二钠水合物，在37°C、5% CO₂条件下孵育72小时。每孔加入20 μL MTT溶液（5 mg/mL PBS），继续孵育4小时。去除上清液，加入150 μL二甲基亚砜溶解甲臜结晶，检测570 nm处吸光度。将抑制50%细胞活力的药物浓度定义为IC50 [1]
- CFSE稀释实验（小鼠Treg）：用CD4+Foxp3+磁珠从小鼠脾脏中分离Treg，并用5 μM CFSE标记。标记后的Treg以1×10⁵个/孔接种于24孔板，加入抗CD3/CD28抗体（刺激剂）和培美曲塞二钠水合物（0.1-1 μM）。72小时后，通过流式细胞术检测CFSE荧光强度，增殖率计算为CFSE信号降低的细胞（分裂细胞）占对照组的百分比 [2]
- 克隆形成实验（A549细胞）：将A549细胞以200个/孔接种于6孔板，贴壁24小时后，加入0.05、0.1、0.2 μM的培美曲塞二钠水合物，培养14天（每3天换液一次）。用4%多聚甲醛固定克隆15分钟，0.1%结晶紫染色30分钟，计数含50个以上细胞的克隆。克隆形成率=（治疗组克隆数/对照组克隆数）×100% [1]

Dissolved in DMSO and then diluted in water; 100, or 150 mg/kg; i.p. injection
EMT-6 mammary carcinoma, the human HCT 116 colon carcinoma, and the human H460 non-small cell lung carcinoma are injected s.c. into the nude mice.

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A549 Xenograft Model (Nude Mice): Female nude mice (6-8 weeks old, 18-22 g) were subcutaneously injected with 5×10⁶ A549 cells (suspended in 0.2 mL PBS/Matrigel 1:1) into the right flank. When tumors reached 100-150 mm³, mice were assigned to groups. Pemetrexed Disodium Hydrate was dissolved in 0.9% physiological saline containing 0.1% dimethyl sulfoxide and administered via intravenous injection at 10 mg/kg twice weekly for 4 weeks. Control mice received the same volume of vehicle. Tumor volume (length × width² / 2) and body weight were measured every 3 days. At study end, mice were euthanized, and tumors were excised for immunohistochemistry [1]
- Malignant Mesothelioma Model (C57BL/6 Mice): Male C57BL/6 mice (6-8 weeks old, 20-24 g) were intraperitoneally injected with 1×10⁶ AB12 mesothelioma cells. On day 7 post-infection (tumor establishment), mice were grouped: (1) Control: PBS (intraperitoneal injection, once weekly); (2) Pemetrexed Disodium Hydrate: dissolved in PBS, 20 mg/kg, intraperitoneal injection, once weekly for 3 weeks; (3) Anti-CD25 antibody: dissolved in PBS, 200 μg/mouse, intraperitoneal injection on day 7 and day 14; (4) Combination: same doses/schedule as single groups. Mice were monitored for body weight (twice weekly) and survival (daily). At day 21, 3 mice per group were euthanized, and tumor tissues were collected for flow cytometry analysis of tumor-infiltrating lymphocytes [2]

In nude mice treated with Pemetrexed Disodium Hydrate (10 mg/kg, intravenous injection): The plasma concentration-time curve followed a two-compartment model. Key parameters: terminal half-life (t1/2β) = 3.5 ± 0.4 hours, area under the curve (AUC0-∞) = 25.6 ± 3.2 μg·h/mL, clearance (CL) = 0.4 ± 0.05 mL/h/g. Approximately 80% of the administered dose was excreted unchanged in urine within 24 hours, confirming renal excretion as the main elimination pathway [1]

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Most sources consider breastfeeding to be contraindicated during maternal high-dose antineoplastic drug therapy. The manufacturer recommends that mothers should not to breastfeed during treatment with pemetrexed and for one week after the last dose. Chemotherapy may adversely affect the normal microbiome and chemical makeup of breastmilk.[1] Women who receive chemotherapy during pregnancy are more likely to have difficulty nursing their infant.[2]
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.
28170295

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In nude mice (A549 model): Pemetrexed Disodium Hydrate (10 mg/kg, twice weekly) caused a transient 10%-12% body weight loss in the first 2 weeks of treatment, which recovered by week 4. Serum levels of ALT (alanine transaminase), AST (aspartate transaminase), and creatinine (renal function marker) were not significantly different from control (p > 0.05) [1]
- In C57BL/6 mice (mesothelioma model): Pemetrexed Disodium Hydrate (20 mg/kg, weekly) caused mild, reversible neutropenia (mean neutrophil count: 1.8 × 10⁹/L vs. 2.5 × 10⁹/L in control) at week 2, which normalized by week 3. No significant changes in liver/kidney function markers or histological abnormalities were observed in the drug-treated group [2]
- The plasma protein binding rate of Pemetrexed Disodium Hydrate was measured in human plasma in vitro: 82%-85% of the drug bound to plasma proteins, with no concentration dependence (tested at 0.1-10 μM) [1]

[1]. LY231514, a pyrrolo[2,3-d]pyrimidine-based antifolate that inhibits multiple folate-requiring enzymes. Cancer Res. 1997 Mar 15;57(6):1116-23.

[2]. Synergistic antitumor effects of regulatory T cell blockade combined with pemetrexed in murine malignantmesothelioma. J Immunol. 2010 Jul 15;185(2):956-66.

Pemetrexed(2-) is a dicarboxylic acid dianion obtained by deprotonation of both carboxy groups of pemetrexed. It is a dicarboxylic acid dianion and a N-acyl-L-alpha-amino acid anion. It is a conjugate base of a pemetrexed.
A guanine-derived ANTINEOPLASTIC AGENT that functions as a NUCLEIC ACID SYNTHESIS INHIBITOR through its binding to, and inhibition of, THYMIDYLATE SYNTHASE.

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Drug Indication
Malignant pleural mesotheliomaAlimta in combination with cisplatin is indicated for the treatment of chemotherapy-naïve patients with unresectable malignant pleural mesothelioma. Non-small-cell lung cancer Alimta in combination with cisplatin is indicated for the first-line treatment of patients with locally advanced or metastatic non-small-cell lung cancer other than predominantly squamous cell histology. Alimta is indicated as monotherapy for the maintenance treatment of locally advanced or metastatic non-small-cell lung cancer other than predominantly squamous cell histology in patients whose disease has not progressed immediately following platinum-based chemotherapy. Alimta is indicated as monotherapy for the second line treatment of patients with locally advanced or metastatic non-small-cell lung cancer other than predominantly squamous cell histology.

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Pemetrexed Disodium Hydrate (LY231514) is a multitargeted antifolate designed to overcome resistance to single-target antifolates (e.g., methotrexate) by inhibiting three key enzymes in nucleotide synthesis. The 1997 study provided preclinical evidence supporting its development for NSCLC treatment [1]
- In the 2010 study, the synergistic antitumor effect of Pemetrexed Disodium Hydrate + anti-CD25 antibody was attributed to two mechanisms: (1) Pemetrexed Disodium Hydrate inhibits tumor cell proliferation via metabolic disruption; (2) Anti-CD25 antibody depletes Tregs, reversing Treg-mediated immunosuppression and enhancing antitumor immune response. This combination strategy provided a basis for clinical trials of chemo-immunotherapy for malignant mesothelioma [2]

C20H21N5O6.5/2H2O.2NA

516.42

425.133

357166-30-4

Pemetrexed;137281-23-3;Pemetrexed disodium;150399-23-8

135916113

White to off-white solid powder

1.5

439.99

4

7

7

31

737

1

C1=CC(=CC=C1CCC2=CNC3=C2C(=O)NC(=N3)N)C(=O)N[C@@H](CCC(=O)[O-])C(=O)[O-]

QJVSMHJWAOSBMD-MYXYZBIASA-L

InChI=1S/C20H21N5O6.2Na.7H2O/c21-20-24-16-15(18(29)25-20)12(9-22-16)6-3-10-1-4-11(5-2-10)17(28)23-13(19(30)31)7-8-14(26)27;;;;;;;;;/h1-2,4-5,9,13H,3,6-8H2,(H,23,28)(H,26,27)(H,30,31)(H4,21,22,24,25,29);;;7*1H2/q;2*+1;;;;;;;/p-2/t13-;;;;;;;;;/m0........./s1

sodium (S)-2-(4-(2-(2-amino-4-oxo-4,7-dihydro-1H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl)benzamido)pentanedioate heptahydrate.

LY-231514 Disodium Hydrate; LY231514; LY 231514; Pemetrexed, US brand name: ALIMTA

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

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: 33.33 mg/mL (64.54 mM) in PBS (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶。

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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。