Pemetrexed disodium   中文名称: 培美曲塞二钠

TS ( Ki = 1.3 nM ); DHFR ( Ki = 7.2 nM ); GARFT ( Ki = 1.3 nM )
Pemetrexed disodium targets three key folate-dependent enzymes involved in 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 [1]
- Pemetrexed disodium inhibits folate-dependent enzymes including TS (IC50 = 0.08-0.5 μM in colorectal cancer cell lines), DHFR (IC50 = 0.8-1.2 μM), and GARFT (IC50 = 0.04-0.06 μM). The IC50 values vary slightly depending on the cell line or enzyme source [2]
- Pemetrexed disodium exerts its effects by binding to TS, DHFR, and GARFT[3]

Pemetrexed disodium 对 CCRF-CEM 白血病、GC3/C1 结肠癌和 HCT-8 回盲部癌细胞具有抗增殖活性，IC50 分别为 25 nM、34 nM 和 220 nM。最近的一项研究表明，顺铂加培美曲塞联合 SOCS-1 基因递送在感染腺病毒表达 SOCS-1 载体的 MPM 细胞中显示出通过抑制细胞增殖、侵袭和诱导细胞凋亡的抗肿瘤作用。激酶测定：使用分光光度法测定 TS 活性，该方法涉及监测因产物 7,8-二氢叶酸形成而导致的 340 nm 处吸光度的增加。测定缓冲液含有 50 mM N-三[羟甲基甲基-2-氨基乙磺酸、25 mM MgCl2、6.5 mM 甲醛、1 mM EDTA 和 75 mM 2-巯基乙醇，pH 7.4。脱氧尿苷酸单磷酸、6R-MTHF 和 hIS 的浓度分别为 100 μM、30μM 和 30 nM（1.7 毫单位/mL）。在 6R-MTHF 浓度下，测定了未抑制的反应和六种浓度的抑制剂。 Ki app 值是通过借助 ENZFITTER 程序使用非线性回归分析将数据拟合到 Morrison 方程来确定的。 Ki 值的计算公式为：Ki app= Ki(1 + [S]/Km)，其中 [S] 等于 30 μM，Km 等于 3 μM。通过在 340 nm 处监测底物 NADPH 和 7,8-二氢叶酸的消失，以分光光度法测定 DHFR 活性。该反应在 25°C 下在 0.5 mL 50 mM 磷酸钾缓冲液中进行，其中含有 150 mM KCl 和 10 nM 2-巯基乙醇，pH 7.5，以及 14 nM（0.34 毫单位毫升）DHFR。 NADPH 浓度为 10 μM，7,8-二氢叶酸浓度为 5、10 或 15 μM。在每个 7,8-二氢叶酸浓度下，检测未抑制的反应和七个浓度的抑制剂。采用ENZFITIER微机程序，通过非线性回归分析将数据拟合到Morrison方程中，得到Ki app 值。 Ki app= Ki(1 + [S]/Km)，其中[S]等于所用7,8-二氢叶酸的浓度，7,8-二氢叶酸的Km等于0.15 μM。 GARFT 活性通过分光光度法监测 295 nm 处产物 5,8-二脱氮杂叶酸形成导致的吸光度增加来测定。反应溶剂含有 75 mM HEPES、20% 甘油和 50 mM α-硫代甘油，pH 7.5，25°C。使用的底物和酶的浓度为 10 μM α,β-甘氨酰胺核糖核苷酸、0-10 μM 10-甲酰基-5,8-二脱氮杂叶酸和 10 nM（1.9 毫单位/mL）GARFT。 Ki 值使用 Beckman DU640 分光光度计的 Enzyme Mechanism 程序计算，该程序使用非线性回归分析将数据拟合到竞争性抑制的 Michaelis-Menten 方程。细胞测定：生成剂量反应曲线以确定 50% 生长抑制 (IC50) 所需的浓度。培美曲塞二钠最初以 4 mg/mL 的浓度溶解在 DMSO 中，并用细胞培养基进一步稀释至所需浓度。将完全培养基中的 CCRF-CEM 白血病细胞添加到 24 孔簇板中，总体积为 2.0 mL。将不同浓度的培美曲塞二钠添加到重复的孔中，使得DMSO的最终体积为0.5%。将板在 37°C、含 5% CO2 的空气中孵育 72 小时。孵育结束时，在 ZBI Coulter 计数器上测定细胞数量。对于多项研究，在 300 μM AICA、5 μM 胸苷、100 μM 次黄嘌呤或 5 μM 乙二苷加 100 μM 次黄嘌呤组合存在下测定每种化合物的 IC50。对于贴壁肿瘤细胞，使用对原始 MTT 比色测定法的修改来测量细胞毒性。将人肿瘤细胞接种在 96 孔平底组织培养板中，每孔 100 μL 测定培养基中。检测培养基含有不含叶酸的 RPMI 1640，辅以 10% FCS 和 2 nM 亚叶酸或 2.3 μM 叶酸作为唯一的叶酸来源。 1A 留空。在 Dulbeccos PBS 中制备 1 mg/mL 的抗叶酸剂储备溶液，随后在 PBS 中进行一系列 2 倍稀释。将每个浓度的 10 μL 等分试样添加到一式三份的孔中。将板在 37°C、空气中含有 5% CO2 的潮湿气氛中孵育 72 小时。将 MTT 以 5 mg/mL 溶解在 PBS 中，向测定的每个孔中添加 10 μL MTF 库存溶液，并将板在 37 °C 下再孵育 2 小时。孵育后，向每个孔中添加 100 μL DMSO。甲臜彻底溶解后，在 Dynatech MR600 读数器上读取板，使用 570 nm 的测试波长和 630 nm 的参考波长。 IC50 确定为与未处理对照相比抑制细胞生长 50% 所需的药物浓度。

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在非小细胞肺癌（NSCLC）细胞系（A549、H460）中，培美曲塞二钠的抗增殖活性IC50值为：A549细胞0.05 μM、H460细胞0.2 μM（MTT法，孵育72小时）。Western blot分析显示，0.1 μM浓度下TS蛋白表达降低40%-60%；PCR检测显示，0.2 μM浓度下DHFR mRNA表达下调30%-50% [1]
- 在人结直肠癌细胞系（HT-29、SW480）中，培美曲塞二钠抑制细胞活力的IC50值为：HT-29细胞0.08 μM、SW480细胞0.3 μM（CCK-8法）。Annexin V/PI染色显示，0.2 μM浓度下凋亡细胞比例达30%-40%；免疫细胞化学检测显示，0.1 μM浓度处理后，60%-70%细胞的嘌呤合成关键酶GARFT核表达降低 [2]
- 在NSCLC细胞系H1299的克隆形成实验中，0.1 μM的培美曲塞二钠使克隆数较对照组减少70%（孵育14天）。此外，0.2 μM浓度处理48小时后，胱天蛋白酶-3（caspase-3）活性升高2.5倍（比色法检测），表明凋亡增强 [3]

在人 H460 非小细胞肺癌异种移植物中，培美曲塞二钠产生持续时间依赖性肿瘤生长延迟 (TGD)。

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在携带A549（NSCLC）异种移植物的裸鼠中，培美曲塞二钠以10 mg/kg剂量静脉注射，每周2次，持续4周。治疗组的平均肿瘤体积比溶媒对照组小60%-70%；肿瘤组织免疫组化分析显示，TS阳性细胞比例降低50%-60% [1]
- 在携带HT-29（结直肠癌）异种移植物的裸鼠中，培美曲塞二钠以15 mg/kg剂量静脉注射，每周1次，持续3周。肿瘤重量抑制率为50%-60%；血清嘌呤代谢物（如次黄嘌呤）水平较对照组降低40%，证实其在体内抑制嘌呤合成 [2]

TS 活性通过分光光度法测量，需要跟踪 7,8-二氢叶酸的产生所带来的 340 nm 处吸光度的增加。测定缓冲液的成分如下：25 mM MgCl₂、6.5 mM 甲醛、1 mM EDTA、75 mM 2-巯基乙醇、50 mM N-三[羟甲基·甲基-2-氨基乙磺酸]。 hIS、6R-MTHF 和脱氧尿苷酸单磷酸的浓度分别为 30 μM、100 μM 和 30 nM（1.7 毫单位/mL）。在 6R-MTHF 浓度下测试未抑制的反应和六种抑制剂浓度。 K_{i app} 值是通过应用非线性回归分析并在 ENZFITTER 程序的帮助下将数据拟合到 Morrison 方程而获得的。 K_{i app}= K_i(1 + [S]/K_m 是用于计算 K_i 的公式> 值，其中 [S] 相当于 30 μM，K_m 相当于 3 μM。使用分光光度法，通过在 340 nm 处追踪 7,8-二氢叶酸和 NADPH 的消失来测量 DHFR 活性. 在 0.5 mL 50 mM 磷酸钾缓冲液中，pH 7.5、150 mM KCl、10 nM 2-巯基乙醇和 14 nM (0.34 milliunitlmL) DHFR 存在于反应过程中，反应温度为 25°C。10，μM 是NADPH 的浓度，而 5、10 或 15 μM 是 7,8-二氢叶酸的浓度。在每个 7,8-二氢叶酸浓度下测量七个抑制剂浓度和一个不受抑制的反应。K_{i app}使用非线性回归分析和 ENZFITI'ER 微机程序将数据拟合到 Morrison 方程即可获得数值。K_{i app}= K_i(1 + [S]/ K_m)，其中 7,8-二氢叶酸的 K_m 等于 0.15 μM，[S] 是所用 7,8-二氢叶酸的浓度。 GARFT 活性通过分光光度法通过追踪 295 nm 处吸光度的升高来测量，该升高是由产物 5,8-二脱氮杂叶酸的形成引起的。在 25°C 和 pH 7.5 下，反应溶剂由 50% α-硫代甘油、20% 甘油和 75 mM HEPES 组成。使用以下浓度的底物和酶：10 μM α,β-甘氨酰胺核糖核苷酸、0-10 μM 10-甲酰基-5,8-二脱氮杂叶酸和 10 nM（1.9 毫单位/mL）GARFT。 Ki 值使用 Beckman DU640 分光光度计的酶机制程序确定，该程序使用非线性回归分析将数据拟合到用于竞争性抑制的 Michaelis-Menten 方程。

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胸苷酸合成酶（TS）活性检测：反应缓冲液含50 mM Tris-HCl（pH 7.5）、10 mM MgCl2和0.1 mM [3H]-dUMP（底物）。将纯化的人TS酶与不同浓度的培美曲塞二钠（0.01-10 μM）在37°C孵育10分钟，随后加入0.5 mM N5,N10-亚甲基四氢叶酸（辅酶）。孵育30分钟后，用5%三氯乙酸终止反应，通过闪烁计数器检测放射性沉淀（产物[3H]-dTMP）的量。根据米氏方程拟合抑制曲线，计算Ki值 [1]
- 二氢叶酸还原酶（DHFR）活性检测：反应体系含100 mM磷酸钾缓冲液（pH 7.0）、0.1 mM NADPH和0.05 mM二氢叶酸（底物）。将纯化的DHFR与培美曲塞二钠（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10分钟。根据药物组相对于对照组（无药物）的酶活性变化，计算Ki值 [1]
- 甘氨酰胺核糖核苷酸甲酰基转移酶（GARFT）活性检测：采用荧光法，以0.1 mM甘氨酰胺核糖核苷酸（GAR）为底物，0.05 mM 10-甲酰四氢叶酸为甲酰供体。反应缓冲液为含5 mM ATP和2 mM MgCl2的50 mM HEPES（pH 7.4）。将培美曲塞二钠（0.01-5 μM）与GARFT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。根据剂量-反应曲线计算IC50值 [2]

为了确定 50% 生长抑制所需的浓度，绘制了剂量反应曲线 (IC50)。首先，将4 mg/mL的培美曲塞二钠溶解在DMSO中，然后用细胞培养基进一步稀释浓度。 24 孔簇板用完全培养基中的 CCRF-CEM 白血病细胞填充至总容量 2.0 mL。重复孔中充满不同浓度的培美曲塞二钠，直到 DMSO 的总体积为 0.5%。将板在 37°C、含有 5% CO₂ 的空气气氛中孵育 72 小时。孵育结束时测量 ZBI Coulter 计数器上的细胞计数。 300 μM 的 AICA、5 μM 的胸苷、100 μM 的次黄嘌呤或 5 μM 乙二苷和 100 μM 次黄嘌呤的组合是在多项研究中发现每种化合物的 IC50 的条件。 MTT 比色测定经过修改，可量化贴壁肿瘤细胞的细胞毒性。使用平底 96 孔组织培养板以 100 μL 检测培养基/孔的密度接种人类肿瘤细胞。在测定培养基中，叶酸的唯一来源是 2.3 μM 或 2 nM 叶酸，以及 10% FCS 和不含叶酸的 RPMI 1640。1A 孔中留空。在 Dulbecco PBS 中制备抗叶酸储备液（每毫升一毫克），然后在 PBS 中连续稀释 2 倍。一式三份的孔中填充有每种浓度的 10 μL 等分试样。将板在空气中含有 5% CO₂ 的湿润气氛中于 37 °C 孵育 72 小时。 MTT 以 5 mg/mL 的浓度溶解在 PBS 中后，将 10 µL MTF 库存溶液添加到测定的每个孔中。然后将板在 37°C 下再孵育两小时。孵育后每孔加入 100 μL DMSO。在甲臜完全溶解后，使用 570 nm 的测试波长和 630 nm 的参考波长在 Dynatech MR600 读数器上读取板。与未经处理的对照相比，阻碍细胞生长 50% 所需的药物量称为 IC50。

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MTT抗增殖实验（A549和H460细胞）：将细胞以5×10³个/孔的密度接种于96孔板，在含10%胎牛血清的RPMI 1640培养基中培养过夜。加入浓度范围为0.01-1 μM的培美曲塞二钠，在37°C、5% CO2条件下孵育72小时。孵育后，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，用酶标仪检测570 nm处吸光度，将抑制细胞活力50%的药物浓度定义为IC50 [1]
- Annexin V/PI凋亡实验（HT-29细胞）：将HT-29细胞以2×10⁵个/孔接种于6孔板，用培美曲塞二钠（0.05、0.1、0.2 μM）处理48小时。用胰酶消化收集细胞，冷PBS洗涤2次，重悬于结合缓冲液中。向细胞悬液中加入Annexin V-FITC和碘化丙啶（PI），室温避光孵育15分钟，通过流式细胞术分析凋亡细胞比例（Annexin V阳性/PI阴性及Annexin V阳性/PI阳性细胞） [2]
- 克隆形成实验（H1299细胞）：将H1299细胞以200个/孔接种于6孔板，贴壁24小时后，加入浓度为0.05、0.1、0.2 μM的培美曲塞二钠，培养14天（每3天换液一次）。孵育结束后，用4%多聚甲醛固定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% [3]

Mice: The mice used are female CBA mice and female NOD/SCID mice (NOD.CB17-Prkdc scid ) that are 6–8 weeks old. In order to investigate the synergistic effect of premetrexed (100 mg/kg) in combination with anti-CD25 Ab or IgG control, tumor-bearing mice receive it intraperitoneally (i.p.) from days 4–8 (5 consecutive days). Based on earlier research conducted on mice, the current study's Pemetrexed dosage and schedule were chosen.

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A549 Xenograft Model in 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 mixed with Matrigel at 1:1 ratio) into the right flank. When tumors reached a volume of 100-150 mm³, mice were randomly divided into two groups (n=6 per group): treatment group and vehicle control group. Pemetrexed disodium was dissolved in 0.9% physiological saline containing 0.1% dimethyl sulfoxide and administered via intravenous injection at a dose of 10 mg/kg twice weekly for 4 weeks. The control group received the same volume of vehicle. Tumor volume was measured every 3 days using a caliper (volume = length × width² / 2), and body weight was recorded weekly to monitor toxicity. At the end of the experiment, mice were euthanized, and tumors were excised for weight measurement and histological analysis [1]
- HT-29 Xenograft Model in Nude Mice: Male nude mice (6-8 weeks old, 20-24 g) were subcutaneously implanted with 1×107 HT-29 cells (in 0.2 mL PBS/Matrigel 1:1) into the left flank. When tumors grew to 200-250 mm³, mice were assigned to two groups (n=5 per group). Pemetrexed disodium was dissolved in 5% glucose solution and administered intravenously at 15 mg/kg once weekly for 3 weeks. The control group received 5% glucose solution. Tumor volume and body weight were measured twice weekly. After treatment, mice were sacrificed, and tumors were collected for immunohistochemistry and metabolite analysis [2]

In nude mice treated with Pemetrexed disodium (10 mg/kg, intravenous injection), the plasma concentration-time profile showed a two-compartment model. The terminal half-life (t1/2β) was 3.5 ± 0.4 hours, and the area under the concentration-time curve (AUC0-∞) was 25.6 ± 3.2 μg·h/mL. Approximately 80% of the administered dose was excreted unchanged in urine within 24 hours, indicating renal excretion as the main elimination pathway [1]
- In a rat pharmacokinetic study (data cited in the article), oral administration of Pemetrexed disodium (20 mg/kg) resulted in an oral bioavailability of less than 10%, due to poor absorption in the gastrointestinal tract. Intravenous administration (10 mg/kg) showed a volume of distribution (Vd) of 0.3 ± 0.05 L/kg, indicating limited tissue distribution [2]

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 the A549 xenograft model (nude mice), Pemetrexed disodium (10 mg/kg, twice weekly) caused a transient 10%-15% decrease in body weight during the first 2 weeks of treatment, but weight recovered by the end of the experiment. No significant changes in serum levels of alanine transaminase (ALT), aspartate transaminase (AST), or creatinine (indicators of liver and kidney function) were observed compared to the control group [1]
- The plasma protein binding rate of Pemetrexed disodium was measured in human plasma in vitro: approximately 80%-85% of the drug bound to plasma proteins, with no concentration-dependent binding (tested at 0.1-10 μM). [2]

[1]. Cancer Res . 1997 Mar 15;57(6):1116-23.

[2]. Int J Cancer . 2013 Jan 15;132(2):459-71.

[3]. Clin Cancer Res . 2000 Mar;6(3):1016-23.

Pemetrexed disodium is an organic sodium salt that is the disodium salt of N-{4-[2-(2-amino-4-oxo-4,7-dihydro-1H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl}-L-glutamic acid. Inhibits thymidylate synthase (TS), 421 dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFT). It has a role as an antineoplastic agent, an antimetabolite, an EC 1.5.1.3 (dihydrofolate reductase) inhibitor, an EC 2.1.2.2 (phosphoribosylglycinamide formyltransferase) inhibitor and an EC 2.1.1.45 (thymidylate synthase) inhibitor. It contains a pemetrexed(2-).
Pemetrexed Disodium is the disodium salt of a synthetic pyrimidine-based antifolate. Pemetrexed binds to and inhibits the enzyme thymidylate synthase (TS) which catalyses the methylation of 2'-deoxyuridine-5'-monophosphate (dUMP) to 2'-deoxythymidine-5'-monophosphate (dTMP), an essential precursor in DNA synthesis.
A guanine-derived ANTINEOPLASTIC AGENT that functions as a NUCLEIC ACID SYNTHESIS INHIBITOR through its binding to, and inhibition of, THYMIDYLATE SYNTHASE.
See also: Pemetrexed (broader).

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Drug Indication
Malignant pleural mesotheliomaPemetrexed Krka in combination with cisplatin is indicated for the treatment of chemotherapy naïve patients with unresectable malignant pleural mesothelioma. Non-small cell lung cancer Pemetrexed Krka 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. Pemetrexed Krka 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. Pemetrexed Krka 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.
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.
Carcinoma of the head and neck (Covered by class waiver: oropharyngeal epithelial carcinoma, excluding nasopharyngeal carcinoma), Malignant pleural mesothelioma

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Pemetrexed disodium is a multitargeted antifolate designed to overcome resistance to single-target antifolates (e.g., methotrexate) by inhibiting multiple enzymes in nucleotide synthesis. The 1997 study was one of the early preclinical investigations supporting its development for NSCLC treatment [1]
- In the 2013 study, Pemetrexed disodium showed higher sensitivity in colorectal cancer cell lines with low TS expression (IC50 0.08 μM) compared to high TS expression lines (IC50 0.5 μM), suggesting TS expression could be a potential predictive biomarker for drug response [2]
- The 2000 study noted that Pemetrexed disodium enhanced the apoptotic effect of cisplatin in H1299 cells (combination index <1), indicating a potential synergistic effect between the two drugs, which later supported clinical trials of pemetrexed plus cisplatin for NSCLC [3]

C20H19N5NA2O6

471.37

471.113

150399-23-8

135413520

Solid powder

160°C 20mm

36-38°C

160°C/20mm

196.92

4

7

7

33

737

1

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

NYDXNILOWQXUOF-GXKRWWSZSA-L

InChI=1S/C20H21N5O6.2Na/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);;/q;2*+1/p-2/t13-;;/m0../s1

disodium;(2S)-2-[[4-[2-(2-amino-4-oxo-3,7-dihydropyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]amino]pentanedioate

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: 50 mg/mL (106.07 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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配方 2 中的溶解度: 100 mg/mL (212.15 mM) in Saline (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.
*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 3 中的溶解度: Saline: 30 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。