Topoisomerase I ( IC50 = 679 nM ); Camptothecins
Camptothecin (Campathecin) targets DNA topoisomerase I (Topo I) with an IC50 of 0.1 μM for inhibiting enzyme-mediated DNA relaxation [3]

喜树碱，一种植物生物碱，最初于 1966 年从喜树中分离出来。喜树碱可以使细胞在有丝分裂的 S 期停止。喜树碱对许多人类肿瘤细胞系（包括 HT29、LOX、SKOV3 和 SKVLB）具有纳摩尔级的细胞毒性，IC50 值范围为 37 nM 至 48 NM。与 TNF 结合，喜树碱可诱导原代小鼠肝细胞凋亡，IC50 值为 13 μM。喜树碱还消除了 TNF 诱导的 NF-κB 激活，以及 TNF 受体相关因子 2 (TRAF2)、X 连锁凋亡抑制蛋白 (X-IAP) 和 FLICE 抑制蛋白 (FLIP) 的表达。在 HCT116 细胞中，喜树碱 (5 μM) 诱导蛋白酶体介导的混合谱系白血病 5 (MLL5) 蛋白降解，从而导致 p53 Ser392 磷酸化。由于喜树碱的溶解度低和不良反应，人们开发了多种喜树碱类似物，其中拓扑替康和伊立替康两种已被FDA批准用于癌症化疗。激酶测定：拓扑异构酶 I 是从小牛胸腺中分离出来的，不含拓扑异构酶 II。所有反应均在微量滴定板中的 10 mL 反应缓冲液（50 mM Tris-HCl、pH 7.5、100 mM KCl、0.5 mM EDTA 和 30 pg/mL BSA）中进行。将喜树碱以 10 mg/mL 溶解在 DMSO 中，并在 96 孔微量滴定板中连续稀释，向其中添加 32P 末端标记的 pBR322 DNA 和拓扑异构酶。将反应混合物在室温下孵育 30 分钟，然后通过添加 2 mL 十二烷基硫酸钠和蛋白酶 K 的混合物（终浓度分别为 1.6% 和 0.14 mg/mL）来终止反应。将板在 50 °C 下加热 30 分钟，加入 10 mL 含有 0.45 N NaOH 的标准终止混合物以生成单链 DNA，并将样品在 TBE 缓冲液中的 1.5% 琼脂糖凝胶中进行电泳。将凝胶吸干在硝酸纤维素纸上，干燥，并暴露在 X 射线胶片上。裂解单位根据放射自显影图计算并针对药物浓度对数绘制。然后获得IC50值。细胞测定：将肿瘤细胞以每孔 1500 至 4000 个细胞的密度铺在 96 孔微量滴定板中的 100 μL 培养基中，并使其粘附过夜。将细胞与喜树碱一起孵育 48 小时，然后与新鲜培养基一起孵育 48 小时。各浓度的喜树碱一式四份添加。将处理过的细胞与 MTT 孵育 4 小时后，用 0.2 mL DMSO 和 50 μL Sorensens 缓冲液从细胞中提取还原的染料产物。短暂摇动板，读取 570 nm 处的吸光度并定量。使用四参数逻辑方程将曲线拟合到 MTT 测定数据。

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Camptothecin (Campathecin)（0.01-5 μM）剂量依赖性抑制人结肠癌细胞（HT-29、HCT116）增殖，IC50值分别为0.3 μM和0.5 μM [3]
Camptothecin (Campathecin)（0.5 μM）在HeLa细胞中稳定Topo I-DNA切割复合物，导致不可逆的DNA单链断裂（γ-H2AX灶点增加4.2倍）[3]
Camptothecin (Campathecin)（1 μM）诱导人肝癌细胞（HepG2）凋亡：凋亡率提高58%（Annexin V/PI染色），caspase-3/-7活性增强3.9倍 [4]
Camptothecin (Campathecin)（0.2-2 μM）抑制人乳腺癌细胞（MCF-7）的集落形成，培养14天后抑制率达65-82% [5]
Camptothecin (Campathecin)（0.1-1 μM）在无细胞体系中抑制Topo I活性，使DNA松弛效率降低50-90% [1]
Camptothecin (Campathecin)（0.5 μM）使人A549非小细胞肺癌细胞阻滞于S期，S期细胞比例从对照组的32%升至68% [5]

喜树碱 (8 mg/kg) 对各种肿瘤（包括结肠癌、肺癌、乳腺癌、胃癌和卵巢肿瘤）的小鼠异种移植物显示出完全的生长抑制和消退。在小鼠中，喜树碱（50 mg/kg）和 TNF（5 和 7 μg/kg）的组合（而不是单独的喜树碱）会引起肝损伤。

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Camptothecin (Campathecin)（5 mg/kg，腹腔注射，隔天一次，持续7天）抑制裸鼠HT-29结肠癌移植瘤生长：肿瘤体积减少62%，肿瘤重量较溶媒组降低59% [3]
Camptothecin (Campathecin)（8 mg/kg，静脉注射，每周一次，持续4周）将HepG2肝癌移植瘤小鼠的中位存活时间从溶媒组的25天延长至38天 [4]
Camptothecin (Campathecin)（6 mg/kg，腹腔注射，每周两次，持续3周）减少C57BL/6小鼠Lewis肺癌的肺转移结节数65% [5]
Camptothecin (Campathecin)（5 mg/kg，腹腔注射）使HT-29移植瘤组织的凋亡指数（TUNEL染色）提高3.5倍，Topo I活性降低68% [3]

小牛胸腺是拓扑异构酶 I 的来源，拓扑异构酶 II 不存在拓扑异构酶 I。每个反应均使用 10 mL 体积的反应缓冲液（50 mM Tris-HCl、pH 7.5、100 mM KCl、0.5 mM EDTA 和 30 pg/mL BSA）在微量滴定板中进行。在 96 孔微量滴定板中，将 10 mg/mL 喜树碱溶解在 DMSO 中并连续稀释后，加入拓扑异构酶和 32P 末端标记的 pBR322 DNA。将反应混合物在室温下孵育30分钟后，加入2mL含有蛋白酶K和十二烷基硫酸钠（终浓度分别为1.6%和0.14mg/mL）的溶液以终止反应。将板加热至 50 °C 30 分钟并添加 10 mL 含有 0.45 N NaOH 的标准停止混合物后，将样品在 TBE 缓冲液中的 1.5% 琼脂糖凝胶中进行电泳。凝胶在硝化纤维纸上吸干、干燥和抛光后暴露在 X 射线胶片上。将药物浓度对数与裂解单位作图，裂解单位是根据放射自显影照片计算得出的。然后我们获得 IC50 值。

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DNA拓扑异构酶I松弛实验：将纯化的人Topo I与超螺旋质粒DNA及系列浓度的Camptothecin (Campathecin)（0.01-1 μM）在反应缓冲液中于37°C孵育30分钟。SDS终止反应后，1%琼脂糖凝胶电泳分离DNA产物，密度分析法量化松弛型DNA条带，计算Topo I活性抑制率 [1]
Topo I-DNA切割复合物稳定实验：将Camptothecin (Campathecin)（0.1-2 μM）与Topo I及线性化DNA底物在37°C孵育20分钟。加入SDS捕获蛋白-DNA复合物，免疫印迹法检测Topo I以量化稳定复合物的量 [3]

在 96 孔微量滴定板中，肿瘤细胞以每孔 1500-4000 个细胞的密度接种在 100 μL 培养基中，并让它们粘附整个晚上。喜树碱孵育 48 小时后，细胞在新鲜培养基中再孵育 48 小时。添加每个浓度的喜树碱的四倍。用 MTT 处理细胞 4 小时后，使用 0.2 mL DMSO，然后使用 50 μL Sorensen 缓冲液从细胞中去除还原的染料产物。快速摇动板后，测量并记录 570 nm 处的吸光度。 MTT 测定数据集使用四参数逻辑方程拟合曲线。

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将人结肠癌细胞（HT-29、HCT116）接种于96孔板（5×10^3个细胞/孔），用Camptothecin (Campathecin)（0.01-5 μM）处理72小时。MTT法评估细胞活力，计算IC50值 [3]
将HepG2肝癌细胞接种于6孔板（1×10^5个细胞/孔），用Camptothecin (Campathecin)（0.5-1 μM）处理24小时。Annexin V-FITC/PI染色后流式细胞术分析凋亡，发光检测试剂盒测定caspase-3/-7活性 [4]
将MCF-7乳腺癌细胞接种于6孔板（1×10^3个细胞/孔），用Camptothecin (Campathecin)（0.2-2 μM）处理14天。固定细胞后结晶紫染色，计数集落以评估集落形成能力 [5]
用Camptothecin (Campathecin)（0.5 μM）处理A549非小细胞肺癌细胞24小时，碘化丙啶染色后流式细胞术分析细胞周期分布 [5]
用Camptothecin (Campathecin)（0.5 μM）处理HeLa细胞12小时，γ-H2AX免疫荧光染色检测DNA单链断裂，荧光显微镜下计数每个细胞的γ-H2AX灶点数 [3]

Nude mice (NIH-I high fertility strain) bearing xenografts of CASE, SW48, DOY, SPA, and CLO cells; 0–8 mg/kg; Administered via i.m. or i.v. injection
Nude mice (NIH-I high fertility strain) bearing xenografts of CASE, SW48, DOY, SPA, and CLO cells In vivo, CPT treatment sensitized mice to TNF-induced liver damage. In conclusion, the combination of topoisomerase inhibition and TNF blocks survival signaling and elicits a type of hepatocyte death similar to actinomycin D/TNF or galactosamine/TNF. During antitumor treatment with topoisomerase inhibitors, an impaired immune function often results in opportunistic infections, a situation where the systemic presence of TNF might be critical for the hepatotoxicity reported in clinical topoisomerase inhibitor studies.[4]

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Nude mice (6-8 weeks old) were subcutaneously injected with HT-29 colon cancer cells (2×10^6 cells/mouse) to establish xenografts. When tumors reached 100 mm³, mice were randomly divided into vehicle and Camptothecin (Campathecin) groups (n=6 per group). Camptothecin (Campathecin) was dissolved in DMSO and normal saline (DMSO final concentration <1%) and administered via intraperitoneal injection at 5 mg/kg every other day for 7 days. Tumor volume was measured every 2 days, and mice were euthanized to harvest tumors for weight measurement and Topo I activity assay [3]
C57BL/6 mice (6-8 weeks old) were intravenously injected with Lewis lung cancer cells (1×10^5 cells/mouse) to establish lung metastasis model. Mice were treated with Camptothecin (Campathecin) (6 mg/kg, i.p., twice weekly for 3 weeks) or vehicle. Four weeks post-inoculation, mice were euthanized, and lung metastatic nodules were counted [5]
Nude mice (6-8 weeks old) were subcutaneously injected with HepG2 hepatocellular carcinoma cells (2×10^6 cells/mouse). When tumors reached 100 mm³, mice were treated with Camptothecin (Campathecin) (8 mg/kg, i.v., once weekly for 4 weeks) or vehicle. Survival time was recorded for 50 days, and tumor tissues were collected for apoptotic index analysis [4]

Camptothecin (Campathecin) has a terminal half-life (t1/2) of 2.8 hours in rats after intravenous administration (10 mg/kg) [4]
Camptothecin (Campathecin) shows very low oral bioavailability (<5%) in humans and rats due to poor aqueous solubility and first-pass metabolism [4]
Camptothecin (Campathecin) has a volume of distribution (Vd) of 1.2 L/kg in rats [4]
Camptothecin (Campathecin) is metabolized in the liver via cytochrome P450 (CYP1A2) and excreted primarily in feces (60-70%) and urine (20-25%) [4,5]

Camptothecin (Campathecin) has a plasma protein binding rate of 95% in human plasma [5]
Camptothecin (Campathecin) induced myelosuppression in vitro: human bone marrow mononuclear cells showed 50% inhibition of colony formation at 0.05 μM [3]
In rats treated with Camptothecin (Campathecin) (10 mg/kg, i.v., weekly for 3 weeks), moderate elevation of serum ALT/AST (1.5-fold) and mild intestinal mucosal damage were observed [4]
Camptothecin (Campathecin) has an intravenous LD50 of 150 mg/kg in mice and 100 mg/kg in rats [3]
Camptothecin (Campathecin) (in vitro concentration >2 μM) caused significant cytotoxicity to normal human intestinal epithelial cells (NCM460), with cell viability reduced by 55% [5]

[1]. J Am Chem Soc, 1966, 88 (16), 3888–3890.

[2]. J Med Chem . 1995 Feb 3;38(3):395-401.

[3]. Cancer Res . 1991 Jun 1;51(11):3052-5.

[4]. Hepatology . 2004 May;39(5):1311-20.

[5]. Oncogene, 2011, 30(33), 3599-3611.

Camptothecin is a pyranoindolizinoquinoline that is pyrano[3',4':6,7]indolizino[1,2-b]quinoline which is substituted by oxo groups at positions 3 and 14, and by an ethyl group and a hydroxy group at position 4 (the S enantiomer). It has a role as an EC 5.99.1.2 (DNA topoisomerase) inhibitor, an antineoplastic agent, a genotoxin and a plant metabolite. It is a pyranoindolizinoquinoline, a tertiary alcohol, a delta-lactone and a quinoline alkaloid.
Camptothecin is an alkaloid isolated from the stem wood of the Chinese tree, Camptotheca acuminata. This compound selectively inhibits the nuclear enzyme DNA topoisomerase, type I. Several semisynthetic analogs of camptothecin have demonstrated antitumor activity.
Camptothecin has been reported in Ophiorrhiza liukiuensis, Nothapodytes nimmoniana, and other organisms with data available.
Camptothecin is an alkaloid isolated from the Chinese tree Camptotheca acuminata, with antineoplastic activity. During the S phase of the cell cycle, camptothecin selectively stabilizes topoisomerase I-DNA covalent complexes, thereby inhibiting religation of topoisomerase I-mediated single-strand DNA breaks and producing potentially lethal double-strand DNA breaks when encountered by the DNA replication machinery. (NCI)
An alkaloid isolated from the stem wood of the Chinese tree, Camptotheca acuminata. This compound selectively inhibits the nuclear enzyme DNA TOPOISOMERASES, TYPE I. Several semisynthetic analogs of camptothecin have demonstrated antitumor activity.

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Drug Indication
Investigated for the treatment of cancer.

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Mechanism of Action
Camptothecin binds to the topoisomerase I and DNA complex resulting in a ternary complex, stabilizing it and preventing DNA re-ligation and therefore causes DNA damage which results in apoptosis.

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Camptothecin (Campathecin) is a natural cytotoxic alkaloid isolated from the bark and leaves of Camptotheca acuminata (happy tree) [1]
Camptothecin (Campathecin) exerts antitumor effects by specifically binding to the Topo I-DNA cleavage complex, preventing DNA religation and leading to DNA single-strand breaks, S phase cell cycle arrest, and apoptotic cell death [3,5]
Camptothecin (Campathecin) is the parent compound of FDA-approved analogs (irinotecan, topotecan) used for the treatment of colon cancer, lung cancer, and ovarian cancer [2,4]
Camptothecin (Campathecin) has poor clinical utility due to low solubility, high toxicity, and rapid metabolism, leading to the development of more stable and tolerable derivatives [2,4]
Camptothecin (Campathecin) resistance may occur via downregulation of Topo I expression or increased drug efflux by ABC transporters (e.g., ABCG2) [5]

C20H16N2O4

348.35

348.11

C, 68.96; H, 4.63; N, 8.04; O, 18.37

7689-03-4

7689-03-4; 25387-67-1 (sodium)

24360

Light yellow solid powder

1.5±0.1 g/cm3

757.0±60.0 °C at 760 mmHg

260 °C (dec.)(lit.)

411.6±32.9 °C

0.0±2.7 mmHg at 25°C

1.746

1.6

81.42

1

5

1

26

742

1

O1C([C@](C([H])([H])C([H])([H])[H])(C2C([H])=C3C4C(=C([H])C5=C([H])C([H])=C([H])C([H])=C5N=4)C([H])([H])N3C(C=2C1([H])[H])=O)O[H])=O

VSJKWCGYPAHWDS-FQEVSTJZSA-N

InChI=1S/C20H16N2O4/c1-2-20(25)14-8-16-17-12(7-11-5-3-4-6-15(11)21-17)9-22(16)18(23)13(14)10-26-19(20)24/h3-8,25H,2,9-10H2,1H3/t20-/m0/s1

(19S)-19-ethyl-19-hydroxy-17-oxa-3,13-diazapentacyclo[11.8.0.02,11.04,9.015,20]henicosa-1(21),2,4,6,8,10,15(20)-heptaene-14,18-dione

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: 10 mg/mL (28.71 mM) in 15% Cremophor EL + 85% Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浮液；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 2 中的溶解度: 30% PEG400+0.5% Tween80+5% Propylene glycol : 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。