SN-38 (NK012)   中文名称: 7-乙基-10-羟基喜树碱

Topoisomerase I; Camptothecins
DNA topoisomerase I (IC50 values ranging from 0.01 to 0.1 μM in various tumor cell lines) [1][2][3]

SN-38，盐酸伊立替康（CPT-11）的生物活性代谢物。 SN-38 对 DNA 拓扑异构酶 I 的抑制作用最强，其次是 CPT，最后是 CPT-11。 CPT-11 剂量依赖性地使松弛 DNA 的位置向带切口 DNA 的方向移动，但 SN-38 和 CPT 对松弛 DNA 的位置没有影响。 SN-38 以剂量依赖性和时间依赖性的方式抑制 DNA 合成。 SN-38 在 DNA 合成中各自的 IC50 值为 0.077 μM。 SN-38对RNA合成的抑制作用小于对DNA合成的抑制作用，并且不抑制蛋白质合成。 SN-38 在 P388 细胞中引起频繁的 DNA 单链断裂。激酶测定：将1单位（本研究条件下0.5μg SV40 DNA完全松弛的最小量）拓扑异构酶I、0.5μL测试化合物和0.5μg SV40 DNA依次加入到反应缓冲液中，即由 25 mM Tris-HCl (pH 7.5)、50 mM KCl、5 mM MgCl2、0.25 mM EDTA 二钠盐、0.25 mM 二硫苏糖醇、15μg/mL 牛血清白蛋白和 5% 甘油组成。然后，将反应混合物 (50 μL) 在 37°C 下孵育 10 分钟，并用 7.5 μL 由 1% 十二烷基硫酸钠、20 mM EDTA 二钠盐和 0.5 mg/L 组成的溶液处理终止反应。 mL 蛋白酶 K 在 37°C 下再反应 30 分钟。将样品与 5 μL 含有 10 mM Na2HPO4、31.3% 蔗糖和 0.3% 溴酚蓝的上样缓冲液混合。在 2 μg/mL 氯喹、10 mM EDTA 存在的情况下，在 0.8% 琼脂糖凝胶上以 50 mA 和 20 V 电泳 17 小时，将松弛（Ir 型）DNA 与超螺旋（I 型）和切口（II 型）DNA 分离、30 mM NaH2PO4 和 36 Mm Tris-HCl (pH 7.8)。电泳后，凝胶用0.05%溴化乙锭染色并用紫外光（302 nm）拍照。 DNA 的量使用密度计进行定量。细胞测定：MTT测定

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在人结肠癌细胞系（HT-29、HCT-116、SW480）中，SN-38 (NK012)表现出强效的浓度依赖性抗增殖活性，IC50值为0.02-0.08 μM。由于细胞摄取增强，NK012的抗增殖效果优于游离SN-38[4]
- SN-38 (NK012)诱导结肠癌细胞发生S期细胞周期阻滞，伴随p21上调和cyclin E下调，从而抑制DNA复制[2][4]
- 该药物可触发肿瘤细胞凋亡，表现为caspase-3激活增强、PARP切割和DNA片段化。相同浓度下，NK012的凋亡诱导效率高于游离SN-38[3][4]
- 在人肺癌A549细胞中，SN-38抑制DNA拓扑异构酶I活性，稳定酶-DNA切割复合物并诱导DNA单链断裂，这是其抗肿瘤作用的重要机制[1]
- NK012作为SN-38负载的纳米粒制剂，在体外可持续释放SN-38，有效药物浓度维持超过72小时[4]

SN-38 (NK012)，抗癌前药伊立替康的活性和有毒代谢物。给药后 30 分钟，Slco1a/1b(−/−) 小鼠中的伊立替康血浆浓度比野生型小鼠高 1.9 倍（分别为 1.89 和 1.01 μM），而 SN-38 (NK012) 血浆浓度Slco1a/1b(−/−) 小鼠的细胞因子比野生型小鼠高 8 倍（分别为 0.4 μg/mL 和 0.05 μg/mL）。 Oatp1a/1b 敲除小鼠中伊立替康的总血浆暴露量 [AUC(5-240)] 比野生型小鼠高 1.7 倍（209.8±6.7 vs. 120.9±4.4 μM/min；P<0.01），并且 2.9- SN-38 的倍数更高（50±2.9 vs. 12±2 μM/min；P<0.001）。

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在携带人结肠癌细胞HT-29异种移植瘤的裸鼠中，以10-30 mg/kg（基于SN-38当量）的剂量静脉注射SN-38 (NK012)，可显著抑制肿瘤生长，治疗4周后肿瘤体积缩小率达60-80%。相同剂量下，NK012的抗肿瘤疗效优于游离SN-38[4]
- 在肺癌小鼠模型中，以5-15 mg/kg剂量静脉注射SN-38，表现出剂量依赖性肿瘤生长抑制，治疗组小鼠存活时间较对照组延长[1]
- NK012在体内具有增强的肿瘤靶向性，与游离SN-38相比，肿瘤组织中SN-38蓄积量更高，而在正常器官（肝、肾、脾）中的分布更低[4]
- 在I期临床试验相关的临床前模型中，SN-38 (NK012)对难治性实体瘤（包括结直肠癌和非小细胞肺癌）表现出活性[3]

反应缓冲液由 25 mM Tris-HCl (pH 7.5)、50 mM KCl、5 mM MgCl₂、0.25 mM EDTA 二钠盐、0.25 mM 二硫苏糖醇、15μg/mL 牛向反应缓冲液中依次加入血清白蛋白、5%甘油1个单位（本研究条件下0.5μg SV40 DNA完全松弛的最低量）、0.5μL测试化合物和0.5μg SV40 DNA。然后将 50 μL 反应混合物在 37°C 下孵育 10 分钟。最后，用 7.5 μL 含有 0.5 mg/mL 蛋白酶 K、20 mM EDTA 二钠盐和 1% 十二烷基硫酸钠的溶液在 37°C 下再处理混合物 30 分钟来终止反应。将样品与 5 μL 上样缓冲液混合，其中含有 0.3% 溴酚蓝、31.3% 蔗糖和 10 mM NaH₂PO₄。使用 0.8% 琼脂糖凝胶在 50 mA 和 20 V 下电泳 17 小时，同时加入 2 μg/mL 氯喹、10 mM EDTA、30，将 I 型（超螺旋）和 II 型（切口）DNA 与松弛（Ir 型）DNA 分离。存在 mM NaH₂PO₄ 和 36 mM Tris-HCl (pH 7.8)。电泳后，对凝胶进行 0.05% 溴化乙锭染色，并进行紫外光 (302 nm) 照相。密度计用于测量 DNA 的数量。

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DNA拓扑异构酶I活性检测：将纯化的人DNA拓扑异构酶I与超螺旋pBR322 DNA在反应缓冲液中于37°C孵育。加入系列浓度（0.001-1 μM）的SN-38，混合物孵育30分钟。加入SDS和蛋白酶K终止反应，随后在50°C孵育1小时。通过1%琼脂糖凝胶电泳分离DNA产物，溴化乙锭染色。定量松弛型DNA条带强度以评估酶抑制效果。结果显示，SN-38可稳定拓扑异构酶I-DNA切割复合物，减少松弛型DNA的形成[1][2]

MTT 测定用于评估 SN-38 (NK012) 的体外敏感性。将细胞接种到 96 孔板后，第二天添加不同浓度的 SN-38 (NK012)。药物暴露 48 小时后丢弃培养基，然后将板在含有 MTT (0.5 mg/mL) 的培养基中孵育 3 小时。通过添加已酸化的 20% 十二烷基硫酸钠 (0.02 M HCl) 来溶解形成的甲臜。使用 570 nm 处的光密度（背景为 670 nm）将细胞活力计算为相对于未处理细胞的百分比。实验三次迭代后得出平均 IC50 值±标准偏差。每个耐药细胞系的相对耐药性的计算包括将其平均IC50值除以相应亲本细胞系的平均IC50值。

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肿瘤细胞抗增殖检测：将结肠癌细胞（HT-29、HCT-116）和肺癌细胞（A549）以3×10³个细胞/孔的密度接种到96孔板中。用游离SN-38或NK012（基于SN-38当量浓度为0.001-1 μM）处理细胞，在37°C、5% CO₂条件下孵育72小时。采用四唑盐比色法检测细胞活力，根据剂量-反应曲线计算IC50值[2][4]
- 细胞周期分析：用0.05 μM的SN-38 (NK012)处理HT-29细胞24-48小时。收集细胞，用70%乙醇固定，碘化丙啶染色后通过流式细胞术分析。测定G0/G1期、S期和G2/M期细胞的百分比，以评估细胞周期阻滞情况[2][4]
- 凋亡检测：用NK012（0.03 μM SN-38当量）处理HCT-116细胞48小时。用膜联蛋白V-FITC和碘化丙啶染色细胞，通过流式细胞术区分早期凋亡细胞（膜联蛋白V阳性/PI阴性）和晚期凋亡细胞（膜联蛋白V阳性/PI阳性）。采用蛋白质印迹法检测caspase-3激活和PARP切割情况[3][4]
- 纳米粒细胞摄取检测：将HT-29细胞与荧光标记的NK012孵育1-4小时。洗涤、固定细胞后，在共聚焦激光扫描显微镜下观察，定量细胞内SN-38蓄积量[4]

Mice: Mice of similar genetic background (>99% FVB) are used, including female wild-type, Slco1a/1b(−/−) (Oatp1a/1b knockout), Slco1a/1b(−/−);1B1(tg), and Slco1a/1b(−/−);1B3(tg) (liver-specific OATP1B1 and OATP1B3 humanized transgenic). The mice are aged 8 to 14 weeks. Mice receive an intravenous injection of 5 μL/g bodyweight of irinotecan (20 mg/mL in a water-based solution containing NaOH, lactic acid, and sorbitol) diluted with saline to a concentration of 10 mg/kg. To achieve a dosage of 1 mg/kg, SN-38 (NK012) is dissolved in DMSO (1 mg/mL) and given intravenously to mice at a rate of 1 μL/g body weight. The experiments end with isoflurane anesthesia, tissue collection, heparin-blood sampling via cardiac puncture, and cervical dislocation. Plasma is collected and kept at -30°C until analysis, while blood samples are centrifuged at 5,200 × g for 5 minutes at 4°C.

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Colon cancer xenograft model: Nude mice were subcutaneously inoculated with HT-29 cells (5×10⁶ cells/mouse) to establish tumor xenografts. When tumors reached a volume of 100-150 mm³, mice were randomly divided into groups (n=6-8 per group). Free SN-38 or NK012 was dissolved in sterile saline and administered intravenously via the tail vein. Doses were 10, 20, or 30 mg/kg (based on SN-38 equivalent), with administration every 7 days for a total of 4 doses. Tumor volume and body weight were measured twice weekly. At the end of the experiment, tumors and major organs were collected for drug concentration analysis and histopathological examination [4]
- Lung cancer model: C57BL/6 mice were intravenously injected with Lewis lung cancer cells (2×10⁵ cells/mouse) to establish metastatic lung tumors. Mice were treated with SN-38 at 5, 10, or 15 mg/kg via intravenous injection once every 5 days for 3 cycles. Mice were euthanized 21 days after tumor cell inoculation, and lung metastatic nodules were counted to evaluate antitumor efficacy [1]
- Pharmacokinetic study: Rats were administered NK012 or free SN-38 intravenously at 20 mg/kg SN-38 equivalent. Blood samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours post-administration. Plasma SN-38 concentrations were measured by HPLC, and pharmacokinetic parameters were calculated [4]

Absorption: SN-38 has poor oral bioavailability (＜10%) due to extensive first-pass metabolism; NK012 is administered intravenously, achieving rapid systemic exposure [3][4]
- Distribution: NK012 shows enhanced tumor distribution via the enhanced permeability and retention (EPR) effect, with SN-38 tumor concentrations 3-5 times higher than free SN-38. Free SN-38 distributes widely in normal tissues, with high levels in the liver and intestines [4]
- Metabolism: SN-38 is metabolized in the liver via glucuronidation to form inactive SN-38G, which is excreted in bile [3]
- Excretion: The majority of SN-38 and its metabolites are excreted via the biliary route (＞70%), with a small portion excreted in urine (＜10%) [3]
- Pharmacokinetic parameters: NK012 prolongs the half-life of SN-38 (t₁/₂ = 8-12 hours) compared to free SN-38 (t₁/₂ = 2-4 hours). The area under the curve (AUC) of SN-38 from NK012 is 2-3 times higher than that of free SN-38 [4]
- Plasma protein binding: SN-38 binds to plasma proteins at a rate of 95-97% [3]

Gastrointestinal toxicity: Free SN-38 induces dose-dependent diarrhea (incidence 30-50% at 15 mg/kg), while NK012 reduces gastrointestinal exposure, lowering diarrhea incidence to 10-15% at the same SN-38 equivalent dose [3][4]
- Myelosuppression: SN-38 (NK012) causes mild to moderate dose-dependent myelosuppression, characterized by leukopenia and thrombocytopenia. The severity is comparable between NK012 and free SN-38 at equivalent doses [3]
- Hepatic toxicity: High doses of free SN-38 (＞30 mg/kg) induce mild elevation of liver transaminases, which is less pronounced with NK012 [4]

[1]. Cancer Res . 1991 Aug 15;51(16):4187-91.

[2]. Cancer Chemother Pharmacol, 1997, 40(3), 259-265.

[3]. Cancer . 2003 May 1;97(9 Suppl):2363-73.

[4]. Int J Nanomedicine . 2018 Dec 20:14:75-85.

SN-38 is a member of the class of pyranoindolizinoquinolines that is (4S)-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14-dione bearing two additional ethyl substituents at positions 4 and 11 as well as two additional hydroxy substituents at positions 4 and 9. It is the active metabolite of irinotecan and is ~1000 times more active than irinotecan itself. It has a role as an apoptosis inducer, an EC 5.99.1.2 (DNA topoisomerase) inhibitor, a drug metabolite and an antineoplastic agent. It is a pyranoindolizinoquinoline, a delta-lactone, a tertiary alcohol and a member of phenols.
7-ethyl-10-hydroxycamptothecin (SN 38) is a liposomal formulation of the active metabolite of Irinotecan [DB00762], a chemotherapeutic pro-drug approved for the treatment of advanced colorectal cancer. SN 38 has been used in trials studying the treatment of Cancer, Advanced Solid Tumors, Small Cell Lung Cancer, Metastatic Colorectal Cancer, and Triple Negative Breast Cancer, among others.
7-Ethyl-10-hydroxycamptothecin has been reported in Apis cerana with data available.
A semisynthetic camptothecin derivative that inhibits DNA TOPOISOMERASE I to prevent nucleic acid synthesis during S PHASE. It is used as an antineoplastic agent for the treatment of COLORECTAL NEOPLASMS and PANCREATIC NEOPLASMS.

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Drug Indication
Investigated for use/treatment in colorectal cancer.

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Mechanism of Action
The entrapment of SN-38 in lipsomes results in a more stable and more soluble form of the drug. This allows for increased affinity of SN-38 to lipid membranes and improved delivery of the drug to tumor sites. SN-38 is a highly effective cytotoxic topoisomerase I inhibitor.

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Pharmacodynamics
SN-38 (7-ethyl-10-hydroxycamptothecin) is the active metabolite of Irinotecan (CPT-11). Irinotecan is a topoisomerase I inhibitor commercially available as Camptosar®. SN-38 has been found to be 200–2000 times more cytotoxic than CPT-11, but has not been used as an anticancer drug due to its poor solubility in pharmaceutically acceptable solvents and low affinity to lipid membranes. SN-38 also undergoes a reversible conversion to an inactive open lactone ring structure at physiological pH. LE-SN-38 is a novel lipsome based formulation containing liposomes of uniform size distribution (<200 nm). Drug entrapment efficiency of the formulation is>95%.

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SN-38 is the active metabolite of irinotecan, a camptothecin derivative, with 100-1000 times higher antitumor activity than irinotecan [1][3]
- Mechanism of action: SN-38 binds to DNA topoisomerase I, stabilizing the enzyme-DNA cleavage complex, preventing DNA religation, and inducing DNA damage, ultimately leading to cell cycle arrest and apoptosis [1][2][3]
- NK012 is a SN-38-loaded polymeric nanoparticle formulation designed to improve solubility, enhance tumor targeting via the EPR effect, and reduce systemic toxicity [4]
- Clinical potential: SN-38 (NK012) has shown activity against irinotecan-refractory tumors, including colorectal, lung, and breast cancer, in preclinical studies [3][4]
- Drug-drug interaction: SN-38 metabolism is inhibited by UGT1A1 inhibitors, which may increase its plasma concentration and toxicity [3]

C22H20N2O5

392.4

392.137

C, 67.34; H, 5.14; N, 7.14; O, 20.39

86639-52-3

104842

Light yellow solid powder

1.5±0.1 g/cm3

810.3±65.0 °C at 760 mmHg

217 °C

443.8±34.3 °C

0.0±3.0 mmHg at 25°C

1.738

2.31

101.65

2

6

2

29

820

1

C(C1C2C=C(C=CC=2N=C2C3=CC4[C@@](C(OCC=4C(=O)N3CC=12)=O)(O)CC)O)C

FJHBVJOVLFPMQE-QFIPXVFZSA-N

InChI=1S/C22H20N2O5/c1-3-12-13-7-11(25)5-6-17(13)23-19-14(12)9-24-18(19)8-16-15(20(24)26)10-29-21(27)22(16,28)4-2/h5-8,25,28H,3-4,9-10H2,1-2H3/t22-/m0/s1

(19S)-10,19-diethyl-7,19-dihydroxy-17-oxa-3,13-diazapentacyclo[11.8.0.02,11.04,9.015,20]henicosa-1(21),2,4(9),5,7,10,15(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

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 (6.37 mM) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 悬浮液；超声助溶。
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 玉米油中，混合均匀。

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配方 2 中的溶解度: 2.08 mg/mL (5.30 mM) 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), 悬浊液; 超声助溶。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL澄清的DMSO储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入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 3 中的溶解度: 2.08 mg/mL (5.30 mM) 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), 悬浊液; 超声助溶。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 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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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。