Topoisomerase I 2.2 μM (IC50)

Exatecan 的 IC50 为 0.975 μg/mL，使其成为拓扑异构酶 I 的强抑制剂。exatecan 甲磺酸盐 (DX- 8951f）[1]。 exatecan mesylate (DX-8951f) 对 PC-6 和 PC-6/SN2-5 细胞的细胞毒作用通过平均 GI50 值分别为 0.186 和 0.395 ng/mL 得到证实。在 PC-6 和 PC-6/SN2-5 细胞中，exatecan mesylate (34 nM) 可稳定 DNA-TopoI 复合物 [3]。

在具有肿瘤细胞但无毒性死亡的小鼠模型中，甲磺酸exatecan（DX-8951f，3.325-50 mg/kg，静脉注射）显示出抗肿瘤功效[1]。在 MIA-PaCa-2 早期模型和 BxPC-3 早期模型中，exatecan Mesylate（15、25 mg/kg，静脉注射）显着抑制 MIA-PaCa 和 BxPC-3 原发肿瘤的生长。在BxPC-3晚期癌症模型中，甲磺酸exatecan（15、25mg/kg，静脉注射）完全根除肺转移并显着减少BxPC-3淋巴转移[2]。

细胞裂解和蛋白质印迹：[3]
使用SDS缓冲液（10 mM HEPES、2 mM正钒酸盐、10 mM NaF、10 mM焦磷酸盐、1 mM PMSF、10µg/mL亮肽、10%2-巯基乙醇、10%甘油、8%SDS、42 mM Tris-HCl、0.002%溴酚蓝，pH 7.4）裂解细胞（5×10⁶）。通过7.5%聚丙烯酰胺凝胶电泳分离全细胞裂解物中的蛋白质，并将其转移到硝化纤维膜上。用抗Topo I人抗体探测膜，然后用辣根过氧化物酶偶联蛋白A孵育。使用ECL试剂观察Topo I特异性条带。

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核提取物制备：[3]
对于核提取物，用冰冷的缓冲液（2 mM K₂HPO₄，5 mM氯化镁，150 mM NaCl、1 mM EGTA、0.1 mM二硫苏糖醇），重新悬浮在含有0.35%Triton-X100和PMSF的缓冲液中，在冰上孵育10分钟。裂解物离心，所得沉淀在4°C下与含有0.35 M NaCl的缓冲液进一步孵育1小时。离心（18000×g，10分钟）后，使用Bradford法和蛋白质测定试剂盒测定上清液（核提取物）的蛋白质浓度。然后用抗Topo I抗体对等量的核蛋白进行蛋白质印迹分析。

生长抑制试验（MTT法）：[1]
使用MTT法在96孔平底微孔板中评估细胞生长抑制。将细胞（500-20000/孔）接种在150μL培养基中，预孵育24小时（P388、CCRF-CEM和K562细胞为4小时）。然后加入试验化合物，包括甲磺酸Exatecan（150μL培养基/孔）或单独培养基（对照），然后孵育3天。处理后，向每个孔中加入20μL MTT溶液（PBS中5mg/mL），并将平板孵育4小时。将平板在800×g下离心5分钟，去除上清液，将甲氮晶体溶解在150μL DMSO中。使用微孔板读数器（3550型）在540nm处测量吸光度。

Experimental Design and Treatment Protocol [2]
Early-Stage Model (3 Weeks Post-Implantation)
Animal Groups: Mice bearing orthotopically implanted BxPC-3-GFP or MIA-PaCa-2-GFP tumors were randomized into five groups (n = 5/group) at 3 weeks post-implantation.
Group 1: Untreated control.
Groups 2 & 3: Treated with Exatecan Mesylate (25 and 15 mg/kg/dose, respectively).
Groups 4 & 5: Treated with gemcitabine (300 and 150 mg/kg/dose, respectively).
Late-Stage Model (6 Weeks Post-Implantation)
Animal Groups: Mice with BxPC-3-GFP tumors were randomized into three groups (n = 20/group) at 6 weeks post-implantation.
Group 1: Untreated control.
Group 2: Treated with Exatecan Mesylate (25 mg/kg/dose).
Group 3: Treated with gemcitabine (300 mg/kg/dose).
Dosing Schedule
Drugs were administered once weekly for 3 weeks, followed by a 2-week break, and then resumed for another 3 weeks.
Monitoring and Analysis
Weekly measurements: Primary tumor size (calculated as a × b² × 0.5, where a = larger diameter, b = smaller diameter) and body weight.
Termination: Mice were sacrificed at study completion, with final tumor weights and GFP imaging (primary tumors and metastases) recorded.

[1]. A new water-soluble camptothecin derivative, DX-8951f, exhibits potent antitumor activity against human tumors in vitro and in vivo. Jpn J Cancer Res. 1995 Aug;86(8):776-82.

[2]. Efficacy of camptothecin analog DX-8951f (Exatecan Mesylate) on human pancreatic cancer in an orthotopic metastatic model. Cancer Res. 2003 Jan 1;63(1):80-5.

[3]. DX-8951f, a water-soluble camptothecin analog, exhibits potent antitumor activity against a human lung cancer cell line and its SN-38-resistant variant. Int J Cancer. 1997 Aug 7;72(4):680-6.

Exatecan mesilate hydrate is a pyranoindolizinoquinoline.
Exatecan Mesylate is a semisynthetic, water-soluble derivative of camptothecin with antineoplastic activity. Exatecan mesylate inhibits topoisomerase I activity by stabilizing the cleavable complex between topoisomerase I and DNA and inhibiting religation of DNA breaks, thereby inhibiting DNA replication and triggering apoptotic cell death. This agent does not require enzymatic activation and exhibits greater potency than camptothecin and other camptothecin analogues. (NCI04)

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Exatecan is a pyranoindolizinoquinoline.
Exatecan has been used in trials studying the treatment of Sarcoma, Leukemia, Lymphoma, Lung Cancer, and Liver Cancer, among others.

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CPT-11, a semisynthetic derivative of camptothecin, exhibited strong antitumor activity against lymphoma, lung cancer, colorectal cancer, gastric cancer, ovarian cancer, and cervical cancer. CPT-11 is a pro-drug that is converted to an active metabolite, SN-38, in vivo by enzymes such as carboxylesterase. We synthesized a water-soluble and non-pro-drug analog of camptothecin, DX-8951f. It showed both high in vitro potency against a series of 32 malignant cell lines and significant topoisomerase I inhibition. The anti-proliferative activity of DX-8951f, as indicated by the mean GI50 value, was about 6 and 28 times greater than that of SN-38 or SK&F 10486-A (Topotecan), respectively. These three derivatives of camptothecin showed similar patterns of differential response among 32 cell lines, that is, their spectra of in vitro cytotoxicity were almost the same. The antitumor activity of three doses of DX-8951f administered i.v. at 4-day intervals against human gastric adenocarcinoma SC-6 xenografts was greater than that of CPT-11 or SK&F 10486-A. Moreover, it overcame P-glycoprotein-mediated multi-drug resistance. These data suggest that DX-8951f has a high antitumor activity and is a potential therapeutic agent.[1]

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We determined the antitumor and antimetastatic efficacy of the camptothecin analogue DX-8951f in an orthotopic metastatic mouse model of pancreatic cancer. DX-8951f showed efficacy against two human pancreatic tumor cell lines in this model. These cell lines were transduced with the green fluorescent protein, enabling high-resolution visualization of tumor and metastatic growth in vivo. The DX-8951f studies included both an early and advanced cancer model. In the early model, using the human pancreatic cancer lines MIA-PaCa-2 and BxPC-3, treatment began when the orthotopic primary tumor was approximately 7 mm in diameter. DX-8951f was significantly effective against both MIA-PaCa-2 and BxPC-3. In contrast, 2', 2'-difluorodeoxycytidine (gemcitabine), the standard treatment for pancreatic cancer, did not have significant efficacy against MIA-PaCa-2. Although gemcitabine showed significant activity against BxPC-3 primary tumor growth, it was not effective on metastasis. In the model of advanced disease, using BxPC-3, treatment started when the orthotopic primary tumor was 13 mm in diameter. DX-8951f was significantly effective in a dose-response manner on the BxPC-3 primary tumor. DX-8951f also demonstrated antimetastatic activity in the late-stage model, significantly reducing the incidence of lymph node metastasis while eliminating lung metastasis. In contrast, gemcitabine was only moderately effective against the primary tumor and ineffective against metastasis at both sites in the late-stage model. Therefore, DX-8951f was highly effective against primary and metastatic growth in this very difficult-to-treat disease and showed significantly higher efficacy than gemcitabine, the standard treatment of pancreatic cancer. DX-8951f, therefore, has important clinical promise and has more positive features than the currently used camptothecin analogue CPT-11, which requires metabolic activation and is toxic.[2]

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We previously reported that DX-8951f, a novel water-soluble camptothecin analog, significantly inhibits the growth of various human and murine tumors in vitro and in vivo. The antitumor effects and topoisomerase I inhibitory activity of DX-8951f are stronger than those of other current camptothecin analogs. In this study, we established an SN-38-resistant cell line, PC-6/SN2-5, from the human oat cell carcinoma PC-6 cell line by a stepwise selection system, investigated the mechanism of resistance of this cell line and then compared the antitumor activity of camptothecin analogs against the cell line. PC-6/SN2-5 cells were resistant to SN-38 (32-fold) and SK&F 104864 (topotecan; 14-fold), but barely resistant to CPT-11 (3-fold) and DX-8951f (2-fold). Topoisomerase I protein levels and topoisomerase I activities of parental cells were similar to those of resistant cells. Determination of the cellular drug concentration by either flow cytometric analysis or the high-performance liquid chromatography method confirmed that the cellular accumulation of SN-38 and topotecan was significantly reduced in PC-6/SN2-5 cells, whereas that of DX-8951f was only slightly reduced. Furthermore, DX-8951f stabilized the cleavable complex formations in intact PC-6/SN2-5 cells as well as in parental cells, but SN-38 and topotecan did not in the resistant cells. Our data suggest that PC-6/SN2-5 cells may have acquired resistance to camptothecin analogs by a decrease in intracellular drug accumulation and that DX-8951f may have the potency to overcome such a type of resistance mechanism induced by camptothecin compounds.[3]

C24H22FN3O4S.CH4O3S.2H2O

567.5838

567.169

197720-53-9

Exatecan;171335-80-1;Exatecan mesylate;169869-90-3;Exatecan-d5 mesylate;2819276-88-3;Dxd;1599440-33-1;Dxd-d5;Deruxtecan;1599440-13-7;Deruxtecan-d6;2760715-89-5;(1S,9R)-Exatecan mesylate;2938875-54-6;(1R,9R)-Exatecan mesylate;(1R)-Deruxtecan;2270986-87-1

151114

Typically exists as solid at room temperature

2.47E-28mmHg at 25°C

3.629

188.65

5

12

1

39

1040

2

O.O.CS(=O)(O)=O.CC[C@]1(C(=O)OCC2C(N3CC4=C5[C@@H](N)CCC6=C5C(N=C4C3=CC1=2)=CC(=C6C)F)=O)O

FXQZOHBMBQTBMJ-MWPGLPCQSA-N

InChI=1S/C24H22FN3O4.CH4O3S.2H2O/c1-3-24(31)14-6-18-21-12(8-28(18)22(29)13(14)9-32-23(24)30)19-16(26)5-4-11-10(2)15(25)7-17(27-21)20(11)19;1-5(2,3)4;;/h6-7,16,31H,3-5,8-9,26H2,1-2H3;1H3,(H,2,3,4);2*1H2/t16-,24-;;;/m0.../s1

(10S,23S)-23-amino-10-ethyl-18-fluoro-10-hydroxy-19-methyl-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16,18,20(24)-heptaene-5,9-dione;methanesulfonic acid;dihydrate

Exatecan mesilate hydrate; 197720-53-9; Exatecan mesylate hydrate; Exatecan mesylate [USAN]; exatecan mesylate dihydrate; DX-8951f; Exatecan methanesulfonate dihydrate; Exatecan mesilate dihydrate;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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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。