Topoisomerase I ( IC50 = 0.975 μg/mL )

Exatecan Mesylate exhibits potent inhibition of topoisomerase I along with potency against a battery of thirty-two malignant cell lines in vitro. Exatecan Mesylate exhibits anti-proliferative activity approximately 6 and 28 times higher than that of SN-38 or SK&F 10486-A, respectively. [1]

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Exatecan has an IC50 of 0.975 μg/mL, making it a strong inhibitor of topoisomerase I. The proliferation of several cancer cell lines, including those from the breast, colon, stomach, and lung regions, is markedly inhibited by exatecan mesylate (DX-8951f) [1]. The cytotoxic action of exatecan mesylate (DX-8951f) against PC-6 and PC-6/SN2-5 cells is demonstrated by average GI50 values of 0.186 and 0.395 ng/mL, respectively. In PC-6 and PC-6/SN2-5 cells, exatecan mesylate (34 nM) stabilizes DNA-TopoI complexes [3].

Three intravenous dosages of Exatecan Mesylate given at 4-day intervals have been shown to have more antitumor activity in mice than either CPT-11 or SK&F 10486-A when used against human gastric adenocarcinoma SC-6 xenografts. Additionally, it defeated P-glycoprotein-mediated multidrug resistance. These findings imply that Exatecan Mesylate is a promising therapeutic agent with strong antitumor activity.[1]

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In a mouse model with tumor cells but no toxic mortality, exatecan mesylate (DX-8951f, 3.325-50 mg/kg, intravenous injection) showed anti-tumor efficacy [1]. In the MIA-PaCa-2 early model and the BxPC-3 early model, exatecan Mesylate (15, 25 mg/kg, iv) significantly suppresses the growth of MIA-PaCa and BxPC-3 primary tumors. In the BxPC-3 advanced cancer model, exatecan mesylate (15, 25 mg/kg, intravenous injection) totally eradicates lung metastasis and dramatically reduces BxPC-3 lymphatic metastasis [2].

SDS buffer (10 mM HEPES, 2 mM orthovanadate, 10 mM NaF, 10 mM pyrophosphate, 1 mMPMSF, 10 µg/mL leupeptin, 10% 2-mercaptoethanol, 10% glycerol, 8% SDS, 42 mM Tris-HCl, 0.002% bromophenol blue, pH 7.4) is used to lyse cells (5×10⁶). Protein from the lysates of the entire cell is separated using a 7.5% polyacrylamide gel and then blotted onto nitrocellulose membrane. First, horseradish peroxidase-conjugated protein A is applied to the membrane, followed by an anti-Topo I human antibody treatment. ECL reagents are used to detect the Topo I-specific band. In order to procure a nuclear extract, five hundred seventy-seven cells are cleaned using an ice-cold buffer (2 mM K₂HPO₄, 5 mM MgCl₂, 150 mM NaCl, 1 mM EGTA, 0.1 mM dithiothreitol), reconstituted in a buffer that contains 0.35% Triton-X100 and PMSF, and subsequently placed on ice for ten minutes. The obtained lysates are centrifuged, and the precipitates are subsequently left to incubate for one hour at 4°C in a buffer containing 0.35 M NaCl. A protein assay kit is used to measure the protein concentration of the supernatant (nuclear extract) following centrifugation (18,000 g, 10 min). Western blotting analysis employing anti-Topo I antibody is used to analyze the same quantity of nuclear protein[3].

MTT assay is used to measure the number of viable cells at the end of the incubation period in growth inhibition experiments, which are carried out in 96-well flat-bottomed microplates. After plating and growing 500–20,000000 cells/well in 150 μL of medium for 24 hours (P388, CCRF–CEM, and K562 cells for 4 hours), the drug (such as Exatecan Mesylate in 150 μL of medium/well) or the medium alone (as a control) is added, and the cells are cultured for an extra 3 days. The plates are incubated for 4 hours and centrifuged at 800 g for 5 minutes after adding MTT (20 μL/well, 5 mg/mL in phosphate-buffered saline). The medium is then removed, and the blue dye that forms is dissolved in 150 μL of DMSO. A Microplate Reader model 3550 is used to measure the absorbance at 540 nm[1].

For therapeutic purposes, mice are randomized into five groups of five at three weeks following orthotopic implantation of BxPC-3-GFP and MIA-PaCa-2-GFP. Group 1 is the negative control, getting no medical attention. Exatecan Mesylate is administered to Groups 2 and 3 at a dose of 25 and 15 mg/kg, respectively. LY 188011 treatments are administered to groups 4 and 5, at doses of 300 and 150 mg/kg, respectively. Mice are randomized into three groups of twenty at six weeks following BxPC-3-GFP orthotopic implantation in order to provide treatment. Group 1 is the negative control, getting no medical attention. Group 3 is administered 300 mg/kg/dose of LY 188011, while Group 2 is treated with 25 mg/kg/dose of Exatecan Mesylate. The dosage for both medications is taken once a week for three weeks, stopped for two weeks, and then resumed for an additional three weeks. Weekly measurements are made of the primary tumor size and body weights in both early and late cancer models. The formula for calculating tumor volumes is a × b² × 0.5, in which a and b stand for the larger and smaller diameters, respectively. When the research is over, mice are killed and examined. We record the final tumor weights and the primary tumor and metastases' direct GFP images for every mouse. TWt and TWc are the mean tumor weights of the treated and control groups, respectively, and the formula for calculating the tumor growth index (IR) is IR (%) = (1 − TWt/TWc) × 100[2].

[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.

Anhydrous ethatecan mesylate is the anhydrous mesylate form of ethatecan, a semi-synthetic, water-soluble camptothecin derivative with antitumor activity. After administration, ethatecan mesylate inhibits topoisomerase I activity by stabilizing the cleavable complex between topoisomerase I and DNA and inhibiting the rejoining of DNA breaks, thereby inhibiting DNA replication and inducing apoptosis.
See also: Exatecan mesylate (note moved to).

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Exatecan is a pyranoindoquinoline compound.
Exatecan has been used in clinical trials to treat various cancers, including sarcoma, leukemia, lymphoma, lung cancer, and liver cancer.

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The semi-synthetic camptothecin derivative CPT-11 has shown potent antitumor activity against lymphoma, lung cancer, colorectal cancer, gastric cancer, ovarian cancer, and cervical cancer. CPT-11 is a prodrug that is converted into the active metabolite SN-38 in vivo by enzymes such as carboxylesterase. We synthesized a water-soluble, non-prodrug form of camptothecin analogue, DX-8951f. This compound exhibited high in vitro activity against 32 malignant cell lines and significant topoisomerase I inhibition. The antiproliferative activity of DX-8951f (expressed as mean GI50 value) was approximately 6-fold and 28-fold higher than that of SN-38 and SK&F 10486-A (topotecan), respectively. These three camptothecin derivatives showed similar differential response patterns in the 32 cell lines, indicating nearly identical in vitro cytotoxicity profiles. In a human gastric adenocarcinoma SC-6 xenograft model, three intravenous injections of DX-8951f at 4-day intervals demonstrated superior antitumor activity compared to CPT-11 or SK&F 10486-A. Furthermore, it overcame P-glycoprotein-mediated multidrug resistance. These data suggest that DX-8951f possesses high antitumor activity and is a potential therapeutic agent. [1]

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We determined the antitumor and antimetastatic efficacy of the camptothecin analog DX-8951f in a mouse model of orthotopic pancreatic cancer. DX-8951f showed efficacy against two human pancreatic cancer cell lines in this model. These cell lines were transduced with green fluorescent protein, allowing for high-resolution observation of tumor and metastatic lesions in vivo. The study of DX-8951f included early and late-stage cancer models. In the early model, human pancreatic cancer cell lines MIA-PaCa-2 and BxPC-3 were used, and treatment was initiated when the diameter of the primary tumor in situ was approximately 7 mm. DX-8951f showed significant efficacy against both MIA-PaCa-2 and BxPC-3. In contrast, the standard treatment for pancreatic cancer, 2',2'-difluorodeoxycytidine (gemcitabine), was ineffective against MIA-PaCa-2. Although gemcitabine significantly inhibited the growth of the primary tumor in BxPC-3, it was ineffective against metastatic lesions. In advanced disease models, BxPC-3 was used, and treatment was initiated when the diameter of the primary tumor in situ was 13 mm. DX-8951f showed significant dose-dependent efficacy against BxPC-3 primary tumors. DX-8951f also showed anti-metastatic activity in advanced models, significantly reducing the incidence of lymph node metastasis and eliminating lung metastasis. In contrast, gemcitabine showed only moderate efficacy against primary tumors and was ineffective against both primary and metastatic sites in advanced models. Therefore, DX-8951f is highly effective against both primary and metastatic growth of this extremely difficult-to-treat disease and is significantly more effective than gemcitabine, the standard treatment for pancreatic cancer. Therefore, DX-8951f has important clinical application prospects and has more advantages than the currently used camptothecin analog CPT-11, which requires metabolic activation and is toxic. [2] We previously reported that the novel water-soluble camptothecin analog DX-8951f significantly inhibited the in vitro and in vivo growth of a variety of human and mouse tumors. DX-8951f exhibited stronger antitumor activity and topoisomerase I inhibitory activity than other existing camptothecin analogues. In this study, a stepwise screening system was used to construct the SN-38-resistant cell line PC-6/SN2-5 from the human oat cancer cell line PC-6, and the drug resistance mechanism of this cell line was investigated. Subsequently, the antitumor activity of camptothecin analogues in this cell line was compared. PC-6/SN2-5 cells showed resistance to SN-38 (32-fold resistance) and SK&F 104864 (topotecan; 14-fold resistance), but lower resistance to CPT-11 (3-fold resistance) and DX-8951f (2-fold resistance). The topoisomerase I protein level and activity in the parental cells were similar to those in the resistant cells. Intracellular drug concentrations were determined by flow cytometry or high performance liquid chromatography, confirming that intracellular accumulation of SN-38 and topotecan was significantly reduced in PC-6/SN2-5 cells, while intracellular accumulation of DX-8951f was only slightly reduced. In addition, DX-8951f was able to stabilize the formation of cleavable complexes in intact PC-6/SN2-5 cells and their parent cells, while SN-38 and topotecan did not have this effect in drug-resistant cells. Our data suggest that PC-6/SN2-5 cells may acquire resistance to camptothecin analogues by reducing intracellular drug accumulation, and DX-8951f may have the potential to overcome this resistance mechanism induced by camptothecin compounds. [3]

C25H26FN3O7S

531.553248882294

531.148

C, 56.49; H, 4.93; F, 3.57; N, 7.91; O, 21.07; S, 6.03

169869-90-3

197720-53-9 (mesylate dihydrate); 169869-90-3 (mesylate); 171335-80-1; 144008-87-7 (HCl); 171335-80-1; 169869-90-3 (mesylate); 197720-53-9 (mesylate dihydrate); 144008-87-7 (HCl)

6918249

Yellow solid powder

3.758

3

10

1

37

1040

2

S(C)(=O)(=O)O.FC1C=C2C3=C(C=1C)CC[C@@H](C3=C1C(C3=CC4=C(COC([C@@]4(CC)O)=O)C(N3C1)=O)=N2)N([H])[H]

BICYDYDJHSBMFS-GRGFAMGGSA-N

InChI=1S/C24H22FN3O4.CH4O3S/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)/t16-,24-;/m0./s1

(1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4'

DX-8951; DX 8951; DX8951

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)

DMSO: 11.8~12.5 mg/mL (22.1~23.5 mM)
Water: ~12.5 mg/mL

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)