TOP I; topoisomerase I

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].

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].

Cell Lysis and Western Blotting: [3]
Cells (5 × 10⁶) were lysed using SDS buffer (10 mM HEPES, 2 mM orthovanadate, 10 mM NaF, 10 mM pyrophosphate, 1 mM PMSF, 10 µg/mL leupeptin, 10% 2-mercaptoethanol, 10% glycerol, 8% SDS, 42 mM Tris-HCl, 0.002% bromophenol blue, pH 7.4). Proteins from the whole-cell lysates were separated by 7.5% polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane. The membrane was probed with an anti-Topo I human antibody, followed by incubation with horseradish peroxidase-conjugated protein A. Topo I-specific bands were visualized using ECL reagents.

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Nuclear Extract Preparation: [3]
For nuclear extracts, cells (5 × 10⁷) were washed with ice-cold buffer (2 mM K₂HPO₄, 5 mM MgCl₂, 150 mM NaCl, 1 mM EGTA, 0.1 mM dithiothreitol), resuspended in buffer containing 0.35% Triton-X100 and PMSF, and incubated on ice for 10 min. The lysates were centrifuged, and the resulting pellet was further incubated with buffer containing 0.35 M NaCl for 1 hour at 4°C. After centrifugation (18,000 × g, 10 min), the protein concentration of the supernatant (nuclear extract) was determined using Bradford’s method with a protein assay kit. Equal amounts of nuclear protein were then subjected to Western blot analysis with anti-Topo I antibody.

Growth Inhibition Assay (MTT Method): [1]
Cell growth inhibition was assessed in 96-well flat-bottom microplates using the MTT assay. Cells (500–20,000/well) were seeded in 150 μL of medium and pre-incubated for 24 hours (4 hours for P388, CCRF-CEM, and K562 cells). Test compounds, including Exatecan Mesylate (in 150 μL medium/well), or medium alone (control) were then added, followed by a 3-day incubation. After treatment, 20 μL of MTT solution (5 mg/mL in PBS) was added to each well, and plates were incubated for 4 hours. The plates were centrifuged at 800 × g for 5 minutes, the supernatant was removed, and the formazan crystals were dissolved in 150 μL of DMSO. Absorbance was measured at 540 nm using a Microplate Reader (Model 3550).

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 is a pyranoindoquinoline compound. Exatecan has been used in clinical trials for the treatment of various cancers, including sarcoma, leukemia, lymphoma, lung cancer, and liver cancer. The semi-synthetic derivative of camptothecin, 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 showed high in vitro activity against 32 malignant cell lines and significant inhibition of topoisomerase I. The antiproliferative activity (expressed as average GI50 value) of DX-8951f 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 32 cell lines, meaning their in vitro cytotoxicity profiles were almost identical. In human gastric adenocarcinoma SC-6 xenografts, DX-8951f showed superior antitumor activity to CPT-11 or SK&F 10486-A when administered intravenously three times at 4-day intervals. Furthermore, it overcame P-glycoprotein-mediated multidrug resistance. These data suggest that DX-8951f has 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 metastasis of 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. Studies of DX-8951f included early and late-stage cancer models. In early models, using human pancreatic cancer cell lines MIA-PaCa-2 and BxPC-3, treatment was initiated when the in situ primary tumor diameter 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 the MIA-PaCa-2 cell line. Although gemcitabine significantly inhibited the growth of primary BxPC-3 tumors, it was ineffective against metastases. In a late-stage BxPC-3 disease model, treatment was initiated when the in situ primary tumor diameter reached 13 mm. DX-8951f showed a dose-dependent and significant inhibitory effect on primary BxPC-3 tumors. DX-8951f also demonstrated anti-metastatic activity in the late-stage model, significantly reducing the incidence of lymph node metastasis and eliminating lung metastasis. In contrast, gemcitabine has only moderate inhibitory effects on primary tumors and is ineffective against both primary and metastatic lesions in advanced models. Therefore, DX-8951f shows extremely high efficacy against both primary and metastatic growth of this 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]

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We previously reported that the novel water-soluble camptothecin analog DX-8951f can significantly inhibit the growth of various human and mouse tumors in vitro and in vivo. The antitumor activity and topoisomerase I inhibitory activity of DX-8951f are stronger than other existing camptothecin analogs. This study used a stepwise screening method to isolate the SN-38 resistant cell line PC-6/SN2-5 from the human oat cancer cell line PC-6, and investigated the drug resistance mechanism of this cell line, comparing the antitumor activity of camptothecin analogues to this cell line. PC-6/SN2-5 cells showed significant 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 a significant decrease in the intracellular accumulation of SN-38 and topotecan in PC-6/SN2-5 cells, while the intracellular accumulation of DX-8951f was only slightly reduced. Furthermore, DX-8951f stabilized 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 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]

C24H22FN3O4

531.55324

435.159

C, 66.20; H, 5.09; F, 4.36; N, 9.65; O, 14.70

171335-80-1

Exatecan mesylate;169869-90-3;Dxd;1599440-33-1;Exatecan mesylate dihydrate;197720-53-9

151115

Gray to brown solid powder

1.5±0.1 g/cm3

818.4±65.0 °C at 760 mmHg

448.7±34.3 °C

0.0±3.1 mmHg at 25°C

1.735

1.81

2

7

1

32

950

2

CC[C@@]1(C2=C(COC1=O)C(=O)N3CC4=C5[C@H](CCC6=C5C(=CC(=C6C)F)N=C4C3=C2)N)O

ZVYVPGLRVWUPMP-FYSMJZIKSA-N

InChI=1S/C24H22FN3O4/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/h6-7,16,31H,3-5,8-9,26H2,1-2H3/t16-,24-/m0/s1

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

DX 8951; DX 8951; Exatecan; 171335-80-1; Exatecan [INN]; Dx 8951; UNII-OC71PP0F89; DX-8951f; OC71PP0F89; DX 8951.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~50 mg/mL (~114.82 mM)

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.)