Topoisomerase 1
Topoisomerase I (Top1) - Belotecan Hydrochloride (CKD602) is a Top1 inhibitor. No IC50/Ki/EC50 values reported.[1]
Belotecan targets DNA topoisomerase I (Top1), an enzyme that relieves torsional strain in DNA during replication and transcription. It binds to and stabilizes the cleavable complex of topoisomerase I-DNA, preventing religation of single-stranded DNA breaks.

Belotecan significantly reduces the viability of YD-8, YD-9, and YD-38 cells in a dose- and time-dependent manner; at 72 hours after treatment, the IC50 values are 2.4, 0.18, and 0.05 μg/mL. These cell lines undergo apoptosis when exposed to belotecan. Oral squamous cell cancer cells undergo G2/M phase arrest upon exposure to belotecan[1]. Belotecan has a noteworthy anticancer effect on glioma cells, as evidenced by its IC50 values of 9.07 nM for LN229, 14.57 nM for U251 MG, 29.13 nM for U343 MG, and 84.66 nM for U87 MG [2].

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Belotecan Hydrochloride (CKD602) exerted time- and dose-dependent antiproliferative effects in YD-8 (tongue), YD-9 (buccal mucosa) and YD-38 (lower gingiva) OSCC cell lines. At 72 h, IC50 values were 2.4 μg/ml for YD-8, 0.18 μg/ml for YD-9, and 0.05 μg/ml for YD-38.[1]
Belotecan Hydrochloride (CKD602) (0.02 μg/ml for 48 and 72 h) induced G2/M phase arrest: at 72 h, G2/M population increased from 11.9±3.8% to 77.6±0.3% in YD-8, from 25.2±3.6% to 54.0±5.4% in YD-9, and from 19.6±3.5% to 78.3±2.6% in YD-38; G1 phase decreased accordingly.[1]
Belotecan Hydrochloride (CKD602) (0.02 μg/ml) increased phosphorylation of histone H2AX (γH2AX Ser139) in all three cell lines. Myt1 protein expression increased at 24 h and decreased at 48 h. Phospho-cdc2 (Tyr15) was significantly increased at 24 h in all lines, and reduced at 72 h in YD-9 and YD-38. Cyclin A2 protein levels increased at 24 and 48 h. Cyclin B1 increased at 24 h in YD-8 and YD-38, with no change in YD-9 at 24 h, and decreased at 48 h in YD-9 and YD-38. Phosphorylation of histone H3 Ser10 was significantly reduced in a time-dependent manner in YD-9 and YD-38 (p<0.05), but not detected in YD-8.[1]
Belotecan Hydrochloride (CKD602) (0.1 and 0.5 μg/ml for 48 h) induced apoptosis in a dose-dependent manner as measured by Annexin V-FITC/PI staining. At 0.5 μg/ml, apoptotic cells (%) were 13.77±2.77% in YD-8, 38.74±14.67% in YD-9, and 26.31±1.20% in YD-38. YD-8 showed lower apoptotic proportion than YD-9 or YD-38.[1]
In vitro, belotecan hydrochloride shows significant time- and dose-dependent cytotoxic effects in various cancer cell lines. At 72 hours after treatment, IC50 values in YD-8, YD-9, and YD-38 cells are 2.4 μg/ml, 0.18 μg/ml, and 0.05 μg/ml, respectively.

Belotecan significantly inhibits the growth of intracerebral gliomas; animals treated with this drug showed notably smaller tumors than animals in the control group[3].
CKD-602 (7-[2-(N-isopropylamino)ethyl]-(20S)-camptothecin, belotecan), a novel synthetic water-soluble camptothecin derivative, is known to have a significant anticancer effect in vitro on human glioma cell lines, including U87MG and U251MG. In the present study, we evaluated the in vivo antitumor effect of CKD-602 in a mouse glioma model. Nude mice with established U87MG glioma were treated with a dose of CKD-602 of 0mg/kg (control group, injection with saline only; n=5), 40 mg/kg (group A) or 60 mg/kg (group B). Thereafter, the dose was repeated once every 4 days for a total of four doses. Tumor volume was measured histologically and apoptosis was detected using the terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL) assay and immunofluorescence analysis with cleaved caspase-3. Mean tumor volume in each group was: control, 145.35 mm(3); group A, 76.51 mm(3); group B, 73.99 mm(3)). Tumor volume was significantly smaller in both groups A and B compared with the control group (group A, p<0.01; group B, p<0.05). Apoptosis of tumor cells was evident to a greater extent in groups A and B relative to the control group, but there were no significant differences in tumor volume or apoptotic index between groups A and B. These results suggest that CKD-602 has a significant anticancer effect on glioma cells in vivo.[1]

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In vivo, belotecan significantly inhibits the growth of intracerebral gliomas; animals treated with this drug showed notably smaller tumors than animals in the control group. It has been studied for the treatment of small cell lung cancer and other cancers.

Cell-free topoisomerase I inhibition assays are performed using purified recombinant topoisomerase I and a DNA substrate. Belotecan is incubated with the enzyme and DNA, and the formation of the cleavable complex is detected by gel electrophoresis or other methods to assess its inhibitory activity (IC50 = 0.119 μg/ml).

For 24, 48, and 72 hours, the cells are exposed to varying concentrations of belotecan (0.01, 0.1, 0.5, 1, 5, and 10 μg/mL). Only medium is applied to control samples of every cell line. Using the MTS assay, cell viability is determined[1].
CKD-602 7-[2-(N-isopropylamino)ethyl]-(20S)-camptothecin, belotecan) is a synthetic water-soluble camptothecin derivative and topoisomerase inhibitor that has been shown to have clinical anticancer effect against ovarian and lung cancer. We studied its anticancer effects on four human glioma cell lines, U87 MG, U343 MG, U251 MG and LN229. Cell viability was quantified by a modified 2-(2-methoxy-4-nitropheyl)-3-(4-nitropheyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt and significant time- and dose-dependent cytotoxicity was observed in all cell lines. Susceptibility to CKD-602 at 48 h after treatment varied among the four cell lines and their IC50 value was as follows: 9.07 nM (95% CI 0.18-37.42) for LN229, 14.57 nM (95% CI 0.86-47.33) for U251 MG, 29.13 nM (95% CI 0.35-101.23) for U343 MG, and 84.66 nM (95% CI 34.63-148.25) for U87 MG. CKD-602 induced cell cycle arrest at G2 phase and produced antiproliferative activity and apoptosis in all cell lines. Thus, CKD-602 showed a significant anticancer effect on glioma cells in vitro and is a promising candidate for further studies on malignant gliomas.[2]

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MTS viability assay: Cells seeded at 2×10⁴ cells/well in 96-well plates, treated with Belotecan Hydrochloride (CKD602) at concentrations 0.01, 0.1, 0.5, 1, 5, 10 μg/ml for 24, 48 and 72 h. After incubation, 20 μl/well MTS reagent added, incubated 1 h at 37°C, absorbance read at 490 nm. IC50 calculated from dose-response curves.[1]
Annexin V-FITC apoptosis assay: Cells plated at 1×10⁶ cells/100 mm dish, treated with 0.1 and 0.5 μg/ml Belotecan Hydrochloride (CKD602) for 48 h, harvested by centrifugation. Cell pellets resuspended in Annexin V binding buffer, stained with FITC-conjugated Annexin V and propidium iodide (PI) for 15 min at room temperature in dark, analyzed by flow cytometry.[1]
Cell cycle analysis: Cells plated at 1×10⁶ cells/100 mm dish, treated with 0.02 μg/ml Belotecan Hydrochloride (CKD602) for 48 and 72 h, harvested, fixed in 70% ethanol at -20°C for ≥1 h, washed, resuspended in PI/RNase staining buffer, analyzed by flow cytometry (≥10,000 events/sample).[1]
Western blot analysis: Cells treated with Belotecan Hydrochloride (CKD602) (0.02 μg/ml), lysed in RIPA buffer containing protease and phosphatase inhibitors, centrifuged at 10,000×g for 20 min at 4°C. Protein concentration determined by BCA assay. 20 μg whole lysate resolved on 10% or 12.5% SDS-PAGE, transferred to PVDF membrane, probed with primary antibodies (anti-β-actin, anti-p53, anti-phospho-H3 Ser10, anti-cyclin B1, anti-cyclin A2, anti-cdc2, anti-phospho-cdc2 Tyr15, anti-Myt1, anti-phospho-H2AX Ser139) overnight at 4°C, then HRP-conjugated secondary antibodies for 1 h, developed with ECL.[1]
In vitro cytotoxicity assays are performed using cancer cell lines such as YD-8, YD-9, and YD-38 (oral cancer cells). Cells are treated with belotecan hydrochloride for various time points (e.g., 72 hours), and cell viability is measured using MTT or similar assays to determine IC50 values.

Mice: Belotecan is administered intraperitoneally to groups A and B at doses of 40 mg/kg, 60 mg/kg, and 0 mg/kg (control group) in order to treat established U87MG gliomas in naked mice. After that, for a total of four doses, the dose is repeated once every four days. Histological measurements of the tumor volume reveal the presence of apoptosis[1].

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In vivo efficacy is evaluated in xenograft models, particularly intracerebral glioma models. Belotecan is administered to tumor-bearing mice, and tumor growth is monitored. The compound's ability to cross the blood-brain barrier and inhibit tumor growth in the brain is assessed.

Belotecan Hydrochloride (CKD602) is a water-soluble camptothecin analogue. Its pharmacokinetics involve transport by P-glycoprotein, multidrug resistance protein 2, and breast cancer resistance protein. It is under investigation for the treatment of small cell lung cancer and other types of cancer.

In phase I/II studies of solid cancers, Belotecan Hydrochloride (CKD602) showed dose-limiting toxicities of neutropenia and thrombocytopenia, but not diarrhea (which is the prototype toxicity of CPT-11). No specific toxicity data provided in this in vitro study. [2]
The toxicity of belotecan is related to its mechanism of action as a topoisomerase I inhibitor. Common side effects include myelosuppression, gastrointestinal toxicity, and alopecia, similar to other camptothecin analogues. It may also cause hepatotoxicity and nephrotoxicity at higher doses.

[1]. Anticancer effects of CKD-602 (Camtobell\u00ae) via G2/M phase arrest in oral squamous cell carcinoma cell lines. Oncol Lett. 2015 Jan;9(1):136-142.

[2]. CKD-602, a camptothecin derivative, inhibits proliferation and induces apoptosis in glioma cell lines. Oncol Rep. 2009 Jun;21(6):1413-9.

[3]. Antitumor activity of CKD-602, a camptothecin derivative, in a mouse glioma model. J Clin Neurosci. 2012 Feb;19(2):301-5

Belotecan hydrochloride is the hydrochloride salt of the semi-synthetic camptothecin analog Belotecan and possesses potential antitumor activity. Belotecan binds to and inhibits the activity of topoisomerase I, stabilizing the cleavable topoisomerase I-DNA complex, thereby inhibiting the rejoining of single-stranded DNA breaks generated by topoisomerase I. When DNA replication encounters the topoisomerase I-DNA complex, fatal double-stranded DNA breaks occur, leading to the interruption of DNA replication and tumor cell apoptosis. Topoisomerase I is an enzyme that mediates reversible single-stranded DNA breaks during DNA replication.
See also: Belotecan (note moved to).

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Belotecan Hydrochloride (CKD602) is a potent topoisomerase I inhibitor that overcomes poor water solubility and toxicity of camptothecin. It has shown antitumor activity against various human tumor cell lines. In this study, it inhibited proliferation of OSCC cell lines derived from Korean patients with different p53 statuses: YD-8 (p53 mutation R273H), YD-9 (p53 protein positive, no mutation), YD-38 (p53 null). The cytotoxic effect was more prominent in YD-9 and YD-38 (no p53 mutation) than in YD-8 (p53 mutation). The drug induced G2/M arrest via upregulation of phospho-cdc2 (Tyr15), cyclin A2, cyclin B1, Myt1 and γH2AX, and downregulation of phospho-histone H3 Ser10.[1]
Belotecan hydrochloride (CKD-602) is a semi-synthetic camptothecin analogue that acts as a DNA topoisomerase I inhibitor. It is marketed under the trade name Camtobell. It is being studied in the treatment of small cell lung cancer and other types of cancer. It blocks enzymes needed for cell division and DNA repair.

C25H28CLN3O4

469.9605

469.177

C, 63.89; H, 6.01; Cl, 7.54; N, 8.94; O, 13.62

213819-48-8

256411-32-2; 213819-48-8 (HCl)

6918340

White to yellow solid powder

772.4ºC at 760mmHg

420.9ºC

4.21E-25mmHg at 25°C

3.813

3

6

5

33

865

1

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

SJKBXKKZBKCHET-UQIIZPHYSA-N

InChI=1S/C25H27N3O4.ClH/c1-4-25(31)19-11-21-22-17(12-28(21)23(29)18(19)13-32-24(25)30)15(9-10-26-14(2)3)16-7-5-6-8-20(16)27-22;/h5-8,11,14,26,31H,4,9-10,12-13H2,1-3H3;1H/t25-;/m0./s1

(19S)-19-ethyl-19-hydroxy-10-[2-(propan-2-ylamino)ethyl]-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;hydrochloride

CKD-602; Camtobell; Belotecan Hydrochloride; 213819-48-8; Camtobell hydrochloride; Belotecan HCl; CKD-602; CKD 602; Belotecan (hydrochloride); (S)-4-ethyl-4-hydroxy-11-(2-(isopropylamino)ethyl)-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dione hydrochloride;CKD 602; CKD602; Belotecan

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: 12.5~47 mg/mL (26.6~100.0 mM)
Water: ~4 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.)