Survivin (IC50 = 0.54 nM)

Survival gene promoter-driven luciferase reporter activity does not affect sepantronium bromide (YM155; 30 μM). Through transcriptional suppression of the survivin gene promoter, sepantronium bromide dramatically reduces endogenous survivin expression in p53-deficient PC-3 and PPC-1 human HRPC cells. The protein expression of c-IAP2, XIAP, Bcl-2, Bcl-xL, Bad, α-actin, and β-tubulin is not impacted by sepatrium bromide (100 nM). With IC50 ranges of 2.3 to 11 nM, sepatrium bromide potently inhibits human cancer cell lines (mutant or truncated p53), including PC-3, PPC-1, DU145, TSU-Pr1, 22Rv1, SK-MEL-5, and A375, respectively. 1]. Gamma radiation sensitivity is increased in NSCLC cells by sepatrium bromide (YM155). The number of apoptotic cells and caspase-3 activity are increased when separantronium bromide and gamma radiation are combined. Furthermore, nuclear DNA double-strand breaks brought on by radiation are not repaired as quickly by sepantronium bromide [2].

In PC-3 xenografts, sepantronium bromide (YM155; 3 and 10 mg/kg) suppresses tumor growth without causing appreciable weight loss or lowering blood cell counts. Serpentine bromide is widely dispersed throughout the body's tumor tissues. In PC-3 orthotopic xenografts, serpentium bromide showed 80% TGI at a dose of 5 mg/kg [1]. When combined with gamma radiation, sepentronium bromide (YM155) exhibits strong anticancer efficacy against H460 or Calu6 xenografts in nude mice [2]. Sepantronium bromide (YM-155) and IL-2 further decreased tumor weight, lung metastasis, and fluorescein-stained tumor pictures in this orthotopic renal tumor and metastatic lung tumor model [3].

A 2,767-bp sequence of human survivin gene promoter is isolated from human genomic DNA by PCR using Pyrobest polymerase and the following primers: 5

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Immunofluorescence staining of γ-H2AX. [2]
Cells were grown to 50% confluence in two-well Lab-Tec Chamber Slides (Nunc) and then cultured for 48 h in the presence of 50 nmol/L YM155 or vehicle before exposure to 3 Gy of γ-radiation. At various times thereafter, they were fixed with 4% paraformaldehyde for 10 min at room temperature, permeabilized with 0.1% Triton X-100 for 10 min at 4°C, and exposed to 5% nonfat dried milk for 10 min at room temperature. The slides were washed with PBS and then incubated at room temperature first for 2 h with mouse monoclonal antibodies to histone γ-H2AX at a dilution of 1:300 and then for 1 h with Alexa 488–labeled goat antibodies to mouse IgG at a dilution of 1:700. The slides were mounted in fluorescence mounting medium, and fluorescence signals were visualized with a confocal laser-scanning microscope equipped with the LSM5 PASCAL system. Three random fields each containing =50 cells were examined at a magnification of × 100. Nuclei containing ≥10 immunoreactive foci were counted as positive for γ-H2AX, as previously described, and percentage of positive cells was calculated[2].

Cells are seeded in 96-well plates at a density of 5-40 × 103. For 48 hours, cells are given DMSO with YM155 dissolved in it. Then, using a sulforhodamine B assay, the cell count is calculated.

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Trypan blue exclusion staining for determination of cell viability. [1]
After culture for 48 h in the absence or presence of YM155, PC-3 and PPC-1 cells were collected by trypsinization and centrifugation (0.05% trypsin-EDTA) and resuspended in DMEM. The cell suspension was diluted in equal volumes of trypan blue (0.4% working solution). Viable (unstained) and dead (stained) cells were counted on a hemocytometer, and the ratio of viable cells to the total number of cells was expressed as viability percentage.

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Measurement of caspase-3 activity. [1]
The caspase-3 activity was measured with a CPP32/Caspase-3 Fluometric Protease Assay Kit (MBL) according to the manufacturer's instructions. After incubation with YM155 for 48 h, PC-3 and PPC-1 cells were lysed in 100 μL of a cell lysis buffer (provided with the kit) and equal volumes (50 μL) of cell lysate were obtained (100 μg of protein). After addition of 2× reaction buffer, the mixture was added to a black 96-well plate. The DEVD-AFC substrate (appended with the kit) was then added at 5 mL/well and the mixture incubated at 37°C for 30 min. Fluorescence emissions were quantified with a spectrofluorometer at an excitation wavelength of 390 nm and an emission wavelength of 460 nm.

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In vitro cell growth inhibition assay. [1]
The antiproliferative activity of YM155 was measured by the method used at the National Cancer Institute. After treatment with YM155 for 48 h, the cell count was determined by sulforhodamine B assay. The GI50 value was calculated by logistic analysis, which is the drug concentration resulting in a 50% reduction in the net protein increase (as measured by sulforhodamine B staining) in control cells during the drug incubation. The assay was done in triplicate, and the mean GI50 value was obtained from the results of four independent assays.

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Time dependency of antitumor activity in vitro. [1]
A549 cells were treated with YM155, methotrexate, or doxorubicin. Each compound was removed after various lengths of exposure time by washing five times with fresh medium. At 72 h of incubation after the beginning of cell treatment, the effect of the selected compound on cell proliferation was determined with the Alamar Blue assay (Serotec; ref. 22), done in duplicate for each concentration (n = 1). The IC50 was calculated by logistic analysis. To understand the in vitro mode of action and pharmacodynamic properties of each compound, the log slope (exposure time) and the reciprocal of the log slope (IC50 of the antiproliferative effect on A549) were plotted on a logarithmic scale for YM155, doxorubicin (as area under the curve–dependent drug), and methotrexate (as time-dependent drug), and the slope of the IC50-exposure time curve was compared among the three agents

PC-3 s.c. (orthotopic) xenografts in male nude mice (BALB/c nu/nu)
5 mg/kg
Subcutaneous injection as a 3-day continuous infusion per week for 3 weeks by an implanted micro-osmotic pump.

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For the in vivo experiments, YM155 was dissolved and diluted in saline immediately before administration.
In vivo antitumor activities against PC-3 s.c. xenograft model.[1]
Five-week-old male nude mice (BALB/c nu/nu) were used. PC-3 cells (2 × 106–3 × 106) were injected into the flanks of the mice and allowed to reach a tumor volume of >100 mm3 in tumor volume (length × width2 × 0.5). YM155 was s.c. administered as a 3-day continuous infusion per week for 2 weeks using an implanted micro-osmotic pump (Alzet model 1003D, Durect) or i.v. administered five times a week for 2 weeks. The percentage of tumor growth inhibition 14 days after initial YM155 administration was calculated for each group using the following formula: MTV = 100 × {1 − [(MTV of the treated group on day 14) − (MTV of the treated group on day 0)] / [(MTV of the control group on day 14) − (MTV of the control group on day 0)]}, where MTV is mean tumor volume. For both the frozen tumors and plasma samples, survivin expression levels were analyzed by Western blotting and YM155 drug concentration by high-performance liquid chromatography/triple quadrupole mass spectrometry (LC/MS/MS) using validated methods.

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In vivo antitumor activities against PC-3 orthotopic xenograft model. [1]
For orthotopic implantation, a PC-3 cell suspension (1 × 106/20 μL per mouse) was injected into the prostate dorsolateral lobe (right side) of 7-week-old male nude mice (BALB/c nu/nu). Two weeks after implantation, YM155 was s.c. administered as a 3-day continuous infusion per week for 3 weeks at 5 mg/kg/d using an implanted micro-osmotic pump (Alzet model 1003D, Durect). Body weight was measured periodically starting from the first day of YM155 administration. Three weeks later, the tumors adhering to the prostate and seminal vesicle were taken and weighed as tumor weight. The antitumor activity of YM155 was expressed as a percentage of tumor growth inhibition calculated using the following formula: MTW = 100 × [1 − (MTW on day 21 of the treated group) / (MTW on day 21 of the control group)].

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Evaluation of tumor growth in vivo. [2]
Tumor cells (2 × 106) were injected s.c. into the right hind leg of 6-week-old female athymic nude mice (BALB/c nu/nu). Tumor volume was determined from caliper measurement of tumor length (L) and width (W) according to the formula LW2/2. Treatment was initiated when the tumors in each group of animals achieved an average volume of ∼200 to 250 mm3. Treatment groups (each containing eight mice) consisted of vehicle control (physiologic saline), YM155 alone, vehicle plus radiation, and YM155 plus radiation. Vehicle or YM155 at a dose of 5 mg/kg of body mass was administered over 7 consecutive days (days 1-7) with the use of an implanted micro-osmotic pump (Alzet model 1003D; Durect). Mice in the radiation groups received 10 Gy of γ-radiation from a cobalt irradiator either as a single fraction on day 3 of drug treatment or fractionated over 5 consecutive days (days 3 to 7); the radiation was targeted to the tumor, with the remainder of the body shielded with lead. Growth delay (GD) was calculated as the time required to achieve a 5-fold increase in volume for treated tumors minus that for control tumors. The enhancement factor was then determined as: (GDcombination − GDYM155)/GDradiation.[2]

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Luciferase-expressing RENCA cells were implanted to the subrenal capsule of the left kidney and tail vein, respectively, to produce a mouse RCC model with an orthotopic tumor and metastatic lung tumors. The mice were randomly divided into four groups with an even distribution of IVIS values. Group 1 received intraperitoneal (IP) injection of 100 μL of phosphate-buffered saline (PBS) as a vehicle control; group 2 received YM155 alone (1 mg/kg body weight per day for 1 week by IP injection); group 3 received IL-2 alone (6000 U of recombinant IL-2 by IP injection on days 0, 4, and 8 of treatment); and group 4, the combination therapy group, received YM155 and IL-2 (dose and dosing schedule the same as in group 2 plus group 3). Tumor imaging was performed and tumor volume was analyzed in all groups on day 14 post-treatment. The mice were sacrificed, and the weights of the orthotopic tumor in the left kidney and sections of bilateral lung tissues with metastatic growth were measured.[3]

[1]. YM155, a novel small-molecule survivin suppressant, induces regression of established human hormone-refractory prostate tumor xenografts. Cancer Res. 2007 Sep 1;67(17):8014-21.

[2]. Radiosensitizing effect of YM155, a novel small-molecule survivin suppressant, in non-small cell lung cancer cell lines. Clin Cancer Res. 2008 Oct 15;14(20):6496-504.

[3]. A combination of YM-155, a small molecule survivin inhibitor, and IL-2 potently suppresses renal cell carcinoma in murine model. Oncotarget. 2015 Aug 28;6(25):21137-47.

Sepantronium bromide is an organic bromide salt consisting of sepantronium cations and bromide anions. It has been found to selectively inhibit survivin (BIRC5) gene promoter activity and to down-regulate survivin in vitro, so leading to induction of apoptosis. It has a role as an antineoplastic agent, a survivin suppressant and an apoptosis inducer. It contains a sepantronium.
Sepantronium Bromide is a small-molecule proapoptotic agent with potential antineoplastic activity. Sepantronium bromide selectively inhibits survivin expression in tumor cells, resulting in inhibition of survivin antiapoptotic activity (via the extrinsic or intrinsic apoptotic pathways) and tumor cell apoptosis. Survivin, a member of the inhibitor of apoptosis (IAP) gene family, is expressed during embryonal development and is absent in most normal, terminally differentiated tissues; upregulated in a variety of human cancers, its expression in tumors is associated with a more aggressive phenotype, shorter survival times, and a decreased response to chemotherapy.

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Various accumulating evidence suggests that survivin, a member of the inhibitor of apoptosis (IAP) family, plays an important role in drug resistance and cancer cell survival in many types of cancer, including hormone-refractory prostate cancer (HRPC). Here, we characterized YM155, a novel small-molecule survivin suppressant, using a survivin gene promoter activity assay. YM155 suppressed expression of survivin and induced apoptosis in PC-3 and PPC-1 human HRPC cell lines at 10 nmol/L. In contrast, YM155 up to 100 nmol/L showed little effect on expression levels of other IAP- or Bcl-2-related proteins. In a s.c. xenografted PC-3 tumor model in mice, 3-day continuous infusions of YM155 at 3 to 10 mg/kg induced massive tumor regression accompanied by suppression of intratumoral survivin. YM155 also completely inhibited the growth of orthotopically xenografted PC-3 tumors. No significant decreases in body weight were observed in mice treated with YM155 during the experimental period. Pharmacokinetic analyses indicated that YM155 is highly distributed to tumors and at concentrations approximately 20-fold higher than those in plasma. Our findings represent the first attempt to show tumor regression and suppression of survivin in p53-deficient human HRPC cells by a single small molecular compound treatment. Further extensive investigation of YM155 in many types of cancer, including HRPC, seems to be worthwhile to develop this novel therapeutic approach.[1]

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Purpose: Survivin, a member of the inhibitor of apoptosis protein family, is an attractive target for cancer therapy. We have now investigated the effect of YM155, a small-molecule inhibitor of survivin expression, on the sensitivity of human non-small cell lung cancer (NSCLC) cell lines to gamma-radiation. Experimental design: The radiosensitizing effect of YM155 was evaluated on the basis of cell death, clonogenic survival, and progression of tumor xenografts. Radiation-induced DNA damage was evaluated on the basis of histone H2AX phosphorylation and foci formation. Results: YM155 induced down-regulation of survivin expression in NSCLC cells in a concentration- and time-dependent manner. A clonogenic survival assay revealed that YM155 increased the sensitivity of NSCLC cells to gamma-radiation in vitro. The combination of YM155 and gamma-radiation induced synergistic increases both in the number of apoptotic cells and in the activity of caspase-3. Immunofluorescence analysis of histone gamma-H2AX also showed that YM155 delayed the repair of radiation-induced double-strand breaks in nuclear DNA. Finally, combination therapy with YM155 and gamma-radiation delayed the growth of NSCLC tumor xenografts in nude mice to a greater extent than did either treatment modality alone. Conclusions: These results suggest that YM155 sensitizes NSCLC cells to radiation both in vitro and in vivo, and that this effect of YM155 is likely attributable, at least in part, to the inhibition of DNA repair and enhancement of apoptosis that result from the down-regulation of survivin expression. Combined treatment with YM155 and radiation warrants investigation in clinical trials as a potential anticancer strategy.[2]

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YM155, a small molecule inhibitor of the antiapoptotic protein survivin, has been developed as a potential anti-cancer drug. We investigated a combination therapy of YM155 and interleukin-2 (IL-2) in a mouse model of renal cell carcinoma (RCC). YM155 caused cell cycle arrest and apoptosis in renal cancer (RENCA) cells. Next, luciferase-expressing RENCA cells were implanted in the left kidney and the lung of BALB/c mice to develop RCC metastatic model. In this orthotopic renal and metastatic lung tumors models, YM155 and IL-2 additively decreased tumor weight, lung metastasis, and luciferin-stained tumor images. Also, the combination significantly suppressed regulatory T cells and myeloid-derived suppressor cells compared with single agent treatment. We suggest that a combination of YM155 and IL-2 can be tested as a potential therapeutic modality in patients with RCC.[3]

C20H19N4O3+.CL-

398.84286

398.115

C, 60.23; H, 4.80; Cl, 8.89; N, 14.05; O, 12.03

355406-09-6

Sepantronium bromide;781661-94-7

23108256

Light brown to brown solid powder

0

6

5

28

571

0

NUZWGSASIPTGSA-UHFFFAOYSA-M

InChI=1S/C20H19N4O3.ClH/c1-13-23(9-10-27-2)17-18(24(13)12-14-11-21-7-8-22-14)20(26)16-6-4-3-5-15(16)19(17)25;/h3-8,11H,9-10,12H2,1-2H3;1H/q+1;/p-1

1-(2-methoxyethyl)-2-methyl-3-(pyrazin-2-ylmethyl)benzo[f]benzimidazol-3-ium-4,9-dione;chloride

YM-155 hydrochloride; 355406-09-6; YM-155 (hydrochloride); 1-(2-methoxyethyl)-2-methyl-4,9-dioxo-3-(pyrazin-2-ylmethyl)-4,9-dihydro-1H-naphtho[2,3-d]imidazol-3-ium chloride; Sepantronium (hydrochloride); 1-(2-methoxyethyl)-2-methyl-3-(pyrazin-2-ylmethyl)benzo[f]benzimidazol-3-ium-4,9-dione;chloride; YM-155hydrochloride; YM-155 (chloride);

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

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.)