Pharmaceutical composition comprising tetraarsenic hexoxide (as 4o 6) for preventing or treating bladder cancer

Abstract

The present invention relates to: a pharmaceutical composition comprising at least 99% of tetraarsenic hexoxide crystalline polymorph a(As4O6-a) for preventing or treating bladder cancer; or a preparation method therefor. The composition of the present invention exhibits an excellent effect of inhibiting the proliferation and metastasis of bladder cancer cells, and thus can be effectively used as an anticancer agent for bladder cancer.

Description

Pharmaceutical composition for preventing or treating bladder cancer containing arsenic tetraoxide (As4O6)

[0001] The present invention relates to a pharmaceutical composition for preventing, treating or inhibiting metastasis of bladder cancer, comprising polymorph a of arsenic hexaoxide (As4O6) as an effective ingredient.

[0002] Cancer is one of the three leading causes of death worldwide, along with infectious diseases and cardiovascular disease. Due to environmental issues, increasing life expectancy, and Westernized dietary habits, the number of cancer-affected individuals is expected to rapidly increase in the future, making it a major disease. Despite extensive research into cancer to develop more effective anticancer drugs, the diversification of cancer pathogenesis necessitates the development of new anticancer drugs with fewer side effects and the ability to overcome drug resistance.

[0003] Cancer cells differ from normal cells in several ways. Normal cells proliferate according to their needs and in the appropriate locations. They adhere to one another and coexist. Furthermore, normal cells self-destruct when they become too old or damaged. However, cancer cells lack all of these functions, ultimately forming a tumor by continuously dividing.

[0004] Bladder cancer (BC) is the tenth most common cancer worldwide, with a high incidence and mortality rate, with an estimated 573,278 new cases and over 212,536 deaths expected in 2020. Bladder cancer is characterized by a high rate of somatic mutations and exhibits clinical and pathological heterogeneity.

[0005] Bladder cancer is broadly classified into non-muscle invasive and invasive bladder cancer, depending on the degree of invasion. Non-invasive bladder cancer is a lesion confined to the mucosa without invading the muscularis propria. It can be treated relatively simply by performing transurethral resection of the bladder tumor followed by intravesical injection of chemotherapy or BCG, depending on risk factors. However, recurrence and progression to invasive cancer are concerns. Invasive bladder cancer, on the other hand, refers to a condition in which the cancer has invaded the muscularis propria. Treatment requires radical cystectomy and complex urinary diversion, which can have fatal consequences for the patient. Therefore, predicting recurrence and progression after primary treatment, as well as early detection and prevention, are crucial.

[0006] Although various methods for diagnosing and treating bladder cancer are being developed, the treatment methods currently used in clinical practice include surgery, chemotherapy drugs (methotrexate, vincristine, doxorubicin, cisplatin, and cytosine), and biological therapy (Bacillus Calmette-Guérin, immunotherapy, and inactivated bacterial solutions). However, these methods are limited due to the high recurrence and mortality rates of bladder cancer, as well as the high cost, serious side effects, and various complications.

[0007] Therefore, frequent recurrence and progression of the disease are frequently raised issues in bladder cancer in these clinical settings, and there is a need to develop a treatment method that can effectively predict recurrence and progression of bladder cancer to invasiveness in clinical settings.

[0008] Meanwhile, as a conventionally known technology, arsenic tetraoxide (As4O6) is known to have an anticancer effect on various cancer types including bladder cancer (Korean Patent No. 0272835), and specific polymorphs of arsenic tetraoxide (As4O6) have been reported to have an anticancer effect on various cancer types (Korean Patent No. 1844050, Korean Patent No. 1834366, Korean Patent No. 1755556). However, the above patents do not disclose any excellent bladder cancer treatment effect in clinical trials targeting bladder cancer patients for polymorphs of arsenic tetraoxide (As4O6).

[0009] Meanwhile, in the field of medicinal chemistry, it is widely known that the same compound can have multiple crystal forms, and that pharmaceutical properties such as solubility and stability can differ depending on the crystal form of polymorphism. It is a common task to examine the existence of polymorphism for the formulation design of a pharmaceutical compound, and it is generally understood that solid-state properties including polymorphism, solvent, and salt formation can have a profound effect on important properties (solubility and stability) essential for the successful development of drug candidates. Such polymorphism not only brings about differences in physical properties due to different lattice energies (and entropies) related to different polymorphisms, but also exhibits different water solubility and dissolution rates, and this difference in solubility affects drug absorption in dosage forms. (Maria Saifee, et al, 2009)

[0010] The importance of drug solubility in water is that drugs must dissolve in order to be absorbed through the body's cell membrane and reach the site of action. Therefore, solubility in water is an essential property of drugs. Consequently, solubility is one of the most crucial and important parameters affecting the bioavailability of drugs. Regardless of the pharmaceutical dosage form and route of administration, solubility is generally understood to correspond to the ability of a drug to be available at an appropriate concentration at the site of drug action (Roberta Censi et al., 2015).

[0011] Accordingly, the inventors of the present invention confirmed that a composition containing a compound having 99% or more of the polymorph a of tetraarsenic hexaoxide with increased water solubility has a killing effect on bladder cancer cell lines, an inhibition of metastasis, and an excellent clinical treatment effect on bladder cancer patients, and completed the present invention.

[0012] The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating bladder cancer, which contains arsenic hexaoxide polymorph a (As4O6-a) as an effective ingredient.

[0013] The purpose of the present invention is to provide a pharmaceutical composition for inhibiting bladder cancer metastasis, which contains arsenic hexaoxide polymorph a (As4O6-a) as an effective ingredient.

[0014] The present invention relates to a pharmaceutical composition for preventing, treating or inhibiting metastasis of bladder cancer, comprising a polymorph of arsenic hexaoxide (As4O6), and comprising 99% or more of polymorph a of arsenic hexaoxide (As4O6-a).

[0015] The above composition may contain less than 1% of polymorph b of arsenic hexaoxide (As4O6-b). The polymorph b may have a purity of 99.9% or higher. The polymorphs As4O6-a and As4O6-b may have the characteristics of (i) to (iii) in Table 1 below:

[0016] Arsenic hexaoxide polymorph a (As4O6-a)Arsenic hexaoxide polymorph b (As4O6-b)(i) Unit cell constants a = b = c = 11.0734 Åα = β = γ = 90°V = 1357.82 Å 3 a = b = c = 11.0600 Åα = β = γ = 90°V = 1352.90 Å 3 (ii) As-O bond length 1.786 Å 2.011 Å (iii) O-As-O bond 98.36° 109.47°

[0017] The above-mentioned arsenic tetraoxide polymorph a (As4O6-a) is a crystalline form characterized by the fact that, in an X-ray powder diffraction spectrum, when a light source wavelength is 1.5406 Å, peaks indicated at 13.84, 27.88, 32.32, 35.3, 39.84, 42.38, 46.34, 48.6, and 49.34 appear in a diffraction angle 2θ range of 10° to 50° at a speed of 1° / min (scan step 0.02°) (see Fig. 1). In addition, it is characterized by the ratio of main peaks appearing at 2θ values ​​of 13.8 and 27.9 being 1:1.3.

[0018] The above As4O6-b is a crystalline form characterized by the fact that, in an X-ray powder diffraction spectrum, when the light source wavelength is 1.5406 Å, the peaks indicated at 13.86, 27.92, 32.36, 35.34, 39.9, 42.44, 46.4, 48.66, and 49.4 appear at a speed of 1° / min (scan step 0.02°) in the diffraction angle 2θ range of 10° to 50° (see Fig. 1). In addition, the As4O6-b is characterized by the ratio of the main peaks appearing at 2θ values ​​of 13.8 and 27.9 being 1:2.5.

[0019] According to another aspect of the present invention, there is provided a pharmaceutical composition for inhibiting bladder cancer metastasis, comprising arsenic hexaoxide polymorph a (As4O6-a) as an effective ingredient, wherein the pharmaceutical composition for inhibiting bladder cancer metastasis comprises 99% or more of arsenic hexaoxide polymorph a (As4O6-a).

[0020] According to another aspect of the present invention, there is provided a method for preparing a pharmaceutical composition for preventing or treating bladder cancer, comprising arsenic hexaoxide polymorph a (As4O6-a) as an effective ingredient,

[0021] The first process is to heat sodium chloride to 100 to 800°C and then cool it;

[0022] The second process involves placing arsenic trioxide (As2O3) on top of sodium chloride, heating it from 100℃ to 1000℃ in a sealed state, and then cooling it;

[0023] A third process for separating crystals crystallized in a filter paper; and

[0024] A fourth process in which the crystals obtained in the third process are used instead of the diarsenic trioxide of the second process, and the second and third processes are repeated 4 to 10 times to obtain tetraarsenic hexaoxide crystals;

[0025] It is manufactured by a method including:

[0026] The present invention relates to a method for producing a pharmaceutical composition for preventing or treating bladder cancer, characterized in that the tetraarsenic hexaoxide crystals obtained in the fourth process contain 99% or more of the tetraarsenic hexaoxide crystal polymorph a (As4O6-a).

[0027] Hereinafter, the present invention will be described in more detail.

[0028] The present invention relates to a composition for preventing or treating bladder cancer, which comprises arsenic hexaoxide polymorph a (As4O6-a) as an effective ingredient, and comprises 99% or more of arsenic hexaoxide polymorph a (As4O6-a).

[0029] The method for manufacturing a composition for preventing or treating bladder cancer of the present invention comprises: a first step of heating sodium chloride to 100 to 800°C and then cooling; a second step of placing arsenic trioxide (As2O3) on the sodium chloride, heating it from 100°C to 1000°C in a sealed state, and then cooling it; a third step of separating crystals crystallized on a filter paper; and a fourth step of using the crystals obtained in the third step instead of the arsenic trioxide of the second step, repeating the second and third steps 4 to 10 times to obtain arsenic hexaoxide crystals.

[0030] A synthesis reactor made of kaolin and a clamp capable of mounting a filter on the upper part of the synthesis reactor are prepared, and sodium chloride is placed in the synthesis reactor and heated and cooled. The reason why sodium chloride is used in the manufacturing method of the present invention is that in the second step, by placing diarsenic trioxide on the sodium chloride and heating it, heat is evenly transferred to the arsenic compound, which helps the arsenic compound to sublimate. In the first step, the sodium chloride is heated at 100 to 800°C for 2 to 6 hours to remove impurities and moisture. In the first step, after heating the sodium chloride, it is cooled at room temperature for 3 to 10 hours.

[0031] Next, a second process is performed in which arsenic trioxide (As2O3) is placed on sodium chloride, then heated from 100℃ to 1000℃ in a sealed state, and then cooled. Here, after placing the arsenic trioxide, 3 to 6 filters (filter paper) capable of capturing arsenic sublimated to the clamp are installed so that the gap between each filter is 2 to 6 mm. It is preferable to use a filter having a basic weight of 70 to 100 g / ㎡, a thickness of 0.17 to 0.25 mm, a filtration speed of 22 to 30 s / 100 ml, and a retention rate of 5 to 10 μm.

[0032] After installing the filter and sealing it, heat the bottom of the synthesis reactor by gradually increasing the temperature from 100℃ to 1000℃ for 3 to 10 hours so that the temperature at the top center of the filter paper is maintained at 150 to 1000℃, and arsenic tetraoxide is crystallized as it passes through the filter paper. Then, cool it at room temperature for more than 5 hours, preferably 5 to 10 hours.

[0033] Next, the third process is performed to separate the white crystals captured on 3 to 6 filters installed in a stacked manner.

[0034] After removing the trace amount of diarsenic trioxide remaining on the sodium chloride in the synthesis reactor, the collected white crystals are placed on top, and the second and third processes are repeated 4 to 10 times under the same conditions to finally obtain tetraarsenic hexaoxide crystals. As a result of confirming the crystal structure obtained according to the manufacturing method of the present invention, it was confirmed that most of them were As4O6-a, and that As4O6-a was 99% or more.

[0035] A pharmaceutical composition comprising a polymorph of tetraarsenic hexaoxide of the present invention can be usefully used for the prevention or treatment of bladder cancer.

[0036] The pharmaceutical composition of the present invention can be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injection solutions, respectively, according to conventional methods. Carriers, excipients, and diluents that can be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulated, it is prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants that are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules, and these solid preparations are prepared by mixing the tetraarsenic hexaoxide of the present invention with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspending agents can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases can include witepsol, macrogol, Tween 61, cocoa butter, laurin butter, and glycerogelatin.

[0037] The dosage of the pharmaceutical composition will vary depending on the age, sex, and body weight of the subject being treated, the specific disease or pathological condition being treated, the severity of the disease or pathological condition, the route of administration, and the prescriber's judgment. Determining the dosage based on these factors is within the skill of those skilled in the art, and the dosage is typically in the range of 0.01 mg / kg / day to approximately 500 mg / kg / day. A more preferred dosage is 0.01 mg / kg / day to 50 mg / kg / day. Administration may be administered once a day or divided into several doses. The above dosage does not limit the scope of the present invention in any way.

[0038] The above pharmaceutical composition can be administered to mammals such as rats, livestock, and humans via various routes. All modes of administration are conceivable, including oral, rectal, intravenous, intramuscular, subcutaneous, intrauterine, or intracerebrovascular injection.

[0039] The composition for preventing or treating bladder cancer of the present invention has an excellent anticancer effect by containing 99% or more of arsenic hexaoxide polymorph a (As4O6-a).

[0040] In addition, the composition comprising the polymorph a of arsenic hexaoxide of the present invention (As4O6-a) has an excellent therapeutic effect in clinical trials of bladder cancer patients.

[0041] Figure 1 is an X-ray powder diffraction spectrum of arsenic hexaoxide polymorph a (As4O6-a) and arsenic hexaoxide polymorph b (As4O6-b) (Figures 1A and 1B).

[0042] Figure 2 is a graph showing the results of a comparative experiment on the proliferation inhibition effect of arsenic tetraoxide polymorph a (As4O6-a) and a comparative substance (HD-2) on bladder cancer cells J82, SiHa, T24, and Caski.

[0043] Figure 3 is a photograph showing the proliferation inhibitory effect of arsenic tetraoxide polymorph a (As4O6-a) on bladder cancer cells J82 at different concentrations (5000, 1000, 100 ng / ml).

[0044] Figure 4 is a photograph showing the growth inhibition effect of arsenic tetraoxide polymorph a (As4O6-a) on bladder cancer cells SiHa at different concentrations (5000, 1000, 100 ng / ml).

[0045] Figure 5 is a photograph showing the proliferation inhibitory effect of arsenic tetraoxide polymorph a (As4O6-a) on bladder cancer cells T24 at different concentrations (5000, 1000, 100 ng / ml).

[0046] Figure 6 is a photograph showing the growth inhibition effect of arsenic tetraoxide polymorph a (As4O6-a) on bladder cancer cells Caski at different concentrations (5000, 1000, 100 ng / ml).

[0047] Figure 7 shows the results of a wound healing assay (7A: cell line migration, 7B; migration inhibition rate) on the migration of 5637 human bladder cancer cells by arsenic hexaoxide polymorph a (As4O6-a).

[0048] Figure 8 is an MRI image of a 70-year-old male patient with bladder cancer (Kim OO) before treatment with arsenic tetraoxide polymorph a (As4O6-a).

[0049] Figure 9 is an MRI image of a 70-year-old male patient with bladder cancer (Kim OO) after treatment with arsenic tetraoxide polymorph a (As4O6-a).

[0050] Figure 10 is an MRI image showing a complete recovery after treatment with arsenic tetraoxide polymorph a (As4O6-a) in a 70-year-old male patient with bladder cancer (Kim OO).

[0051] Figure 11 is an MRI image of a 65-year-old male patient with bladder cancer (Shin OO) before treatment with arsenic tetraoxide polymorph a (As4O6-a).

[0052] Figure 12 is an MRI image of a 65-year-old male patient with bladder cancer (Shin OO) after treatment with arsenic tetraoxide polymorph a (As4O6-a).

[0053] To facilitate understanding of the present invention, experimental examples and examples will be provided for a detailed description. However, the following experimental examples and examples merely illustrate the scope of the present invention and are not intended to limit the scope of the present invention. These experimental examples and examples are provided to more fully explain the present invention to those of average skill in the art.

[0054] Hereinafter, the present invention will be described in detail through examples.

[0055] <Example: Preparation of arsenic tetraoxide polymorph a (As4O6-a)>

[0056] It was manufactured using the manufacturing method of arsenic tetraoxide crystal polymorph-a disclosed in Korean Patent No. 1755556.

[0057] Prepare a synthetic reactor (100 mm high, 190 mm in diameter) specially manufactured with kaolin and clamps that can accommodate 3 to 6 filters. The primary clamp is installed 50 mm away from the synthetic reactor, and the secondary to 6th clamps are installed above the primary clamp at intervals of 2 to 6 mm. The specifications of each clamp are 210 mm in diameter and 10 mm in thickness.

[0058] 400 to 600 g of coarse salt (moisture content of 10% or less) is placed in a synthetic reactor, spread evenly to a thickness of about 20 mm, tamped, and gradually heated at 100 to 800°C for 3 hours while continuously heating until the salt surface temperature inside the reactor reaches 290±30°C to remove moisture and impurities, and then cooled at room temperature for 5 hours.

[0059] 100g of the raw material As2O3 (purity 98% or higher, manufacturer YUNNAN WENSHAN JINCHI ARSENIC CO., LTD.) is placed on the coarse salt in the synthesis reactor, and 3 to 6 clamps are installed on the filters (filter paper) capable of capturing sublimated arsenic located at the upper part of the synthesis reactor so that the gap between each filter is 2 to 6mm. It is preferable to use a filter having a basic weight of 70 to 100g / ㎡, a thickness of 0.17 to 0.25mm, a filtration speed of 22 to 30 s / 100ml, and a retention rate of 5 to 10㎛.

[0060] Fix the filter using a clamp and apply heat to the bottom of the synthesis reactor to gradually increase the temperature from 100℃ to 1,000℃. First, heat for 1 hour so that the external temperature of the bottom of the synthesis reactor is approximately 350℃±100℃, and then heat to approximately 600-650℃ and 700-1,000℃ so that the external temperature of the bottom of the synthesis reactor is approximately 600-650℃ and 700-1,000℃, and heat for a total of 5-10 hours so that the temperature at the center of the top filter paper of the filter is maintained at 150℃±100℃, and then cooled to room temperature for approximately 5-7 hours. During this process, the powder As2O3 placed on the salt in the synthesis reactor turns into a gas inside the synthesis reactor and rises, but since the upper temperature outside the synthesis reactor is relatively low, it turns into a liquid and then crystallizes into a solid to form white crystals on the filter.

[0061] The collected white crystals were placed on coarse salt in a synthesis reactor, heated and cooled again, and the process of collecting crystals was repeated four more times, ultimately obtaining 12.0 g of crystals. The structure of the obtained arsenic compound crystals was confirmed to be mostly As4O6-a, with As4O6-a accounting for more than 99% and As4O6-b accounting for less than 1%.

[0062] When the DSC (Differential Scanning Calorimetry) value was heated at a rate of 10℃ / min, it was confirmed that As4O6-a had an endothermic peak (melting point) at 282.67℃, and As4O6-b had an endothermic peak (melting point) at 286.77℃. The powder X-ray diffraction spectra of As4O6-a and As4O6-b are shown in Fig. 1, and the diffraction data of arsenic hexaoxide polymorph a (As4O6-a) and arsenic hexaoxide polymorph b (As4O6-b) are as shown in Table 2.

[0063] As4O6-aAs4O6-b2θ (°)Diffraction intensity2θ (°)Diffraction intensity13.847631.0113.864012.0927.881000027.921000032.322801.7432.362130.2335.33369.8235.34251139.84623.24239.9447.42242.381551.542.441431.8646.342345.246.44159.848.6447.6948.66564.99549.34502.76149.4375.571

[0064]

[0065] <Experimental Example 1: Water solubility test of arsenic tetraoxide polymorphs a and b>

[0066] The experiment was conducted using a test method to measure the water solubility (g / L) of pure chemicals that are stable and nonvolatile in water.

[0067] - Measurement method and measurement range: Flask method (1~1,000 mg / L), performed at 20±0.5℃

[0068] - Guideline: OECD Guideline 105 (Water solubility)

[0069] The results of comparison of the water solubility of arsenic tetraoxide polymorphs a, b and a comparative substance (crystalline form HD-2 of patent registration no. 0272835) are shown in Table 3.

[0070] Sample Test Item Unit Test Item Test Result Arsenic hexaoxide polymorph a (As4O6-a) Solubility g / L ECD TEST Guideline 105 (Flask method) 0.596 Arsenic hexaoxide polymorph b (As4O6-b) Solubility g / L 0.205 Reference substance (HD-2) Solubility g / L 0.195

[0071]

[0072] As shown in Table 3, arsenic hexaoxide polymorph a (As4O6-a) showed a water solubility that was more than three times higher than that of arsenic hexaoxide polymorph b (As4O6-b) and the comparative material (Korean Patent No. 0272835, HD-2).

[0073] <Experimental Example 2: Experiment on the Inhibitory Effect of As4O6-a on the Proliferation of Urinary Bladder Cancer Cells>

[0074] [Media used according to bladder cancer cell type]

[0075] -T24, CasKi: RPMI1640(WELGENE), (+10% Fetal Bovine Serum,+1% Penicillin-Streptomycin)

[0076] -J82, SiHa: MEM(WELGENE), (+10% Fetal Bovine Serum,+1% enicillin-Streptomycin)

[0077] -RT4: McCoy's 5A (WELGENE), (+10% Fetal Bovine Serum,+1% Penicillin-Streptomycin)

[0078] [Brand and catalog number of reagent used]

[0079] - RPMI1640(WELGENE, Cat#011-01)

[0080] - MEM (WELGENE, Cat#007-01)

[0081] - McCoy's 5A (WELGENE, Cat#005-02)

[0082] - FBS(GenDEPOT, Cat#F0900-050)

[0083] - Penicillin-Streptomycin, 100X(WELGENE, Cat#LS202-02)

[0084] - 0.25% trypsin-EDTA (1X) (Gibco, Cat#25200-072)

[0085] [Experimental Method]

[0086] -Cell stocks were each thawed and cultured in the appropriate medium.

[0087] -Cell culture was performed on Mondays, Wednesdays, and Fridays using 0.25% trypsin-EDTA (Gibco).

[0088] -5×10 in 96 well palte 3 After seeding with cells / well (50 μL), the cells were stabilized for 4 hours.

[0089] - 50 μL of distilled water solution of arsenic hexaoxide polymorph a (As4O6-a) and arsenic hexaoxide polymorph b (As4O6-b) was treated to the concentrations below and reacted for 72 hours.

[0090] ① The final concentrations were processed to be 20000, 4000, 800, 160, 32, 6.4, 1.28, 0.256, 0.0512, 0.01024, and 0.002048 ng / mL.

[0091] ② The manufacturing concentration was manufactured to be twice the final concentration above.

[0092] -EZ-Cytox (Duzen Bio Co., Ltd.) and Media (ratio 15:35) were mixed in advance, and 50 μL of diluted EZ-Cytox was treated and the absorbance was measured at 450 nm.

[0093] [Experimental Results]

[0094] -T24 cells showed no cytotoxicity at 160 ng / mL.

[0095] -Caski cells showed no cytotoxicity from 160 ng / mL.

[0096] -J82 cells showed no cytotoxicity from 32 ng / mL.

[0097] -SiHa cells showed no cytotoxicity from 160 ng / mL.

[0098] <Experimental Example 3: Cell Migration Inhibition Experiment of As4O6-a in 5637 Human Bladder Cancer Cells>

[0099] To observe the inhibitory effect of As4O6-a on the migration of 5637 human bladder cancer cells, the effect was observed through a cell migration assay (Wound healing assay).

[0100] Collect 5637 cells from the logarithmic growth phase and seed 5x10 in a 6-well plate. 5 Each well was set to a density of 10 cells / well, 2 ml per well, and cultured in an incubator at 37°C, saturated humidity, and 5% CO2. After culturing the cells on the plate, the supernatant was discarded, and a 20 μL pivot tip was used to draw a “L” horizontal line in a 6-well plate to separate the cells into two sides, and the plates were washed twice with PBS. Three As4O6-a solutions (0 μmol / L, 0.39 μmol / L, 0.78 μmol / L, 1.56 μmol / L) diluted with 1% FBS were added to each well. Pictures were taken at 0 and 24 h using a 100x magnification microscope.

[0101] Movement inhibition rate (%) = (width before drug action - width after drug action) / width before drug action × 100%

[0102] As shown in Fig. 7, after 24 hours, in the control group without As4O6-a intervention, the gap between the cell groups that had been separated on both sides decreased, but under the action of As4O6-a, the gap between the cell groups that had been separated on both sides decreased to a lesser extent depending on the concentration. Here, after 24 hours, As4O6-a It was observed that the gap between the two cell groups at a concentration of 1.56 μmol / L was significantly different compared to the control group. This observation indicated that As4O6-a effectively inhibited cell migration (metastasis).

[0103] <Experimental Example 4: Clinical Trial of As4O6-a in Bladder Cancer Patients>

[0104] A clinical trial was conducted using the arsenic tetraoxide polymorph a (As4O6-a) of Example 1 as follows.

[0105] (1) Clinical trial-1: 70-year-old male patient (Kim OO):

[0106] At Samsung Hospital, bladder cancer was diagnosed by CT scan and confirmed by cystoscopy biopsy. After the bladder cancer diagnosis, 15 mg of arsenic hexaoxide polymorph a (As4O6-a) of Example 1 was administered orally three times a day (5 mg per time) for two months. The MRI image of this patient before treatment is shown in Fig. 8, and the images after 45 days of administration of arsenic hexaoxide polymorph a (As4O6-a) are shown in Fig. 9 and Fig. 10 (complete cure reading), respectively.

[0107] (1) Clinical trial-2: 65-year-old male patient (Shin OO)

[0108] Bladder cancer was confirmed through a tissue biopsy at Eulji Hospital, and after the diagnosis of bladder cancer, 15 mg of arsenic hexaoxide polymorph a (As4O6-a) of Example 1 was orally administered three times a day (5 mg per time) for 10 months. The MRI image of this patient before treatment is shown in Fig. 10, and the images of arsenic hexaoxide polymorph a (As4O6-a) 10 months later are shown in Figs. 11 and 12, respectively.

[0109] Through clinical trial results, it was confirmed that the composition of the present invention has an effect of effectively inhibiting the proliferation of bladder cancer.

Claims

1. A pharmaceutical composition for preventing or treating bladder cancer, comprising arsenic hexaoxide a (As4O6-a) as an active ingredient, A pharmaceutical composition for preventing or treating bladder cancer, characterized in that the above arsenic hexaoxide has 99 wt% or more of the crystal polymorph a of arsenic hexaoxide having the following characteristics (i) to (iii). (i) Unit cell constants: a = b = c = 11.0734 Å, α = β = γ = 90°, V = 1357.82 Å 3 (ii) As-O bond length: 1.786 Å (iii) O-As-O bond angle: 98.36° 2. In paragraph 1, A pharmaceutical composition for preventing or treating bladder cancer, characterized in that the above arsenic tetraoxide has a purity of 99.9% or higher.

3. In paragraph 1, The above polymorph a is a pharmaceutical composition for preventing or treating bladder cancer, characterized in that, in an X-ray powder diffraction spectrum, when the light source wavelength is 1.5406 Å, the peaks indicated at a speed of 1° / min (scan step 0.02°) in a diffraction angle 2θ range of 10° to 50° are 13.84, 27.88, 32.32, 35.3, 39.84, 42.38, 46.34, 48.6, and 49.

34.

4. A pharmaceutical composition for inhibiting bladder cancer metastasis containing arsenic hexaoxide a (As4O6-a) as an active ingredient, A pharmaceutical composition for inhibiting bladder cancer metastasis, characterized in that the above arsenic hexaoxide has 99 wt% or more of the crystal polymorph a of arsenic hexaoxide having the following characteristics (i) to (iii). (i) Unit cell constants: a = b = c = 11.0734 Å, α = β = γ = 90°, V = 1357.82 Å 3 (ii) As-O bond length: 1.786 Å (iii) O-As-O bond angle: 98.36° 5. In paragraph 4, A pharmaceutical composition for inhibiting bladder cancer metastasis, characterized in that the above arsenic hexaoxide has a purity of 99.9% or higher.

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