Pharmaceutical composition for preventing or treating cancer, comprising tetraarsenic hexoxide crystalline polymorph

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating cancer and a method for producing same, the composition comprising tetraarsenic hexoxide in which the content of tetraarsenic hexoxide crystalline polymorph a (As.sub.4O.sub.6-a) is 99% or more. The composition of the present invention exhibits an excellent cancer cell proliferation inhibition effect and thus can be useful as an anticancer drug.

Claims

1. A pharmaceutical composition containing tetraarsenic hexoxide (As406) as an active ingredient for treatment of cancer, wherein the tetraarsenic hexoxide includes 99 wt % or more of tetraarsenic hexoxide crystalline polymorph a having features (i) to (iii) below: (i) Cell parameters: a=b=c=11.0734 Å α=β=γ=90° V=1357.82 Å.sup.3 (ii) As-O bond length: 1.786 Å (iii) O-As-O bond angle: 98.36°, wherein the pharmaceutical composition further comprises at least one of a carrier, a diluent, and an excipient, wherein in an X-ray powder diffraction spectrum of the crystalline polymorph a, obtained by using a light source wavelength of 1.5406 Å within a diffraction angle (2θ) of 10° to 50° at a rate of 1°/min (can step of 0.02°), peaks are shown at 2θ values of 13.84, 27.88, 32.32, 35.3, 39.84, 42.38, 46.34, 48.6, and 49.34, wherein the tetraarsenic hexoxide is prepared by: a first step of heating sodium chloride at 100˜800° C., followed by cooling; a second step of placing arsenic trioxide (As.sub.2O.sub.3) on the sodium chloride, followed by heating from 100° C. to 1000° C. in an airtight state and then cooling; a third step of separating crystals crystallized in a filter bed collecting sublimated arsenic; and a fourth step of repeating the second and third steps four to ten times using the crystals obtained in the third step instead of the arsenic trioxide in the second step, thereby obtaining the tetraarsenic hexoxide including 99 wt % or more of tetraarsenic hexoxide crystalline polymorph a, and wherein the carrier, the diluent, and the excipient do not include water.

2. The pharmaceutical composition of claim 1, wherein the tetraarsenic hexoxide has a purity of 99.9% or higher.

3. The pharmaceutical composition of claim 1, wherein the cancer is selected from the group consisting of lung cancer, esophageal cancer, gastric cancer, colorectal cancer, prostate cancer, pancreatic cancer, cervical cancer, ovarian cancer, and endometrial cancer.

4. The pharmaceutical composition of claim 1, wherein the carrier, the diluent, or the excipient is at least one of lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, propylene glycol, polyethylene glycol, vegetable oils, injectable esters, wax, polyethylene glycol, polysorbate 61, cacao butter, laurin butter, and glycerogelatin.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1A-B show X-ray powder diffraction spectra of As.sub.4O.sub.6-a and As.sub.4O.sub.6-b.

(2) FIGS. 2A-B show graphs depicting the results of assessing cell proliferation inhibitory effects after the lung cancer cell line NCL-H226 was treated with Example 1 and Comparative Examples 1 to 3 and incubated for 48 hours (FIG. 2A) and 72 hours (FIG. 2B).

(3) FIG. 3 shows a graph depicting the results of assessing lung cancer cell proliferation inhibitory effects according to treatment time when the lung cancer cell lines NCL-H226 and SK-MES-1 were treated with 10 μM Example 1.

(4) FIGS. 4A-B show graphs depicting the results of assessing esophageal cancer cell proliferation inhibitory effects according to treatment time when the esophageal cancer cell lines KYSE-150 and TE-1 were treated with 10 μM Example 1 (FIG. 4A) and cell proliferation inhibitory effects after TE-1 was treated with Example 1 and Comparative Examples 1 to 3 and then incubated for 72 hours (FIG. 4B).

(5) FIGS. 5A-B show graphs depicting the results of assessing gastric cancer cell proliferation inhibitory effects according to concentration of Example 1 and treatment time when the gastric cancer cell line AGS was treated with Example 1 (FIG. 5A) and cell proliferation inhibitory effects after AGS was treated with Example 1 and Comparative Examples 1 to 3 and then incubated for 72 hours (FIG. 5B).

(6) FIGS. 6A-B show graphs depicting the results of assessing colorectal cancer cell proliferation inhibitory effects according to treatment time when the colorectal cancer cell lines HT29 and HCT116 were treated with 10 μM Example 1 (FIG. 6A) and cell proliferation inhibitory effects after HT29 was treated with Example 1 and Comparative Examples 1 to 3 and then incubated for 72 hours (FIG. 6B).

(7) FIGS. 7A-B show graphs depicting the results of assessing cell proliferation inhibitory effects after the prostate cancer cell line PC-3 was treated with Example 1 and Comparative Examples 1 to 3 and incubated for 48 hours (FIG. 7A) and 72 hours (FIG. 7B).

(8) FIGS. 8A-B show graphs depicting the results of assessing cell proliferation inhibitory effects after the pancreatic cancer cell line BxPC-3 was treated with Example 1 and Comparative Examples 1 to 3 and incubated for 72 hours (FIG. 8A) and cell proliferation inhibitory effects after the pancreatic cancer cell lines BxPC-3 and PANC-1 were treated with Example 1 of different concentrations and incubated for 72 hours (FIG. 8B).

(9) FIGS. 9A-C show graphs depicting the results of assessing the cell proliferation inhibition after the ovarian cancer cell line SK-OV-3 (FIG. 9A), the human cervical cancer cell line HeLa (FIG. 9B), and the human endometrial cancer cell line HEC-1B (FIG. 9C) were treated with Example 1 and Comparative Examples 1 to 3 and then incubated for 72 hours.

(10) FIG. 10 shows a graph depicting the results of comparing, through IC.sub.50 values, cell proliferation inhibition degrees of Example 1 in human lung, esophageal, gastric, colorectal, prostate, pancreatic, cervical, ovarian, and endometrial cancer cell lines.

MODE FOR CARRYING OUT THE INVENTION

(11) Hereinafter, preferable examples of the present invention will be described in detail. However, the present invention is not limited to the examples described herein, and thus may be embodied into different forms. Rather, these examples are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

Example 1: Preparation of Present Tetraarsenic Hexoxide

(12) A synthesis reactor (100 mm in height and 190 mm in diameter) specially manufactured using kaolin and three to six clamps capable of mounting filters thereon were prepared. A first clamp was installed at a distance of 50 mm from the synthesis reactor, and second to sixth clamps were installed above the first clamp at intervals of 2-6 mm from the first stamp, and the dimension of each clamp was 210 mm in diameter and 10 mm in thickness.

(13) Coarse salt weighing 400-600 g (a moisture content of 10% or less) was introduced into the synthesis reactor, and then evenly spread out and packed to a thickness of about 20 mm. The synthesis reactor was slowly heated at 100-800° C. for 3 hours, and continuously heated such that the surface temperature of the salt was 290±30° C. inside the reactor, thereby removing moisture and impurities. Then, cooling was carried out at room temperature for 5 hours.

(14) Then, 100 g of a raw material, As.sub.2O.sub.3 (a purity of 98% or higher, prepared by YUNNAN WENSHAN JINCHI ARSENIC CO., LTD.) was placed on the coarse salt inside the synthesis reactor, and filters (filter beds) capable of collecting sublimated arsenic were mounted on the three to six clamps installed above the synthesis reactor such that the intervals between the filters were 2-6 mm. The filters used herein preferably had a basic weight of 70-100 g/m.sup.2, a thickness of 0.17-0.25 mm, a filtration speed of 22-30 s/100 ml, and a retention rate of 5-10 μm.

(15) The filters were fixed using the clamps, and then heat was applied to the bottom portion of the synthesis reactor to gradationally raise the temperature from 100° C. to 1,000° C. First, the bottom portion of the synthesis reactor was heated for 1 hour such that the temperature outside the bottom portion of the synthesis reactor was about 350±100° C., and thereafter, heating was carried out such that the temperature outside the bottom portion of the synthesis reactor was about 600-650° C. and about 700-1,000° C., so the temperature of the center portion of the highest filter bed was maintained at 150±100° C. through heating for a total of 5-10 hours. Then, cooling was carried out at room temperature for 5-7 hours. In this procedure, the As.sub.2O.sub.3 powder placed on the salt inside the synthesis reactor transformed into a gas inside the synthesis reactor, and the gas moved up, and then transformed into a liquid since the upper temperature outside the synthesis reactor was relatively low, and thereafter, the liquid was crystallized as a solid, and thus white crystals were generated on the filters.

(16) The collected white crystals were placed on the coarse salt inside the synthesis reactor, and the heating, cooling, and crystal collecting processes were again repeated four times, thereby finally obtaining 12.0 g of the crystals. As a result of checking the structure of the obtained arsenic compound crystals, it was confirmed that most of the crystals were As.sub.4O.sub.6-a while 99 wt % or more of As.sub.4O.sub.6-a and less than 1 wt % of As.sub.4O.sub.6-b were obtained.

(17) It was confirmed that as for the differential scanning calorimetry (DSC) value at a temperature rise rate of 10° C./min, As.sub.4O.sub.6-a showed an endothermic peak (melting point) at 282.67° C. and As.sub.4O.sub.6-b showed an endothermic peak (melting point) at 286.77° C.

(18) X-ray powder diffraction spectra of As.sub.4O.sub.6-a and As.sub.4O.sub.6-b are shown in FIGS. 1A-B, and diffraction data of As.sub.4O.sub.6-a and As.sub.4O.sub.6-b are shown in Table 2 below.

(19) TABLE-US-00002 TABLE 2 As.sub.4O.sub.6-a As.sub.4O.sub.6-b 2θ (°) Diffraction intensity 2θ (°) Diffraction intensity 13.84 7631.01 13.86 4012.09 27.88 10000 27.92 10000 32.32 2801.74 32.36 2130.23 35.3 3369.82 35.34 2511 39.84 623.242 39.9 447.422 42.38 1551.5 42.44 1431.86 46.34 2345.2 46.4 4159.8 48.6 447.69 48.66 564.995 49.34 502.761 49.4 375.571

(20) As confirmed in FIGS. 1A-B and Table 2, the ratio of main peaks shown at 2θ values of 13.8 and 27.9 was 1:1.3 in As.sub.4O.sub.6-a, and the ratio of main peaks shown at 2θ values of 13.8 and 27.9 was 1:2.5 in As.sub.4O.sub.6-b. DSC analysis, structure determination, and X-ray diffraction analysis of the prepared compounds were carried out by the following methods.

(21) (1) DSC Analysis

(22) Using a DSC system (SDT Q600 V20.9 Build 20), 20.0 mg of a sample was analyzed while the temperature was raised to 310° C. at a temperature rise rate of 10° C./min with N.sub.2 flowing out at 100 mL/min.

(23) (2) X-Ray Crystallography

(24) Single crystals of tetraarsenic hexoxide (As.sub.4O.sub.6, MW=395.6) were placed on a glass fiber and then an X-ray beam was applied thereto, to observe diffraction patterns on photographic films and the presence or absence of the organization of diffraction data, thereby determining space groups and cell parameters. Diffraction intensities were collected in the range of 10°<2θ<50°. The crystal structure of As.sub.4O.sub.6 was determined from the data by the Patterson method by using a structure determination program (SHELXTL program).

(25) (3) X-Ray Diffractometry

(26) A sample was prepared by pulverizing the obtained crystals into particles having a size of 10-30 μm (−325 mesh), filling a glass holder for X-ray diffraction analysis (20 mm×16 mm×1 mm) with the particles, compressing the particles by a glass slide or the like, and flattening the particles to allow a sample surface to be parallel with a holder surface. The X-ray diffraction spectrum of the crystals was drawn using Cu Kα.sub.1 (1.54060 Å) of XRD within a diffraction angle (2θ) of 10° to 50° at a rate of 1°/min (scan step of 0.02°).

Comparative Example 1: Preparation of Tetraarsenic Hexoxide

(27) A synthesis reactor (100 mm in height and 190 mm in diameter) specially manufactured using kaolin and three to six clamps capable of mounting filters thereon were prepared. A first clamp was installed at a distance of 50 mm from the synthesis reactor, and second to sixth clamps were installed above the first clamp at intervals of 2-6 mm from the first stamp, and the dimension of each clamp was 210 mm in diameter and 10 mm in thickness.

(28) Coarse salt weighing 400-600 g (a moisture content of 10% or less) was introduced into the synthesis reactor, and then evenly spread out and packed to a thickness of about 20 mm. The synthesis reactor was slowly heated at 100-800° C. for 3 hours, and continuously heated such that the surface temperature of the salt was 290±30° C. inside the reactor, thereby removing moisture and impurities. Then, cooling was carried out at room temperature for 5 hours.

(29) Then, 100 g of a raw material, As.sub.2O.sub.3 (a purity of 98% or higher, prepared by YUNNAN WENSHAN JINCHI ARSENIC CO., LTD.) was placed on the coarse salt inside the synthesis reactor, and filters (filter beds) capable of collecting sublimated arsenic were mounted on the three to six clamps installed above the synthesis reactor such that the intervals between the filters were 2-6 mm The filters used herein preferably had a basic weight of 70-100 g/m.sup.2, a thickness of 0.17-0.25 mm, a filtration speed of 22-30 s/100 ml, and a retention rate of 5-10 μm.

(30) The filters were fixed using the clamps, and then heat was applied to the bottom portion of the synthesis reactor to gradationally raise the temperature from 100° C. to 1,000° C. First, the bottom portion of the synthesis reactor was heated for 1 hour such that the temperature outside the bottom portion of the synthesis reactor was about 350±100° C., and thereafter, heating was carried out such that the temperature outside the bottom portion of the synthesis reactor was about 600-650° C. and about 700-1,000° C., so the temperature of the center portion of the highest filter bed was maintained at 150±100° C. through heating for a total of 5-10 hours. Then, cooling was carried out at room temperature for 5-7 hours. In this procedure, the As.sub.2O.sub.3 powder placed on the salt inside the synthesis reactor transformed into a gas inside the synthesis reactor, and the gas moved up, and then transformed into a liquid since the upper temperature outside the synthesis reactor was relatively low, and thereafter, the liquid was crystallized as a solid, and thus white crystals were generated on the filters. 48.5 g of crystals were collected from the filters. As a result of checking the crystal structure of the collected arsenic compounds, it was confirmed that As.sub.4O.sub.6-b accounted for 99 wt % or more.

Comparative Examples 2 to 4: Preparation of Tetraarsenic Hexoxide

(31) Comparative Examples 2 and 3 were prepared by mixing Example 1 (composition having 99% or more of crystalline polymorph As.sub.4O.sub.6-a) and Comparative Example 1 (composition having 99% or more of crystalline polymorph As.sub.4O.sub.6-b) at 4:1 and 1:1, respectively.

Test Example 1: Test on Human Cancer Cell Proliferation Inhibitory Effects

(32) (1) Materials and Cell Culture

(33) Fetal bovine serum (FBS) and cell culture medium were prepared (Hyclone), and dimethyl sulfoxide (DMSO) and 3-(4,5-dimethyl-thiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT, Amresco LLC, USC) were prepared.

(34) As human cancer cell lines, human lung cancer cell lines NCL-H226 and SK-MES-1, human esophageal cancer cell lines KYSE-150 and TE-1, human gastric cancer cell line AGS, human colorectal cancer cell lines HT29 and HCT116, human prostate cancer cell line PC-3, human pancreatic cancer cell lines BxPC-3 and PANC-1, human cervical cancer cell lines SiHa and HeLa, human ovarian cancer cell line SK-OV-3, and human endometrial cancer cell lines Ishikawa, HEC-1A, and HEC-1B were obtained from the Shanghai Cell Bank of Chinese Academy of Sciences. The cells were incubated in media supplemented with 10% FBS, 50 U/ml penicillin, and 50 μg/ml streptomycin and suitable for incubation of respective cells (RPMI-1640 (NCL-H226, TE-1, BxPC-3, Ishikawa), MEM (SK-MES-1, SiHa, HeLa, HEC-1B), Ham's F-12K (AGS), Ham's F-12 (PC-3), McCOY's 5A (HT29, HCT116, SK-OV-3, HEC-1A), RPMI-1640:Ham's F-12=1:1(v:v) (KYSE-150), and DMEM (PANC-1)) in a humidified incubator with 5% CO.sub.2 and 95% air. The media were exchanged every three days.

(35) (2) Cell Proliferation Assay (MTT Assay)

(36) The effects of Example 1 and Comparative Examples 1 to 3 on cell proliferation were assessed using MTT assay. MTT assay is based on the ability of viable cells against MTT to produce insoluble dark blue formazan products. After the cells were suspended in the medium by trypsin treatment and collected, the cells were dispensed at a density of 4×10.sup.3 cells/well in a 96-well culture dish (Costar, Cambridge, Mass., USA). After 24 hours, the cells in the media containing 10% FBS were treated with Example 1 and Comparative Examples 1 to 3, at 0, 0.625, 1.25, 2.5, 5, 10, 20, 40, or 80 μM, and then incubated. Here, stock solutions obtained by dissolving Example 1 and Comparative Examples 1 to 3 at 5×10.sup.−2 M in 1 M sodium hydroxide was used. For MTT assay for cell proliferation, supernatants were removed from the cells incubated for 24 hours, 48 hours, and 72 hours after the sample treatment, and 20 μl of 5 mg/ml MTT solution was added per well, and the cells were incubated at 37° C. for 4 hours to form formazan crystals. After the incubation, supernatants were again removed, followed by addition of 100 μl of DMSO to every well, and then mixing was carried out to completely dissolve dark blue crystals. All the crystals were completely dissolved by standing at room temperature for 15 minutes, and the absorbance was measured using a micro-plate reader at a wavelength of 570 nm (A.sub.570 nm).

(37) (3) Statistical Analysis

(38) The absorbance value of the control group treated without the sample was calculated as 100, and the absorbance value of the treatment group treated with the sample, compared with that of the control group, was calibrated, and the percentage of inhibition of cell proliferation was calculated according to the following equation.
Percentage (%) of inhibition of cell proliferation=((mean absorbance of control group cells−mean absorbance of treatment group cells)/mean absorbance of control group cells)×100

(39) All data were expressed as mean±standard error of the mean (mean±SEM). One-way analysis of variance (ANOVA) followed by Dunnett's post-test was used to perform multiple comparison. Statistical significance was defined as p<0.05, and each test was repeated three times.

(40) (4) Results of Investigating Inhibition of Lung Cancer Cell Line Proliferation

(41) The human lung cancer cell lines NCL-H226 and SK-MES-1 were treated with Example 1 and Comparative Examples 1 to 3, and incubated for 24, 48, and 72 hours, followed by MTT assay. The results obtained using NCL-H226 are shown in FIGS. 2A-B and 3.

(42) All of the test results obtained by treating NCL-H226 with Example 1 and Comparative Examples 1 to 3 and then incubating the cells for 48 hours (FIG. 2A) and 72 hours (FIG. 2B) confirmed that the percentages of inhibition of the lung cancer cell line NCL-H226 proliferation were higher in the treatment with Example 1 than the treatment with Comparative Examples 1 to 3.

(43) In addition, as a result of investigating the inhibition of cancer cell proliferation according to treatment time when the lung cancer cell lines NCL-H226 and SK-MES-1 were treated with 10 μM Example 1 (FIG. 3), it was confirmed that the percentage of inhibition of cell proliferation was increased with the treatment time in both of NCL-H226 and SK-MES-1.

(44) (5) Results of Investigating Inhibition of Esophageal Cancer Line Proliferation

(45) The human esophageal cancer cell lines KYSE-150 and TE-1 were treated with Example 1 and Comparative Examples 1 to 3, and incubated for 24, 48, and 72 hours, followed by MTT assay. The results are shown in FIGS. 4A-B.

(46) As a result of investigating the inhibition of cancer cell proliferation according to treatment time after KYSE-150 and TE-1 were treated with 10 μM Example 1 (FIG. 4A), it was confirmed that in the case of the esophageal cancer cell line TE-1, the percentage of inhibition of cell proliferation was increased with the treatment time, and in the case of the esophageal cancer cell line KYSE-150, there is no great difference in percentage of inhibition of cell proliferation between the treatment for 24 hours and the treatment for 48 hours, but the percentage of inhibition of cell proliferation was significantly increased in the treatment for 72 hours.

(47) In addition, all of the test results obtained by treating TE-1 with Example 1 and Comparative Examples 1 to 3 and then incubating the cells for 72 hours (FIG. 4B) confirmed that the percentage of inhibition of the esophageal cancer cell line TE-1 proliferation was higher in the treatment with Example 1 than the treatment with Comparative Examples 1 to 3.

(48) (6) Results of Investigating Inhibition of Gastric Cancer Line Proliferation

(49) The human gastric cancer cell line AGS was treated with Example 1 and Comparative Examples 1 to 3, and incubated for 24, 48, and 72 hours, followed by MTT assay. The results are shown in FIGS. 5A-B.

(50) As a result of investigating the inhibition of cancer cell proliferation according to treatment time when the gastric cancer cell line AGS was treated with Example 1 of different concentrations (FIG. 5A), it was confirmed that the inhibition of cell proliferation was increased with the treatment time at each treatment concentration, and the percentage of inhibition of gastric cancer cell proliferation was high in spite of the treatment for only 48 hours.

(51) In addition, all of the test results obtained by treating AGS with Example 1 and Comparative Examples 1 to 3 and then incubating the cells for 72 hours (FIG. 5B) confirmed that the percentage of inhibition of gastric cancer cell line AGS proliferation was higher in the treatment with Example 1 than the treatment with Comparative Examples 1 to 3.

(52) (7) Results of Investigating Inhibition of Colorectal Cancer Cell Line Proliferation

(53) The human colorectal cancer cell lines HT29 and HCT116 were treated with Example 1 and Comparative Examples 1 to 3, and incubated for 24, 48, and 72 hours, followed by MTT assay. The results are shown in FIGS. 6A-B.

(54) As a result of investigating the inhibition of cancer cell proliferation according to treatment time when HT29 and HCT116 were treated with 10 μM Example 1 (FIG. 6A), it was confirmed that the percentage of inhibition of cell proliferation was increased with the treatment time in both HT29 and HCT116.

(55) In addition, all of the test results obtained by treating HT29 with Example 1 and Comparative Examples 1 to 3 and then incubating the cells for 72 hours (FIG. 6B) confirmed that the percentage of inhibition of the colorectal cancer cell line HT29 proliferation was higher in the treatment with Example 1 than the treatment with Comparative Examples 1 to 3.

(56) (8) Results of Investigating Inhibition of Prostate Cancer Cell Line Proliferation

(57) The prostate cancer cell line PC-3 was treated with Example 1 and Comparative Examples 1 to 3, and incubated for 48 and 72 hours, followed by MTT assay. The results are shown in FIGS. 7A-B.

(58) All of the test results obtained by treating PC-3 with Example 1 and Comparative Examples 1 to 3 and then incubating the cells for 48 hours (FIG. 7A) and 72 hours (FIG. 7B) confirmed that the percentage of inhibition of the prostate cancer cell line PC-3 proliferation was higher in the treatment with Example 1 than the treatment with Comparative Examples 1 to 3.

(59) (9) Results of Investigating Inhibition of Pancreatic Cancer Cell Line Proliferation

(60) The human pancreatic cancer cell lines BxPC-3 and PANC-1 were treated with Example 1 and Comparative Examples 1 to 3, and incubated for 24, 48, and 72 hours, followed by MTT assay. The results are shown in FIGS. 8A-B.

(61) All of the test results obtained by treating BxPC-3 with Example 1 and Comparative Examples 1 to 3 and then incubating the cells for 72 hours (FIG. 8A) confirmed that the percentage of inhibition of the pancreatic cancer cell line BxPC-3 proliferation was higher in the treatment with Example 1 than the treatment with Comparative Examples 1 to 3.

(62) In addition, the test results obtained by treating the pancreatic cancer cell lines BxPC-3 and PANC-1 with Example 1 according to concentration of Example 1 and then incubating the cells for 72 hours (FIG. 8B) confirmed that the percentage of inhibition of cell proliferation was increased dependent on the concentration of Example 1 in both BxPC-3 and PANC-1.

(63) (10) Results of Investigating Inhibition of Ovarian, Cervical, and Endometrial Cancer Cell Proliferation

(64) The human ovarian cancer cell line SK-OV-3, the human cervical cancer cell lines SiHa and HeLa, and the human endometrial cancer cell lines Ishikawa, HEC-1A, and HEC-1B were treated with Example 1 and Comparative Examples 1 to 3, and incubated for 72 hours, followed by MTT assay. The results are shown in FIGS. 9A-C.

(65) All of the test results obtained by treating the human ovarian cancer cell line SK-OV-3 (FIG. 9A), the human cervical cancer cell line HeLa (FIG. 9B), and the human endometrial cancer cell line HEC-1B (FIG. 9C) with Example 1 and Comparative Examples 1 to 3 and then incubating the cells for 72 hours confirmed that the percentages of inhibition of the ovarian, cervical, and endometrial cancer cell proliferation were higher in the treatment with Example 1 than the treatment with Comparative Examples 1 to 3.

(66) (11) Comparison of Cell Proliferation Inhibitory Activity in Various Cancer Cell Lines

(67) IC.sub.50 values of Example 1 were analyzed on the basis of the test results obtained by the treatment with Example 1 for 72 hours in the test results obtained through the tests of inhibition of the human lung, esophageal, gastric, colorectal, prostate, pancreatic, ovarian, cervical, and endometrial cancer cells proliferation. The analysis results are shown in Table 3 and FIG. 10.

(68) TABLE-US-00003 TABLE 3 Cancer cell growth inhibition Cancer type Cell line (IC.sub.50, μM) Lung cancer NCL-H226 5.12 SK-MES-1 2.47 Esophageal cancer KYSE-150 9.85 TE-1 2.35 Gastric cancer AGS 0.9 Colorectal cancer HT29 1.27 HCT116 5.07 Prostate cancer PC-3 4.98 Pancreatic cancer BxPC-3 4.98 PANC-1 5.62 Cervical cancer SiHa 2.8 HeLa 1.63 Ovarian cancer SK-OV-3 5.02 Endometrial cancer Ishikawa 2.75 HEC-1A 2.47 HEC-1B 4.61

(69) As can be seen from Table 3 and FIG. 10, the IC.sub.50 values of Example 1, which are associated with cancer cell proliferation inhibitory activity, had low concentrations in all of the types of cancer.

(70) Although not shown in the present invention, the tetraarsenic hexoxide of the present invention was confirmed to inhibit the proliferation of various cancer cells, such as brain cancer, breast cancer, liver cancer, and skin cancer.

(71) Therefore, it can be seen that Example 1 showed excellent anticancer effects by inhibiting the growth of cancer cells in various cancers, such as lung cancer, esophageal cancer, gastric cancer, colorectal cancer, prostate cancer, pancreatic cancer, cervical cancer, ovarian cancer, and endometrial cancer.