Pharmaceutical composition for treating liver cancer, comprising tetraarsenic hexoxide crystalline polymorph
11154530 · 2021-10-26
Assignee
Inventors
Cpc classification
C01P2002/88
CHEMISTRY; METALLURGY
C01P2002/77
CHEMISTRY; METALLURGY
C01P2002/72
CHEMISTRY; METALLURGY
A61P1/16
HUMAN NECESSITIES
C01P2002/74
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
Abstract
The present invention relates to a pharmaceutical composition for preventing or treating liver 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 (As.sub.4O.sub.6) as an active ingredient for treatment of liver 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.
2. The pharmaceutical composition of claim 1, 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 tetraarsenic hexoxide crystals.
3. The pharmaceutical composition of claim 1, wherein the tetraarsenic hexoxide has a purity of 99.9% or higher.
4. The pharmaceutical composition of claim 1, wherein in the 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 (scan 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.
5. 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, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, propylene glycol, polyethylene glycol, vegetable oils, injectable esters, Witepsol, Macrogol, Tween 61, cacao butter, laurin butter, and glycerogelatin.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1)
(2)
(3)
(4)
(5)
(6)
MODE FOR CARRYING OUT THE INVENTION
(7) 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
(8) 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.
(9) 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.
(10) 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.
(11) 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.
(12) 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.
(13) 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.
(14) X-ray powder diffraction spectra of As.sub.4O.sub.6-a and As.sub.4O.sub.6-b are shown in
(15) 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
(16) As confirmed in
(17) (1) DSC Analysis
(18) 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.
(19) (2) X-Ray Crystallography
(20) 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).
(21) (3) X-Ray Diffractometry
(22) 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
(23) 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.
(24) 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.
(25) 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.
(26) 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
(27) 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 Liver Cancer Cell Proliferation Inhibitory Effects
(28) (1) Materials and Cell Culture
(29) 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.
(30) As human cancer cell lines, human liver cancer cells SMMC-7721 and HCCLM3 were obtained from the Shanghai Cell Bank of Chinese Academy of Sciences. The SMMC-7721 cells were incubated in RPMI-1640 medium supplemented with 10% FBS, 50 U/ml penicillin, and 50 μg/ml streptomycin and the HCCLM3 cells were incubated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin in a humidified incubator with 5% CO.sub.2 and 95% air. The media were exchanged every three days.
(31) (2) Cell Proliferation Assay (MTT Assay)
(32) 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 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.570nm).
(33) (3) Statistical Analysis
(34) 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
(35) 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.
(36) (4) Results of Test Using SMMC-7721 Cells
(37) The human liver cancer cell line SMMC-7721 cells were 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
(38) (5) Results of Test Using HCCLM3 Cells
(39) The human liver cancer cell line HCCLM3 cells were 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
Test Example 2: In Vivo Efficacy Test in Animal Models Transplanted with Human Liver Cancer Cell Line H22 Cells
(40) (1) Methods
(41) Human liver cancer cell line H22 cells (purchased from the Shanghai Cell Bank of Chinese Academy of Sciences) were incubated in RPMI-1640 medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin in an incubator under conditions of 37° C., 5% CO.sub.2, and 95% air. The medium was exchanged every three days.
(42) Fifty 6-week-old ICR female and male mice, which were safe from specific pathogens and respiratory diseases and had a body weight of 18-20 g, were used as experimental animals. The mice were allowed free access to food and water, and were bred in a 12-hr light/12-hr dark cycle.
(43) Out of the 50 mice, 10 mice were classified as the normal group, and the remaining 40 animals were subcutaneously inoculated with H22 cells at 1×10.sup.7 cells per mouse, and bred for 7 days. On 7 days after the cell inoculation, the mice were randomly divided into 10 mice per group, and then test groups were designated as shown in Table 3 below, and respective corresponding samples were orally administered daily for 11 days. 5-Fluorouracil (5-FU), which is used as an anticancer drug, was treated for a positive control group.
(44) TABLE-US-00003 TABLE 3 Treatment information H22 cell Group inoculation Reagent treatment Normal control group Untreated Untreated Cancer model group Treated Distilled water Test Group 1 Treated Example 1 - 4.5 mg/kg Test Group 2 Treated Example 1 - 2.25 mg/kg Positive control Treated 5-Fluorouracil - 20 mg/kg group
(45) (2) Investigation of Cancer Growth Inhibition
(46) After the administration of the drug for each group, cancer sizes were measured daily using a Vernier caliper, and cancer volumes were calculated using the equation V=0.5×ab.sup.2 (a is the longest diameter of cancer and b is the shortest diameter of cancer). The results are shown in Table 4 and
Percentage of inhibition of cancer growth=(1−(sample treatment group/cancer model group))×100
(47) TABLE-US-00004 TABLE 4 Cancer volume (mm.sup.3) according to number of days of administration Group 5 days 7 days 9 days 11 days Cancer model group 224.29 399.47 664.71 1812.69 Positive control group 165.62 212.78 318.07 661.97 Test Group 1 168.09 238.22 388.85 880.55 Test Group 2 216.75 302.66 388.56 987.61
(48) TABLE-US-00005 TABLE 5 Percentage (%) of inhibition of cancer growth Group 5 days 7 days 9 days 11 days Positive control group 26.16 46.73 52.15 63.48 Test Group 1 25.06 40.36 41.50 51.42 Test Group 2 3.36 24.23 41.54 45.52
(49) As can be seen from Table 4 above and
(50) In addition, as shown in Table 5 above and
(51) It can be seen through these results that Example 1 of the present invention inhibits the growth of liver cancer.
Test Example 3: In Vivo Efficacy Test in Animal Models Transplanted with Human Liver Cancer Cell Line SMMC-7721 Cells
(52) (1) Methods
(53) Human liver cancer cell line SMMC-7721 cells (purchased from the Shanghai Cell Bank of Chinese Academy of Sciences) were incubated in RPMI-1640 medium supplemented with 10% FBS, 50 U/ml penicillin, and 50 μg/ml streptomycin in an incubator under conditions of 37° C., 5% CO.sub.2, and 95% air. The medium was exchanged every three days.
(54) Twenty five 5-week-old babl/c-nu male mice, which were safe from specific pathogens and respiratory diseases and had a body weight of 18-20 g, were used as experimental animals. The mice were allowed free access to food and water, and were bred in a 12-hr light/12-hr dark cycle.
(55) The twenty five mice were subcutaneously inoculated into the neck site with SMMC-7721 cells at 2×10.sup.7 cells per mouse, and bred for 7 days. On 7 days after the cell inoculation, the mice were randomly divided into 5 mice per group, and then test groups were designated as shown in Table 6 below, and respective corresponding samples were orally administered daily for 7 days. 5-Fluorouracil (5-FU), which is used as an anticancer drug, was treated for a positive control group.
(56) TABLE-US-00006 TABLE 6 Group Reagent treatment Cancer model group Distilled water Positive control group 5-Fluorouracil - 20 mg/kg Test Group 1 Example 1 - 4.5 mg/kg Test Group 2 Example 1 - 2.25 mg/kg Test Group 3 Example 1 - 1.125 mg/kg
(57) (2) Investigation of Inhibition of Cancer Growth
(58) Cancer tissues were isolated from the mice after seven days of administration of the drug for each group, and visually observed. Cancer weights were measured. In addition, the inhibition of cancer growth was calculated on the basis of the measured weights of the cancer tissues by using the following equation. The results are shown in Table 7 and
Percentage of inhibition of cancer growth=(1−(sample treatment group/cancer model group))×100
(59) TABLE-US-00007 TABLE 7 Percentage (%) Cancer of inhibition Group weight (g) of cancer growth Cancer model group 1.07 ± 0.52 — Positive control group 0.72 ± 0.27 32.5 Test Group 1 0.65 ± 0.17 39.7 Test Group 2 0.70 ± 0.10 34.3 Test Group 3 0.77 ± 0.12 28.0
(60) As can be seen from Table 7 above, it was confirmed that the cancer weight was reduced in Test Groups 1 to 3 administered with Example 1 compared with the cancer model group, and the cancer weight was reduced depending on the dose of Example 1.
(61) It can be seen through these results that Example 1 of the present invention inhibits the growth of liver cancer.