KR20230070096A - Pharmaceutical composition for cacner treatment comprising salt of meta arsenite - Google Patents

Abstract

The purpose of the present invention is to provide a novel combination composition that can be used in cancer treatment by exploring ingredients that have excellent anti-cancer effects when used together with meta-arsenite salt. One embodiment of the present invention provides a pharmaceutical composition for treating cancer comprising at least one selected from a group consisting of sodium meta-arsenite, and a brocooli extract.

Description

Pharmaceutical composition for cancer treatment containing meta arsenite salt {PHARMACEUTICAL COMPOSITION FOR CACNER TREATMENT COMPRISING SALT OF META ARSENITE}

The present invention relates to a pharmaceutical composition for treating cancer containing a meta-arsenite salt, and more particularly, to a pharmaceutical composition for treating cancer containing sodium meta-arsenite and beta-carotene or a broccoli extract.

Cancer refers to a disease in which cells ignore the death cycle and proliferate abnormally, destroying the function of the human body. Because it is caused by the uncontrolled growth and division of abnormal cells (cancer cells), it can occur in any living tissue, and there are numerous causes such as carcinogens, viruses, and heredity, so the pathogenesis has not yet been clearly identified. Cancer cells can migrate to other organs in the body through the blood or lymph, which is called metastasis. Pathologically, cancer includes epithelial cell-derived carcinoma, connective tissue-derived sacroma, and hematological cancer.

Cancer is the number one cause of death in Korea, and the mortality rate due to cancer is very high worldwide. In many cases, the effect of surgical operation cannot be expected unless the cancer is in its early stages, and even if detected early, the prognosis is often poor. In a state where the cancer has already progressed to a severe degree, the human body must be hit with anti-cancer drugs on a regular basis, and in some cases, radiation therapy must also be continued. Despite the administration of anticancer drugs and irradiation of radiation, it is very difficult to treat because it grows and metastasizes at a rapid rate.

Accordingly, studies on compounds or compositions capable of treating cancer are being actively conducted, and one of several compounds is meta arsenite salt (Salt of meta asenite, AsO ₂ ^- ).

Since anticancer drug compositions containing meta-arsenite salts were registered in 2001, anti-cancer drug compositions for various uses using meta-arsenite salts have been studied.

An object of the present invention is to provide a composition of a new combination that can be used for cancer treatment by searching for a component having a very excellent anticancer effect when used together with meta arsenite salt.

One embodiment of the present invention, sodium meta arsenite; And it provides a pharmaceutical composition for the treatment of cancer containing at least one selected from the group consisting of beta-carotene and broccoli extract.

The cancer may be any one selected from the group consisting of colon cancer, leukemia, promyelocytic leukemia, lymphoma, melanoma, non-small cell lung cancer, renal cancer, uterine cancer, prostate cancer, ovarian cancer, breast cancer, and stomach cancer.

Based on 1mL of the pharmaceutical composition, the sodium metaarsenite may be included in an amount of 1 to 100 μg, and at least one selected from the group consisting of beta-carotene and broccoli extract may be included in a concentration of 1 to 20 μM.

The pharmaceutical composition may further include a pharmaceutically acceptable adjuvant.

The pharmaceutical composition may be in the form of an oral dosage form or a subcutaneous injection form.

The pharmaceutical composition may be administered at 1 mg/kg to 30 mg/kg.

sodium meta arsenite according to the present invention; And a pharmaceutical composition for cancer treatment containing at least one selected from the group consisting of beta-carotene and broccoli extract, can maximize the cancer treatment effect.

1 is a diagram showing cell proliferation over time when lymphoma U937 was treated with the compositions of the control group (test group 0) and test groups 1 to 6 of the present invention.
Figure 2 is a diagram showing cell proliferation over time when lymphoma U937 was treated with the compositions of the control group (test group 0) and test groups 7 to 16 of the present invention.

The present invention will be described in more detail through the following test examples or examples. However, the following test examples or examples are intended to illustrate the present invention, and the present invention is not limited thereto.

Test Example 1: Investigation of cell proliferation of tumor cell lines in vitro

In order to find a component having an excellent anticancer effect when combined with metaarsenite salt, the anticancer effect by combination with various components was confirmed. Specifically, in vitro to investigate the anticancer effect of the combination of sodium metaarsenite (NaAsO ₂ ) as metaarsenite salt, potassium polygamma-glutamate, arabinoxylane, histidine, beta-carotene, and broccoli extract Direct cell proliferation of tumor cells was measured and investigated.

1) Material

Human tumor cell lines were used to measure in vitro cytotoxicity, which is as follows.

Colon cancer: HT29, DLD1

Leukemia: CCRF-CEM

Promyelocytic leukemia: K562

Lymphoma: U937

Melanoma: MEXF 462L, MEXF 514L

Non-small cell lung cancer: LXFL 529L

Renal cancer: RXF 944L, RXF 486L

Uterine Cancer: UXF 1138

Prostate Cancer: PC3

Ovarian Cancer: OVCAR3

Breast Cancer: MXAF401NL

Gastric cancer: GXF251L

2) Cell culture

Human tumor cells were grown at 37 °C in humid conditions (95% air, 5% carbon dioxide) using monolayer cultures in RPMI 1640 medium with 10% heat-inactivated New born Calf Serum and 1% glutamine gentamycine. .

3) Cell proliferation assay

In order to examine the anti-proliferation ability of the compound used in the experiment, cell proliferation was confirmed through colorimetric ELISA BrdU assay.

Cells were obtained after logarithmic growth in 1640 medium supplemented with 10% calf serum by trypsinization, plated in a 96-well microplate (100 uL cell suspension, 1 × 106 cells/mL), and then measured.

After 24 hours of recovery to allow cells to logarithmic growth, 50 uL of culture medium (6 controls per plate) or culture medium containing test drugs (test group information, see Table 1 below) was added to the wells.

After preservation for 72 hours, absorbance was measured using a microtiter plate reader (wavelength: 450 nm) to determine the degree of cell proliferation.

4) sample

Dimethyl sulfoxide ("DMSO") was obtained from Sigma Aldrich. Table 2 below provides the DMSO properties supplied.

Poly-L-γ-glutamic acid potassium was obtained from Sigma Aldrich. Table 3 below provides the supplied potassium polygamma-glutamate properties.

Arabinoxylan was obtained from Sigma Aldrich. Table 4 below provides the supplied arabinoxylane properties.

Histidine (L-Histidine) was obtained from Sigma Aldrich. Table 5 below provides the supplied histidine properties.

Beta-carotene was obtained from Sigma Aldrich. Table 6 below provides the beta carotene properties supplied.

As the broccoli extract, broccoli methanol extract is used. The method of obtaining is as follows. Commercially purchased broccoli is dried in the shade and ground. Using an appropriate amount of methanol for the pulverized broccoli, extraction is performed at 50° C. under reduced pressure.

The above samples were stored below 4 °C. For in vitro studies, samples were dissolved in DMSO and diluted with culture medium so that the final concentration of DMSO was 0.3% or less.

Example 1: Results of Cell Proliferation of Meta-Arsenite Salt

In the same manner as in Test Example 1, the cell proliferation of meta-arsenite salt was confirmed through absorbance (OD value) in each tumor cell line for 72 hours, and the results are shown in Table 7 below.

At this time, DMSO (final concentration 0.3%) was used as a control group.

As a result of screening, in most tumor cell lines, the group treated with meta-arsenite salt and beta-carotene (test group 5), the group treated with meta-arsenite salt and broccoli extract (test group 6), and meta-arsenite It was confirmed that the group treated with salt, beta-carotene, and broccoli extract showed high activity (test group 16).

In addition, it was confirmed that the highest activity was shown in lymphoma U937. Based on this, in order to confirm cell proliferation over time when additionally treated with lymphoma U937, after preservation for 24, 48, and 72 hours in each group, the results of measuring absorbance (OD value) are shown in Table 8, Figures 1 and 2 below. shown in

At this time, DMSO was used as a control group.

As shown in Table 8 and FIGS. 1 and 2, when stored for 72 hours, the group treated with meta-arsenite salt, beta-carotene, and broccoli extract was more than treated with meta-arsenite salt (Test Group 1) alone (Test Group 16), the group treated with meta-arsenite salt and beta-carotene (Test Group 5), and the group treated with meta-arsenite salt and broccoli extract (Test Group 6). could

Test Example 2: Investigation of cytotoxicity against tumor cell lines in vitro

In order to find out the clearer anticancer effect of the combination of sodium metaarsenite, beta-carotene, and broccoli extract, the effect of which was confirmed through cell proliferation investigation, direct cytotoxicity against tumor cells was measured and investigated in vitro.

1) Material

Human tumor cell lines were used to measure in vitro cytotoxicity, which is as follows.

Colon cancer: HT29, DLD1

Leukemia: CCRF-CEM

Promyelocytic leukemia: K562

Lymphoma: U937

Melanoma: MEXF 462L, MEXF 514L

Non-small cell lung cancer: LXFL 529L

Renal cancer: RXF 944L, RXF 486L

Uterine Cancer: UXF 1138

Prostate Cancer: PC3

Ovarian Cancer: OVCAR3

Breast Cancer: MXAF401NL

Gastric cancer: GXF251L

2) Cell culture

Human tumor cells were grown in monolayer cultures in RPMI 1640 medium supplemented with 10% fetal bovine serum (Life Technologies, Karlsruhe, Germany) at 37°C under humid conditions (95% air, 5% carbon dioxide).

3) Cytotoxicity assay

An enhanced propidium iodide assay was used to examine the ability of the compounds used in the experiment to prevent proliferation. Cells were obtained after logarithmic growth in 1640 medium supplemented with 10% fetal bovine serum by trypsinization, and the plates were plated in 96-well microplates (100 μl cell suspension, 1 Х10 ⁵ and 5 Х10 ⁴ cells/mL). After that, it was measured.

After 24 hours of recovery for logarithmic growth of the cells, 50 μl of culture medium (6 control wells per plate) or test drug (see test group information, test groups 1, 5, 6, 16 in Table 1) Culture medium was added to the wells. According to the cell doubling period, the medium was changed to a fresh medium containing 6 μg/ml of propidium iodide after about 5 days of culture. Then, the microplate was preserved for 24 hours at -18 °C to kill the whole cells. After warming the plate, fluorescence was measured using a Millipore CytoFluor 2350-microplate reader (excitation wavelength 530 nm, emission wavelength 620 nm) to quantify the total number of cells. The growth inhibition rate is shown in Equation 1 below.

[Equation 1]

In Equation 1, T represents the number of tumor cells after treatment of the test group, and C represents the number of tumor cells after treatment of the control group.

IC ₅₀ and IC ₇₀ values are also determined by plotting compound concentration against cell number. The average IC ₅₀ and IC ₇₀ values are determined by Equation 2 below.

[Equation 2]

In Equation 2, x is a specific tumor cell line, and n represents the total number of cell lines.

If IC ₅₀ and IC ₇₀ values could not be determined within the investigated single dose range, the lowest or highest concentration studied was used for the calculation.

The assay is considered valid only if the positive control (5-FU) causes a tumor growth inhibition of T/C < 30% and if the fluorescence intensity is greater than 500 units in the case of treatment of cells in the control.

4) sample

Obtained in the same manner as in Test Example 1, and they were stored at 4 ° C or less. For in vitro studies, samples were dissolved in DMSO and diluted with culture medium so that the final concentration of DMSO was less than 0.3%.

Example 2: Results of cytotoxicity investigation of meta-arsenite salt

The results of measuring the cytotoxicity of metaarsenite salt using Equation 1 in the same manner as in Test Example 2 are shown in Tables 9 to 12 below. Table 9 below shows the survey results for test group 1, Table 10 shows the survey results for test group 6, Table 11 shows the survey results for test group 5, and Table 12 shows the survey results for test group 16. At this time, 5-FU was used as a control group. In addition, the concentrations of beta-carotene and broccoli extract in all groups were fixed at 10 uM, and the activity was observed according to the meta-arsenite salt concentration.

As can be seen in Table 9, metaarsenite salt showed remarkable activity in the leukemia strain CCRF-CEM and lymphoma U937 at a concentration of 10 μg/ml, and in most tumor cell lines at a concentration of 100 μg/ml. there was an active

As shown in Table 10, it was confirmed that test group 6 showed higher activity in all tumor cell lines than the group treated only with meta-arsenite salt (test group 1).

As shown in Table 11, it was confirmed that test group 5 exhibited higher activity than the group (test group 6) treated with meta-arsenite salt and broccoli extract in all tumor cell lines.

As shown in Table 12, it was confirmed that test group 16 showed superior activity in all tumor cell lines compared to other test groups. This is a result that can be seen that it is very effective in vitro than when only metaarsenite salt (test group 1) was treated alone.

Example 3: Toxicity test

A toxicity test was performed on the combination of metaarsenite salt, beta-carotene, and broccoli extract according to one embodiment of the present invention as follows.

After dissolving the composition of test groups 1, 5, 6, and 16 in dimethylsulfoxide (DMSO) and diluting it with water, the mice (10 per group, test group information, test group 1, 5, 6, 16 of Table 1) Reference) was administered at 20 mg/kg each and observed for 7 days, but no mice died. At this time, beta-carotene and broccoli extract of each group (test groups 5, 6, and 16) were administered at 10 mM/kg.

Formulation Example 1: Preparation of tablets

In one tablet (200 mg), 100 mg of the composition of test groups 1, 5, 6 or 16, 51 mg of lactose, 40 mg of corn starch, and 2 mg of colloidal silicon dioxide were mixed. A 3% polyvinylprolidone solution was added to the mixture, and then pulverized and passed through a 14 mesh sieve. After drying and passing through a 14-mesh sieve again, 1 mg of magnesium stearate was added to make the obtained mixture into tablets.

Formulation Example 2: Preparation of injection

Dissolve 10 mg of the composition of test groups 1, 5, 6 or 16 in 10 ml of one ampoule in 200 mg of polyoxyethylene hydrogenated castor oil, add distilled water for injection to make 10 ml, and contain 1 mg in 1 ml An injection was prepared.

Example 4: In vitro test using meta-arsenite salt, beta-carotene and broccoli extract

Metaarsenite salts can cause telomere damage. Therefore, a combination of metaarsenite salt and beta-carotene, broccoli extract was tested to see if it showed evidence of a synergistic effect in vitro in non-small cell lung cancer cell lines.

1) Material

H460 (4 kb, IC ₅₀ , IC ₅₀ =10 μM) and A549 (6 kb, IC ₅₀ =13 μM) were selected because they have relatively short telomeres and are part of the International Cancer Institute (NCI) 60 cell line panel . IC ₅₀ concentrations for metaarsenite salts were determined using the MTT assay and the widely accepted median effect method by Chou and Talalay, based on fixed IC ₅₀ ratios used for the detection of synergy, causticity or antagonism. (1984), 22:27-55).

2) Cell culture and MTT assay

Cells were grown in their respective recommended media such as RMPI 1640, Iscove's or DMEM (Invitrogen) under standard conditions (5% CO ₂ /37° C. constant temperature and humidity) and routinely passaged. For the MTT proliferation assay, exponentially growing cells were harvested and seeded in a 96 well plate (2 x 10 ³ cells/well). Since each cell type and treatment period are different, the amount of cells and the treatment period were determined according to the cell line so that an appropriate absorbance (0.2 to 0.8) was obtained in advance, and then proceeded.

To evaluate the drug's growth inhibition potential, test drugs were added to each well at concentrations ranging from 1 to 100 μM. Incubate at 5% CO ₂ /37°C for an appropriate time (1, 2, 3, 4, 5 days) to ensure sufficient exposure of the test drug. After that, add 20 uL of 5 mg/mL MTT solution to each well and leave it for 4 hours at 5% CO ₂ /37℃. At this time, the MTT solution (Sigma, St, Louis, MO) is made by dissolving it in PBS (Phosphate Buffered Saline) at 5 mg/mL, and since it is sensitive to light, light exposure should always be minimized during use. After use, wrap in aluminum foil and refrigerate (4℃). As a control, PBS was added instead of wells containing cells. After removing all the supernatant, dispense 100 uL of DMSO (Sigma, St, Louis, MO) into each well and react while shaking the plate sufficiently for 10 to 30 minutes while blocking light. After all procedures are completed, absorbance is measured at 550 nm using a plate reader.

However, in order to measure the cell growth at the time of drug addition (d0), which enables calculation of actual cell death, one of the 96 well plates was developed immediately after drug addition. The others were cultured for 5 days before 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added, and the conversion to purple formazan by spiny cells was measured. Measured using a SynergyHT plate reader (550 nm) and K4C software (BioTEK). Formazan was dissolved in DMSO. Growth curves were generated in MSExcel, and total cell death as well as 50% and 100% growth inhibitory concentrations were detected.

3) Analysis

In A549 (Table 13) and H460 (Table 14), the group treated with meta-arsenite salt only (Test Group 1), the group treated with meta-arsenite salt, beta-carotene, and broccoli extract (Test Group 16), meta-arsenite salt Cell growth inhibitory effects were observed in the group treated with Senite salt and beta-carotene (Test Group 5) and the group treated with meta-arsenite salt and broccoli extract (Test Group 6). The cell viability (%) was calculated as in Equation 3 below.

[Equation 3]

Cell viability (%) = (Test group absorbance - Blank absorbance) / (Control group absorbance - Blank absorbance) x 100

4) sample

Phosphate Buffered Saline ("PBS") and Dimethyl sulfoxide ("DMSO") were obtained from Sigma Aldrich.

As shown in Table 13, when the O.D. value was measured in each test group (control group, test group 1, test group 5, test group 6, test group 16) after culturing in A549 for 1 to 5 days, metaar The group treated with meta-arsenite salt, beta-carotene, and broccoli extract (Test Group 16), and the group treated with meta-arsenite salt and beta-carotene (test Group 5), and the group treated with meta-arsenite salt and broccoli extract (test group 6) showed high cell growth inhibitory effect.

That is, when judging by the cell viability (%), about 60% of cells were treated with meta-arsenite salt, beta-carotene, and broccoli extract (Test Group 16) compared to the meta-arsenite salt alone treatment group. The growth inhibitory effect was shown, and in test group 5 and test group 6, 43% and 40% of cell growth inhibitory effects were confirmed, respectively.

As confirmed in Table 14, when the O.D. value was measured in each test group (control group, test group 1, test group 5, test group 6, test group 16) after culturing in H460 for 1 to 5 days, in A549 showed a similar pattern to In other words, the group treated with meta-arsenite salt, beta-carotene, and broccoli extract (Test Group 16), treated with meta-arsenite salt and beta-carotene together, compared to the treatment with meta-arsenite salt (Test Group 1) alone. One group (test group 5) and the group treated with meta-arsenite salt and broccoli extract (test group 6) showed low cell viability (%) in that order.

In other words, compared to the group treated with meta-arsenite salt alone (Test Group 1), the group treated with meta-arsenite salt, beta-carotene, and broccoli extract (Test Group 16) showed about 65% of cell growth inhibitory effect. In Test Group 5 and Test Group 6, 48% and 46% of cell growth inhibitory effects were confirmed, respectively.

Through this, it can be inferred that the combined use of meta-arsenite salt, beta-carotene, and broccoli extract enhances the effect of meta-arsenite salt, so antitumor effects are expected even at low doses.

In addition, as shown in Tables 15 and 16 below, A549 and H460 have IC ₅₀ of 1.4 μM and 1 μM for beta-carotene, respectively, and IC ₅₀ of 1.8 μM and 1.5 μM for broccoli extract, respectively, but are relatively meta It was insensitive to arsenite salts. H460 cells with shorter telomeres (4 kb) have a lower IC ₅₀ than A549 cells with relatively long telomeres (6 kb). However, when the three drugs were combined, very significant synergistic effects were obtained at most effect levels. Therefore, metaarsenite salts are thought to make it possible to sensitize lung cancer cell lines to beta-carotene and broccoli extract.

Claims (6)

sodium meta arsenite; and
A pharmaceutical composition for treating cancer comprising at least one selected from the group consisting of beta-carotene and broccoli extract. According to claim 1,
The cancer is any one selected from the group consisting of colon cancer, leukemia, promyelocytic leukemia, lymphoma, melanoma, non-small cell lung cancer, renal cancer, uterine cancer, prostate cancer, ovarian cancer, breast cancer and stomach cancer, a pharmaceutical composition for the treatment of cancer . According to claim 1 or 2,
Based on 1 mL of the pharmaceutical composition, the sodium metaarsenite is included in an amount of 1 to 100 μg, and at least one selected from the group consisting of beta-carotene and broccoli extract is included in a concentration of 1 to 20 μM. pharmaceutical composition. According to claim 1 or 2,
The pharmaceutical composition for the treatment of cancer further comprises a pharmaceutically acceptable adjuvant. According to claim 1 or 2,
The pharmaceutical composition is a pharmaceutical composition for the treatment of cancer in the form of an oral dosage form or subcutaneous injection formulation. According to claim 1 or 2,
The pharmaceutical composition is a pharmaceutical composition for the treatment of cancer that is administered at 1mg / kg ~ 30mg / kg.

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