WO2024147560A1 - Immuno-anticancer composition comprising toxicodendron vernicifluum extract and cytokine killer cells - Google Patents

Abstract

Previously, it has been known that a Toxicodendron vernicifluum extract is effective in treating arthritis and the like by suppressing immunity. However, in the present invention, it has been experimentally demonstrated that treatment of cytokine killer cells with a Toxicodendron vernicifluum extract actually increases the immune sensitivity of immune cells, resulting in improved cancer cell killing ability. Therefore, the immune anticancer composition comprising the Toxicodendron vernicifluum extract and cytokine killer cells as active ingredients of the present invention has the advantage of effectively treating lung cancer or breast cancer by improved cancer cell killing ability, and if an anticancer immune cell therapy product comprising same is prepared, the anticancer immune cell therapy product can be administered to lung cancer and breast cancer patients to effectively treat lung cancer and breast cancer.

Description

Immuno-anticancer composition containing sumac tree extract and cytokine killer cells

The present invention provides an anti-cancer immunocomposition containing sumac extract and cytokine killer cells as active ingredients, and an anti-cancer immune cell therapy composition containing the same.

Lacquer tree (Toxicodendron Vernicifluum or Rhus Veniciflua Strokes) is a deciduous broad-leaved small tree belonging to the Anacardiaceae family and is known to be native to the Central Asian highlands and the Himalayas.

The sap of the lacquer tree is called lacquer, and in oriental medicine, dried lacquer has been used since ancient times to eliminate stagnant blood and promote blood circulation, and to treat deworming, abdominal pain, excessive gastric acid, cough, pulmonary tuberculosis, pain, constipation, diabetes, mucus, and preservatives. It is known to be effective in treating the stomach, stomach, and pain in the female meridian.

Sumac contains a large amount of antioxidants. According to known results, it has been reported that antioxidant substances present in sumac tree bark extract show strong antioxidant activity in rat brain cells and inhibit the growth of blood cancer cells.

In addition, Korean Patent Application No. 10-1997-0013163, Korean Patent No. 10-1997-0004193, and Korean Patent No. 10-0251526 report that the urushiol component of lacquer extract has an anticancer effect; Korean Patent No. 10-0257448 reported an anticancer composition containing fustin, fisetin, sulphuretin, and butein as active ingredients in a composition purified from sumac extract using a silica column.

On the other hand, it has been reported in Korean Patent No. 10-0807169 that lacquer tree extract was used as a composition for preventing and treating arthritis. In the above registered patent, it has been reported that sumac extract has immunosuppressive effects, synovial cell activity inhibition effects, anti-inflammatory effects, and pharmacological effects such as preventive and therapeutic effects in animal models of arthritis.

The above results confirm that the lacquer tree extract has an anti-cancer effect by ingesting it in a cancer animal model or directly treating it with cell lines, and that the lacquer tree extract suppresses the immune response and has an anti-inflammatory effect and an effect in treating arthritis. shows that

Therefore, it has been determined that lacquer tree extract induces the death of cancer cells directly rather than through an immune response or induces an anticancer effect through a mechanism other than activating an immune response.

Patent documents and references mentioned herein are herein incorporated by reference to the same extent as if each individual document was individually and specifically identified by reference.

The present invention experimentally demonstrated that treating cytokine killer cells with sumac tree extract improves the cancer killing ability of cytokine killer cells. Therefore, the purpose of the present invention is to provide an immuno-anticancer composition containing sumac extract and cytokine killer cells as active ingredients, and an anti-cancer immune cell therapy composition containing the same.

Other objects and technical features of the present invention are presented in more detail by the following detailed description, claims, and drawings.

The present invention provides an immuno-anticancer composition containing sumac extract and cytokine killer cells as active ingredients.

The sumac extract is characterized in that it enhances the killing ability of cytokine killing cells against cancer cells, and the cancer cells are characterized in that they are lung cancer or breast cancer cells.

When the cancer cells (cells/ml) are treated with 5 to 10 times the amount of cytokine killing cells (cells/ml) and the sumac extract is simultaneously treated at 10 to 100 μg/ml, in preparation for the case where the sumac extract is not treated, It is characterized in that the activity of the cancer cells is further reduced by 5 to 30%.

The cancer cells are characterized in that they are lung cancer or breast cancer cells; When lung cancer cells 5x10 ⁴ cells/ml are treated simultaneously with 5x10 ⁴ to 5x10 ⁵ cells/ml of the cytokine-induced killing cells and 10 μg/ml of the sumac tree extract, the activity of the cancer cells increases by 20 compared to the case where the sumac tree extract is not treated. characterized by a further decrease of 30%; When 5x10 ⁴ to 2.5x10 ⁵ cells/ml of the cytokine-induced killing cells and 10 to 100 ㎍/ml of the sumac tree extract are simultaneously treated with 5x10 ⁴ cells/ml of the breast cancer cells, compared to the case where the sumac tree extract is not treated, the cancer cells It is characterized by a further decrease in activity by 5 to 20%.

Previously, it was known that lacquer tree extract was effective in treating arthritis and other conditions by suppressing immunity. However, in the present invention, it was experimentally proven that when cytokine killer cells are treated with sumac tree extract, the immune sensitivity of immune cells actually increases, showing improved cancer cell killing ability.

Therefore, the immuno-anticancer composition containing lacquer tree extract and cytokine killer cells of the present invention as active ingredients has the advantage of effectively treating lung cancer or breast cancer due to its improved cancer cell killing ability, and when an anti-cancer immune cell therapy containing the same is manufactured, lung cancer and When administered to breast cancer patients, it can effectively treat lung and breast cancer.

Figure 1 shows changes in the killing effect of lung cancer cell line (H441) of cytokine-induced killer cells treated with the sumac extract (RVS extract) of the present invention.

Figure 2 shows changes in the killing effect of cytokine-induced killer cells treated with the RVS extract of the present invention on breast cancer cell line (MCF7).

Figure 3 shows changes in the killing effect of cytokine-induced killer cells treated with the lacquer tree extract of the present invention on a breast cancer cell line (MDA-MB231).

The present invention provides an immuno-anticancer composition containing sumac extract and cytokine killer cells as active ingredients.

The lacquer tree extract can be prepared by hot water extraction of the bark of lacquer tree using a solvent selected from water, methanol, ethanol, butanol, or a mixed solvent thereof. In detail, the bark of the lacquer tree was cut into pieces, dried, extracted with water at 95°C for 2 hours, cooled, and the supernatant was filtered through a filter with a diameter of 0.2 μm, concentrated under reduced pressure, and freeze-dried.

The cytokine killer cell is a type of immune cell that recognizes infected cells or cancer cells, attaches to them, and secretes enzymes that destroy the outer membrane of the cells.

The lacquer tree extract of the present invention is characterized by enhancing the killing ability of cytokine killer cells against cancer cells. The cancer cells are not limited to any cancer that is monitored and killed by cytokine killer cells, but are preferably lung cancer or breast cancer cells. .

According to an embodiment of the present invention, the cancer cells (cells/ml) are treated with 5 to 10 times the amount of cytokine killing cells (cells/ml), and the sumac tree extract is simultaneously treated with 10 to 100 μg/ml. It is characterized in that the activity of the cancer cells is further reduced by 5 to 30% compared to the case where the sumac extract is not treated.

In detail, when 5x10 ⁴ to 5x10 ⁵ cells/ml of the cytokine-induced killing cells and 10 ㎍/ml of the lacquer tree extract are simultaneously treated with 5x10 ⁴ cells/ml of lung cancer cells, the cancer cells are treated in contrast to the case where the sumac tree extract is not treated. Characterized by a further decrease in activity by 20 to 30%; When 5x10 ⁴ to 2.5x10 ⁵ cells/ml of the cytokine-induced killing cells and 10 to 100 ㎍/ml of the sumac tree extract are simultaneously treated with 5x10 ⁴ cells/ml of the breast cancer cells, compared to the case where the sumac tree extract is not treated, the cancer cells It is characterized by a further decrease in activity by 5 to 20%.

The anticancer immunocomposition of the present invention can be prepared at a ratio of 5x10 ⁴ to 5x10 ⁵ cells/ml of cytokine-induced killer cells and 10 to 100 ㎍/ml of lacquer extract, and if the ratio is maintained at all times, a buffer solution to maintain cell activity There is no problem if more of this appropriate carrier is used.

The anticancer immunocomposition of the present invention contains cytokine-induced killing cells that kill cancer cells and sumac extract that improves the cancer cell killing ability of the cytokine killing cells, and can be manufactured as an anticancer immune cell therapy agent containing the anticancer immunocomposition.

The anti-cancer immune cell therapy agent of the present invention includes cytokine-induced killer cells activated by sumac extract. The cytokine-induced killer cells can be manufactured by isolating peripheral blood mononuclear cells (PBMC) from peripheral blood, isolating the cytokine-induced killer cells from the isolated peripheral blood mononuclear cells, and then activating them by treating them with a sumac extract. The activation is performed by treating the isolated cytokine-induced killer cells with lacquer extract, and cultivating the cytokine-induced killer cells by administering 10 to 100 μg/ml of lacquer extract to 5x10 ⁴ to 5x10 ⁵ cells/ml. It is desirable to do so.

The anti-cancer immune composition of the present invention can be prepared as an anti-cancer immune cell therapy composition. The anticancer immune cell therapy composition of the present invention is not limited to any cancer known to be treatable by cytokine-induced killer cells, but lung cancer or breast cancer is preferred.

The anti-cancer immune cell therapy composition of the present invention can be prepared in unit dosage form by formulating using pharmaceutically acceptable excipients according to a method that can be easily performed by those skilled in the art. there is. At this time, the formulation may be in the form of suspension in a cell freezing solution or a buffer solution, and may additionally contain a stabilizer. The anticancer immune cell therapy composition of the present invention can be administered parenterally, and can also be administered by intravenous injection, subcutaneous injection, intraperitoneal injection, or transdermal administration. The appropriate dosage of the anti-cancer immune cell therapy composition of the present invention can be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, sex, administration time, and administration route.

The present invention will be described in detail below through examples.

Example

1. Cytokine-induced killer cell-mediated lung cancer cell line killing effect of sumac tree extract

Changes in the killing effect of cytokine-induced killer cells (CIK) on lung cancer cell lines (H441) by extracts of sumac (Toxicodendron Vernicifluum or Rhus Veniciflua Strokes) were confirmed.

The lacquer tree extract (RVS extract) was prepared by hot water extraction followed by filtration, concentration under reduced pressure, and freeze-drying; The CIK was prepared by treating human PBMC with CD3 antibody and IL-2 in T-cell culture medium and culturing them for 2 weeks; The lung cancer cell line (H441) was cultured in RPMI 1604 medium with 10% FBS and seeded at a concentration of 5x10 ⁴ cells/ml in a 96 well plate one day before CIK co-culture.

The activity of lung cancer cells was analyzed through RFP killing assay. The RFP killing assay measures cell killing ability through immune cells by manufacturing an RFP stable cell line using H441, MCF7, and MDA-MB-231 and then measuring the number of RFP positive cells that are reduced due to the killing effect when co-cultured with CIK. This is how to do it.

Figure 1 shows changes in the killing effect of cytokine-induced killer cells treated with the lacquer extract of the present invention on a lung cancer cell line (H441).

A lung cancer cell line (H441, 5x10 ⁴ cells/ml) treated with 10㎍/ml of RVS extract and cultured for 48 hours, and a lung cancer cell line (H441, 5x10 ⁴ cells/*?*) cultured for 48 hours without treatment of RVS extract. As a result of comparing, it was confirmed that the cell activity was the same (see ns in Figure 1). Therefore, it is believed that simply treating the lung cancer cell line (H441) with the RVS extract of the present invention does not change the activity of the lung cancer cell line (H441).

The lung cancer cell line (H441, 5x10 ⁴ cells/ml) was treated with cytokine-induced killer cells (5x10 ⁴ cells/ml) and cultured for 48 hours. As a result, only the lung cancer cell line (H441, 5x10 ⁴ cells/ml) was cultured for 48 hours. Compared to the results, it was confirmed that lung cancer cell line activity (H441, RFP count) decreased by about 13%.

The lung cancer cell line (H441, 5x10 ⁴ cells/ml) was simultaneously treated with cytokine-induced killer cells (5x10 ⁴ cells/ml) and RVS 10㎍/ml, and cultured for 48 hours. The result was a lung cancer cell line (H441, 5x10 ⁴ cells/ml). It was confirmed that lung cancer cell line activity (H441, RFP count) decreased by about 21% compared to the results of treating only cytokine-induced killing cells (5x10 ⁴ cells/ml) and culturing them for 48 hours (see ** in Figure 1). ).

In addition, the lung cancer cell line (H441, 5x10 ⁴ cells/ml) was simultaneously treated with cytokine-induced killer cells (5x10 ⁵ cells/ml) and RVS extract 10㎍/ml and cultured for 48 hours ^. Lung cancer cell line activity (H441, RFP count) compared to the results of treating only cytokine-induced killer cells (5x10 ⁵ cells/ml) (lung cancer cell line: cytokine-induced killer cells = 1:10) and culturing for 48 hours. It was confirmed that this was further reduced by about 25% (see * in Figure 1).

Therefore, the RVS extract of the present invention does not have direct apoptosis ability against lung cancer cell line (H441), but when used together with cytokine-induced killer cells, it is believed to improve the apoptosis ability of lung cancer cell line (H441) by cytokine-induced killer cells.

2. Cytokine-induced killer cell-mediated breast cancer cell line killing effect of sumac tree extract

Changes in the effect of the RVS extract of the present invention on killing cytokine-induced killer cells (CIK) in a breast cancer cell line (MCF7) were confirmed.

Figure 2 shows changes in the killing effect of cytokine-induced killer cells treated with the RVS extract of the present invention on breast cancer cell line (MCF7).

Comparison of a breast cancer cell line (MCF7, 5x10 ⁴ cells/ml) treated with 10㎍/ml of RVS extract and cultured for 48 hours with a breast cancer cell line (MCF7, 5x10 ⁴ cells/ml) cultured for 48 hours without treatment with RVS extract. As a result, it was confirmed that the cell activity was the same.

Therefore, it is believed that simply treating the breast cancer cell line (MCF7) with the RVS extract of the present invention does not change the activity of the breast cancer cells (MCF7).

Breast cancer cell line (MCF7, 5x10 ⁴ cells/ml) was treated with cytokine-induced killing cells (2.5x10 ⁵ cells/ml) and cultured for 48 hours. Only breast cancer cell line (MCF7, 5x10 ⁴ cells/ml) was cultured for 48 hours. Compared to the previous results, it was confirmed that breast cancer cell line activity (MCF7, RFP count) decreased by about 62%.

Breast cancer cell line (MCF7, 5x10 ⁴ cells/ml) was simultaneously treated with cytokine-induced killing cells (2.5x10 ⁵ cells/ml) and RVS extract 10㎍/ml and cultured for 48 hours. Breast cancer cell line (MCF7, 5x10 ⁴ cells/ml) It was confirmed that breast cancer cell line activity (MCF7, RFP count) decreased by about 5% compared to the results of treating cytokine-induced killing cells (2.5x10 ⁵ cells/ml) and culturing them for 48 hours (Figure 2). * reference).

Figure 3 shows changes in the killing effect of cytokine-induced killer cells treated with the lacquer extract of the present invention on a breast cancer cell line (MDA-MB231).

The breast cancer cell line (MDA-MB231) is different from the breast cancer cell line (MCF7) in origin and level of receptor expression. The breast cancer cell line (MDA-MB231) is derived from adenocarcinoma and has the characteristics of not expressing estrogen receptor, progesterone receptor, or HER2 (CD340) and having a P53 mutation. In contrast, the breast cancer cell line (MCF7) is derived from invasive ductal carcinoma and expresses the estrogen receptor and progesterone receptor, but does not express HER2 (CD340) and has no confirmed P53 mutation. There is a characteristic.

As a result of the experiment, a breast cancer cell line (MDA-MB231, ^5x10 cells/㎖) was treated with 100㎍/㎖ of RVS extract and cultured for 48 hours, and a breast cancer cell line (MDA-MB231, 5x10 ^cells/㎖) was cultured for 48 hours without treatment of RVS extract. cells/mL), it was confirmed that the cell activity was the same.

Breast cancer cell line (MDA-MB231, 5x10 ⁴ cells/ml) was simultaneously treated with cytokine-induced killing cells (5x10 ⁴ cells/ml) and RVS extract 0㎍/㎖, RVS extract 10㎍/㎖, or RVS extract 100㎍/㎖. and cultured for 48 hours to analyze cell activity.

As a result of the experiment, when the breast cancer cell line (MDA-MB231, 5x10 ⁴ cells/ml) was treated with only cytokine-induced killing cells (5x10 ⁴ cells/ml), only the breast cancer cell line (MDA-MB231, 5x10 ⁴ cells/ml) was cultured for 48 hours. The activity of the breast cancer cell line (MDA-MB231, RFP count) decreased by about 35% compared to the results of culturing the breast cancer cell line (MDA-MB231, ^5x104 cells/ml) and RVS extract at 100㎍/ml. Confirmed.

Breast cancer cell line (MDA-MB231, 5x10 ⁴ cells/ml) was simultaneously treated with cytokine-induced killing cells (5x10 ⁴ cells/ml) and RVS 10 ㎍/ml and cultured for 48 hours. It was confirmed that breast cancer cell line activity (MDA-MB231, RFP count) decreased by about 5% compared to the results of treating only cytokine-induced killer cells (5x10 ⁴ cells/ml) and culturing them for 48 hours ^; Breast cancer cell line (MDA-MB231, 5x10 ⁴ cells/ml) was simultaneously treated with cytokine-induced killing cells (5x10 ⁴ cells/ml) and RVS extract 100㎍/ml and cultured for 48 hours. Breast cancer cell line (MDA-MB231) It was confirmed that breast cancer cell line activity (MDA-MB231, RFP count) decreased by about 15% compared to the results of treating only cytokine-induced killing cells (5x10 ⁴ cells/ml) and culturing ^them for 48 hours. .

Breast cancer cell line (MDA-MB231, 5x10 ⁴ cells/ml) was treated with cytokine-induced killing cells (2.5x10 ⁵ cells/ml) and RVS extract 0㎍/㎖, RVS extract 10㎍/㎖ or RVS extract 100㎍/㎖. and cultured for 48 hours to analyze cell activity.

As a result of the experiment, when the breast cancer cell line (MDA-MB231, 5x10 ⁴ cells/ml) was treated with only cytokine-induced killing cells (2.5x10 ⁵ cells/ml), only the breast cancer cell line (MDA-MB231, 5x10 ⁴ cells/ml) was treated for 48 hours. Compared to the culture results, it was confirmed that the activity of the breast cancer cell line (MDA-MB231, RFP count) decreased by about 62%.

Breast cancer cell line (MDA-MB231, 5x10 ⁴ cells/ml) was simultaneously treated with cytokine-induced killing cells (2.5x10 ⁵ cells/ml) and RVS extract 10 ㎍/ml and cultured for 48 hours. Breast cancer cell line (MDA- Breast cancer cell line activity (MDA-MB231, RFP count) decreased by about 13% compared to the results of treating MB231, 5x10 ⁴ cells/ml) with only cytokine-induced killing cells (2.5x10 ⁵ cells/ml) and culturing them for 48 hours. Confirmed; Breast cancer cell line (MDA-MB231, 5x10 ⁴ cells/ml) was simultaneously treated with cytokine-induced killer cells (2.5x10 ⁵ cells/ml) and RVS extract 100㎍/ml and cultured for 48 hours. Breast cancer cell line (MDA- Breast cancer cell line activity (MDA-MB231, RFP count) decreased by about 17% compared to the results of treating MB231, 5x10 ⁴ cells/ml) with only cytokine-induced killing cells (2.5x10 ⁵ cells/ml) and culturing them for 48 hours. Confirmed.

Therefore, the RVS extract of the present invention has no direct apoptotic ability against breast cancer cell lines (MCF7 or MDA-MB231), but when used together with cytokine-induced killer cells, it kills breast cancer cell lines (MCF7 or MDA-MB231) by cytokine-induced killer cells. It is believed that the killing ability of breast cancer cell lines (MCF7 or MDA-MB231) by cytokine-induced killer cells is improved depending on the concentration of RVS extract.

The specific embodiments described in this specification are meant to represent preferred embodiments or examples of the present invention, and are not intended to limit the scope of the present invention. It will be apparent to those skilled in the art that modifications and other uses of the present invention do not depart from the scope of the invention as set forth in the claims herein.

The immuno-anticancer composition containing sumac extract and cytokine killer cells of the present invention as active ingredients has the advantage of being able to effectively treat lung cancer or breast cancer due to its improved cancer cell killing ability. When an anti-cancer immune cell therapy containing the same is manufactured, lung cancer and breast cancer can be prevented. When administered to patients, it can effectively treat lung cancer and breast cancer.

Claims (6)

An immune-anticancer composition containing sumac tree extract and cytokine killer cells as active ingredients. The immuno-anticancer composition according to claim 1, wherein the lacquer tree extract enhances the killing ability of cytokine killer cells against cancer cells. The method of claim 2, wherein the cancer cells (cells/ml) are treated with 5 to 10 times the amount of cytokine killing cells (cells/ml) and the sumac extract is simultaneously treated at 10 to 100 μg/ml. An immuno-anticancer composition characterized in that the activity of the cancer cells is further reduced by 5 to 30% compared to the case where the cancer cells are not activated. The immuno-anticancer composition according to claim 2, wherein the cancer cells are lung cancer or breast cancer cells. According to claim 2, when 5x10 ⁴ to 5x10 ⁵ cells/ml of the cytokine-induced killing cells and 10 ㎍/ml of the lacquer tree extract are simultaneously treated with 5x10 ⁴ cells/ml of the lung cancer cells, in preparation for the case where the lacquer tree extract is not treated. An immuno-anticancer composition, characterized in that the activity of the cancer cells is further reduced by 20 to 30%. The method of claim 2, wherein 5x10 ⁴ to 2.5x10 ⁵ cells/ml of the cytokine-induced killer cells and 10 to 100 ㎍/ml of the lacquer tree extract are simultaneously treated on 5x10 ⁴ cells/ml of the breast cancer cells when the lacquer tree extract is not treated. An immuno-anticancer composition characterized in that the activity of the cancer cells is further reduced by 5 to 20% compared to.

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