KR102024322B1 - A composition for enhancing radiation sensitivity and an anticancer adjuvent composition comprising ginsenoside rg3 - Google Patents

Abstract

One embodiment of the present invention provides a pharmaceutical composition for enhancing radiation sensitivity comprising ginsenoside Rg3 as an active ingredient.

Description

A composition for enhancing radiation sensitivity and an anticancer adjuvant composition comprising ginsenoside Rg3 {A COMPOSITION FOR ENHANCING RADIATION SENSITIVITY AND AN ANTICANCER ADJUVENT COMPOSITION COMPRISING GINSENOSIDE RG3}

The present invention relates to the use of ginsenoside Rg3 in adjuvant anticancer treatment.

Cancer is one of the incurable diseases that humanity has to solve, and huge capital is invested in the development to cure it all over the world.In Korea, it is the number one disease death cause and more than 100,000 people a year It is diagnosed, and about 60,000 or more die.

Carcinogens that cause cancer include smoking, ultraviolet rays, chemicals, foods, and other environmental factors. However, the causes of various cancers are difficult to develop, as well as the effects of treatments vary depending on the site of occurrence.

Despite the recent rapid development of medicine and various forms of cancer treatment through surgery, radiation therapy, and chemotherapy, many cancer patients still suffer from complete removal of cancer cells and prevention of metastasis.

Radiation therapy is now known as an essential treatment method for various types of cancers, but it has been pointed out that obtaining radiation resistance of cancer cells and damaging normal tissues in high dose radiation treatments lowers the efficiency of radiation therapy. Therefore, researches on radiation therapy sensitizers to improve the efficiency of radiation therapy have been attempted, but currently reported radiation therapy sensitizers are mainly anticancer agents, for example, Taxol and cisplatin are reported. It is.

There is no anti-cancer agent, but the radiation therapy enhancer used only for radiation therapy is tirapazamine, but it is effective only in tumor cells of hypoxia and drug delivery into tumor tissue due to the hypoxic specific tumor pressure. Poorly effective in clinical radiation therapy.

When used in combination with radiation therapy, chemotherapy agents used to enhance radiotherapy can be combined with the side effects of radiation therapy, such as inflammation, gastrointestinal disorders, nausea, vomiting, and diarrhea. Because of the limitations of use.

Radiation therapy causes various side effects by irradiating patients with high doses of radiation, which side effects are dependent on radiation dose. Therefore, there is an urgent need for the development of a technology that can show an improved anticancer effect while lowering the radiation dose.

On the other hand, ginseng is a perennial plant belonging to the genus Panax , and the genus Panax is a perennial root of the genus Araliaceae . In particular, saponins contained in large amounts in ginseng include ginsenosides Rb1, Rb2, Rc, Rd, Rg1, Re, etc., in particular, memory due to damage to animal models damaged by scopolamine or hippocampus neurons. Ginseng is known to alleviate the decline.

Nevertheless, ginsenosides have not been studied for the purpose of increasing sensitivity in radiation therapy, and the present inventors have confirmed that ginsenoside Rg3 can be effectively used for anticancer treatment by increasing sensitivity in radiation therapy. The present invention has been completed.

 Molecular Medicine Reports, 12, 609-614, 2015.  Journal of International Medical Research, 42 (3), 628-640, 2014.  Biomedicin & Pharmacotherapy, 96, 619-625, 2017.  BMC Cancer, 9, 250 (1-11), 2009.

It is an object of the present invention to provide an anticancer adjuvant composition which can minimize side effects of biosafety and anticancer treatment.

According to one aspect of the invention, there is provided a pharmaceutical composition for enhancing radiation sensitivity comprising ginsenoside Rg3 as an active ingredient.

In one embodiment, the radiation may be 1Gy to 4Gy low level radiation.

According to another aspect of the present invention, there is provided an anticancer adjuvant composition for enhancing anticancer activity comprising ginsenoside Rg3 as an active ingredient.

In one embodiment, the anticancer agent may be a targeted therapeutic.

In one embodiment, the targeted therapeutic agent may be gefitinib or erlotinib.

In one embodiment, the cancer may be at least one selected from the group consisting of liver cancer, stomach cancer, colon cancer, lung cancer, breast cancer, rectal cancer and pancreatic cancer.

According to the invention, the composition not only significantly enhances the anticancer effect of the radiation treatment, but also does not cause side effects on human administration.

It is to be understood that the effects of the present invention are not limited to the above effects, and include all effects deduced from the configuration of the invention described in the detailed description or claims of the present invention.

Figure 1 shows the synergistic effect of gefitinib (gefitinib) and Rg3 combined treatment.
Figure 2 confirms the radiation sensitivity enhancement effect of Rg3 in radiation therapy.
Figure 3 confirms the effect of increasing apoptosis (apoptosis) by radiation and Rg3 combined treatment.
Figure 4 shows the signaling system involved in apoptosis by radiation and Rg3 combined treatment.

The terminology used herein is to select general terms that are currently widely used as possible in consideration of the functions in the present invention, but may vary according to the intention or precedent of the person skilled in the art, the emergence of new technologies and the like. In addition, in certain cases, there is also a term arbitrarily selected by the applicant, in which case the meaning will be described in detail in the description of the invention. Therefore, the terms used in the present invention should be defined based on the meanings of the terms and the contents throughout the present invention, rather than the names of the simple terms.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art. Terms such as those defined in the commonly used dictionaries should be construed as having meanings consistent with the meanings in the context of the related art, and are not construed in ideal or excessively formal meanings unless expressly defined in this application. Do not.

The numerical range includes the numerical values defined in the range. All maximum numerical limits given throughout this specification include all lower numerical limits as if the lower numerical limits were clearly written. All minimum numerical limits given throughout this specification include all higher numerical limitations as if the higher numerical limit were clearly written. All numerical limitations given throughout this specification will include all better numerical ranges within the broader numerical range, as the narrower numerical limitations are clearly written.

When a part is said to "include" a certain component, this means that it may further include other components, without excluding other components, unless specifically stated otherwise.

Hereinafter, with reference to the accompanying drawings will be described the present invention. As those skilled in the art would realize, the described embodiments may be modified in various different ways, all without departing from the spirit or scope of the present invention.

According to one aspect of the invention, there is provided a pharmaceutical composition for enhancing radiation sensitivity comprising ginsenoside Rg3 as an active ingredient.

Said “cancer”, “tumor” or “malignant” generally refers to or describes the physiological state of a mammal that is characterized by unregulated cell growth. Examples of the cancer include, but are not limited to, carcinoma, lymphoma, leukemia, blastoma, and sarcoma, and may be selected, for example, from the group consisting of liver cancer, gastric cancer, colon cancer, lung cancer, breast cancer, rectal cancer, and pancreatic cancer.

The "ginsenoside Rg3" is a major component of red ginseng has been found to be effective in the treatment of various diseases through a number of clinical experiments and animal experiments, but the effect of increasing the sensitivity of radiation therapy has not been studied yet.

The ginsenoside Rg3 may be normally separated from ginseng or red ginseng, but may also have a chemically synthesized effect. In addition, derivatives of ginsenoside Rg3 obtained by addition or substitution reaction of substituents commonly practiced in the art may also be included in the scope of the present invention.

The "comprising as an active ingredient" may mean that it contains an effective amount that can exhibit a radiosensitivity enhancing effect, for example, enhance the death of cancer cells or inhibit the proliferation of cancer cells following radiation therapy.

The ginsenoside Rg3 may be involved in cancer cell death and induce cancer cell suicide when irradiated, and may reduce radiation survival and radiation resistance of cancer cells, thereby improving radiation sensitivity.

The term "radiation resistance" means that the treatment of a disease using radiation does not kill or kill the abnormal cells despite irradiation. Alternatively, in the case of radiation therapy, it means that there is no therapeutic effect from the beginning of the treatment or the disease treatment effect at the beginning but the therapeutic effect is lost in the continuous treatment process.

The term "enhancing radiation sensitivity" means enhancing the sensitivity of cells to radiation in radiation therapy. This can increase the radiation treatment efficiency, especially when used in combination with cancer treatment to increase the radiation sensitivity of the tumor cells can produce a killing effect of the radiation on the tumor cells and inhibit the proliferation.

Said "radiation treatment" includes irradiation of radiation after administration of the composition of the present invention to cancer cells, wherein "radiation irradiation" refers to ionizing radiation, in particular, commonly used linear accelerators or radionuclides. May refer to gamma radiation emitted by

Irradiation by the radionuclide may be performed externally or internally, and the amount of siRNA or antiviral agent administered, the amount of radiation and the amount of radiation intermittent, the patient's response to the type, location, chemotherapy or radiation therapy of the tumor It can be changed by a series of variables such as

Techniques for the irradiation include brachytherapy, radionuclide therapy, external beam radiation therapy, thermotherapy (including cryoablation and hyperthermia), radiation surgery, charged particle radiation therapy (charged-particle radiotherapy), neutron radiation therapy and photodynamic therapy.

The radiation sensitivity enhancement and radiation treatments are applicable to any animal that can cause cancer, and can include livestock such as cattle, pigs, sheep, horses, dogs, cats, as well as humans and primates.

The radiation may be 1Gy to 4Gy low level radiation.

The radiation dose range of the radiation is not particularly limited, but may be irradiated at a dose of 0.5 to 10 Gy, preferably at a dose of 1 to 4 Gy. When irradiated with a dose in the above range can greatly maximize the effect of concomitant administration by greatly reducing the secondary problems such as lowering immunity due to radiation side effects and the like.

According to another aspect of the present invention, there is provided an anticancer adjuvant composition for enhancing anticancer activity comprising ginsenoside Rg3 as an active ingredient.

The "anticancer adjuvant" means an agent that can improve, enhance or enhance the anticancer effect of the anticancer agent. For example, when the anticancer adjuvant is used in combination with an anticancer agent, the anticancer effect of the anticancer agent may be improved, improved or increased.

As another example, a concentration-dependent anticancer agent may be used as an anticancer adjuvant that can improve, enhance or enhance the anticancer effect of the anticancer agent when used in combination with an anticancer agent at a level that does not exhibit anticancer activity by itself. .

The anticancer adjuvant may be used as an anticancer agent or an anticancer adjuvant according to the treatment concentration, and may be used as an anticancer adjuvant in the treatment concentration range which does not exhibit anticancer activity by itself.

The anticancer agent may be a targeted therapeutic agent, and the target therapeutic agent may be gefitinib or erlotinib.

Non-small cell lung cancer, which accounts for the majority of cancers, is very difficult to diagnose early and has a high mortality rate due to early distant metastasis. About 40% to 50% of patients relapse and die even though they are found in stage 1 and undergo surgical treatment.

Conventional anticancer drugs are used as the main therapeutic agents, and epidermal growth factor receptor (EGFR) target therapeutic agents such as gefitinib and erlotinib have recently been used as target therapeutic agents. The anticancer effect can be maximized when administered with cenoside Rg3.

On the other hand, the composition may be administered in the form of oral delivery, parenteral delivery. The composition can be administered systemically or topically, and the administration can include oral and parenteral administration. The composition may be formulated with a suitable amount of pharmaceutically acceptable vehicle or carrier to provide a suitable dosage form.

The composition may further comprise a carrier, excipient and diluent used in the manufacture of the pharmaceutical composition.

The carrier, excipient and diluent may include 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, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil, but are not limited thereto.

In addition, the composition may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, suppositories, and sterile injectable solutions.

Solid preparations for oral administration may be used in tablets, pills, powders, granules, capsules, and the like, wherein the solid preparations include at least one excipient such as starch, calcium carbonate, sucrose, lactose in the compound and fractions thereof. Or gelatin can be mixed and prepared. In addition to the above excipients, lubricants such as magnesium styrate and talc may be used.

As a liquid preparation for oral administration, suspending agents, solvents, emulsions, syrups, and the like may be used, and various excipients such as wetting agents, sweeteners, fragrances, preservatives, and the like may be used in addition to water and liquid paraffin, which are simple diluents.

For parenteral administration, sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, suppositories can be used. The non-aqueous solvent and suspension may be propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerogelatin may be used.

The composition may be administered in a pharmaceutically effective amount sufficient to treat the disease at a reasonable benefit / risk ratio applicable to the medical treatment, and the effective dose level may be of the type and severity of the subject, age, sex, type of disease, drug Activity, sensitivity to drug, time of administration, route of administration and rate of release, duration of treatment, factors including concurrently used drugs, and other factors well known in the medical arts.

The composition may be administered in combination with other therapeutic agents, such as anticancer agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered in single or multiple doses.

The anticancer agent is cisplatin, oxaliplatin, carboplatin, carcarplatin, procarbazine, mechlorethamine, cyclophosphamide, ifosfamide, Melphalan, chlorambucil, bisulfan, nitrosourea, diactinomycin, daunorubicin, doxorubicin, bleomycin , Plicomycin, mitomycin, etoposide, tamoxifen, taxol, transplatinum, 5-fluorouracil , Vincristin, vinblastin, methotrexate, but are not limited thereto.

Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, and can be easily determined by those skilled in the art.

The preferred dosage of the composition may vary depending on the condition and weight of the patient, the severity of the disease, the form of the drug, the route of administration, and the duration of time, and a suitable total daily dose may be determined by the practitioner within the correct medical judgment. .

Generally, an amount of 0.001 to 1000 mg / kg, preferably 0.05 to 200 mg / kg, more preferably 0.1 to 100 mg / kg, may be administered once to several times daily.

The composition is not particularly limited as long as it is an object for the purpose of preventing or treating cancer, and any object may be applied. For example, it can be applied to any individual such as non-human animals such as monkeys, dogs, cats, rabbits, marmots, rats, mice, cows, sheep, pigs, goats, and humans, and the like, and the mode of administration is limited if it is a conventional method in the art. Includes without.

Those skilled in the art to which the present invention pertains will be able to apply by changing the type, introduction ratio, etc. of each configuration based on the description of the present invention, if the equivalent technical effect is implemented in spite of the above modification, the present invention It will be included in the technical idea of the.

Through the following examples, the present invention will be described in more detail, but the present invention is not limited by the following examples.

Cell line culture

Lung cancer cell lines (A549, PC9, HCC827) were sold from Korea Cell Line Bank (KCLB) and used in this experiment.

Each cancer cell line was cultured in a constant temperature cell incubator at 37 ° C., 5% CO 2, using RPMI-1640 medium containing 10% fetal bovine serum (FBS) and 1% anti-biotics. Incubated.

Experimental Example  1: targeted therapeutic agent and Rg3  Anticancer effect test by combined treatment

Lung cancer cells were inoculated into 96 well-culture plates at 5 × 10 ⁴ cells per well and incubated for 24 hours.

Gefitinib alone or in combination with Rg3 was further incubated for 24 hours under normal oxygen (normoxic) conditions.

Tetrazolium compounds [3- (4,5-dimethylthiazol-2-yl) -5 (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium, inner salt; MTS (a)] cell viability was analyzed using an assay kit (Promega).

20 μL MTS solution was added to each well and incubated for 4 hours and absorbance was measured at 490 nm wavelength.

Referring to FIG. 1, the cell viability was decreased when Rg3 was simultaneously administered in all cell lines, and the result suggests that the anticancer activity of the target therapeutic agent may be further increased when administered with Rg3.

Experimental Example  2: radiation and Rg3  Study on the effect of increasing radiation sensitivity by combined treatment

Lung cancer cell lines were seeded at 1 × 10 ⁵ cells / ml in a T-25 culture flask (culture flask, NuncProducts, Denmark) and incubated for 4 hours.

The cultured lung cancer cell lines were irradiated with gamma rays using ⁶⁰ Co, and cell viability was analyzed by varying the radiation dose.

20 μL MTS solution was added to each well and incubated for 4 hours before absorbance was measured at 490 nm wavelength.

Referring to FIG. 2, the cell viability of the radiotherapy treated with Rg3 was reduced in all cell lines, and the results suggest that Rg3 may further increase the anticancer therapeutic effect by enhancing the sensitivity of radiotherapy.

Experimental Example  3: radiation and Rg3  According to combined treatment Apoptosis  Increase effect test

Apoptosis was confirmed by a change in the appearance of apoptotic cells in the process of apoptosis.

The cultured lung cancer cell line treated with Rg3 for 24 hours and untreated group was stained with Hoechst 33342 dye to observe the change in appearance.

During cell death, phosphatidylserine (PS), a membrane phospholipid of the cell, is moved out of the plasma membrane to be exposed to the extracellular environment, and the membrane imbalance can be detected using the Annexin V protein.

In order to detect the Annexin V protein, flow cytometry was performed after staining the Rg3 treated group and the untreated group of the cultured lung cancer cell line using the FITC Annexin V Apoptosis Detection kit (BD Pharmigen ™, CA).

Referring to FIG. 3, the cells treated with Rg3 in radiotherapy significantly increased apoptosis compared to untreated cells.

Experimental Example  4: radiation and Rg3  Signaling System Change Test by Combined Therapy

Many studies have been conducted on intracellular signaling systems that control cell survival and death. Among the signaling molecules, phosphoinositide 3-kinase (PI3K), Akt, and mitogen-activated protein kinase (MAPK) [extracellular Related protein kinases (ERK), p38 and C-Jun N-terminal kinases (JNK)] and IKK (IκB kinase) / IκB / NF (nuclear factor) -κB are p21 ^{WAF1 / Cip1} , Bcl-2, p53, cyclin It is considered to be an important factor in regulating the expression level and activation of cell death regulatory and cell cycle regulatory proteins, including E and CDK.

In order to confirm the change in the signaling protein according to the Rg3 combination treatment, protein expression levels of the Rg3 treated group and the untreated group were compared, and confirmed by Western blot using PARP, p53 and JNK as the primary antibodies.

Referring to FIG. 4, the expression level of the signal protein involved in apoptosis was significantly increased according to the Rg3 concurrent treatment, and the result suggests that Rg3 may promote apoptosis following radiotherapy.

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined form.

The scope of the invention is indicated by the following claims, and it should be construed that all changes or modifications derived from the meaning and scope of the claims and their equivalents are included in the scope of the invention.

Claims (6)

delete delete As an anticancer adjuvant composition for enhancing the activity of gefitinib anticancer agent comprising ginsenoside Rg3 as an active ingredient,
The cancer is lung cancer anticancer adjuvant composition. delete delete delete

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