KR20200100913A - Pharmaceutical Composition for enhancing the electrotherapy of cancer and method using the same - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for enhancing electric field treatment on cancer and a method using the same, comprising an autophagy inhibitor as an active ingredient. The present invention provides a more excellent cancer treatment effect by enhancing an effect of the electric field treatment on the cancer.

Description

Pharmaceutical Composition for enhancing the electrotherapy of cancer and method using the same

It relates to a pharmaceutical composition capable of enhancing the effect of electric field treatment and a method using the same.

In 2015, the National Statistical Office announced that 27.9% of Korea's deaths died from cancer, which accounts for the highest percentage of the causes of death, and that the death rate from cancer continues to increase. Many studies are active to treat these cancers, but nonetheless, incurable or incurable cancers still exist. One example is glioblastoma (GBM), in which more than half of the patients die within 1-2 years after diagnosis. Glioblastoma accounts for one-third of glioma and is characterized by a very high degree of malignancy and rapid development. Since it is easy to cause intratumoral bleeding, it is difficult to extract it by surgical operation, and it is not easy to treat because it is highly resistant to anticancer drug therapy, and shows high resistance to radiation therapy.

Electric field therapy, a new cancer treatment, shows the potential of treating the aforementioned glioblastoma. Electric field therapy applies an electric field to cancer cells to prevent the division of cancer cells, resulting in apoptosis. Therefore, electric field therapy showed new therapeutic potential for incurable cancer, which was difficult to treat with conventional cancer treatments such as surgery, radiation therapy, and chemotherapy. However, as the mechanism for electric field treatment is currently unclear, a study on this is required (Korean Patent Laid-Open No. 10-2016-0134469).

One aspect provides a pharmaceutical composition for enhancing electric field treatment for cancer.

Another aspect provides a method of electric field treatment for cancer.

One aspect provides a pharmaceutical composition for enhancing electric field treatment for cancer comprising an autophagy inhibitor as an active ingredient.

The term “prevention” refers to any action that inhibits the progression or delays the onset of cancer by administration of the pharmaceutical composition according to the present invention.

The term "treatment" refers to any action in which symptoms of cancer are improved or advantageously changed by administration of the pharmaceutical composition according to the present invention.

The cancer is colorectal cancer, liver cancer, gastric cancer, colon cancer, breast cancer, lung cancer, squamous cell carcinoma, fibroma, non-small cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, melanoma in the skin or eye, uterine cancer, ovarian cancer, rectal cancer, Anal cancer, fallopian tube death, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer , Penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, kidney cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, brain stem It may be one or more cancers selected from the group consisting of glioma, pituitary adenoma, or glioblastoma. In one embodiment, the cancer may be glioblastoma.

The term'autophagy', autophagy, or autophagy refers to a mechanism of destruction that naturally degrades unnecessary or non-functional cellular components in the process of regulation. Autophagy plays a role in destroying or recycling cellular components. During autophagy, the target cytoplasmic component is isolated from other components in the cell and surrounded by a double membrane, which is called an autophagosome. The autologous vesicle then fuses with the lysosome, and the contents are decomposed and recycled. Autophagy generally acts in three forms, consisting of macroautophagy, microautophagy, and chaperone-mediated autophagy. Autophagy can be associated with apoptosis in connection with disease. Apoptosis refers to a step leading to death by genetic properties while maintaining a functional role of a cell. As the entire cell is atrophy, a new protein is synthesized and the cell's suicide gene can induce death. Such apoptosis can occur when stress is extreme within the cell. Apoptosis can occur mainly by activation of caspase at high levels. Caspase is a protease and degrades proteins such as ATG3 and Beclin1, which play an important role in autophagy. When the proteins are degraded, autophagy activation can no longer occur, and a reaction can proceed in the direction of cell death. In addition, when autophagy proteins are degraded by caspase, autophagy proteins promote cell death. For example, when Beclin1 is degraded, it moves to the mitochondria and increases mitochondrial permeability, thereby promoting endogenous cell death. Thus, inhibitors of autophagy can induce the cell death.

The inhibitor of autophagy may be one selected from the group consisting of an inhibitor of kinase that induces autophagy, or microRNA and combinations thereof. Kinases that induce autophagy may include Akt kinase or phosphatidylinositol3-kinase (PI3K) kinase. Akt kinase is an enzyme that phosphorylates serine and threonine, and is a protein that plays a central role in cell signaling. Akt is activated by PI3K activated by EGF receptor activity and can inhibit apoptosis. PI3K kinase can regulate autophagy and apoptosis through growth stimulation signals by the PI3K-Akt axis. Therefore, inhibitors of Akt kinase or PI3K kinase can inhibit autophagy and increase apoptosis. In one embodiment, the Akt may be one selected from the group consisting of Akt1, Akt2, Akt3, and combinations thereof.

The microRNA (or miRNA) refers to a small non-expressing RNA molecule composed of about 22 nucleotides found in plants, animals, viruses, and the like. This RNA can perform functions such as RNA silencing and regulation of gene expression after transcription. miRNAs can function through complementary base pairs in mRNA molecules, encoded by DNA in the nucleus of eukaryotes such as plants and animals, and viral DNA in certain viruses whose genomes are based on DNA. miRNAs can resemble small interfering RNAs (siRNAs) in the interfering RNA (RNAi) pathway, except for microRNAs from regions of RNA transcripts that fold and fold to form short hairpins. In one embodiment, the microRNA may be miR-29b.

According to one embodiment, miR-29b, one of the microRNAs, decreases the expression of Akt2 protein, which is believed to inhibit the expression of Akt2 protein by a silencing effect. When miR-29b is co-treated in electric field therapy as in one embodiment, the inhibition of Akt2 may increase. Therefore, in electric field therapy, microRNA can be an effective substance that can enhance the effect of treatment on cancer.

The term'electric field therapy' is a treatment regimen in which an electric field is applied to a cell to prevent cell division and result in apoptosis. The electric field treatment is based on the principle that the electric field applied from the outside of the cell can control the movement of microtubules to form the spindle, and the cells in which the spindle is not properly formed due to the electric field are prevented from normal cell division. In one embodiment, the electric field may be an AC electric field.

According to an embodiment, the electric field treatment may inhibit the expression of Akt kinase related to the autophagy. Electric field therapy can induce apoptosis by reducing the expression of Akt kinase and inhibiting the occurrence of autophagy. Accordingly, the inhibitor of autophagy according to an aspect further enhances the inhibitory effect of autophagy that occurs during electric field treatment, and thus may be administered simultaneously or sequentially with electric field treatment to enhance the effect of cancer treatment. That is, the autophagy inhibitor according to an embodiment may be used as an effective material for enhancing an electric field treatment effect on cancer.

The pharmaceutical composition may contain a pharmaceutically acceptable carrier in addition to the active ingredient. At this time, the pharmaceutically acceptable carrier is commonly used in the formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia, rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose , Polyvinyl pyrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil, but are not limited thereto. In addition, a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like may be additionally included in addition to the above components.

The pharmaceutical composition may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically applied) according to a desired method, and, for example, subcutaneous (i.e., topical preparation for skin) or oral ( That is, it may be oral) administration. The dosage varies depending on the condition and weight of the patient, the degree of disease, the form of the drug, the route and time of administration, but may be appropriately selected by those skilled in the art.

The pharmaceutical composition may be administered in a therapeutically effective amount. The term "therapeutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type of disease, severity, drug activity, drug sensitivity, It can be determined by the time of administration, route of administration and rate of excretion, duration of treatment, factors including concurrent drugs and other factors well known in the medical field. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or administered in combination with another therapeutic agent, may be administered sequentially or simultaneously with a conventional therapeutic agent, and may be administered single or multiple. It is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all the above factors, and this can be easily determined by a person skilled in the art.

The therapeutically effective amount of the pharmaceutical composition may vary depending on the patient's age, sex, condition, body weight, absorption of the active ingredient in the body, the inactivation rate and excretion rate, the type of disease, and the drug to be used in combination, and generally 1 kg in weight 0.1 to 500 mg per day may be administered daily or every other day, or divided into 1 to 5 times a day. However, since it may increase or decrease depending on the route of administration, the severity of obesity, sex, weight, age, etc., the dosage amount is not limited in any way.

Another aspect provides a method of treating an electric field for cancer comprising administering a therapeutically effective amount of the pharmaceutical composition according to one aspect while applying an alternating current electric field to an individual. In one embodiment, administering the therapeutically effective amount of the pharmaceutical composition to the subject may be performed simultaneously or sequentially with applying an alternating electric field to the subject.

The term "individual" means any mammal, including humans, who have or are likely to develop cancer.

In one embodiment, the voltage of the AC electric field may be 0.5 to 1.5 V/cm or 0.5 to 1.0 V/cm. In addition, the frequency of the AC electric field may be 100 to 300 kHz, 100 to 200 kHz, 100 to 150 kHz. The time to apply the alternating current electric field may be 1 to 72 hours, 1 to 48 hours, 1 to 36 hours, or 1 to 24 hours. In one embodiment, the time to apply the AC electric field may be 48 hours. In addition, the electric field may be continuously applied.

Among the terms or elements mentioned in the electric field treatment method for cancer, the same as those mentioned in the pharmaceutical composition is understood to be as mentioned in the preceding description.

In the electric field treatment method for cancer according to an embodiment, the suppression of autophagy by the electric field treatment and the suppression of autophagy by the pharmaceutical composition according to one aspect generate a synergistic effect with each other, resulting in a greater effect of cell death. have. Accordingly, the treatment method using the electric field treatment and the pharmaceutical composition according to one aspect can produce a more enhanced cancer treatment effect than performing the electric field treatment alone.

Each description and embodiment disclosed in the application may be applied to each other description and embodiment. That is, all combinations of various elements disclosed in the present application belong to the scope of the present application. In addition, it cannot be considered that the scope of the present application is limited by the specific description described below.

The pharmaceutical composition according to an aspect may be used as an effective material that can be used in combination with electric field therapy by further enhancing the therapeutic effect of electric field therapy in the treatment of cancer such as glioblastoma.

In addition, the electric field treatment method for cancer according to another aspect may further enhance the effect of the electric field treatment on cancer by synergistic action according to the pharmaceutical composition.

1 is a photograph of the results of western blotting measuring the effect on autophagy in glioblastoma cell lines after electric field treatment.
Figure 2a is a photograph showing the results of Western blotting measuring the expression level of Akt2 in glioblastoma cell lines after electric field treatment.
2B is a diagram showing the results of micro-assays for the expression of Akt2 and miR-29b in electric field treatment in glioblastoma cell lines after electric field treatment.
3 is a graph showing the results of a 3′UTR luciferase reporter assay performed to measure the expression level of Akt2 protein when miR-29b is administered to a glioblastoma cell line.
4A is a photograph of Western blotting results for the therapeutic effect when the electric field treatment and miR-29b are administered in combination with the glioblastoma cell line.
FIG. 4B is a graph showing cell viability measurement results and photographs of cells with respect to changes in treatment effects when electric field treatment for glioblastoma cell lines and miR-29b are administered in combination.
5 is a graph showing the weight and volume of a tumor and a photograph of the tumor in order to measure the effect of the electric field treatment on a mouse transplanted with glioblastoma.
6A is an optical micrograph taken to observe the level of Akt2 expression in order to measure the effect of electric field treatment on mice implanted with glioblastoma.
6B is a photograph showing the results of Western blotting in order to measure the effect of electric field treatment on mice implanted with glioblastoma, and to observe the level of Akt2 expression.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1. Induction of autophagy of electric field treatment in vitro

Human glioblastoma cell lines U373 and U87 were provided from the Cell Bank of Korea (Seoul, Korea), and to measure the effect on the autophagy of electric field treatment, a medium containing various growth factors necessary for growth was used to measure 37 degrees Celsius. Also, it was cultured in an incubator under 5% CO ₂ conditions. Then, an electric field was applied to the cells at 0.9 V/cm and 150 kHz for 48 hours. And the proteins separated by electrophoresis were transferred to a nitrocellulose membrane, and antibodies against p-mTOR, Akt1, mTOR, p-P70S6K, P70S6K, p-4EBP1 and 4EBP1 (Cell Signaling Technology, Danvers, MA, USA), Akt2 and Western blotting was performed using an antibody against Akt3 (Abcam, Cambridge, UK) and β-actin (Santa Cruz Biotechnology, Dallas, TX, USA).

Through this, the expression level of phosphorylated mTOR and the phosphorylation of proteins (P70S5K, 4EBP1) were examined. The results for this are shown in FIG. 1.

As shown in Figure 1, the electric field treatment reduced the degree of phosphorylation of mTOR, and accordingly, increased the degree of phosphorylation of P70S5K and 4EBP1. These results indicate that electric field therapy has the effect of inducing autophagy.

Example 2. Reduction of Akt2 expression in glioblastoma cells after electric field treatment

After performing electric field treatment (0.9 V/cm, 150 kHz) in vitro using glioblastoma cells for 24, 48, and 72 hours, changes in the protein expression levels of Akt1, Akt2, and Akt3 at each time were determined through Western blotting. Confirmed. The results are shown in FIG. 2A.

As shown in Figure 2a, it was confirmed that the electric field treatment significantly reduced the expression of Akt2 compared to other proteins. This indicates that electric field treatment decreases the expression of Akt2, inhibits autophagy and induces apoptosis, thereby necrosis of cancer cells.

2.1. In vitro electric field therapy and miR-29b regulates Akr2

Using U373 and U87 glioblastoma cell lines, electric field treatment was performed to confirm that the electric field treatment down-regulates Akt2, known as oncogene, and at the same time miR-29b (up-regulation). After implementation, microarray (Affymetrix GeneChip miRNA 4.0 Array) analysis was performed for the electric field treatment experimental group and the control group for confirmation. The results are shown in Fig. 2b.

As shown in FIG. 2B, electric field treatment was shown to down-regulate the expression of Akt2 protein known to induce autophagy, and up-regulate miR-29b, known as a factor that inhibits Akt2. This indicates that electric field therapy inhibits the induction of autophagy and induces apoptosis of cancer cells.

Example 3. Confirmation of Akt2 inhibition by miR-29b

In order to confirm whether miR-29b, known as a factor that down-regulates Akt2 in uterine cancer cell lines, has the same effect in glioblastoma cell lines, a 3'UTR luciferase reporter assay was performed. The results are shown in FIG. 3.

As shown in FIG. 3, when miR-29b was injected into cancer cells (transfection), the expression of Akt2 luciferase was decreased. This indicates that miR-29b also inhibits Akt2 in glioblastoma cell lines.

Example 4. Reduction of Akt2 expression level and cell survival rate when combined treatment with electric field treatment and miR-29b

In order to confirm the synergistic effect on the reduction of Akt2 expression level when combined treatment with electric field treatment and miR-29b in U87, U373 glioblastoma cell lines, an electric field was applied to the cells at 0.9 V/cm, 150 kHz for 48 hours, and then miR -29b (Bioneer Accession No. MI0000107) was injected. And, the amount of Akt2 protein was confirmed through Western blotting. In addition, in order to compare the efficacy of cancer cell treatment, cell viability was measured through colony forming assay and trypan blue assay. The results are shown in FIGS. 4A and 4B, respectively.

As shown in Figs. 4A and 4B, it was confirmed that the cell division of glioblastoma was significantly suppressed when the treatment in combination with miR-29b was more than the single treatment of the electric field. Through this, it can be confirmed that the treatment efficiency of cancer increases when the electric field treatment and miR-29b are combined.

Example 5. Reduction of tumor size in mouse experiments through electric field treatment

In order to examine the effect of electric field therapy on mice, human glioblastoma cells (U373, U87) were cultured, and then the glioblastoma cells were injected subcutaneously into the back of a 5-week-old male mouse Balb/c-nude. And the mice with tumors were separated into groups, and an electric field generator was connected by attaching electrodes to the tumor site. An electric field of 1 V/cm and 150 kHz was applied to the tumor of the mouse for about 12 days. After the electric field treatment was completed, the mouse was euthanized and the tumor was removed, and the weight and volume of the tumor were measured. The results are shown in FIG. 5.

As shown in FIG. 5, it can be seen that the weight of the tumor was reduced to less than half compared to the control group during the electric field treatment, and the volume of the tumor was also reduced to half. Through this, it can be seen that electric field therapy inhibits the growth of glioblastoma cells in vivo.

Example 6. Reduction of Akt2 expression in tumor tissues in vivo

After electric field treatment, in order to measure the degree of change in Akt2 expression in vivo, as in Example 5, after injecting the tumor into the subcutaneous area of the mouse, 1 V/cm from when the tumor reached about 100 mm ³ , An electric field of 150 kHz was applied for about 14 days. After the electric field treatment was completed, control and treatment mice were euthanized, tumors were excised, and the Akt2 expression level was observed through immunohistochemistry. Then, after performing PAGE (polyacrylamide gel electrophoresis) containing sodium dodecyl sulfate (SDS), Western blotting was performed to confirm the expression level of Akt2. Each result is shown in Figures 6a and 6b.

As shown in Figs. 6a and 6b, it could be directly confirmed that Akt2 expression in the treatment group receiving electric field treatment was significantly reduced compared to the control group. This is consistent with the experimental results of Example 3. In addition, it was confirmed that the Akt2 expression level in the treatment group was significantly reduced in the measurement of the Akt2 expression level by Western blotting.

Claims (11)

A pharmaceutical composition for enhancing electric field treatment for cancer comprising an autophagy inhibitor as an active ingredient.
The pharmaceutical composition of claim 1, wherein the inhibitor is one selected from the group consisting of an inhibitor of a kinase or microRNA and a combination thereof that induces autophagy.
The pharmaceutical composition of claim 2, wherein the kinase inducing autophagy comprises Akt kinase or PI3K kinase.
The pharmaceutical composition of claim 2, wherein the microRNA is miR-29b.
The pharmaceutical composition according to claim 1, wherein the electric field treatment is an alternating current electric field treatment.
The method according to claim 1, wherein the cancer is colon cancer, liver cancer, gastric cancer, colon cancer, breast cancer, lung cancer, squamous cell carcinoma, fibroma, non-small cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, melanoma in the skin or eye, uterine cancer, Ovarian cancer, rectal cancer, anal muscle cancer, fallopian tube death, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, Soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma , Spinal cord tumor, brain stem glioma, pituitary adenoma, or a pharmaceutical composition for treating one or more cancers selected from the group consisting of glioblastoma.
The pharmaceutical composition according to claim 6, wherein the cancer is glioblastoma.
An electric field treatment method for cancer comprising the step of administering a therapeutically effective amount of the pharmaceutical composition of claim 1 while applying an alternating electric field to an individual.
The method of claim 8, wherein the administering step is performed simultaneously or sequentially with an alternating electric field.
The method of claim 8, wherein the voltage of the electric field is 0.5 to 1.5 V/cm.
The method of claim 8, wherein the frequency of the electric field is 100 to 300 kHz.

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