KR20170001598A - Pharmaceutical composition for treating cancer or metabolic syndrome - Google Patents

Abstract

The present invention relates to a pharmaceutical composition comprising gracillin or a pharmaceutically acceptable salt thereof as an active component for preventing or treating cancer or a metabolic disease. The gracillin-containing composition of the present invention has almost no side effects, and has excellent effects of treating lung cancer, thereby being useful for a pharmaceutical composition and/or health food for preventing, alleviating, and treating a lung disease such as non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma of lung, or large cell carcinoma of lung. According to the present invention, provided are a novel medicine surpassing a conventional lung cancer medicine, and a therapeutic method thereof.

Description

[0001] The present invention relates to a pharmaceutical composition for treating cancer or metabolic syndrome,

The present invention relates to a pharmaceutical composition for preventing or treating cancer or metabolic diseases, which comprises gracillin or a pharmaceutically acceptable salt thereof as an active ingredient.

Lung cancer is rapidly growing in Korea due to the increase of smoking population and industrial pollution. The incidence of lung cancer is second only to gastric cancer in men, followed by uterine cancer, gastric cancer, lung cancer and colon cancer in women. And the mortality rate is the number one cancer death rate in both men and women.

The incidence of lung cancer and mortality due to lung cancer is expected to continue for a considerable period of time based on current smoking status. Lung cancer usually causes symptoms such as invasion of surrounding tissues or airway obstruction due to the growth of cancer cells, and lymph node metastasis. In addition, most of the lung cancer is diagnosed as advanced stage III (3) stage at the time of diagnosis, so it is urgent to reduce the mortality by early treatment of lung cancer because it is difficult to cure.

Currently, the chemotherapy by the administration of the anticancer drug is dependent on the administration of about 40 kinds of strong cytotoxic anticancer drugs, which causes many side effects. Therefore, it is required to develop new anticancer drugs that can reduce the adverse effects and increase the rate of amelioration. In order to solve such a problem, research has been conducted to develop an anticancer agent from natural products, especially native plants and plant herbal medicines. Taxol (paclitaxel), which is isolated from a tree of interest, is known to be a natural cancer-derived anticancer agent. However, since it acts not only on cancer cell death but also on other normal cells in the body, it has a problem that it can induce other diseases. Toxicity and side effects are serious.

mTOR (mammalian Target of Rapamycin) is a ser / thr kinase that is known to be involved in cell growth, proliferation, mobility and survival. Its activity is mainly regulated by growth factors, nutrients, energy and stress, Disease, cancer, immune response, brain disease, and the like. In particular, the activation of mTOR in cancer is associated with the mutation of several oncogenes and tumor suppressor genes located upstream. PI3KCA amplification, PTEN loss, PI3K signaling, RAS and NF1 mutation activate MAPK, respectively, and LKB1 and TP53 truncation Because it is known to inhibit AMPK and activate mTOR, it will be possible to develop multi-target anticancer drugs through mTOR inhibition.

ATP-competitive mTOR kinase inhibitors (KI) and mTOR / PI3K dual inhibitors, including rapalogs (rapamycin and its analogs), are known to directly inhibit mTOR. However, It is a problem. In addition, AMPK agonists or metabolic inhibitors which contribute to the activation of AMPK have been reported as indirect inhibitors of mTOR. However, development of anticancer drugs is limited due to low efficacy and induction of cell survival by autophagy.

In addition, drugs targeted to mitochondria as metabolic inhibitors have excellent cell killing effect but at the same time have a strong toxicity problem. Typically, rotenoid-based drugs pass the blood brain barrier (BBB) and cause a brain disease such as Parkinson's disease .

On the other hand, gracillin has been reported as a component of steroid saponin compound such as Dioscorea Rhizoma . Dioscorea batatas Decaisne or yams (Dioscorea japonica Thunberg) are removed from the root of Dioscoreaceae rootstock and dried or steamed. The main ingredient of starch is 15 ~ 20% starch and 1 ~ 1.5% (Mucin), which is a viscous glycoprotein. Other components include steroidal saponins such as dioscin and gracillin.

As described above, it has been reported that it is possible to treat intestinal dysfunction by using an extract of Ganoderma lucidum containing Gracilin (Korean Patent Registration No. 10-0811683), a therapeutic use for a single compound of Gracylin, There is no known application for the treatment of lung cancer or metabolic diseases of compounds.

The inventors of the present invention have conducted intensive studies to find a herbal medicine ingredient having excellent anticancer effect and little side effect than known anticancer drugs. As a result, it has been found that the gracillin compound derived from natural products has excellent anticancer effect in various carcinomas including lung cancer The present invention has been completed.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, which comprises gracillin or a pharmaceutically acceptable salt thereof as an active ingredient.

It is also an object of the present invention to provide a pharmaceutical composition for preventing or treating metabolic diseases, which comprises gracillin or a pharmaceutically acceptable salt thereof as an active ingredient.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to solve the above-mentioned problems, the present invention provides a pharmaceutical composition for preventing or treating cancer or metabolic diseases, which comprises, as an active ingredient, gracillin or a pharmaceutically acceptable salt thereof represented by the following formula .

[Chemical Formula 1]

The present invention also provides a method of preventing or treating cancer or metabolic diseases, comprising administering to a subject gracillin or a pharmaceutically acceptable salt thereof.

The present invention also provides a method of preventing or treating cancer or metabolic diseases of gracillin or a pharmaceutically acceptable salt thereof.

In one embodiment of the present invention, the glycylin is characterized by inducing apoptosis of cancer cells.

In another embodiment of the present invention, the glycine is characterized by inducing apoptosis by inhibiting the function of the mitochondrial complex II.

In another embodiment of the present invention, the above-mentioned gracillin induces apoptosis by binding to succinate dehydrogenase (SDHA), which is a constitutive protein of mitochondrial complex II.

In another embodiment of the present invention, the cancer is lung cancer.

In another embodiment of the present invention, the cancer is characterized by breast cancer, prostate cancer or colon cancer.

In another embodiment of the present invention, the lung cancer is characterized by non-small cell lung cancer, small cell lung cancer, lung cancer, lung squamous cell cancer or hyaline cell cancer.

In another embodiment of the present invention, the metabolic disease is hyperglycemia, hyperlipidemia, obesity, diabetes, or arteriosclerosis.

The composition containing the glycylin of the present invention has little side effects and is particularly effective for treating lung cancer. Therefore, the composition for preventing, ameliorating and treating lung cancer diseases such as non-small cell lung cancer, small cell lung cancer, lung cancer, lung squamous cell carcinoma, The present invention can provide a novel therapeutic agent and a therapeutic method that goes beyond conventional lung cancer therapeutic agents.

Further, according to the present invention, it can be usefully used for the treatment of metabolic diseases such as hyperglycemia, hyperlipidemia, obesity, diabetes, or arteriosclerosis.

In addition, according to the present invention, it is possible to anticipate a wide range of anticancer effects by effectively inhibiting mTOR and downstream signaling by activated AMPK, thereby overcoming the limit of a single target anticancer drug. Furthermore, since it binds to the succinate dehydrogenase, a constituent protein of the mitochondrial complex II, and inhibits the function of the complex II, it induces apoptosis by increasing the intracellular ROS, and exhibits apoptosis even at a low concentration of μM. And the low drug efficacy of the metabolic inhibitor can be solved.

In addition, according to the present invention, by activating AMPK, it is possible to alleviate side effects of the existing mTOR inhibitor and to prevent passage of BBB (blood brain barrier) due to low lipophilicity, Can reduce the risk.

FIG. 1 shows the result of evaluating the AMPK / mTOR modulating activity of thirteen kinds of compounds having excellent cancer cell growth inhibitory activity in the natural substance-derived substance library through Western blotting.
Fig. 2 shows the result of evaluating the activity of inhibiting ATP formation in cancer cells against the 13 kinds of compounds shown in Fig. 1 above.
FIG. 3 shows a process for finally selecting a gracillin (GRA) compound as a study target in a natural material-derived material library.
Figure 4 shows that the activation of mTOR downstream S6 (p-S6) is inhibited by GRacillin (GRA).
FIG. 5 is a Western blot result indicating that AMPK activation (p-AMPK) is induced in lung cancer cell lines by GRACIN and that mTOR and downstream signaling are inhibited.
Figure 6 shows that GRACA inhibits the formation of nonadherent colonies of lung cancer cells at a concentration range of about 1-1.5 [mu] M.
Figure 7 shows that GRacillus (GRA) inhibits the formation of adherent colonies of lung cancer cells at a concentration range of about 1-1.5 [mu] M.
Figure 8 shows that GRacillus (GRA) inhibits sphere formation of lung cancer cells at a concentration range of about 1-1.5 [mu] M.
FIG. 9 shows the result of performing cell viability tests on conventional anticancer drug resistant cell lines to confirm whether the glycylin exhibits cross-reactivity with conventional anticancer drugs.
FIG. 10 is a graph showing that the lung tumor volume is significantly reduced by treatment with gracillin (GRA) in a xenograft mouse model (H1299 xenograft / PDX) implanted with a lung cancer cell line or a patient lung cancer tissue.
Fig. 11 is a CT image showing that pulmonary tumor formation is significantly reduced by the treatment with ^gracillin (GRA) in the Kras ^{G12D / +} mouse model.
Figure 12 shows that tumor growth (T) growth is significantly inhibited by gracillin (GRA) treatment in a xenograft mouse model transplanted with a breast cancer cell line (MDA-MB-231), a prostate cancer cell line (DU145), or a colon cancer cell line (HCT116) Fig.
FIG. 13 shows the result of flow cytometry analysis to confirm the effect of gracillin (GRA) on lung cancer cell killing.
14 is a Western blot showing the effect of gracillin (GRA) on the expression of cleaved caspase-3 and cleaved PARP, which are markers of apoptosis.
FIG. 15 shows the results of DCFDA / MitoSOX / TMRM / NBDG staining showing the effect of gracillin (GRA) on cytoplasmic ROS (DCF-DA), mitochondrial ROS (MitoSOX) and mitochondrial membrane potential (TMRM).
16 (A) shows Western blotting results showing the effect of gracillin (GRA) on the activation of apoptosis mediators p38 and JNK (p-p38, p-JNK) ) Or antacid (NAC), the expression of cleaved caspase-3 and cleaved PARP was reduced.
FIG. 17 is a FACS analysis result showing that apoptosis caused by gracillin is reduced when the polysaccharide is treated in the presence of a p38 inhibitor (P38i) or an antioxidant (NAC).
FIG. 18 shows the result of MitoSOX staining showing that mitochondrial ROS (MitoSOX) increases specifically when succinate and gracaine, which are substrates of mitochondrial complex II, are treated together.
FIG. 19 shows flow cytometry results showing that the increase of subG1 phase by gracillin is the highest when the succinate and the gracillin (GRA + SUC) of the mitochondrial complex II substrate are treated together.
Fig. 20 shows in silico computational modeling analysis results indicating that gracillin stably binds to the constitutive protein SDHA (succinate dehydrogenase) of the mitochondrial complex II.
Figure 21 shows the results of a DARTS assay showing that the glycylline binds to the constitutive protein SDHA (Succinate dehydrogenase) of the mitochondrial complex II.
Figure 22 shows IHC and Western blot results indicating SDHA (Succinate dehydrogenase) expression levels in lung tissue of mice overexpressing mutated Kras (Kras ^{G12D / +} ).
23 is a ^graph showing the results of a ^comparison between mice that overexpress mutant Kras (Kras ^G12V ) or cigarette smoking condensates (CSC), NNK / benzo [a] pyrene (B [a] P) or urethane IHC results indicating the expression level of SDHA (succinate dehydrogenase) in the lung tissue of treated mice.
24 is a ^graph showing the expression level of SDHA ( ^{succinate dehydrogenase} ) in human lung cancer cells overexpressing mutated KRAS (KRAS ^G12V ) or in human-derived lung cancer cells in which KRAS is activated by growth factors such as EGF and IGF-1. Results.
FIG. 25 is a ^graph showing the increase in the stability of SDHA ( ^{succinate dehydrogenase} ) in human lung cancer cells overexpressing mutated KRAS (KRAS ^G12V ) or in human-derived lung cancer cells in which KRAS is activated by growth factors such as EGF and IGF- Blot results.
Fig. 26 shows the result of MTT assay showing that the selectivity for the KRAS / LKB1 mutant cancer is high.
Figure 27 shows the glucose uptake / lactate production assay results indicating that the potential for side effects such as glycyl lactic acidosis is low.
28 shows the results of a xenograft mouse model and a xenograft mouse model (PDX) transplanted with prostate cancer cell line (DU145), a breast cancer cell line (MDA-MB-231), a colon cancer cell line (HCT116) ; patient-derived xenografts) were measured for changes in body weight due to administration of glycine (GRA).
Figure 29 shows the evaluation of liver and kidney function, biliary duct, and systemic inflammation to evaluate long-term toxicity following administration of GRACA in mice It is a graph.
FIG. 30 is a graph showing changes in blood glucose concentration, lung, liver, spleen, kidney, and cerebral peroxidation level of mice administered with GRA (GRA).

The present invention provides a pharmaceutical composition and / or a health functional food composition for preventing, ameliorating or treating a cancer or metabolic disease containing as an active ingredient gracillin or a pharmaceutically acceptable salt thereof. In this case, the gracyline compound has a structure represented by the following formula (1).

[Chemical Formula 1]

In addition, the gracillin compound according to the present invention can be used in the form of a pharmaceutically acceptable salt. The salt is preferably an acid addition salt formed by a pharmaceutically acceptable free acid, and the free acid may be an organic acid or an inorganic acid. The organic acids include, but are not limited to, citric, acetic, lactic, tartaric, maleic, fumaric, formic, propionic, oxalic, trifluroacetic, benzoic, gluconic, methosulfonic, glycolic, succinic, Glutamic acid and aspartic acid. The inorganic acid includes, but is not limited to, hydrochloric acid, bromic acid, sulfuric acid, and phosphoric acid.

The composition of the present invention is not limited to the carcinoma that can be prevented, ameliorated or treated, but includes lung cancer, breast cancer, prostate cancer, colon cancer and the like, preferably lung cancer. At this time, there is no limitation on the kind of lung cancer disease, but it may include non-small cell lung cancer, small cell lung cancer, lung cancer, lung squamous cell cancer or hyaline cell cancer.

The metabolic diseases which can be prevented, ameliorated or treated by the composition according to the present invention are not limited, but may include hyperglycemia, hyperlipidemia, obesity, diabetes, arteriosclerosis and the like.

The therapeutic compositions of the present invention may further comprise components such as conventional therapeutically active ingredients, other adjuvants, pharmaceutically acceptable carriers, and the like. The pharmaceutically acceptable carrier includes saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and the like.

The term "individual" as used herein refers to a subject in need of treatment for a disease, and more specifically refers to a human or non-human primate, mouse, rat, dog, cat, It means mammals.

The term "pharmaceutically effective amount" as used herein refers to the type and severity of the disease to be administered, the age and sex of the patient, sensitivity to the drug, administration time, administration route and rate of release, Can be readily determined by those skilled in the art in an amount that is determined by factors well known in the medical arts, and can be maximized without adverse effects, taking into account all of the above factors.

The composition of the present invention is not limited as long as it can reach the target tissues. For example, oral administration, arterial injection, intravenous injection, percutaneous injection, intranasal administration, transbronchial administration, or intramuscular administration. The daily dose is about 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg, and is preferably administered once a day or divided into several times a day.

The composition of the present invention can be used variously for medicines, foods and beverages for prevention and improvement of cancer or metabolic diseases, and can be used in powder, granule, tablet, capsule or beverage form.

In the present invention, first, thirteen compounds having the best cell proliferation inhibitory effect were firstly selected from about 500 natural product libraries, and among them, Gracylin, which is superior in AMPK / mTOR signal transduction regulatory activity and ATP production inhibitory activity, (GRA) was finally selected as a study substance (see Example 2).

AMP-activated protein kinase (AMPK) is a cancer-like protein enzyme. MTOR plays an important role in tumor cell division, vascular growth, and cancer cell metabolism. Is known. In other words, since AMPK activated by cancer cells has been reported to inhibit the growth and proliferation of cancer cells by lowering the activity of mTOR enzyme that controls cancer cell metabolism, GRA (GRA) was selected by verifying the activity of regulating AMPK / mTOR signaling Respectively.

Then, not only determine the inhibitory effect on various cancer cell lines on the in vitro to identify specific tumor inhibitory effect of the graphene cylinder, using a Xenograft model was confirmed that an excellent cancer treatment effect even on in vivo, breast cancer, prostate cancer, And colorectal cancer (see Examples 3 and 4).

In addition, in the present invention, it was confirmed that the effect of treatment with lung cancer was caused by apoptosis by confirming that the expression of cleaced caspase-3 and cleaved PARP was induced by the glycyrin. The expression of cleaved caspase-3 and cleaved PARP indicates that apoptosis occurs because PARP is an enzyme that helps repair damaged DNA and is known to be cleaved by cleaved caspase-3 when apoptosis occurs (see Example 5 ).

Also, in the present invention, it was confirmed that the glycylin increases mitochondrial reactive oxygen species (ROS), which activates the p38 pathway and eventually induces apoptosis. Active oxygen (ROS) is a byproduct of mitochondrial respiration that produces energy, and can induce cell death depending on the amount and duration of production. Apoptosis induced by mitochondrial ROS is generally mediated by p38 or JNK (See Example 6).

Also, in the present invention, as a result of in silico computational modeling analysis and DARTS assay, it was found that gracillin binds to succinate dehydrogenase (SDHA), which is a constituent protein of mitochondrial complex II, in a very stable form to inhibit the function of complex II, RTI ID = 0.0 > ROS < / RTI > can induce apoptosis (see Example 7).

In addition, in the present invention, SDHA (Succinate dehydrogenase) was expressed at high levels in lung cancer tissues due to various causes in mice and humans, thereby confirming that there is a correlation between lung cancer and SDHA (see Example 8).

In the present invention, the possibility of neurotoxicity due to passage of the blood brain barrier (BBB) is low through analysis of physical properties of the glycine, and the risk of side effects such as lactic acidemia through glucose uptake / lactate production assays Of the total population. Generally, biguanides drugs such as phenformin lower mitochondrial function and cause cytosol glycolysis. Thus, side effects such as lactic acidosis are problematic. However, Cylinders increased glucose uptake while decreased lactate production.

In addition, it was confirmed that gracillin did not induce toxicity by evaluating weight change, long-term toxicity, and systemic inflammatory response by administration of gracillin in vivo , and it was confirmed that it did not induce changes in blood glucose concentration and peroxidation of various organs (See Example 9).

Therefore, the composition containing the glycine according to the present invention can be used as an effective therapeutic agent for cancer diseases, particularly lung cancer diseases.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[Example]

Example 1: Experimental method

1-1. MTT assay

Cells were attached to a 96-well plate at a number of 1500 cells per well, and the next day, gracillin was treated at each concentration and cultured for 3 days. After incubation, the MTT solution was treated to a final concentration of 200-500 μg / ml. After culturing for 2-4 hours, the medium was removed, and the resulting formazan was dissolved in DMSO and absorbance was measured at 570 nm.

1-2. Anchorage independent colony formation assay

Cells were diluted to a number of 1000 cells per well in 0.4% agar medium, coated on top of bottom agar (bottom agar), coated on 24% , And gracylin was treated at each concentration for about 2 weeks. The colony formed after the drug treatment was stained with 200-500 μg / ml MTT solution and the number of colonies was confirmed by microscope.

1-3. Anchorage dependent colony formation assay

Cells were plated in 12 wells at a density of 150 cells per well and treated with gracillin at the corresponding concentration for about 2 weeks from the next day. After completion of drug treatment, colony formation was observed by staining with crystal violet.

1-4. Sphere formation assay

Cells were diluted in 100 μl of the medium at a concentration of 2 × 10 ⁵ cells / ml, treated with each concentration of gracillin, and 20 μl of each was treated with dropping into the lid of a 60-mm dish. A sphere form formed about 3 days later was observed under a microscope.

1-5. Tumor xenograft model

H1299 cells were transplanted into NOD / SCID mice at about 5 × 10 ⁶ cells per spot in matrigel or transplanted subcutaneously with chopped patient-derived lung cancer tissue. Approximately 10 days after tumor size reached 150-200 mm ³ , each concentration of gracillin was administered intraperitoneally for about 2 weeks. Tumor volume was calculated by the following formula, and mouse weight was measured at regular intervals to confirm toxicity.

Tumor volume (mm ³ ) = Long axis × Short axis × Short axis / 2

1-6. MMP fluorescence-combining CT imaging

8-week-old Kras ^{G12D / +} transgenic mice were intraperitoneally administered 10 mg / kg of ^gracillin for about 2 months. After completion of the drug treatment, 150 μl of MMPsence 680 (PerkinElmer) was intravenously administered to the tail vein. After about 24 hours, fluorescence-combining CT images were obtained using the FLIT (Fluorescent Imaging topography) module of the IVIS SpectrumCT In Vivo Imaging System.

1-7. Flow cytometry

After 5 × 10 ⁵ cells were attached to a 60 mm dish, the next day, gracillin was treated with each concentration and time, and cells were removed with trypsin and fixed with 80% ethanol for one day. Cells were stained with 25 μM of PI (Propidium Iodide) for 30 min with 10 μg / ml of RNase, and the cells were stained with FACScalibur BD science).

1-8. DCF-DA / MitoSOX / TMRM staining

6, the cover slip on the well plate was attached to the peeled back, about 10 ⁵ cells. After 24 hours, each fluorescent reagent was treated for 30 minutes, and DCF-DA (ex 485 nm, em 535 nm), MitoSOX, TMRM (ex 510 nm, em 580 nm) were observed by fluorescence microscope for each wavelength.

1-9. in silico computational modeling

Using Swissdock, an online docking web server, the expected molecular interactions of mitochondrial complex II subunit SDHA and gracillin were analyzed. SDHA used a known protein structure (PDB; 1NEK), and binding models selected clusters with full fitness less than -2,500 kcal / mol. The analysis result was schematized using 'UCSF Chimera'.

1-10. Glucose uptake assay

2-NBDG is a fluorescent glucose analog used to monitor glucose uptake of living cells as a cell survival marker. To measure the intracellular glucose influx, 6 well plate to the peeled back cover slip, it was attached to about 10 ⁵ cells. After 24 hours, the glycine was treated and each fluorescent reagent was treated for 30 minutes. NBDG was observed under fluorescence microscope at ex 485 nm and em 535 nm wavelength.

1-11. Lactate production assay

After attaching 10 ⁴ cells to a 96 well plate, the medium was treated with gracillin the next day, and the lactate secreted from the cells was analyzed at 450 nm absorbance using Biovision 's lactate production kit. Quantitative values of each sample were calculated by lactate standard curve.

1-12. DARTS assay

Using the principle that when a small molecule compound binds to a target protein, the target protein is prevented from being recognized and decomposed by proteolytic enzymes, it is confirmed that the glycerin binds directly to SDHA. Gracilin (final 1 μM) or DMSO (vehicle control) was added to 20 μl of 15 μg cell lysate and incubated on ice for 30 min. After adding 1 μl of 0.05% trypsin-EDTA and incubating on ice for a certain period of time, proteolysis was induced. 5 μl of 5x sample buffer was added and the mixture was boiled at 95 ° C for 5 minutes. SDHA degradation was detected using Western blotting.

Example 2: Selection of a Graciline Compound

2-1. Primary selection of compounds

First, thirteen compounds with the highest inhibitory activity against cancer cell proliferation were firstly selected from about 500 natural substance-derived substance libraries, and the AMPK / mTOR modulating activity of these compounds was evaluated by Western blotting.

As a result, as shown in Fig. 1, the expression of the phosphorylation form (pAMPK) of AMPK markedly increased and the phosphorylation form (pmTOR) of mTOR was remarkably decreased in the case of the compound No. 13, Gracillin (GRA) The most effective inhibition of < RTI ID = 0.0 >

Furthermore, ATP production inhibitory activities of the above 13 candidate compounds having the best cancer cell proliferation inhibitory activity were evaluated. As a result, as shown in Fig. 2, it was confirmed that the generation of ATP was most remarkably reduced in the case of the compound No. 13, i.e., glycylin.

2-2. Final compound selection

Based on the results of Example 2-1, gracillin (GRA) having excellent AMPK / mTOR regulatory activity and ATP production inhibitory activity was finally selected as a study substance (see FIG. 3).

Specifically, it was confirmed that the activation (p-S6) of S6 downstream of mTOR in the RTK (Receptor Tyrosine Kinase) array kit was inhibited by GRA treatment compared to the control (CT) (see FIG. 4).

Western blot analysis revealed that AMPK activation (p-AMPK) was induced in lung cancer cell line (H460, A549) within 1-6 hours after treatment with GRA, and sequential inhibition of mTOR and downstream signaling was observed 5).

Example 3: in vitro Of tumor growth inhibition

Since gracillin was finally selected as an anticancer agent candidate in Example 2, it was first confirmed in vitro to inhibit the proliferation of an actual lung cancer cell line.

3-1. MTT assay

First, MTT assay was performed according to the method of Example 1-1. As shown in the following Table 1, it was found that the growth of lung cancer cells was suppressed at an IC _{50 of} about 5 μM. In particular, the inhibitory activity of glycine on cell proliferation of KRAS or LKB1 mutant cell line (H460) was significantly increased.

[Table 1]

3-2. Colony formation assays

As a result of performing anchorage independent colony formation assay and anchorage dependent colony formation assay according to the methods of Examples 1-2 and 1-3, 7, respectively, it was found that the gracillin inhibits the formation of nonadherent / adherent colonies of lung cancer cells at a level of about 1-1.5 [mu] M.

In addition, sphere formation assay was performed according to the method of Example 1-4, and as shown in FIG. 8, the glycine inhibited sphere formation of lung cancer cells at about 1-1.5 [mu] M level .

3-3. Cross-reactivity analysis

Cell survival experiments were performed on anticancer drug resistant cell lines in order to confirm whether the glycerin cross - reacts with conventional anticancer drugs. As a result, as shown in Fig. 9, paclitaxel-resistant lung cancer cell lines H226B / R, SK-MES / R, and geftinib / erlotinib resistant lung cancer cell lines PC9 / / ER showed better ability to inhibit cell proliferation than non-resistant cell lines.

Thus, it can be seen that these anticancer drug and gracillin show little cross-reactivity.

3-4. Analysis of various carcinomas

In addition, as shown in Table 2 below, it was confirmed that gracillin inhibits the proliferation of lung cancer cells in Example 3-1. In addition, as shown in Table 2, And 5 [mu] M, respectively.

[Table 2]

Example 4: in vivo Of tumor growth inhibition

Since in Example 3, the inhibition of the proliferation of lung cancer cell lines was confirmed only in vitro , the in vivo experiment was carried out in the following manner.

4-1. Xenograft model

For each of the xenograft mouse model (H1299 xenograft) transplanted with the H1299 lung cancer cell line prepared according to the method of Example 1-5 and the xenograft mouse model (PDX; patient-derived xenograft) transplanted with the patient-derived lung cancer tissue, 20 mg / kg was administered intraperitoneally once a day. As a result, as shown in Fig. 10, tumor formation was significantly reduced within about two to three weeks in both models.

4-2. MMP fluorescence-combining CT imaging

In the Kras ^{G12D / +} transgenic mouse model, 10 mg / kg of ^{gracine was} intraperitoneally administered for 8 weeks. As shown in FIG. 11, it was confirmed that the formation of lung tumors was effectively reduced.

4-3. Analysis of various carcinomas

MDA-MB-231, a breast cancer cell line, DU145, a prostate cancer cell line, and HCT116, a colon cancer cell line, were prepared in the same manner as in Example 1-3, and then each xenograft mouse model was prepared. 20 mg / kg of gracaine was intraperitoneally administered once a day and tumor size changes were measured for 21 to 25 days. As a result, as shown in Fig. 12, it was confirmed that the formation of each of the above breast cancer, prostate cancer, and colorectal cancer tumors was significantly reduced within 2 to 3 weeks.

Example 5: Confirmation of induction of apoptosis by gracillin

5-1. Flowcytometry

As a result of flow cytometry analysis according to the method of Example 1-7, as shown in Fig. 13A, the generation of subG1 phase (cell death) due to the gracillin was increased depending on the concentration (1-10 [mu] M) As shown in Fig. 13B, it was found that the cell death due to the gracillin occurred very rapidly within 24 hours.

In addition, as shown in Fig. 13C, when caspase inhibitor (CASi) was treated with both of glycylin and caspase inhibitor, apoptosis by gracillin was decreased.

5-2. Western blot

Western blot analysis showed that cleaved form of caspase-3 and PARP (poly- (ADP-ribose) polymerase) was observed by gracillin as shown in Fig. 14A, inhibitor (CASi) were treated in the same manner. As a result, as shown in Fig. 14B, the truncated form was remarkably reduced.

Therefore, the above results suggest that apoptosis is mainly caused by apoptosis.

Example 6: Confirmation of Mitochondrial Inhibitory Activity by Gracilin

Through the above examples, it has been confirmed that since the glycine activates AMPK within 1-6 hours and induces apoptosis within 12-24 hours, it is possible to use the following method under the assumption that the gracillin drug action point can be present in the mitochondria The mitochondrial inhibitory activity by gracillin was observed.

6-1. DCF-DA / MitoSOX / TMRM staining

Since reactive oxygen species (ROS) is known to be mainly produced as a byproduct of mitochondrial respiration that produces energy and can induce apoptosis according to the amount and duration of production, DCF-DA / MitoSOX / TMRM staining was performed. As a result, as shown in Fig. 15, gracillin increased cytoplasmic ROS (DCF-DA), mitochondrial ROS (MitoSOX) and lowered mitochondrial membrane potential (TMRM) simultaneously.

6-2. Western blot

Since apoptosis induced by mitochondrial ROS is generally known to be mediated by p38 or JNK, Western blotting was performed on lung cancer cell lines (H460, A549) to confirm the phosphorylation of these proteins after treatment with glycine. As a result, as shown in Fig. 16A, the expression of phosphorylated p38 and JNK (p-p38, p-JNK) was observed.

In addition, as shown in Fig. 16B, the expression of cleaved caspase-3 and cleaved PARP was reduced in the presence of p38 inhibitor (P38i) or antacid (NAC) in the presence of NAC, It was confirmed that apoptosis was decreased.

6-3. FACS analysis

As a result of FACS analysis, as shown in Fig. 17, it was confirmed that when glycine was treated in the presence of p38 inhibitor (P38i) or antioxidant (NAC), apoptosis caused by the glycine was reduced, Which is consistent with the Western blot results.

This result implies that the mitochondrial ROS produced by the glycine activates the p38 pathway and ultimately induces apoptosis.

Example 7 Confirmation of Mitochondrial Complex II Inhibitory Activity by Gracilin

Since it was confirmed through the above Example 6 that the glycylin induces apoptosis by increasing the mitochondrial ROS (MitoSOX), the following experiment was further carried out to specifically confirm the generation mechanism of the mitochondrial ROS.

7-1. MitoSOX staining

The succinate and gracaine, which are substrates of the complex II among the four electron transporting enzyme complexes constituting the mitochondrial electron transport system, were treated together and then subjected to MitoSOX staining according to the method of Example 1-8. As a result, , Mitochondrial ROS (MitoSOX) was observed specifically, while mitochondrial ROS (MitoSOX) was not observed at the same time in glutamate / malate treatment condition, which is a substrate of mitochondrial complex I.

7-2. Flowcytometry

As shown in Fig. 19, flow cytometry analysis showed that the increase of subG1 phase by gracillin was highest at 60 to 70%, especially in the presence of succinate (GRA + SUC).

7-3. in silico computational modeling

As shown in FIG. 20, in-silico computational modeling analysis according to the method of Example 1-9 showed that gracillin bound to SDHA (succinate dehydrogenase (succinate dehydrogenase)), which is a constituent protein of mitochondrial complex II, In particular, it was found that there is a high possibility that the binding site of the glycine is present in the SDHAF2 binding site involved in the flavinylation of SDHA.

These results imply that the glycylin inhibits the function of the mitochondrial complex II and that the ROS produced thereby can induce apoptosis.

7-4. DARTS assay

In addition to the in silico computational modeling results of Example 7-3 above, the results of the DARTS assays again confirmed that the glycylin binds to SDHA. As shown in FIG. 21, in the control group treated with DMSO, The expression of SDHA was degraded and the expression of SDHA was inhibited. In the case of treatment with glycine, the degradation of SDHA by trypsin was inhibited, and the expression of SDHA was maintained, indicating that the glycine was bound to SDHA.

Example 8: Confirmation of association between SDHA and lung cancer

As a result of the above Example 7, it was confirmed that the complex of ROS produced by the binding of GRACIL to the SDHA, which is a constitutive protein of the mitochondrial complex II, inhibited the apoptosis of the cell, And SDHA expression in lung cancer.

8-1. Expression of SDHA in mouse lung cancer tissue

First, the expression of SDHA in lung tissues of mice overexpressing mutated Kras (Kras ^{G12D / +} ), one of the genetic mutations occurring in lung cancer, was confirmed by IHC and Western blotting. As a result, as shown in FIG. 22, it was observed that SDHA was expressed in the lung tissue of the transgenic mouse through IHC, and that the expression of SDHA protein in the lung tissue of the mouse compared to the normal control (WT) Was found to be highly expressed.

In addition, a tumor tissue produced by transplanting human lung cancer cells overexpressing another mutant KRAS (Kras ^G12V ), or a cigarette smoking condensate (CSC), a cause of cancer, NNK / benzo [a] pyrene ] P) or urethane in the lung tissue of the mice. As a result of IHC, as shown in FIG. 23, it was found that each mouse lung tissue expresses SDHA at a higher level than the normal control group.

8-2. Expression of SDHA in Human Lung Cancer Tissue

In addition to the above results, the expression level of SDHA in lung cancer cells overexpressing mutant KRAS (KrasG12V) or lung cancer cells derived from human-induced lung cancer cells by KRAS-activated growth factors such as EGF and IGF-1 was evaluated by Western blot Respectively. As a result, as shown in FIG. 24, the expression of SDHA was significantly increased in lung cancer tissues overexpressing mutated KRAS as compared with the normal control (EV). In the case of cancer cells overexpressing EGF or IGF-1 The expression of SDHA was also increased.

Further, as a result of observing the change of expression of SDHA with time in the above lung cancer tissues, as shown in Fig. 25A, in the lung cancer tissue overexpressing mutated KRAS as compared with the normal control group, the expression of SDHA was maintained Whereas, when normal KRAS was overexpressed, the expression of SDHA again decreased over time.

In addition, as shown in Fig. 25B, when KRAS was activated by treating EGF in lung cells of a normal control (EV), expression of SDHA was increased as in the case of KRAS mutation, and the expression level was continuously maintained .

These results indicate that SDHA is highly expressed in lung cancer tissues or cells induced by various causes and the stability is increased.

Example 9: Verification of Side Effects of Gracilin

9-1. Properties of Gracylin

As shown in the following Table 1, since the content of clogP, which indicates the drug permeability of the drug, is less than 2, the molecular weight is more than 400, and the polar surface area is more than 90, the blood brain barrier of drugs such as rotenoids (mitochondrial complex I inhibitor) The potential for neurotoxicity through the blood brain barrier (BBB) is expected to be low.

[Table 3]

9-2. MTT assay

As shown in Fig. 26, the growth inhibitory activity of glycine was much better than phenformin, which is a biguanide drug, in KR4 / mutant cancer cell line H460 of KRKB / LKB1, Suggesting a high selectivity for cancer.

9-3. glucose uptake / lactate production assay

In general, biguanides drugs induce cytosol glycolysis through diminished mitochondrial function, and side effects such as lactic acidosis are known.

However, as shown in FIG. 27, it is expected that the risk of side effects such as lactic acidosis may be lowered because the glycine increases glucose uptake (A in FIG. 27) but rather reduces lactate production (FIG. 27B) do.

9-4. in vivo Of Toxicity by Gracillin

In order to examine the physical toxicity by gracillin administration in vivo , xenograft mouse model, which was prepared by transplanting prostate cancer cell line DU145, breast cancer cell line MDA-MB-231, colon cancer cell line HCT116, and patient- We compared the body weight changes of grafted xenograft mouse models (PDXs) with tissue-derived xenografts.

As a result, as shown in Fig. 28, it was confirmed that there was no difference in body weight between the control group in which the glycilin was not administered and the mouse model in which the glycylin was administered. These results indicate that the anticancer effect of glycyrrhizin does not show toxicity.

In addition, long-term toxicity by the administration of glycerin was verified. As a result, as shown in FIG. 29, there was no change in liver function and kidney function and biliary duct function, and no systemic inflammatory reaction was observed. As a result, Was not observed.

Furthermore, as shown in Fig. 30, the change in blood glucose level was observed in the case of administration of glycine, compared with the control group in which the glycine was not administered. As shown in Fig. 30, , Spleen, kidney, and brain.

From the above results, it was found that the toxicity due to the administration of the glycine was not observed in the mouse.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention. There will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (8)

A pharmaceutical composition for preventing or treating cancer or metabolic diseases, which comprises, as an active ingredient, gracillin represented by the following formula (1) or a pharmaceutically acceptable salt thereof.
[Chemical Formula 1]

The pharmaceutical composition according to claim 1, wherein the glycine induces apoptosis of cancer cells.
The pharmaceutical composition according to claim 2, wherein the glycine induces apoptosis by inhibiting the function of mitochondrial complex II.
[Claim 5] The pharmaceutical composition according to claim 3, wherein the gracillin induces apoptosis by binding to succinate dehydrogenase (SDHA), which is a constitutive protein of mitochondrial complex II.
2. The pharmaceutical composition according to claim 1, wherein the cancer is lung cancer.
The pharmaceutical composition according to claim 1, wherein the cancer is breast cancer, prostate cancer or colon cancer.
[Claim 7] The pharmaceutical composition according to claim 5, wherein the lung cancer is non-small cell lung cancer, small cell lung cancer, lung cancer, lung squamous cell cancer or hyaline cell cancer.
The pharmaceutical composition according to claim 1, wherein the metabolic disease is hyperglycemia, hyperlipidemia, obesity, diabetes or arteriosclerosis.

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