KR101831779B1 - Composition for protecting radiation comprising nisoldipine - Google Patents

Abstract

The present invention relates to a composition for protection against radiation containing nisoldipine as an active ingredient, wherein nisoldipine can inhibit the expression of insulin-forming growth factor binding protein (IGFBP5), p53, and p21, which are increased by radiation in vascular endothelial cells. In addition, nisoldipine can inhibit increased cell senescence, gene damage and calcium absorption by radiation, and can inhibit intestinal damage by radiation. Therefore, according to the present invention, the composition containing nisoldipine as an active ingredient having the above-mentioned effect can be used to treat patients with radiation exposure by protecting cells from radiation damage in various cells including vascular endothelial cells or by promoting cell regeneration after the damage.

Description

[0001] The present invention relates to a composition for protecting a radiation containing a nisol dipine as an active ingredient,

The present invention relates to a composition for radioprotectants containing nisoldipine as an active ingredient.

Ionizing radiation (IR) was first used by Helsper in 1967 for cancer treatment purposes. At present, radiation and radiation isotopes are used in various fields such as diagnosis and treatment, aerospace, semiconductor, biotechnology and food production as well as various industrial uses, and are closely related to our lives. Particularly, in Korea, a large part of electric power production is dependent on nuclear power generation, and small-sized radiation irradiation devices are used in the industrial field for diagnosis of non-destructive structure of buildings. Accordingly, . This increase in the use of radiation, which is closely related to life, is also increasing human exposure.

In addition, there are increasing incidents of radiation exposure unintentionally, such as the accident of heavy water leakage at Wolsong nuclear power plant in 1999 or the accident at a uranium factory in Tokai-mura, Japan. Radiation exposure causes DNA repair, cell cycle abnormality, cell death leading to apoptosis and chromosomal aberrations in peripheral blood lymphocytes (PBL), tissue and organ damage, malformations and cancer It is known. However, there has not yet been developed a method for monitoring the human body effect caused by radiation exposure or for quickly estimating the radiation obstacle in case of unexpected accident such as nuclear accident.

If patients are exposed to radiation, accurate and accurate identification of the radiation dose received by the patient is essential to determine the patient's treatment plan and to determine the prognosis. In addition, confirming the radiation dose will play an important role in predicting future chronic effects of radiation and in identifying the cause of the disease that may be caused by radiation exposure.

Although the dose range can be clinically estimated using the patient's symptoms and signs after radiation exposure, the more objective and accurate method is to use a physiological method to calculate the radiation dose by using a losing dosimeter or reconstructing the exposure situation, A biological method has been used to estimate the radiation dose by measuring changes in the body induced by the human body. However, it is necessary to develop a damage regulator or exposure biomarker that can diagnose radiation exposure more conveniently and accurately.

In recent years, there has been a growing interest in radioprotective agents for protecting the body from radiation exposure. The synthesis and activity of various derivatives of sulfhydryl group compounds, which are highly effective in eliminating reactive oxygen species, have been investigated from the 1950s to the 1980s, but the oral administration effect is insufficient and toxicity is high and the half-life of the system is too short It is not widely used. Since then, protease inhibitors, vitamins, metallo-elements and calcium antagonists have emerged. In the 1990s, attention was paid to internal defense systems. Granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF) We investigated the effects of various cytokines, polysaccharides and prostaglandins (PG) such as TNF-α, IL-6 and stem cell factor (SCF) Are increasingly being studied. However, synthetic materials are highly toxic and, in the case of an immuno-hematopoietic agent, not only have many side effects, but also are expensive and practically limited in their use.

Nisoldipine is a calcium channel blocker known as a therapeutic agent for essential hypertension and stable angina. The in vivo half-life of nisol dipine is about 7 to 12 hours. If necessary, take 5 mg twice a day, or 20 mg once a day for patients with chronic stable angina pectoris. The effect of NISOLDIPIN as a therapeutic agent for hypertension and angina pectoris has been successfully demonstrated in many studies, but the effect of NISOLDIPIN as a composition for radioprotectant has not been disclosed to date.

Korean Patent No. 10-1358810 (Apr. 28, 2014)

It is an object of the present invention to provide a composition for radioprotectants containing nisol dipine as an active ingredient.

Another object of the present invention is to provide a composition for preventing or treating radiation damage or radiation damage caused by irradiation containing nisole dipine as an active ingredient.

It is still another object of the present invention to provide a pharmaceutical composition for reducing an anticancer drug-induced toxicity, which comprises nisol dipine as an active ingredient.

It is still another object of the present invention to provide a pharmaceutical composition for an anticancer agent, which comprises the nisol dipine and the anticancer agent as an active ingredient, and the nisol dipine reduces the anticancer drug-induced toxicity.

In order to accomplish the above object, the present invention provides a composition for radioprotectants containing nisol dipine as an active ingredient.

In addition, the present invention provides a composition for preventing or treating cancer damage by radiation exposure or irradiation containing nisol dipine as an active ingredient.

The present invention also provides a pharmaceutical composition for reducing an anticancer drug-induced toxicity comprising nisol dipine as an active ingredient.

Still another object of the present invention is to provide a pharmaceutical composition for an anticancer agent, which comprises nisol dipine and an anticancer agent as an active ingredient, wherein the nisol dipine reduces anticancer drug-induced toxicity.

The present invention relates to a radioprotectant composition containing nisol dipine as an active ingredient, wherein the nisol dipine inhibits the expression of insulin-forming growth factor binding protein (IGFBP5), p53 and p21 increased by radiation in vascular endothelial cells have. In addition, NISOL DIPHIN can inhibit increased cell senescence, gene damage and calcium absorption by radiation, and can inhibit intestinal damage by radiation.

Accordingly, the composition of the present invention containing the nisol dipine having the above-mentioned effect as an active ingredient can be used for the treatment of radiation exposure patients by protecting cells from radiation damage in various cells including vascular endothelial cells or promoting cell regeneration after damage .

Figure 1 shows a map of the pDrive5Lucia vector.
2A shows a method of screening a substance or drug that inhibits the expression of IGFBP5 by irradiating HAEC, an arterial vascular endothelial cell, with a reporter vector containing an IGFBP5 gene promoter .
FIG. 2B is a graph showing the inhibitory effect of IGFBP5 on the expression of simvastatin or nisol dipin selected in FIG. 2A after treatment with HAEC, an arterial vascular endothelial cell, and irradiation with radiation.
Figures 2C and 2D show the effect of inhibition of the expression of IGFBP5 and p53 protein by gene toxicity (radiation or doxorubicin) by nisol dipine in Western blot and polymerase chain reaction.
3A shows the effect of suppressing the expression of p53 phosphorylation and p21 protein by nisol dipine by western blot, which is increased by gene toxicity (radiation or doxorubicin).
FIG. 3B shows the MTT assay showing the effect of increasing the cell survival rate reduced by the toxicity of the toxin (radiation or doxorubicin) by nisol dipine.
FIG. 3C shows the effect of inhibiting cell senescence induced by gene toxicity (radiation or doxorubicin) by nisol dipine by staining SA-beta-gal, a cell senescence marker.
FIG. 4A shows the effect of decreasing angiogenesis and blood vessel length by nisol dipine by genotoxicity (radiation or doxorubicin) by an angiogenic ability assay.
FIG. 4B shows the effect of suppressing the increase of gene damage caused by irradiation with nisol dipine by staining with γ-H2AX, a gene damage marker.
FIG. 4C shows the effect of suppressing the increase of calcium intake by gene toxicity (radiation or doxorubicin) by nisol dipine by calcium infiltration assay.
FIG. 5A is a graph showing an effect of suppressing the increase in calcium absorption by irradiation with nisol dipine and another calcium channel inhibitor telenzepine by calcium infiltration assay.
Figure 5B shows the effect of telenegraft on cell viability reduced by gene toxicity (radiation or doxorubicin) by MTT assay.
Figure 5C shows the effect of telenegraft on cell senescence increased by genotoxicity (radiation or doxorubicin) by staining SA-beta-gal, a cell senescence marker.
FIG. 6A shows the effect of increasing radiation-induced mouse survival rate by nisol dipine. FIG.
Fig. 6B shows the effect of nisol dipine on changes in mouse body weight by radiation.
FIG. 7A shows the effect of increasing the intestinal blood vessel reduced by radiation by nisol dipine by fluorescence staining with CD31, a blood vessel marker.
FIG. 7B shows the effect of suppressing the apoptosis of mouse intestinal tissue induced by radiation by nisol dipine by staining with a TUNEL assay.
FIG. 8A shows the effect of inhibiting or increasing the killing and radiation sensitivity of cancer cells, which are increased by radiation, by nisol dipine by clonogenic assay.
FIG. 8B shows the effect of suppressing or increasing the radiation sensitivity of cancer cells, which are increased by radiation in an animal model, by nisol dipine by measuring tumor size after xenogeneic xenotransplantation.

The inventors of the present invention have confirmed that NISOL DIPHINE inhibits the expression of insulin-forming growth factor binding protein (IGFBP5), p53 and p21 increased by radiation, inhibits cell senescence, gene damage and calcium absorption, The present inventors confirmed that Nissol dipin increases mouse survival rate and inhibits intestinal damage by radiation.

As used herein, the term " prophylactic " means any action that inhibits radiation damage or delays damage by nisol dipine.

As used herein, the term " treatment " refers to any action that alleviates or alleviates the symptoms of radiation damage by the nisol dipine.

The term " nisoldipine " as used herein is a calcium channel blocker known as a therapeutic agent for essential hypertension and stable angina.

The term " insulin-like growth factor binding protein (IGFBP5) " used in the present invention is known to play an important role in various cell functions including cell proliferation. IGFBP5 has a nuclear localization signal (NLS), which is located in the nucleus, and the function of IGFBP5 secreted out of the cell and the function of the secreted protein and nucleus in the nucleus is not yet well studied. However, in a recent paper it has been reported that the function of IGFBP5 in the nucleus has a function of transactivation. IGFBP5 is composed of three regions (N-terminus, L-domain, and C-terminus), with the IGF binding site at the N-terminus and NLS at the C-terminus. The L-domain and C-terminus also have heparin binding sites. Although glycosylation and phosphorylation of IGFBP5 are known to inhibit heparin binding of IGFBP5, the precise biological significance of glycation and phosphorylation has not yet been elucidated.

As used herein, the term " radiation irradiation " refers to exposure to the action of electron beam radiation or alpha or beta particles, including, for example, X-rays, ultraviolet rays, alpha particles and gamma rays. Radiation can affect both cancer cells and normal cells, but using a variety of methods and techniques, cancer cells are destroyed by treating cancer with fewer effects on normal cells. It does not kill cancer cells immediately, but destroys the ability of cancer cells to divide and multiply, preventing new cancer cells from dividing and producing, and the cancer cells that no longer divide will die out at the end of their lives. Each treatment kills more cells, dead cells are degraded and transported to the blood, drained out of the body, and the size of the tumor is reduced. Most of the normal cells are recovered, but some normal cells are not recovered, resulting in side effects of radiation therapy. Changes in cells and tissues that can be caused by side effects include changes in cell division timing, cell death and necrosis, tissue damage, and immune function.

As used herein, the term " Gray (Gy) " refers to a dose of radiation that can release one line (Joule) in 1 kg of biological tissue.

The present invention provides a pharmaceutical composition for radioprotectants containing nisol dipine as an active ingredient.

Preferably, the nisol dipine can inhibit the expression of radiation-induced insulin-like growth factor binding protein (IGFBP5), p53 and p21.

Preferably, the nisol dipine can inhibit increased cellular aging, gene damage and calcium uptake by radiation.

Preferably, the Nisodipine can inhibit increased intestinal damage by radiation.

Preferably, the pharmaceutical composition is a lubricant, a release control agent, a surfactant, an enhancer, a polymer, a carrier, a diluent, a filler, a weight agent, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, ≪ / RTI > and a binding agent.

In addition, the present invention provides a pharmaceutical composition for preventing or treating cell damage by radiation exposure or irradiation comprising nisol dipine as an active ingredient.

The present invention also provides a pharmaceutical composition for reducing an anticancer drug-induced toxicity comprising nisol dipine as an active ingredient.

Preferably, the anticancer drug-induced toxicity may be, but is not limited to, genetic damage.

Preferably, the anticancer agent may be selected from the group consisting of doxorubicin, cisplatin and paclitaxel, but is not limited thereto.

In addition, the present invention provides a pharmaceutical composition for an anticancer agent, which comprises nisol dipine and an anticancer agent as an active ingredient, wherein the nisol dipine reduces anticancer drug-induced toxicity.

When the composition of the present invention is a pharmaceutical composition, it may contain a pharmaceutically acceptable carrier in addition to the above-mentioned active ingredients. Such pharmaceutically acceptable carriers are those conventionally used in pharmaceutical preparations, and include lactose, dextrose, The present invention relates to a process for the preparation of a medicament for the treatment and / or prophylaxis of cancer, comprising administering a therapeutically effective amount of a compound selected from the group consisting of cross, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, Carboxymethylcellulose, carboxymethylcellulose, hydroxybenzoate, talc, magnesium stearate, mineral oil, and the like. The pharmaceutical composition may further contain a filler, a weight agent, a binder, a lubricant, a wetting agent, a disintegrant, a sweetener, a flavoring agent, an emulsifying agent, a suspending agent, a fragrance, a preservative and the like as an additive and an auxiliary agent.

The pharmaceutical composition may be formulated into tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, nonaqueous solutions, emulsions, lyophilized preparations, suppositories and sterile injectable solutions.

The pharmaceutical composition may be administered by intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, subcutaneous, intradermal, intranasal, intrapulmonary, intraocular, intramuscular, Rectally, topically, orally, and by inhalation.

An effective amount of the active ingredient of the above pharmaceutical composition means an amount required for prevention or treatment of the disease. Accordingly, the present invention is not limited to the particular type of the disease, the severity of the disease, the kind and amount of the active ingredient and other ingredients contained in the composition, the type of formulation and the patient's age, body weight, general health status, sex and diet, But are not limited to, various factors, including, for example, the rate of administration, the rate of administration, the duration of the treatment, the drugs used concurrently.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example  One : IGFBP5  Discovery of the expression inhibitor

1. Cell culture and irradiation

Human aortic endothelial cells (HAEC) and human brain microvascular endothelial cells (HBMVEC) were cultured in EBM-2 medium supplemented with 2% FBS and various growth factors at 37 ° C , And incubated in a 5% CO ₂ incubator. Radiation was irradiated with gamma rays and 137Cs gamma-ray source (Atomic Energy of Canada, Ltd., Canada) was used. The cells growing at an irradiation intensity of 2.82 Gy / min were irradiated with gamma rays of 4 Gy (1 min 25 sec) and used in the experiment.

2. IGFBP5  Promoter vectors and vectors using them IGFBP5  Selection of expression inhibitor

Of the IGFBP5 gene, the region from -2086 bp to + 142 bp was amplified by PCR and cloned into pDrive5 Lucia vector (InvivoGen, USA) using SdaI and NcoI restriction enzymes. The vector was introduced into arterial vascular endothelial cells, HAEC, and vascular endothelial cells without a vector were removed with zeocin. Cells with the vector introduced into a 96-well plate were dispensed in 3000 wells per well, treated with a drug library (10 μM), and irradiated one hour later. Twenty-four hours later, 20 ul of the supernatant was taken from each well, and each supernatant was added to a new 96-well plate to which 200 ul of QAUNTI-Blue (InvivoGen, USA) reagent was added and reacted at 37 DEG C for 30 minutes. Thereafter, the absorbance at 630 nm was measured in a microplate reader.

FIG. 1 shows a map of the pDrive5Lucia vector. FIG. 2A shows the result of irradiating HAEC with a reporter vector containing an IGFBP5 gene promoter, and then selecting a substance or a drug that inhibits the expression of IGFBP5 . As a result, simvastatin and nisoldipine selected as inhibitors of IGFBP5 expression were treated with HAEC, an arterial vascular endothelial cell, and irradiated with radiation. As a result, referring to Fig. 2B, simvastatin and nisol dipine showed a concentration- Lt; / RTI >

3. Nissol Dipin  by IGFBP5  And p53 protein expression analysis Western Blat )

HAEC, an arterial vascular endothelial cell, was treated with 10 μmol / ml of nisol dipine and treated with 4 Gy of radiation or 1 μg / ml doxorubicin after one hour and then cultured for 48 hours. Then, proteins were extracted from the cells using RIPA solution, which is a cell lysis reagent, and quantitated. Proteins of the same amount were separated by SDS (sodium dodecyl sulfate) -PAGE (polyacrylamide gel electrophoresis), transferred to a nitrocellulose membrane, and subjected to Western blotting to analyze protein expression levels of IGFBP5 and p53.

As a result, referring to FIG. 2C, it was confirmed that the treatment of radiation or doxorubicin markedly increased the expression of IGFBP5 and p53 protein, and the pretreatment of Nisol dipine inhibited the expression of IGFBP5 and p53 protein.

4. Nissol Dipin  by IGFBP5  And p53 gene expression analysis ( Reverse transcription  Polymerase chain reaction)

HAEC, an arterial vascular endothelial cell, was treated with 10 μmol / ml of nisol dipine and treated with 4 Gy of radiation or 1 μg / ml of doxorubicin for one hour and then cultured for 48 hours. 1 ml of trizol (Trizol, Invitrogen, Calif., USA) was added to the plate, and then 0.2 ml of chloroform was added to extract the RNA. The sample was shaken for 15 to 20 seconds, allowed to stand at room temperature for 2 to 3 minutes, treated with cold isopropanol to separate the aqueous layer, and washed with cold 70% ethanol. The RNA pellet was air dried and resuspended in diethyl pyrocarbonate (DEPC) -treated water and analyzed for agarose gel analysis and spectrophotometry quantitation using a small amount of sample Respectively.

Using gene expression was quantified RNA, RT-PCR kit ^{(One Step SYBR PrimeScript TM RT-} PCR Kit, takara, Osaka, Japan) and the PCR machine (DNA Engine Opticon 2 system, MJ Reserch, Massachusetts, United States) Reverse transcription-polymerase chain reaction (RT-PCR) was performed. Expression levels of the genes were normalized by GAPDH analysis.

As a result, referring to FIG. 2D, it was confirmed that treatment with radiation or doxorubicin increased the expression of IGFBP5 and p53 genes, and pretreatment of Nisoldipine inhibited the expression of IGFBP5 and p53 genes.

Example  2: Inhibitory effect of vascular endothelial cells

One. Nissol Dipin  Of p53 phosphorylation and p21 protein expression

Nasal dipin was treated with 10 μmol / ml of HAEC in the arterial vascular endothelial cells and HBMVEC in the brain capillary endothelial cells. After one hour, the cells were treated with 4 Gy of radiation or 1 μg / ml of doxorubicin and then cultured for 48 hours. Then, proteins were extracted from the cells using RIPA solution, which is a cell lysis reagent, and quantitated. Proteins of the same amount were separated by molecular weight through SDS-PAGE, transferred to a nitrocellulose membrane, and subjected to Western blotting to analyze p53 phosphorylation and p21 protein expression.

As a result, referring to FIG. 3A, it was confirmed that treatment with radiation or doxorubicin increased phosphorylation of p53 and protein expression of p21, and pretreatment of nisol dipine inhibited phosphorylation of p53 and protein expression of p21 .

It is known that the phosphorylation of p53 and the increase of p21 protein expression due to the gene damage reaction stops the cell division cycle in the interphase and progresses to cell senescence.

2. Cell survival analysis

Nasal dipin was treated with 10 μmol / ml of HAEC in the arterial endothelial cells and HBMVEC in the brain capillary endothelial cells. After one hour, the cells were treated with 4 Gy of radiation or 1 μg / ml of doxorubicin and then cultured for 48 hours. After that, trypsin was treated to measure the number of living cells to analyze cell growth or cell viability.

As a result, referring to FIG. 3B, it was confirmed that treatment of radiation or doxorubicin reduced cell survival rate, and pretreatment of nicol dipine increased cell survival rate.

3. Cell aging Marker (SA-beta-gal) staining

The vascular endothelial cells were dispensed on a 35 mm plate at 4 × 10 ⁴ and treated with 4 Gy of radiation or 1 μg / ml of doxorubicin, followed by treatment with Nisodipine and incubation for 2 to 4 days. The plates were then washed and the cells fixed with 3.7% (v / v) paraformaldehyde solution. To stain SA-beta-gal, a cell senescence marker, 150 mM sodium chloride, 2 mM magnesium chloride, and 5 mM sodium chloride were added to 40 mM citrate-sodium phosphate (pH 6.0) mM potassium ferricyanide, 5 mM potassium ferrocyanide and 1 mg / ml of X-gal were added, and the mixture was allowed to stand at 37 캜 for 24 hours. Thereafter, cell aging was analyzed by counting the number of blue stained cells using an optical microscope.

As a result, referring to FIG. 3C, it was confirmed that the treatment of radiation or doxorubicin increased the cell senescence marker SA-beta-gal 2 to 3 times compared to the control group, Respectively.

4. Capillary formation assay

HAEC, an arterial vascular endothelial cell, was treated with radiation or 1 μg / ml doxorubicin, followed by treatment with Nisodipine, and cultured for 2 to 4 days. Matrigel (BD Biosciences, USA) was added to a 24 well plate at a concentration of 500 [mu] l / well to solidify the gel for 30 minutes. The vascular endothelial cells were plated on each gel at 1.5 × 10 ⁵ , and cultured for about 20 hours. Then, the lengths and the tube points of the formed tubes were counted, and the difference in angiogenesis was analyzed.

As a result, referring to FIG. 4A, it was confirmed that the treatment of radiation or doxorubicin significantly reduced angiogenesis or vascular length compared with the control group, and that the treatment of nisol dipine restored reduced angiogenesis or vessel length Respectively.

5. Analysis of gene damage reaction

A slide of 8 mm in diameter was placed on a 12-well plate, and HAEC (arterial blood vessel endothelial cell) was dispensed thereon at 4 × 10 ⁴ . One hour after treatment of vascular endothelial cells with nisol dipine and 4 Gy radiation, the slides were washed and fixed with 3.7% (v / v) paraformaldehyde solution. The cells were reacted with the γ-H2AX antibody, which is a gene damage marker, and stained with Alexa-596, a secondary antibody, and stained with DAPI. The stained cells were observed with a fluorescence microscope at a magnification of 40.

As a result, referring to FIG. 4B, it was confirmed that irradiation increased γ-H2AX, a gene damage marker, compared to the control group, and that treatment with Nisol dipine inhibited increased γ-H2AX.

According to the above results, although the cell growth was inhibited by irradiation, cell senescence was accelerated, the angiogenic ability was decreased, and the gene damage reaction of vascular cells was increased, but the cell growth and angiogenesis ability Restoration of senescence, inhibition of senescence, and reduction of gene damage by radiation. Therefore, it has been confirmed that the treatment of nisol dipine can protect against vascular cell damage caused by DNA damage reaction factors such as radiation or doxorubicin.

6. Analysis of intracellular calcium inflow

96 well plate HAEC, an arterial vascular endothelial cell, was dispensed into each well at 1 × 10 ⁴ and cultured for one day. Thereafter, radiation or 1 μg / ml doxorubicin was treated, followed by treatment with nicotinipine and incubation for 2 to 4 days. An equal volume of 2X Fluo-4 calcium assay reagent was added to each well and cultured at 37 ° C for 1 hour. The fluorescence was then measured at a wavelength of 494 nm / 516 nm.

As a result, referring to FIG. 4C, it was confirmed that the treatment of radiation or doxorubicin increased the amount of calcium intake compared to the control group, and the treatment of the nisol dipine suppressed the calcium intake and showed a calcium inflow similar to that of the control group.

Example  3: Relationship between Calcium Inflow and Radiation Vascular Injury

1. Calcium channel inhibitor effect

Because nisol dipine is a dihydropyrimidine calcium channel inhibitor, it has been compared by administering another calcium channel blocker, telenzepine.

The vascular endothelial cells were plated at 1 × 10 ⁴ in each well of a 96-well plate and cultured for one day. Then, Nysol diphene and telenzepine were treated at 0, 1, 10, and 100 μmol / ml, irradiated with 4 Gy radiation, and calcium inflow was measured after 24 hours.

As a result, referring to FIG. 5A, it was confirmed that the nisole dipine and the telenzepine inhibited calcium influx in a concentration-dependent manner and showed the same effect as the concentration treatment in the control group in the irradiated cells.

2. Telenjipin  Cell survival analysis

The vascular endothelial cells were divided into 6-well plates in the same number, treated with telenegraft, treated with 4 Gy radiation or 1 μg / ml doxorubicin for 1 hour and then cultured for 48 hours. After that, trypsin was treated to measure the number of living cells to analyze cell growth or cell viability.

As a result, referring to FIG. 5B, it was confirmed that administration of telenegraft to the decrease of cell survival rate by doxorubicin increased cell survival rate, but administration of telenegipine was not effective for reduction of cell survival rate by radiation.

3. Cell aging Marker (SA-beta-gal) staining

HAEC, 4 × 10 ⁴ arterial vascular endothelial cells, were treated with 4 Gy of radiation or 1 μg / ml doxorubicin on 35 mm plates, followed by treatment with telenegrin for 2 days. After that, SA-beta-gal, a cell senescence marker, was stained and cell aging was analyzed by counting the number of blue stained cells using an optical microscope.

As a result, referring to FIG. 5C, it was confirmed that the treatment of telenegraft did not inhibit the aging of vascular cells promoted by doxorubicin or radiation treatment.

Example  4: In mouse animal models Nissol Diphin  Radiation protection effect

1. Mouse survival analysis

8-week-old mice were intraperitoneally injected with nicotine dipine at a concentration of 50 mg / kg two days before irradiation. The radiation was irradiated at 8 Gy, and the status of the mice was confirmed twice a day every day.

As a result, referring to FIG. 6A, it was confirmed that the survival rate of mice was decreased in the group irradiated with radiation, but the survival rate of mice was significantly increased in the group treated with nisol dipine.

2. Mouse weight change analysis

8-week-old mice were intraperitoneally injected with nicotine dipine at a concentration of 50 mg / kg two days before irradiation. The radiation was irradiated at 8 Gy, and the body weight of the mice was measured daily.

As a result, referring to FIG. 6B, it was found that there was no significant difference in body weight between the group irradiated with radiation and the group treated with radiation and nisol dipine.

3. Radiation-induced field In damage Nissol Diphin  Protection effect

8-week-old mice were intraperitoneally injected with Nisol dipine at a concentration of 50 mg / kg two days before irradiation, and irradiated at 8 Gy and 13 Gy, respectively. After 3 days, the mice were harvested and lysed with 1% paraformaldehyde The tissue was fixed and frozen ablation was carried out. The section of the tissue slide was fluorescently stained with CD31, a blood vessel marker, and the anatomical shape of the intestine was observed.

As a result, referring to FIG. 7A, it was confirmed that the villi of the intestine was broken and the intestinal tissue was lost in the group irradiated with 8 Gy radiation. In the group administered with 8 Gy radiation, the intestinal villi was less damaged I was able to identify an organized organization.

In 13 Gy irradiated group, intestinal villus apoptosis was marked and most of the intestinal tissue was disappeared. In the group treated with 13 Gy radiation enisol dipine, most of the intestinal villi were severed, It was found that the intestinal tissue was lost or the cell death was less than that of the group.

8 Gy and 13 Gy showed significantly reduced intestinal blood vessels. In the group treated with 8 Gy radiation and nisol dipine, intestinal blood vessels were similar to those of the control group, and 13 Gy radiation and nisol dipine In the treated group, the intestinal blood vessels disappeared more in the control group than in the control group, but much more blood vessels were observed than in the irradiated group.

FIG. 7B is a photograph of a section taken after fluorescence staining with TUNEL (green) and CD31 (red) after 3 days from the administration of Nissol dipine in a mouse and 8 Gy of radiation.

In the irradiated group, the cells around the blood vessels showed apoptosis, but the apoptosis was significantly reduced in the group treated with the nisol dipine.

4. The radiation sensitivity of cancer cells Nissol Diphin  effect

NCI-A549 cells, a human lung cancer cell line, were inoculated on a 100 mm plate and cultured in a 5% CO ₂ incubator at 37 ° C. Then, NCI-A549 lung cancer cell lines were divided into 100, 200, 500, And cultured for 24 hours. Twenty-four hours later, each cell group was treated with 0, 10, and 20 μmol / ml of Nisol dipine and irradiated with 0, 1, 2, and 4 Gy radiation for 1 hour and then cultured for 10 to 14 days. When a suitable size of colonies were formed, the colonies were stained with 0.4% trypan blue and the number of colonies was measured.

As a result, referring to FIG. 8A, it was confirmed that the formation of colonies was slightly decreased in the lung cancer cell line treated with the nisol dipine compared with the control cells, and thus it was confirmed that the nisol dipine maintained the radiation sensitivity of the cancer cells.

Next, in order to confirm the radiation sensitivity of the cancer cells in the animal model, 1 x 10 ⁶ A549 lung cancer cell lines were injected into the hind legs of nude mice and the mice were grown until the tumor size reached 150 mm ³ . Then, the tumor size was measured for 5 weeks by dividing into the group treated with 50 mg / kg of nisol dipine or the group treated only with radiation, the group treated with both nisol dipine and radiation.

As a result, referring to FIG. 8B, it was confirmed that the size of tumor did not increase but rather decreased in the group treated with Nisol dipine. Therefore, it was confirmed that the nitrosyl dipine has an effect of maintaining or slightly increasing the radiation sensitivity of the tumor.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is clearly understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

The scope of the present invention is defined by the appended claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included within the scope of the present invention.

Claims (9)

Wherein the pharmaceutical composition contains nisoldipine as an active ingredient and inhibits intestinal injury or vascular injury caused by radiation. 2. The pharmaceutical composition according to claim 1, wherein the nicotinic diphin inhibits the expression of radiation-induced insulin-like growth factor binding protein (IGFBP5), p53 and p21. The pharmaceutical composition according to claim 1, wherein the nicotinic diphin inhibits increased cell senescence, gene damage and calcium absorption by radiation. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is at least one selected from the group consisting of a lubricant, a release controlling agent, a surfactant, a thickening agent, a polymer, a carrier, a diluent, a filler, a weight, a wetting agent, a sweetener, a flavoring agent, ≪ / RTI > further comprising a pharmaceutically acceptable additive selected from the group consisting of lubricants, lubricants, and binding agents. A reagent composition containing nisoldipine as an active ingredient and inhibiting the intestinal injury, vascular injury, and insulin-like growth factor binding protein (IGFBP5), p53 and p21 expressed by radiation. A method of inhibiting intestinal damage, vascular injury, and insulin-forming growth factor binding protein (IGFBP5), p53 and p21 expression, which comprises treating nisole dipine in a cell in vitro, . A pharmaceutical composition for reducing an anticancer drug-induced toxicity comprising nisoldipine as an active ingredient. The pharmaceutical composition according to claim 7, wherein the anticancer agent is selected from the group consisting of doxorubicin, cisplatin, and paclitaxel. A nisoldipine and an anticancer agent as an active ingredient, wherein the nisol dipine reduces anticancer drug-induced toxicity.

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