An Anticancer Agent Comprising Sodium Butyrate as an Active Ingredient

专利摘要:
PURPOSE: Provided is an anticancer agent which comprises sodium butyrate as an active ingredient and pharmaceutically acceptable carrier, inhibits angiogenesis-promoting factor and is used as a therapeutic for angiogenesis related solid cancer. CONSTITUTION: The anticancer agent is characterized by comprising sodium butyrate as an active ingredient, and pharmaceutically acceptable carrier. It specifically inhibits angiogenesis-promoting factor and vasculogenesis, and is thus as a therapeutic for angiogenesis related solid cancer.
公开号:KR20030020482A
申请号:KR1020010052415
申请日:2001-08-29
公开日:2003-03-10
发明作者:김규원
申请人:대한민국(서울대학교)；
IPC主号:

专利说明:

Anticancer agent comprising sodium butyrate as an active ingredient {An Anticancer Agent Comprising Sodium Butyrate as an Active Ingredient}
[4] The present invention relates to an anticancer agent comprising sodium butyrate as an active ingredient and a pharmaceutically acceptable carrier. More specifically, the present invention relates to an anticancer agent having sodium butyrate as an active ingredient and capable of treating cancer by inhibiting angiogenesis induced in a hypoxic state.
[5] Survival of all the cells of human tissues requires the supply of oxygen and nutrients by blood vessels and the release of metabolites. Microstructures of blood vessels exist in each tissue of the human body, and these blood vessels are absolutely necessary for normal function and metabolic activity of each organ and tissue of the human body. However, the mechanism of angiogenesis is a field that has hardly been studied. In 1991, it was divided into two different pathways and the concept of angiogenesis began to be established.
[6] One of the two known angiogenesis pathways is vasculogenesis, a de novo angiogenesis process in which blood vessels and blood cells are formed by the division and differentiation of angioblasts, the vascular progenitor cells. This process occurs only in early development, and most organs produce blood vessels by this process and do not occur in adults. Another pathway is angiogenesis, in which existing blood vessels germinate by stimulation, creating new blood vessels.
[7] In angiogenesis, the walls of the blood vessels are relaxed to increase the permeability of the walls of blood vessels, and the contraction of cells occurs due to the breakdown of vascular endothelial cells. Then, various proteolytic enzymes are activated to break down the basement membrane, and vascular endothelial cells move and proliferate from the vascular wall to peripheral tissues in the direction of stimulation to form loops, and the formed loops are differentiated to generate a vascular network.
[8] This angiogenesis occurs very actively during organ formation and tissue growth during development, and this mechanism is responsible for angiogenesis from birth to adulthood. Thus, throughout life, from developmental stages to aging, angiogenesis acts as a key mechanism for creating new blood vessels, and its dysregulation is directly linked to the development of numerous diseases. In other words, excessive angiogenesis is associated with diseases such as solid cancer, diabetic retinopathy, rheumatoid arthritis, psoriasis, purulent granulomas, and renal vascular glaucoma. It is associated with the development of senile diseases such as stroke and atherosclerosis. However, since the fundamental treatment of these diseases has not yet been developed, it is urgent to identify the molecular mechanism of angiogenesis and to develop a new treatment method based on this.
[9] On the other hand, it was found that the induction of angiogenesis is closely related to the oxygen deficiency of the microstructure. Various cellular reactions to maintain intracellular oxygen partial pressure due to lack of oxygen include increased respiration, vasodilation, increased red blood cell production, increased angiogenesis, decreased your life, and reduced ATP production. The most effective way to overcome in the body is angiogenesis. In particular, when cell proliferation is vigorous, such as cancer tissue, the number of cells is increased spatially, and the distance that oxygen supplied by blood vessels can reach by diffusion is known to be only 150 μm in the case of spherical tumor tissue. Therefore, in order for oxygen to be delivered to cells existing within the spatially expanded cancer tissues, angiogenesis must occur and supply oxygen. Therefore, the growth of early solid cancers, including liver cancer, depends on the self-growth factors secreted by the cancer cells themselves, and the growth of cancer tissues larger than 1-2 mm ³ leads to oxygen deficiency. Many cancer cells that do not acquire resistance to oxygen or lack of oxygen may die from necrosis or apoptosis. In order to overcome the lack of oxygen in this process, it is assumed that cancer cells secrete angiogenesis factors and cause new angiogenesis from surrounding vascular tissues. Thus, the blood vessels thus formed will be a pathway for metastasis of cancer cells. In other words, when metastatic cancer cells with active cell division are generated by activating oncogenes such as Myc and Ras, and then apoptosis genes such as Bcl-2 and p53 are damaged, the proliferation of cells continues, resulting in an oxygen deficiency state. Signaling by deficiency increases the expression of hypoxia-inducible factor-1α (HIF-1) to increase the expression of angiogenic genes.
[10] To date, studies on cancer show that oxygen deficiency in cancer tissues increases the stability of the transcription factor HIF-1α and also increases the expression of AP-1. It has been found that VEGF (vascular endothelial growth factor), bFGF (basic fibroblast growth factor), and IGF-II (insulin-like growth factor-II) and its major triggers for the expression of its receptors. Angiogenesis caused by the lack of oxygen is known to occur not only cancer but also myocardial infarction and retinal ischemia. This HIF-1α increases the expression of several genes involved in increasing oxygen transfer and decreasing oxygen consumption. Therefore, HIF-1α plays a key role in regulating gene expression due to oxygen deficiency. Therefore, specifically inhibiting HIF-1α increased by oxygen deficiency can effectively suppress angiogenesis caused by oxygen deficiency.
[11] Accordingly, the present inventors conducted various pharmacological experiments on the function of sodium butyrate in inhibiting angiogenesis induced in the hypoxic state, and therefore, in the hypoxic state because it has a specific inhibitory function on angiogenesis factors induced in the hypoxic state It was confirmed that the present invention can be used as a therapeutic agent for solid tumors induced by angiogenesis.
[12] After all, the main object of the present invention is to provide an anticancer agent comprising sodium butyrate as an active ingredient that inhibits the expression of angiogenic promoters induced in the hypoxic state.
[1] 1 is a photograph showing Western blots of cell extracts isolated from sodium butyrate-treated cancer cells to selectively inhibit the expression of HIF-1α in normal and hypoxic states.
[2] FIG. 2 is a photograph showing treatment of sodium butyrate with RNA isolated from cancer cells according to the RT-PCR method, thereby selectively suppressing the expression of VEGF in the normal and hypoxic states.
[3] FIG. 3 is a photograph showing treatment of sodium butyrate on venous vascular endothelial cells of a human umbilical cord and selectively inhibiting tube formation in normal partial pressure and hypoxic state using a phase contrast microscope.
[13] According to the conventional report, the hypoxic state in the center of solid cancer caused by the continuous proliferation of solid cancer acts as a powerful angiogenesis promoter. At this time, due to the hypoxic state occurring in the center, the stability of HIF-1α, an angiogenic factor, is rapidly increased, which induces the expression of other angiogenic factors, resulting in angiogenesis induced by hypoxic state. . It is a pathway that supplies oxygen and nutrients to cancer cells in the center of solid cancer, and plays a role in continuing the progression of cancer. Therefore, the present inventors anticipated that sodium butyrate would inhibit the angiogenesis and suppress the expression of angiogenesis-induced angiogenesis induced hypoxia. As a result, it was confirmed that sodium butyrate has an effect of suppressing the expression of angiogenesis-promoting factors induced by hypoxia.
[14] In the present invention, in order to confirm that HIF-1α protein whose expression is increased in the hypoxic state is suppressed by sodium butyrate, after culturing human sarcoma cancer cells in the hypoxic state, the cell extract separated by treatment with sodium butyrate is used. Western blot was performed. As a result, it was confirmed that sodium butyrate inhibits the expression of HIF-1α that is increased in the hypoxic state.
[15] In addition, in order to confirm the inhibitory effect of sodium butyrate on the expression of VEGF, the most representative factor among angiogenesis promoters, RNA was isolated from human muscle sarcoma cells treated with sodium butyrate in normal and hypoxic states. RT-PCT. In RT-PCR, sodium butyrate of the present invention was confirmed to specifically inhibit the expression of VEGF only in the hypoxic state.
[16] In addition, we used venous endothelial cells of human umbilical cord as an in vitro experimental model to verify the inhibitory effect on angiogenesis. In order to verify the inhibitory effect on the tube forming ability of vascular endothelial cells, vascular endothelial cells were cultured on matrigel and treated with sodium butyrate to observe the degree of tube formation. Inhibition was confirmed.
[17] Sodium butyrate has the effect of specifically inhibiting angiogenesis induced in the hypoxic state, so in the case of solid cancers such as liver cancer where excessive angiogenesis is induced in the hypoxic state and accelerated further malignancy, It can be used particularly effectively in clinical areas where it inhibits and promotes healing. In addition, sodium butyrate did not show any cytotoxicity after long-term treatment with high concentrations in animal cells, so the side effects would be less. In clinical use of sodium butyrate, the dosage should be determined by a specialist, depending on various factors, such as the patient's condition, age, and complications, but generally at a dose of 10 μg to 10 mg per day on a 60 kg adult basis, Preferably at a dose of 20 μg to 5 mg.
[18] If sodium butyrate is to be used clinically, this component can be formulated into conventional pharmaceutical formulations according to conventional methods in the pharmaceutical art. Preferred pharmaceutical preparations for this purpose include oral preparations such as tablets, hard, soft capsules, solutions, suspensions and the like, topical administration external preparations such as injectable solutions, ointments, creams, gels, lotions and the like. These pharmaceutical preparations are conventionally pharmaceutically acceptable carriers, for example, for oral preparations, excipients, binders, disintegrants, suspending agents, solubilizing agents, suspending agents, preservatives, extenders, stabilizers for injections , Preservatives, dissolution aids, buffers, isotonic agents, external preparations, can be prepared using aqueous and oily research bases, antioxidants, preservatives, extenders, and the like.
[19] Hypoxia-induced angiogenesis inhibitors comprising sodium butyrate according to the present invention include, but are not limited to, the above-mentioned daily doses per unit dosage form or half, 1/3, 1/4, 1/5 or 1/6 of Such unit dosage forms are administered 1 to 6 times per day so that a dose is included.
[20] Below. Through the examples will be described the present invention in more detail. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .
[21] Example 1 Inhibition of HIF-1α Expression by Sodium Butyrate
[22] In order to confirm that HIF-1α, which is increased in hypoxia, was inhibited by sodium butyrate, Western blotting was performed using cell extracts of human sarcoma cancer cells. First, human sarcoma cancer cells were cultured in a normal pressure and hypoxic state in a culture dish having a diameter of 100 mm, respectively. Then, sodium butyrate was treated for 16 hours at a concentration of 20 mM, and cell extracts were separated from each cell. 30 µg of the isolated cell extract was dissolved in 20 µl of gel loading buffer, followed by bathing for 3 minutes, electrophoresis, and proteins were transferred from the gel to the nitrocellulose filter using an electric kit. The nitrocellulose filter to which proteins were attached was reacted at 4 ° C. for 16 hours with a primary antibody against HIF-1α capable of recognizing HIF-1α. After the reaction was washed, the reaction with a secondary antibody that can recognize the primary antibody for 1 hour at room temperature. Subsequently, the reaction was performed in a dark room with a solution capable of recognizing such an antibody (ECL Western blotting detection reagent, Amersham, USA) for 5 minutes, and then exposed to an X-ray film to investigate the expression level of the protein. At this time, the amount of HIF-1α was corrected by Western blotting with an antibody against tubulin, a protein that is known to be expressed in the same amount in cells with the same filter. FIG. 1 is a photograph showing Western blot of cell extracts isolated from sodium butyrate-treated cancer cells to suppress HIF-1α expression in a hypoxic state.
[23] As shown in FIG. 1, it was observed that HIF-1α expression was hardly expressed in cells cultured under normal partial pressure. However, the cells cultured in the hypoxic state was found to significantly increase the expression of HIF-1α, an angiogenesis-promoting factor, and when treated with sodium butyrate, the expression rapidly decreases. Sodium butyrate is induced in the hypoxic state. It was confirmed that the effect of reducing the expression of HIF-1α.
[24] Example 2 Inhibition of Expression of VEGF by Sodium Butyrate
[25] To confirm that expression of VEGF, which is increased in hypoxia, is suppressed by sodium butyrate, total RNA was isolated from the cells. Cells incubated in a 100 mm dish were treated with 1 ml of Tri-zol reagent (GIBCO-BRL, USA), left at room temperature for 5 minutes, then 200 μl of chloroform was added, shaken vigorously for 15 seconds, and stirred at room temperature for 3 minutes. It was left. After centrifugation at 4 ° C. and 15,000 rpm for 15 minutes, the supernatant was transferred to a new tube, and 0.5 ml of isopropyl alcohol was added thereto, left at room temperature for 10 minutes, and centrifuged at 4 ° C. 15,000 rpm for the bottom of the tube. A white precipitate was obtained as total RNA. The total RNA precipitate was simply washed with 75% ethanol, dried at room temperature for 5-10 minutes, and the RNA was dissolved in an appropriate amount of distilled water. At this time, the total RNA was prepared in a group not treated with sodium butyrate and treated with cancer cells cultured in a normal partial pressure state and a hypoxic state, and all four types of RNA were isolated. 5 μg of total RNA, 1 μl of 12-18mer oligo (dT), and distilled water were mixed with 12 μl, and then allowed to stand at 70 ° C. for 10 minutes and cooled on ice for 2 minutes. 4 μl of 5 × buffer solution, 1 μl of 10 mM dNTP mix, 2 μl of 0.1 M DTT, 1 μl of SuperScript II RT (reverse transcriptase) was added to the prepared RNA sample, and then reacted at 42 ° C. for 50 minutes and left on ice. CDNA was made. cDNA and PCR reaction solution (10XPCR buffer bath, 200uM dNTP mix, VEGF specific primer, 2.5units Taq DNA polymerase) were mixed and 25 cycles of PCR were performed at 94 ° C for 1 minute, 65 ° C for 1 minute, and 72 ° C for 1 minute. Next, DNA fragments amplified by RT-PCR were electrophoresed. In this case, PCR experiments were performed with primers for beta actin (β-actin), which are known to be genes that are expressed in the same cell, for the same cDNA, thereby correcting VEGF expression. 2 is a photograph showing that the expression of VEGF is suppressed in the hypoxic state by performing RT-PCR with the cell extract isolated from the sodium butyrate treated cancer cells of the present invention.
[26] As shown in Figure 2, the treatment of sodium butyrate at normal pressure did not change the expression of VEGF. However, treatment of sodium butyrate in a hypoxic state was confirmed to decrease the expression of VEGF. Gene expression of VEGF, a representative angiogenesis promoter, is increased by HIF-1α. At this time, treatment with sodium butyrate reduces the expression of HIF-1α which increases expression in the hypoxic state, affecting VEGF regulated expression in the lower part thereof, thereby reducing the expression specifically in the hypoxic state. It could be confirmed that.
[27] Example 3 Inhibition of Tube Formation of Endothelial Cells by Sodium Butyrate
[28] In order to observe the in vivo angiogenesis inhibitory effect by sodium butyrate, tube formation experiments of vascular endothelial cells on metrigel similar to the most in vivo conditions were performed in vitro. 300 μl of 10 mg / ml concentration of methagel was dropped on a 24 well-plate and allowed to stand at 37 ° C. for 30 minutes to gel. When the gel is formed, trypsin is treated to 4x10 ⁵ cells / well by incubating vascular endothelial cells cultured to a level where the bottom is not visible. After treating sodium butyrate at a concentration of 20 mM, the cells are incubated at normal oxygen partial pressure and hypoxic state, respectively. The phase contrast microscope observed the formation of a tube, a vessel-like structure in the gel, at different time periods. FIG. 3 is a photograph showing that sodium butyrate is treated to venous vascular endothelial cells of human umbilical cord, and tube formation is suppressed at normal partial pressure and hypoxic state using a phase contrast microscope.
[29] As shown in FIG. 3, it was found that sodium butyrate inhibits vascular endothelial tube formation in hypoxia, which can be used as an effective inhibitor and therapeutic agent for angiogenesis induced in hypoxia such as solid cancer. It means that there is.
[30] Example 4 Preparation of Composition
[31] If sodium butyrate is to be used clinically, this component can be used as formulated in conventional pharmaceutical formulations, according to conventional methods in the pharmaceutical art, as follows.
[32] Composition 1 : Tablet
[33] According to the method for preparing a tablet in the Korean Pharmacopoeia General Formulation using the following ingredients, a tablet containing 10 mg of active ingredient per tablet was prepared.
[34] Component content (mg / tablet)
[35] Sodium Butyrate 10
[36] Lactose 285
[37] Magnesium Stearate 5
[38] Calcium Carboxymethylcellulose 25
[39] Light silicic anhydride 75
[40] Total amount 400mg
[41] Composition 2 : Soft Capsule
[42] According to the method for preparing a capsule in the Korean Pharmacopoeia General Formulation using the following ingredients, a soft capsule containing 10 mg of active ingredient per capsule is prepared.
[43] Component Content (mg / Capsule)
[44] Sodium Butyrate 10
[45] Gelatin 140
[46] Glycerin 60
[47] Paraoxymethylbenzoate 0.3
[48] Paraoxypropylmethylbenzoate 0.3
[49] Soybean oil q.s.
[50] Total amount 400mg
[51] Composition 3 : Injection
[52] Injectables containing 2 mg of active ingredient per ampoule are prepared according to the preparation of injectables in the Korean Pharmacopoeia General Formulation using the following ingredients.
[53] Component Content (mg / ampoule)
[54] Sodium Butyrate 2
[55] Sodium Citrate 500
[56] Distilled water for injection
[57] 5 ml total
[58] Composition 4 : Ointment
[59] According to the method for preparing an ointment in the Korean Pharmacopoeia General Formulation using the following ingredients, an ointment containing 10 mg of active ingredient per g is prepared.
[60] Component Content (mg / g)
[61] Sodium Butyrate 10
[62] Hard Fluid Paraffin 100
[63] Stearyl Alcohol 80
[64] Cetostearyl Alcohol 13
[65] Propylene Glycol 50
[66] Sorbitan monostearate 30
[67] Monostearic acid polyoxyethyl sorbitan 40
[68] Butylated Hydroxytoluene 0.4
[69] Paraoxybenzoic Acid Methyl Ester 0.9
[70] Paraoxybenzoic Acid Butyl Ester 0.9
[71] Purified water q.s.
[72] Total amount 1 g
[73] As described and demonstrated in detail above, the present invention provides an anticancer agent comprising sodium butyrate as an active ingredient and a pharmaceutically acceptable carrier. An anticancer agent comprising sodium butyrate of the present invention as an active ingredient and a pharmaceutically acceptable carrier specifically inhibits angiogenesis-promoting factors induced in a hypoxic state and inhibits angiogenesis of vascular endothelial cells in a hypoxic state. It may be used as a treatment for solid cancer by induced angiogenesis.

权利要求:
Claims (2)
[1" claim-type="Currently amended] An anticancer agent comprising sodium butyrate as an active ingredient and a pharmaceutically acceptable carrier.
[2" claim-type="Currently amended] The method of claim 1,
Pharmaceutically acceptable carriers include excipients, binders, disintegrants, suspending agents, solubilizing agents
Agents, suspending agents, preservatives, extenders, stabilizers, preservatives, dissolution aids, buffers,
Tonicity agents, aqueous and oily research bases, antioxidants, preservatives, extenders, or
Characterized in that the mixture
Anticancer drugs.

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引用文献:

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法律状态:
2001-08-29|Application filed by 대한민국(서울대학교)

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2001-08-29|Priority to KR1020010052415A

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2003-03-10|Publication of KR20030020482A

优先权:

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申请号 | 申请日 | 专利标题

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KR1020010052415A|KR20030020482A|2001-08-29|2001-08-29|An Anticancer Agent Comprising Sodium Butyrate as an Active Ingredient|