KR20100083579A - Compositions comprising hemin for the preventing or treating cancer - Google Patents

Abstract

PURPOSE: A composition containing hemin for preventing and treating cancer is provided to control or block STAT5b protein and to prevent and treat STAT5b-mediated cancer. CONSTITUTION: A composition for preventing and treating cancer contains hemin as a main ingredient and pharmaceutically acceptable additive. The cancer is solid cancer such as breast cancer, prostate cancer, or bladder cancer. The hemin is a hemin derivative or salt thereof. The composition is produced in a pharmaceutical formulation by oral, skin, vagina, or parenteral administration.

Description

Compositions Comprising Hemin for the Preventing or Treating Cancer}

The present invention relates to a composition for the prevention and treatment of cancer diseases, including hemin, and more particularly, to hemin (hemin), a kind of heme-iron, and a pharmaceutically acceptable additive. It relates to a composition for preventing and treating cancer diseases.

Breast cancer is a mass of cancerous cells in the breast, and generally refers to cancers that occur in the breast ducts and lobules. The epithelial cells of the breast grow and divide under the stimulation of female hormones such as estrogen. The longer the epithelial cells of the breast are exposed to the female hormone estrogen, i.e. no childbirth or breastfeeding, or the faster Women with long menstruation due to late menopause have a higher risk of developing breast cancer. In addition, women who are obese after menopause increases the risk of breast cancer, which increases female hormones. It is known that 5-10% of breast cancer patients have a genetic predisposition, and mutations in the BRCA1 and BRCA2 genes are known to cause hereditary breast cancer.

The 5-year survival rate for breast cancer is close to 100% in stage 0 cancer but less than 20% in stage 4. Therefore, the best way to improve the survival rate of breast cancer is to detect it early in the absence of symptoms. The most basic treatment for breast cancer is surgical resection of the lesion, and all patients without metastases to other organs need surgery. Breast cancer has been proven to be effective in postoperative adjuvant therapy. Adjuvant therapy includes chemotherapy, radiation therapy, anti-hormonal therapy, and molecular targeted therapy. The use of such adjuvant therapy is determined by the stage of cancer, the expression of receptors, the type of surgery, and the like.

Insulin-like growth factor-1 receptor (IGF-IR) is a transmembrane receptor tyrosine kinase that is activated by insulin and ligands called IGF-I and IGF-II. It is known that overexpression of these proteins is associated with various types of carcinoma. (Ken Garber, et al., JNCI, 97 (11), 2005). In particular, IGF-IR is highly expressed in pre-menopausal female breast cancer patients, male prostate cancer patients, and colon cancer patients. Therefore, IGF-IR, which was discovered and cloned in 1986, has become a target for new anticancer drugs.

There are about 10 IGF-IR inhibitors currently being developed or under development around the world, which are mainly effective only in combination with conventional chemotherapy and are not as effective as single-agents. There are disadvantages.

According to recent research papers, the function of STAT5b (signal transducer and activator of transcription 5b) as a cell signal transduction and transcription factor in breast cancer cells is becoming increasingly important. In other words, kinase enzymes such as Jak2 and Brk / PTK6 play an important role in activating and recruiting STAT5b, and thus activated STAT5b is known to be a major regulator of breast cancer cell proliferation, cyclin D1, IGF-I and IGF. -Binding directly to promoters of IR genes to regulate their gene and protein expression.

Therefore, finding a variety of methods or drugs that can control or block these STAT5b proteins with these key functions will be of great help in the prevention and treatment of solid cancers, especially breast cancer, prostate cancer, colon cancer and bladder cancer, especially invasive and metastatic solid cancer. will be.

In particular, the recent increase in breast cancer (about 50% of the total breast cancer occurrence) in the pre-menopausal young women population in Korea, the research on the prevention and treatment is urgently urgent.

Accordingly, the present inventors made efforts to prepare a composition for preventing and treating cancer diseases by regulating or blocking STAT5b protein, and inhibiting the activity of major proteins involved in cell proliferation of solid cancers such as breast cancer, prostate cancer, colon cancer and bladder cancer. As a result, the present invention has been completed.

After all, the present invention is to provide a composition for the prevention and treatment of cancer diseases, including hemin (hemin) as a main component, and a pharmaceutically acceptable additive.

In order to achieve the above object, the present invention provides a composition for the prevention and treatment of cancer diseases, the hemin (hemin) as a main component, and includes a pharmaceutically acceptable additive.

In the present invention, the cancer disease may be characterized in that the solid cancer.

In the present invention, the solid cancer may be any one selected from the group consisting of breast cancer, prostate cancer, colon cancer and bladder cancer.

In the present invention, the breast cancer may be characterized as a pre-menopausal female breast cancer.

In the present invention, the hemin may be a derivative of hemin or a salt thereof.

In the present invention, the pharmaceutically acceptable additives are nutrients such as proteins, carbohydrates, sugars, talc, magnesium stearate, cellulose, calcium carbonate, starch-gelatin paste, pharmaceutically acceptable carriers, excipients, diluents and solvents. It may be characterized in that any one selected from the group consisting of.

The present invention also provides a composition for preventing and treating cancer diseases is prepared in a pharmaceutical formulation suitable for oral, rectal, nasal mucosa, external use, skin, vaginal or parenteral administration, capsules, cassettes, powders, granules or tablets It provides a therapeutic agent for cancer diseases, characterized in that it is prepared in a suitable oral dosage form as a separate unit.

The present invention has the effect of providing a composition for preventing and treating cancer diseases, the main component of hemin (hemin) is a kind of heme-iron.

The composition of the present invention can regulate or block STAT5b proteins that mediate expression of IGF-I, cyclin D1 and IGF-IR genes that contribute to cancer in hypoxic conditions, thereby preventing and treating cancer diseases mediated by the STAT5b. It works.

Hereinafter, the present invention will be described in more detail.

The present invention provides a composition for preventing and treating cancer diseases, which contains hemin as a main component and includes a pharmaceutically acceptable additive. Hemin (hemin), an essential component of the present invention, is a type of heme-iron, a human breast cancer cell (MDA) which is an estrogen-receptor α-independent and aggressive cancer cell to invasive and metastatic. In the hypoxia condition of -MB 231 cells, the expression of IGF-I gene and cyclin D1 and IGF-IR protein, which contribute to cancer, is inhibited through STAT5b.

In the present invention, the hemin may be a derivative of hemin or a salt thereof. The derivatives refer to similar compounds obtained by chemically changing a part of hemin. In addition, the term pharmaceutically acceptable salt of hemin means that derived from pharmaceutically acceptable inorganic or organic acids and bases. Examples of suitable acids include hydrochloric acid, bromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, malic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-P-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, Benzoic acid, malonic acid, naphthalene-2-sulfonic acid and benzenesulfonic acid.

In the present invention, the pharmaceutically acceptable additives are nutrients such as proteins, carbohydrates, sugars, talc, magnesium stearate, cellulose, calcium carbonate, starch-gelatin paste, pharmaceutically acceptable carriers, excipients, diluents and solvents. It may be characterized in that any one selected from the group consisting of.

The present invention is also limited to the pharmaceutical formulation suitable for oral, rectal, nasal mucosa, external (bucal or sublingual), skin, vaginal or parenteral (root, subcutaneous and intravenous) administration of the composition for preventing and treating cancer diseases. And suitable formulations for administration by inhalation or gas injection are also included. Suitable for these formulations are convenient in separate dosage units and may be prepared by methods well known in the art of pharmacy. All of the methods according to these aspects include processes involving associating the active ingredient with the liquid carrier, or subdividing it into solid carriers, or both processes, which may then be shaped into the desired formulation if desired.

According to another aspect, suitable pharmaceutical formulations for oral administration are conveniently in discrete units such as capsules, cassettes or tablets each containing a reserve amount of the active ingredient, and may be powders or granules. In another embodiment, the formulation is present as a solution, suspending agent or emulsion. In another aspect, the active ingredient may be formulated as a bolus, pill, or paste. Tablets and capsules for oral administration include conventional excipients such as binders, fillers, lubricants, disintegrants, or wetting agents. Tablets may be coated by methods well known in the art. Oral liquid preparations can be made, for example, in the form of aqueous or oily suspensions, solutions, emulsions, syrups or ericssons, or as powder products which can be combined with water or other suitable vehicle used. Such liquid preparations may include conventional additives or preservatives such as suspending agents, emulsifiers, water-insoluble vehicles (including cooking oil).

According to an aspect of the present invention, a composition based on hemin may be formulated for parenteral administration (eg, injection, eg, borus injection or sustained injection), and may be used in ampoules, prefilled syringes, It may be present in a multi-dose container with an infusion or preservative attached. The compositions may be prepared in the form of suspensions, solutions, or emulsions in oily or aqueous vehicles, and may include formulation formers such as suspending agents, stabilizers and / or dispersants. Alternatively, the active ingredient may be in powder form, obtained by sterile separation of sterile solids or by freezing the solution for combination with a suitable vehicle, eg, sterile, sterile, pre-use.

For topical administration to the skin, hemin-based compositions can be formulated as ointments, creams or lotions, or transdermal patches, in one aspect of the invention. Such transdermal patches include linalool, carvacrol, thymol, citral, menthol and t-anethol. And penetration enhancers. Ointments or creams may, for example, be formulated with a water soluble or oily base with the addition of suitable thickening and / or gelling agents. Lotions may be formulated with a water soluble or oily base and also generally include one or more emulsifiers, stabilizers, dispersants, suspending agents, thickening agents or pigments. Formulations for topical application in the mouth include, as a fragrance base, active ingredients in inactive bases such as lozenges, gelatin and glycerin or sucrose and acacia, which normally comprise the active ingredient in sucrose and acacia or tragacanth. PASTILLES with and mouthwashes with the active ingredient in a suitable liquid carrier.

As a pharmaceutical preparation for rectal administration wherein the carrier is a solid, in one other embodiment, a single dose of suppository is preferred. Suitable carriers may include cocoa butter or other materials commonly used in the art, and suppositories may be conveniently formed by mixing and cooling the active compound with a softened or molten carrier and then shaping the mold. Formulations suitable for vaginal administration include pessaries, tampons, creams, gels, pastes, foams or sprays, and the active ingredient additionally contains a carrier known in the art.

In one embodiment of the present invention for nasal administration, the compounds may be used in liquid spray or dispersed powder or drop form. Drops may be formulated with an aqueous or non-aqueous base consisting of one or more dispersing agents, solubilizing agents or suspending agents. Liquid sprays can conveniently be administered in pressurized packs.

Inhalation administration of a compound in one embodiment of the present invention may be by means of a convenient device capable of an insufflator, nebulizer or pressurized pack or other aerosol spray. Pressurized packs may contain dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or a suitable gas as a suitable propellant. In another embodiment, pressurized aerosols are defined by having a valve for administering a fixed amount. In another variation, the hemin according to the invention can be in the form of a dry powder composition, for inhalation or insufflation administration, for example a mixed powder of a compound with a suitable powder base such as lactose or starch. You can do that. In another embodiment, the powder composition is present in single dose form in a gelatin or blister pack, which may be administered as a powder, for example by addition of a capsule, cartridge or for example an inhalator or insufflator. In another aspect, the formulations described above can be applied to enable sustained release of the active ingredient.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Example 1: Cell Culture

MDA-MB 231 human breast cancer cells were cultured in L-15 medium (Sigma Chemical, USA) containing 10% fetal bovine serum (FBS) and 100 U / ml penicillin.

In addition, HC11 mouse mammary epithelial cells were cultured in RPMI 1640 medium (Gibco-BRL, USA) containing 10% fetal bovine serum (FBS), 5 μg / ml insulin and 10 ng / ml EGF.

COS-7 monkey kidney cells and CHO Chinese hamster ovary cells were also treated with DMEM (Gibco-) containing 10% fetal bovine serum (FBS), 2 mM glutamine and 100 U / ml penicillin and streptomycin at 37 ° C. and 5% CO ₂ conditions. BRL, USA).

The cultured cells were resuspended in an appropriate medium at a density of 2.5 × 10 ⁵ cells / ml every experiment. For hypoxic conditions, the cells were transferred to airtight chambers (NuAire, Plymouth, MN) and flowed a 5% carbon dioxide / 95% nitrogen mixture until the oxygen concentration reached 2%.

Example 2: Investigating the Effect of Hemin on the Viability of Breast Cancer Cells Under Hypoxic Conditions

In order to determine the effect of hemin on the viability of breast cancer cells in hypoxic conditions, the experiment was as follows.

First, spread the breast cancer cell line MDA-MB 231 in a 96-well plate, incubate for 6 hours with various concentrations of hemin (0, 10, 20 and 40 μM), and then inactivate only the living cells. After treatment with the stained (blue formazan) MTT was incubated for 4 hours at 37 ℃. The process was performed under 2% O ₂ hypoxia conditions.

RIPA lysis buffer (Gibco-BRL, USA) was added to the cultured cells, and the cells were lysed and lysed by centrifugation at 15,000 rpm. Protein concentration was measured at (Fig. 1). As a result, as shown in Figure 1, when treated with 10μM hemin, the cell growth inhibition was about 30% compared to untreated cells, when treated with 20μM hemin, 50% of cells Growth inhibition was shown.

That is, when hemin was treated with breast cancer cells, it was found that dose-dependent inhibition of cell growth.

Example 3 Investigation of the Effect of Hemin on the Expression of STAT5b, IGF-IR and Cyclin D1 Proteins

To investigate the effect of hemin on the expression of various hypoxia-regulated proteins (STAT5b, IGF-IR and cyclin D1 protein), the following experiments were performed.

Treatment of hemin according to the concentration (0, 10, 20 and 40 μM) in 2% O ₂ hypoxic condition using the breast cancer cell line MDA-MB 231, incubated for 6 hours, the degree of expression of each protein regulated by hypoxia Was investigated.

In addition, to determine the effect of the hemin treatment time, 20 μM of hemin was treated, incubated for a time (0, 3, 6 and 12 h), and the expression level of each protein controlled by hypoxia was investigated.

That is, the cell lysate obtained by adding RIPA lysis buffer (Gibco-BRL, USA) to each of the cultured cells was developed on an 8-12% SDS-polyacrylamide gel and again transferred onto a nitrocellulose membrane, followed by STAT5b, Blots using antibodies against IGF-IR and cyclin D1 protein and labeled using anti-β-actin antibody (FIG. 2).

As shown in FIG. 2, the protein levels of STAT5b, IGF-IR and cyclin D1 were dose- and time-dependently down-regulated by hemin.

Example 4 Inhibition of Binding Sites and Mutual Adhesion Between STAT5b Protein and IGF-I and Cyclin D1 Genes

Certain stimulants are required for STAT5a and STAT5b protein synthesis in COS-7 cell lines. The synthesized STAT5a and STAT5b proteins enter the nucleus and bind to the STAT5 binding site of IGF-I to show activity. An electrophoretic mobility shift assay (EMSA) was performed to confirm the binding activity of STAT5 and IGF-I.

First, transfection of STAT5b expression vector (pMX / STAT5b, Kyoto University, Japan) and STAT5a expression vector (pMX / STAT5a, Kyoto University, Japan) to COS-7 cells using FuGene 6 (Roche, Switzerland). Was prepared.

In the transfected COS-7 cells, STAT5a and STAT5b expressed in the cytoplasm under hypoxia (2% O ₂ ) and normal oxygen (normoxia; 20% O ₂ ) conditions bind to the IGF-I promoter in the nucleus. EMSA (kit: Promega Corp, WI) was conducted to determine if the activation is affected. In particular, the treatment was performed at peak time and dose to determine the effects of fac and hemin. In other words, under hypoxic condition, fac concentration was 200μM, time was 1h and hemin concentration was 20μM, time was 6h.In normal oxygen condition, fac concentration was 50μM, time was 0.5h, hemin concentration was 20μM and time was 6h. Treated with.

As a result, it was found that hemin weakens the binding force between the IGF-I gene and STAT5b in hypoxic conditions of COS-7 cells transfected with STAT5b (FIG. 3A).

In the same way, we investigated the effects of STAT5a and STAT5b proteins on cyclin D1 synthesis. In other words, STAT5a and STAT5b expressed in the cytoplasm under normal oxygen (20% O ₂ ) and hypoxic (2% O ₂ ) conditions in transfected COS-7 cells grown more than 80% enter the nucleus and cyclin D1 promoter (GAS1) EMSA was carried out to determine if the activation affects activation by binding to.

As a result, it was found that the binding force between the cyclin D1 gene and STAT5b was weakened by hemin under hypoxic conditions of COS-7 cells transfected with STAT5b (FIG. 3B).

Example 5: Investigation of the promoter of IGF-I and cyclin D1

With the Cyclin D1 Promoter by the STAT5b Protein To investigate whether the IGF-I promoter is activated, CHO cells were used to examine the STAT5bCA / 700bpIGF-I-pGL2 promoter (Dr. Honglin Jiang, Virginia Polytechnic Institute and State University, USA) under 2% O ₂ hypoxic conditions. The degree of activation was investigated. The 700bpIGF-I-pGL2 region of the promoter is 700bp from the 5'-end and includes the STAT5 binding site of the IGF-I gene.

Specifically, the degree of activation of the STAT5bCA / 700bpIGF-I-pGL2 promoter under hypoxia and normoxia conditions, respectively, was compared by measuring relative luciferase activity. In addition, when treated with fac and hemin (hemin), in order to investigate whether the IGF-I promoter is activated, the concentration of fac in hypoxic conditions, 200μM, time 1h and hemin concentration 20μM, time was 6h, Under normal oxygen conditions, fac concentration was 50 μM, time 0.5 h, hemin concentration 20 μM and time 6 h, followed by measuring luciferase activity (Fig. 4a). As a result, as shown in Figure 4a, it was found that the STAT5b / 700bp IGF-I pGL2-P activity of the hemin-treated under hypoxic conditions.

In addition, the human cyclin D1 promoter-luciferase construct was used to investigate the effects of fac, hemin, and normal / hypoxia on cyclin D1 activity in transcriptional activation. CHO cells were co-transfected with STAT5a, STAT5b and cyclin D1 (GAS1) constructs. Fac and hemin (hemin) were treated in normal or hypoxic conditions for different times and cultured in serum-free media (FIG. 4B). As a result, as shown in Figure 4b, it was found that the luciferase activity of the STAT5b / cyclin D1 promoter significantly decreased ( *** p <0.001 ) when hemin was treated under hypoxic conditions.

Example 6: Investigation of Increasing and Decreasing Expression of IGF-I Gene

In order to investigate the increase in IGF-I gene expression by STAT5b protein or the decrease (inhibition) of IGF-I gene expression by hemin, it was confirmed that STAT5b protein is required for IGF-I gene expression under hypoxic conditions as follows. It was.

First, transcript mRNA of the IGF-I gene was measured by real-time polymerase chain reaction (PCR) under 2% O ₂ hypoxia and normoxia conditions using mammary epithelial cell line HC11 cells. The real time PCR was performed using an ABI 7900 HT real time PCR system for human IGF-I and β-actin mRNAs according to the TaqMan Gene Expression Assays. Each measurement was obtained by repeating the measurement three times by normalizing the β-actin mRNAs level. As a result, as shown in Figure 5, when hemin treatment in hypoxic conditions, it was found that IGF-I gene expression was most reduced.

Example 7: Inhibition of expression of IGF-I gene by interfering RNA (si-STAT5b)

In order to investigate the degree of inhibition (STAT5b knock down) of the expression of IGF-I gene by interfering RNA (si-STAT5b) to the STAT5b protein, STAT5b protein is required for IGF-I gene expression under hypoxic conditions. It was confirmed as well.

First, the control group (pKD-NegCon, Lake placid, NY) and the interfering RNA siSTAT5b (pKD-siSTAT5b, Lake placid, NY) were treated with 2% O ₂ hypoxic and normal oxygen conditions using breast cancer cell line MDA-MB 231, respectively. It was. The treated cells were harvested after 24 hours and subjected to immunoprecipitation against STAT5b protein and β-actin to measure their respective expression levels (FIGS. 6A and 6B).

As a result, as shown in Figure 6a, it was found that siRNA-STAT5b reduces the expression of the IGF-I gene. Specifically, as shown in FIG. 6B, breast cancer cells transformed with pKD-siSTAT5b under the hypoxic condition reduced STAT5b transcript levels by more than 70% while the control (mock: pKD-NegCon) had little effect. .

Having described the specific part of the present invention in detail, it will be apparent to those of ordinary skill in the art that such a specific description is merely a preferred embodiment, thereby not limiting the scope of the present invention. will be. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

1 shows that when hemin is treated at various concentrations in the breast cancer cell line MDA-MB 231 under hypoxia conditions, cell growth is inhibited in a dose-dependent manner.

Figure 2 is a photograph showing the results of Western blotting performed to investigate the effect of hemin on the expression of various proteins regulated by hypoxia.

Figure 3a is a photograph showing the EMSA results carried out to determine the effect of oxygen concentration and hemin on the interaction between the STAT5b protein and IGF-I.

Figure 3b is a photograph showing the EMSA results carried out to determine the effect of oxygen concentration and hemin on the interaction between the STAT5b protein and cyclin D1.

Figure 4a is a result of comparing the activation of the STAT5bCA / 700bpIGF-I-pGL2 promoter in the hypoxic and normal oxygen conditions by measuring luciferase activity, respectively.

Figure 4b is a result of measuring the luciferase activity of the activation of the STAT5b / cyclin D1 promoter in hypoxic and normal oxygen conditions, respectively. An asterisk (*) shows a statistically significant decrease in promoter activation according to the ANOVA test.

Figure 5 is the result of measuring the increase and decrease the expression of IGF-I gene in hypoxic and normal oxygen conditions according to TaqMan gene expression analysis. Asterisk (*) shows statistically significant decrease in expression following ANOVA test (FIG. 5).

Figure 6a is a result of electrophoresis performed to determine the effect of siRNA-STAT5b on the expression of IGF-I gene, Figure 6b is a graph showing the value measured. Asterisk (*) shows statistically significant decrease in expression according to ANOVA test (FIG. 6B).

Claims (7)

Hemin (hemin) as a main component, and comprising a pharmaceutically acceptable additive for cancer disease prevention and treatment composition. The method of claim 1, The cancer disease is a composition for preventing and treating cancer diseases, characterized in that the solid cancer. The method of claim 2, The solid cancer is any one selected from the group consisting of breast cancer, prostate cancer, colon cancer and bladder cancer composition for preventing and treating cancer diseases. The method of claim 3, The breast cancer is a composition for the prevention and treatment of cancer diseases, characterized in that the pre-menopausal female breast cancer. The method of claim 1, The hemin is a derivative of hemin or a salt thereof, the composition for preventing and treating cancer diseases. The method of claim 5, The pharmaceutically acceptable additive is selected from the group consisting of proteins, carbohydrates, sugars, talc, magnesium stearate, cellulose, calcium carbonate, starch-gelatin paste, pharmaceutically acceptable carriers, excipients, diluents and solvents. A composition for preventing and treating cancer diseases, characterized in that any one. The composition for preventing and treating cancer diseases according to claim 6 is prepared in a pharmaceutical formulation suitable for oral, rectal, nasal mucosa, external use, skin, vaginal or parenteral administration, capsules, cassettes, powders, granules or tablets Cancer disease treatment agent, characterized in that it is prepared in a suitable oral dosage form as a separate unit.

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