JP5150091B2 - Cell and heart preservatives under hypoxic conditions - Google Patents

Description

The present invention relates to cells and cardiac preservatives under hypoxic conditions . More specifically, the present invention relates to a cell under hypoxia and a heart preservative containing 1,5-D-anhydrofructose as an active ingredient.

1,5-D-anhydrofructose (hereinafter referred to as 1,5-AF) acts on starch or starch degradation products of α-1,4-glucan lyase, an enzyme derived from certain ascomycetes and red algae. Can be produced. 1,5-AF has a double bond in the molecule, and is a highly reactive sugar compared to other monosaccharides.
Since 1,5-AF has antioxidant activity and antibacterial activity, it is an antioxidant that can be safely added to foods (see Patent Document 1), and an antibacterial agent that is particularly effective against Bacillus subtilis and lactic acid bacteria (see Patent Document 2). The use as is disclosed. This monosaccharide is also a precursor of antibiotic microtesin (see Patent Document 3).
Recently, anti-caries action (see Patent Document 4), hypoglycemic action (see Patent Document 5), anti-inflammatory action, and platelet aggregation inhibitory action (see Patent Document 6) have also been reported. AF is a carbohydrate that is expected to be used in various fields such as health foods and pharmaceuticals having further functionality.
Furthermore, a technique for producing a sugar chain (G- (G) _n -AF) containing 1,5-AF as a constituent sugar as a 1,5-AF derivative has also been proposed (see Patent Document 7).

  By the way, not only in Japan, but also in the world, diseases caused by ischemic disorders, such as cerebral infarction and myocardial infarction, are steadily increasing, and ischemic disorders in organs indispensable for life support such as heart, brain and kidney are Serious illness or death can be contributed through the irreversible cellular damage that rapidly results from a lack of oxygen carried by the bloodstream and / or a decrease in cellular respiration. Ischemic tissue damage is mainly caused by hypoxia-induced cell necrosis, and further inflammatory reaction after ischemia (release of inflammatory mediators such as ATP and HMGB-1 from necrotic cells, or hypoxia − Inflammatory signals and cytokine production in living cells due to oxidative stress accompanying reoxygenation. The latter mechanism may also cause secondary systemic inflammatory disorders.

  Currently, antiplatelet drugs and anticoagulants are commonly used as therapeutic agents in addition to surgical treatment for the treatment of ischemic diseases such as myocardial infarction and cerebral infarction. The target is a thrombus, which prevents the disease from worsening, and is not a direct therapeutic agent. In addition, since aspirin and the like conventionally used have antiplatelet and anticoagulant actions, they cannot be used in patients who are bleeding, patients with liver disorders, and patients with leukopenia. Panaldine (Daiichi Pharmaceutical Co., Ltd., trade name) may cause agranulocytosis, jaundice, etc. as side effects. Therefore, reducing side-effects and reducing cell necrosis under ischemic (hypoxic) conditions is considered the most effective treatment and prevention method for ischemic diseases, and the development of such drugs is eagerly desired. Yes. As drugs for improving the survival rate of cells under ischemic conditions, drugs containing quinolone, quinone, aminoglycoside or chloramphenicol antibacterial agents as active ingredients have been proposed (see Patent Document 8). Has been suggested to have possible side effects in the central nervous system, such as convulsions, in combination with drugs that have anti-blood small-plate action, such as aspirin, and is unlikely to be sufficient in terms of efficacy Therefore, safer and more effective drugs are desired.

Also in organ transplantation, an organ is removed from the organ donor and is ischemic (hypoxic) until transplantation, and is reperfused (reoxygenated) after transplantation. In addition, the longer the time required for transplantation, the greater the degree of organ damage. This disorder is thought to be caused by ischemia-reperfusion to generate active oxygen, which attacks the components that make up the cell. As an organ preservative for this phenomenon, peptides (see Patent Document 9) and antioxidant compositions (see Patent Document 10) have been proposed as substances exhibiting redox properties. However, for these peptides, the antigenicity of peptides will be a problem, and the antioxidant composition having a more complex chemical structure than 1,5-AF has a problem of toxicity in vivo. It can happen.
Japanese National Patent Publication No. 9-505988 JP 2001-89377 A French Patent Application Publication No. 2617502 JP 2004-123604 A Special table 2003-519660 gazette JP 2006-16301 A JP 2001-204490 A WO01 / 051614 Japanese Patent Laid-Open No. 5-139992 JP-A-8-26902

The object of the present invention is to prevent or treat myocardial infarction caused by vascular occlusion and other serious ischemic heart diseases, and to treat myoblasts or myocardial cells or living bodies under hypoxic conditions. It is to provide the use of 1,5-AF for preservation of the external heart .

  Still other objects and advantages of the present invention will become apparent from the following description.

Thus, for the clinical management of ischemic heart disease, suggesting the potential use of 1, 5-AF will be given a new insight into the treatment concept using 1, 5-AF.

According to the present invention, the above objects and advantages of the present invention are achieved firstly by a preservative for myoblasts or cardiomyocytes under hypoxic conditions characterized in that it contains 1,5-AF. Is done.
The above objects and advantages of the present invention are secondly achieved by a preservative for the heart under hypoxia characterized by containing 1,5-D-anhydrofructose.
Furthermore, the above objects and advantages of the present invention are thirdly the use of 1,5-D-anhydrofructose for protecting or storing cells in vitro under hypoxic conditions, Myoblasts or cardiomyocytes and the protection or preservation is achieved by the above use which does not affect the production of VEGF (vascular endothelial growth factor).

By using 1,5-AF, it is suggested to prevent or treat ischemic heart disease and it is possible to preserve the isolated heart.

1,5-AF used in the present invention can be prepared by a known method, for example, the method described in Patent Document 1, when used as a pharmaceutical composition .
The ischemic diseases where Ru is suggested the use of 1, 5-AF, for example, ischemic cardiomyopathy, myocardial infarction, etc. ischemic heart failure like.
A pharmaceutical composition containing 1,5-AF can be administered according to its dosage form by various methods known per se. The dosage, administration site, administration interval, period, etc. It can be determined in consideration of the age, weight, medical condition, or other drugs or treatments. The administration method can be, for example, oral administration, direct injection into the body, such as intravenous, subcutaneous, intraperitoneal, or external use, such as injection or infusion, and is not particularly limited.

The dose of drug compositions, the dosage form, administration method, or preventing or varies by condition to be treated, for example, 0.1Mg～100,000mg in terms of active ingredient as the dose per body weight 1 kg, preferably Can be 10 mg to 1,000 mg, and can be administered at an appropriate dosing frequency such as once or several times a day or once every several days.
The form of the drug composition, e.g., tablets, capsules, drip preparations, powders, granules, although injection and the like are not particularly limited. In addition, components necessary for preparing a preparation, such as a preparation carrier, an excipient, and a stabilizer, can also be contained.

Furthermore, the pharmaceutical composition Ru also der include nutrients, such as preventive or therapeutic agent or any other pharmaceutical agent or glucose ischemic heart disease.
Drug composition is Ru can also be administered to a non-human mammal.

Excised heart coercive Sonzai of the present invention can be used in a state of 1, 5-A F is dissolved in the storage solution. The preservation solution as saline, buffers, such as isotonic solution and an organ preservation solution that has been used conventionally Ru available.
Below, further details the present invention by way of examples. The present invention is not limited to the embodiment.

In an ischemic disease, blood flow is interrupted, and the tissues and cells beyond that fall into a hypoxic state, leading to cell death. The hypoxic culture model assumes an ischemic state in the acute phase in clinical practice, and is widely used as a model for cerebral infarction, for example, when cerebral nerve cells are used. Here, the action of 1,5-AF on ischemic diseases was evaluated from the viewpoint of cell protection (cell death suppression) under hypoxic culture conditions in various cells. The cells used were myocardial cells as a model of myocardial infarction, cerebral neurons as a model of cerebral infarction, and myoblasts and keratinocytes as models of limb ischemic disease. Examples 2 and 3 are reference examples.

Example 1 (Cytoprotective effect of 1,5-AF on cardiomyocytes)
H9c2 cells, a cell line derived from rat myocardium, were seeded in a 24-well microplate (1 × 10 ⁵ cells / well) and cultured for 24 hours, and then metabolized with 1,5-AF and 1,5-AF in vivo. A certain amount of 1,5-D-anhydroglucitol (hereinafter referred to as 1,5-AG) was added in a predetermined amount, and hypoxia was immediately performed for 72 hours using Aneropac Kenki / Ischemic System (Mitsubishi Gas Chemical Co., Ltd.). Culture (O ₂ concentration 1% or less, CO ₂ concentration around 5%) was performed {medium: D-MEM (Low glucose) containing 10% FBS and 2% penicillin / streptomycin}. After hypoxic culture, adherent cells were fixed with 80% ethanol and then stained with 0.4% crystal violet. After staining, the plate was washed well with water, dye (dye) was extracted with methanol, and absorbance was measured at a wavelength of 570 nm using a microplate reader. The cell viability was calculated with the absorbance before the start of hypoxic culture as 100%.
The results are shown in FIG. The viable cell rate after 72-hour hypoxic culture is about 5% in the 1,5-AF 0, 125, 250 μg / ml group, and the 1,5-AG group (1,000 μg / ml). It turns out that it is almost dead. However, the viable cell rates in the group of 1,5-AF of 500 to 1,000 μg / ml are 28.8 ± 2.6% and 11.2 ± 1.1%, respectively. Cell necrosis was significantly suppressed. From this, it was confirmed that 1,5-AF has a cytoprotective effect on cardiomyocytes under hypoxic culture conditions.

Example 2 (Investigation of cytoprotective effect of 1,5-AF on nerve cells)
PC12 cells (rat adrenal medullary pheochromocytoma) commonly used as a model of neural progenitor cells are seeded in a 24-well microplate at a rate of 1 × 10 ⁵ cells / well and cultured under conditions of 37 ° C. and CO ₂ 5%. {Medium: D-MEM (Low glucose) containing 10% FBS and 2% penicillin / streptomycin}. Subsequently, the medium was replaced with the same medium containing dibutylyl cyclic cAMP 2 mM (bucladecin sodium: Actocin injection, manufactured by Daiichi Pharmaceutical Co., Ltd.), and cultured for 24 hours to differentiate into nerve cells. Thereafter, the supernatant is removed, a medium containing predetermined amounts of 1,5-AF and 1,5-AG is added, and immediately, 48 hours using Aneropac Kenki / Ischemic System (Mitsubishi Gas Chemical Co., Ltd.) Hypoxic culture (O ₂ concentration 1% or less, CO ₂ concentration around 5%) was performed. After hypoxic culture, adherent cells were fixed with 80% ethanol and then stained with 0.4% crystal violet. After staining, the plate was washed well with water, dye (dye) was extracted with methanol, and absorbance was measured at a wavelength of 570 nm using a microplate reader. The cell viability was calculated with the absorbance before the start of hypoxic culture as 100%.
The results are shown in FIG. The viable cell rate after 48 hours of hypoxic culture was approximately 10% in the 1,5-AF 0, 125, and 1,5-AG groups (1,000 μg / ml). However, when 1,5-AF is 250, 500, and 1,000 μg / ml, the viable cell rates are 45.9 ± 3.1%, 69.4 ± 4.9%, and 54.9 ± 10.5%, respectively. Yes, cell death in hypoxic culture was significantly suppressed. From this, it was found that 1,5-AF has a cytoprotective effect on neurons under hypoxic culture conditions.

Example 3 (Examination of cytoprotective effect of 1,5-AF on keratinocytes)
Human keratinocyte (HaCaT) cells are seeded in a 24-well microplate (1 × 10 ⁵ cells / well) and cultured for 24 hours, and then predetermined amounts of 1,5-AF and 1,5-AG are added. Hypoxic culture (O ₂ concentration 1% or less, CO ₂ concentration around 5%) was performed for 48 hours using an ischemic system (Mitsubishi Gas Chemical Co., Ltd.) {medium: FBS 10% and penicillin streptomycin 2% Including D-MEM (Low glucose)}. After hypoxic culture, adherent cells were fixed with 80% ethanol and then stained with 0.4% crystal violet. After staining, the plate was washed well with water, dye (dye) was extracted with methanol, and absorbance was measured at a wavelength of 570 nm using a microplate reader. The cell viability was calculated with the absorbance before the start of hypoxic culture as 100%.
The results are shown in FIG. After 48 hours of hypoxic culture, the viable cell rate of 1,5-AF at 500 and 1,000 μg / ml was significantly higher than the other groups (500 μg / ml: 55.7 ± 10.9%, 1,000 μg / ml: 73.4 ± 14.8%), cell death in hypoxic culture was significantly suppressed. This indicates that 1,5-AF has a cytoprotective effect against keratinocytes under hypoxic culture conditions.

Example 4 (Study of cytoprotective effect of 1,5-AF on myoblasts)
Mouse myoblasts (C2C12) were seeded in a 24-well microplate (1 × 10 ⁵ cells / well) and cultured for 24 hours, and then predetermined amounts of 1,5-AF and 1,5-AG were added. / Hypoxic culture (O ₂ concentration 1% or less, CO ₂ concentration around 5%) was performed for 24 hours using an ischemic system (Mitsubishi Gas Chemical Co., Ltd.) {medium: FBS 10% and penicillin streptomycin 2% D-MEM (Low glucose)}. After hypoxic culture, adherent cells were fixed with 80% ethanol and then stained with 0.4% crystal violet. After staining, the plate was washed well with water, dye (dye) was extracted with methanol, and absorbance was measured at a wavelength of 570 nm using a microplate reader. The cell viability was calculated with the absorbance before the start of hypoxic culture as 100%. At the same time, VEGF (vascular endothelial growth factor) in the culture supernatant was measured using an ELISA kit (manufactured by BioSource), and the lactic acid level was measured using Lactate Pro LT-1710 (manufactured by Arkray).

  The cell viability results are shown in FIG. Viable cells after 24-hour hypoxic culture were hardly observed in 1,5-AF 0, 125, and 1,5-AG groups (1,000 μg / ml). However, when 1,5-AF is 250, 500, and 1,000 μg / ml, the viable cell rate is about 60 to 70% (250 μg / ml: 62.3 ± 4.8%, 500 μg / ml: 68.8). ± 2.9%, 1,000 μg / ml: 66.2 ± 8.5%), and cell death in hypoxic culture was significantly suppressed. From this, it was found that 1,5-AF has a cytoprotective effect on myoblasts under hypoxic culture conditions.

  The VEGF content in the supernatant after 24 hours of hypoxic culture is shown in FIG. It was found that low oxygen culture significantly increased VEGF in the culture supernatant, but 1,5-AF did not affect the production of VEGF by hypoxic culture. This tendency was also observed in cardiomyocytes. The decrease in VEGF production in hypoxic tissue at the ischemic site prolongs the remodeling of blood vessels such as angiogenesis and neovascularization as a supplement to the blood supply, so that 1,5-AF produces VEGF. It seems to be very useful not to affect. On the other hand, the lactic acid level in the culture supernatant was significantly increased by the hypoxic culture, whereas 1,5-AF was found to suppress the production of lactic acid in a concentration-dependent manner (C in FIG. 4). ).

Example 5 (Examination of cytoprotective effect of 1,5-AF by pretreatment)
Mouse myoblasts (C2C12) and rat myocardial cell line (H9c2) were seeded in a 24-well microplate (1 × 10 ⁵ cells / well) and cultured for 24 hours, and then 1,5-AF and 1,5-AG were added. A predetermined amount was added and further cultured for 24 hours {Medium: D-MEM (Low glucose) containing 10% FBS and 2% penicillin / streptomycin}. Thereafter, the culture supernatant is washed to remove 1,5-AF and 1,5-AG, the same medium as above is added, and low using an aneropack-kenki / ischemic system (Mitsubishi Gas Chemical Co., Ltd.). Oxygen culture (O ₂ concentration 1% or less, CO ₂ concentration around 5%) was performed. After hypoxic culture, adherent cells were fixed with 80% ethanol and then stained with 0.4% crystal violet. After staining, the plate was washed well with water, dye (dye) was extracted with methanol, and absorbance was measured at a wavelength of 570 nm using a microplate reader. The cell viability was calculated with the absorbance before the start of hypoxic culture as 100%.

  The survival rate of C2C12 cells after 24 hours of hypoxic culture is shown in FIG. Pretreatment with 1,5-AF at 500 μg / ml and 1,000 μg / ml significantly suppressed cell necrosis associated with hypoxic culture. In addition, the same result was obtained with 1,000 μg / ml 1,5-AF in H9c2 cells after 72 hours of hypoxic culture (FIG. 5B). These results suggest that taking 1,5-AF in advance leads to prevention of ischemic disease.

Evaluation of cytoprotective action of 1,5-AF under cardiomyocyte hypoxic culture conditions Evaluation of cytoprotective action of 1,5-AF under neuronal hypoxic culture conditions Evaluation of cytoprotective action of 1,5-AF under keratinocyte hypoxic culture conditions Evaluation of cytoprotective effect of 1,5-AF under myoblast hypoxia culture condition, VEGF content and lactic acid level in culture supernatant Evaluation of cytoprotective effect of 1,5-AF pretreatment under myoblast and cardiomyocyte hypoxic culture conditions

Claims (3)

A preservative for myoblasts or cardiomyocytes under hypoxic conditions, comprising 1,5-D-anhydrofructose. A preservative for the heart under hypoxic conditions, comprising 1,5-D-anhydrofructose. Use of 1,5-D-anhydrofructose for the protection or preservation of cells in vitro under hypoxic conditions, wherein the cells are myoblasts or cardiomyocytes and the protection or preservation is VEGF Use as described above, which does not affect the production of (vascular endothelial growth factor).

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