KR20040042579A - Therapeutic angiogenic agents containing sphingomyelin - Google Patents

Abstract

PURPOSE: Provided is a pharmaceutical composition containing sphingomyelin as an active ingredient for the promotion of angiogenesis. The composition is useful for the prevention and treatment of angiogenesis related diseases, such as ischemia, wound healing, ulcer, arteriosclerosis, Coronary thrombosis, vascular disease or depilation. CONSTITUTION: A pharmaceutical composition for the promotion of angiogenesis is characterized by containing an effective amount of sphingomyelin of the formula(1), as an active ingredient, wherein the effective amount thereof is 0.01-50 mg/kg. In the formula, R2 is the side chain of the fatty acid, preferably stearic acid or nervonic acid.

Description

Therapeutic angiogenic agents containing sphingomyelin

The present invention relates to an angiogenesis promoter, and more specifically, an angiogenesis promoting agent containing sphingomyelin as an active ingredient for promoting angiogenesis, and for the treatment and prevention of diseases and diseases requiring angiogenesis, including the same. It relates to a pharmaceutical composition.

Angiogenesis is a series of processes in which new blood vessels form in existing blood vessels, but in addition to physiological processes such as ovulation and wound healing, pathological conditions such as cancer cell proliferation and metastasis, diabetic retinopathy and rheumatoid arthritis Hemangioma and deeply involved. In normal cases, angiogenic factors such as basic fibroblast growth factor (bFGF), vascular endothelial growth factor, thrombospondin-1, angiostatin, Although angiogenesis inhibitors such as endostatin have a strict regulation in the body, angiogenesis is not induced, but when an injury such as a wound or cancer occurs, it promotes angiogenesis for regeneration of the wound or metastasis and growth of the wound. The balance of factors and inhibitors breaks down and new blood vessels are formed (Folkman, J., Nat. Med., 1: 27-31, 1995; Folkman, J., J. Natl. Cancer Inst., 82: 4-6 , 1990; Folkman, J. et al., J. Biol. Chem., 267: 10931-10934, 1992).

Angiogenesis is a process in which new capillaries are formed from existing blood vessels. Vascular vessels, such as cancer, rheumatoid arthritis, ulcers, ischemia, arteriosclerosis, myocardial infarction, cerebrovascular disease, or hair loss using factors that inhibit or induce angiogenesis. Attempts to treat angiogenesis-related diseases and disorders are being studied worldwide. As an agent for inhibiting angiogenesis, clinical trials include thalidomide, CM101, TNP-470, angiostatin, and endostatin. Clinical and mechanistic studies are also underway for the use of ischemic heart disease, wound treatment and hair regrowth. However, since these growth factors are proteins, there are disadvantages in that they are denatured by temperature, difficult to separate and purified, and relatively expensive. Therefore, it is an urgent task to develop angiogenesis promoters that can be manufactured more stably and economically.

The history of the study of sphingolipids began with the study of cell membrane components, but gradually it was demonstrated that these substances have a function of regulating cellular activity (Hannun, YA, et al., Science, 235: 670-674, 1987). Among the sphingolipids, sphingosine-1-phosphate, sphingosyl phosphorylcholin, sphingosine, etc. play various roles in cell proliferation, differentiation, migration and apoptosis effects. It is also known to promote angiogenesis (Goetzl, EJ, et al., Cancer Res., 59: 4732-4737, 1999; Alessenko, AV Membr. Cell Biol., 13: 303-320, 2000; Goetzl, EJ, J. Immunol., 162: 2049-2056, 1999; Lee, OH, et al., Biochem. Biophys.Res.Commun., 264: 743-750, 1999; Boguslawski, G., et al., Biochem. Biophys. Res. Commun., 272: 603-609, 2000). Sphingomyelin, which belongs to such sphingolipids, is one of the major membrane lipids that form cell membranes and is known to be highly distributed in metastatic cancer cell membranes rather than non-metastatic cancer cells (Dahiya, R., et al., Biochem. Cell Biol., 70: 548-554, 1992). However, no known angiogenic activity of sphingomyelin is known.

It is an object of the present invention to provide a therapeutic and preventive agent for diseases and conditions requiring angiogenesis such as wound healing, ulcers, ischemia, arteriosclerosis, myocardial infarction, cerebrovascular disease or hair loss.

1 is a graph showing the effect of sphingomyelin on the migration of vascular endothelial cells.

Figure 2 is a graph showing the effect of sphingomyelin on the differentiation of vascular endothelial cells.

Figure 3a is a photograph showing the effect of promoting angiogenesis in vivo of the control group not used sphingomyelin.

Figure 3b is a photograph showing the effect of promoting angiogenesis in vivo when 0.3 μg of sphingomyelin.

Figure 3c is a photograph showing the effect of promoting angiogenesis in vivo when 0.6 μg of sphingomyelin is used.

4 is a graph quantitatively showing the number of blood vessels newly formed in each of FIGS. 3A, 3B, and 3C.

In order to achieve the above object, the present invention finds that sphingomyelin has angiogenic activity, and provides a pharmaceutical composition containing the same as an active ingredient.

Hereinafter, the present invention will be described in detail.

First, the present invention provides an angiogenesis promoter containing sphingomyelin as an active ingredient.

The structure of sphingomyelin used in the present invention is the same as that of Chemical Formula 1, and the one purchased from Sigma was used.

<Formula 1>

Wherein R ₂ is a fatty acid side chain, preferably stearic acid or nervonic acid.

In order to confirm the effect of sphingomyelin on angiogenesis, the present invention tested the effect of sphingomyelin on the movement of vascular endothelial cells, an essential process of angiogenesis. It was confirmed that the induction effectively.

In addition, as a result of testing the effect of sphingomyelin on endothelial cell differentiation during angiogenesis, it was confirmed that sphingomyelin effectively induces the differentiation of human angiogenesis, and through CAM (Chorioallantoic Membrane) experiment using egg It was found that sphingomyelin has a strong activity of inducing angiogenesis in vivo.

The present invention also provides a pharmaceutical composition for the treatment or prevention of diseases and disorders that require angiogenesis containing sphingomyelin as an active ingredient.

Diseases and diseases requiring angiogenesis may include wound healing, ulcers, ischemia, arteriosclerosis, myocardial infarction, cerebrovascular disease, or alopecia.

The composition may further comprise a pharmaceutically acceptable carrier or excipient, which may be used orally with a pharmaceutically acceptable carrier or the like, or may be administered as an injection with an appropriate solvent and diluent.

Oral preparations

The sphingomyelin according to the present invention can be used as a capsule or tablet during oral administration. In the case of a capsule, general excipients such as starch, lactose, talc, and magnesium stearate can be used. Excipients can all be used. In addition, all other conventional carriers can be used.

In addition, as an additive, starch, crystalline cellulose, hydroxypropyl methyl cellulose, polyethylene glycol, lactose, polyvinylpyrrolidone, glyceryl behanate, and the like may be used. As a diluent, glucose, spray dried lactose, Fast-flolactose, anhydrous lactose, white sugar, starch, stark 1500, calcium dihydrogen phosphate, emcompress, crystalline cellulose (avicel) and the like can be added.

Wet binders and granules can be water, ethanol, gum arabic, tragacantha, gelatin solution, starch solution, white sugar syrup, povidone, cellulose derivatives, and the like. Polyethylene glycol 4000, 6000, 8000 Sodium lauryl sulfate, lauryl magnesium sulfate, sodium benzoate, polyethylene monostearate, glyceryl triacetate, magnesium stearate, zinc stearate, calcium, stearic acid, talc, hardened vegetable oil, liquid paraffin or paraffin can be added.

In addition, starch, talc, silicon dioxide, magnesium carbonate, magnesium oxide, etc. may be used as the fluidizing agent, and starch, talc, etc. may be used as an anti-sticking agent, and hydroxypropyl methyl cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose as additives. , Ethyl cellulose, methyl cellulose, carboxymethyl cellulose, polyacrylic acid, acrylic acid, acrylic acid derivatives, polyvinylpyrrolidone, polyethylene glycol and the like can be added.

Injection

On the other hand, when used for injection, it is preferable to use alcohols, higher fatty acid esters, etc. as a solvent, and dilute using phosphate buffered saline or physiological saline, and sodium benzoate, methyl as an antiseptic. Parabens or propylparabens and the like can be added.

For example, when preparing a pharmaceutical composition according to the present invention in the form of an injection, sphingomyelin may be dissolved in a solvent such as alcohol, and diluted with physiological saline or phosphate buffered saline to be administered to the human body.

Dosage

The effective dose of sphingomyelin of the present invention is generally from 0.01 to 50 mg / kg in adults. It may be administered several times a day, preferably 1 to 6 times divided by a predetermined time interval, depending on the judgment of the doctor or pharmacist.

Acute toxicity

Toxicity of oral administration to sphingomyelin showed that 50% lethal dose (LD ₅₀ ) by oral toxicity test was at least 1000 mg / kg or more.

Hereinafter, the present invention will be described in detail by examples. However, the examples are only to illustrate the invention and the present invention is not limited by the following examples.

Example 1: Effect of sphingomyelin on vascular endothelial cell migration

The migration of vascular endothelial cells is an essential process of angiogenesis, and it has been confirmed by the Bodendenhammer method how sphingomyelin affects the migration of vascular endothelial cells (Gho, YS, et al., Cancer Res., 59). : 5128-5132, 1999). The polycarbonate membrane was coated with 0.1% gelatin for 10 minutes and then dried at room temperature for 1 hour. Human Umbilical Vein Endothelial Cells (HUVEC) 1 × 10 ⁶ cells / ml were placed in 30 μl of the lower chamber, dried polycarbonate membrane, the upper chamber was raised and screwed. The chamber was inverted to allow cells to adhere to the membrane and then incubated for 2 hours in a 37 ° C. CO ₂ incubator. After 2 hours, 50 μl of sphingomyelin was added to the upper chamber.

Sphingomyelin was purchased from Sigma, and sphingomyelin was dissolved in chloroform, chloroform was removed with nitrogen, and then dissolved again with dimethyl sulfoxide. Then, incubated for another 2 hours, the polycarbonate membrane was stained with Difquick staining reagent, and the number of endothelial cells migrated through the polycarbonate membrane was counted at 20 × magnification under a microscope (see FIG. 1).

As can be seen in Figure 1, sphingomyelin effectively induced the migration of human vascular endothelial cells and showed the best activity at 0.5 [mu] g / ml. In addition, unlike other conventional angiogenesis-promoting factors such as fibroblast growth factor and vascular endothelial growth factor, sphingomyelin did not change activity after 10 minutes boiling at 100 ° C.

Example 2 effect of sphingomyelin on differentiation of vascular endothelial cells

In order to investigate the effect of sphingomyelin on endothelial cell differentiation in angiogenesis, the following experiment was performed (Gho, Y. S., et al., Cancer Res., 59: 5128-5132, 1999). 0.3 ml aliquots of RPMI 1640 medium 1: 1 with no matrigel and serum were added to each well of a 24-well plate, and then solidified in a 37 ° C. incubator for 1 hour. Thereafter, 40,000 human umbilical vascular endothelial cells were placed in each well and treated with sphingomyelin at concentrations of 0, 0.1, 0.5 and 2.5 μg / ml. After incubation for 3 hours at 37 ℃ incubator and observed the degree of the tube (tube) made under a microscope and taking a picture to measure the tube area formed.

As can be seen in Figure 2, sphingomyelin, compared to the control without sphingomyelin, it was observed that effectively induce differentiation of human vascular endothelial cells to increase the tube area significantly. In addition, sphingomyelin vascular endothelial differentiation-inducing activity was similar to fibroblast growth factor known as a potent angiogenesis inducer.

Example 3 Angiogenesis-promoting Effect of Sphingomyelin in Vivo

Whether sphingomyelin has an angiogenic effect in vivo was examined by experiments using egg CAM (Gho, Y. S., et al., Cancer Res., 59: 5128-5132, 1999). The fertilized egg was placed in a 37 ° C. incubator and incubated for 2 days. After removing 4 ml of albumin, 4 days later, eggshells of about 3 cm × 3 cm were removed to form a window, and the culture was continued. 18 μl of a mixture of Type I collagen (Rat tail, Becton Dickinson, USA) and sphingomyelin was inoculated onto a 1/4 piece of Thermanox disc, dried, and then incubated for 3 days in a CAM of 10-day pear eggs. The presence of blood vessels leading to the sample was checked and the number of newly formed vessels was counted (see FIGS. 3 and 4). Experiments were conducted using 10-14 eggs per each sample.

The control group treated only with Type I collagen without sphingomyelin did not induce angiogenesis (see FIG. 3A), whereas the group treated with Type I collagen containing 0.3 μg and 0.6 μg of sphingomyelin had strong angiogenesis. (See FIGS. 3B and 3C). Figure 4 is a graph quantitatively showing the number of blood vessels newly formed by each sample treatment, 0.3 μg and 0.6 μg of sphingomyelin about 5 times than the control group treated only with Type I collagen without sphingomyelin It has been observed to induce a degree of angiogenesis. These results, in addition to the results shown in Examples 1 and 2, show that sphingomyelin has a potent activity inducing angiogenesis in vivo.

As described above, sphingomyelin, a lipid present in vivo, efficiently induces the migration and differentiation of vascular endothelial cells and strongly promotes angiogenesis in vivo, as well as fibroblast growth factor and blood vessels. Compared with other existing angiogenic factors such as endothelial growth factor, it was confirmed that it is a new angiogenic factor that can be economically prepared without degeneration by temperature. Accordingly, sphingomyelin and pharmaceutical compositions containing the same are useful for the purpose of treating and preventing diseases and disorders in which angiogenesis is essential, such as heart disease, cerebrovascular disease or hair loss, such as wounds and ulcers and ischemic diseases. Can be used.

Claims (4)

Angiogenesis promoter containing an effective amount of sphingomyelin as an active ingredient. The method of claim 1, The effective amount is angiogenesis promoter, characterized in that 0.01 to 50 mg / kg. Pharmaceutical composition for the treatment or prevention of diseases requiring angiogenesis, characterized in that it contains sphingomyelin as an active ingredient. The method of claim 3, wherein The disease in which the angiogenesis is required is a wound composition, ulcer, ischemia, arteriosclerosis, myocardial infarction, cerebrovascular disease or alopecia.