KR20020078147A - Use of acharan sulfate for prevention and treatment of hyperlipidemia - Google Patents

Abstract

PURPOSE: Acharan sulfate having an effect on increasing HDL-cholesterol levels while reducing triglyceride levels and total cholesterol levels is provide which can be effectively used for prevention and treatment of hyperlipidemia. CONSTITUTION: A pharmaceutical composition for prevention and treatment of hyperlipidemia contains Acharan sulfate separated from the body of Achatina fulica Bowdich hyperlipidemia and a pharmaceutically acceptable salt thereof as an effective ingredient. The composition can be applied to parenteral administration in practical clinical therapeutic application and used in the shape of pharmaceutical formulations.

Description

Use of acharan sulfate for prevention and treatment of hyperlipidemia}

The present invention relates to the use of acaran sulfate enzyme, specifically to the use of acaran sulfate in the prevention and treatment of hyperlipidemia.

Glycosaminoglycans are structurally diverse and complex through partial changes in the biosynthesis process. GAGs have a basic backbone that is partially sulfided by the repeated uronic acid and glucosamine, and these complex chemical structures allow various kinds of biological activity. As is known to date, these various biological activities appear to be due to the presence of specific sequences of their own.

Animal GAGs include hyaluronic acid, chondroitin sulfate, keratin sulfate, heparan sulfate, heparin, and the like. Akaran sulfuric acid was discovered and patented (Korean Patent 170064). Among these GAGs, heparin is isolated in mast cells along the arteries in the liver, lungs, and skin mucosa, and the other GAGs are distributed in connective tissue. Chondroitin sulfate, used as a medicine, is obtained from shark cartilage and bovine bronchus. In addition, GAGs are also found in molluscs, which are inferior, such as the presence of GAGs with antithrombin binding sites with 3-0-sulfuric acid groups in shellfish (Pejler et al., J. Biol. Chem ., 262, 11413-11421, 1986), chondroitin sulfates substituted with sulfuric acid have also been found in sea cucumbers and sea urchins (Mourao et al., TIGG , 7, 235-246, 1995). It is also known that chondroitin sulfate is present in the cornea of cuttlefish (Karamanos et al ., Int. J. Biochem ., 23, 67-72, 1991), and acaran sulfate is found to be abundant in African king snails.

Acaran sulfate is structurally similar to heparin and heparan sulfuric acid, and the structure of the disaccharide → 4) -α-D-GlcNAc (1 → 4) -α-L-IdoA2S (1 → is repeated. Sulfuric acid GAG is connected by a 1 → 4 bond in which glucosamine and uronic acid groups are repeated.Heparan sulfuric acid has a single sulfation of disaccharides N-acetyl-D-glucosamine and D-glucuronic acid. Heparin has a monosulfated structure, while heparin has a trisulfated structure mainly on the disaccharides N-sulfonyl-D-glucosamine and L-iduronic acid.

GAGs exist in the form of proteoglycans in vivo, and these proteoglycans are cleaved by protease and glycosidase to release glycosides. The physiological role of GAGs is not known yet, but it seems to show physiological activity by binding to proteins in vivo. Proteins that bind GAGs in vivo can be largely classified into five types, including protease inhibitors, plasma lipoproteins, growth factors, lipolytic enzymes, and extracelluar matrix proteins. These proteins bind to GAGs and act on their specific biological activities.

The first GAGs exhibit physiological activity in combination with protease inhibitors. Representative protease inhibitors that bind GAGs include antithrombin III and heparin cofactor II. Heparin binds to antithrombin III to inhibit the action of blood factors Xa and thrombin, and binds to heparin cofactor II to inhibit the action of thrombin to prevent blood clotting.

The second GAG class binds to lipoproteins of plasma. Representative plasma lipoproteins that bind to GAG class are apolipoprotein E and apolipoprotein B. GAGs that bind to plasma lipoproteins include heparin and chondroitin, and these heparin and chondroitin interact with sites in which the acid and basic amino acids of apolipoprotein E and apolipoprotein B are concentrated to regulate the occurrence of atherosclerosis.

The third GAG class binds to the growth factor, and the growth factor that binds to the GAG class is basic fibroblast growth factor (bFGF), and the GAG class that binds to the growth factor includes heparin and heparan sulfate. Heparin and heparan sulfate play an important role in cell growth and differentiation by binding to bFGF and preventing the degradation of bGFG.

The fourth GAG binds to lipolytic enzymes. Intravenous injection of heparin and heparan sulfate releases low-density lipoproteinases and triglycerides, which are lipolytic enzymes, into plasma cells. Free lipolytic enzymes break down fat in the blood and play a major role in preventing atherosclerosis.

Fifth GAGs bind to enveloped cell conjugates, and proteins of the enveloped cell conjugates that interact with GAGs include fibronectin, vitronectin, laminin, and thrombospondin. . Proteins that bind to these GAGs are important for cell adhesion, information transfer between cells, cell differentiation, and cell growth.

As such, GAGs can be used for various purposes because they show various physiological activities in combination with proteins in vivo. As an example in which GAGs are used as drugs, chondroitin sulfate is also used in eye drops, arthritis drugs, and recently, tonic drinks. Hiyal uronic acid is also widely used as an additive in cosmetics. Heparin is widely used as a glycan drug in the clinic, and heparin is widely used as a anticoagulant for thrombosis, after surgery, kidney dialysis, artificial blood vessels, extracorporeal circulation, and the like. Recently, in addition to anticoagulant action, various biological activities such as complement activity of immune system, inhibition of peripheral angiogenesis, interaction of basic fibroblast growth factor, and antiviral action have been reported.

Acaran sulfate exhibits a variety of biological activities, including the above-mentioned bioactivity, and is characterized by the low side effects of anticoagulants that are structurally modified (eg deacetylation, sulfated, new oligo GAG). It is possible that sulfated oligosaccharides of acaran sulfate provide the structural features necessary for binding to fibroblast growth factors and thus play an important role in cell growth and differentiation. In fact, the compound that was sulfated after deacetylation of -NH ₂ is expected to have a 6-fold increase in cell growth compared to the control group. Acaran sulfate is also ideal as a model compound for reactions using sulfate-based transferases because of its simple structure of repeating disaccharides. In addition, acaran sulfate is very effective for liver function and menopausal disorders, including osteoporosis.

Although acaran sulfate exhibits various pharmacological effects as described above, it is difficult to handle due to their large molecular weight and there is a problem of causing side effects. Therefore, there is a great need to prepare acaran sulfate having low molecular weight. Recently, the present inventors have discovered that the bacteroids stecoris, a microorganism that synthesizes GAG degrading enzymes in the human intestine HJ-15 was isolated and identified and patented and registered (Korean Patent Registration No. 10-0257168), and a new acaran sulfate degrading enzyme was isolated from it (Korean Patent Application No. 2000-75605) .

Accordingly, the present inventors revealed that the acaran sulfate can be effectively used for the prevention and treatment of hyperlipidemia by effectively lowering the TG (triglyceride) and cholesterol levels associated with hyperlipidemia while studying the function of the acaran sulfate. The present invention has been completed.

It is an object of the present invention to provide the use of acaran sulfate for the prevention and treatment of hyperlipidemia.

1 is a graph showing the 1 H-NMR analysis of the acaran sulfate of the present invention,

2 is a graph showing the effect of acaran sulfate on total cholesterol, triglyceride (TG) and HDL-cholesterol in serum,

■; Normal, □; Control,

▨; Chitosan oligosaccharide, ▧; Acaran sulfate

*; p <0.01, **; p <0.005

FIG. 3 is a graph showing the effect of acaran sulfate on total cholesterol and serum TG amounts in hyperlipidemia induced by poloxamer-407.

■; Normal group; Poloxamer-407 injection group,

▨; Lovastatin injection group, ▧; Acaran sulfate-administered group (100 mg / kg),

▤; Acaran sulfate administration group (300 mg / kg)

A; Total cholesterol, B; Serum TG Levels

*; p <0.05, **; p <0.02, ***; p <0.002

In order to achieve the above object, the present invention provides acaran sulfate and pharmaceutically acceptable salts thereof for preventing and treating hyperlipidemia, which effectively lower TG and cholesterol levels.

The present invention also provides a pharmaceutical composition for preventing and treating hyperlipidemia containing acaran sulfate and a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the present invention provides the use of acaran sulfate and its pharmaceutically acceptable salts for the prevention and treatment of hyperlipidemia.

Hereinafter, the present invention will be described in detail.

The present invention provides acaran sulfate and pharmaceutically acceptable salts thereof for preventing and treating hyperlipidemia that very effectively lower TG and cholesterol levels.

The acaran sulfate of the present invention may be used in the form of a pharmaceutically acceptable salt, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. Acaran sulfuric acid can form pharmaceutically acceptable acid addition salts according to methods conventional in the art. Organic acids and inorganic acids may be used as the free acid, hydrochloric acid, bromic acid, sulfuric acid or phosphoric acid may be used as the inorganic acid, and citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, or fumaric acid may be used as the organic acid. (fumaric acid), formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, galluxuronic acid, embonic acid, glutamic acid or aspartic acid Can be used.

The present invention also provides a pharmaceutical composition for preventing and treating hyperlipidemia containing acaran sulfate and a pharmaceutically acceptable salt thereof as an active ingredient.

The acaran sulfate and its pharmaceutically acceptable salts can be administered parenterally during clinical administration and can be used in the form of general pharmaceutical formulations.

That is, the acaran sulfate and pharmaceutically acceptable salts thereof of the present invention may be administered in various parenteral formulations, and when formulated, fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are commonly used, are formulated. It is prepared using diluent or excipient. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like can be used. As a suppository base, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

In addition, the acaran sulfate and pharmaceutically acceptable salts thereof may be used in admixture with various carriers as pharmaceutically acceptable, such as physiological saline or organic solvents, glucose, sucrose or Carbohydrates such as dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, small molecule proteins or other stabilizers can be used as medicaments.

The effective dose of the acaran sulfate and its pharmaceutically acceptable salt is 10-30 mg / kg for injection, 100-300 mg / kg for oral administration and can be administered once or twice a day.

The total effective amount of acaran sulfate and pharmaceutically acceptable salts thereof in the pharmaceutical composition of the present invention may be administered to the patient in a single dose by bolus form or by infusion for a relatively short period of time. Multiple doses may be administered by a fractionated treatment protocol with long term administration. Since the concentration of the acaran sulfate and its pharmaceutically acceptable salt is determined in consideration of various factors such as the age and health of the patient, as well as the route of administration and the number of treatments of the drug, it is considered that One of ordinary skill in the art will be able to determine appropriate effective dosages for the particular use of the acaran sulfate and pharmaceutically acceptable salts thereof as pharmaceutical compositions.

In addition, the present invention provides the use of acaran sulfate and its pharmaceutically acceptable salts for the prevention and treatment of hyperlipidemia.

In order to confirm the effect of the acaran sulfate of the present invention on hyperlipidemia, the present inventors first isolated and purified acaran sulfate from Achatina fulica Bowdich and confirmed its structure using ¹ H-NMR analysis. (See Table 1 and FIG. 1 ).

We also identified the effects of the acaran sulfate of the present invention on total cholesterol, HDL-cholesterol and TG concentrations in hyperlipidemic rat animal models induced by cholesterol-containing mixtures.

As a result, it was observed that the administration of cholesterol-containing mixtures significantly increased serum cholesterol and TG levels, resulting in relative hyperlipidemia compared with the normal group, whereas HDL-cholesterol was decreased. In the case of serum cholesterol, both chitosan oligosaccharide and acaran sulfate treated group showed a significant decrease (p <0.01) and elevated TG levels also showed a significant decrease (p <0.01, p <0.001). On the other hand, in the case of HDL-cholesterol reduced by the administration of the cholesterol-containing mixture, the value was increased by the administration of acaran sulfate (see FIG. 2 ).

Next, the present inventors confirmed the effect of the acaran sulfate of the present invention on total cholesterol, HDL-cholesterol and TG concentration in hyperlipidemic rat animal model induced by poloxamer-407 (Pluronic F-127) solution.

As a result, in the present invention, serum cholesterol and TG levels were increased by 15 and 50 times, respectively, by poloxamer-407 administration. In comparison, the lovastatin-administered group, compared with the control group, lowered cholesterol and TG levels by 36.9% and 35.1%, respectively, and showed excellent hypolipidemic effect. On the other hand, even in the acaran sulfate-administered group showed a significant decrease in cholesterol and TG levels, respectively (see Figure 3 ).

The mechanism by which poloxamer-407 induces hyperlipidemia has not been elucidated, but recent reports suggest that the activity of HMG-CoA (3-hydroxyl-3-methylglutaryl coenzyme) reductase and lipoprotein lipase (LPL) Is known to be inhibited (Thomas P., Johnston and Warren K. Palmer, Biochemical Pharmacology , 1993 , 46, 1037-1042). The results suggest that acaran sulfate is likely to be an effective substance for improving arteriosclerosis as well as general lipid metabolism.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Determination of the Effect of Acaran Sulfate on Hyperlipidemia Induced by Cholesterol-containing Mixtures

<1-1> Separation and Purification of Acaran Sulfate

In order to confirm the effect of the acaran sulfate of the present invention on hyperlipidemia, the present inventors first separated the acaran sulfate from an African chameleon ( Achatina fulic a Bowdich).

African snails used in the present invention were purchased from a snail farm in Yongin, Gyeonggi-do, Kim et al. (Kim YS., JO YY., Chang IM., Toida T., Park Y., J. Biol. Chem. , 1996 , 271, 11750-5), acaran sulfate was isolated and purified, and the structure was confirmed by ¹ H-NMR analysis ( Table 1 and Figure 1 ).

¹ H-NMR data of acaran sulfate L-IdoAp2S D-GlcNpAc H-1 5.189 5.114 H-2 4.345 4.020 H-3 4.284 3.74 H-4 4.027 3.74 H-5 4.930 3.867 H-6 3.87, 3.90 NAc 2.083

As a result, the present inventors separated the acaran sulfuric acid represented by the following formula (1 ) and used in subsequent experiments.

<1-2> Measurement of effects on cholesterol-induced hyperlipidemia

We have identified the effects of acaran sulfate of the invention on total cholesterol, HDL-cholesterol and TG concentrations in hyperlipidemic rat animal models induced by cholesterol-containing mixtures.

About 220 g of male SD (Sprague-Dawley) rats were used for the experiment, and were used at the experimental animal breeding environment for at least one week after being sold at the experimental animal breeding ground of Seoul National University. The kennel was adjusted to 12 hours from 8 o'clock in the morning to 8 o'clock by artificial lighting, and the room temperature was maintained at 18 ° C to 23 ° C. Feed was used as a solid feed composition containing 21.0% crude protein, 3.5% crude fat, 5.0% crude cellulose and 8.0% minerals.

First, the corn oil mixture containing 1% cholesterol and 1% sodium cholate was orally administered to male SD rats for 5 consecutive days. Lan sulfate (100 mg / kg) was orally administered once a day for 5 days. Animals not administered acaran sulfate were used as controls. Blood was taken from animals 12 hours after the last sample administration and the total cholesterol, HDL-cholesterol and TG concentrations in serum were measured and compared with normal and control groups. In this case, chitosan oligosaccharides were used as a comparative drug at a concentration of 100 mg / kg / day.]

Serum total cholesterol was measured using the cholesterolenolenzyme-V (Eiken Chem. Co.) kit, and neutral lipid was measured by the enzyme method by Kim et al. Using TG-V (Eiken Chem. Co.) kit. , HDL total cholesterol was measured by the precipitation method by Kim et al. Using the HDL-C 555 (Eiken Chem. Co.) kit (Kim JC., 臨 床 檢査 法 提要, 1983 , 29, 456-474).

All experimental results were expressed using the mean and standard error. The comparison between the groups was Student's t- test, and it was statistically significant when the p value was less than 0.05 compared to the control group.

As a result, it was observed that the administration of cholesterol-containing mixtures significantly increased serum cholesterol and TG levels, resulting in relative hyperlipidemia compared with the normal group, whereas HDL-cholesterol was decreased. In the case of serum cholesterol, both chitosan oligosaccharide and acaran sulfate treated group showed a significant decrease (p <0.01) and elevated TG levels also showed a significant decrease (p <0.01, p <0.001). On the other hand, in the case of HDL-cholesterol reduced by the administration of the cholesterol-containing mixture, the value was increased by the administration of acaran sulfate ( FIG. 2 ).

Example 2 Measurement of the Effect of Acaran Sulfate on Hyperlipidemia Induced by Poloxamer-407

Next, the present inventors confirmed the effect of the acaran sulfate of the present invention on total cholesterol, HDL-cholesterol and TG concentration in hyperlipidemic rat animal model induced by poloxamer-407 (Pluronic F-127) solution.

First, male SD rats (380 ± 20 g) bred under the same conditions as in Example <1-2> were used as experimental animals. Acaranic sulfate at 100 mg / kg and 300 mg / kg concentrations dissolved in physiological saline was orally administered once a day for 3 days, and prepared on the fourth day according to the cold method (Schmollka IR., Polymers and Controlled Drug Delivery , 1991 , 189, CRC press) 1 ml of poloxamer-407 solution, previously dissolved in secondary distilled water and refrigerated overnight, was intraperitoneally administered. The lovastatin used as a comparative drug was mixed well in secondary distilled water with 0.4% of carboxymethylcellulose (w / v) and 3.3% of glycerin (v / v) before administration. Suspension was used. The sample was administered again for 2 consecutive days, and the rats fasted for 12 hours before the experiment were collected from the abdominal vein under ether anesthesia to obtain serum.

Intraperitoneal administration of 1 ml of poloxamer-407 solution in normal rats is reported to increase serum cholesterol by 10-15 fold and TG by 40-50 fold (M. Sugano, T. Fujikawa, Y. Hiratsuji, K). Nakashima, N. Fukuda, Y. Hasegawa, Am. J. Clin.Nutr., 1980, 33, 787-793; Warren K. Palmer, Euge E. Emeson, Thomas P. Johnston, Atherosclerosis , 1998, 136, 115. -123). In the present invention, poloxamer-407 administration increased serum cholesterol and TG levels by 15 and 50 times, respectively. In comparison, the lovastatin-administered group, compared with the control group, lowered cholesterol and TG levels by 36.9% and 35.1%, respectively, and showed excellent hypolipidemic effect. On the other hand, acaran sulfate administration group also showed a significant reduction in cholesterol and TG levels respectively ( FIG. 3 ).

The mechanism by which poloxamer-407 induces hyperlipidemia has not been elucidated, but recent reports have suggested that the activity of HMG-CoA (3-hydroxyl-3-methylglutaryl coenzume) reductase and kipoprotein lipase (LPL) Is known to be inhibited (Thomas P., Johnston and Warren K. Palmer, Biochemical Pharmacology , 1993 , 46, 1037-1042). The results suggest that acaran sulfate is likely to be an effective substance for improving arteriosclerosis as well as general lipid metabolism.

Example 3 Parenteral Administration Acute Toxicity in Rats

Acute toxicity test was performed using 6-week-old SPF SD rats. Two animals per group were suspended parenterally in a dose of 1 g / kg / 15 ml by suspending the acaran sulfate of the invention in a 0.5% methylcellulose solution. After administration of the test substance, mortality, clinical symptoms, and changes in body weight were observed. Hematological and hematological examinations were performed. Necropsy was performed to observe abdominal and thoracic organ abnormalities. As a result, there were no clinical symptoms or deaths in all animals treated with the test substance, and no toxicity change was observed in weight change, blood test, blood biochemistry test, autopsy findings, etc. As a result, the tested acaran sulfate of the present invention did not show a toxicity change in rats up to 3,000 mg / kg, and the parenteral administration minimum dose (LD ₅₀ ) was determined to be a safe substance of 500 mg / kg or more.

As described above, acaran sulfate, a glycosaminoglycan isolated from the body of an African king snail of the present invention, lowers the levels of TG and total cholesterol in serum, while raising HDL-cholesterol levels, thereby preventing hyperlipidemia and It has been confirmed that it can be usefully used for treatment.

Claims (3)

A pharmaceutical composition for preventing and treating hyperlipidemia comprising acaran sulfate and a pharmaceutically acceptable salt thereof as an active ingredient. The pharmaceutical composition for preventing and treating hyperlipidemia according to claim 1, wherein the pharmaceutical composition lowers the level of triglyceride (TG). The pharmaceutical composition for preventing and treating hyperlipidemia according to claim 1, wherein the pharmaceutical composition lowers the level of total cholesterol.