WO2004058245A1

WO2004058245A1 - Tanshinone iia for prophylaxising or treating atherosclerosis

Info

Publication number: WO2004058245A1
Application number: PCT/CN2002/000944
Authority: WO
Inventors: Lianquan Gu; Peiqing Liu; Guihua Li; Min Huang
Original assignee: Zhongshan Univertsity
Priority date: 2002-12-31
Filing date: 2002-12-31
Publication date: 2004-07-15
Also published as: AU2002362159A1

Abstract

This invention related to the use of Tanshinone IIA for preparing drugs for prophylaxising or treating atherosclerosis and those correlating diseases. The test indicated that Tanshinone IIA could be used to reduce the deposit of cholesterol in the arterial walls, thus avaoiding the formation of atheromatous plaques and preventing atherosclerosis. The chemical formula of Tanshinone IIA was showed as follow.

Description

Tanshinone ΠΑ for preventing and treating arteriosclerosis TECHNICAL FIELD

The present invention relates to the use of tanshinone IIA for the preparation of a medicament for preventing and treating atherosclerosis and atherosclerosis-related diseases, and a method for preventing and treating atherosclerosis and atherosclerosis-related diseases with tanshinone ΠΑ. Background of the invention

Atherosclerosis is a systemic disease related to disorders of lipid metabolism. The lesions are characterized by liposomes in the blood, especially cholesterol deposited in the intima of the arterial wall to form atherosclerotic plaques, leading to arteriosclerosis. . When atherosclerosis occurs in important organs such as the heart and brain, it will cause ischemic changes in tissues and cause serious consequences, such as angina pectoris, myocardial infarction, cerebral infarction, renal tissue infarction, and arterial gangrene of the limbs. It is a serious threat to human health today. Common diseases (pharmacology, edited by Li Yuling, People's Medical Publishing House, 2000, p 82-88).

The causes of atherosclerosis are complex and the pathogenesis is not fully understood. Factors that have been identified as being associated with arteriosclerosis include:

1. Hyperlipidemia: Hyperlipidemia caused by pathological changes or diet, especially LDL content is too high (containing the most cholesterol, and the lipoprotein molecules are small, easy to enter the lining of the blood vessel wall), which can easily cause cholesterol in the Deposition of Intima of Arterial Vessel Wall.

2. Hypertension: The blood vessel wall of patients with hypertension is easily damaged, which facilitates the entry of lipoproteins into the lining of the vascular wall and causes the deposition of cholesterol on the lining of the arterial vascular wall.

3. Diabetes: Hyperglycemia can cause dyslipidemia, which helps LDL to enter the intima of blood vessel walls.

It can be seen that the high cholesterol concentration in the blood and its intimal deposition in the arterial vessel wall are the most important causes of atherosclerosis.

The current drugs for treating atherosclerosis are mainly to reduce the concentration of cholesterol in the blood by reducing the biosynthesis of cholesterol or accelerating the breakdown of cholesterol. For example: Lifibmte's role is to reduce the synthesis of cholesterol, while Simvastatin and Colestyramine are used to promote the breakdown of cholesterol. Since these drugs are mainly achieved by affecting metabolic activities in the body, they have significant side effects and are not suitable for long-term use. Moreover, the treatment effect is not ideal, and the cholesterol deposition rate is generally reduced by 20-30% [Drug, Wang Rulong, Chief Editor of Yuan Zhengping, Chemical Industry Press, Third Edition (1999), p 544-559].

Salvia miltiorrhiza is the dry roots and rhizomes of the salvia salvia 0¾ / Wa m Utiorrhiza Bunge, which is bitter, slightly cold, attentive to heart, and liver meridian. As a traditional Chinese medicine, it is widely used in clinical prescriptions. It is mainly used for removing stasis and pain, promoting blood circulation, clearing the heart and removing annoyance. Tanshinone IIA is the main component of fat-soluble extract of Salvia miltiorrhiza. To date, there has been no report on the use of a single component of tanshinone IIA for the prevention and treatment of atherosclerosis. The object of the present invention is to provide a new medical application of tanshinone IIA or a pharmaceutical composition thereof as shown in formula I for the prevention and treatment of atherosclerosis and atherosclerosis-related diseases. Tanshinone IIA represented by formula I provided by the present invention refers to a compound isolated from natural salvia or a compound obtained by applying a chemical synthesis method.

The inventors have found through research that tanshinone IIA can enter the bloodstream, and by competing with cholesterol, it can reduce and clear the deposition of cholesterol on the intima of the blood vessel wall to avoid the formation of atheromatous plaques, showing that tanshinone IIA has the ability to prevent and treat atheroma Good prospects for sclerosis and atherosclerosis-related diseases.

The present invention is a new type for the prevention and treatment of atherosclerosis and atherosclerosis-related diseases, which is developed based on the mechanism of action of tanshinone IIA having the ability to compete with cholesterol for the binding (deposition) site of the arterial wall intima drug. Summary of the Invention

An object of the present invention is to provide a method for preventing and treating arteriosclerosis caused by the deposition of cholesterol on the inner wall of arterial blood vessels, and diseases related to atherosclerosis, using tanshinone IIA or a pharmaceutical composition thereof shown in Formula 1 below. Use in medicine.

The structure of tanshinone IIA according to the present invention is shown in Formula I.

I

Our research shows that tanshinone ΠΑ compounds have a good similarity to cholesterol in terms of structure and shape, physical properties, properties of water-soluble groups and hydrophobic groups, and distribution in the molecule. By competing with cholesterol, Reduce and clear the deposition of cholesterol on the intima of the blood vessel wall, avoid the formation of atheromatous plaques, and prevent and treat atherosclerosis and atherosclerosis-related diseases.

By conducting in vitro experiments and animal pharmacological experiments, the present invention determines that tanshinone ΠΑ can reduce the deposition of cholesterol on the intima of the blood vessel wall and avoid the formation of atheromatous plaques. Effective medicine for sclerosis and atherosclerosis-related diseases.

In the present invention, the tanshinone IIA compound or a pharmaceutical composition containing the compound can be used in the form of an injection, a tablet, a pill, a capsule, a solution, a suspension, or an emulsion.

In the present invention, the administration route of the tanshinone IIA compound or the pharmaceutical composition containing the compound includes oral administration, transdermal administration, intravenous administration, or intramuscular administration.

In the present invention, the oral dose of the tanshinone IIA compound is 50-200 mg / kg body weight / day. The present invention also provides a medicament for preventing and treating atherosclerosis, which medicament contains the above tanshinone IIA compound and any one or more pharmaceutically acceptable adjuvants or carriers. The present invention also provides a method for preventing and treating atherosclerosis, which method comprises treating a patient with an effective amount of a tanshinone IIA compound or a pharmaceutical composition containing the compound.

The tanshinone ΠA compound according to the present invention is a compound obtained by extraction and isolation from Salvia miltiorrhizae [Salvia-biology and its application, edited by Xu Rensheng, Science Press, 1990, p 83-87], or a compound obtained by chemical synthesis [Yoshinobu Inouye and Hiroshi Kakisawa, Total syntheses of tansliinone-I, tanshinone-II and cryptotanshinone, Bulletin of the Chemical Society of Japan, 1969, 42, 3318-3323]. BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a photographic schematic diagram of a normal control group in the observation of tanshinone IIA on plaque formation in rabbits with dyslipidemia.

Fig. 2 is a schematic diagram of a control group of dyslipidemia model in the observation of tanshinone IIA on arterial wall plaque formation in rabbits with hyperlipidemia.

Fig. 3 is a photographic view of the control group of the positive drug cholestyramine in the observation of tanshinone III on the arterial wall plaque formation in rabbits with hyperlipidemia.

FIG. 4 is a schematic photograph of the control group of the positive drug, neovastatin, in the observation of tanshinone IIA on the arterial wall plaque formation in rabbits with hyperlipidemia.

FIG. 5 is a schematic diagram of a control group of a high-dose tanshinone III group in the observation of tanshinone IIA on arterial wall plaque formation in rabbits with hyperlipidemia.

FIG. 6 is a photographic schematic diagram of a medium-dose group of tanshinone IIA in the observation of tanshinone IIA on arterial wall plaque formation in rabbits with hyperlipidemia.

FIG. 7 is a photographic schematic diagram of the tanshinone IIA low-dose group in the observation of tanshinone IIA on arterial wall plaque formation in rabbits with hyperlipidemia. The present invention is further described below through examples. These examples are only used to illustrate the implementation method and effect of the present invention, and do not limit the present invention in any form. Example 1: Extraction, Isolation and Purification of Tanshinone ΠΑ in Salvia

Dry 1 kg of Salvia miltiorrhizae and extract 3 times with 95% ethanol for 24 hours each time. The extract was concentrated to 500 mL under reduced pressure, then 500 mL of water was added, and extracted with 1,000 mL of chloroform four times. The extract was concentrated under reduced pressure. Analytical separation, silica gel is 100-200 mesh, the eluent is a petroleum ether / ethyl acetate mixed solution containing 1% -10% ethyl acetate, and gradient elution is performed. Tanshinone IIA was obtained at about 0.85 g. Example 2: Experiment of Tanshinone ΠΑ Reducing Cholesterol Deposits in Arterial Vessel Wall of Hyperlipemia Animal Model (Rat)

The experimental animals were 48 adult male rats (Sprague-Dawley), weighing 150-200 g. Minute Normal control group, hyperlipidemia model control group, positive drug cholestyramine (500 mg / kg) control group, tanshinone IIA high dose (300 mg / kg), medium dose (100 mg / kg), low dose (30 mg / kg) administration group. Except for the normal control group, the other groups were fed with high cholesterol and lipid materials for 45 consecutive days.

High cholesterol and lipid feed formula: 3% cholesterol, 10% lard, 0.2% methylthiouracil and 88% basic corpus.

Arterial vessel wall cholesterol determination method: isopropanol extraction, high-iron-acetic acid-sulfuric acid colorimetric method (Xu Shuyun, "Pharmacological Experimental Methodology" 2nd edition, P1031 "Determination of blood lipids", People's Medical Publishing House 1994). The experimental results are shown in Table 1. Table 1. Effects of tanshinone ΠΑ on cholesterol content in arterial vessels of hyperlipidemia rats

* Calculation method of reduction rate in this table and the following tables: Reduction rate (%) = [1-(content of drug treatment group-normal control group content) / (hyperlipidemia model control group content-normal control group content]] X 100% ** High dose has a certain effect on rat appetite, and the measurement data has a large error. Example 3: Effect of tanshinone ΠΑ on serum cholesterol concentration in an animal model of hyperlipidemia (rat). The experimental animals were 48 adult male rats (Sprague-Dawley), weighing 150-200 g. Normal control group, hyperlipidemia model control group, positive drug cholestyramine (500 mg / kg) control group, tanshinone ΠΑ 酮 dose (300 mg / kg), medium dose (100 mg / kg), low dose ( 30 mg / kg) administration group. Except the normal control group, the other groups were fed with high cholesterol and lipid feed for 45 consecutive days.

High cholesterol and lipid feed formula: 3% cholesterol, 10% lard, 0.2% methylthiouracil and 88% basal feed.

Determination method of serum cholesterol content: High iron-acetic acid-sulfuric acid colorimetric method. The experimental results are shown in Table 2. Table 2. Effects of Tanshinone ΠΑ on serum cholesterol concentration in hyperlipidemia rats

Group Serum cholesterol concentration reduction rate (%)

(mg / dL)

Normal control group 37.5 ± 10.8

Hyperlipidemia model control group 574.6 ± 95. 3 —— Positive drug cholestyramine control group 379.2 ± 88. 5 34-Tanshinone ΠΑ high-dose group 418. 9 ± 104. 7 Animal intake of less tanshinone ΠA medium-dose group 552.8 8 ± 84. 6 No significant reduction in tanshinone ΠΑ The low-dose group 564.7 ± 91.8 did not decrease significantly. The results of this experiment showed that tanshinone IIA did not affect the metabolism of cholesterol. It is shown that tanshinone IIA reduces the intimal cholesterol deposition in arterial blood vessel walls through a new mechanism of action, that is, the competition mechanism. Example 4: Experiment on Tanshinone ΠΑ Reducing Cholesterol Deposition in Arterial Vessel Wall of Hyperlipidemia Animal Model (Rabbit)

The experimental animals were 105 healthy ordinary New Zealand rabbits, half male and half female, weighing 2.0-2.5 kg. Normal control group, hyperlipidemia model control group, positive drug cholestyramine (500 mg / kg) control group, positive drug simvastatin (simvastatin, Shu Jiangzhi, 5 mg / kg, distributed by Merck China Ltd.) In the control group, tanshinone IIA was administered in a high dose (300 mg / kg), a medium dose (100 mg / kg), and a low dose (30 mg / kg). Except for the normal control group, the other groups were fed with high cholesterol and lipid feed for 90 consecutive days.

Arterial vessel wall cholesterol determination method: isopropanol extraction, high iron-acetic acid-sulfuric acid colorimetry. The experimental results are shown in Table 3. Table 3. Effects of tanshinone ΠΑ on cholesterol content in arterial wall of hyperlipidemia rabbits

* High doses have an effect on appetite in rats, and the measurement data have large errors. Example 5: Effect of Tanshinone ΠΑ on serum cholesterol concentration in an animal model of hyperlipidemia (rabbit)

The experimental animals were 36 healthy New Zealand rabbits, half male and half male, weighing 2.0-2.5 kg. Normal control group, hyperlipidemia model control group, positive drug cholestyramine (500 mg / kg) control group, positive drug neovastatin (5 mg / kg) control group, tanshinone ΠΑ high dose (300 mg / kg :), medium dose (100 mg / kg), Low dose (30mg / kg) administration group. Except for the normal control group, the other groups were fed with cholesterol and lipid materials for 45 consecutive days.

High cholesterol and lipid corpus formula: 3% cholesterol, 10% lard, 0.2% methylthiouracil and 88% basic feed.

Determination of serum cholesterol content: High iron-acetic acid-sulfuric acid colorimetric method. The experimental results are shown in Table 4. Table 4.Effect of Tanshinone ΠΑ on serum cholesterol concentration in hyperlipidemia rabbits

The results of this experiment show that tanshinone IIA does not significantly affect cholesterol metabolism. It is shown that tanshinone IIA P low-arterial vascular wall intimal cholesterol deposition is achieved through a new mechanism of action, that is, the competition mechanism. Example 6: Effect of Tanshinone ΠΑ on Plaque Formation in Arterial Wall of Rabbit Model of Lipidemia

According to the conditions of the fourth embodiment. The middle segment of the thoracic aorta of the rabbits was taken, and the optical density of the arterial strips was scanned by the IBAS image processing system and integrated. The results are shown in Table 5. Table 5.Effect of Tanshinone ΠΑ on Arterial Wall Integral Optical Density in Rabbits with Hyperlipidemia

Relative integral optical density reduction rate of arterial wall in group (%) Normal control group 0.31 ± 0.07 — Hyperlipidemia model control group 1.06 ± 0.25 — Positive drug cholestyramine control group 0.78 ± 0.19 37.3 Positive drug neovastatin control group 0.51 ± 0.15 73.3 Tanshinone ΠΑ high-dose group 0.57 ± 0.13 65.3 Tanshinone ΠA medium-dose group 0.64 ± 0.16 56.0 Tanshinone ΠA low-dose group 0.82 ± 0.30 32.0 The results showed that the tanshinone IIA high and medium dose groups could significantly reduce the cholesterol sensitivity density of the arterial strips (relative optical density was 0.57 ± 0.13 and 0.64 ± 0.16, respectively). 0.51 ± 0. 15) similar.

In this experiment, in order to observe the effect of tanshinone ΠΑ on the formation of plaques in the aortic wall of hyperlipidemia rabbits, the middle section of the thoracic aorta of rabbits in each group was photographed. . Figures 1 to 7 show that tanshinone IIA high and medium dose groups can significantly reduce plaque formation in the arterial wall, and the results are similar to those of the lipid-lowering drug simvastatin group. Example 7: Effect of Tanshinone IIA on Aortic Tube Wall Plaque Formation in Apolipoprotein E Deficient Mice

Experimental method: 150 apolipoprotein E-deficient mice (male, provided by School of Public Health, Sun Yat-sen University) were divided into 3 periods of 14 weeks, 24 weeks, and 34 weeks, and each week was divided into 6 groups. : Tanshinone II A group (300mg / kg / day, 100mg / kg / day and 30mg / kg / day), the lipid-lowering drug neovastatin control group (5mg / kg / day) and cholestyramine (500mg / kg / Day) control group, positive control group (perfusion of normal saline). The amount of gastric juice was 0.1 ml / 10 g body weight. Dosing time: 4 weeks. At 14 weeks, 24 weeks and 34 weeks of age, 0.5% to 1.0ml of 1% pentobarbital was administered to the mice by intraperitoneal anesthesia. Blood was taken from the heart under sterile conditions, and total cholesterol was measured by conventional biochemical methods (see Table 6 for results) Take the aortic root blood vessels and thoracic aorta, and perform a conventional histochemical examination of the aortic (sinus) root continuously in sections, taking 1 at 5 intervals and HE staining. The sections were analyzed with Leica Q550 IW image analyzer and Qwin image analysis and processing software for morphological measurement. The average of 6 consecutive sections of each mouse blood vessel was used for statistical analysis. Measurements include vascular lumen area (LA), plaque area (PA), and plaque area to lumen area ratio (PA / LA). Computer image scanning quantitative analysis (see Table 7 for results).

Results: Lipid streaks were noticeable in 50% of apolipoprotein E-deficient mice in the 14-week-old positive control group, and no obvious lesions were detected in the tanshinone ΠΑ 髙, medium-dose and neovastatin groups. % Small plaques appear. Small plaque appeared in 10% of the lipid-lowering drug cholestyramine control group. At 24 weeks of age, 80% of control rats had plaque formation, while small plaque formation rates of the tanshinone II A high, medium, and low dose groups and the neovastatin and cholestyride control groups were 40%, 50%, and 60%, respectively. And 40%, 50%. At 34 weeks of age, plaque formation was seen in both the treatment group and the control group, but compared with the control group, the area of the aortic plaque and the area of the lumen in the treatment group were significantly reduced, but it had a role in reducing cholesterol. Wireless association.

Table 6.Effect of Tanshinone ΠΑ on total cholesterol (g / L) in plasma of apolipoprotein E-deficient mice (X soil s)

Group 14 weeks 24 weeks 34 weeks Normal mice group 24. 3 ± 4. 2 26. 4 ± 5. 1 27. 9 ± 6. 2 Apolipoprotein E-deficient mouse group 38.9 ± 5. 6 50. 3 ± 6. 2 54. 2 ± 5. 7 Positive drug cholestyramine control group 34.7 ± 3. 9 42. 5 ± 4. 3 46. 2 ± 5. 4

• Positive drug simvastatin control group 31.5 ± 4 4 36. 0 ± 5. 1 41. 3 ± 6. 0 Tanshinone ΠΑ high-dose group 34.2 ± 5. 8 45.1 ± 4. 0 49. 1 ± 6. 8 Tanshinone IIA medium dose group 36.7 ± 5. 3 45. 8 ± 6. 9 51. 4 ± 5. 6 Tanshinone IIA low dose group 37.1 ± 4. 6 47. 6 ± 6. 5 52 2 ± 4. 9 Table 7. Effect of Tanshinone ΠΑ on the formation of atherosclerotic lesions in apolipoprotein E-deficient mice (mni ² , x ± s)

Lumen area (LA), plaque area (PA), and plaque area to lumen area ratio (PA / LA) Example 8: Tanshinone ΠΑ Toxicology Test

Acute toxicity test in mice: The experimental animals were healthy male mice.试验 Tested by gavage. At the dose of 25 g / kg, no symptoms of poisoning and death were found in the test mice. The test showed that cryptotanshinone LD ₅ . > 25 g / kg.

Mice 30-day toxicity test: The experimental animals were healthy male mice. The test was performed by gavage, and the dose was 100 mg / kg / day for 30 consecutive days. The test showed that there was no death, no significant abnormal changes or toxic reactions in the growth and development, hematopoietic function, and biochemical indicators of the rats; no pathological changes were found in the anatomy and tissue microscopy of important organs. Example 9: Test of Tanshinone IIA Entering Blood

The experimental animals were healthy male rats.试验 Tested by gavage. The dose was 100 mg / kg / day. 5 consecutive days. One hour after the 5th day of administration, blood was sacrificed and extracted with chloroform. A significant amount of tanshinone IIA could be detected by HPLC. Industrial applicability

The above experiments of the present invention show that:

(1) The effects of tanshinone ΠΑ on the deposition of cholesterol in the intima of arterial blood vessels in an animal model of hyperlipidemia have shown that tanshinone ΠΑ can reduce and eliminate the deposition of cholesterol on the lining of blood vessels.

(2) Experiments on the effects of tanshinone ΙΙΑ on atherosclerotic plaque formation in animal models of hyperlipidemia show that tanshinone ΠΑ can effectively inhibit cholesterol from forming plaques on the intima of arterial blood vessel walls.

(3) The effects of tanshinone ΠΑ on serum cholesterol concentration in animal models of hyperlipidemia showed that tanshinone ΙΙΑ did not affect the metabolism of cholesterol. It shows that tanshinone ΠΑ can reduce the intimal cholesterol deposition of arterial blood vessel wall through a new mechanism of action, that is, the competition mechanism.

(4) The tanshinone IIA shown in Formula I of the present invention, its toxicological test shows that at normal dosage, its effect It is safe for drug application.

(5) The normal oral dose of tanshinone IIA or its pharmaceutical composition according to Formula I of the present invention is 50-200 mg of tanshinone IIA / kg body weight / day.

(6) Compared with the existing commonly used drugs for treating hyperlipidemia, the compound of the present invention has the characteristics of better therapeutic effect, smaller side effects and wider application range.

(7) The tanshinone IIA shown in Formula I of the present invention can enter the bloodstream, and through the competition with cholesterol, it can reduce and clear the deposition of cholesterol on the intima of the blood vessel wall to avoid the formation of atheromatous plaques. It can be used for preparing medicines for preventing and treating atherosclerosis and atherosclerosis-related diseases.

Claims

Rights request

1. Use of a tanshinone IIA compound represented by the following formula I or a pharmaceutical composition containing the compound for the manufacture of a medicament for treating atherosclerosis and atherosclerosis-related diseases.

2. The use according to claim 1, wherein the tanshinone IIIA is a compound isolated or purified from natural salvia miltiorrhiza or a chemically synthesized compound.

3. The use according to claim 1 or 2, wherein the tanshinone ΠΑ can enter the blood, and through the competition with cholesterol, it can reduce and eliminate the deposition of cholesterol on the intima of the blood vessel wall, avoid the formation of atheromatous plaques, and achieve prevention And the purpose of treating atherosclerosis and atherosclerosis-related diseases.

4. The use according to any one of claims 1 to 3, wherein the medicament is used in the form of an injection, a tablet, a pill, a capsule, a solution, a suspension, an emulsion.

5. The use according to any one of claims 1 to 4, wherein the route of administration of the drug comprises oral, transdermal, intravenous or intramuscular.

6. The use according to any one of claims 1 to 5, wherein the oral dose of tanshinone III is 50-200 mg / kg body weight / day.

7. A medicament for preventing and treating atherosclerosis, comprising tanshinone IIA according to claim 1 and any one or more pharmaceutically acceptable adjuvants or carriers.

8. A method for preventing and treating atherosclerosis, comprising treating a patient with an effective amount of tanshinone IIA or a pharmaceutical composition thereof according to claim 1.