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Application of kaempferol-3-O-rutinoside to preparation of medicine for treating PCSK9 (Proprotein convertase subtilisin/kexin type 9)-mediated disease

A technology of rutinoside and kaempferol, applied in the field of treating PCSK9-mediated diseases, can solve problems such as pain and allergies, poor compliance of PCSK9 monoclonal antibody injections, and high price

Inactive Publication Date: 2018-09-25
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, PCSK9 monoclonal antibody drugs are all injections, and clinical adverse reactions such as swelling at the injection site, pain, and allergies are often shown, and the use compliance is poor.
The research and development costs of monoclonal antibody drugs are high and expensive. For example, alirocumab and evolocumab require high treatment costs of US$14,300 / year and US$14,523 / year, respectively, and long-term use makes patients overwhelmed
In recent years, people have gradually focused on the development of small molecule PCSK9 inhibitors to overcome the disadvantages of poor patient compliance and high price of PCSK9 monoclonal antibody injections

Method used

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  • Application of kaempferol-3-O-rutinoside to preparation of medicine for treating PCSK9 (Proprotein convertase subtilisin/kexin type 9)-mediated disease
  • Application of kaempferol-3-O-rutinoside to preparation of medicine for treating PCSK9 (Proprotein convertase subtilisin/kexin type 9)-mediated disease
  • Application of kaempferol-3-O-rutinoside to preparation of medicine for treating PCSK9 (Proprotein convertase subtilisin/kexin type 9)-mediated disease

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1, the influence of kaempferol-3-O-rutinoside on TG, TC, HDL-C and LDL-C of hyperlipidemia mice

[0020] 1. Experimental animals

[0021] Healthy female ICR mice, clean grade, weighing 18-22 g, were provided by the Animal Center of Yangzhou University. The experimental animals were raised in an independent environment with 12h-12h alternation of day and night, the room temperature was maintained at 24±2°C, water and food were free to drink, and the experiment was carried out after 1 week of adaptation to the environment. All handling of animals followed the requirements of the Ethics Committee of the International Association for the Study of Pain.

[0022] 2. Experimental materials

[0023] Kaempferol-3-O-rutinoside (Chengdu Ruifensi Biotechnology Co., Ltd.). Basic feed and high-fat feed (containing 3% cholesterol). Triglyceride (TG) test kits, total cholesterol (TC) test kits, low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein chol...

Embodiment 2

[0035] Embodiment 2, expression and purification of PCSK9 and EGF-A protein

[0036]PCSK9 mainly binds to the EGF-A domain of LDLR, leading to the degradation of LDLR, reducing the ability of cells to absorb LDL, and exerting its activity in regulating lipid metabolism. In order to facilitate purification and subsequent detection, the expression and purification of PCSK9 and EGF-A proteins were performed separately. A His tag was added to the N-terminal of PCSK9, and a GST tag was added to the N-terminal of EGF-A. His-PCSK9 [12] and GST-EGF-A [13] The expression and purification of protein were carried out according to literature. His-PCSK9 crude bacterial solution was adsorbed by Ni column, and imidazole was eluted sequentially to obtain His-PCSK9 protein ( figure 1 ). The GST-EGF-A crude bacterial liquid is captured by the GST adsorption column, and glutathione is eluted to obtain the GST-EGF-A protein ( figure 2 ).

Embodiment 3

[0037] Example 3. Research on the mechanism of kaempferol-3-O-rutinoside inhibiting PCSK9

[0038] 3.1 PCSK9-induced LDLR degradation cell model and compound intervention

[0039] 3.1.1 Experimental materials

[0040] Kaempferol-3-O-rutinoside (Chengdu Ruifensi Biotechnology Co., Ltd.). Tris, TEMED, β-Mercaptoethanol, glycine, Tween-20, SDS, 30% Acrylamide / Bis, Ammonium Persulfate, GAPDH were purchased from sigma; Marker was purchased from Thermo; bromophenol blue, glycerin, Bovine SerumAlbumin were purchased from Sunshine; methanol, Sodium chloride and glycerol were purchased from Nanjing Chemical Reagent Co., Ltd.; LDLR antibody and anti-rabbit were purchased from cellsignaling; RIPA lysate was purchased from Beyotime Biotechnology; ECL luminescent liquid was purchased from Pierce.

[0041] HepG2 human liver cancer cells were purchased from ATCC, USA.

[0042] 3.1.2 Experimental grouping

[0043] Take HepG2 human liver cancer cells in the logarithmic growth phase, digest...

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Abstract

The invention relates to the lipid-lowering field of kaempferol-3-O-rutinoside, and relates to application of kaempferol-3-O-rutinoside to preparation of PCSK9 (Proprotein convertase subtilisin / kexintype 9) target inhibition medicine. Kaempferol-3-O-rutinoside is firstly found to have specific activity for inhibiting the binding of PCSK9 to LDLR (Low-Density Lipoprotein Receptor), can be subjected to affinity binding with an active pocket area of a PCSK9 crystal structure, and form stable hydrogen bonds with a plurality of amino acid residues, thereby being a novel small molecule inhibitor for PCSK9. The kaempferol-3-O-rutinoside can reduce the levels of TC (Total Cholesterol), TG (Triglyceride) and LDL-C (Low-Density Lipoprotein Cholesterol) in plasma of hyperlipidemia model mice, and improve the level of HDL-C (High-Density Lipoprotein cholesterol). The kaempferol-3-O-rutinoside has the effect of alleviating lipid metabolism disorder of hyperlipidemia and can be used for preventingor treating cardiovascular diseases (including the hyperlipidemia).

Description

technical field [0001] The invention relates to the application of kaempferol-3-O-rutinoside in the preparation of PCSK9 inhibitors, which can be used for treating diseases mediated by PCSK9. Background technique [0002] Hyperlipidemia, mainly elevated low-density lipoprotein cholesterol (LDL-C), is one of the risk factors for cardiovascular disease. The current lipid-lowering drugs of choice in clinical practice are statins. However, long-term clinical use of statins is likely to cause side effects such as liver toxicity and myalgia. [1] . And more than 50% of patients with coronary heart disease, after using intensive statin therapy, LDL-C still fails to reach the target value recommended by relevant guidelines, and the effect on reducing the incidence and mortality of coronary heart disease is not significant [2] . In order to make up for the limitations of the application of statins and solve the clinical needs, the research of new lipid-lowering drugs will become a...

Claims

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Application Information

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IPC IPC(8): A61K31/7048A61P3/06
CPCA61K31/7048A61P3/06
Inventor 刘吉华刘秀峰李丽
Owner CHINA PHARM UNIV
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