WO2016115840A1 - Uses of 2,3,5,4'-tetrahydroxystilbene-2-o-β-d-glucoside in preparation of inhibitor for tlr4 innate immune system - Google Patents

Abstract

Uses of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside and a pharmaceutically acceptable salt or ester thereof in the preparation of an inhibitor for a TLR4 innate immune system. The 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside can alleviate a TLR4 innate immune response system. A result of ELISA protein detection shows that 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside can make the content of TLR4 reduce by 56% and make the expression of NF-κB reduce by 23% compared with those of a model group, and accordingly effectively restrains the release of inflammatory factors.

Description

Application of stilbene glycoside in the preparation of inhibitors of TLR4 innate immune system Technical field

The present invention relates to pharmacological activity studies, and in particular to the inhibitory effect of stilbene glycoside on the innate immune system of TLR4.

Background technique

With the increasing understanding of homology, metabolic interaction, and immune association between liver and intestinal anatomy, the concept of liver-gut-axis has been proposed and accepted. The concept of "liver-gut axis" was officially proposed by Marshall in 1998. It clarifies the close relationship between substances, cells, cytokines, etc. between the liver and the intestine through the inherent portal vein system. Depicts the anatomical and functional interactions between the two organs of the liver and the intestine, especially the cytokines and the hepatoenteral circulation of the cells.

The application of "liver-gut axis" theory can partially explain the cause of nonalcoholic fatty liver disease (NAFLD). Intestinal flora disorder and intestinal mucosal barrier damage activate liver innate immunity, destroy liver homeostasis and aggravate liver inflammation. Frasinariu OE and others believe that food can change the intestinal flora and increase the intestinal flora and its products through the portal vein translocation to the liver. Pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) activate Toll-like receptor-4 (TLR4), and TLRs-mediated signaling induces innate immune responses such as cytokines and IFN-α. Excessive production of cytokines is associated with elevated levels of LPS-binding proteins produced by endotoxin stimulation in the plasma, promoting endotoxin binding to the CD14/TLR4 complex. In the liver, the LPS/TLR4 pathway promotes the release of TGF-β from liver Kupffer cells by sensing the signals produced by transforming growth factor-β (TGF-β) in hepatic stellate cells, leading to the development of liver fibrosis.

Stilbene glycoside (2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, TSG) has been widely demonstrated to improve liver lipid accumulation and to reduce serum, The content of AST and ALT in liver tissue homogenate has a certain inhibitory effect on inflammation.

Zhang Youzhi ("Anti-inflammatory action of stilbene glycoside and its mechanism", Huazhong University of Science and Technology) studied the acute inflammatory effect of stilbene glycoside against mouse auricle, and pointed out that this effect may be related to the inhibition of acute phase Non-specific inflammatory responses to local vasodilation and increased capillary permeability in the rat ear Guanidine; stilbene glycoside has anti-swelling effect on rat toe, and this effect may be related to inhibiting the release of local inflammatory mediators such as prostaglandins and serotonin in rat toes; THSG can directly inhibit PGE2 production, and this effect It may be related to inhibition of gene level COX-2 expression or direct inhibition of COX-2 enzyme activity. However, Zhang Youzhi also pointed out that "the anti-inflammatory effect of THSG on animal models has not been reported."

Zeng Cheng ("The effect of stilbene glycoside on inflammatory bowel disease: induction of PPAR-γ and inhibition of NF-κB inflammatory pathway", Ph.D., Huazhong University of Science and Technology) showed that stilbene glycoside up-regulated PPAR-γ gene and The expression of protein level inhibits the expression of NF-κB and inflammatory mediators TNF-α, IL-6 and COX-2, and reduces the production of lipid oxidation products MPO and MDA to protect IBD.

Xiong Zhange et al. ("Effects of stilbene glycoside on inflammation-related factors in mice with acute alcoholic liver injury", World Chinese Journal of Digestion, December 28, 2012, 20(36): 3649-3655) Studies show that TSG is small Rats have a good protective effect on acute alcoholic liver injury. The mechanism may be to inhibit the activation of Kupffer cells induced by endotoxin, thereby inhibiting the activation of downstream nuclear factor NF-κB, and reducing the trigger mechanism of inflammatory cascade amplification. Liver function protection.

However, the role of stilbene glycoside in inhibiting the innate immune system of TLR4 has not been reported in the literature, so the use of stilbene glycoside as an inhibitor of the innate immune system of TLR4 has a good development prospect.

Summary of the invention

The inventors have found through experiments that stilbene glycoside and its pharmaceutically acceptable salt or ester can act as inhibitors of the TLR4 innate immune system. This discovery has good prospects for development.

It is an object of the present invention to provide the use of stilbene glucoside and a pharmaceutically acceptable salt or ester thereof for the preparation of an inhibitor of the TLR4 innate immune system.

Further, the pharmaceutically acceptable salt is selected from various inorganic or organic acid salts such as hydrochloride, hydrobromide, phosphate, sulfate, citrate, lactate, tartrate, Malay. Acid salts, fumarates, mandelates and oxalates; various inorganic or organic base salts such as sodium hydroxide, trishydroxymethylaminomethane and N-methyl-glucosamine.

Further, the pharmaceutically acceptable ester is selected from a C1-C6 alkyl ester or a benzyl ester.

Further, in the application, stilbene glucoside and a pharmaceutically acceptable salt or ester thereof may be used singly or in the form of a pharmaceutical composition.

Further, the pharmaceutical composition includes stilbene glycoside as an active ingredient and its pharmaceutically acceptable Accepted salt or ester, and one or more pharmaceutically acceptable excipients.

Further, pharmaceutically acceptable excipients include, but are not limited to, fillers such as starch, sucrose, dextrin, lactose, compressible starch, microcrystalline cellulose, sorbitol, etc.; binders such as cellulose derivatives (methyl fibers) , ethyl cellulose, hydroxypropyl cellulose, hypromellose, sodium carboxymethyl cellulose), sodium alginate, gelatin, polyvinylpyrrolidone, etc.; wetting agents such as water, ethanol, glycerin, etc.; disintegrating agents such as Dry starch, sodium carboxymethyl starch, hypromellose, crospovidone, effervescent disintegrant, agar, calcium carbonate, sodium hydrogencarbonate, etc.; absorption enhancer such as quaternary ammonium compound; surfactant such as ten Hexadecanol; adsorption carrier such as kaolin and soap clay; lubricants such as talc, calcium stearate, magnesium stearate and polyethylene glycol; injection solvents such as water for injection, vegetable oil, ethyl oleate, propylene glycol, poly Ethylene glycol, ethanol, glycerin, benzyl alcohol, etc.; buffers such as acetic acid-sodium acetate, sodium citrate-sodium citrate, tartaric acid-sodium tartrate, disodium hydrogen phosphate-sodium dihydrogen phosphate, sodium bicarbonate-sodium carbonate Etc.; bacteriostatic agents such as benzyl alcohol, hydroxypropyl butyrate/methyl ester, Phenol, chlorobutanol, etc.; local anesthetic agent lidocaine, benzyl alcohol, chlorobutanol, etc.; isotonicity adjusting agents such as sodium chloride, glucose, glycerol, etc.; antioxidants such as sodium sulfite, sodium hydrogen sulfite , sodium metabisulfite, etc.; suspending agents such as gelatin, methyl cellulose, pectin, etc.; protective agents such as lactose, sucrose, maltose, etc.; emulsifiers such as Tween, Span, Poloxamer, sodium lauryl sulfate, Fatty acid glycerides; bases such as PEG6000, PEG4000, sodium stearate, hydrogenated vegetable oil, insect wax, and the like. Also included are pigments, flavoring agents, sweeteners, solubilizers, absorption enhancers, and the like.

Further, the content of the stilbene glycoside and the pharmaceutically acceptable salt or ester thereof in the pharmaceutical composition is from 0.1 to 99.9% by weight, preferably from 1 to 90% by weight, from 5 to 80% by weight, 10 -70% by weight, 20-60% by weight or 30-50% by weight.

Further, the stilbene glucoside and a pharmaceutically acceptable salt or ester thereof and the pharmaceutical composition can be administered by enteral or parenteral route. The administration preparations include tablets, capsules, granules, injections, creams, ointments, patches, sprays, dropping pills, and the like. Routes of administration include subcutaneous, intradermal, intraarterial, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional, intracranial injection or infusion, or oral, topical, rectal, menstrual Nasal, buccal, vaginal, sublingual, intradermal, mucosal, tracheal, urethral administration, or by inhalation of aerosol or implant accumulation or acupuncture.

Further, the preparation may be prepared according to a conventional production method in the pharmaceutical field, for example, by mixing the active ingredient with one or more carriers, and then preparing the desired dosage form.

Further, the stilbene glycoside and a pharmaceutically acceptable salt or ester thereof are administered at a daily dose 0.01-200 mg/kg, preferably 0.1-100 mg/kg, 1-80 mg/kg or 10-50 mg/kg, further preferably 12-15 mg/kg, 22-25 mg/kg or 45-50 mg/kg; Treat with medication or combination therapy.

The invention induces SD rat model by high-fat diet, induces simultaneous administration of aqueous solution of stilbene, and analyzes the expression of main signal molecules of TLR4 pathway by liver whole gene scanning technology, including CD14 and myeloid differentiation factor 88 (My D88). ), IL-1 related protein kinases (IRAKs), tumor necrosis factor receptor activating factor 6 (TRAF6), transforming growth factor-β (TGF-β), interferon (IFN), etc., and using enzyme-linked reaction to detect fetal Protein content of globulin A (FetA), type 4 Toll-like receptor (TLR4), NF-кB, tumor necrosis factor TNF-α, interleukin-1α (IL-1α), interleukin-6 (IL-6), evaluation Inhibition of stilbene glycoside in the innate immune system of TLR4.

The results showed that the high fat diet fed rats for 12 weeks activated the TLR4/NF-кB signaling pathway, induced the expression of pro-inflammatory cytokines, stimulated the TLR4 innate immune response system, causing chronic low-grade inflammation, and simultaneously administered In rats with aqueous stilbene glycoside, the innate immune response system of TLR4 was alleviated. The results of ELISA protein showed that TSG could down-regulate TLR4 content by 56% and NF-кB expression by 23% compared with the model group, thus effectively inhibiting the release of inflammatory factors. . Therefore, stilbene glycoside can be used as a potent inhibitor of the TLR4 innate immune system.

DRAWINGS

Figure 1. Structural formula of stilbene glycoside (2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, TSG).

Figure 2. Key gene clustering tree in the TLR4 innate immune response system.

Figure 3. Statistical diagram of key gene data in the TLR4 innate immune response system.

Figure 4. Stilbene glycoside against fetuin A (FetA), type 4 Toll-like receptor (TLR4), NF-кB, tumor necrosis factor TNF-α, interleukin-1 alpha (IL-1 alpha), interleukin-6 ( Effects of IL-6) and interleukin-10 (IL-10) levels: *p<0.05, **p<0.01, ***p<0.001 compared with the normal group, #p< compared with the model group 0.05, ##p<0.01, ###p<0.001.

detailed description

1. Objects and methods

1.1 Subject: 21 healthy SD rats, weighing 180-200g, purchased from Chengdu, Sichuan, qualified Certificate number: 0016254, animal ethics number: R-062014012.

1.2 method

1.2.1 Establishment of a rat model of chronic low-grade inflammation induced by TLR4 innate immune system and group administration

Twenty-one SD rats, weighing 180-200 g, were randomly divided into three groups: normal control group (CON), high fat model group (MOD), and stilbene glycoside (24.28 mg/kg) administration group (TSG.). In addition to the normal control group, each group was fed a high-fat diet (cholesterol 1%, lard 10%, cooked egg yolk 10%) for 12 weeks until the end of the experiment.

1.2.2 Liver sample preservation method

At the end of the 12th week of the experiment, the rat liver was removed, weighed, washed with 0.9% saline, and part of the liver was cut into a cryotube and quickly stored in liquid nitrogen for whole-genome detection.

1.2.3 Liver homogenate sample processing method

Part of the liver was cut and weighed 1 g of the broken liver. Placed in a 50 ml centrifuge tube, 9 ml of 0.9% physiological saline was added, and homogenized with a motorized glass homogenizer. After homogenization, the centrifuge tube was placed in a desktop high-speed refrigerated centrifuge, centrifuged at 4000 °C for 4 min at 4 °C, and the supernatant was taken. The Elisa method was used to detect fetuin A (FetA) and type 4 Toll in liver cells. Changes in receptor (TLR4), NF-кB, tumor necrosis factor TNF-α, interleukin-1α (IL-1α), interleukin-6 (IL-6), and interleukin-10 (IL-10).

1.2.3 Statistical analysis method of data

表示，并采用单因素方差分析(ANOVA)。The data analysis results of the invention are all adopted

Expressed and used one-way analysis of variance (ANOVA).

2. Results

2.1 Effect of stilbene glycoside on the expression of major genes in TLR4 innate immune response system

Agilent's rat 4x44K gene expression chip was used to perform whole-genome scanning on the liver tissues of three groups of rats. The main expression genes in the TLR4 innate immune response system were selected, and the key gene expression clustering tree and corresponding standardized signal values were obtained. Figure 2, 3). The results showed that the bacterial lipopolysaccharide brought by the high-fat diet for 12 weeks in SD rats was used as an exogenous ligand or fetuin secreted by the liver as an endogenous ligand, which increased the expression of CD14 and TLR4 signals and promoted the distribution. Formation of the combination with CD14/TLR4 The complex, which causes the recruitment of downstream signaling molecules, is mainly expressed in the protein kinase-binding protein Tab2, transforming growth factor-β (Tgfb1), IL-1α, IL-6 and interferon, which cause tumor necrosis factor Tnf and β transforming growth factor activation. Up-regulation of the expression of the γ receptor 1 (Ifngr1) signaling molecule stimulates the TLR4 innate immune response system. In the TSG group administered with aqueous solution of stilbene, it is better to inhibit LR4 activation of leucine-rich region expression (Tril), down-regulate the expression of CD14 and TLR4 signaling molecules, and effectively inhibit the signal expression of downstream cytokines, among which IL -6. The down-regulation of Tgfb1 signaling molecules is significant, which inhibits the TLR4 innate immune response system.

2.2 Effect of stilbene glycoside on key protein content

As shown in the results in Figure 4, FetA, TLR4, NF-кB, TNF-α, IL-1α, IL- in the liver homogenate of the high-fat model group were compared with the normal group. The content of 6 was significantly increased, and the expression of TLR4 protein was increased by nearly 1.5 times. It indicates that free fatty acid (FFA) in high-fat diet can bind to FetA secreted by liver, as an endogenous ligand of TLR4, mediating TLR4/NF-кB signaling pathway and promoting release of inflammatory factors; IL-10 as a kind of Anti-inflammatory and immunosuppressive cytokines showed a significant downward trend in the model group (p < 0.05), further deepening the level of inflammation. At the same time, the aqueous solution of stilbene was given to reduce the content of TLR4 by 56% compared with the model group, increase the content of IL-10, and inhibit the protein content of FetA and NF-кB to some extent, thereby inhibiting the release of related inflammatory factors. It shows that stilbene glycoside has a certain inhibitory effect on TLR4 innate immune response system.

Example 1 Injectable powder injection

Weigh 100 g of stilbene glycoside, 0.5 g of sodium metabisulfite, 9 g of sodium chloride, 40 g of mannitol, 20 g of lactose, dissolve stilbene glycoside, sodium metabisulfite, sodium chloride, mannitol and lactose with appropriate amount of water for injection, filter, adjust The solution was sterilized by filtration to pH 6.7-7.4, 0.22 μm filter, diluted to 1000 ml with sterile water for injection, filled in a 10 ml brown glass bottle, 4 ml per bottle, and lyophilized to obtain a finished product.

Example 2 dropping pills

Weigh 180g of stilbene glycoside, add PEG40002Kg as matrix, and apply proper amount of liquid paraffin as coolant; the temperature of liquid medicine is 50°C, the dropping speed is 12 drops per minute, the dropping distance is 4.5cm, the length of cooling column is 30cm, and the inner diameter of drip is 2.5cm. The coolant temperature is 0 ° C (ice water bath).

Example 3 injection

Weigh 50g of stilbene glycosides, add 9g of sodium chloride, add water for injection to 1000ml, adjust the pH to 7.0-7.4, filter, and seal the filtrate in 2, 5 or 10ml ampoules, sterilize at 100 °C for 30min, and get the injection. liquid.

Example 4 soft capsule

Weigh 10g of stilbene glycoside and 60g of edible corn oil, stir well to prepare the content of soft capsule; weigh the appropriate amount of gelatin, glycerin and water (mass ratio: gelatin: glycerol: water = 1:0.5:1) The capsule material glue; using the rotary molding method, the contents and the capsule glue are loaded into the automatic rotary bag making machine, and the soft capsule is pressed, and the appropriate amount of liquid paraffin is used as the lubricant.

Example 5 tablet

Weigh 25g of stilbene glycoside, 50g of starch, 13g of hypromellose, 10g of poloxamer, and sieved through 80 mesh, mix well, add 0.5% povidone ethanol solution to make soft material, use 14 mesh sieve After granulation, it was dried in an oven at 60 ° C, sieved with a 12 mesh sieve, and mixed with 2 g of magnesium stearate, and then compressed.

The present invention can be better understood from the above embodiments. However, those skilled in the art will readily appreciate that the specific material ratios, process conditions, and results described in the examples are merely illustrative of the invention and are not intended to limit the invention as described in the claims. .

Claims (9)

Use of stilbene glucoside and a pharmaceutically acceptable salt or ester thereof for the preparation of an inhibitor of the TLR4 innate immune system. The use according to claim 1, wherein the pharmaceutically acceptable salt is selected from the group consisting of inorganic or organic acid salts, inorganic or organic base salts, and the pharmaceutically acceptable ester is selected from the group consisting of C1-C6 alkyl groups. Ester or benzyl ester. The use according to claim 2, wherein the inorganic or organic acid salt is selected from the group consisting of hydrochloride, hydrobromide, phosphate, sulfate, citrate, lactate, tartrate, and Malay. One or more combinations of an acid salt, a fumarate, a mandelate, and an oxalate selected from the group consisting of sodium hydroxide, tris, and N-methyl-glucosamine One or more combinations. The use according to any one of claims 1 to 3, characterized in that the stilbene glycoside and a pharmaceutically acceptable salt or ester thereof are used singly or in the form of a pharmaceutical composition. The use according to Claim 4, characterized in that the pharmaceutical composition comprises, as an active ingredient, stilbene glycoside and a pharmaceutically acceptable salt or ester thereof, and one or more pharmaceutically acceptable excipients. The use according to claim 5, wherein the pharmaceutically acceptable adjuvant is selected from the group consisting of a filler, a binder, a wetting agent, a disintegrant, a surfactant, an adsorption carrier, a lubricant, an injection solvent, and a buffer. One or more combinations of bacteriostatic agents, local anesthetics, isotonicity adjusting agents, suspending agents, protective agents, emulsifiers, matrices, coloring agents, flavoring agents, sweeteners, solubilizers, and absorption enhancers. The use according to Claim 4, characterized in that the content of the stilbene glycoside and the pharmaceutically acceptable salt or ester thereof in the pharmaceutical composition is from 0.1 to 99.9% by weight, preferably from 1 to 90% by weight. Percent, 5-80% by weight, 10-70% by weight, 20-60% by weight or 30-50% by weight. The use according to Claim 4, characterized in that the pharmaceutical composition is selected from the group consisting of tablets, capsules, granules, injections, creams, ointments, patches, sprays and pills. The use according to claim 1 or 4, wherein the stilbene glucoside and a pharmaceutically acceptable salt or ester thereof are administered at a dose of 0.01 to 200 mg/kg per day, preferably 0.1 to 100 mg/ Kg, 1-80 mg/kg or 10-50 mg/kg.

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