Application of demethylwedelolactone in preparation of uric acid reducing medicine

Abstract

The invention discloses application of demethylwedelolactone in preparation of uric acid lowering drugs, and belongs to the technical field of medicines. The invention discloses an effect of demethylwedelolactone in lowering uric acid and an action mechanism of demethylwedelolactone in lowering the uric acid level. In-vivo and in-vitro pharmacological experiment results prove that the demethylwedelolactone can inhibit the activity of xanthine oxidase, promote the expression of an OAT1 gene, inhibit the generation of uric acid and promote the excretion of uric acid at the same time, so that the demethylwedelolactone plays a role in reducing the uric acid, which prompts that the demethylwedelolactone has a new application as a medicine for treating or improving the hyperuricemia.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and particularly relates to an application of norwedelic acid in the preparation of a uric acid-lowering drug. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Hyperuricemia (HUA) is a metabolic disease caused by blood uric acid concentration exceeding normal values. Hyperuricemia is not only a prerequisite for the onset of gout, but can also further develop into a series of diseases such as hypertension, cardiovascular disease, diabetes, and hyperlipidemia. Prospective epidemiological studies have pointed out that hypertension, dietary factors, and chronic kidney disease are risk factors for hyperuricemia. In recent years, with the continuous improvement of people's living standards and changes in dietary structure, the incidence of hyperuricemia has also been rising.

[0004] Uric acid in the human body is mainly synthesized in the liver. Hypoxanthine and xanthine are oxidized by xanthine oxidase (XOD) to produce uric acid. The liver and kidneys of most mammals contain uricase, which can convert uric acid into allantoin. However, the human body lacks uricase, so uric acid easily accumulates in the body, causing tissue lesions and pathological reactions. The kidney is a key organ for uric acid excretion. The excretion of uric acid requires specific transporters located in renal tubular cells and intestinal epithelial cells. The three urate transporters URAT1, GLUT9 and OAT4 are mainly involved in the reabsorption of uric acid in the renal tubules. Urate transporters such as ABCG2, OAT1, and OAT2 are involved in the secretion of uric acid. Several transporters play an important role in regulating serum uric acid, and their abnormal expression can lead to abnormal urate transport.

[0005] At present, xanthine oxidase inhibitors are the first choice for uric acid-lowering treatment. Commonly used drugs include allopurinol and febuxostat. However, allopurinol is prone to allergic reactions, bone marrow suppression, intestinal reactions and other adverse reactions after use; febuxostat can cause adverse reactions such as increased liver enzyme levels, nausea, cough, and rash. Drugs that promote uric acid excretion can also be selected to play a uric acid-lowering role. Commonly used drugs are mainly benzbromarone and probenecid. Benzbromarone is a uricosuric drug that has been used for a long time in clinical practice, but it has been withdrawn from the US and some European markets due to its high liver toxicity. Probenecid is a URAT1 inhibitor that can promote uric acid excretion by reducing the reabsorption of uric acid. Its adverse reactions mainly include hemolytic anemia and liver and kidney adverse reactions. Uric acid excretion drugs may cause uric acid deposition in the urinary tract and cause kidney damage. Drinking more water and properly alkalizing urine are required. It can be seen that the uric acid-lowering drugs commonly used in clinical practice all have a certain degree of adverse reactions, and there is an urgent need to find a drug with high efficacy and low toxicity and side effects to treat hyperuricemia.

[0006] Demethylwedelolactone (DWEL) is a natural product extracted from the traditional Chinese medicine Eclipta prostrata. Studies have shown that demethylwedelolactone is an effective trypsin inhibitor that can treat arthritis (patent CN 1634037 A), chronic bronchitis and emphysema (patent CN 1810809 A), inhibit the migration and invasion of breast cancer cells, and protect against cell damage. Although literature studies have shown that demethylwedelolactone has a certain therapeutic effect on complications of hyperuricemia such as nephrotic syndrome, hyperlipidemia, and inflammation, there are no reports on its uric acid-lowering activity. Summary of the invention

[0007] In order to solve the deficiencies of the prior art, the object of the present invention is to provide an application of norwedeholactone in the preparation of uric acid-lowering drugs. The present invention discloses the effect of norwedeholactone in lowering uric acid and its mechanism of action for lowering uric acid levels.

[0008] In order to achieve the above object, the technical solution of the present invention is:

[0009] The first aspect of the present invention provides the use of a compound represented by formula I or a pharmaceutically acceptable salt thereof in the preparation of a product having a uric acid-lowering effect; the product is a medicine or an experimental reagent for scientific experiments;

[0010]

[0011] Wherein, R1, R2 and R3 are each independently selected from hydroxyl, halogen, C1-C2 alkyl; the C1-C2 alkyl is unsubstituted or substituted by one or more substituents selected from alkyl, alkoxy, halogen, cyano, nitro;

[0012] The pharmaceutically acceptable salt is an inorganic acid salt or an organic acid salt of the compound represented by formula I.

[0013] In some embodiments of the present invention, the drug is a xanthine oxidase inhibitor and / or a drug that promotes uric acid excretion.

[0014] Preferably, the drug for promoting uric acid excretion is an OAT1 gene expression promoter.

[0015] In some embodiments of the present invention, the drug is a drug for treating diseases caused by high uric acid, and the diseases caused by high uric acid include but are not limited to gout, hyperuricemia or uric acid nephropathy.

[0016] In some embodiments of the present invention, the inorganic acid salt is selected from hydrochloride, hydrobromide and hydroiodide; the organic acid salt is selected from formate, acetate, propionate, malonate, 1,4-succinate, benzoate, nicotinate, fumarate, malate, maleate, gluconate and citrate.

[0017] In some embodiments of the present invention, the substituent is selected from halogen, methyl, methoxy, cyano and nitro; and the substitution is mono-substitution or di-substitution.

[0018] Preferably, when the substitution is disubstitution, the substituent is halogen or methyl.

[0019] In a preferred embodiment of the present invention, R1, R2 and R3 are each independently selected from hydroxyl, methyl, ethyl, monochloromethyl, monobromomethyl, 2-chloroethyl, 2-bromoethyl.

[0020] Further, as an example, the present invention provides a series of compounds represented by formula I and pharmaceutically acceptable salts thereof, selected from the following compounds:

[0021] Compound 1: Wedelinolone, the structural formula is:

[0022]

[0023] The second aspect of the present invention provides a uric acid-lowering drug comprising wedelaractone or a pharmaceutically acceptable salt thereof.

[0024] In some embodiments of the present invention, the uric acid lowering drug contains weedelolide or a pharmaceutically acceptable salt thereof as a main active ingredient.

[0025] In some embodiments of the present invention, the uric acid lowering drug contains one or more pharmaceutically acceptable excipients.

[0026] In some embodiments of the present invention, the pharmaceutically acceptable excipient is selected from one or more of glycerol, butylene glycol, trehalose, betaine, allantoin, carbomer, dextrin, starch, gel cellulose, methyl cellulose, sodium carboxymethyl cellulose or polyvinyl pyrrolidone.

[0027] In some embodiments of the present invention, the dosage form of the uric acid-lowering drug is a cream, an ointment, a patch, a gel, a lotion, a spray, a tincture or a film.

[0028] The beneficial effects of the present invention are:

[0029] At present, there is no research report that norwegian chrysanthemum lactone has the effect of lowering uric acid. The present invention screens monomer compounds based on the zebrafish hyperuric acid model and finds that norwegian chrysanthemum lactone has the effect of lowering uric acid. Norwegian chrysanthemum lactone was evaluated using an in vitro xanthine oxidase model and molecular biology techniques, and it was found that the drug can effectively inhibit the activity of xanthine oxidase and promote the expression of the OAT1 gene. Therefore, it is judged that norwegian chrysanthemum lactone can inhibit the production of uric acid while promoting the excretion of uric acid, thereby exerting a uric acid-lowering effect, and can be applied to the development of uric acid-lowering drugs to treat hyperuricemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0031] Figure 1 The inhibitory effect of the positive drug allopurinol (APL) in Example 1 of the present invention on the zebrafish hyperuricemia model;

[0032] Figure 2 The effect of demethylwedeholactone on uric acid levels in zebrafish in Example 1 of the present invention;

[0033] Figure 3 The effect of demethylwedeholactone on xanthine oxidase in Example 2 of the present invention;

[0034] Figure 4 It is the relative quantitative level of the OAT1 gene in zebrafish in Example 2 of the present invention. DETAILED DESCRIPTION

[0035] The present invention provides the use of demethylwedeholactone, and those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0036] At present, there is no research report that norwedelactone has the effect of lowering uric acid. The present invention proves through in vivo and in vitro experiments that norwedelactone has a good uric acid lowering effect on the zebrafish hyperuric acid model induced by potassium oxonate and sodium xanthine. The in vitro experimental results prove that norwedelactone can inhibit xanthine oxidase and thus inhibit the formation of uric acid. The molecular biology experimental results prove that norwedelactone can promote OAT1 gene expression and thus promote the excretion of uric acid, and has concentration dependence. It has a new medical use as a uric acid lowering therapeutic drug, and can be applied to the research and development of uric acid lowering drugs to treat hyperuricemia.

[0037] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0038] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0039] The abbreviations of the reagents used in the following examples are shown in Table 1.

[0040] Table 1 Reagent abbreviations

[0041]

[0042]

[0043] Example 1. Zebrafish hyperuricemia model

[0044] Normal and well-developed 5dpf wild AB zebrafish larvae were placed in a 6-well plate, with 40 larvae in each well. The fish water was prepared with ultrapure water, which contained 5mM sodium chloride, 0.17mM potassium chloride, 0.33mM calcium chloride, and 0.33mM magnesium sulfate. A blank group, a model group, and a positive drug group were set up, and the positive drug was allopurinol (APL). In the model group, 1000μM potassium oxonate (PO) and 50μM xanthine sodium salt (XSS) were added to the fish water for modeling. In addition to the treatment of the model group, 5mM, 10mM, and 20mM APL were added to the positive drug group, respectively. After drug administration, the zebrafish larvae were placed in a constant temperature incubator at 28.5℃ for incubation. After 24h, the uric acid level in the zebrafish was measured using a uric acid determination kit (Nanjing Jiancheng Bioengineering Institute). The results are shown in Figure 1 Different concentrations of APL can inhibit the increase of uric acid levels in zebrafish in a concentration-dependent manner. This confirms that this zebrafish hyperuricemia model can be used for the evaluation of the uric acid-lowering pharmacological effects of compounds.

[0045] According to the above method, the positive drug group was replaced with the drug administration group to verify the uric acid-lowering effect of demethylwedeholactone (30 μM, 10 μM and 3 μM). Figure 2 When the concentration of norwedeholactone was 30μM, 10μM and 3μM, its inhibition rate on uric acid level in zebrafish was 45.28%, 39.38% and 30.27% respectively, indicating that norwedeholactone can significantly reduce uric acid level in zebrafish in a concentration-dependent manner. The above results provide a reliable theoretical basis for the development of norwedeholactone's uric acid-lowering effect.

[0046] According to the above method, the demethyl wedelagolide was replaced with an inorganic acid salt (any one of hydrochloride, hydrobromide and hydroiodide) and an organic acid salt (formate, acetate, propionate, malonate, 1,4-succinate, benzoate, nicotinate, fumarate, malate, maleate, gluconate and citrate) of demethyl wedelagolide to verify the uric acid-lowering effect of the pharmaceutically acceptable salt of demethyl wedelagolide. It has been verified that the pharmaceutically acceptable salt of demethyl wedelagolide can also significantly reduce the uric acid level in zebrafish and is concentration-dependent.

[0047] During the experiment, no deaths occurred in the zebrafish fry in the drug-treated group, which indicates that wedeliolide and its pharmaceutically acceptable salts have low toxicity and side effects and are highly safe.

[0048] Example 2. Mechanism of action of demethylwedelactone in lowering uric acid

[0049] 1. In vitro xanthine oxidase activity assay

[0050] Xanthine oxidase is a key enzyme that catalyzes purine metabolism. Inhibiting its activity can effectively reduce the formation of uric acid in serum. Use PBS (pH = 7.5) to prepare 0.05U / mL XOD solution and 2mM xanthine solution (using a small amount of NaOH for dissolution); use DMSO to prepare 3mM, 1mM and 0.3mM demethylwedelactone solutions. Take a 96-well plate, set up the drug group and DMSO group respectively, and set up two sub-wells for each group. Operate according to the method in Table 2 to observe the changes in the absorbance values ​​of each group.

[0051] The results are as follows Figure 3 As shown in the data, when the concentration of demethylwedeholactone was 30μM, 10μM and 3μM, its inhibition rate on uric acid level in zebrafish was 70.37%, 40.59% and 20.44% respectively, indicating that demethylwedeholactone has an inhibitory effect on xanthine oxidase in a concentration-dependent manner. This suggests that demethylwedeholactone may play a role in lowering uric acid by inhibiting the activity of xanthine oxidase and inhibiting the production of uric acid.

[0052] Table 2 In vitro XOD inhibition assay method

[0053]

[0054]

[0055] 2. Quantitative PCR experiment

[0056] The OAT1 gene plays an important role in regulating uric acid excretion, and its increased expression promotes uric acid excretion. Total RNA was extracted using a column-type animal tissue total RNA extraction and purification kit (Sangon Biotechnology Co., Ltd.). cDNA was synthesized using the AMeasy 1st Strand cDNA Synthesis Kit IV Pro (Beijing Aomei Biotechnology Co., Ltd.). q-PCR analysis was performed using the 2*PerfectHS SYBR QPCR Mixture (ROX) kit (Beijing Aomei Biotechnology Co., Ltd.). The two-step PCR reaction program used was: 95℃30s, 95℃10s, 60℃30s for 40 cycles. The specific information of the primers is shown in Table 3. The 2-△△Ct method was used to calculate the relative quantification of the mRNA levels of each gene between the groups and the β-actin gene was used as a control for normalization.

[0057] Effects of norwedeholide on OAT1 gene expression in zebrafish Figure 4. Compared with the normal group zebrafish, the level of OAT1 in the model group zebrafish was significantly reduced. Wedelagolide promoted the expression of OAT1 in the model group zebrafish, and as the concentration of the drug continued to increase, the expression of OAT1 in the zebrafish continued to increase. This suggests that wedelagolide may promote the excretion of uric acid by increasing the expression of OAT1 and thus exerting its uric acid-lowering effect.

[0058] Table 3 Primer information

[0059] Gene name Sequence 5′-3′ bp β-actin-F TCG AGC AGG AGA TGG GAA CC 20 β-actin-R CTC GTG GAT ACC GCA AGA TTC 21 OAT1-F GGACGA TAT CCT GCC AGC TC 20 OAT1-R CGT CCT GTA AGG CCA GAT CC 20

[0060] According to the above method, the demethyl wedelagolide was replaced with an inorganic acid salt (any one of hydrochloride, hydrobromide and hydroiodide) and an organic acid salt (any one of formates, acetates, propionates, malonates, 1,4-succinates, benzoates, nicotinates, fumarates, malates, maleates, gluconates and citrates) of demethyl wedelagolide to verify the uric acid-lowering mechanism of the pharmaceutically acceptable salt of demethyl wedelagolide. It has been verified that the pharmaceutically acceptable salt of demethyl wedelagolide also has an inhibitory effect on xanthine oxidase in a concentration-dependent manner; it promotes the expression of OAT1 in zebrafish in the model group in a concentration-dependent manner.

[0061] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of the compound represented by formula I or a pharmaceutically acceptable salt thereof in the preparation of a product having a uric acid-lowering effect; the product is a medicine or an experimental reagent for scientific experiments; in, R1, R2 and R3 are each independently selected from hydroxyl, halogen, C1-C2 alkyl; the C1-C2 alkyl is unsubstituted or substituted by one or more substituents selected from alkyl, alkoxy, halogen, cyano, nitro; The pharmaceutically acceptable salt is an inorganic acid salt or an organic acid salt of the compound represented by formula I.

2. The use according to claim 1, characterized in that The drug is a xanthine oxidase inhibitor and / or a drug that promotes uric acid excretion; Preferably, the drug for promoting uric acid excretion is an OAT1 gene expression promoter; Preferably, the drug is a drug for treating gout, hyperuricemia or uric acid nephropathy.

3. The use according to claim 1, characterized in that The inorganic acid salt is selected from hydrochloride, hydrobromide and hydroiodide; the organic acid salt is selected from formate, acetate, propionate, malonate, 1,4-succinate, benzoate, nicotinate, fumarate, malate, maleate, gluconate and citrate.

4. The use according to claim 1, characterized in that The substituent is selected from halogen, methyl, methoxy, cyano and nitro; the substitution is mono-substitution or di-substitution; Preferably, when the substitution is disubstitution, the substituent is halogen or methyl.

5. The use according to any one of claims 1, characterized in that: The R1, R2 and R3 are each independently selected from hydroxyl, methyl, ethyl, monochloromethyl, monobromomethyl, 2-chloroethyl, 2-bromoethyl.

6. The use according to any one of claims 1 to 5, characterized in that: The compound represented by formula I or a pharmaceutically acceptable salt thereof is selected from: Compound 1: Wedelinol, the structural formula of which is:

7. A uric acid lowering drug, characterized in that: Containing norwedeholactone or a pharmaceutically acceptable salt thereof; Preferably, the uric acid lowering drug contains weedelolide or a pharmaceutically acceptable salt thereof as a main active ingredient.

8. The uric acid lowering drug according to claim 7, characterized in that: The uric acid lowering drug contains one or more pharmaceutically acceptable excipients.

9. The uric acid lowering drug according to claim 8, characterized in that: The pharmaceutically acceptable excipient is selected from one or more of glycerol, butylene glycol, trehalose, betaine, allantoin, carbomer, dextrin, starch, gel cellulose, methyl cellulose, sodium carboxymethyl cellulose or polyvinyl pyrrolidone.

10. The uric acid lowering drug according to any one of claims 7 to 9, characterized in that: The dosage form of the uric acid lowering drug is cream, ointment, patch, gel, lotion, spray, tincture or film.

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