Application of salinomycin in the preparation of drugs for lowering uric acid and / or treating hyperuricemia

Abstract

The present invention discloses the use of salinomycin in the preparation of a medicament for reducing uric acid and / or treating hyperuricemia. In addition, the present invention also claims the use of salinomycin in the preparation of a medicament for treating and / or preventing gout or gout complications caused by hyperuricemia, and the use of salinomycin in the preparation of a medicament for treating renal damage caused by hyperuricemia. The salinomycin can effectively reduce uric acid in the serum of an animal, can play a role in treating hyperuricemia, can play a role in alleviating renal damage, and has no obvious side effects.

Description

Technical Field

[0001] The present invention belongs to the technical field of antibiotics, and more specifically, relates to the use of salinomycin in the preparation of drugs for lowering uric acid and / or treating hyperuricemia. Background Art

[0002] In the field of chemical medicine, uric acid is the final metabolite of purine compounds in humans. Purines produced endogenously and exogenously ingested by the human body are ultimately metabolized to uric acid by xanthine oxidase in the liver. Due to the evolutionary absence of urate oxidase, primates and humans are unable to metabolize uric acid to allantoin, so human serum uric acid levels are five to six times higher than those of other mammals.

[0003] Under normal purine diet, fasting blood uric acid levels of men higher than 416μmol / L and women higher than 360μmol / L on two different days are called hyperuricemia. Hyperuricemia (HUA) is caused by an imbalance between uric acid synthesis and uric acid excretion. Large amounts of urate deposits in the body can easily induce joint and tissue damage, inflammatory reactions, and subsequently gout, cardiovascular disease, diabetes and other metabolic diseases.

[0004] Usually, there are no symptoms in a state of hyperuricemia, but if it lasts for a long time, the urate in the blood will crystallize and deposit in the joints, subcutaneous tissue, kidneys and other parts, leading to a series of clinical manifestations such as gout and gout complications. Today, gout has become the second largest metabolic disease in my country after diabetes, and it seriously endangers people's lives and health.

[0005] In addition, a large number of studies have shown that hyperuricemia is closely related to kidney disease. High uric acid levels in the body can cause varying degrees of renal tubular damage, interstitial fibrosis, glomerular sclerosis, etc. 70% of uric acid is excreted through the kidneys and 30% through the intestines; the kidneys are the main route for uric acid excretion, and reduced excretion and increased reabsorption will cause hyperuricemia.

[0006] Uric acid transporters in the kidney are an important part of uric acid excretion and reabsorption, and can be roughly divided into reabsorption-related proteins and secretion-related proteins. Reabsorption-related proteins, such as glucose transporter 9 (GLUT9) and uric acid transporter 1 (URAT1); secretion-related proteins, such as organic anion transporter (OAT) 1 and ATP-binding transporter G2 (ABCG2).

[0007] At present, drugs for treating hyperuricemia mainly target transporters that inhibit uric acid production and promote uric acid excretion. However, although currently marketed drugs such as allopurinol, topiroxetine, and benzbromarone have strong uric acid-lowering effects, they can cause adverse reactions such as liver and kidney damage; Resinade is the first selective inhibitor of uric acid reabsorption, which can specifically inhibit the URAT1 transporter, but it cannot be used to treat patients with liver damage or renal dialysis. Therefore, how to provide a uric acid-lowering drug that can lower uric acid, treat hyperuricemia, has few side effects, and has a protective effect on organs has become a technical problem that needs to be solved urgently. Summary of the invention

[0008] In view of the above-mentioned existing technical problems, the primary purpose of the present invention is to provide the use of salinomycin in the preparation of drugs for lowering uric acid and / or treating hyperuricemia, wherein the salinomycin can effectively reduce uric acid in the serum of animals and can play a role in treating hyperuricemia without obvious side effects.

[0009] The second object of the present invention is to provide the use of salinomycin in the preparation of a drug for treating and / or preventing gout or gout complications caused by hyperuricemia.

[0010] The third object of the present invention is to provide the use of salinomycin in the preparation of a drug for treating renal damage caused by hyperuricemia.

[0011] The fourth object of the present invention is to provide a pharmaceutical composition comprising a therapeutically effective amount of salinomycin and optionally a pharmaceutically acceptable carrier or excipient for use in the preparation of a drug for lowering uric acid and / or treating hyperuricemia.

[0012] The fifth object of the present invention is to provide a pharmaceutical composition comprising a therapeutically effective amount of salinomycin and a pharmaceutically acceptable carrier or excipient selected from a pharmaceutically acceptable source for use in the preparation of a medicament for treating renal injury caused by hyperuricemia.

[0013] In order to achieve the above object, the present invention is implemented by the following technical solutions:

[0014] Application of salinomycin in the preparation of drugs for lowering uric acid and / or treating hyperuricemia.

[0015] The inventor first constructed a hyperuricemia mouse model by intraperitoneal injection of potassium oxonate and oral administration of hypoxanthine, observed the histopathological changes of hyperuricemia model mice and salinomycin-administered mice by HE staining, and studied the effect of salinomycin on lowering uric acid and treating hyperuricemia. The inventor found through experiments that salinomycin can effectively reduce uric acid in mouse serum and can play a role in treating hyperuricemia; in addition, the inventor also found that salinomycin can improve the damage caused by renal tubular dilatation, reduce necrosis of renal tubular epithelial cells, and thus play a role in reducing kidney damage. The inventor found through further research that salinomycin may achieve the effect of lowering uric acid, treating hyperuricemia, and reducing kidney damage by effectively inhibiting the expression of URAT1 in animals.

[0016] Furthermore, the present invention also seeks to protect the use of salinomycin in the preparation of a drug for treating and / or preventing gout or gout complications caused by hyperuricemia.

[0017] Preferably, the gout complication is one or more of gouty arthritis, urinary tract stones or cardiovascular disease. More specifically, gout can be acute gout or chronic gout; the cardiovascular disease includes but is not limited to cardiovascular diseases such as thrombosis and atherosclerosis.

[0018] Furthermore, based on the research of the present invention, the present invention also requests protection for the use of salinomycin in the preparation of a drug for treating renal damage caused by hyperuricemia.

[0019] Preferably, the renal injury refers to renal tubular dilatation and / or renal tubular epithelial cell necrosis.

[0020] Preferably, the treatment of hyperuricemia refers to reducing the reabsorption of uric acid.

[0021] Preferably, the reducing the reabsorption of uric acid refers to reducing the reabsorption of uric acid by inhibiting the expression of URAT1.

[0022] Preferably, the hyperuricemia is hyperuricemia caused by potassium oxonate and / or hypoxanthine.

[0023] Furthermore, the present invention also claims the use of a pharmaceutical composition in the preparation of a drug for lowering uric acid and / or treating hyperuricemia, wherein the pharmaceutical composition comprises a therapeutically effective amount of salinomycin and optionally a pharmaceutically acceptable carrier or excipient.

[0024] Furthermore, the present invention also seeks to protect the use of a pharmaceutical composition in the preparation of a drug for treating renal damage caused by hyperuricemia, wherein the pharmaceutical composition comprises a therapeutically effective amount of salinomycin and a pharmaceutically acceptable carrier or excipient.

[0025] Compared with the prior art, the present invention has the following beneficial effects: the present invention has found through experiments that salinomycin can effectively reduce uric acid in mouse serum, thereby playing a role in treating hyperuricemia; in addition, salinomycin can also improve the damage caused by tubular dilatation, reduce necrosis of tubular epithelial cells, and thereby play a role in alleviating kidney damage. Salinomycin may achieve the effects of lowering uric acid, treating hyperuricemia, and alleviating kidney damage by inhibiting the expression of URAT1 in animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a schematic diagram of the effect of salinomycin on the levels of serum uric acid, blood urea nitrogen and blood creatinine in hyperuricemia model mice; Figure 1 Figure A is a schematic diagram showing the effect of salinomycin on serum uric acid levels in hyperuricemia model mice; Figure B is a schematic diagram showing the effect of salinomycin on blood urea nitrogen levels in hyperuricemia model mice; Figure C is a schematic diagram showing the effect of salinomycin on blood creatinine levels in hyperuricemia model mice.

[0027] Figure 2 Schematic diagram of the effect of salinomycin on renal pathological damage in hyperuricemia model mice. Figure 2 Figure A is a schematic diagram of the kidneys of mice in the blank group; Figure B is a schematic diagram of kidney pathological damage in mice with hyperuricemia model; Figure C is a schematic diagram of the effect of salinomycin on kidney pathological damage in mice with hyperuricemia model.

[0028] Figure 3 Schematic diagram of the effect of salinomycin on the expression level of URAT1 protein in the kidneys of hyperuricemia mice; Figure 3 In the figure, Figure A is a schematic diagram of the band development of URAT1 protein expression levels in different treatment groups, and Figure B is a schematic diagram of URAT1 protein expression levels in different treatment groups. DETAILED DESCRIPTION

[0029] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0030] Example 1 In vivo uric acid lowering activity experiment of salinomycin

[0031] (1) Experimental animal grouping

[0032] The present invention selects male KM mice weighing about 20±2g, and the mice are raised under standard conditions in a controlled room with a temperature of 25±1°C and a humidity of 50±10%, and a light / dark cycle of 12 hours. All animals are allowed to freely eat laboratory diets and water. The mice are randomly divided into six groups, each with 6 mice. They are: a blank group (control group), a hyperuricemia model group (HN group), a low-dose salinomycin administration group (0.5mg / kg, HN+SAL 0.5mg group), a high-dose salinomycin administration group (1mg / kg, HN+SAL 1mg group), an allopurinol group (HN+AP group), and a single salinomycin administration group (1mg / kg, SAL 1mg group).

[0033] (2) Establishment and drug administration of hyperuricemia model mice

[0034] Except for the blank group and the single drug administration group which were given 0.5% sodium carboxymethylcellulose (CMC-Na), the remaining four groups were given potassium oxonate and hypoxanthine for modeling. Half an hour after the model was established, drugs were administered according to the groups for a total of 7 days.

[0035] (3) Animal sacrifice and biochemical index testing

[0036] On the seventh day, two hours after administration, the mice were anesthetized and blood was collected from their orbits, then they were killed, and the kidneys were removed by dissection and fixed for subsequent pathological staining analysis. Organ tissues were stored in a -80°C refrigerator. After centrifugation of the blood samples, the supernatant was taken and the relevant biochemical indicators were tested using a biochemical analyzer.

[0037] Figure 1 The figure is a schematic diagram of the effect of salinomycin on the serum uric acid, blood urea nitrogen and blood creatinine levels of hyperuricemia model mice; Figure A is a schematic diagram of the effect of salinomycin on the serum uric acid level of hyperuricemia model mice. As shown in Figure A, the blood uric acid level of mice in the hyperuricemia model group (HN group) was about 250μM, which was significantly higher than that in the blank group (control group), while the uric acid levels of mice in the different doses of salinomycin administration groups (HN+SAL 0.5mg group, HN+SAL 1mg group) were significantly lower than those in the hyperuricemia model group (HN group), indicating that salinomycin has a better effect in lowering uric acid.

[0038] Figure B is a schematic diagram of the effect of salinomycin on the blood urea nitrogen level of hyperuricemia model mice. As shown in Figure B, the blood urea nitrogen level of mice in the hyperuricemia model group (HN group) was significantly higher than that in the blank group (control group), and the blood urea nitrogen levels of mice in the different doses of salinomycin administration groups (HN+SAL 0.5mg group, HN+SAL 1mg group) were significantly lower than those in the hyperuricemia model group (HN group); and the blood urea nitrogen level of the positive drug allopurinol group (HN+AP group) was even higher than that in the hyperuricemia model group (HN group), indicating that the drug has certain damage to the kidneys of mice, while salinomycin has a certain protective effect on the kidneys.

[0039] Figure C is a schematic diagram of the effect of salinomycin on the blood creatinine level of hyperuricemia model mice. As shown in Figure C, the blood creatinine level of mice in the hyperuricemia model group (HN group) was significantly higher than that in the blank group (control group), and the blood creatinine levels of mice in the different doses of salinomycin administration groups (HN+SAL 0.5mg group, HN+SAL 1mg group) were significantly lower than those in the hyperuricemia model group (HN group); while the blood creatinine level of the positive drug allopurinol group (HN+AP group) was equivalent to that of the hyperuricemia model group (HN group), indicating that the drug had certain damage to the kidneys of mice, while salinomycin had a certain protective effect on the kidneys.

[0040] Figure 2 The figure is a schematic diagram of the effect of salinomycin on renal pathological damage in hyperuricemia model mice. Figure 2 As shown, the hyperuricemia model (HN group) can cause tubular dilatation and tubular epithelial cell necrosis in the mouse kidneys, while the salinomycin administration group can improve the damaging effects of tubular dilatation and thus play a protective role on the kidneys.

[0041] Example 2 Experiment on the effect of salinomycin on URAT1 expression in vivo

[0042] (1) Mouse kidney protein extraction

[0043] Take about 5 mg of mouse kidney tissue from step (3) of Example 1, add lysis solution, protease inhibitor (100×) and phosphatase inhibitor (50×), put in 2 steel balls, and grind ultrasonically on a grinder. Vortex the grinding solution for about 5 minutes once, for a total of 6 times. Centrifuge at low temperature, 13000 RCF, 15 minutes, and take the supernatant to a new centrifuge tube.

[0044] (2) Protein quantification

[0045] Take 2 μL of tissue supernatant and place it in a 96-well plate. Add 18 μL of dd HO. 2O, and then add 200μL BCA protein quantification working solution. Incubate in an incubator for 30 minutes. Take the sample and measure the absorbance at 570mm on the microplate reader. Substitute the absorbance value into the standard curve to calculate the concentration of the sample, and at the same time calculate the amount of RIPA and loading buffer to be added to dilute and quantify the protein. Boil the prepared protein sample in a 100℃ metal bath for 5-8 minutes. Store the cooked protein in a -80℃ refrigerator.

[0046] (3) Western blot verification of URAT1 expression

[0047] ① Electrophoresis: Prepare the SDS-PAGE gel according to the proportion, add enough electrophoresis solution, pull out the comb of the gel, and load the protein samples in order. Start electrophoresis, use 80V voltage to concentrate the protein, and then switch to 100V voltage to separate the protein.

[0048] ② Transfer: After electrophoresis, soak the PVDF membrane in methanol for activation. Then transfer the gel to the PVDF membrane using the sandwich method, and transfer the protein on the SDS-PAGE to the PVDF membrane. Add enough transfer solution and transfer at 90V. After transfer, block the PVDF membrane with 5% skim milk for 1 hour. After blocking, cut the PVDF membrane according to the molecular weight of URAT1 protein.

[0049] ③ Antibody incubation: URAT1 antibody was diluted at a ratio of 1:1000, and then the URAT1 band was placed in the URAT1 antibody tube for overnight incubation. After the primary antibody incubation, the band was washed with TBST solution for about 5 minutes once, for a total of 6 times. Use 5% skim milk to prepare the secondary antibody solution of the corresponding species of the primary antibody at a ratio of 1:5000. Place the band in the secondary antibody incubation box and incubate for about 1 hour. After the secondary antibody incubation, the band was washed with TBST solution for about 10 minutes once, for a total of 3 times.

[0050] ④ Development: Prepare TCL luminescent liquid in a 1:1 ratio, evenly add 200 μL of luminescent liquid to each strip, and then place it in a multifunctional imaging analysis system for strip development.

[0051] Figure 3 Schematic diagram of the effect of salinomycin on the expression level of URAT1 protein in the kidneys of hyperuricemia mice, Figure 3 Figure A is a schematic diagram of the band development of URAT1 protein expression levels in different treatment groups, and Figure B is a schematic diagram of URAT1 protein expression levels in different treatment groups. Figure 3As shown in the data, the expression level of URAT1 in the hyperuricemia model group was significantly increased, while the URAT1 levels in the mice in the different doses of salinomycin administration groups were decreased, indicating that salinomycin may play a role in lowering uric acid and alleviating kidney damage by effectively inhibiting the expression of URAT1.

[0052] Based on the above experiments and their experimental results, the present invention proves that salinomycin can effectively reduce uric acid in mouse serum, thereby playing a role in treating hyperuricemia; in addition, salinomycin can also improve the damage caused by tubular dilatation, reduce necrosis of tubular epithelial cells, and thus play a role in reducing kidney damage. Salinomycin may achieve the effect of reducing uric acid, treating hyperuricemia, and reducing kidney damage by effectively inhibiting the expression of URAT1 in animals.

[0053] The foregoing embodiments are merely illustrative and are used to explain some features of the method of the present invention. The appended claims are intended to require the widest possible range that can be imagined, and the embodiments presented herein are demonstrated by the applicant's actual test results. Therefore, the applicant's intention is that the appended claims are not limited by the selection of examples that illustrate the features of the present invention. Some numerical ranges used in the claims also include sub-ranges within them, and changes in these ranges should also be interpreted as being covered by the appended claims where possible.

Claims

1. The use of salinomycin in the preparation of a drug for treating renal damage caused by hyperuricemia, characterized in that: Salinomycin treats renal injury caused by hyperuricemia by inhibiting the expression of URAT1.

2. The application according to claim 1, characterized in that: The renal injury refers to renal tubular dilatation and / or renal tubular epithelial cell necrosis.

3. The application according to claim 1, characterized in that: The hyperuricemia is hyperuricemia caused by potassium oxonate and / or hypoxanthine.

4. Use of a pharmaceutical composition in the preparation of a drug for treating renal damage caused by hyperuricemia, characterized in that: The pharmaceutical composition comprises a therapeutically effective amount of salinomycin and optionally selected pharmaceutically acceptable excipients; salinomycin treats renal damage caused by hyperuricemia by inhibiting the expression of URAT1.

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