Use of L-galactose in the preparation of a drug for preventing and / or treating hyperuricemia related diseases and a prevention and treatment drug

Abstract

This invention discloses the application of L-galactose in the preparation of drugs for the prevention and / or treatment of hyperuricemia-related diseases, as well as the use of L-galactose in preventive and therapeutic drugs. This invention is the first to discover and confirm that L-galactose has the activity of lowering uric acid levels in the body, representing a pioneering discovery of a new use for a known substance. It is anticipated that the use of L-galactose to treat hyperuricemia will have good biocompatibility, low toxicity, and high safety, making it particularly suitable as a therapeutic drug or daily health care ingredient for chronic diseases requiring long-term use (such as hyperuricemia and gout).

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of L-galactose in the preparation of drugs for the prevention and / or treatment of hyperuricemia-related diseases and related preventive and therapeutic drugs. Background Technology

[0002] The statements herein provide only background information in relation to this invention and do not necessarily constitute prior art.

[0003] Hyperuricemia (HUA) is a common metabolic disease caused by disordered purine metabolism and / or impaired uric acid excretion, leading to elevated uric acid levels in the blood. It is a direct cause of acute gout attacks and is closely associated with the risk of developing many major chronic diseases, including chronic kidney disease, cardiovascular disease, hypertension, diabetes, and metabolic syndrome, making it a significant threat to public health.

[0004] When blood uric acid levels exceed the saturation point of monosodium urate in the joints, causing it to precipitate and deposit in the peripheral joints and surrounding tissues, it is called gout. Therefore, hyperuricemia is the underlying cause of gout.

[0005] Meanwhile, hyperuricemia can also lead to acute uric acid nephropathy, chronic uric acid nephropathy, and kidney stones, increasing the risk of kidney failure.

[0006] Currently, uric acid-lowering drugs in clinical practice are mainly divided into two categories:

[0007] Uric acid production inhibitors, such as allopurinol and febuxostat, reduce uric acid production by inhibiting the activity of xanthine oxidase (XOD). However, these drugs may cause serious adverse skin reactions (such as Stevens-Johnson syndrome), liver and kidney damage, and long-term use carries cardiovascular risks (such as febuxostat).

[0008] Uric acid excretion promoters, such as benzbromarone and probenecid, work by inhibiting the reabsorption of uric acid in the renal tubules, thus promoting its excretion in urine. However, these drugs may increase the risk of kidney stones and should be used with caution in patients with renal insufficiency. Furthermore, benzbromarone has been reported to cause fulminant hepatitis.

[0009] The limitations of existing drugs mainly lie in their significant safety concerns and limited target specificity. Therefore, there is a huge clinical need and market potential in developing novel uric acid-lowering drugs or functional health products with novel mechanisms of action, high safety profiles, and natural sources.

[0010] L-galactose is a naturally occurring hexose and an important intermediate in the biosynthesis of vitamin C. Recent studies have found that L-galactose and its derivatives possess various biological activities, including antioxidant, anti-aging, and immunomodulatory effects. However, to date, no literature or patents, either domestically or internationally, have reported that L-galactose can regulate uric acid metabolism or lower blood uric acid levels. Summary of the Invention

[0011] In view of the shortcomings of the existing technology, the purpose of this invention is to provide the application of L-galactose in the preparation of medicaments for the prevention and / or treatment of hyperuricemia-related diseases.

[0012] To achieve the above objectives, the present invention provides the following technical solution:

[0013] As a first aspect of the invention, it provides the use of L-galactose in the preparation of a medicament for the prevention and / or treatment of hyperuricemia-related diseases, said medicament using L-galactose as the active pharmaceutical ingredient and further comprising a pharmaceutically acceptable carrier.

[0014] The high uric acid-related diseases include hyperuricemia and gout.

[0015] Furthermore, the gout includes acute gout and chronic gout.

[0016] The research of this invention found that L-galactose can rapidly reduce serum uric acid levels and has high safety, making it suitable for the treatment of acute gout as well as the long-term treatment of chronic gout.

[0017] Current research indicates that approximately two-thirds of the uric acid produced by the liver is excreted through the kidneys. Blood flowing through the kidneys is first filtered by the glomeruli, which filter uric acid into the urine (primary urine). When the primary urine flows through the proximal convoluted tubule, 98%-100% of the uric acid is reabsorbed back into the bloodstream by the epithelial cells. This is the most crucial step in regulating blood uric acid levels. In this process, the URAT1 protein, located on the apical membrane side of renal tubular cells, is responsible for transporting uric acid from the primary urine into the cells; the GLUT9 protein, located on the basement membrane side of renal tubular cells, is responsible for transporting uric acid from the cells into the bloodstream, completing the reabsorption process. Therefore, URAT1 and GLUT9 are key genes for renal uric acid reabsorption and are also the targets of clinical drugs such as benzbromarone and probenecid. These drugs reduce renal reabsorption of uric acid by inhibiting the expression of URAT1 or GLUT9 in the kidneys, thereby lowering blood uric acid levels.

[0018] The mechanism of action of L-galactose in lowering uric acid provided by this invention is that L-galactose can inhibit the expression of GLUT9 or URAT1 in renal cells, suggesting that it may lower uric acid by inhibiting the reabsorption of uric acid by the kidneys.

[0019] The study of this invention found that L-galactose did not affect the function or pathological indicators of the liver and kidneys in hyperuricemic model mice or healthy mice, and did not cause significant liver and kidney damage, indicating that L-galactose has high safety.

[0020] This invention has found that L-galactose significantly reduces serum urea levels in hyperuricemic mice and can improve kidney function. Based on this research, the hyperuricemia-related diseases also include uric acid nephropathy caused by hyperuricemia.

[0021] As a second aspect of the invention, a drug for the prevention and treatment of hyperuricemia-related diseases is also provided, using L-galactose as the active pharmaceutical ingredient.

[0022] In some embodiments of the present invention, the dosage form of the drug is selected from tablets, capsules, injections, pills, granules, ointments, mixtures, or suspensions.

[0023] Furthermore, the injectable agent is an injection solution or a sterile powder for injection.

[0024] In some embodiments of the present invention, L-galactose in the drug is dissolved in sterile saline.

[0025] Compared with the prior art, the beneficial effects achieved by one or more embodiments of the present invention are as follows:

[0026] 1. This invention is the first in the world to discover and confirm that L-galactose has a novel pharmacological activity—it can reduce uric acid levels in the body. This is a pioneering discovery of new uses for known substances and expands the application of L-galactose.

[0027] 2. The technical solution provided by this invention has excellent safety advantages: L-galactose is an intermediate in the synthesis pathway of vitamin C and is expected to have good biocompatibility, low toxicity and side effects, and high safety. It is particularly suitable as a treatment drug or daily health care ingredient for chronic diseases (such as hyperuricemia and gout) that require long-term use.

[0028] 3. The L-galactose involved in this invention has a wide range of sources and diverse applications: L-galactose can be obtained through chemical synthesis, enzymatic conversion, or extraction from natural plants (such as certain algae and traditional Chinese medicine). This invention can prepare it into various forms of pharmaceutical preparations (such as tablets, capsules, oral liquids, and injections). Attached Figure Description

[0029] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0030] Figure 1 This is a statistical graph showing the effect of L-galactose on the survival rate of 293T cells.

[0031] Figure 2 The following are statistical graphs showing the effects of L-galactose on cellular gene expression; where a represents the transcriptional level of the GLUT9 gene in 293T cells; b represents the expression level of the GLUT9 gene in 293T cells; and c represents the transcriptional level of the GLUT9 gene in HK2 cells. * indicates... p <0.05, ** indicates p <0.01.

[0032] Figure 3 The following are statistical graphs showing the effects of L-galactose on body weight and serum uric acid levels in mice with hyperuricemia; where a represents body weight over time; b represents serum uric acid levels on day 8; c represents serum uric acid levels on day 16; and d represents serum uric acid levels on day 24. * indicates comparison with the model group. p <0.05, ** indicates p <0.01, *** indicates p <0.001, **** indicates p <0.0001.

[0033] Figure 4 The following are statistical graphs showing the effects of L-galactose on serum liver and kidney function indicators in mice with hyperuricemia. Specifically, a) represents serum urea levels; b) represents serum creatinine levels; c) represents serum alanine aminotransferase (ALT) levels; d) represents serum aspartate aminotransferase (AST) levels; e) represents serum total bilirubin levels; f) represents serum alkaline phosphatase (ALP) levels; g) represents serum albumin levels; and h) represents serum total protein levels. * indicates comparison with the model group, and # indicates comparison with the allopurinol group. * and # indicate... p <0.05, ** and ## indicate p <0.01, *** indicates p <0.001, **** indicates p <0.0001.

[0034] Figure 5The following is a statistical graph showing the effects of L-galactose on the pathological damage of the liver and kidneys in a mouse model of hyperuricemia. In the graph, a represents the H&E staining of liver tissue from the control group; b represents the H&E staining of liver tissue from the model group; c represents the H&E staining of liver tissue from the allopurinol group; d represents the H&E staining of liver tissue from the low-dose L-galactose group; e represents the H&E staining of liver tissue from the high-dose L-galactose group; f represents the H&E staining of kidney tissue from the control group; g represents the H&E staining of kidney tissue from the model group; h represents the H&E staining of kidney tissue from the allopurinol group; i represents the H&E staining of kidney tissue from the low-dose L-galactose group; and j represents the H&E staining of kidney tissue from the high-dose L-galactose group.

[0035] Figure 6 The following are statistical graphs showing the effects of L-galactose on body weight and serum uric acid levels in healthy mice; where a represents body weight over time; b represents serum uric acid levels on day 8; c represents serum uric acid levels on day 16; and d represents serum uric acid levels on day 24.

[0036] Figure 7 The following are statistical graphs showing the effects of L-galactose on liver and kidney function indicators in the serum of healthy mice: a) serum creatinine level; b) serum alanine aminotransferase (ALT) level; c) serum aspartate aminotransferase (AST) level; d) serum total bilirubin level; e) serum alkaline phosphatase (ALP) level; f) serum albumin level; and g) serum total protein level.

[0037] Figure 8 The following is a statistical graph showing the effects of L-galactose on the pathological damage of the liver and kidneys of healthy mice; where a is the H&E staining image of the liver tissue of the control group; b is the H&E staining image of the liver tissue of the L-galactose group; c is the H&E staining image of the kidney tissue of the control group; and d is the H&E staining image of the kidney tissue of the L-galactose group.

[0038] Figure 9 This is a statistical graph showing the effect of L-galactose on the transcription of uric acid transporter genes in the kidneys of hyperuricemic mouse models; where a represents the transcriptional level of the GLUT9 gene; and b represents the transcriptional level of the URAT1 gene. * indicates comparison with the model group. p <0.05, ** indicates p <0.01, *** indicates p <0.001, **** indicates p <0.0001.

[0039] Figure 10This is a statistical graph showing the effect of L-galactose on the transcription of uric acid transporter genes in the kidneys of healthy mice; where a represents the transcriptional level of the GLUT9 gene; and b represents the transcriptional level of the URAT1 gene. * indicates comparison with the control group. ** indicates... p <0.01. Detailed Implementation

[0040] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0041] The present invention will be further described below with reference to the embodiments.

[0042] Example 1

[0043] Effects of L-galactose on cell viability

[0044] 293T and HK2 cells were purchased from the Stem Cell Bank of the Chinese Academy of Sciences (https: / / www.cellbank.org.cn / ejianjie.php). In this study, 293T cells were cultured in high-glucose DMEM (Gibco) medium supplemented with 10% (v / v) fetal bovine serum and 1% (v / v) penicillin-streptomycin (Gibco). HK2 cells were cultured in DMEM / F-12 medium (Gibco) containing 10% (v / v) fetal bovine serum and 1% (v / v) penicillin-streptomycin. Cells were seeded in 96-well plates at a density of 5000 cells per well with 0.1 mL of medium. After 16 hours, cells were treated for 12 hours with different concentrations of L-galactose (0, 0.1 mM, 0.4 mM, 2.0 mM, 10 mM, and 50 mM, L-galactose dissolved in sterile physiological saline). The toxicity of the drug to cells was assessed using a Cell-Counting kit (CCK8, 40203ES60, Yeasen, Shanghai, China).

[0045] As a result, the analysis results of the Cell-Counting kit (CCK8) showed that ( Figure 1 In this experiment, all concentrations of L-galactose used had no significant effect on the viability of 293T cells, indicating that L-galactose has no obvious cytotoxicity.

[0046] Example 2

[0047] Effects of L-galactose on cellular gene expression

[0048] 293T and HK2 cells were seeded in 24-well plates at a density of 1 × 10⁻⁶ cells per well.5 Cells were cultured in 0.5 mL medium. After 16 hours, cells were treated with different concentrations of L-galactose (0, 0.4 mM, 2.0 mM, and 10 mM, L-galactose dissolved in sterile saline) for 12 hours. The culture medium was then removed, and RT-qPCR and Western blot analyses were performed. Total RNA was extracted from the cells using the SteadyPure RNA Extraction Kit (AG21017, Accurate Biology). Genomic DNA (gDNA) was removed and complementary DNA (cDNA) was synthesized using the Evo M-MLVRT Mix Kit with gDNA Clean for qPCR Ver.2 (AG11728, Accurate Biotechnology). qPCR detection: The expression of the uric acid transporter GLUT9 (solute carrier family 2, member 9, also known as SLC2A9) gene was detected using the SYBR Green kit (AG11701, Accurate Biotechnology) on a Roche LightCycler® 480II real-time quantitative PCR system. The primer sequences used for RT-qPCR are shown in Table 1. Relative expression levels were calculated using the 2-ΔΔCt formula.

[0049] Table 1. Primer sequences used in this study

[0050]

[0051] Western blot analysis:

[0052] Total cellular protein was extracted and separated by 10% SDS-PAGE. Protein samples were transferred to activated PVDF membranes and blocked with 5% skim milk for 1 hour. The membranes were then incubated overnight at 4°C with the following primary antibodies: anti-β-actin (1:4000 dilution, AC026, ABclonal) and anti-GLUT9 (1:1000 dilution, 26486-1-AP, Proteintech). The following day, the membranes were incubated with the corresponding secondary antibodies for 1.5 hours. Protein bands were visualized using a chemiluminescence imaging system (Analytik Jena, Germany) and analyzed for grayscale using ImageJ.

[0053] Figure 2 The results in (a) and (b) indicate that L-galactose dose-dependently inhibits the transcription and expression levels of the GLUT9 gene in 293T cells. Meanwhile, Figure 2 c indicates that L-galactose inhibits the transcription level of the GLUT9 gene in HK2 cells.

[0054] Example 3

[0055] L-galactose-mediated hyperuricemia model mice

[0056] After one week of acclimatization, 50 male C57BL / 6JNifdc mice aged 6-8 weeks and weighing 21-24g were randomly divided into 5 groups (n=10 per group), with no significant difference in weight between the groups. The treatments for each group were as follows: (1) Control group: 0.5% sodium carboxymethyl cellulose (CMC-Na) solution was administered by gavage; (2) Model group: hypoxanthine (300 mg / kg / d, H108384, Aladdin, Shanghai) and potassium oxonate (300 mg / kg / d, A601238, Sangon Biotech, Shanghai) mixed solution was administered by gavage; (3) Allopurinol group: Allopurinol solution (10 mg / kg / d, TA09075G, Sinopharm Chemical Reagent Co., Ltd., Shanghai) was administered by gavage on the basis of the model group; (4) Low-dose L-galactose group (Gal-L group): L-galactose (20 mg / kg / d, L812550, Maclean Biochemical, Shanghai) solution was administered by gavage on the basis of the model group; (5) High-dose L-galactose group (Gal-H group): L-galactose (40 mg / kg / d) solution was administered by gavage on the basis of the model group. Drug intervention began 2 hours after administration of the modeling drug. Patients were weighed every 6-8 days, and blood was collected from the retro-orbital venous plexus after a 12-hour fast.

[0057] Mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and housed in a standard laboratory animal barrier environment (12 h / 12 ​​h light / dark cycle, humidity 50±15%, temperature 22±2℃). All procedures followed the "Guidelines for the Household and Use of Laboratory Animals" (8th edition, ISBN-10: 0-309-15396-4). The experimental protocol was approved by the Bioethics and Biosafety Review Board (BGI-IRB) of the Jinan Microecology Biomedical Shandong Laboratory.

[0058] Figure 3 The results showed that low and high doses of L-galactose did not significantly affect the body weight of mice with hyperuricemia. Figure 3 The results showed that on day 8, high-dose L-galactose significantly reduced serum uric acid levels in mice. Figure 3 Results in c and d showed that, on days 16 and 24, both low and high doses of L-galactose significantly reduced serum uric acid levels in mice.

[0059] Example 4

[0060] L-galactose intervention in healthy mice

[0061] After one week of acclimatization, 20 male C57BL / 6JNifdc mice aged 6-8 weeks and weighing 21-24g were randomly divided into two groups (n=10 per group), with no significant difference in weight between the groups. The treatments for each group were as follows: (1) Control group: administered 0.5% sodium carboxymethyl cellulose (CMC-Na) solution by gavage; (2) L-galactose (Gal group): administered L-galactose (40 mg / kg / d) solution by gavage in addition to the treatment for the control group. After the experiment, the mice were anesthetized and their serum was collected.

[0062] like Figure 6 The results, shown in a fortified table (a), b for day 8 (b), c for day 16 (c), and d for day 24 (d), indicate that gavage administration of 40 mg / kg / d L-galactose did not affect the body weight or serum uric acid levels in healthy mice.

[0063] Example 5

[0064] Effects of L-galactose on serum uric acid and other blood biochemical parameters in mice

[0065] Whole blood was collected from euthanized mice, and serum was separated by centrifugation at 3000 rpm for 15 min at 4°C. The following indicators were strictly measured according to the kit instructions: uric acid (E-BC-K016-M, Elabscience) and urea (E-BC-K183-M, Elabscience). The following indicators were measured using a Mindray BS800M blood biochemistry analyzer: alanine aminotransferase (ALT, 105-001389-00), aspartate aminotransferase (AST, 105-001390-00), alkaline phosphatase (ALP, 105-001391-00), total bilirubin (T-bil-V, 105-001401-00), creatinine (105-001404-00), albumin (ALB, 105-001397-00), and total protein (105-015580-00).

[0066] In a mouse model of hyperuricemia Figure 4 The results showed that, compared with the model group and the allopurinol group, low-dose and high-dose L-galactose significantly reduced serum urea levels, indicating improved renal function. Figure 4 Results b and c showed that low and high doses of L-galactose did not affect the levels of creatinine and alanine aminotransferase in mouse serum. Figure 4 The results showed that, compared with the model group, high-dose L-galactose significantly reduced the serum aspartate aminotransferase content. Figure 4The results (e, f, g, h) showed that low and high doses of L-galactose did not affect the levels of total bilirubin, alkaline phosphatase, albumin, and total protein in mouse serum. These results indicate that low (20 mg / kg / d) and high (40 mg / kg / d) L-galactose did not cause significant liver and kidney damage.

[0067] In healthy mice, such as Figure 7 The results, shown in a fork of serum creatinine (a), serum alanine aminotransferase (b), serum aspartate aminotransferase (c), serum total bilirubin (d), serum alkaline phosphatase (e), serum albumin (f), and serum total protein (g), indicate that oral administration of 40 mg / kg / d L-galactose did not affect the levels of serum creatinine, alanine aminotransferase, aspartate aminotransferase, total bilirubin, alkaline phosphatase, albumin, and total protein in healthy mice. This suggests that L-galactose does not affect the function of the liver and kidneys in healthy mice.

[0068] Example 6

[0069] Histopathological analysis of mouse liver and kidney tissue sections

[0070] Liver and kidney tissues were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, stained with hematoxylin and eosin (H&E), and observed under a microscope to assess the degree of pathological damage.

[0071] In a mouse model of hyperuricemia Figure 5 H&E staining images of liver tissue from control group (a), model group (b), allopurinol group (c), low-dose L-galactose group (d), and high-dose L-galactose group (e); H&E staining images of kidney tissue from control group (f), model group (g), allopurinol group (h), low-dose L-galactose group (i), and high-dose L-galactose group (j) show that low-dose (20 mg / kg / d) and high-dose (40 mg / kg / d) L-galactose did not cause significant pathological damage to liver and kidney tissues, indicating the high safety of L-galactose.

[0072] In healthy mice, such as Figure 8 The H&E staining images of liver tissue in the control group (a), liver tissue in the L-galactose group (b), kidney tissue in the control group (c), and kidney tissue in the L-galactose group (d) show that gavage administration of 40 mg / kg / d L-galactose did not cause significant pathological damage to the liver and kidney tissues of healthy mice, indicating the high safety of L-galactose.

[0073] Example 7

[0074] Analysis of transcriptional levels of uric acid transporter gene in mouse kidney

[0075] Kidney tissues were collected from mice, and total RNA was extracted using the SteadyPure RNA Extraction Kit (AG21017, AccurateBiology). The differences in transcriptional levels of the uric acid transporter genes GLUT9 and URAT1 in kidney tissues were detected using RT-PCR. Primer sequences used for RT-qPCR are shown in Table 2. Relative expression levels were calculated using the 2-ΔΔCt formula.

[0076] Table 2. Primer sequences used in this study

[0077]

[0078] In a mouse model of hyperuricemia Figure 9 The results of GLUT9 gene transcription level shown in a and URAT1 gene transcription level shown in b show that low dose (20 mg / kg / d) and high dose (40 mg / kg / d) of L-galactose significantly reduced the transcription levels of GLUT9 and URAT1 genes in kidney tissue.

[0079] In healthy mice, Figure 10 The results of GLUT9 gene transcription level shown in a and URAT1 gene transcription level shown in b show that gavage administration of 40 mg / kg / d L-galactose significantly reduced the transcription levels of GLUT9 and URAT1 genes in kidney tissue.

[0080] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Use of L-galactose for the preparation of a medicament for the treatment of hyperuricemia-related diseases, characterized in that, The drug uses L-galactose as its active pharmaceutical ingredient and also includes a pharmaceutically acceptable carrier. The high uric acid-related diseases mentioned are hyperuricemia, gout, or uric acid nephropathy.

2. The use of L-galactose according to claim 1 in the preparation of a medicament for treating hyperuricemia-related diseases, characterized in that, The gout mentioned includes acute gout and chronic gout.

3. The use of L-galactose according to claim 1 in the preparation of a medicament for treating hyperuricemia-related diseases, characterized in that, L-galactose inhibits the expression of GLUT9 or URAT1 in the kidneys.

4. The use of L-galactose according to claim 1 in the preparation of a medicament for treating hyperuricemia-related diseases, characterized in that, L-galactose inhibits the reabsorption of uric acid by the kidneys, thereby lowering uric acid levels.

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