Application of composition containing uridine or derivatives thereof and praziquantel in preparation of medicine for preventing or treating liver injury

Abstract

The invention discloses application of a composition containing uridine or a derivative thereof and praziquantel in preparation of a medicine for preventing or treating liver injury. Uridine or derivatives thereof and praziquantel cooperate to further inhibit hepatic stellate cell activation, so that the uridine or derivatives thereof and praziquantel can be used as a medicine for inhibiting hepatic stellate cell activation and preventing or treating liver injury. Particularly, the compound can be used for preparing medicines for treating hepatic fibrosis caused by schistosomiasis.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and more specifically, to the use of a composition comprising uridine or a derivative thereof and praziquantel in the preparation of a medicament for preventing or treating liver damage. Background Art

[0002] Liver fibrosis refers to a pathological process in which abnormal proliferation of connective tissue in the liver is caused by various pathogenic factors, leading to excessive deposition of diffuse extracellular matrix in the liver. Hepatocyte damage and necrosis caused by various pathogenic factors is the initial link in the occurrence of liver fibrosis, and its further development will lead to cirrhosis and even liver cancer.

[0003] Liver fibrosis may be caused by a variety of chronic liver diseases, such as hepatitis B and C, alcoholic hepatitis, non-alcoholic fatty liver disease, schistosomiasis, autoimmune liver disease, drug-induced liver injury, etc. Among them, schistosomiasis is an important zoonosis caused by schistosome infection, especially in developing countries. Current treatment mainly relies on praziquantel, a broad-spectrum anti-schistosomal drug that can effectively eliminate schistosomes in the body. Although praziquantel has significant efficacy in the treatment of schistosomiasis, when used alone, its therapeutic effect is limited because it cannot effectively inhibit the fibrotic response of the body after infection. In addition, the use of praziquantel alone may also lead to a decrease in efficacy due to the development of drug resistance. Current studies have shown that monotherapy may not be able to fully solve the side effects caused by schistosome infection, especially in the intervention of fibrosis, and traditional therapies often cannot meet clinical needs. Summary of the invention

[0004] In order to overcome the above-mentioned shortcomings, the present invention provides a new medicine for preventing or treating liver damage.

[0005] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0006] Application of uridine or its derivatives as synergists of praziquantel.

[0007] Specifically, the synergist refers to an agent that can increase the efficacy of praziquantel in treating schistosomiasis, especially in reducing the area of ​​liver fibrosis caused by schistosomiasis infection and reducing liver cell apoptosis.

[0008] The present invention also provides use of a composition comprising uridine or its derivatives and praziquantel in preparing a drug for inhibiting the activation of hepatic stellate cells.

[0009] Through experimental research, the inventors found that a composition comprising uridine or its derivatives and praziquantel has a good effect on inhibiting the activation of hepatic stellate cells caused by schistosoma japonicum infection. Compared with praziquantel, uridine or its derivatives, the effect of inhibiting the activation of hepatic stellate cells caused by schistosoma japonicum infection is significantly improved. The composition can be used to prepare drugs for preventing or treating liver damage, especially for preparing drugs for preventing or treating liver fibrosis caused by schistosoma japonicum infection.

[0010] Use of a composition comprising uridine or its derivatives and praziquantel in preparing a medicament for preventing or treating liver damage.

[0011] Specifically, the liver damage is liver fibrosis.

[0012] More specifically, the liver damage is liver fibrosis caused by schistosomiasis.

[0013] In the present invention, the uridine derivative includes one or more of uracil, uridine monophosphate, uridine diphosphate, uridine triphosphate, triacetyl uridine, tribenzoyl uridine or 5-ethyl uridine.

[0014] A pharmaceutical composition contains uridine or its derivatives and praziquantel for simultaneous or separate administration.

[0015] Furthermore, the pharmaceutical composition also contains a pharmaceutically acceptable carrier, diluent or excipient as an auxiliary agent.

[0016] In preparing these compositions, the active ingredient is usually mixed with an excipient, or diluted with an excipient, or enclosed in a carrier which may be in the form of a capsule or sachet. When the excipient acts as a diluent, it may be a solid, semisolid or liquid material which serves as an excipient, carrier or medium for the active ingredient.

[0017] Examples of suitable excipients include lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup and methylcellulose. In addition, the composition may also include lubricants (such as talc, magnesium stearate and mineral oil), wetting agents, emulsifiers and suspending agents, preservatives (such as methyl hydroxybenzoate and propyl hydroxybenzoate), sweeteners or flavoring agents.

[0018] The pharmaceutical composition of the present invention can be prepared into various known dosage forms. Specifically, the dosage form prepared by the pharmaceutical composition is an oral dosage form or an injection dosage form.

[0019] Oral dosage forms include solid dosage forms such as tablets, dripping pills, quick-release dripping pills, capsules (such as liquid gel capsules and solid gel capsules), granules or powders. Tablets can be compressed tablets, ground tablets, enteric-coated tablets, sugar-coated tablets, film-coated tablets or multilayer compressed tablets. As the suitable auxiliary agent type for preparing tablets, include but are not limited to adhesives, lubricants, diluents, disintegrants, coloring agents, flavoring agents, drainage agents or melting agents. Oral dosage forms can also be liquid oral dosage forms, such as aqueous solutions, emulsions, suspensions, solutions made of non-effervescent particles and / or suspensions or effervescent particles. As the suitable auxiliary agent type for preparing liquid oral agents, include solvents, preservatives, emulsifiers, suspending agents, diluents, sweeteners, melting agents, coloring agents or flavoring agents.

[0020] The injection dosage forms include: injection solution (normal), lyophilized powder injection, powder injection (normal), injection tablets, etc.

[0021] Specifically, the pharmaceutical composition can be in the form of tablets, pills, powders, elixirs, suspensions, emulsions, solutions, syrups, soft and hard gelatin capsules, suppositories, sterile injectable solutions and sterile packaged powders.

[0022] Furthermore, in the pharmaceutical composition, the mass ratio of uridine or its derivative to praziquantel is 1:3 to 3:1.

[0023] Furthermore, the pharmaceutical composition is prepared in the form of an oral dosage form or an injection dosage form.

[0024] Specifically, the pharmaceutical composition is a formulated injection, a formulated tablet, a formulated capsule, a formulated pill or a formulated pill.

[0025] The pharmaceutical composition is used in preparing a medicine for treating liver fibrosis.

[0026] Furthermore, the pharmaceutical composition is used in the preparation of a drug for treating liver fibrosis caused by schistosomiasis.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention provides the use of uridine or its derivatives as a synergist of praziquantel. It is found that the use of uridine or its derivatives together with praziquantel can significantly improve the effect of inhibiting hepatic stellate cell activation and reducing hepatocyte apoptosis. The composition containing uridine or its derivatives and praziquantel can be used as a drug for inhibiting hepatic stellate cell activation, preventing or treating liver damage. The present invention also provides a new pharmaceutical composition, which contains uridine or its derivatives and praziquantel as active ingredients administered simultaneously or separately. The pharmaceutical composition can be used to prepare a drug for inhibiting hepatic stellate cell activation or treating liver damage, especially for preparing a drug for treating liver fibrosis caused by schistosomiasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a data graph showing the effect of each group on the survival rate of L-O2 cells;

[0030] Figure 2 The data graphs show the effects of each group on hepatic stellate cell activation markers;

[0031] Figure 3 The data graphs show the effects of each group on the liver function of mice infected with Schistosoma japonicum;

[0032] Figure 4 This is a data graph showing the effects of each group on the pathological examination results of liver fibrosis in mice infected with Schistosoma japonicum;

[0033] Figure 5 The data graph shows the effects of each group on the activation markers of hepatic stellate cells in mice infected with Schistosoma japonicum. DETAILED DESCRIPTION

[0034] The technical solutions in the embodiments of the present invention will be described clearly and completely below, but the implementation manner of the present invention is not limited thereto.

[0035] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0036] 1. Uridine: purchased from Shanghai Bio-Tech Biotechnology Co., Ltd., catalog number ST1735;

[0037] 2. Praziquantel: purchased from Shanghai MacLean Biochemical Technology Co., Ltd., catalog number P823033;

[0038] 3. Schistosoma japonicum: cercariae (Jiangsu strain) were provided by the Oncomelania Ecology Laboratory, Jiangsu Institute of Schistosomiasis Control;

[0039] 4. ICR mice (age: 6 weeks, weight 20 ± 2 g) were purchased from Weitonglihua Experimental Animal Technology Co., Ltd., Zhejiang Province, China;

[0040] 5. Annexin-V apoptosis detection kit: purchased from Nanjing Novozyme Biotechnology Co., Ltd., catalog number A211;

[0041] 6. Trizol, reverse transcription kit, and real time qPCR kit were purchased from Nanjing Novozyme Biotechnology Co., Ltd., with catalog numbers R711, R333, and Q711, respectively;

[0042] 7. Recombinant human TGF-β1 protein: purchased from Aibixin (Shanghai) Biotechnology Co., Ltd., catalog number abs04204.

[0043] Example 1 Effects of Uridine and Praziquantel on the Survival Rate of L-O2 Cells

[0044] 1. Experimental Methods

[0045] Liver cells include hepatocytes and non-parenchymal cells, among which hepatocytes account for the largest proportion. Adverse effects such as schistosoma japonicum eggs can cause apoptosis of hepatocytes. Hepatocytes in the exponential growth phase (i.e., L-O2 cells) were selected, and after trypsin digestion of the cells, a single cell suspension was prepared with DMEM complete medium at 1.0×10 per well. 5 The cells were seeded at a density of 100 cells / well in a 6-well plate and incubated overnight in a cell culture incubator (37°C, 5% CO2). The cells were divided into five groups: control group, model group, praziquantel intervention group, uridine intervention group, and uridine / praziquantel combined intervention group. Soluble egg antigen (SEA) (final concentration 20 μg / mL) was added to the model group, praziquantel intervention group, uridine intervention group, and uridine / praziquantel combined intervention group; complete medium containing praziquantel (final concentration 500 μg / mL) was added to the praziquantel intervention group, complete medium containing uridine or its derivatives (final concentration 500 μg / mL) was added to the uridine intervention group, and complete medium containing uridine and praziquantel (the mass ratio of uridine to praziquantel was 1:1, and the final concentration was 500 μg / mL) was added to the uridine / praziquantel combined intervention group; complete medium was added to the control group and culture was continued for 24 hours. The apoptosis of L-O2 cells was detected using the Annexin-V apoptosis detection kit. The results are shown in Figure 1 shown.

[0046] 2. Experimental results

[0047] from Figure 1It can be seen that compared with the control group, the proportion of cell apoptosis in the model group was significantly increased (1.07±1.10% vs 25.30±2.36%, p<0.0001), while compared with the model group, the proportion of cell apoptosis was significantly reduced after uridine or praziquantel intervention (25.30±2.36% vs 8.18±1,12%, p<0.0001; 25.30±2.36% vs 10.43±2.27%, p<0.0001), but compared with the uridine and praziquantel combined intervention group, the proportion of cell apoptosis in the uridine and praziquantel combined intervention group was further reduced (8.18±1,12% vs 1.09±0.32%, p<0.001; 10.43±2.27% vs 1.09±0.32%, p<0.01).

[0048] Example 2 Effects of Uridine and Praziquantel on Hepatic Stellate Cell Activation Markers

[0049] 1. Experimental Methods

[0050] Among the many factors that activate hepatic stellate cells, TGF-β1 is the most effective profibrotic cytokine. Activated hepatic stellate cells highly express α-smooth muscle actin (α-SMA) and secrete a variety of collagens, including type I collagen (Collagen I) and type III collagen (Collagen III). The effects of uridine intervention on the mRNA and protein levels of α-SMA, Collagen I and Collagen III in model cells (i.e., TGF-β-treated LX-2 cells) were detected by RT-qPCR and Western blot. LX-2 cells were prepared into single cell suspensions according to the operating procedures of Example 1, with 1.0×10 5 Cells were seeded at a density of 100 cells / well in a 6-well plate and incubated overnight in a cell culture incubator (37°C, 5% CO2). The cells were divided into five groups: control group, model group, praziquantel intervention group, uridine intervention group, and uridine / praziquantel combined intervention group. After the model group, praziquantel intervention group, uridine intervention group, and uridine / praziquantel combined intervention group were stimulated with TGF-β (final concentration 25 ng / mL) for 12 hours, the culture medium was removed and the cells were washed with sterile PBS. The praziquantel intervention group was added with complete culture medium containing praziquantel (final concentration 500 μg / mL), the uridine intervention group was added with complete culture medium containing uridine (final concentration 500 μg / mL), the uridine / praziquantel combined intervention group was added with complete culture medium containing uridine and praziquantel (the mass ratio of uridine to praziquantel was 1:1, final concentration 500 μg / mL), and the control group and model group were added with complete culture medium and cultured for 24 hours. The total RNA of cells was extracted by Trizol method, and the expression levels of α-SMA, Collagen I and Collagen III mRNA were detected by RT-qPCR. The results are shown in Figure 2shown.

[0051] 2. Experimental results

[0052] from Figure 2 It can be seen that compared with the control group, the mRNA expression levels of α-SMA, Collagen I and Collagen III in the model group were significantly increased (p < 0.0001), and compared with the model group, the mRNA expression levels of α-SMA, Collagen I and Collagen III were significantly decreased after intervention with uridine or praziquantel (p < 0.01), but compared with the combined intervention group of uridine and praziquantel, the mRNA expression levels of α-SMA, Collagen I and Collagen III in the combined intervention group of uridine and praziquantel were further decreased (p < 0.05).

[0053] Example 3 Effects of uridine and praziquantel on liver function in mice infected with Schistosoma japonicum

[0054] 1. Experimental Methods

[0055] ICR mice were randomly divided into five groups, namely control group, infection group, infection + uridine group, infection + praziquantel group, infection + praziquantel + uridine group. Except for the control group, the rest of the mice were infected with 15±2 cercariae of Schistosoma japonicum through the abdominal skin. From the fifth week of infection, mice in the infection + praziquantel group were given 300 mg / kg of praziquantel every other day, mice in the infection + uridine group were given 300 mg / kg of uridine, mice in the infection + uridine + praziquantel group were given 300 mg / kg of praziquantel + uridine, and the control group was given the same volume mass concentration of 1% sodium carboxymethyl cellulose solution; it should be noted that praziquantel, uridine, and praziquantel + uridine were all administered in a suspension state (for example, praziquantel is a suspension obtained by dispersing praziquantel in a mass concentration of 1% sodium carboxymethyl cellulose solution). All mice were killed on the 84th day to obtain the required samples. Blood was collected from mice in each group, placed at room temperature for two hours, and then centrifuged at 3000 rpm for 15 minutes. The supernatant was collected and the levels of alanine aminotransferase, aspartate aminotransferase, albumin, and alkaline phosphatase in the serum were detected using an automatic biochemical analyzer.

[0056] 2. Experimental results

[0057] The experimental results are as follows Figure 3As shown in the figure, compared with the control group, the serum alanine aminotransferase and aspartate aminotransferase of the infected mice were significantly increased (p < 0.0001), while compared with the infected group, the serum alanine aminotransferase and aspartate aminotransferase were significantly decreased after uridine or praziquantel treatment (ALT: p < 0.01; AST: p < 0.05), indicating that uridine and praziquantel can alleviate the hepatic cell necrosis caused by schistosomiasis, and the combined use of uridine and praziquantel is more effective than that of single use (ALT: p < 0.05; AST: p < 0.05); compared with the control group, the serum albumin of the infected mice was significantly decreased (p < 0.0001), while compared with the infected group Compared with the control group mice, serum alkaline phosphatase in the infected group mice was significantly increased (p<0.001), while serum alkaline phosphatase in the infected group mice was decreased after uridine or praziquantel treatment (p<0.01), and the difference was statistically significant, indicating that uridine and praziquantel can help relieve hepatitis and bile duct obstruction caused by schistosomiasis, and the combined use of uridine and praziquantel has a better effect than that of either of them (p<0.05).

[0058] Example 4 Effects of uridine and praziquantel on pathological examination results of liver fibrosis in mice infected with Schistosoma japonicum

[0059] 1. Experimental Methods

[0060] The liver of the mouse in Example 3 was collected, and liver tissue of appropriate size at the same position of the left lobe of the liver was fixed in a 4% paraformaldehyde solution for 24 hours. After gradient dehydration, transparency, wax immersion, embedding, slicing, drying, dewaxing, rehydration and Masson staining, the slices were sealed, observed under an optical microscope and photographed.

[0061] 2. Experimental results

[0062] The experimental results are as follows Figure 4 As shown, compared with the control group, there were a large number of worm egg granulomas and collagen deposition (Masson staining positive) areas in the liver of the infected mice (p < 0.0001), and compared with the infected group, the area of ​​collagen deposition was significantly reduced after treatment with uridine or praziquantel (p < 0.0001), indicating that uridine and praziquantel can effectively alleviate the liver fibrosis caused by Schistosoma japonicum, and when the two are used together, the area of ​​collagen deposition is further reduced (p < 0.05), indicating that uridine and praziquantel can further alleviate the liver fibrosis caused by Schistosoma japonicum infection when used together.

[0063] Example 5 Effects of Uridine and Praziquantel on Activation Markers of Hepatic Stellate Cells in Mice Infected with Schistosoma japonicum

[0064] 1. Experimental Methods

[0065] The mouse liver in Example 3 was collected, and liver tissue of appropriate size at the same position of the liver was taken, Trizol and zirconium oxide grinding beads were added, and the tissue was ground at 4°C for 25 minutes to fully lyse the tissue. The supernatant was taken to a new centrifuge tube for RNA extraction, and the RT-qPCR method was used to detect the mRNA level of the α-SMA gene in the liver tissue.

[0066] 2. Experimental results

[0067] The experimental results are as follows Figure 5 As shown, compared with the control group, the relative expression of α-SMA gene mRNA in the liver of mice in the infection group was increased (p < 0.0001), while compared with the infection group, the relative expression of α-SMA gene mRNA was significantly decreased after treatment with uridine or praziquantel (p < 0.01), indicating that uridine and praziquantel can effectively reduce the hepatic stellate cell activation caused by Schistosoma japonicum, and when the two are used together, the relative expression of α-SMA is further reduced (p < 0.05), indicating that uridine and praziquantel can further reduce the hepatic stellate cell activation caused by Schistosoma japonicum infection when used together.

[0068] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the embodiments here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. The use of uridine or its derivatives as a synergist of praziquantel.

2. Use of a composition comprising uridine or its derivatives and praziquantel in the preparation of a drug for inhibiting the activation of hepatic stellate cells.

3. Use of a composition comprising uridine or its derivatives and praziquantel in the preparation of a medicament for preventing or treating liver damage.

4. The use according to claim 3, characterized in that: The liver damage is liver fibrosis.

5. The application according to claim 3, characterized in that: The liver damage is liver fibrosis caused by schistosomiasis.

6. A pharmaceutical composition, characterized in that The invention comprises uridine or its derivatives and praziquantel as active ingredients to be administered simultaneously or separately.

7. The pharmaceutical composition according to claim 6, characterized in that: The mass ratio of the uridine or its derivative to praziquantel is 3:1 to 1:

3.

8. The pharmaceutical composition according to claim 6, characterized in that: The pharmaceutical composition further contains a pharmaceutically acceptable carrier, diluent or excipient as an auxiliary agent.

9. The pharmaceutical composition according to claim 6, characterized in that: The dosage form of the pharmaceutical composition is an oral dosage form or an injection dosage form.

10. The pharmaceutical composition according to claim 6, characterized in that: The pharmaceutical composition is used in preparing a medicine for treating liver fibrosis caused by schistosomiasis.

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