Use of composition containing uridine or derivative thereof and praziquantel in preparation of medicament for preventing or treating liver injury

Abstract

Disclosed is a use of a composition comprising uridine or a derivative thereof and praziquantel in preparation of a medicament for preventing or treating liver injury, and in particular in preparation of a medicament for treating hepatic fibrosis caused by schistosomiasis.

Description

Use of compositions comprising uridine or its derivatives and praziquantel in the preparation of medicaments for the prevention or treatment of liver injury. Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more specifically, to the use of compositions comprising uridine or its derivatives and praziquantel in the preparation of medicaments for the prevention or treatment of liver injury. Background Technology

[0002] Liver fibrosis refers to the pathological process caused by abnormal proliferation of connective tissue in the liver due to various pathogenic factors, resulting in excessive deposition of diffuse extracellular matrix in the liver. Hepatocellular damage and necrosis caused by various pathogenic factors are the initial steps in the development of liver fibrosis, which can lead to cirrhosis and even liver cancer.

[0003] Liver fibrosis can be caused by various chronic liver diseases, such as hepatitis B and C, alcoholic hepatitis, non-alcoholic fatty liver disease, schistosomiasis, autoimmune liver disease, and drug-induced liver injury. Schistosomiasis, caused by infection with schistosomes, is a significant zoonotic disease, particularly prevalent in developing countries. Current treatment primarily relies on praziquantel, a broad-spectrum anti-schistosomiasis drug that effectively eliminates schistosomes from the body. While praziquantel is highly effective in treating schistosomiasis, its efficacy is limited when used alone because it cannot effectively inhibit the post-infection fibrotic response. Furthermore, monotherapy with praziquantel may lead to decreased efficacy due to drug resistance. Current research indicates that monotherapy may not fully address the side effects of schistosomiasis infection, particularly in the intervention of fibrosis, where traditional therapies often fail to meet clinical needs. Summary of the Invention

[0004] In order to overcome the above-mentioned shortcomings, the present invention provides a new drug for the prevention or treatment of liver damage.

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

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

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

[0008] The present invention also provides the use of a composition comprising uridine or a derivative thereof and praziquantel in the preparation of a medicament for inhibiting hepatic stellate cell activation.

[0009] Through experimental research, the inventors discovered that the composition containing uridine or its derivatives and praziquantel has a better inhibitory effect on the activation of hepatic stellate cells caused by schistosomiasis infection. Compared with the inhibitory effect of praziquantel, uridine or its derivatives on the activation of hepatic stellate cells caused by schistosomiasis infection, the composition has been significantly improved. It can be used to prepare drugs for the prevention or treatment of liver damage, especially drugs for the prevention or treatment of liver fibrosis caused by schistosomiasis infection.

[0010] Use of compositions comprising uridine or its derivatives and praziquantel in the preparation of medicaments for the prevention or treatment of liver injury.

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

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

[0013] In this invention, the uridine derivative includes one or more of uracil, uridine monophosphate, uridine diphosphate, uridine triphosphate, triacetyluridine, tribenzoyluridine, or 5-ethyluridine.

[0014] A pharmaceutical composition comprising uridine or a derivative thereof for simultaneous or separate administration and praziquantel.

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

[0016] In preparing these compositions, the active ingredient is typically mixed with an excipient, diluted with an excipient, or encapsulated in a carrier that may be in the form of a capsule or pouch. When the excipient acts as a diluent, it can be a solid, semi-solid, or liquid material serving as the medium for the excipient, carrier, or active ingredient.

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

[0018] The pharmaceutical composition described in this invention can be prepared into various known dosage forms. Specifically, the dosage form prepared from the pharmaceutical composition is an oral dosage form or an injectable dosage form.

[0019] Oral dosage forms include solid dosage forms such as tablets, pellets, immediate-release pellets, 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. Suitable adjuvants for tablet preparation include, but are not limited to, binders, lubricants, diluents, disintegrants, colorants, flavoring agents, flow diverters, or melting agents. Oral dosage forms can also be liquid oral dosage forms, such as aqueous solutions, emulsions, suspensions, solutions and / or suspensions made from non-effervescent particles, or effervescent tablets made from effervescent particles. Suitable adjuvants for liquid oral dosage forms include solvents, preservatives, emulsifiers, suspending agents, diluents, sweeteners, melting agents, colorants, or flavoring agents.

[0020] The injectable dosage forms include: injection solutions (ordinary), lyophilized powder for injection, powder for injection (ordinary), and tablets for injection.

[0021] Specifically, the pharmaceutical composition may be 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 dosage form of the pharmaceutical composition is an oral dosage form or an injectable dosage form.

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

[0025] The use of the pharmaceutical composition in the preparation of a medicament for treating liver fibrosis.

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

[0027] Compared with the prior art, the beneficial effects of the present invention are:

[0028] This invention provides the application of uridine or its derivatives as an synergist for praziquantel. It has been found that the use of uridine or its derivatives in conjunction with praziquantel significantly enhances the effects of inhibiting hepatic stellate cell activation and reducing hepatocyte apoptosis. Compositions containing uridine or its derivatives and praziquantel can be used as drugs for inhibiting hepatic stellate cell activation and preventing or treating liver damage. This invention also provides a novel pharmaceutical composition comprising uridine or its derivatives and praziquantel as active ingredients, administered simultaneously or separately. This pharmaceutical composition can be used to prepare drugs for inhibiting hepatic stellate cell activation or treating liver damage, particularly for treating liver fibrosis caused by schistosomiasis. Attached Figure Description

[0029] Figure 1 shows the data on the effect of each group on the survival rate of L-O2 cells;

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

[0031] Figure 3 shows the data on the effects of each group on liver function in mice infected with Schistosoma japonicum.

[0032] Figure 4 shows the data on the impact of each group on the pathological examination results of liver fibrosis in mice infected with Schistosoma japonicum.

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

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

[0035] Unless otherwise specified, the reagents, methods and equipment used in this invention are all conventional reagents, methods and equipment in this technical field.

[0036] 1. Urate: Purchased from Shanghai Beyotime Biotechnology Co., Ltd., product number ST1735;

[0037] 2. Praziquantel: Purchased from Shanghai Maclean Biochemical Technology Co., Ltd., product number P823033;

[0038] 3. Schistosomiasis: Cercariae (Jiangsu strain) were provided by the Snail Ecology Laboratory of Jiangsu Institute of Schistosomiasis Control;

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

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

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

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

[0043] Example 1: Effects of uridine and praziquantel on L-O2 cell survival

[0044] 1. Experimental Methods

[0045] Hepatic cells include parenchymal cells and non-parenchymal cells, with parenchymal cells making up the largest proportion. Adverse effects such as schistosome eggs can induce apoptosis in parenchymal cells. Hepatic parenchymal cells in the exponential growth phase (i.e., L-O2 cells) were selected, and after trypsin digestion, single-cell suspensions were prepared using DMEM complete medium at a concentration of 1.0 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells / well in 6-well plates and incubated overnight in a cell culture incubator (37°C, 5% CO2). 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; praziquantel intervention group was additionally added to complete medium containing praziquantel (final concentration 500 μg / mL); uridine intervention group was additionally added to complete medium containing uridine or its derivatives (final concentration 500 μg / mL); and uridine / praziquantel combined intervention group was additionally added to complete medium containing uridine and praziquantel (uridine to praziquantel mass ratio 1:1, final concentration 500 μg / mL); the control group was added to complete medium. Cells were cultured for another 24 hours. Apoptosis of L-O2 cells was detected using the Annexin-V apoptosis detection kit. The results are shown in Figure 1.

[0046] 2. Experimental Results

[0047] As shown in Figure 1, the apoptosis rate in the model group was significantly higher than that in the control group (1.07±1.10% vs 25.30±2.36%, p<0.0001). Compared with the model group, the apoptosis rate was significantly lower after intervention with uridine or praziquantel (25.30±2.36% vs 8.18±1.12%, p<0.0001; 25.30±2.36% vs 10.43±2.27%, p<0.0001). However, compared with the combined intervention group of uridine and praziquantel, the apoptosis rate was further lower in the combined intervention group of uridine and praziquantel (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 markers of hepatic stellate cell activation

[0049] 1. Experimental Methods

[0050] Among the many factors that activate hepatic stellate cells, TGF-β1 is the most potent pro-fibrotic cytokine. Activated hepatic stellate cells highly express α-smooth muscle actin (α-SMA) and secrete various 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 procedure in Example 1, at a density of 1.0 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells / well in 6-well plates and incubated overnight in a cell culture incubator (37°C, 5% CO2). Cells were divided into five groups: control group, model group, praziquantel intervention group, uridine intervention group, and uridine / praziquantel combined intervention group. After stimulation with TGF-β (final concentration 25 ng / mL) for 12 hours in the model group, praziquantel intervention group, uridine intervention group, and uridine / praziquantel combined intervention group, the culture medium was removed and the cells were washed with sterile PBS. The praziquantel intervention group was treated with complete culture medium containing praziquantel (final concentration 500 μg / mL), the uridine intervention group with complete culture medium containing uridine (final concentration 500 μg / mL), and the uridine / praziquantel combined intervention group with complete culture medium containing uridine and praziquantel (uridine to praziquantel mass ratio 1:1, final concentration 500 μg / mL). The control group and model group were treated with complete culture medium. All cells were cultured for another 24 hours. Total RNA was extracted from cells using the 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 2.

[0051] 2. Experimental Results

[0052] As shown in Figure 2, compared with the control group, the expression levels of α-SMA, Collagen I, and Collagen III mRNA in the model group were significantly increased (p < 0.0001). Compared with the model group, the expression levels of α-SMA, Collagen I, and Collagen III mRNA were significantly decreased after intervention with uridine or praziquantel (p < 0.01). However, compared with the combined intervention group of uridine and praziquantel, the expression levels of α-SMA, Collagen I, and Collagen III mRNA were further decreased in the combined intervention group of uridine and praziquantel (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: control group, infection group, infection + uridine group, infection + praziquantel group, and infection + praziquantel + uridine group. Except for the control group, all other mice were infected with 15±2 cercariae of Schistosoma japonicum via abdominal skin infection. Starting from the fifth week of infection, mice in the infection + praziquantel group were given praziquantel 300 mg / kg every other day; mice in the infection + uridine group were given uridine 300 mg / kg; and mice in the infection + uridine + praziquantel group were given praziquantel + uridine 300 mg / kg. The control group received the same volume of 1% sodium carboxymethyl cellulose solution. It should be noted that praziquantel, uridine, and praziquantel + uridine were all administered in suspension form (e.g., praziquantel was dispersed in a 1% sodium carboxymethyl cellulose solution to obtain a suspension). All mice were sacrificed on day 84 to obtain the required samples. Blood was collected from each group of mice and left at room temperature for two hours. The mice were 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 automated biochemical analyzer.

[0056] 2. Experimental Results

[0057] The experimental results are shown in Figure 3. Compared with the control group, the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in the infected group were significantly increased (p < 0.0001). However, compared with the infected group, serum ALT and AST levels were significantly decreased after treatment with uridine or praziquantel (ALT: p < 0.01; AST: p < 0.05), indicating that uridine and praziquantel can alleviate hepatocellular necrosis caused by schistosomiasis. The combined use of uridine and praziquantel was more effective than their individual use (ALT: p < 0.05; AST: p < 0.05). Compared with the control group, the serum albumin level in the infected group was significantly decreased (p < 0.0001), while the serum albumin level was significantly decreased after treatment with uridine or praziquantel (ALT: p < 0.01; AST: p < 0.05). Compared with the infected group, serum albumin levels significantly increased after treatment with uridine or praziquantel (p < 0.0001), indicating that uridine and praziquantel help restore liver protein synthesis function in schistosomiasis-infected mice. The combined use of uridine and praziquantel was more effective than either treatment alone (p < 0.01). Compared with the control group, serum alkaline phosphatase levels were significantly increased in the infected group (p < 0.001). However, compared with the infected group, serum alkaline phosphatase levels decreased after treatment with uridine or praziquantel (p < 0.01), and the difference was statistically significant. This indicates that uridine and praziquantel help alleviate hepatitis and bile duct obstruction caused by schistosomiasis, and the combined use of uridine and praziquantel was more effective than either treatment alone (p < 0.05).

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

[0059] 1. Experimental Methods

[0060] Mouse livers from Example 3 were collected, and liver tissue of appropriate size from the same location in the left lobe of the liver was fixed in 4% paraformaldehyde solution for 24 hours. After gradient dehydration, clearing, paraffin embedding, embedding, sectioning, drying, dewaxing, rehydration, and Masson staining, the sections were sealed and observed and photographed under an optical microscope.

[0061] 2. Experimental Results

[0062] The experimental results are shown in Figure 4. Compared with the control group, the infected mice had a large number of granulomas of parasite eggs and areas of collagen deposition (Masson staining positive) in their livers (p < 0.0001). Compared with the infected group, the area of ​​collagen deposition area was significantly reduced after treatment with uridine or praziquantel (p < 0.0001), indicating that uridine and praziquantel can effectively alleviate liver fibrosis caused by Schistosoma japonicum. When the two were used together, the area of ​​collagen deposition area was further reduced (p < 0.05), indicating that the use of uridine and praziquantel together can further alleviate liver fibrosis caused by Schistosoma japonicum infection.

[0063] Example 5: Effects of uridine and praziquantel on biomarkers of hepatic stellate cells in mice infected with Schistosoma japonicum

[0064] 1. Experimental Methods

[0065] The livers of mice in Example 3 were collected, and liver tissue of appropriate size from the same location 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 tissue was centrifuged at 12000×g at 4°C for 5 minutes, and the supernatant was transferred to a new centrifuge tube for RNA extraction. The α-SMA gene mRNA level in the liver tissue was detected by RT-qPCR.

[0066] 2. Experimental Results

[0067] The experimental results are shown in Figure 5. Compared with the control group, the relative expression level of α-SMA gene mRNA in the liver of infected mice was increased (p < 0.0001). Compared with the infected group, the relative expression level of α-SMA gene mRNA was significantly reduced after treatment with uridine or praziquantel (p < 0.01), indicating that uridine and praziquantel can effectively reduce the activation of hepatic stellate cells caused by Schistosoma japonicum. When the two were used together, the relative expression level of α-SMA was further reduced (p < 0.05), indicating that the use of uridine and praziquantel together can further reduce the activation of hepatic stellate cells caused by Schistosoma japonicum infection.

[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 implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. Application of uridine or its derivatives as synergists for praziquantel.

2. Use of a composition comprising uridine or its derivative and praziquantel in the preparation of a medicament for inhibiting hepatic stellate cell activation.

3. Use of a composition comprising uridine or its derivatives and praziquantel in the preparation of a medicament for the prevention or treatment of liver injury.

4. The application according to claim 3, characterized in that, The liver damage described is liver fibrosis.

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

6. A pharmaceutical composition, characterized in that, It contains uridine or its derivatives and praziquantel as active ingredients, administered simultaneously or separately.

7. The pharmaceutical composition according to claim 6, characterized in that, The mass ratio of 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 also contains a pharmaceutically acceptable carrier, diluent, or excipient as an adjuvant.

9. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutical composition is formulated into an oral dosage form or an injectable dosage form.

10. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutical composition is used in the preparation of a drug for treating liver fibrosis caused by schistosomiasis.

Images