Application of sematinib in preparation of medicine for treating hepatic fibrosis

Abstract

The invention discloses application of sematinib in preparation of a medicine for treating hepatic fibrosis, and belongs to the technical field of biological medicines. The hepatic fibrosis disclosed by the invention is MAFLD related fibrosis. The results of an in-vivo LX-2 cell experiment and an in-vitro CDAA diet induced hepatic fibrosis mouse model show that the sematinib significantly reduces the contractility and migration potential of LX-2 cells, and can significantly improve liver inflammation and reduce collagen deposition. Therefore, the sematinib can be used for preparing the medicine for treating the hepatic fibrosis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a use of selutinib in preparing a medicament for treating liver fibrosis. Background Art

[0002] Metabolic-associated fatty liver disease (MAFLD) is a chronic disease prevalent worldwide. Its incidence has exploded over the past 20 years, driven by rising obesity and other related factors. MAFLD is a clinical and pathological syndrome characterized by hepatic steatosis and lipid accumulation in patients without a history of excessive alcohol consumption, often accompanied by hyperlipidemia, hyperglycemia, and insulin resistance. If not effectively treated, MAFLD can gradually progress to non-alcoholic steatohepatitis (MASH) and liver fibrosis. While intervention can restore the liver to normal, once cirrhosis develops, 1-4% of patients will develop hepatocellular carcinoma (HCC).

[0003] Fibrosis is only one of the processes involved in MAFLD. Many other pathological processes, such as hepatocyte damage and macrophage inflammatory infiltration, contribute to its development. Currently, the molecular mechanisms underlying the development of MAFLD remain unclear, and effective treatments are lacking.

[0004] Therefore, strengthening relevant research on MAFLD is of great significance to promoting the prevention and treatment of MAFLD.

[0005] Selutinib is an oral multi-target receptor tyrosine kinase inhibitor that has been used as an anti-tumor drug to treat hepatobiliary malignancies such as HCC, gallbladder cancer, and intrahepatic bile duct carcinoma.

[0006] Currently, there are no reports on the use of selutinib in the treatment of liver fibrosis. Summary of the Invention

[0007] Purpose of the invention: The purpose of the present invention is to provide a use of selutinib in the preparation of a medicament for treating liver fibrosis.

[0008] Technical solution: The purpose of the present invention is achieved through the following technical solution:

[0009] The present invention provides a use of selutinib in preparing a medicament for treating liver fibrosis.

[0010] The selutinib is used as the sole active ingredient in preparing a drug for treating liver fibrosis.

[0011] The selutinib is used in combination with other drugs to prepare a drug for treating liver fibrosis.

[0012] The liver fibrosis is MAFLD-related fibrosis.

[0013] The selutinib can improve liver inflammation and reduce collagen deposition.

[0014] The drug includes selutinib and a pharmaceutically acceptable carrier or excipient.

[0015] The excipients include one or more of antioxidants, wetting agents, diluents, emulsifiers, preservatives, disintegrants or adhesives.

[0016] The antioxidant is selected from at least one of ascorbic acid, sulfite, bisulfite, gallic acid and lipids thereof.

[0017] The wetting agent is selected from at least one of water and ethanol.

[0018] The diluent is selected from at least one of starches, sugars, celluloses or inorganic salts.

[0019] The emulsifier is selected from at least one of Tweens, Spans, glycerol fatty acid esters, pectin, agar, sodium alginate or silicon dioxide.

[0020] The preservative is selected from at least one of benzoic acid and its salts, sorbic acid and its salts, or parabens.

[0021] The disintegrant is selected from at least one of starch, sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone, low-substituted hydroxypropyl cellulose or cross-linked polyvinyl pyrrolidone.

[0022] The binder is selected from at least one of starch slurry, sodium carboxymethyl cellulose, povidone, hydroxypropyl cellulose, methyl cellulose or ethyl cellulose.

[0023] The dosage form of the medicine is granules, tablets, capsules, suspensions, oral solutions or injections.

[0024] The medicament of the present invention can be administered by various known means, such as oral administration, injection, etc. A preferred embodiment of the present invention is that the selutinib is administered orally. The medicament of the present invention can be administered alone or in combination with other drugs. The oral composition can be in any orally acceptable dosage form, including but not limited to capsules, tablets, granules, suspensions, and oral solutions.

[0025] Actual dosage levels of the active ingredients in the medicaments of the present invention can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend on a variety of factors, including the route of administration, time of administration, rate of excretion, duration of treatment, other drugs, compounds, and / or materials used in combination with selutinib, the age, sex, weight, general health, and previous medical history of the patient being treated, and similar factors well known in the medical arts.

[0026] Beneficial effects:

[0027] The selutinib described in the present invention can be used as an active ingredient in the treatment of non-alcoholic fatty liver fibrosis, opening up new uses for selutinib and providing a new option for the preparation of drugs for the treatment of non-alcoholic fatty liver fibrosis. The present invention has found that the use of selutinib improves the level of fibrosis in metabolic-associated fatty liver disease (MAFLD, i.e., non-alcoholic fatty liver disease). Therefore, selutinib can be used to prepare a potential drug for the treatment of non-alcoholic fatty liver fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the spatial structure of selutinib;

[0029] Figure 2 This is the result of the LX-2 cell collagen gel contraction experiment;

[0030] Figure 3 This is the result of Transwell experiment of LX-2 cells;

[0031] Figure 4 The results of Sirius red and HE staining experiments are shown in Figure 2.

[0032] Figure 5 This is the result of determination of serum triglyceride and total cholesterol in mice. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is described in detail below through specific embodiments, but the protection scope of the present invention is not limited to the embodiments.

[0034] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0035] The selutinib of the present invention is derived from TargetMol Chemicals Inc., with a purity of 99.85%. Its spatial structure is as follows Figure 1 shown.

[0036] Choline-methionine deficient diet (CDAA) was purchased from Deci Biotechnology (Wuxi) Co., Ltd.

[0037] Example 1 TGF-β stimulates LX-2 cells

[0038] The present invention uses recombinant protein TGF-β1 to induce liver fibrosis cell model to conduct in vitro experiments, confirming the improvement effect of selutinib on fibrosis.

[0039] 1. Cell seeding: LX-2 cells (ATCC) in the logarithmic growth phase were seeded into 6-well culture plates. The culture conditions were the same as those in the conventional procedure.

[0040] 2. Starvation culture: When the cells grow to about 60%-70% confluence, replace the culture medium with DMEM containing 1% FBS and culture under this condition for 4 hours.

[0041] 3. TGF-β stimulation: TGF-β (R&D Company, USA) was dissolved in Gibico high glucose medium and then diluted to 2 ng / mL with DMEM medium containing 1% FBS.

[0042] The cells were divided into five groups: a control group, a TGF-β-stimulated group, and selutinib dose groups (prepared with double-distilled water) with 1 μM, 10 μM, and 100 μM TGF-β. The five groups were starved for 2 hours with 1 mL of DMEM medium containing 1% FBS (Jiangsu KeyGen Biotech Co., Ltd.). Subsequently, 1 μL of 1 μM, 10 μM, and 100 μM selutinib was added to the three selutinib dose groups, respectively. After 2 hours, the medium was removed and the control group was treated with 1 mL of DMEM medium containing 1% FBS. The TGF-β-stimulated group and the different selutinib dose groups were stimulated with 1 mL of 1% DMEM medium containing TGF-β for 24 hours.

[0043] Example 2 Collagen gel contraction experiment

[0044] 1. Cell preparation: After the TGF-β stimulation in Example 1 is completed, the cells are digested and collected as usual, and then resuspended in 1 mL of DMEM medium containing 10% FBS. The cells are counted and the cell concentration in each well is adjusted to 1×10 6 / mL for future use.

[0045] 2. Prepare the gel: Take 200 μL of pre-cooled type I rat tail collagen (Corning, USA), slowly add 20 μL of 0.1% acetic acid solution, then mix with 400 μL of cell suspension, and then add an appropriate amount of pre-cooled 1 mol / L NaOH solution. Immediately and gently mix. If the cell suspension does not change color, repeat the addition of the above NaOH solution until the solution turns pink.

[0046] 3. Seed plate: Take an equal amount of the above mixture and slowly add it into a 48-well plate, 300 μL per well, to ensure even distribution.

[0047] 4. Solidification: Place the culture plate in a 37°C, 5% CO2 incubator and incubate for about 30 minutes.

[0048] 5. Photography: Add 300 μL of starvation medium (Jiangsu KeyGen Biotech Co., Ltd.) and take microscopic images at 0 and 24 hours after seeding to record the contraction of the collagen gel.

[0049] The results of the collagen gel contraction experiment are shown in Figure 2 As can be seen from the figure: the collagen gel initially appeared in a uniform, transparent disc shape with a smooth surface without cracks. After 24 hours, the collagen gel in the TGF-β group significantly shrank compared with the control group; and compared with the TGF-β group, the collagen gel shrinkage trend of the selutinib dose groups stimulated with TGF-β (i.e., TGF-β1μM group, TGF-β10μM group, and TGF-β100μM group) slowed down, indicating that after the use of selutinib, collagen reduced the contractile ability of LX-2 cells.

[0050] Example 3 Transwell migration assay

[0051] 1. Cell preparation: Take the remaining cells from the collagen gel contraction experiment and dilute an appropriate amount of cells to 1×10 4 Take 200 μL of the cell suspension and evenly inoculate it into the upper chamber of the Transwell plate.

[0052] 2. Culture: Add 500 μL of DMEM medium without TGF-β to the lower chamber of the control group, and add 500 μL of DMEM medium containing TGF-β to the lower chamber of the other four groups. Place the culture plate in a 37°C, 5% CO2 incubator and culture for 24 hours.

[0053] 3. After incubation, remove the cells from the incubator and discard the culture medium in the upper chamber. Rinse the cells three times with 1 mL of PBS and then fix them with 1 mL of 4% paraformaldehyde for 30 minutes.

[0054] 4. Discard 4% paraformaldehyde, add 500 μL 0.1% crystal violet, and let it stand at room temperature for 1 hour.

[0055] 5. Discard the crystal violet and add 500 μL PBS to each well to wash the cells. Wash three times at room temperature on a shaker for 5 minutes each time.

[0056] 6. Photography: After staining, place the chamber under an inverted microscope for observation, and randomly select 5 fields of view at 10× magnification for photography.

[0057] Transwell migration assay results are shown in Figure 3 As can be seen from the figure: compared with the control group, crystal violet staining of the lower surface of the Transwell membrane in the TGF group showed that cells densely covered the membrane pores and appeared uniformly purple; while only sporadic stained cells were seen in the control group; compared with the TGF group, the number of migrating cells in the selutinib dose groups (i.e., TGF-β1μM group, TGF-β10μM group, and TGF-β100μM group) was significantly reduced, indicating that selutinib significantly reduced the migration ability of LX-2 cells.

[0058] Example 4 Establishment of a mouse model of liver fibrosis

[0059] Experimental animals: 8-week-old C57BL / 6WT male mice weighing 20±1.5 g were purchased from the laboratory of Nanjing Medical University and randomly divided into 5 groups: control group, model group, low-dose group, medium-dose group, and high-dose group, with 8 mice in each group.

[0060] The control group mice were fed a regular diet (Jiangsu Qinglongshan Biotechnology Co., Ltd.), while the model group and the low-, medium-, and high-dose groups were fed a CDAA diet. Simultaneously, the low-, medium-, and high-dose groups were given 10 mg / kg, 20 mg / kg, and 40 mg / kg of selutinib by gavage, respectively. Both the control and model groups were given 0.2 mL of 0.9% saline by gavage once every 1.5 days for 8 weeks.

[0061] Selutinib was dissolved in a mixed solvent of 60% 0.9% saline and 40% PEG (polyethylene glycol), and prepared into different doses of 10 mg / kg, 20 mg / kg, and 40 mg / kg, respectively. The patients were gavaged at 0.1 mL / 10 mg, once every 1.5 days, for 8 weeks.

[0062] Example 5 Preparation of liver tissue paraffin sections

[0063] 1. Tissue Fixation: After modeling, all mice were anesthetized with 2% isoflurane inhalation and the livers were isolated. The liver tissues were fixed in 4% paraformaldehyde for at least 24 hours. The liver tissues were then removed and immersed in distilled water for 15 minutes, repeated twice.

[0064] 2. Dehydration: Immerse the tissue in 50%, 70%, 80%, and 90% ethanol solutions for 30 minutes each, followed by 60 minutes each in 100% ethanol (I) and 100% ethanol (II). Remove the liver tissue and trim it flat.

[0065] 3. Transparent treatment: Soak the dehydrated liver tissue in xylene I and xylene II for 10 minutes each time.

[0066] 4. Wax immersion: Place the transparentized liver tissue in 60°C liquid paraffin and perform two rounds of immersion treatment, each round lasting 2 hours, to fully replace the xylene in the tissue.

[0067] 5. Embedding: Transfer the paraffin-soaked liver tissue into an embedding container and completely wrap the tissue with melted paraffin for subsequent sectioning.

[0068] 6. Sectioning and Fixation: Cut the embedded liver tissue into 5 μm thick continuous slices, flatten them in a 42°C water bath, transfer them to glass slides, and bake them in a 60°C oven for 30 minutes for fixation. Finally, dry and store them at room temperature.

[0069] Example 6 Sirius Red and HE Staining Experiment

[0070] 1. Picrosirius Red staining

[0071] Steps:

[0072] (1) Dewax the paraffin sections prepared in Example 5 to water: the sections were sequentially placed in xylene solution (dewaxing time was 10 minutes), fresh xylene solution (dewaxing time was 10 minutes), anhydrous ethanol (5 minutes), 95% ethanol (5 minutes), 85% ethanol (5 minutes), 75% ethanol (5 minutes), and distilled water (5 minutes).

[0073] (2) Sirius red staining: Add Sirius red staining solution to the slices and stain for 1 hour. Then, rinse the slices gently with running water to remove the staining solution on the slice surface.

[0074] (3) Dehydration and clearing: Dehydrate the stained sections in 95% ethanol I for 2 min, then dehydrate in 95% ethanol II (2 min), anhydrous ethanol I (3 min), and anhydrous ethanol II (3 min). Clear the sections with xylene I (5 min) and xylene II (5 min), then seal the sections with neutral gum.

[0075] (4) Observe under a microscope.

[0076] 2. HE staining

[0077] Steps:

[0078] (1) Dewax the paraffin sections prepared in Example 5 to water: dewax the sections in xylene solution for 10 minutes, fresh xylene solution for 10 minutes, anhydrous ethanol for 5 minutes, 95% ethanol for 5 minutes, 85% ethanol for 5 minutes, 75% ethanol for 5 minutes, and distilled water for 5 minutes.

[0079] (2) Hematoxylin staining: Slices were stained with hematoxylin solution for 1 minute, washed with distilled water to remove floating color, differentiated with differentiation solution for 30 seconds, and rinsed with tap water twice, each time for 3 minutes.

[0080] (3) Eosin staining: Drop eosin staining solution on the tissue section, stain for 1 minute (adjust the time according to the actual situation), and then quickly dehydrate.

[0081] (4) Dehydration and sealing: The sections were placed in ethanol gradient for dehydration in sequence. The immersion time in 75% ethanol, 85% ethanol, 95% ethanol and anhydrous ethanol was 3 seconds, the immersion time in clean anhydrous ethanol was 1 minute, and the sections were transparentized in xylene solution twice, each time for 1 minute.

[0082] (5) Seal the slides with neutral gum and dry them in a cool place.

[0083] (6) Observe under a microscope.

[0084] The results of Sirius red and HE staining experiments are shown in Figure 4 As can be seen from the figure: HE staining showed that compared with the control group (sham group), the livers of the four groups of mice fed with CDAA feed showed macrovesicular and microvesicular fatty degeneration; picrosirius red staining showed that compared with the control group, the collagen fibers of the model group (CDAA group) mice were dark red and fibrosis was severe, with fibrosis mostly located in the central periphery and perisinusoidal areas. In contrast, the red color of the collagen fibers in the liver tissues of the mice in the selutinib dose groups (i.e., 10 mg / kg group, 20 mg / kg group, and 40 mg / kg group) became significantly lighter, indicating that after the use of selutinib, the collagen deposition in the mouse liver was significantly reduced and fibrosis was alleviated.

[0085] Example 7 Determination of triglyceride and serum total cholesterol levels in mice

[0086] 1. Serum triglycerides

[0087] Reagents and instruments: serum triglyceride (Nanjing Jiancheng A110-1-1 triglyceride assay kit 96T), microplate reader, 96-well microplate.

[0088] Steps:

[0089] (1) Prepare samples: Take out serum from the sample bank (blood from the ocular vein).

[0090] (2) The sample addition process is shown in Table 1:

[0091] Table 1 Sample addition process

[0092]

[0093] Perform the assay according to the kit instructions.

[0094] 2. Serum total cholesterol

[0095] Reagents and instruments: total cholesterol determination kit (Nanjing Jiancheng A111-1-1 total cholesterol determination kit 96T), microplate reader, 96-well microplate.

[0096] The operating steps are the same as those for the determination of serum triglycerides.

[0097] The results of triglyceride and total serum cholesterol are shown in Figure 5 .in, Figure 5 A is triglyceride, Figure 5 B is total cholesterol. As can be seen from the figure: compared with the control group, the model group (CDAA group) showed a significant increase in serum triglyceride and total cholesterol levels, while the serum triglyceride and total cholesterol levels in the three selutinib dose groups (i.e., CDAA 10 mg / kg group, CDAA 20 mg / kg group, and CDAA 40 mg / kg group) decreased, indicating that selutinib improved liver metabolism in mice.

[0098] These results indicate that selutinib has a significant therapeutic effect in alleviating liver fibrosis.

[0099] The present study found that selutinib has the potential to be a promising therapeutic intervention for the treatment of liver fibrosis and deserves further study in the clinical setting.

[0100] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes may be made to it in form and detail without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. Use of selutinib in the preparation of a medicament for treating liver fibrosis.

2. The use according to claim 1, characterized in that The selutinib is used as the sole active ingredient in preparing a drug for treating liver fibrosis.

3. The use according to claim 1, characterized in that The selutinib is used in combination with other drugs to prepare a drug for treating liver fibrosis.

4. The use according to claim 1, characterized in that The liver fibrosis is MAFLD-related fibrosis.

5. The use according to claim 1, characterized in that The selutinib can improve liver inflammation and reduce collagen deposition.

6. The use according to claim 1, characterized in that The drug includes selutinib and a pharmaceutically acceptable carrier or excipient.

7. The use according to claim 6, characterized in that The dosage form of the medicine is granules, tablets, capsules, suspensions, oral solutions or injections.

8. The use according to claim 1, characterized in that The selutinib is administered orally.

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