Application of rezantinib in preparation of medicine for treating non-alcoholic steatohepatitis

Abstract

The invention discloses an application of rezantinib in preparation of a medicine for treating non-alcoholic steatohepatitis. The invention discovers that the compound shows a good treatment effect in a non-alcoholic steatohepatitis (NASH) model for the first time. In-vivo experiments prove that in NASH model mice, the Razitinib can significantly reduce pathological characteristics such as liver weight, liver fat level and liver index. Meanwhile, the level of transaminase (ALT / AST) in serum can be remarkably reduced through treatment of the rezantinib, and it is indicated that the liver injury and the liver function are effectively improved through the rezantinib. The invention provides a brand-new and effective drug treatment strategy for clinical treatment of NASH, and has remarkable clinical application value and market prospect.

Description

Technical Field

[0001] This invention relates to the application of rizotinib in the preparation of a drug for treating non-alcoholic steatohepatitis, and belongs to the field of biomedical technology. Background Technology

[0002] Globally, the prevalence of non-alcoholic fatty liver disease (NAFLD) has risen sharply due to the increasing prevalence of metabolic syndrome, diabetes, and obesity. This disease is irreversible and cannot resolve spontaneously, making it a major cause of liver-related morbidity and mortality. NAFLD is now renamed metabolic dysfunction-associated steatotic liver disease (MASLD). Non-alcoholic steatohepatitis (NASH) is a more severe form of NAFLD, causing not only severe liver damage but also cardiovascular disease, chronic liver and kidney disease, and extrahepatic malignancies. NAFLD accounts for 10% of all hepatocellular carcinoma (HCC) cases, and the incidence of NAFLD-HCC in China is projected to increase by 82% between 2016 and 2030. Given the rapid progression of the disease and unmet treatment needs, there is an urgent need to develop drugs that can effectively treat NASH.

[0003] Significant breakthroughs have been made in basic research on NASH. In addition to treatments such as diet, exercise, or bariatric surgery, GLP-1 receptor agonists have recently been approved for clinical treatment of NASH. However, other effective intervention targets and strategies for NASH still lack.

[0004] Rezitinib Mesylate Capsules (trade name: Rezazinib is an irreversible, highly selective third-generation small-molecule epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TK1) developed by Beierda Pharmaceuticals. Its currently approved indications include first-line treatment of adult patients with locally advanced or metastatic non-small cell lung cancer (NSCLCc) with EGFR exon 19 deletion or exon 21 (L858R) substitution mutations, and treatment of adult patients with locally advanced or metastatic NSCLCc who have experienced disease progression after prior EGFR-TKi treatment and are also EGFR T790M mutation-positive. Rezazinib demonstrates excellent efficacy and safety. However, there are currently no reports on whether rezazinib has any therapeutic effect on non-alcoholic steatohepatitis (NASH). Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing drugs for treating NASH by providing the application of retezilinib in the preparation of drugs for treating non-alcoholic steatohepatitis.

[0006] In order to achieve the above-mentioned purpose, the present application provides the use of regorafenib and / or a pharmaceutically acceptable salt form thereof in the preparation of a medicament for treating non-alcoholic steatohepatitis.

[0007] The present application also provides the use of regorafenib and / or a pharmaceutically acceptable salt form thereof in the preparation of a medicament for treating simple fatty liver.

[0008] Preferably, the medicament contains the active ingredient of regorafenib and / or a pharmaceutically acceptable salt form thereof and at least one pharmaceutically acceptable carrier or excipient.

[0009] Preferably, the dosage form of the medicament includes tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, oral liquids, oral suspensions, granules, powders, pills, beads, suspensions, wine, tincture or drops.

[0010] Compared with the prior art, the present application has the following beneficial effects:

[0011] The regorafenib provided by the present application can be used for clinically treating fatty hepatitis. Animal experiments show that regorafenib has good therapeutic effect on fatty hepatitis, can reduce the pathological characteristics such as liver weight, liver fat level and liver index, and at the same time, regorafenib treatment can significantly reduce the serum transaminase (ALT / AST) level, indicating that it effectively improves liver damage and liver function. The present application provides a new indication of regorafenib, which has good clinical application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 It is shown that regorafenib has good therapeutic effect in the CDAHFD-induced fatty hepatitis model, wherein A shows the liver color of control group mice, regorafenib-low-dose treatment group and regorafenib-high-dose treatment group mice in the high-fat diet-induced fatty liver model; B shows the liver weight of mice in each group, and regorafenib can significantly reduce the liver weight of mice, and the regorafenib-high-dose treatment group has lower liver weight; C shows the liver weight ratio of mice in each group, and the regorafenib-high-dose treatment group has lower liver weight ratio, suggesting lower fatty liver level; D shows the triglyceride TG level in the liver of mice in each group; E-G show the triglyceride TG level (E), aspartate aminotransferase AST (F) and alanine aminotransferase level ALT (G) in the serum of mice in each group.

[0013] Figure 2Rezivertinib showed no therapeutic activity in CDK12-deficient fatty hepatitis mice, wherein A shows the liver color of control mice, wild-type mice-rezivertinib group, CDK12 knockout mice-control group and CDK12 knockout mice-rezivertinib group in a high-fat diet-induced fatty liver model; B shows the liver weight of mice in each group; C shows the liver weight ratio of mice in each group; D shows the triglyceride TG level in the liver of mice in each group; E-G show the triglyceride TG level (E), aspartate aminotransferase AST (F) and alanine aminotransferase level ALT (G) in the serum of mice in each group.

[0014] Figure 3 The binding mode of Cdk12 protein and Rezivertinib is shown, wherein A is a cartoon diagram of the binding of Cdk12 protein and Rezivertinib; B is a surface diagram of the binding of Cdk12 protein and Rezivertinib; C is a 2D diagram of the binding of Cdk12 protein and Rezivertinib; D is a 3D diagram of the binding of Cdk12 protein and Rezivertinib.

[0015] Figure 4 Rezivertinib inhibits the formation of CDK12 / CCNK complex. DETAILED DESCRIPTION

[0016] In order to make the present application more apparent and easy to understand, the preferred embodiments are described in detail below with the accompanying drawings.

[0017] EMBODIMENT

[0018] I. Animal source and rearing environment

[0019] The C57BL / 6 wild-type mice involved in the present application are purchased from Shanghai Southern Model Organisms Technology Co., Ltd. All mice are reared in a specific pathogen free (SPF) level environment, maintaining a 12 / 12 hour light / dark rhythm (light time: 8:00-20:00), an environmental temperature of 22±2℃ and a humidity of 40%-60%.

[0020] II. Experimental method

[0021] 1.1 Construction of CDK12 gene knockout mice

[0022] The present application uses a Cre-loxP system to construct a mouse model with liver-specific knockout of CDK12 gene, and through Flox mice (flox mice with Neo gene removed, CDK12 flox / + :Flp +) and alb-cre tool mice, and the flox mice are constructed by Shanghai Southern Model Organisms Technology Co., Ltd. The mating strategy of the flox mice and alb-cre tool mice is as follows:

[0023] 1) The flox mice with Neo gene removed (CDK12 flox / + : Neo + ) are obtained by mating with wild-type mice, and the flox mice with Flp genotype removed (the mice are simply written as: CDK12 flox / + ) are obtained;

[0024] 2) The obtained flox positive heterozygote mice with Neo and Flp removed (CDK12 flox / + ) are divided into two parts: one part of the flox mice is mated with Cre tool mice, and flox positive and Cre positive mice (the mice are simply written as: CDK12 flox / + : Cre + ) and flox positive and Cre negative mice (CDK12 flox / + ) are obtained; one part of the flox mice is self-crossed to obtain flox homozygote (the mice are simply written as: CDK12 flox / flox ) and flox heterozygote mice (CDK12 flox / + ).

[0025] 3) To obtain flox homozygote and Cre heterozygote mice, the obtained flox and Cre double positive heterozygote mice (CDK12 flox / + : Cre+) are mated with flox homozygote mice (CDK12 flox / flox ), and finally the experimental group mice of flox homozygote and Cre positive (CDK12 flox / flox : Cre + ) and the control group mice of flox homozygote and Cre negative (CDK12 flox / flox ) are obtained for subsequent experiments. The mice with genotype of wild type (CDK12 + / + ) born in the same litter are used as control group to ensure the consistency of genetic background.

[0026] 1.2 Diet-induced fatty liver hepatitis model

[0027] Mice were first subjected to overnight fasting treatment (free water drinking), and then fed with normal chow diet (normal control group) or CDAHFD diet (choline-deficient, L-amino acid-defined high-fat diet, A06071302, Research Diet) (NASH group) for 8 weeks. After the end of modeling, 4 mice (for CDK12 knockout combined with repotrectinib treatment) or 12 mice (for repotrectinib dose gradient treatment) were taken from each group for subsequent experiments. In the repotrectinib dose gradient treatment experiment, mice were treated with normal saline, 25 mg / kg or 50 mg / kg repotrectinib, respectively. In the CDK12 knockout combined with repotrectinib treatment experiment, 50 mg / kg repotrectinib was used for treatment.

[0028] 2. Measurement of serum indicators of mice

[0029] The mice were inhaled anesthetized with isoflurane, and whole blood was collected by enucleation. The blood sample was placed at 4°C for 30 minutes, and then centrifuged at room temperature at 3000 rpm for 15 minutes. The supernatant was aspirated as serum, and was stored at -80°C after aliquoting. The levels of triglyceride (TG), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum were strictly operated and measured according to the corresponding kit instructions.

[0030] 2.1 Alanine aminotransferase (ALT) determination

[0031] The -80°C frozen serum was thawed on ice and diluted with sterile PBS at a ratio of 1:1. Alanine aminotransferase assay kit (Shenzhen Leidu, item number R01502) was used for detection: R1 and R2 reagents were mixed according to the instructions to prepare the working solution. 15 μl of diluted serum was taken from each sample, and was loaded in order for determination using a fully automatic biochemical analyzer.

[0032] 2.2 Aspartate aminotransferase (AST) determination

[0033] Aspartate aminotransferase assay kit (Shenzhen Leidu, item number R01702) was used, and R1 and R2 mixed working solution was prepared according to the instructions. 15 μl of serum was taken from each sample, labeled with serial numbers, and then determined using a fully automatic biochemical analyzer.

[0034] 2.3 Triglyceride (TG) determination

[0035] Triglyceride assay kit (Shenzhen Leidu, item number R02802) was used, and R1 and R2 working solution was prepared according to the instructions. 5 μl of serum was taken from each sample, loaded in order, and detected on a fully automatic biochemical analyzer.

[0036] 3. Detection of liver-related indicators

[0037] 3.1 Liver weight index

[0038] After blood collection, mice were euthanized, fixed, and the abdominal cavity was opened to remove the liver in its entirety. After rinsing with pre-cooled PBS, the liver was dried with filter paper and weighed accurately (to 0.01 g). The liver weight index was calculated: Liver weight index (%) = [liver wet weight (g) / body weight (g)] x 100%.

[0039] 3.2 Liver triglyceride (TG) content determination

[0040] Detection was performed using a tissue triglyceride enzyme method kit (Beijing Puli Lei, item number E1013-105).

[0041] 1) Tissue lysis: About 50 mg of liver tissue was accurately weighed, 1 ml of pre-cooled lysis solution was added, and a glass homogenizer was used to homogenize on ice. After standing for 10 minutes, 500 μl of the homogenate was transferred to a 1.5 ml EP tube and incubated in a 70°C water bath for 10 minutes. After centrifugation at 2000 rpm for 5 minutes at room temperature, the supernatant was collected for determination.

[0042] 2) Heat treatment: 500 μl of the homogenate was transferred to a 1.5 ml EP tube and incubated in a 70°C water bath for 10 minutes. After centrifugation at 2000 rpm for 5 minutes at room temperature, the supernatant was collected for determination.

[0043] 3) Preparation of working solution: The TG determination working solution was prepared according to the kit instructions.

[0044] 4) Preparation of standard curve: The standard provided by the kit was diluted according to the instructions.

[0045] 5) Determination: The sample, standard, and working solution were added to a 96-well plate, and the reaction was carried out at 37°C for 15 minutes. The absorbance (OD value) was measured at a wavelength of 550 nm, and the liver TG content was calculated according to the standard curve (unit: mmol / g or mg / g of tissue).

[0046] 4.1 Cell culture

[0047] Human embryonic kidney cells HEK293T were cultured in DMEM high glucose medium (Gibco, 11995065) containing 10% fetal bovine serum (CLARK, FB25015) and 1% penicillin / streptomycin double antibody (Beyotime, C0222) in a 37°C, 5% CO2 incubator (Thermo). All experimental cells were confirmed to be free of mycoplasma contamination by PCR and DAPI staining. Subsequent experiments were performed after the cells were cultured for 24 hours.

[0048] 4.2 Plasmid construction

[0049] 1) RNA extraction: About 5 x 10 6HEK293T cells, add 1 mL TRIzol reagent lysis, fully blow and mix, and stand at room temperature for 5 min; add 200 μL chloroform, shake vigorously for 30 s, stand at room temperature for 2 min; centrifuge at 4°C, 12000 g for 15 min, carefully transfer the upper aqueous phase to a new RNase-free centrifuge tube; add an equal volume of isopropanol, mix well by inverting, stand at room temperature for 10 min; centrifuge at 4°C, 12000 g for 10 min, discard the supernatant; add 1 mL of pre-cooled 80% ethanol to wash the precipitate, centrifuge at 7500 g for 5 min; discard the supernatant, dry the RNA precipitate at room temperature, then dissolve in 39 μL of DEPC water, and determine the concentration and purity for standby use.

[0050] 2) Reverse transcription: use reverse transcription kit (Vazyme, R211-01), prepare 20 μL reaction system according to the instructions: take 1 μg total RNA, add 2xRT Mix 10 μL, HiScript II Enzyme Mix 2 μL, Oligo(dT) 23 VN (50 μM) 1 μL, Random hexamers (50 ng / μl) 1 μL, and RNase-free ddH2O to 20 μL. The reaction conditions are: 25°C for 5 min, 50°C for 15 min, and 85°C for 5 min.

[0051] 3) Plasmid construction: use the reverse transcription product as a template, PCR amplify the full-length coding sequence of CDK12 and CCNK genes, purify after enzyme digestion, and clone into PCDH and pcDNA3.1 vectors respectively, construct recombinant plasmids PCDH-Flag-CDK12 and pcDNA3.1-HA-CCNK, and verify the correctness by sequencing.

[0052] 4.3 Cell transfection

[0053] Take well-grown HEK293T cells, inoculate at a density of 2x10 6 per dish in a 10 cm culture dish, and culture overnight until the cell confluence reaches 70%-80%. Co-transfect PCDH-Flag-CDK12 and pcDNA3.1-HA-CCNK plasmids into cells by PEI transfection method, and replace the fresh complete culture medium after 6 h of transfection.

[0054] 4.4 Drug treatment

[0055] After 24 h of transfection, treat the cells with different concentrations of THZ531, Mavelertinib (Mavelertinib) and Rezivertinib (Rezivertinib) (gradient concentration), and collect the cell samples after 24 h of drug treatment for subsequent analysis.

[0056] 4.5 Co-Immunoprecipitation (Co-IP)

[0057] 1) Aspirate the medium, wash the cells twice with pre-cooled PBS, add 1 mL of RIPA lysis buffer (containing protease inhibitors) per 6 cm dish, and lyse for 10 min;

[0058] 2) Collect the lysate into a 1.5 mL centrifuge tube, centrifuge at 14000 rpm for 5 min at 4°C;

[0059] 3) Take 80 μL of the supernatant as the Input group, add 20 μL of 5x SDS loading buffer, and boil for use;

[0060] 4) Add 20 μL of anti-Flag magnetic beads (Yeasen, 20584ES08) to the remaining supernatant, and incubate overnight at 4°C with rotation;

[0061] 5) The next day, wash the magnetic beads with pre-cooled RIPA lysis buffer for 6 times, 5 min each time;

[0062] 6) Add 40 μL of 1x SDS loading buffer to the bound magnetic beads, denature at 100°C metal bath for 10 min, and after centrifugation, take the supernatant for Western blot analysis or store at -80°C.

[0063] 4.6 Western Blot

[0064] After the protein sample is separated by SDS-PAGE electrophoresis, it is transferred to a PVDF membrane, which is blocked with 5% skim milk at room temperature for 1 h; HA-tag antibody (Proteintech, 51064-2-AP, 1:2000) or Flag-tag antibody (Proteintech, 80801-2-RR, 1:2000) is added and incubated overnight at 4°C; TBST is washed for 3 times, 10 min each time; HRP-labeled goat anti-rabbit secondary antibody (Proteintech, SA00001-4, 1:5000) is added and incubated at room temperature for 1 h; after washing with TBST, ECL chemiluminescence reagent is used for development, and the image is collected by ImageQuant LAS4000 imaging system, and the gray value is analyzed by Image J software.

[0065] 4.7 Molecular Docking

[0066] The binding mode of Rezivertinib (docking score: -6.76664) with Cdk12 protein is plotted in 2D and 3D.

[0067] III. Experimental Results

[0068] Rociletinib has a good therapeutic effect in a high-fat diet-induced non-alcoholic steatohepatitis model, such as Figure 1 as shown in FIG. 1, wherein, Figure 1 A shows the liver color of control group mice, rociletinib-low dose treatment group and rociletinib-high dose treatment group mice in a high-fat diet-induced fatty liver model; Figure 1 B shows the liver weight of mice in each group, and rociletinib can significantly reduce the liver weight of mice, and the liver weight of the rociletinib-high dose treatment group is lower; Figure 1 C shows the liver weight ratio of mice in each group, and the rociletinib-high dose treatment group has a lower liver weight ratio, indicating a lower level of fatty liver; Figure 1 D shows the triglyceride TG level in the liver of mice in each group; Figure 1 E-G show the triglyceride TG level, aspartate aminotransferase AST and alanine aminotransferase level ALT in the serum of mice in each group, which shows that rociletinib can significantly improve NASH, reduce the serum and liver triglyceride level of mice, and reduce the level of aspartate aminotransferase AST and alanine aminotransferase level ALT in the serum of mice, and it is dose-dependent.

[0069] Rociletinib has no therapeutic activity in CDK12 deletion fatty hepatitis mice, such as Figure 2 as shown in FIG. 2, wherein, Figure 2 A shows the liver color of control group mice, wild type mice-rociletinib group, CDK12 knockout mice-control group and CDK12 knockout mice-rociletinib group mice in a high-fat diet-induced fatty liver model; Figure 2 B shows the liver weight of mice in each group; Figure 2 C shows the liver weight ratio of mice in each group; Figure 2 D shows the triglyceride TG level in the liver of mice in each group; 2E-G shows the triglyceride TG level (E), aspartate aminotransferase AST (F) and alanine aminotransferase level ALT (G) in the serum of mice in each group.

[0070] Molecular docking shows the interaction mode of rociletinib with Cdk12, such as Figure 3 A-D, in which the Cdk12 protein is shown as a light blue cartoon, and rociletinib is shown as a yellow stick. In the 3D diagram, the C skeleton of the Cdk12 protein is shown in light blue, the N atom is shown in blue, the O atom is shown in dark red, the H atom is shown in white, and the S atom is shown in gold. Rociletinib is shown as a yellow stick. Salt bridge interaction is shown as a blue dotted line, and hydrogen bond interaction is shown as a purple dotted line, wherein the longer the hydrogen bond length, the weaker the hydrogen bond interaction. Rociletinib can form 3 hydrogen bond interactions and 1 salt bridge interaction with the Cdk12 protein: the amino group as a hydrogen bond donor forms 2 hydrogen bond interactions with ASP819 and MET816, with a distance of The nitrogen on the six-membered ring acts as a hydrogen bond acceptor to form one hydrogen bond with MET816 with a distance of 2.6 A In addition, Rezafetinib can also form one salt bridge with ASP819.

[0071] Co-IP experiment shows that Rezafetinib inhibits the formation of CDK12 / CCNK complex in a dose-dependent manner, while THZ531 and Mavelertinib have no such effect, as shown in Figure 4

[0072] The above description is only the preferred embodiment of the present application, not any form and substantial limitation of the present application. It should be noted that for ordinary skilled in the art, without departing from the present application, a number of improvements and supplements can also be made, which should be considered as the protection scope of the present application.​

Claims

1. Use of Rezafetini and / or a pharmaceutically acceptable salt form thereof in the manufacture of a medicament for the treatment of non-alcoholic steatohepatitis.

2. Use of Rezafetini and / or a pharmaceutically acceptable salt form thereof in the manufacture of a medicament for the treatment of simple fatty liver.

3. Use according to claim 1 or 2, characterized in that, The medicament contains the active ingredient Rezafetini and / or a pharmaceutically acceptable salt form thereof and at least one pharmaceutically acceptable carrier or excipient.

4. Use according to claim 1 or 2, characterized in that, The dosage form of the medicament includes tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, oral liquids, oral chewable tablets, granules, instant powder, pills, beads, suspensions, wine, tincture or drops.

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