Use of sotorasib in the preparation of a medicament for treating nonalcoholic steatohepatitis

Abstract

The application discloses application of Rezafetin in preparation of a medicine for treating non-alcoholic fatty liver disease. The application first discovers that the compound has good treatment effect in a non-alcoholic fatty liver disease (NASH) model. Through in-vivo experiments, it is confirmed that in a NASH model mouse, the Rezafetin can significantly reduce pathological characteristics such as liver weight, liver fat level and liver index. Meanwhile, the Rezafetin treatment can significantly reduce the level of serum transaminase (ALT / AST), indicating that the Rezafetin effectively improves liver damage and liver function. The application provides a brand-new and effective drug treatment strategy for clinically treating NASH, and has significant 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] To achieve the above objectives, the present invention provides the use of rizotinib and / or its pharmaceutically acceptable salt form in the preparation of a medicament for treating non-alcoholic steatohepatitis.

[0007] The present invention also provides the use of rizotinib and / or its pharmaceutically acceptable salt form in the preparation of a medicament for the treatment of simple fatty liver.

[0008] Preferably, the drug contains an active ingredient and at least one pharmaceutically acceptable carrier or excipient, wherein the active ingredient is rizatinib and / or its pharmaceutically acceptable salt form.

[0009] Preferably, the dosage form of the drug includes: tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, oral liquids, lozenges, granules, powders, pills, elixirs, suspensions, tinctures, or drops.

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

[0011] The retzatinib provided by this invention can be used clinically to treat steatohepatitis. Animal experiments have shown that retzatinib has a good therapeutic effect on steatohepatitis, and can reduce pathological features such as liver weight, liver fat level and liver index. At the same time, retzatinib treatment can significantly reduce serum transaminase (ALT / AST) levels, indicating that it effectively improves liver damage and liver function. This invention provides a new indication for retzatinib and has good prospects for clinical application. Attached Figure Description

[0012] Figure 1 This study demonstrates the excellent therapeutic effect of rizatinib in a CDAHFD-induced fatty liver model. A shows the liver color of mice in the control group, the low-dose rizatinib group, and the high-dose rizatinib group in a high-fat diet-induced fatty liver model. B shows the liver weight of each group; rizatinib significantly reduced liver weight, with the high-dose rizatinib group showing even lower liver weight. C shows the liver weight ratio of each group; the high-dose rizatinib group had a lower liver weight ratio, indicating a lower level of fatty liver. D shows the triglyceride (TG) level in the liver of each group. EG shows the serum triglyceride (TG) level (E), aspartate aminotransferase (AST) level (F), and alanine aminotransferase (ALT) level (G) of each group.

[0013] Figure 2This study demonstrates that rizatinib has no therapeutic activity in CDK12-deficient steatohepatitis mice. A shows the liver color of mice in a high-fat diet-induced fatty liver model, comparing control, wild-type, CDK12 knockout, and CDK12 knockout mouse-rizatinib groups. B shows the liver weight of each group. C shows the liver weight ratio of each group. D shows the triglyceride (TG) level in the liver of each group. EG shows the serum triglyceride (TG) level (E), aspartate aminotransferase (AST) level (F), and alanine aminotransferase (ALT) level (G) of each group.

[0014] Figure 3 The binding patterns of Cdk12 protein and Rezivertinib are shown, where A is a cartoon image of Cdk12 protein binding with Rezivertinib; B is a surface image of Cdk12 protein binding with Rezivertinib; C is a 2D image of Cdk12 protein binding with Rezivertinib; and D is a 3D image of Cdk12 protein binding with Rezivertinib.

[0015] Figure 4 This demonstrates that rizatinib inhibits the formation of the CDK12 / CCNK complex. Detailed Implementation

[0016] To make the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0017] Example

[0018] I. Animal source and breeding environment

[0019] The C57BL / 6 wild-type mice used in this invention were all purchased from Shanghai Southern Model Biotechnology Co., Ltd. All mice were housed in a specific pathogen-free (SPF) environment, maintaining a 12 / 12-hour light / dark rhythm (light time: 8:00–20:00), with an ambient temperature of 22±2℃ and humidity of 40%–60%.

[0020] II. Experimental Methods

[0021] 1.1 Construction of CDK12 gene knockout mice

[0022] This invention uses the Cre-loxP system to construct a liver-specific CDK12 gene knockout mouse model, specifically using Flox mice (Neo gene-removed Flox mice, CDK12 knockout mice). flox / + :Flp +Flox mice were obtained through a mating strategy with alb-cre mice. Flox mice have loxP sites flanking the target gene sequence. When the Cre recombinase recognizes the loxP sites, it can cleave and recombine them. Flox mice were constructed by Shanghai Southern Model Biotechnology Co., Ltd. The mating strategy between Flox mice and alb-cre mice is as follows:

[0023] 1) Flox mice with Neo gene removed (CDK12) flox / + :Flp + By mating with wild-type mice, flox mice (abbreviated as CDK12) with the Flp genotype segregated were obtained. flox / + );

[0024] 2) The obtained Neo- and Flp-depleted flux-positive heterozygous mice (CDK12) flox / + The mice were divided into two groups: one group of flux mice were mated with Cre tool mice to obtain flux-positive and Cre-positive mice (abbreviated as CDK12). flox / + Cre + ) and flux-positive and Cre-negative mice (CDK12) flox / + A subset of flux mice were self-crossed to obtain flux homozygous mice (abbreviated as CDK12). flox / flox ) and flox heterozygous mice (CDK12) flox / + ).

[0025] 3) To obtain mice that are homozygous for flux and heterozygous for Cre, the resulting double-positive heterozygous mice for flux and Cre (CDK12) were... flox / + Cre+) and flux homozygous mice (CDK12) flox / flox Mice were mated to obtain experimental group mice (CDK12) that were homozygous for flux and positive for Cre. flox / flox Cre + ) and flux-homozygous and Cre-negative control mice (CDK12) flox / flox ), used for subsequent experiments. All experiments used littermates with wild-type genotype (CDK12). + / + Mice were used as a control group to ensure the consistency of genetic background.

[0026] 1.2 Diet-induced steatohepatitis model

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

[0028] 2. Measurement of mouse serum markers

[0029] Mice were anesthetized by inhalation with isoflurane, and whole blood was collected by enucleation. Blood samples were incubated at 4°C for 30 minutes, then centrifuged at 3000 rpm for 15 minutes at room temperature. The supernatant was collected as serum, aliquoted, and stored at -80°C for later analysis. Serum triglyceride (TG), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels were measured strictly according to the respective kit instructions.

[0030] 2.1 Alanine aminotransferase (ALT) assay

[0031] Take serum frozen at -80℃, thaw on ice, and dilute 1:1 with sterile PBS. Detect the alanine aminotransferase using a kit (Shenzhen Raydu, catalog number R01502): mix reagents R1 and R2 according to the instructions to prepare the working solution. Take 15 μl of diluted serum for each sample, load it sequentially, and perform the analysis using a fully automated biochemical analyzer.

[0032] 2.2 Aspartate aminotransferase (AST) assay

[0033] The aspartate aminotransferase assay kit (Shenzhen Raydu, catalog number R01702) was used, and the mixed working solution of R1 and R2 was prepared according to the instructions. 15 μl of serum was taken from each sample, labeled with a serial number, and then measured using a fully automated biochemical analyzer.

[0034] 2.3 Triglyceride (TG) Determination

[0035] Using a triglyceride assay kit (Shenzhen Raydu, catalog number R02802), prepare working solutions R1 and R2 according to the instructions. Take 5 μl of serum for each sample, load the samples sequentially, and perform the analysis on a fully automated biochemical analyzer.

[0036] 3. Liver-related indicator testing

[0037] 3.1 Liver weight index

[0038] Mice were euthanized after blood collection, fixed, and their livers were completely removed from the abdominal cavity. The livers were rinsed with pre-cooled PBS, blotted dry with filter paper, and weighed precisely (to 0.01 g). The liver weight index was calculated as follows: Liver weight index (%) = [liver wet weight (g) / body weight (g)] × 100%.

[0039] 3.2 Determination of liver triglyceride (TG) content

[0040] The tissue triglyceride enzyme assay kit (Beijing Pulilai, catalog number E1013-105) was used for detection.

[0041] 1) Tissue lysis: Accurately weigh about 50mg of liver tissue, add 1ml of pre-cooled lysis buffer, homogenize thoroughly on ice using a glass homogenizer, and let stand for 10 minutes;

[0042] 2) Heat treatment: Transfer 500 μl of homogenate to a 1.5 ml EP tube, incubate at 70℃ for 10 minutes, centrifuge at 2000 rpm for 5 minutes at room temperature, and collect the supernatant for testing;

[0043] 3) Preparation of working solution: Prepare the TG assay working solution according to the kit instructions;

[0044] 4) Preparation of standard curve: Use the standards provided in the kit to perform serial dilutions according to the instructions;

[0045] 5) Measurement: Add sample, standard and working solution to 96-well plate, react at 37℃ for 15 minutes, and measure absorbance (OD value) at 550nm wavelength. Calculate liver TG content (unit: mmol / g or mg / g tissue) according to standard curve.

[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 antibiotics (Beyotime, C0222), and routinely cultured in a 37°C, 5% CO2 incubator (Thermo). All cells used in experiments were confirmed to be free of mycoplasma contamination by PCR and DAPI staining. Subsequent experiments were performed 24 hours after cell passage.

[0048] 4.2 Plasmid Construction

[0049] 1) RNA extraction: Take approximately 5 × 10⁻⁶ RNA samples. 6One HEK293T cell was lysed with 1 mL of TRIzol reagent, thoroughly mixed by pipetting, and incubated at room temperature for 5 min. 200 μL of chloroform was added, and the mixture was vigorously shaken for 30 s, then incubated at room temperature for 2 min. The cells were centrifuged at 12000 g for 15 min at 4 °C, and the supernatant was carefully transferred to a new RNase-free centrifuge tube. An equal volume of isopropanol was added, and the mixture was inverted and incubated at room temperature for 10 min. The cells were centrifuged at 12000 g for 10 min at 4 °C, and the supernatant was discarded. The precipitate was washed with 1 mL of pre-cooled 80% ethanol, and centrifuged at 7500 g for 5 min. The supernatant was discarded, and the RNA precipitate was dried at room temperature and dissolved in 39 μL of DEPC water. The concentration and purity were then determined for later use.

[0050] 2) Reverse transcription: Using a reverse transcription kit (Vazyme, R211-01), prepare a 20 μL reaction system according to the instructions: Take 1 μg of total RNA, add 10 μL of 2×RT Mix, 2 μL of HiScript II Enzyme Mix, and Oligo(dT). 23 1 μL of VN (50 μM) and 1 μL of Random hexamers (50 ng / μL) were added, and the volume was brought to 20 μL with RNase-free ddH2O. The reaction conditions were: 25℃ for 5 min, 50℃ for 15 min, and 85℃ for 5 min.

[0051] 3) Plasmid construction: Using the reverse transcription product as a template, the full-length coding sequences of CDK12 and CCNK genes were amplified by PCR, digested and purified, and then cloned into PCDH and pcDNA3.1 vectors, respectively, to construct recombinant plasmids PCDH-Flag-CDK12 and pcDNA3.1-HA-CCNK, and their correctness was verified by sequencing.

[0052] 4.3 Cell transfection

[0053] Take HEK293T cells in good growth condition, at 2×10⁻⁶ 6 The cells were seeded at a density of 10 cm in 10 cm culture dishes and cultured overnight until the cell confluence reached 70%–80%. The PCDH-Flag-CDK12 and pcDNA3.1-HA-CCNK plasmids were co-transfected into the cells using the PEI transfection method. The culture medium was replaced with fresh complete medium 6 hours after transfection.

[0054] 4.4 Drug treatment

[0055] 24 hours after transfection, cells were treated with different concentration gradients of THZ531, Mavelertinib, and Rezivertinib (gradient concentrations). Cell samples were collected 24 hours after drug treatment for subsequent analysis.

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

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

[0058] 2) Collect the lysis buffer into a 1.5 mL centrifuge tube and centrifuge at 4 °C and 14,000 rpm for 5 min;

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

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

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

[0062] 6) Add 40 μL of 1×SDS loading buffer to the magnetic beads, denature in a metal bath at 100℃ for 10 min, centrifuge, and take the supernatant for Western blot analysis or store at -80℃.

[0063] 4.6 Western Blot

[0064] After separation by SDS-PAGE, protein samples were transferred to a PVDF membrane and blocked with 5% skim milk at room temperature for 1 h. HA-tagged antibody (Proteintech, 51064-2-AP, 1:2000) or Flag-tagged antibody (Proteintech, 80801-2-RR, 1:2000) was added and incubated overnight at 4°C. The samples were washed three times with TBST for 10 min each time. HRP-labeled goat anti-rabbit secondary antibody (Proteintech, SA00001-4, 1:5000) was added and incubated at room temperature for 1 h. After thorough washing with TBST, the samples were developed using ECL chemiluminescence reagent. Images were acquired using an ImageQuant LAS4000 imaging system, and grayscale analysis was performed using ImageJ software.

[0065] 4.7 Molecular docking

[0066] 2D and 3D mapping was performed to visualize the binding pattern of Rezivertinib (docking score: -6.76664) to Cdk12 protein.

[0067] III. Experimental Results

[0068] Rezazinib showed good therapeutic efficacy in a high-fat diet-induced non-alcoholic steatohepatitis model, such as... Figure 1 As shown, where, Figure 1 A shows the liver color of mice in the control group, the low-dose rizatinib treatment group, and the high-dose rizatinib treatment group in a high-fat diet-induced fatty liver model. Figure 1 B shows the liver weight of mice in each group. Rezatinib can significantly reduce the liver weight of mice, and the liver weight of the high-dose rezatinib treatment group is even lower. Figure 1 C shows the liver weight ratio of mice in each group. The rizatinib-high-dose treatment group had a lower liver weight ratio, suggesting a lower level of fatty liver. Figure 1 D shows the triglyceride (TG) levels in the livers of mice in each group; Figure 1 EG showed that serum triglyceride (TG), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels were present in each group of mice. The results showed that rizatinib significantly improved NASH, reduced serum and liver triglyceride levels, and decreased serum AST and ALT levels in mice in a dose-dependent manner.

[0069] Rezazinib showed no therapeutic activity in CDK12-deficient steatohepatitis mice. Figure 2 As shown, where, Figure 2 A shows the liver color of mice in a high-fat diet-induced fatty liver model, including control mice, wild-type mice in the rizatinib group, CDK12 knockout mice in the control group, and CDK12 knockout mice in the rizatinib group. 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 show the triglyceride (TG) level (E), aspartate aminotransferase (AST) (F), and alanine aminotransferase (ALT) level (G) in the serum of mice in each group.

[0070] Molecular docking revealed the interaction mode between rezazantinib and Cdk12, such as... Figure 3 As shown in AD, Cdk12 protein is displayed as a light blue cartoon, and rezatinib as a yellow stick. In the 3D image, the C-backbone of Cdk12 protein is displayed as light blue, N atoms as blue, O atoms as bright red, H atoms as white, S atoms as gold, and rezatinib as a yellow stick. Salt bridge interactions are shown as blue dashed lines, and hydrogen bonding interactions as purple dashed lines. The longer the hydrogen bond length, the weaker the hydrogen bond interaction. Rezatinib can form three hydrogen bonds and one salt bridge interaction with Cdk12 protein: the amino group acts as a hydrogen bond donor, forming two hydrogen bonds with ASP819 and MET816, with distances of [missing information]. The nitrogen on the six-membered ring acts as a hydrogen bond acceptor, forming a hydrogen bond with MET816 at a distance of [missing information]. In addition, rizatinib can also form a salt bridge with ASP819.

[0071] Co-immunoprecipitation (Co-IP) experiments showed that renzatinib inhibited CDK12 / CCNK complex formation in a dose-dependent manner, while THZ531 and Walkertinib did not have this effect. Figure 4 As shown.

[0072] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make several improvements and additions without departing from the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. Use of rizatinib and / or its pharmaceutically acceptable salt form in the preparation of medicaments for the treatment of non-alcoholic steatohepatitis.

2. The application as described in claim 1, characterized in that, The drug contains an active ingredient and at least one pharmaceutically acceptable carrier or excipient, wherein the active ingredient is rizatinib and / or its pharmaceutically acceptable salt form.

3. The application as described in claim 1, characterized in that, The dosage forms of the medicine include: tablets, capsules, oral liquids, lozenges, granules, powders, pills, elixirs, suspensions, tinctures, or drops.

Images