Drug for treating drug-induced liver injury by taking DDR1 gene or protein as target spot

Abstract

The invention discloses a medicine for treating drug-induced liver injury by taking a DDR1 gene or protein as a target spot, and belongs to the technical field of medicines. The drug-induced liver injury is caused by abnormal activation or accumulation of DDR1 protein, and the pathological characteristics of the drug-induced liver injury comprise nesting apoptosis of liver cells. The invention discloses that DDR1 is a key molecular target for inducing hepatotoxicity by the Platinib for the first time, and the Platinib can lead to obvious increase of DDR1 protein and phosphorylation level thereof at a treatment concentration, so that cell-ECM and cell-cell adhesion are damaged, and anoikis is activated. By inhibiting the expression of the DDR1 gene or inhibiting the activity and accumulation of the DDR1 protein, the hepatocyte apoptosis caused by the Platinib can be effectively reversed. The invention provides a new molecular target for intervening in the induced hepatotoxicity of the prilatinib, and provides a new strategy for clinically and safely treating RET fusion positive tumors by preparing a drug for preventing or treating drug-induced liver injury by utilizing a substance for inhibiting the targeted DDR1.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to Discoidin Domain Receptor 1 (DDR1) as an inhibitory target for the development of drugs for the prevention or treatment of drug-induced liver injury, especially Pralsetinib-induced hepatotoxicity. BACKGROUND

[0002] Pralsetinib (BLU-667) is a highly selective RET (Rearranged during Transfection) tyrosine kinase inhibitor, used for the treatment of non-small cell lung cancer and thyroid cancer with positive RET fusion. With its high objective response rate (about 55%) and significant progression-free survival (median 22 months), the drug has become a first-line treatment for RET-driven tumors. However, clinical trial data shows that Pralsetinib has significant hepatotoxicity, manifested as elevated AST and ALT levels (incidence rates of 69% and 46%, respectively), with 2.1% of patients experiencing severe liver injury (grade III / IV) (Markham A. Pralsetinib: First Approval [J]. Drugs. 2020, 80(17): 1865-70.).

[0003] Currently, there is a lack of effective intervention means for such liver injury in clinical practice, and patients often need to adjust the dose or interrupt the treatment, which seriously affects the anti-tumor efficacy and treatment process. Therefore, it is of great clinical significance to further explore the mechanism of Pralsetinib-induced hepatotoxicity and develop corresponding intervention strategies, which also provides a reference for the rational application and subsequent development of similar RET inhibitors.

[0004] Discoidin domain receptor 1 (DDR1) belongs to the non-integrin type of collagen receptors, which plays a key role in cell differentiation, proliferation, adhesion and extracellular matrix remodeling. Recent studies have shown that DDR1 exhibits cell type-specific dual regulation characteristics in different pathological models. For example, in tumor models, inhibition of DDR1 can promote apoptosis of cancer cells, while in dry eye and ulcerative colitis models, DDR1 inhibition can protect normal epithelial cells and reduce ferroptosis and apoptosis (Dai Y, et al. A highly selective inhibitor of discoidin domain receptor-1 (DDR1-IN-1) protects corneal epithelial cells from YAP / ACSL4-mediated ferroptosis in dry eye [J]. Br J Pharmacol. 2024, 181(21):4245-61.; Li X, et al. Discoidin domain receptor 1 (DDR1) promote intestinal barrier disruption in Ulcerative Colitis through tight junction proteins degradation and epithelium apoptosis [J]. Pharmacol Res. 2022, 183:106368.). These findings suggest that DDR1 has complex functions in maintaining tissue homeostasis, but its role in drug-induced liver injury has not been reported, especially its role in platinib hepatotoxicity is still unknown.

[0005] Curcumin is a natural polyphenolic compound extracted from turmeric roots, which has been shown to regulate a variety of cellular signaling molecules, including oxidative stress factors, inflammatory factors and apoptosis-related proteins. One of its most classic mechanisms of action is to activate the NRF2 signaling pathway and enhance the ability of cells to resist oxidative stress, which has been widely reported and fully verified. At the same time, curcumin has also been shown to induce tumor cell anoikis, thereby increasing the efficacy of drug anti-tumor therapy. However, whether it regulates anoikis in normal liver cells and whether it mediates liver protection through DDR1 has not been reported to date. SUMMARY

[0006] The purpose of the present application is to reveal the mechanism of platinib-induced hepatotoxicity and to provide a safe and effective intervention drug.

[0007] To achieve the above purpose, the technical scheme adopted by the present application is as follows: The present application research found that abnormal activation of discoidin domain receptor 1 (DDR1) is a key molecular event of pralsetinib-induced hepatocyte anoikis leading to liver toxicity, and targeting inhibition of DDR1 can play a significant liver protection role.

[0008] Therefore, the present application provides the use of a substance targeting DDR1 in the preparation of a drug for preventing or treating drug-induced liver injury caused by abnormal activation or accumulation of DDR1 protein, which manifests as hepatocyte anoikis.

[0009] DDR1 The nucleotide sequence of the gene is shown as SEQ ID NO. 1, and the amino acid sequence of the DDR1 protein is shown as SEQ ID NO. 2.

[0010] Further, the drug-induced liver injury is pralsetinib-induced liver toxicity.

[0011] Further, the RET inhibitor is pralsetinib.

[0012] The present application research found that pralsetinib at a therapeutic concentration can significantly up-regulate the protein and phosphorylation level of DDR1 in hepatocytes, and induce down-regulation of the expression of key adhesion molecules such as cadherins (e.g. CDHs) and integrins (e.g. ITGAs), thereby destroying cell adhesion and ultimately activating the mitochondrial-dependent apoptosis pathway. Knocking down DDR1 can specifically reduce the liver toxicity and anoikis of pralsetinib, while overexpression of DDR1 can exacerbate its toxic effects, confirming the core regulatory position of DDR1. Therefore, inhibiting the expression of DDR1 gene / protein can be used as a means to intervene in pralsetinib-induced liver toxicity.

[0013] Specifically, the substance targeting DDR1 is an inhibitor targeting DDR1 gene expression or inhibiting the activity or protein accumulation of DDR1 protein.

[0014] In the present application research, RNA interference technology is used to down-regulate the expression of DDR1 in hepatocytes, thereby reversing pralsetinib-induced hepatocyte apoptosis, which is manifested as a decrease in hepatocyte apoptosis induced by pralsetinib after using siRNA. Therefore, the present application provides a new therapeutic target for intervening in pralsetinib-induced liver toxicity. DDR1 Corresponding drug preparations for knocking down the expression of the gene are developed, and the drug achieves the purpose of treating pralsetinib-induced liver toxicity side effects by down-regulating DDR1 the expression of the gene.

[0015] As a preferred, the substance targeting DDR1 is siRNA or shRNA targeting DDR1 gene expression.

[0016] As a specific embodiment of the present application, the nucleotide sequence of the siRNA is: 5'-CUGACAUGAAGGGACAUUUdTdT-3'. The siRNA is targeted to DDR1 The siRNA of the gene reverses the liver toxicity induced by pralsetinib by inhibiting the expression of DDR1

[0017] Preferably, the substance targeting DDR1 is curcumin or a pharmaceutically acceptable salt, ester or derivative thereof.

[0018] The derivative is derived from the curcumin core structure and can function by inhibiting the transcription level or reducing the stability of DDR1 protein or directly inhibiting the phosphorylation of DDR1. DDR1

[0019] The present application provides an effective therapeutic drug for pralsetinib-induced liver toxicity. The results of cell and animal experiments show that, compared with the pralsetinib alone group, the combination of curcumin can reverse the increase of serum liver enzymes in mice caused by pralsetinib and the abnormal increase of DDR1 protein and its phosphorylation level in hepatocytes, restore the expression of adhesion molecules and improve the cell adhesion function, thereby antagonizing anoikis and restoring liver damage.

[0020] In the present application, the drug for treating drug-induced liver injury also includes a pharmaceutically acceptable excipient, including a filler, a wetting agent, a binder, a disintegrant or a lubricant.

[0021] The preparation form of the drug can be a solid preparation or a liquid preparation. Preferably, the preparation form of the drug is an oral preparation.

[0022] The present application also provides DDR1 The gene or DDR1 protein as a target for screening drugs for treating pralsetinib-induced liver toxicity.

[0023] Another object of the present application is to provide an antitumor combination drug composition, which comprises a first preparation of pralsetinib and a pharmaceutically acceptable carrier, and a second preparation of curcumin or a salt, ester or derivative thereof and a pharmaceutically acceptable carrier.

[0024] The combination of curcumin or a salt, ester or derivative thereof and pralsetinib down-regulates the accumulation of DDR1 protein caused by pralsetinib to intervene in the liver toxicity side effects of pralsetinib.

[0025] The present application shows that curcumin has no antagonistic effect on the antitumor effect of pralsetinib and has a synergistic effect.

[0026] Preferably, the mass ratio of curcumin to pralsetinib in the drug composition is 1:1.

[0027] ​​The pharmaceutically acceptable carrier may be, but is not limited to, a filler, wetting agent, binder, disintegrant, or lubricant.

[0028] This invention also provides the use of the pharmaceutical composition in the preparation of a medicament for treating RET fusion-positive tumors. The tumors may be, but are not limited to, non-small cell lung cancer and thyroid cancer.

[0029] The beneficial effects of this invention are as follows: (1) This invention reveals for the first time that DDR1 is a key molecular target for pralatinib-induced hepatotoxicity. At therapeutic concentrations, pralatinib can significantly increase the levels of DDR1 protein and its phosphorylation, thereby disrupting cell-ECM and cell-cell adhesion and activating anodic apoptosis. This is achieved by inhibiting... DDR1 The expression of the gene or the inhibition of the activity and accumulation of the DDR1 protein can effectively reduce hepatocyte apoptosis induced by pralatinib, establishing the core regulatory role of DDR1 in drug-induced liver injury. This invention provides a new molecular target for intervening in drug-induced liver injury caused by abnormal activation or accumulation of the DDR1 protein.

[0030] (2) This invention reveals that curcumin can improve cell adhesion function and antagonize anodic apoptosis by reducing the level of abnormal DDR1 protein. Its protective effect is different from the traditional antioxidant stress pathway, but relies on a novel mechanism that targets the DDR1 signaling axis. This invention provides a new use for curcumin in the preparation of drugs for treating drug-induced liver injury caused by abnormal activation or accumulation of DDR1 protein, expanding the application scope of curcumin.

[0031] (3) The combination therapy of curcumin and pralatinib provided by the present invention can significantly reduce its liver toxicity, improve patient tolerance and drug safety, without affecting or even slightly enhancing the anti-tumor efficacy of pralatinib, and provide a safer and more effective strategy for the clinical treatment of RET fusion-positive tumors. Attached Figure Description

[0032] Figure 1 The effects of pralatinib at certain concentration gradients on hepatocytes are shown, where A represents survival rate, B represents the level of apoptosis-related proteins, C represents the apoptosis rate, D represents the level of anti-apoptosis-related proteins, and E represents the level of DDR1 protein.

[0033] Figure 2 The effects of pralatinib on mouse modeling are shown in Figure A, which shows the changes in liver weight-to-body weight ratio, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, and Figure B shows the histopathological staining results of mouse liver tissue.

[0034] Figure 3Effect of loss of anchorage-dependent apoptosis-related proteins on liver cell apoptosis induced by pralsetinib, A is the effect of ROCK inhibitor Y-27632 on the survival rate of liver cells under the action of pralsetinib, B is the effect of pralsetinib on the transcription level of ITGA6, CDH1 and RND3 genes, C is the effect of integrin allosteric activator Mn 2+ Effect on apoptosis, E is the effect of CDH1 and CDH5 overexpression on apoptosis.

[0035] Figure 4 Effect of DDR1 on apoptosis induced by pralsetinib, A and B are the effect of DDR1 gene-specific siRNA and overexpression plasmid on liver cell apoptosis induced by pralsetinib.

[0036] Figure 5 Effect of curcumin on liver toxicity induced by pralsetinib, A is the apoptosis rate, B is the effect on c-PARP and DDR1 protein level, C is the effect of NRF2 gene-specific siRNA and overexpression plasmid on apoptosis, D and E are the intervention effect of curcumin on liver damage of pralsetinib modeling mice.

[0037] Figure 6 Effect of curcumin on the effect of pralsetinib on TPC-1, a thyroid cancer cell positive for RET fusion.

[0038] Figure 7 Effect of curcumin on the effect of pralsetinib on Lc-2 / ad, a non-small cell lung cancer cell positive for RET fusion. DETAILED DESCRIPTION

[0039] The application will be further described below in conjunction with specific examples. The following examples are only used to illustrate the application and are not intended to limit the scope of the application. Modifications or replacements of the methods, steps or conditions of the application, without departing from the spirit and essence of the application, all belong to the scope of the application.

[0040] The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.

[0041] Specifically, C57BL / 6J mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.; 2-hydroxypropyl-β-cyclodextrin was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; human normal hepatocyte cell line THLE-2 and human non-small cell lung cancer cell Lc-2 / ad were purchased from Wuhan Ponsay Life Science Co., Ltd.; TPC-1 human papillary thyroid carcinoma cells were purchased from Shanghai Gaining Biotechnology Co., Ltd.; β-Actin, GAPDH antibodies were purchased from Hangzhou Dig Biotechnology Co., Ltd.; phospho-DDR1 antibody was purchased from Sigma-Aldrich Company, DDR1 antibody was purchased from Cell Signaling Technology; cleaved-PARP antibody was purchased from Shanghai Aibio Trading Co., Ltd.; Y-27632 was purchased from MedChemExpress; siRNA was purchased from Beijing Genview Biotech Co., Ltd., the negative control (NC) sense strand sequence was 5'-UUCUCCGAACGUGUCACGUdTdT-3'; si DDR1 The sense strand was 5'-CUGACAUGAAGGGACAUUUdTdT-3'; si NRF2 The sense strand of siRNA #1 was 5'-AUUAUUAUGACUGUUAAAUdTdT-3', and the antisense strand was 5'-UUAUUAACAGUCAGAUAAUdTdT-3'; NRF2 The sense strand of siRNA #2 was 5'-AGUGUCAGUAUGUUGAAUCdTdT-3'; DDR1 CDH1 CDH5 NRF2 Plasmids were purchased from Wuhan Moli Bioscience and Technology Co., Ltd.; transfection reagent jetPRIME was purchased from Polyplus Transfection Company; qPCR reagent TB Green® Premix Ex Taq™ II was purchased from Takara; apoptosis kit was purchased from Hangzhou Link Biological Technology Co., Ltd.

[0042] Platinib, CAS No. 2097132-94-8, chemical name cis-N-[(1S)-1-[6-(4-fluoro-1H-pyrazol-1-yl)-3-pyridyl]ethyl]-1-methoxy-4-[4-methyl-6-[(5-methyl-1H-pyrazol-3-yl)amino]-2-pyrimidinyl cyclohexylcarbonyl amide, molecular formula C 27 H 32 FN902, molecular weight 533.6, purchased from Shanghai Taotuo Biochemical Technology Co., Ltd. The structural formula is as follows: .

[0043] Curcumin, CAS No. 458-37-7, molecular formula C 21 H​​​20 O6, with a molecular weight of 368.38, was purchased from MedChemExpress. Its structural formula is as follows: .

[0044] Example 1 1. Human normal hepatocytes THLE-2 were seeded at a density of 8,000 cells / well in 96-well plates and treated with a certain concentration gradient of pralatinib (final concentrations of 3.125, 6.25, 12.5, 25, 37.5 and 50 μM) for 24 h. After treatment, the cells were fixed with 10% trichloroacetic acid at 4℃ for 1 h, dried, stained with SRB and the absorbance was measured to examine the viability.

[0045] The results are as follows Figure 1 As shown in Figure A, pralatinib significantly reduces human hepatocyte survival as the effective concentration increases.

[0046] 2. Human normal hepatocytes (THLE-2) were seeded at a density of 100,000 per well in 12-well plates and treated with different concentrations of pralatinib (final concentrations of 0, 12.5, 25, and 50 μM) for 24 h or with 25 μM pralatinib for different durations (0, 3, 6, 9, 12, and 24 h). Cells were harvested, and cell cycle was detected using PI staining combined with flow cytometry. Proteins were extracted and quantified, and then Western blot was used to detect protein levels.

[0047] The results are as follows Figure 1 As shown in Figure B, with increasing concentration and duration of action, pralatinib significantly increased the levels of apoptosis-related proteins cleaved-PARP and cleaved-CASP3.

[0048] like Figure 1 As shown in C, pralatinib significantly upregulated the level of apoptosis in human hepatocytes with increasing concentration.

[0049] like Figure 1 As shown in Figure D, with increasing concentration, pralatinib reduced the levels of anti-apoptotic proteins MCL-1 and BCL-XL.

[0050] like Figure 1 As shown in E, with increasing concentration, pralatinib significantly increased the DDR1 baseline and its phosphorylated protein levels.

[0051] The above results suggest that pralatinib can induce apoptosis in normal human hepatocytes.

[0052] Example 2 Twenty-four male C57BL / 6J mice were randomly divided into three groups: a control group, a 1-fold clinical dose group, and a 2-fold clinical dose group, with eight mice in each group. Praltinib was administered via gavage at doses of 50 mg / kg / day and 100 mg / kg / day. The control group received 5% β-cyclodextrin instead of β-cyclodextrin. After four weeks of continuous administration, blood samples were collected via orbital sampling to examine serum ALT and AST levels. Liver tissue was dissected, dehydrated with 4% paraformaldehyde, and then coated with paraffin to prepare paraffin blocks. Sections were then stained with hematoxylin and eosin (HE), stained with TUNEL fluorescent dye, and immunohistochemically stained with c-PARP antibody.

[0053] The results are as follows Figure 2 As shown in A, both 1-fold and 2-fold clinical equivalent doses of pralatinib caused elevated serum ALT and AST levels in mice, and the animal model results were consistent with clinical liver toxicity.

[0054] like Figure 2 As shown in Figure B, staining of liver pathological sections revealed that pralatinib induced apoptosis in mouse hepatocytes.

[0055] Example 3 1. Rho kinase (ROCK) regulates cytoskeleton contraction and reorganization, acting as the executor of anodic apoptosis. The ROCK inhibitor Y-27632 inhibits cell contraction and subsequent anodic apoptosis by suppressing ROCK1 / 2 activity. To verify that pralatinib-induced hepatocyte apoptosis is anodic apoptosis, the following experiment was conducted: Human normal hepatocytes THLE-2 were seeded at a density of 8,000 per well in 96-well plates and treated with pralatinib at a final concentration of 25 μM and Y-27632 at a final concentration of 20 μM for 24 h. After fixation with 10% trichloroacetic acid at 4°C for 1 h, the cells were dried, stained with SRB, and the absorbance was measured to assess the viability.

[0056] The results are as follows Figure 3 As shown in Figure A, Y-27632 significantly improved the survival rate of human hepatocytes under pralatinib treatment, indicating that the main form of hepatocyte apoptosis induced by pralatinib is anodic apoptosis.

[0057] 2. Integrin receptors (ITGAs) mediate cell-ECM connectivity and the transduction of pro-survival signals and the inhibition of pro-apoptotic signals; cadherins (CDHs) mediate cell-cell adhesion; RND3 is a member that inhibits the RhoA / ROCK signaling pathway. To verify that pralatinib weakens cell-ECM connectivity and cell-cell adhesion, activates ROCK, and further induces anodic apoptosis in hepatocytes, the following experiments were conducted: Human normal liver cells THLE-2 were seeded in 6-well plates at a density of 240,000 cells per well, and after overnight stable adhesion, 25 μM pralsetinib was given for 24 h, mRNA was extracted, and RT-qPCR was performed to investigate the transcription level.

[0058] The results are shown in B of Figure 3 Under the action of pralsetinib, the transcription levels of CDH1, ITGA6 and RND3 were significantly reduced, indicating that pralsetinib disrupted cell-cell adhesion, cell-ECM connection, and activated the executor ROCK protein of the anoikis pathway.

[0059] 3, MnCl2 is an allosteric activator of integrin receptors, which can strongly activate integrin receptors and activate downstream pro-survival signaling pathways. In order to verify that restoring cell-ECM connection and cell-cell adhesion can inhibit pralsetinib-induced anoikis, the following experiments were performed: Human normal liver cells THLE-2 were seeded in 12-well plates at a density of 120,000 cells per well, and after overnight stable adhesion, the overexpression plasmids of CDH1 and CDH5, which mediate cell-cell adhesion, were introduced into THLE-2 cells by jetPRIME transfection reagent, and stable for 12 h, 25 μM pralsetinib was given for 24 h, or 100 μM MnCl2 was combined with pralsetinib for 24 h, and the cells were collected. PI / Annexin V staining combined with flow cytometry was used to detect cell apoptosis; protein was extracted and quantified. CDH1 Gene ID: 999, CDH5 Gene ID: 1013) and negative control vector were introduced into THLE-2 cells by jetPRIME transfection reagent, and stable for 12 h, 25 μM pralsetinib was given for 24 h, or 100 μM MnCl2 was combined with pralsetinib for 24 h, and the cells were collected. PI / Annexin V staining combined with flow cytometry was used to detect cell apoptosis; protein was extracted and quantified.

[0060] The results are shown in C of Figure 3 MnCl2 allosterically activates integrin receptors, which can reduce the apoptosis rate induced by pralsetinib, indicating that activating cell integrin receptors can inhibit pralsetinib-induced anoikis.

[0061] As shown in D and E of Figure 3 After overexpression of CDH1 , CDH5 in cells, the apoptosis rate induced by pralsetinib decreased, indicating that increasing the level of cell-cell adhesion can inhibit pralsetinib-induced anoikis.

[0062] The above results suggest that pralsetinib can lead to a decrease in the levels of cell-cell adhesion and cell-ECM connection proteins, activate ROCK, and thus induce anoikis.

[0063] Example 4 To verify whether the target point DDR1 of pralsetinib plays a key role in the anokis of liver cells induced by pralsetinib, the following experiment was conducted. Human normal liver cells THLE-2 were inoculated in a 12-well plate at a density of 80,000 cells per well, and after overnight stable adhesion, siRNA targeting DDR1 (sense strand: 5'-CUGACAUGAAGGGACAUUUdTdT-3') and negative control NC (sense strand: 5'-UUCUCCGAACGUGUCACGUdTdT-3') of the gene sequence ID: NM_001202521.1) or overexpression plasmid (original vector: pCDNA3.0-3'HA, encoding sequence Gene ID: 780) and negative control vector were introduced into THLE-2 cells by jetPRIME transfection reagent, and after stable expression, 25 μM pralsetinib was given for 24 h, and the cells were collected, and PI / Annexin V staining combined with flow cytometry was used to detect cell apoptosis. DDR1 (NCBIsite DDR1 Gene sequence ID: NM_001202521.1) and negative control NC (sense strand: 5'-UUCUCCGAACGUGUCACGUdTdT-3') or overexpression plasmid (original vector: pCDNA3.0-3'HA, encoding sequence Gene ID: 780) and negative control vector were introduced into THLE-2 cells by jetPRIME transfection reagent, and after stable expression, 25 μM pralsetinib was given for 24 h, and the cells were collected, and PI / Annexin V staining combined with flow cytometry was used to detect cell apoptosis. DDR1 Gene sequence ID: NM_001202521.1) and negative control NC (sense strand: 5'-UUCUCCGAACGUGUCACGUdTdT-3') or overexpression plasmid (original vector: pCDNA3.0-3'HA, encoding sequence Gene ID: 780) and negative control vector were introduced into THLE-2 cells by jetPRIME transfection reagent, and after stable expression, 25 μM pralsetinib was given for 24 h, and the cells were collected, and PI / Annexin V staining combined with flow cytometry was used to detect cell apoptosis. DDR1 Gene sequence ID: NM_001202521.1) and negative control NC (sense strand: 5'-UUCUCCGAACGUGUCACGUdTdT-3') or overexpression plasmid (original vector: pCDNA3.0-3'HA, encoding sequence Gene ID: 780) and negative control vector were introduced into THLE-2 cells by jetPRIME transfection reagent, and after stable expression, 25 μM pralsetinib was given for 24 h, and the cells were collected, and PI / Annexin V staining combined with flow cytometry was used to detect cell apoptosis.

[0064] The results are shown in FIG. A. Figure 4 After the intracellular knockdown of DDR1, the cell apoptosis rate decreased. DDR1 The results are shown in FIG. B.

[0065] After overexpression of DDR1, the apoptosis rate increased. Figure 4 DDR1 The above results suggest that the increase in DDR1 level is a key factor in mediating the anokis of liver cells induced by pralsetinib.

[0066] Example 5 1. Human normal liver cells THLE-2 were inoculated in a 12-well plate at a density of 120,000 cells per well, and after overnight stable adhesion, 25 μM pralsetinib was given for 24 h, and the cells were collected, and PI / Annexin V staining combined with flow cytometry was used to detect cell apoptosis; the protein was extracted and quantified, and then Western blot was used for protein level detection.

[0067] The results are shown in FIG. A. Curcumin significantly reduced the level of cell apoptosis induced by pralsetinib.

[0068] Figure 5 The results are shown in FIG. B. Curcumin reduced the levels of c-PARP, DDR1 protein and phosphorylated DDR1 induced by pralsetinib.

[0069] Figure 5 The results are shown in FIG. B.

[0070] ​2、To investigate whether the intervention of curcumin on the hepatotoxicity of pralsetinib is related to the classic NRF2 antioxidant stress pathway, the following experiment was designed: Human normal liver cells THLE-2 were seeded in 12-well plates at a density of 80,000 cells per well, and after overnight stable adhesion, the target NRF2 (#1 sense strand 5'-AUUAUUAUGACUGUUAAAUdTdT-3', #2 sense strand 5'-AGUGUCAGUAUGUUGAAUCdTdT-3') and negative control NC (sense strand 5'-UUCUCCGAACGUGUCACGUdTdT-3') were introduced into THLE-2 cells using jetPRIME transfection reagent, and after stable expression, 25 μM pralsetinib was given for 24 h, then the cells were collected, the protein was extracted and quantified, and then Western blot was used for protein level detection.

[0071] Human normal liver cells THLE-2 were seeded in 12-well plates at a density of 120,000 cells per well, and after overnight stable adhesion, the target NRF2 Overexpression plasmid (original vector: pCDNA3.0-3'HA, NRF2 Gene ID: 4780) and negative control vector were introduced into THLE-2 cells by jetPRIME transfection reagent, and after 12 h of stable expression, 25 μM pralsetinib was given for 24 h, then the cells were collected, the protein was extracted and quantified, and then Western blot was used for protein level detection.

[0072] The results are shown in Figure 5 , the cell NRF2 knockdown and overexpression do not affect pralsetinib-induced apoptosis, indicating that curcumin does not intervene in cell apoptosis through the classic NRF2 pathway.

[0073] 3, 20 male C57BL / 6J mice were randomly divided into 4 groups, namely control group, pralsetinib group, curcumin group, and pralsetinib+curcumin combination group, 5 mice in each group. The drug was given by gavage. The dose of pralsetinib was 100 mg / kg / day, and the dose of curcumin was 100 mg / kg / day. The control group was replaced with 5% β-cyclodextrin, and continuous administration was given for 4 weeks. Blood samples were collected using the orbital blood sampling method, and the levels of serum liver damage biomarkers ALT and AST were detected. The liver tissue was dissected, dehydrated with 4% paraformaldehyde, and then wrapped in paraffin to make wax blocks. After slicing, HE dye was used for staining.

[0074] The results are shown in Figure 5 , under the action of curcumin, the serum ALT and AST levels of pralsetinib gavage model mice decreased significantly.

[0075] As Figure 5 shown in E of FIG. 1, liver pathological section HE staining showed that the inflammatory infiltration and cell vacuolization induced by pralsetinib under the action of curcumin were improved.

[0076] Example 6 RET fusion positive thyroid cancer cells TPC-1 and non-small cell lung cancer cells Lc-2 / ad were inoculated in 96-well plates at a density of 8 thousand per hole, and a certain concentration gradient of pralsetinib (the final concentration of pralsetinib on TPC-1 cells was 1, 10, 50, 100, 500, 1000 nM, and the final concentration of pralsetinib on Lc-2 / ad cells was 0.01, 0.1, 1, 10, 100, 1000 nM) was combined with curcumin at a final concentration of 10 μM for 24 h, TPC-1 cells were fixed with 10% trichloroacetic acid at 4°C for 1 h, dried, and then SRB staining and absorbance determination were performed to investigate the survival rate; Lc-2 / ad cells were added with CCK8 reagent, incubated at 37°C for 30 min, and then absorbance determination was performed to investigate the cell survival rate.

[0077] As Figure 6 and Figure 7 shown, curcumin had no antagonistic effect on pralsetinib killing cancer cells, and showed a slight synergistic effect.

[0078] The above merely describes specific embodiments of the present application, which aims to enable those skilled in the art to understand the content of the present application and implement it, and cannot limit the protection scope of the present application. Any equivalent transformation or replacement made according to the essence of the present application should be covered within the protection scope of the present application.

Claims

1. The use of a substance that targets and inhibits discoid domain receptor 1 in the preparation of a medicament for the prevention or treatment of drug-induced liver injury, characterized in that, The drug-induced liver injury is caused by abnormal activation or accumulation of the discoid domain receptor 1 protein, manifested as hepatocyte anolysm.

2. The application as described in claim 1, characterized in that, The drug-induced liver injury was liver toxicity induced by RET inhibitors.

3. The application as described in claim 2, characterized in that, The RET inhibitor is pralatinib.

4. The application as described in any one of claims 1-3, characterized in that, The substance that targets and inhibits discoid domain receptor 1 is an inhibitor that targets and inhibits the expression of discoid domain receptor 1 gene or inhibits the activity or accumulation of discoid domain receptor 1 protein.

5. The application as described in claim 4, characterized in that, The substance that targets and inhibits discoid domain receptor 1 is siRNA or shRNA that targets and inhibits the expression of the discoid domain receptor 1 gene.

6. The application as described in claim 5, characterized in that, The nucleotide sequence of the siRNA is: 5'-CUGACAUGAAGGGACAUUUdTdT-3'.

7. The application as described in claim 4, characterized in that, The substance that targets and inhibits discoid domain receptor 1 is curcumin or its pharmaceutically acceptable salts, esters, or derivatives.

8. A combination antitumor drug composition, characterized in that, The formulation includes a first formulation of pralatinib and a pharmaceutically acceptable carrier, and a second formulation of curcumin or its salts, esters, derivatives and a pharmaceutically acceptable carrier.

9. The use of the pharmaceutical composition of claim 8 in the preparation of a medicament for treating RET fusion-positive non-small cell lung cancer or thyroid cancer.

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