A medicine for preventing or treating liver toxicity caused by sotorasib and application thereof

Abstract

The application discloses a medicine for preventing or treating liver toxicity caused by sotorasib and application thereof, and belongs to the technical field of medicines.The application first discloses that "Rab7a up-regulation-lipophagy activation-autophagy-dependent ferroptosis" is a key mechanism chain of liver toxicity induced by sotorasib.The medicine can play a significant liver protection role by targeting and inhibiting the Rab7a-mediated lipophagy-dependent ferroptosis pathway.The application screens and verifies a new use of deferasirox (an iron chelator) and hydroxychloroquine (an autophagy inhibitor) in preventing and treating liver toxicity side effects of sotorasib, which can significantly reduce liver pathological damage and transaminase elevation caused by sotorasib while not antagonizing the treatment effect of sotorasib on KRAS G12C mutant tumors.The application provides a brand-new molecular target and a safe and effective combined drug strategy for solving the clinical liver toxicity problem of sotorasib, and has important clinical conversion value.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the development of drugs and their uses for the prevention or treatment of liver toxicity caused by sotorasib, targeting the Rab7a-mediated lipopygy-dependent ferroptosis signaling axis. Background Technology

[0002] Sotorasib (also known as Sotorasib) is a type of KRAS G12C Sotorexane, a mutation inhibitor with a pyridopyrimidine core structure, exerts significant antitumor activity by covalently binding to the 12th cysteine ​​residue, locking KRAS into an inactive state. Clinically, sotorexane is widely used to treat KRAS. G12C Mutant non-small cell lung cancer (NSCLC). Clinical trial data showed that, compared with docetaxel, sotorexane significantly improved progression-free survival and objective response rate in patients (PMID: 36764316).

[0003] However, despite its significant efficacy, sotorarece's hepatotoxicity severely limits its clinical application. Clinical data shows that approximately 25% of patients experience hepatotoxicity, with nearly half reaching grade 3-4, often leading to dose reduction, treatment interruption, or even permanent discontinuation, thus affecting the continuity of anti-tumor therapy (PMID: 34357500) and greatly impacting the clinical efficacy of sotorarece.

[0004] Currently, there are no effective interventions for this type of liver injury in clinical practice. The usual approach is to reduce or discontinue medication, leading to delays in treatment for cancer patients. Therefore, in-depth research into the mechanism of sotorexide-induced hepatotoxicity and the development of corresponding intervention strategies are of significant clinical importance and will also provide a basis for similar KRAS treatments. G12C This provides a reference for the rational application and subsequent development of mutation inhibitors.

[0005] Ferroptosis is a form of programmed cell death mediated by iron-dependent lipid peroxidation, characterized by the peroxidation of polyunsaturated fatty acids (PUFAs) in the cell membrane, generating lipid peroxidation end products (PMID: 22632970). Recent studies have found a close link between autophagy, particularly lipophage (the autophagic degradation of lipid droplets), and ferroptosis. Excessive activation of lipophage can lead to increased PUFA release, thereby exacerbating lipid peroxidation and promoting ferroptosis (PMID: 39243450). Previous studies have shown that lipophage-mediated autophagy-dependent ferroptosis plays a crucial role in sepsis-induced kidney damage and some tumor models (PMID: 38583680). However, whether ferroptosis and lipophage are involved in sotorèse-induced hepatotoxicity has not yet been reported.

[0006] Ras-associated protein 7a (Rab7a), belonging to the Rab subfamily of the RAS superfamily, is a small GTPase that plays a crucial regulatory role in intracellular membrane transport, late endosome maturation into lysosomes, and autophagy flux (PMID: 30463228). Studies have shown that Rab7a is a key driver of lipophage, and its aberrant activation can mediate lipid droplet degradation and trigger downstream oxidative stress and cell death (PMID: 38583680). Although the role of Rab7a in various disease models has been extensively studied, its role in drug-induced liver injury, particularly whether it acts as a key regulatory node in sotorexide-induced hepatotoxicity, remains unknown. Summary of the Invention

[0007] The purpose of this invention is to reveal the molecular mechanism of sotorexide-induced liver toxicity and to provide a safe and effective intervention drug to solve the problem of limited clinical application of sotorexide.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides the use of substances that target and inhibit the lipophage-dependent ferroptosis pathway mediated by Ras-associated protein 7a (Rab7a) in the preparation of medicaments for the prevention or treatment of sotorexe-induced hepatotoxicity, which is caused by sotorexe-induced upregulation of Rab7a expression leading to abnormal activation of lipophage and subsequently triggering autophagy-dependent ferroptosis.

[0009] This invention reveals that sotorexide induces abnormal upregulation of Rab7a in hepatocytes, thereby driving excessive lipophage and leading to autophagy-dependent ferroptosis, which is the key molecular mechanism by which sotorexide causes hepatotoxicity. Targeting and inhibiting the Rab7a-mediated lipophage-dependent ferroptosis pathway can exert a significant hepatoprotective effect. Therefore, this invention proposes to apply substances that target and inhibit the Rab7a-mediated lipophage-dependent ferroptosis pathway to the preparation of drugs for the prevention or treatment of sotorexide-induced hepatotoxicity.

[0010] Sotorexane-induced hepatotoxicity manifests as elevated serum ALT and AST levels, inflammatory infiltration and fibrosis of liver tissue, and at least one of hepatocellular damage or death. Mechanistic studies show that sotorexane at therapeutic concentrations significantly upregulates Rab7a protein levels in hepatocytes and induces excessive activation of lipoplasmosis. During lipoplasmosis, lipid droplets are encapsulated in lysosomes and degraded, releasing large amounts of polyunsaturated fatty acids (PUFAs), leading to increased levels of cell membrane lipid peroxidation and iron accumulation, ultimately inducing autophagy-dependent ferroptosis in hepatocytes.

[0011] Furthermore, information on the human Rab7a gene can be found in the NCBI Sequence Database under number NM_004637.6, with the nucleotide sequence shown in SEQ ID NO.1, and the amino acid sequence of the Rab7a protein shown in SEQ ID NO.2; information on the mouse Rab7a gene (corresponding to Rab7) can be found in the NCBI Sequence Database under number NM_001293652.1, with the nucleotide sequence shown in SEQ ID NO.3, and the amino acid sequence of the corresponding protein shown in SEQ ID NO.4.

[0012] Furthermore, the substance that targets and inhibits the Rab7a-mediated lipophage-dependent ferroptosis pathway exerts its effect through at least one of the following mechanisms: (1) Inhibit Rab7a gene expression or inhibit Rab7a protein activity; (2) Inhibit lipophagy or block autophagy flow; (3) Inhibit ferroptosis or reduce ferroptosis-related responses.

[0013] Specifically, the substance is a nucleic acid molecule that inhibits the expression of the Rab7a gene. Furthermore, the nucleic acid molecule can be, but is not limited to, siRNA, shRNA, antisense oligonucleotides, miRNA mimics, or pharmaceutically acceptable derivatives thereof.

[0014] Preferably, the nucleic acid molecule is siRNA that targets and inhibits the expression of the Rab7a gene.

[0015] In one specific embodiment of the present invention, the nucleotide sequence of the siRNA is 5'-GGCUAGUCACAAUGCAGAUdTdT-3'. The siRNA targeting the Rab7a gene inhibits Rab7a expression, blocks lipophagy, and thus reverses Sotorexe-induced liver toxicity.

[0016] Alternatively, the substance is an inhibitor that inhibits the activity or subcellular localization of Rab7a protein. The inhibitor blocks the lipophage process by inhibiting the GTP binding or hydrolysis of Rab7a, inhibiting the interaction between Rab7a and its effector proteins, and / or inhibiting Rab7a-mediated autophagosome-lysosome fusion, thereby reversing sotorexe-induced liver toxicity.

[0017] Alternatively, the substance is a lipophage inhibitor that reduces lipophage flux by inhibiting the lipidation of key autophagy factors ATG7 and / or LC3, inhibiting the activity of lysosomal acid hydrolases, and / or inhibiting lipid droplet-lysosomal fusion, thereby blocking the lipophage process and reversing sotorexe-induced liver toxicity.

[0018] Preferably, the lipophage inhibitor can be, but is not limited to, hydroxychloroquine or chloroquine, or a pharmaceutically acceptable salt, ester, or derivative thereof. This invention demonstrates that hydroxychloroquine or chloroquine can effectively intervene in sotorexe-induced liver toxicity by blocking autophagic flux and inhibiting lipophage.

[0019] Alternatively, the substance may be a ferroptosis inhibitor. The ferroptosis inhibitor may be, but is not limited to, an iron chelating agent.

[0020] Preferably, the ferroptosis inhibitor is deferasirox or a pharmaceutically acceptable salt, ester, or derivative thereof. This invention demonstrates that deferasirox, as an iron chelating agent, can effectively intervene in sotorelese-induced liver toxicity by reducing intracellular free iron levels and inhibiting ferroptosis.

[0021] Another object of the present invention is to provide an antitumor combination pharmaceutical composition comprising a first formulation formed of sotorexane and a pharmaceutically acceptable carrier, and a second formulation formed of hydroxychloroquine or chloroquine or derarosi and a pharmaceutically acceptable carrier.

[0022] This invention demonstrates that the combined use of deferasirox or hydroxychloroquine with sotorexide can significantly inhibit sotorexide-induced lipid peroxidation, mitochondrial damage, and elevated transaminase (ALT / AST) levels in hepatocytes. More importantly, both animal and cell experiments confirmed that while deferasirox and hydroxychloroquine effectively protect the liver, they did not antagonize the effects of sotorexide on KRAS. G12CIt exhibits cytotoxic activity against mutant tumor cells without introducing additional toxic reactions.

[0023] In the pharmaceutical composition, the dosage of sotorexane to derarosi or hydroxychloroquine or chloroquine is proportioned according to the clinically standard dosage or the optimal ratio determined experimentally.

[0024] In one specific embodiment of the present invention, in a mouse model, sotorexane was used in combination with derarosi at a dose of 5 mg / kg / day at a dose of 300 mg / kg / day.

[0025] In one specific embodiment of the present invention, in a mouse model, sotorexane was used in combination with hydroxychloroquine at a dose of 300 mg / kg / day.

[0026] Another object of the present invention is to provide the pharmaceutical composition described herein in the preparation of a treatment for KRAS. G12C Application in drugs for mutant tumors.

[0027] Furthermore, the tumor can be, but is not limited to, non-small cell lung cancer.

[0028] This invention also provides the application of the Rab7a gene or protein as a target in screening drugs for the prevention or treatment of sotorexe-induced liver toxicity.

[0029] The beneficial effects of this invention are as follows: (1) Mechanism Innovation: This invention reveals for the first time that "Rab7a upregulation-lipophage activation-autophagy-dependent ferroptosis" is the key mechanism chain of sotorèrese-induced hepatotoxicity. Sotorèrese promotes lipophage by upregulating Rab7a, leading to the accumulation of lipid peroxides and ferroptosis. This provides a novel molecular target (Rab7a) and theoretical basis for intervening in sotorèrese hepatotoxicity, which is different from traditional non-specific hepatoprotective strategies.

[0030] (2) Repurposing existing drugs with great potential for translation: This invention screened and verified the novel uses of clinically marketed drugs deferasirox (an iron chelator) and hydroxychloroquine (an autophagy inhibitor) in the prevention and treatment of sotorexide hepatotoxicity. Compared with the development of entirely new compounds, these two drugs have clear pharmacokinetic characteristics and safety records, and are highly feasible for clinical translation, providing a rapid and effective solution to the problem of clinical limitations of sotorexide.

[0031] (3) Safe and effective combination strategy: The combination drug regimen (sotorexane + deirarosi / hydroxychloroquine) provided by this invention can significantly reduce the hepatotoxicity of sotorexane (reduce serum transaminase and improve liver tissue pathological damage) without affecting or even potentially enhancing its effect on KRAS. G12CThe therapeutic effect on mutated tumors. This not only improves patients' tolerance to sotorarece, but also ensures the continuity and effectiveness of anti-tumor treatment. Attached Figure Description

[0032] Figure 1 To investigate the effect of different concentrations of sotorexane (Soto) on the survival rate of hepatocytes using the CCK-8 assay.

[0033] Figure 2 To detect changes in ALT and AST levels in mouse serum after treatment with sotorexide.

[0034] Figure 3 The effect of HE staining on the pathological structure of mouse liver tissue was investigated.

[0035] Figure 4 Masson staining (Masson trichrome staining) was used to examine the degree of sotorèse-induced liver fibrosis.

[0036] Figure 5 Sirius red staining was used to examine the degree of Sotorexe-induced liver fibrosis.

[0037] Figure 6 To observe, via transmission electron microscopy, the formation of numerous encapsulated lipid droplet structures in the liver of Sotorexe-induced mice.

[0038] Figure 7 To observe the changes in autophagy in the liver of mice induced by sotorèse using transmission electron microscopy, the yellow arrows indicate the increased number of autophagosomes with double membrane structures after sotorèse treatment.

[0039] Figure 8 To detect the expression level of autophagy marker protein LC3-II in the liver of sotorexe-induced mice by immunohistochemistry.

[0040] Figure 9 To detect changes in the expression of the antioxidant enzyme GPX4 protein in the liver of Sotorexe-induced mice using immunohistochemistry.

[0041] Figure 10 To detect changes in the expression of ferroptosis-promoting protein ACSL4 in the liver of sotorexe-induced mice using immunohistochemistry.

[0042] Figure 11 The kit was used to detect changes in malondialdehyde (MDA) content in the liver tissue of sotorexai-induced mice.

[0043] Figure 12 The kit was used to detect ferrous ions (Fe2+) in the liver tissue of sotorexane-induced mice. 2+ () Changes in level.

[0044] Figure 13To monitor the activation of autophagic flux in hepatocytes by sotorecase using the mCherry-GFP-LC3 dual fluorescence system.

[0045] Figure 14 The expression level of Rab7a protein in hepatocytes and mouse liver tissue under the action of sotorexide was detected by Western blot.

[0046] Figure 15 To detect the expression and distribution of Rab7 protein in mouse liver tissue after administration of sotorexide via immunohistochemistry.

[0047] Figure 16 To investigate the effect of Rab7a knockdown on sotorexe-induced cell viability using the CCK-8 assay after siRNA technology.

[0048] Figure 17 To investigate the effect of Rab7a knockdown on the reversal of sotorexe-induced lipid droplet degradation, BODIPY 493 / 503 staining was used.

[0049] Figure 18 To investigate the effects of co-administration of pan-Caspase inhibitors (Z-VAD-FMK), ferroptosis inhibitors (deferrasirox, DFX), necrosis inhibitors (Necrostatin-1), autophagy inhibitors (chloroquine, CQ) with sotorexide on hepatocyte survival using the CCK-8 assay.

[0050] Figure 19 To investigate the inhibitory effect of autophagy inhibitor chloroquine on sotorexe-induced lipid droplet reduction, BODIPY 493 / 503 staining was used.

[0051] Figure 20 To investigate the inhibitory effect of the iron chelator derarosi on sotorexe-induced lipid droplet reduction, BODIPY 493 / 503 staining was used.

[0052] Figure 21 To investigate the effect of chloroquine on sotorexe-induced lipid peroxidation levels using the C11-BODIPY 581 / 591 fluorescent probe.

[0053] Figure 22 To investigate the effect of the iron chelator derarosix on sotorexe-induced lipid peroxidation levels using the C11-BODIPY 581 / 591 fluorescent probe.

[0054] Figure 23 The effects of delafloxacin (DFX) and hydroxychloroquine (HCQ) on the amelioration of sotorexe-induced liver histopathological damage in mice were investigated using HE staining.

[0055] Figure 24To investigate the effects of delarose and hydroxychloroquine on serum ALT levels in sotorexe-induced mice for blood biochemistry assays.

[0056] Figure 25 To investigate the effects of delarose and hydroxychloroquine on serum AST levels in sotorexe-induced mice for blood biochemistry assays.

[0057] Figure 26 The kit was used to detect ferrous ions (Fe2+) in mouse liver tissue induced by sotorexide in patients with deirarosi and hydroxychloroquine. 2+ () Changes in level.

[0058] Figure 27 The kit was used to detect changes in malondialdehyde (MDA) content in mouse liver tissue induced by sotorexide in response to deirarosi and hydroxychloroquine.

[0059] Figure 28 To observe the effect of derarosi on the antitumor activity of sotorexide in the NCI-H2122 cell line using microscopy.

[0060] Figure 29 To observe the effect of hydroxychloroquine on the antitumor activity of sotorexide in the NCI-H2122 cell line using microscopy.

[0061] Figure 30 The effect of combined administration of deraxisol and sotorexe on the survival rate of NCI-H2122 cells was investigated using the CCK-8 assay.

[0062] Figure 31 The effect of combined hydroxychloroquine and sotorexane on the survival rate of NCI-H2122 cells was investigated using the CCK-8 assay. Detailed Implementation

[0063] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0064] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0065] Specifically, C57BL / 6J mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.; HL-7702 (human hepatocyte cell line) was purchased from Guangzhou Genio Biotechnology Co., Ltd.; and NCI-H2122 (carrying KRAS cells) was purchased from Guangzhou Genio Biotechnology Co., Ltd. G12CThe mutant human non-small cell lung cancer cell line was purchased from Shanghai Beyotime Biotechnology Co., Ltd.; Sotorexe was purchased from Shanghai Taosu Biochemical Technology Co., Ltd.; Z-VAD-FMK was purchased from Selleck; Necrostatin-1 was purchased from Shanghai Taosu Biochemical Technology Co., Ltd.; 2-hydroxypropyl-β-cyclodextrin was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; chloroquine was purchased from Selleck Biotechnology Co., Ltd.; deferasirox was purchased from MedChemExpress; hydroxychloroquine was purchased from Tokyo Chemical Industry Co., Ltd.; β-Actin antibody was purchased from Hangzhou Daige Biotechnology Co., Ltd.; LC3-II antibody was purchased from Cell Signaling Technology Co., Ltd.; Rab7a antibody was purchased from Hangzhou Huaan Biotechnology Co., Ltd.; ACSL4 and GPX4 antibodies were purchased from Affinity; siRNA was purchased from Beijing Qingke Biotechnology Co., Ltd.; negative The positive strand sequence of the control (NC) was 5'-UUCUCCGAACGUGUCACGUTT-3', and the positive strand sequence of siRab7a was 5'-GGCUAGUCACAAUGCAGAUdTdT-3'. HE staining kit, CCK-8 (Cell Counting Kit-8), BODIPY 581 / 591 C11 fluorescent probe, and BODIPY493 / 503 fluorescent probe detection kit were purchased from Shanghai Beyotime Biotechnology Co., Ltd.; mCherry-GFP-LC3 adenovirus was purchased from Shandong Weizhen Biotechnology Co., Ltd.; Sirius Red staining kit, ferrous ion content detection kit, and MDA content detection kit were purchased from Beijing Solarbio Science & Technology Co., Ltd.; Masson staining kit was purchased from Zhuhai Beso Biotechnology Co., Ltd.; and the jetPRIME® transfection reagent was purchased from Polyplus Transfection.

[0066] Sotorexe, CAS number 2296729-00-3, chemical name 6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-1-(4-methyl-2-(propyl-2-yl)pyridin-3-yl)-4-[(2S)-2-methyl-4-(propyl-2-enoyl)piperazin-1-yl]pyrido[2,3-d]pyrimidin-2(1H)-one, molecular formula C 30 H 30 F2N6O3, with a molecular weight of 560.594, was purchased from Shanghai Taosu Biochemical Technology Co., Ltd. Its structural formula is as follows: .

[0067] Deferrasirox, CAS No. 201530-41-8, chemical name 4-[3,5-bis(2-hydroxyphenyl)-1,2,4-triazol-1-yl]benzoic acid, molecular formula C 21 H 15 N3O4, with a molecular weight of 373.36, was purchased from MedChemExpress. Its structural formula is as follows: .

[0068] Hydroxychloroquine (hydroxychloroquine sulfate), CAS number 747-36-4, chemical name 2-[4-[(7-chloroquinoline-4-yl)amino]pentylamino]ethanol sulfate, molecular formula C 18 H 28 ClN3O5S, with a molecular weight of 433.95, was purchased from Tokyo Chemical Industry Co., Ltd. Its structural formula is as follows: .

[0069] Example 1 Human normal liver parenchymal cells (HL-7702) were seeded at a density of 5000 kJ / well in 96-well plates according to their growth characteristics and cultured in RPMI-1640 medium (containing 10% fetal bovine serum). After stabilization overnight, cells were treated with a gradient of sotorexide (final concentrations of 0, 5, 10, 20, 40, and 80 μM) for 24 h. Cell viability was assessed using CCK-8 working solution.

[0070] The results are as follows Figure 1 As shown, sotorexane significantly reduces the survival rate of human hepatocytes with increasing concentration.

[0071] Example 2 This study used 6-8 week old male C57BL / 6J mice as experimental subjects to construct a sotorexane liver toxicity evaluation model. Twelve test animals were randomly divided into a control group and a treatment group. The control group was administered 20% β-cyclodextrin solution by gavage, while the treatment group was administered 300 mg / kg of sotorexane by gavage. After four weeks of continuous administration, blood samples were collected via orbital sampling to examine the serum ALT and AST levels. Liver tissue was dissected, fixed in 4% paraformaldehyde, and then paraffin-coated to prepare paraffin blocks. After sectioning, liver tissue structure was examined by HE staining, and liver fibrosis level was examined by Masson staining and Sirius Red staining.

[0072] Serum marker results as follows Figure 2 As shown, sotorexane can cause elevated serum ALT and AST levels in mice, and the animal model results are consistent with clinical liver toxicity.

[0073] HE staining results are as follows Figure 3As shown, HE staining of the livers of mice in the sotorexide-treated group revealed typical pathological features of liver injury, such as hepatocyte cytoplasmic vacuolation, indistinct cell outlines, and inflammatory cell infiltration, indicating that sotorexide can induce liver tissue damage in mice.

[0074] Masson staining results are as follows: Figure 4 As shown, Masson staining revealed a slight increase in blue collagen fiber signal in the livers of mice in the drug-treated group. Sirius Red staining results are as follows... Figure 5 As shown, Sirius Red staining revealed that the red signal intensity in the livers of mice in the treatment group was slightly higher than that in the control group. These findings suggest that sotorexide can induce mild liver fibrosis in mice.

[0075] Example 3 1. The mouse liver toxicity evaluation model constructed in Example 2 was used. Fresh liver tissue was taken and ultrathin sections were prepared by fixation with glutaraldehyde, osmium tetroxide, dehydration, embedding, and sectioning. Transmission electron microscopy (TEM) was used to observe the ultrastructure of cells and analyze the morphological changes of lipid droplets in hepatocytes.

[0076] The results are as follows Figure 6 As shown, lipid droplets in the liver cells of mice in the control group were regular and round with clear edges; while in the liver cells of mice in the sotorexide-treated group, the lipid droplets were observed to be wrapped by the autophagosome membrane structure, which is a typical feature. This ultrastructural evidence suggests that sotorexide activates the lipophagy process in liver cells, leading to the degradation of lipid droplets through the autophagy pathway.

[0077] The results are as follows Figure 7 As shown, a large number of autophagic vacuoles were observed to aggregate in hepatocytes of the sotorexide-treated group, and autophagosomes with typical double membrane structures were clearly visible (as shown by the yellow arrows), indicating that sotorexide induced a significant increase in the number of autophagosomes in liver tissue.

[0078] 2. Take liver tissue from the mouse liver toxicity model (300 mg / kg sotorexane) constructed in Example 2, prepare paraffin sections, perform immunohistochemical staining, and detect the expression levels of autophagy marker protein LC3-II and ferroptosis key proteins GPX4 and ACSL4.

[0079] The results are as follows Figure 8 As shown, compared with the control group (Vehicle), the positive staining of LC3-II in the liver tissue of mice in the sotorexide-treated group was significantly enhanced, confirming the increased level of autophagy in the liver tissue in vivo.

[0080] The results are as follows Figure 9 and Figure 10 As shown, sotorexane induced a decrease in GPX4 protein levels and an increase in ACSL4 protein levels in mouse liver, confirming that sotorexane treatment induced ferroptosis in mouse liver tissue.

[0081] 3. The mouse liver toxicity evaluation model constructed in Example 2 was used. Mouse liver tissue was collected, and the malondialdehyde (MDA) content in the tissue was measured using an MDA detection kit. Fe... 2+ The test kit measures the content of ferrous ions in tissues.

[0082] The results are as follows Figure 11 As shown, the MDA content in the liver tissue of mice in the sotorexide-treated group was significantly higher than that in the control group, indicating severe lipid peroxidation damage in vivo.

[0083] The results are as follows Figure 12 As shown, compared with the control group, the Fe in the liver tissue of mice in the sotorexide-treated group was significantly lower. 2+ The significantly increased levels confirmed that Sotorexe can induce hepatic iron overload in vivo.

[0084] Example 4 Human normal hepatocytes (HL-7702) were seeded in confocal culture dishes. After cell adhesion, mCherry-GFP-LC3 adenovirus was added for infection for 12 h. The culture medium was then replaced with fresh medium and treated with 40 μM sotorexate for 24 h. mCherry-GFP-LC3 is a dual-fluorescent labeling system. When autophagosomes fuse with lysosomes to form autophagolysosomes (in an acidic environment), the green fluorescence of GFP is quenched, leaving only the red fluorescence of mCherry, thus indicating the patency of autophagic flux. After washing with PBS, autophagic flux was observed using a laser confocal microscope.

[0085] The results are as follows Figure 13 As shown, the red and green fluorescence in the control group was uniformly distributed, showing a yellow signal; while in the sotorexe-treated group, the intracellular LC3 protein changed from diffuse distribution to punctate aggregation, and a large number of individual red fluorescent spots (indicating autolysosomes) were observed, indicating that sotorexe significantly promoted autophagic flux in hepatocytes.

[0086] Example 5 Human normal liver parenchymal cells (HL-7702) were seeded at an appropriate density in 6-well plates and treated with different concentration gradients (0, 10, 20, 40 μM) of sotorexide for 24 h. Cells were harvested, total protein was extracted, and the expression level of Rab7a protein was detected by Western blot.

[0087] The results are as follows Figure 14 As shown, with increasing sotorexate concentration, the protein level of Rab7a in hepatocytes exhibited a clear gradient increasing trend, suggesting that Rab7a responds to sotorexate treatment and is upregulated.

[0088] Example 6 The mouse liver toxicity evaluation model constructed in Example 2 was used. Paraffin sections of mouse liver tissue were prepared and subjected to immunohistochemical staining to specifically detect the expression and localization of Rab7a protein.

[0089] The results are as follows Figure 15 As shown, compared with the control group, the positive staining of Rab7a in the liver tissue of mice in the sotorexide-treated group was significantly enhanced, indicating that sotorexide also induced the upregulation of Rab7a protein expression at the in vivo level.

[0090] Example 7 1. Human normal liver parenchymal cells HL-7702 were seeded in 6-well plates. Using jetPRIME transfection reagent, siRab7a targeting Rab7a (sense strand sequence 5'-GGCUAGUCACAAUGCAGAUdTdT-3') or negative control NC (sense strand sequence 5'-UUCUCCGAACGUGUCACGUTT-3') were transfected into the cells. After 24 h of transfection, the cells were treated for another 24 h with or without the addition of 40 μM sotorexate. After that, CCK-8 reagent was added to each well to detect cell viability.

[0091] The results are as follows Figure 16 As shown, the CCK-8 assay results indicate that knocking down Rab7a significantly improved the survival rate of hepatocytes in the sotorexe treatment group.

[0092] 2. Repeat the transfection and drug administration process in step 1, then incubate with BODIPY 493 / 503 fluorescent probes to label intracellular lipid droplets and observe using a fluorescence microscope.

[0093] The results are as follows Figure 17 As shown, in the control group transfected with NC, sotorexed treatment resulted in a significant reduction in green fluorescent spots representing lipid droplets in the cells; while in the experimental group transfected with siRab7a, knockdown of Rab7a significantly blocked the reduction of lipid droplets caused by sotorexed, and the number of green fluorescent spots was restored.

[0094] Example 8 1. Human normal liver parenchymal cells (HL-7702) were seeded at an appropriate density in 96-well plates. After overnight cell adhesion, the cells were treated with 40 μM sotorexide alone, or with 40 μM sotorexide in combination with different cell death inhibitors (including 20 μM pan-caspase inhibitor Z-VAD-FMK, 40 μM ferroptosis inhibitor deferasirox, 40 μM necroptosis inhibitor Necrostatin-1, and 15 μM autophagy inhibitor chloroquine) for 24 h. Cell viability was assessed using CCK-8 working solution.

[0095] The results are as follows Figure 18 As shown, the cell survival rate was significantly reduced in the sotorexane treatment group alone; the combination with Z-VAD-FMK or Necrostatin-1 did not significantly improve the survival rate; however, the cell survival rate was significantly restored when the sotorexane was combined with deirarosi or chloroquine. This result indicates that sotorexane-induced hepatocyte death mainly depends on ferroptosis and autophagy.

[0096] 2. Human normal hepatocytes (HL-7702) were seeded in culture plates and treated with 40 μM sotorexane, or in combination with 15 μM chloroquine, or in combination with 40 μM deferasirox for 24 h. Subsequently, the cells were incubated with the BODIPY 493 / 503 fluorescent probe, which specifically binds to neutral lipids and emits green fluorescence to label intracellular lipid droplets.

[0097] The results are as follows Figure 19 As shown, the number of green fluorescent spots on labeled lipid droplets decreased in the sotorexane-only treatment group, while the number of lipid droplets returned to normal in the chloroquine-treated group.

[0098] The results are as follows Figure 20 As shown, the number of green fluorescent spots of labeled lipid droplets decreased in the sotorexe-only treatment group, while the number of lipid droplets returned to normal levels after combined treatment with delaros.

[0099] 3. Human normal hepatocytes (HL-7702) were seeded in culture plates and treated with 40 μM sotorexane, or in combination with 15 μM chloroquine, or in combination with 40 μM derarosi for 24 h. Subsequently, the cells were incubated with the C11-BODIPY 581 / 591 fluorescent probe, whose fluorescence changed from red to green upon lipid peroxidation. After washing with PBS, the cells were observed using a fluorescence microscope.

[0100] The results are as follows Figure 21 As shown, compared with the sotorexane monotherapy group, the chloroquine combination therapy group showed reduced green fluorescence and increased red fluorescence.

[0101] The results are as follows Figure 22 As shown, compared with the sotorexide monotherapy group, the derarosixidide combination therapy group showed a decrease in green fluorescence and an increase in the proportion of red fluorescence.

[0102] Example 9 1. Forty-eight male C57BL / 6J mice aged 6-8 weeks were randomly divided into eight groups (n=6 per group): control group (VEC), sotorexide monotherapy group (Soto), deferasirox monotherapy group (DFX), sotorexide and deferasirox combination group (Soto+DFX), hydroxychloroquine monotherapy group (HCQ), sotorexide and hydroxychloroquine combination group (Soto+HCQ), hydroxychloroquine and deferasirox combination group (HCQ+DFX), and three-drug combination group (Soto+HCQ+DFX). The administration regimen was by gavage. The sotorexide dose was 300 mg / kg, and the deferasirox and hydroxychloroquine doses were 5 mg / kg and 30 mg / kg, respectively, once daily for 28 consecutive days. After 4 weeks of continuous administration, blood samples were collected via orbital sampling to examine serum ALT and AST levels. Partial liver tissue was fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE). The pathological structural changes of the liver were observed under a microscope.

[0103] The results are as follows Figure 23 As shown, the sotorexane monotherapy group exhibited significant hepatocyte cytoplasmic vacuolation and inflammatory cell infiltration, among other damaging characteristics. In contrast, the intervention groups treated with derarosiform, hydroxychloroquine, and a combination of derarosiform and hydroxychloroquine showed significant improvement in these pathological changes, with hepatocytes exhibiting more regular arrangement and a more normal morphology, further confirming the protective effect of the drugs on liver tissue.

[0104] The results are as follows Figure 24 and Figure 25 As shown, ALT and AST levels in mice treated with sotorexide alone were significantly higher than in the control group, indicating impaired liver function. However, when combined with deferasirox or hydroxychloroquine, ALT and AST levels significantly decreased to near-normal levels, and the three-drug combination group also showed excellent protective effects. This indicates that deferasirox and hydroxychloroquine can effectively alleviate liver dysfunction induced by sotorexide.

[0105] 2. Take fresh liver tissue and use Fe... 2+ The test kit measures the ferrous ion content in tissues to assess iron overload.

[0106] The results are as follows Figure 26 As shown, the liver Fe in mice in the sotorexane monotherapy group 2+ The levels were significantly elevated. Compared with the single-drug groups, the Fe levels in the sotorexane and deferrasiro combination group, the sotorexane and hydroxychloroquine combination group, and the three-drug combination group were significantly higher. 2+ The levels were significantly reduced. This indicates that derarosiform and hydroxychloroquine can effectively inhibit sotorexe-induced accumulation of iron in the liver.

[0107] 3. Use an MDA detection kit to measure the malondialdehyde (MDA) content in liver tissue to assess lipid peroxidation levels.

[0108] The results are as follows Figure 27 As shown, the MDA content in the liver of mice treated with sotorexide alone was significantly increased, indicating severe oxidative damage. However, after intervention with deferasirox or hydroxychloroquine, MDA levels were significantly inhibited, and the combined use of the three drugs also showed significant effects. These results confirm that deferasirox and hydroxychloroquine exert their hepatoprotective effects by inhibiting lipid peroxidation.

[0109] Example 10 1. Select the option carrying KRAS G12C The mutant human non-small cell lung cancer cell line NCI-H2122 was used as an in vitro drug efficacy evaluation model. NCI-H2122 cells were seeded in 96-well or 6-well plates and cultured in NCI-H2122-specific medium. After cell adhesion or the formation of stable growth clusters, the cells were divided into groups: control group (DMSO), sotorexide alone (40 μM), deferasirox alone (40 μM), hydroxychloroquine alone (15 μM), sotorexide and deferasirox combination group, and sotorexide and hydroxychloroquine combination group. The drug treatment time was 24 h for all groups. After the drug treatment, the morphological changes of cancer cells in each group were first observed using an optical microscope to visually assess the drug's killing effect.

[0110] The results are as follows Figure 28 and Figure 29 As shown, NCI-H2122 cells treated alone exhibited significant cell fragmentation and reduced cell number, indicating cell death. Notably, when delarose or hydroxychloroquine was used in combination with sotorarece, the morphological damage to cancer cells was not reduced; instead, significant cell death persisted, suggesting that delarose and hydroxychloroquine did not antagonize the killing effect of sotorarece on cancer cells.

[0111] 2. The CCK-8 assay was used to quantitatively detect the cell viability of each group in order to accurately assess the effect of combined drug therapy on the antitumor efficacy of sotorexide.

[0112] The results are as follows Figure 30 As shown, derarosi not only did not weaken the efficacy of sotorexane, but also significantly enhanced its killing effect on lung cancer cells.

[0113] The results are as follows Figure 31 As shown, hydroxychloroquine not only did not weaken the efficacy of sotorexane, but also significantly enhanced its killing effect on lung cancer cells.

[0114] The above description is merely a specific embodiment of the present invention, intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and should not be construed as limiting the scope of protection of the present invention. All equivalent modifications or substitutions made based on the essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. The use of a substance that targets and inhibits the Rab7a-mediated lipophage-dependent ferroptosis pathway in the preparation of a medicament for the prevention or treatment of sotorexide-induced hepatotoxicity, characterized in that, The liver toxicity was caused by Sotorexe-induced upregulation of Rab7a expression, which triggered abnormal lipophage activation and subsequently autophagy-dependent ferroptosis.

2. The application as described in claim 1, characterized in that, The manifestations of liver toxicity include at least one of the following: elevated serum ALT and AST levels, inflammatory infiltration and fibrosis of liver tissue, and hepatocellular damage and death.

3. The application as described in claim 1, characterized in that, The substance exerts its effects through at least one of the following mechanisms: (1) inhibiting Rab7a gene expression or inhibiting Rab7a protein activity; (2) inhibiting lipophagy or blocking autophagic flux; and (3) inhibiting ferroptosis or reducing ferroptosis-related responses.

4. The application as described in claim 3, characterized in that, The substance is a nucleic acid molecule that inhibits the expression of the Rab7a gene, and the nucleic acid molecule is siRNA, shRNA, antisense oligonucleotide, miRNA mimic, or a pharmaceutically acceptable derivative thereof.

5. The application as described in claim 3, characterized in that, The substance is an inhibitor that inhibits the activity or subcellular localization of Rab7a protein. The inhibitor is used to inhibit GTP binding or hydrolysis of Rab7a, inhibit the interaction between Rab7a and its effector proteins, and / or inhibit Rab7a-mediated autophagosome-lysosome fusion.

6. The application as described in claim 3, characterized in that, The substance is a lipophage inhibitor, which is hydroxychloroquine, chloroquine, or a pharmaceutically acceptable salt, ester, or derivative thereof.

7. The application as described in claim 3, characterized in that, The substance is a ferroptosis inhibitor, which is deferasirox or a pharmaceutically acceptable salt, ester or derivative thereof.

8. A combination antitumor drug composition, characterized in that, The composition comprises a first formulation formed of sotorexane and a pharmaceutically acceptable carrier, and a second formulation formed of hydroxychloroquine or chloroquine or derarosi and a pharmaceutically acceptable carrier.

9. The pharmaceutical composition of claim 8 in the preparation of a treatment for KRAS G12C Application in drugs for mutant tumors.

10. The application as described in claim 9, characterized in that, The tumor is non-small cell lung cancer.

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