Medicine for preventing or treating liver toxic and side effects of osimertinib
By combining autophagy inhibitors with osimertinib, the autophagy pathway of hepatocytes is inhibited, which solves the problem of liver damage caused by osimertinib, improves the survival rate of hepatocytes and reduces liver damage, ensuring that the anti-tumor effect is not affected.
Patent Information
- Application Number
- CN202510751955.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-06
- Publication Date
- 2025-10-03
AI Technical Summary
Existing technologies cannot effectively address the severe drug-induced liver injury caused by osimertinib in the treatment of non-small cell lung cancer. Conventional treatments such as glycyrrhizic acid and silymarin drugs are ineffective, resulting in some patients having to interrupt anti-cancer treatment and a decline in their quality of life.
Autophagy inhibitors such as bafilomycin A1, hydroxychloroquine or S-adenosylmethionine are used in combination with osimertinib to reverse osimertinib-induced liver cell damage by inhibiting the autophagy pathway of hepatocytes.
It significantly improves the survival rate of liver cells and reduces liver damage during osimertinib treatment. The combination drug shows biosafety both in vivo and in vitro and does not affect the anti-tumor effect.
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Figure CN120732880A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of medical technology, and in particular to a novel use of hydroxychloroquine or S-adenosylmethionine in preparing a drug for treating liver toxic side effects caused by osimertinib. Background Art
[0002] Osimertinib (AZD9291), a third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), has demonstrated remarkable efficacy in the treatment of non-small cell lung cancer, significantly prolonging patient survival and becoming a key treatment option for many patients with advanced lung cancer. Osimertinib can be used as a first-line drug in the treatment of non-small cell lung cancer, or as a second-line alternative after resistance to other EGFR-TKIs develops (OuYamaguchi, et al . Severe hepatotoxicity due to osimertinib after nivolumabtherapy in patients with non-small cell lung cancer harboring EGFRmutation. Thoracic Cancer . 2020,11(4):1045–1051.). However, osimertinib can cause severe drug-induced liver injury during the treatment of non-small cell lung cancer.
[0003] Drug-induced liver injury is classified into hepatocellular injury, cholestatic, and mixed types based on the serum elevation of routine liver biochemical indicators, such as aspartate aminotransferase and alanine aminotransferase. Clinically, routine liver biochemical indicators are an important basis for determining drug discontinuation. In osimertinib treatment cases, there are often reports of patients experiencing a sharp increase in aspartate aminotransferase and alanine aminotransferase after osimertinib treatment, indicating severe liver injury and requiring drug discontinuation (Iván González, et al . Histopathological Features of Drug-Induced Liver Injury Secondary to Osimertinib. ACG Case Rep J . 2019,6(2):e00011.). However, in clinical practice, after patients stop taking osimertinib, there are usually no anti-tumor drugs available for subsequent treatment.
[0004] Currently, clinical treatment for osimertinib-induced liver injury often involves symptomatic liver protection measures, such as the routine use of glycyrrhizic acid and silymarin drugs. However, these measures have been less than ideal and have been unable to effectively curb the progression of liver damage. This has resulted in some patients being forced to discontinue anticancer treatment due to worsening liver function, leading to a sharp decline in quality of life and a worrying prognosis. Therefore, further investigation into the mechanism of action of osimertinib-induced liver injury and the identification of effective protective agents to address this issue are technical challenges facing those skilled in the art.
[0005] Hydroxychloroquine (HCQ) is a 4-aminoquinoline compound. It is formed by introducing a hydroxyl group (-OH) onto a nitrogen atom in the chemical structure of chloroquine. As a classic antimalarial and immunomodulatory drug, HCQ has diverse pharmacological properties. It can not only precisely embed itself into biomembranes to interfere with pathogen metabolism, but also subtly regulate intracellular inflammatory signaling pathways and autophagy (Paulo Michel Pinheiro Ferreira, et al . Chloroquine and hydroxychloroquine in antitumor therapies based on autophagy-related mechanisms. Pharmacol Res . 2021,168:105582.). To date, there have been no reports on the use of hydroxychloroquine as a treatment for osimertinib-induced hepatotoxicity.
[0006] S-adenosyl-L-methionine (SAM) is a methyl donor and is clinically an effective dietary supplement for antidepressants and pain relief. In addition, S-adenosyl-L-methionine can also be used as a liver protective agent in the treatment of intrahepatic cholestasis of pregnancy, alcoholic liver disease and chronic hepatitis C (Shelly C Lu, et al . S-ADENOSYLMETHIONINE IN LIVER HEALTH, INJURY, AND CANCER. Physiol Rev .2012,92(4):1515–1542.). However, its protective mechanism as a hepatoprotective agent is still unclear. To date, there are no reports of its use as a therapeutic drug for osimertinib-induced hepatotoxicity. Summary of the Invention
[0007] The purpose of the present invention is to provide a drug that can effectively improve liver damage caused by the anti-tumor drug osimertinib, and to reduce the liver toxicity caused by osimertinib alone through combined use.
[0008] To achieve the above object, the present invention adopts the following technical solutions: The present invention provides the use of an autophagy inhibitor in the preparation of a medicament for preventing or treating osimertinib-induced liver toxicity and side effects, wherein the liver toxicity and side effects include hepatocellular damage or death caused by osimertinib-induced activation of the hepatocellular autophagy pathway.
[0009] Specifically, the manifestations of the liver toxic side effects include the production of intracytoplasmic vacuoles in hepatocytes, the vacuolar structure of which is autophagolysosome-like vacuoles; and elevated aspartate aminotransferase and alanine aminotransferase in serum.
[0010] Research conducted in this study demonstrates that osimertinib can activate the autophagy pathway in normal hepatocytes during tumor treatment, leading to cytoplasmic vacuolation and ultimately hepatocyte cell death. Combining osimertinib with an autophagy inhibitor to inhibit osimertinib-induced autophagy activation in hepatocytes can reverse the hepatotoxicity caused by osimertinib as an anti-tumor drug. Therefore, autophagy inhibitors can be used to prevent and treat osimertinib-induced liver damage.
[0011] In the present invention, the autophagy inhibitor is a substance used to inhibit the autophagy pathway of hepatocytes. Preferably, the autophagy inhibitor is bafilomycin A1, hydroxychloroquine or S-adenosylmethionine. The CAS number of the bafilomycin A1 is 88899-55-2, and the molecular formula is C 35 H 58 O9; the CAS number of the hydroxychloroquine is 747-36-4, and the molecular formula is C 18 H 26 ClN3O; the CAS number of S-adenosylmethionine is 29908-03-0, and the molecular formula is C 15 H 22 N6O5S.
[0012] The present study demonstrates that combining bafilomycin A1, hydroxychloroquine, or S-adenosylmethionine with osimertinib can inhibit autophagy in hepatocytes and reverse osimertinib-induced hepatotoxicity. Therefore, bafilomycin A1, hydroxychloroquine, or S-adenosylmethionine can serve as hepatoprotective agents during osimertinib treatment.
[0013] The present invention provides an effective therapeutic drug for the liver toxicity caused by osimertinib. Experimental results show that compared with the group treated with osimertinib alone, the combination of autophagy inhibitors can significantly improve the survival rate of hepatocytes and reduce the liver damage caused by osimertinib treatment, indicating that the combination can significantly inhibit the hepatotoxicity of osimertinib. Mechanistic studies have shown that the specific mechanism of osimertinib-induced hepatotoxicity is that osimertinib can significantly activate the autophagy pathway of hepatocytes, thereby inducing the formation of vacuoles in hepatocytes and ultimately leading to hepatocyte death. Bafilomycin A1, hydroxychloroquine or S-adenosylmethionine can reverse hepatocyte death by inhibiting the autophagy activation caused by osimertinib.
[0014] Animal experiments have shown that the effective dose of hydroxychloroquine in the drug is 10-15 mg / kg, and the effective dose of S-adenosylmethionine is 25-30 mg / kg. These dosages are safe and effective.
[0015] Another object of the present invention is to provide an anti-tumor combination drug comprising a first formulation of osimertinib and a pharmaceutically acceptable carrier, and a second formulation of hydroxychloroquine or S-adenosylmethionine and a pharmaceutically acceptable carrier. The present invention utilizes the second formulation to alleviate hepatotoxicity caused by the first formulation.
[0016] Osimertinib is an anti-tumor drug, and its clinical indications may include but are not limited to lung cancer.
[0017] The experimental results showed that compared with the osimertinib monotherapy group, there was no significant difference in the killing effect of the combination drug group on lung cancer cells, indicating that the combination drug will not reduce the anti-tumor effect of osimertinib.
[0018] For the mouse model, the effective dose of hydroxychloroquine in the drug is 10 mg / kg; the effective dose of S-adenosylmethionine is 25 mg / kg.
[0019] Furthermore, the pharmaceutically acceptable carrier includes at least one of a filler, a disintegrant, a lubricant, and a binder. Specifically, the filler is selected from starch, lactose, and microcrystalline cellulose; the disintegrant is selected from sodium carboxymethyl starch and cross-linked polyvinylpyrrolidone; the lubricant is selected from magnesium stearate and talc; and the binder is selected from hypromellose and povidone.
[0020] Furthermore, the drug is in the form of an oral preparation, which is a tablet, capsule, granule or oral liquid preparation.
[0021] The present invention also provides the use of the anti-tumor combination drug in the preparation of a drug for treating lung cancer. The combination drug provided by the present invention can alleviate the hepatotoxicity of osimertinib in the treatment of lung cancer.
[0022] Furthermore, the lung cancer may be, but is not limited to, non-small cell lung cancer. Still further, the non-small cell lung cancer is EGFR mutation non-small cell lung cancer.
[0023] Furthermore, the lung cancer is lung adenocarcinoma.
[0024] The present invention has the following beneficial effects: (1) The present invention provides the mechanism of hepatotoxicity induced by the anti-tumor drug osimertinib, namely, hepatotoxicity is induced by activating hepatocyte autophagy, which provides a theoretical basis for clinical research on hepatotoxicity induced by osimertinib and is conducive to the screening and development of therapeutic drugs.
[0025] (2) The present invention provides an effective therapeutic drug for the liver toxicity caused by osimertinib. The combination of autophagy inhibitors and osimertinib can significantly improve the survival rate of hepatocytes and reduce the liver damage caused by osimertinib treatment.
[0026] (3) The present invention provides a novel use of hydroxychloroquine or S-adenosylmethionine in the preparation of a drug for preventing or treating the hepatotoxic side effects of osimertinib. Hydroxychloroquine or S-adenosylmethionine inhibits osimertinib-activated autophagy, thereby alleviating the death of hepatocytes caused by excessive activation of the autophagic pathway, thereby alleviating the hepatotoxicity caused by osimertinib. The present invention shows that the combination of hydroxychloroquine or S-adenosylmethionine and osimertinib can alleviate the hepatotoxicity caused by osimertinib, and the combined drug has biosafety and can be used in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 This is the survival rate of the human hepatocyte cell line MIHA after being treated with gradient concentrations of osimertinib (0.25 μM to 16 μM, 2-fold dilution) for 48 hours in Example 1.
[0028] Figure 2 In Example 2, the human hepatocyte cell line MIHA was treated with gradient concentrations of osimertinib (2 μM, 4 μM, 8 μM) for 48 hours, and then Annexin V-FITC / PI double dye staining was performed to detect the proportion of apoptotic cells.
[0029] Figure 3 The protein expression levels of c-PARP (cleaved-PARP), cc3 (cleaved-caspase 3), and β-Actin in the human hepatocyte cell line MIHA after treatment with 4 μM osimertinib for 6 hours, 12 hours, 24 hours, and 48 hours in Example 2.
[0030] Figure 4The cell survival rate is shown in Example 2 after the human hepatocyte cell line MIHA was treated with osimertinib alone or co-treated with the apoptosis inhibitor Z-VAD-FMK (Z-VAD), the cell necrosis inhibitor Necrostatin (Necro-1), the para-apoptosis inhibitor cycloheximide (CHX), and the autophagy activator Rapamycin (Rapa).
[0031] Figure 5 The cell morphology of the human hepatocyte cell line MIHA treated with gradient concentrations of osimertinib (2 μM, 4 μM, 8 μM) for 48 hours in Example 3 was shown. The number and diameter of vacuoles in the cells were counted using Image J. The scale bar is 10 μm.
[0032] Figure 6 This is the vacuolar structure photographed using an electron microscope after the human hepatocyte cell line MIHA was treated with 4 μM osimertinib for 24 hours in Example 3.
[0033] Figure 7 In Example 3, the human hepatocyte line MIHA was co-transfected with LC3-GFP (green fluorescence) and BFP-LAMP1 (blue fluorescence), followed by treatment with 4 μM osimertinib for 6 hours. Fluorescence distribution and vacuolar locations were imaged under a fluorescence microscope. The white arrows indicate the colocalization of LC3-GFP and BFP-LAMP1, and the black arrows indicate the location of vacuoles. LC3 and BFP colocalize with vacuoles.
[0034] Figure 8 This figure shows the protein expression levels of LC3, p62, and GAPDH in the human hepatocyte cell line MIHA after 48 hours of treatment with a gradient of osimertinib (2 μM, 4 μM, and 8 μM) in Example 3. GAPDH serves as the internal reference protein. From left to right, the figure shows the western blot results, the p62:GAPDH statistical plot, and the LC3:GAPDH statistical plot. The statistical plots are grayscale analysis of the western blot results using Image J.
[0035] Figure 9The protein expression levels of Lamp1, LC3, p62, and β-Actin are shown in Example 4 after 12 hours of treatment with 2 μM osimertinib in the human hepatocyte cell line MIHA, either in combination or alone with bafilomycin A1. β-Actin is used as the internal reference protein. From left to right in the figure are the western blot results, the Lamp1:β-Actin statistical graph, the p62:β-Actin statistical graph, and the LC3:β-Actin statistical graph. The statistical graphs are statistical analysis graphs obtained after grayscale analysis of the western blot results using Image J.
[0036] Figure 10 The cell survival rate in the human hepatocyte cell line MIHA in Example 4 was treated with osimertinib, combined with or alone with bafilomycin A1 for 48 hours.
[0037] Figure 11 Schematic diagram of the animal experiment operation process in Example 5.
[0038] Figure 12 The hematoxylin-eosin staining images of liver tissue and the statistical analysis of necrosis area in Example 5. From left to right in the figure are the staining images and the statistical graph of necrosis area.
[0039] Figure 13 The concentrations of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum after the use of hydroxychloroquine and osimertinib alone or in combination in Example 5. From left to right in the figure are the ALT results and AST results.
[0040] Figure 14 The survival rate of the lung cancer cell line NCI-H1975 after single or combined use of hydroxychloroquine and osimertinib in Example 6.
[0041] Figure 15 The cell survival rate in the human hepatocyte cell line MIHA in Example 7 was measured after treatment with osimertinib alone or co-treated with S-adenosyl-L-methionine (SAM), Tiopronin (Tio), Glycyrrhizic acid (GA), Silymarin (Sily), and N-Acetyl-L-cysteine (NAC).
[0042] Figure 16 This figure shows the cell morphology of the human hepatocyte line MIHA treated with 2 μM osimertinib, either alone or in combination with 5 μM SAM, for 12 hours in Example 7. The number and diameter of vacuoles were counted using Image J. Scale bar, 10 μm. From left to right in the figure are microscopic images and statistical analysis graphs.
[0043] Figure 17 This figure shows the protein expression levels of LAMP1, LC3, p62, and β-Actin in the human hepatocyte cell line MIHA treated with 2 μM osimertinib, either in combination with or alone, for 12 hours. β-Actin serves as the internal reference protein. From left to right, the figure shows the western blot results, the statistical graph for Lamp1:β-Actin, the statistical graph for p62:β-Actin, and the statistical graph for LC3:β-Actin. The statistical graphs are grayscale analysis results using Image J.
[0044] Figure 18 Schematic diagram of the animal experiment operation process in Example 8.
[0045] Figure 19 The hematoxylin-eosin staining images of liver tissue and the statistical analysis of necrosis area in Example 8. From left to right in the figure are the staining images and the statistical graph of necrosis area.
[0046] Figure 20 The concentrations of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum after administration of SAM and osimertinib alone or in combination in Example 8. The ALT and AST results are shown from left to right.
[0047] Figure 21 The cell survival rate after SAM and osimertinib were used alone or in combination in lung cancer cells NCI-H1975 in Example 9. DETAILED DESCRIPTION
[0048] The present invention will be further described below in conjunction with specific examples. The following examples are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the inventive method, steps or conditions all fall within the scope of the present invention.
[0049] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available reagents and materials unless otherwise specified.
[0050] Hydroxychloroquine, molecular weight 335.87, molecular formula C 18 H 26 ClN3O, the structural formula is: .
[0051] S-adenosylmethionine, molecular weight 398.44, molecular formula C 15 H 22 N6O5S, the structural formula is: .
[0052] Example 1 1. Reagents and Materials The human hepatocyte cell line MIHA was a gift from the research group of Zheng Xiaohui at Wenzhou Medical University. MIHA cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) high-glucose medium, 10% fetal bovine serum, and 1% penicillin / streptomycin solution. Cells were cultured at 37°C in a 5% CO2 incubator. Osimertinib was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number A302849. CCK-8 reagent was purchased from Beijing Lambolide Trading Co., Ltd., catalog number CK001. DMEM high-glucose medium and penicillin / streptomycin solution were purchased from Hangzhou Keyi Biotechnology Co., Ltd., catalog numbers C0006 and CP011, respectively. Fetal bovine serum is catalog number SH30396.03.
[0053] 2. Experimental methods Cell administration: MIHA cells were seeded at 3500 cells per well in a 96-well plate and cultured overnight to allow attachment. Osimertinib was dissolved in dimethyl sulfoxide (DMSO) to a 16 mM stock solution. The solution was then prepared and diluted two-fold to a final dosing concentration of 0.25 μM to 16 μM. After osimertinib treatment for 48 hours, CCK-8 reagent was added at a 1:100 ratio. The cells were incubated at 37°C for 1.5 hours, and absorbance was measured at 450 nm.
[0054] Calculation of cell viability: Divide the absorbance value at each concentration by the absorbance value of the no-drug group to calculate the corresponding cell viability. Cell viability curves were generated using Gradpad Prism, with osimertinib concentration as the horizontal axis and cell viability at each concentration as the vertical axis.
[0055] 3. Experimental results Depend on Figure 1 As shown in Figure 3, the survival rate of MIHA cells decreased significantly with increasing doses of osimertinib, indicating the toxicity of osimertinib on hepatocytes.
[0056] Example 2 1. Reagents and Materials The Annexin V-FITC / PI dual-dye staining kit was purchased from Lianke Biotechnology, catalog number AP101. The apoptosis inhibitor Z-VAD-FMK (Z-VAD), the necrosis inhibitor Necrostatin (Necro-1), and the autophagy activator Rapamycin (Rapa) were purchased from Taoshu Biotechnology, catalog numbers T89262, T1847, and T1537, respectively. The para-apoptosis inhibitor cycloheximide (CHX) was purchased from MedChemExpress (Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd.), catalog number HY-12320. Cisplatin (DDP) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number D109812. All other reagents and materials were the same as in Example 1.
[0057] 2. Experimental methods Cell administration: MIHA cells were seeded at an appropriate density in 12-well plates and cultured overnight to allow attachment. Osimertinib was diluted in DMSO to 2 mM, 4 mM, and 8 mM and treated at a ratio of 1:1000 for 48 hours, resulting in final concentrations of 2 μM, 4 μM, and 8 μM. Alternatively, MIHA cells were treated with 4 μM osimertinib for 6, 12, 24, and 48 hours. Cisplatin (DDP) was also administered for 48 hours to serve as a positive control for induction of apoptosis.
[0058] Annexin V-FITC / PI staining: Follow the instructions provided with the kit. Digest and harvest cells with trypsin, resuspend in the buffer provided with the kit, add an appropriate amount of dye, incubate in the dark for 5 minutes, and analyze using a flow cytometer.
[0059] Extract cell protein: discard the supernatant, extract the protein with loading buffer, denature at 95°C for 20 minutes, and then detect the protein by Western Blot.
[0060] Cell viability assay: 3500 MIHA cells were seeded per well in a 96-well plate and cultured overnight to allow attachment. Osimertinib was dissolved in dimethyl sulfoxide (DMSO) to a 16 mM stock solution, and the drug solution was prepared to a final dosing concentration of 6 μM. Inhibitors were used in combination to adjust the final concentrations of Z-VAD to 10 μM, Necrostatin-1 to 10 μM, Cycloheximide to 20 μg / mL, and Rapamycin to 10 μM. After 48 hours of osimertinib treatment alone or co-treatment with inhibitors, CCK-8 reagent was added at a 1:100 ratio. After incubation at 37°C for 1.5 hours, absorbance was measured at 450 nm.
[0061] 3. Experimental results like Figure 2 As shown in the data, in normal hepatocytes MIHA, the proportion of apoptotic cells did not increase significantly with increasing doses of osimertinib.
[0062] like Figure 3 As shown, the DDP group, a positive group for inducing cell apoptosis, showed significant detection of the production of cleaved PARP and caspase 3, which are hallmarks of cell apoptosis. However, no cleaved PARP and caspase 3 were detected in the osimertinib-treated group, indicating that MIHA cells did not undergo apoptosis.
[0063] like Figure 4 As shown in the results, compared with the osimertinib alone group, the survival rate of the group treated with osimertinib in combination with the apoptosis inhibitor Z-VAD-FMK (Z-VAD), the cell necrosis inhibitor Necrostatin (Necro-1), the para-apoptosis inhibitor cycloheximide (CHX), and the autophagy activator Rapamycin (Rapa) was not improved, indicating that the cell death caused by osimertinib was not caused by apoptosis, necrosis or para-apoptosis.
[0064] joint Figure 1 Cell viability assay showed that when the concentration of osimertinib reached 8 μM, half of the cells died.
[0065] These results indicate that osimertinib-induced hepatotoxicity is not caused by apoptosis, necrosis, or paraapoptosis.
[0066] Example 3 1. Reagents and Materials The reagents and materials were the same as those in Example 1. The transmission electron microscope was a Spirit 120 kV electron microscope, and the confocal laser scanning microscope was a Leica TCS-SP8.
[0067] 2. Experimental Design The cell administration method is the same as in Example 2.
[0068] Cell photography and data statistics: Cell morphology was photographed under a normal light microscope, and the number and radius of vacuoles were counted using Image J.
[0069] Sample preparation and imaging for electron microscopy: After harvesting cells, fix them with 2.5% glutaraldehyde solution and then post-fix them with 1% OsO₄ solution. After staining with uranyl acetate and lead citrate solution, the specimens were cut into slides and imaged under an electron microscope.
[0070] Cell transfection and fluorescence imaging: MIHA cells were plated at an appropriate density in confocal microplates one day in advance. LC3-GFP and BFP-LAMP1 plasmids were co-transfected into MIHA cells using lipofection. Upon protein expression, LC3 localized to autophagolysosomes, and LAMP1 to lysosomes. Twenty-four hours after protein expression, cells were treated with 4 μM osimertinib for 12 hours, and fluorescence distribution was imaged using a confocal laser scanning microscope.
[0071] Extraction of cell protein: The protein extraction method is the same as that in Example 1.
[0072] 3. Experimental results like Figure 5 As shown, treatment with MIHA-osimertinib induced the formation of vacuoles in cells, and the size and diameter of these vacuoles increased in a concentration-dependent manner.
[0073] like Figure 6 As shown in Figure 2, electron microscopy results show that the vacuolar structures induced by osimertinib in MIHA cells are autolysosome-like vacuoles. The blue arrow indicates the lysosome, and the red arrow indicates the autolysosome.
[0074] like Figure 7 As shown, LC3-GFP-labeled autolysosomes co-localize with BFP-LAMP1-labeled lysosomes and vacuoles. The white arrows indicate the fluorescent signal, and the black arrows indicate the co-localization of the fluorescent signal with the vacuoles.
[0075] like Figure 8 As shown in the figure, treatment with MIHA osimertinib induces the activation of autophagy pathway proteins p62 and LC3 in a concentration-dependent manner.
[0076] These results indicate that osimertinib can induce cell death by stimulating the production of cytoplasmic vacuoles in normal human hepatocytes MIHA. This vacuolar cell death is associated with the generation of lysosomes and autophagolysosomes.
[0077] Example 4 1. Reagents and Materials Bafilomycin A1 was purchased from Taoshu Biological Company, item number: T6740; hydroxychloroquine was purchased from Taoshu Biological Company, item number: T9287. Other reagents and materials were the same as those in Example 1.
[0078] 2. Experimental Design Cell Administration: MIHA cells were plated at an appropriate concentration in 12-well plates and allowed to adhere. MIHA cells were treated with osimertinib alone or co-treated with autophagy inhibitors. Bafilomycin A1 and hydroxychloroquine, a clinically approved drug, were used as the autophagy inhibitors. Bafilomycin A1 is a late autophagy inhibitor that inhibits lysosomal acidification, thereby inhibiting autophagosome-lysosome fusion. Upon administration, the inhibition of autophagosome-lysosome fusion prevents autophagosome degradation, leading to an increase in the autophagosome marker LC3B-II and the autophagy substrate p62. Drug treatment was administered in addition to bafilomycin A1. If the drug activated autophagy, LC3B-II and p62 levels would increase despite bafilomycin A1 treatment. If the drug inhibited or had no effect on autophagy, LC3B-II and p62 levels would remain unchanged despite bafilomycin A1 treatment. Like bafilomycin A1, hydroxychloroquine increases the pH of lysosomes and inhibits the degradation of autophagosomes after entering lysosomes.
[0079] Bafilomycin A1 was administered as follows: MIHA cells were pre-treated with bafilomycin A1 to a final concentration of 50 nM. After 2 hours of treatment, the medium was replaced with fresh medium and osimertinib was administered simultaneously to a final concentration of 2 μM. After 16 hours of treatment, the next step of the experiment was performed.
[0080] The administration of hydroxychloroquine was as follows: MIHA cells were treated with osimertinib alone or co-treated with hydroxychloroquine to a final concentration of 2 μM osimertinib and 25 μM hydroxychloroquine. After 16 hours of treatment, the next step of the experiment was performed.
[0081] Cell photography and data statistics: same as in Example 3.
[0082] Extraction of cell protein: The protein extraction method is the same as that in Example 1.
[0083] Cell activity detection: same as Example 2.
[0084] 3. Experimental results like Figure 9 As shown in Figure 3, p62 and LC3 protein expression levels were higher in the group co-treated with bafilomycin A1 and osimertinib compared to the group treated with osimertinib alone, indicating that lysosomal degradation function was not impaired. The accumulation of p62 and LC3 proteins is caused by the activation of autophagy.
[0085] like Figure 10 As shown in Figure 3, the survival rate of cells in the group co-treated with bafilomycin A1 and osimertinib was higher than that in the group treated with osimertinib alone.
[0086] The above results indicate that osimertinib induces MIHA cell death in human hepatocytes by activating autophagy, and inhibiting autophagy can alleviate the hepatocellular toxicity caused by osimertinib.
[0087] Example 5 1. Animal experiments Twenty male C57BL / 6 mice were randomly divided into four groups: blank control group, hydroxychloroquine group (HCQ group), osimertinib (OSI) group, and OSI+HCQ group, with 5 mice in each group.
[0088] 2. Experimental Design like Figure 11 As shown, OSI was administered at a dose of 50 mg / kg / day via oral gavage, while HCQ was administered at a dose of 10 mg / kg / day via intraperitoneal injection. Mice in each group were treated according to the above-mentioned groupings. Serum and liver tissue were collected 21 days later for pathological analysis.
[0089] 3. Experimental results like Figure 12 As shown in the hematoxylin-eosin staining, necrosis is manifested by nuclear condensation, nuclear rupture or dissolution, loss of cell tissue outlines, tissue vacuolation, and accompanied by apoptosis and immune cell infiltration. Compared with the blank control group, the area of necrosis in the liver tissue of mice in the OSI group was significantly increased, while the area of necrosis in the liver tissue of mice treated with HCQ was significantly reduced.
[0090] like Figure 13 As shown, compared with the blank control group, the concentrations of ALT and AST in the serum of mice in the OSI group were increased, and the concentrations of ALT and AST in the serum of mice in the HCQ treatment group were decreased.
[0091] These results indicate that hydroxychloroquine can treat osimertinib-induced hepatotoxicity in vivo.
[0092] Example 6 1. Reagents and Materials NCI-H1975 was purchased from Zhejiang Meisen Cell Technology Co., Ltd. The remaining reagents and materials were the same as those in Example 1.
[0093] 2. Experimental methods Cellular administration: Lung cancer cells NCI-H1975 were seeded at an appropriate density in 96-well plates and cultured overnight to allow attachment. Osimertinib was dissolved in dimethyl sulfoxide (DMSO) to a 16 mM stock solution, and the drug solution was prepared and diluted 2-fold to a final administration concentration of 0.01 μM to 10 μM. In combination with hydroxychloroquine, the final hydroxychloroquine concentration was 25 μM. After treating the cells for 48 hours, CCK-8 reagent was added at a ratio of 1:100. After incubation at 37°C for 1.5 hours, the absorbance was measured at 450 nm.
[0094] The calculation of survival rate is the same as in Example 1.
[0095] 3. Experimental results Depend on Figure 14 As shown in the data, lung cancer cells NCI-H1975 are sensitive to osimertinib, and the combined use of hydroxychloroquine does not affect the anti-tumor effect of osimertinib.
[0096] Example 7 1. Reagents and Materials S-adenosyl-L-methionine (SAM), Tiopronin (Tio), Glycyrrhizic acid (GA), Silymarin (Sily), and N-Acetyl-L-cysteine (NAC) were purchased from Taoshu Biological Company with catalog numbers T7475, T1630, T2741, T6670, and T0875, respectively. The remaining reagents and materials were the same as in Example 1.
[0097] 2. Experimental methods Cell administration: MIHA were seeded at an appropriate density in 96-well plates and cultured overnight to allow attachment. After dissolving osimertinib in dimethyl sulfoxide (DMSO) to a 16 mM stock solution, the drug solution was prepared and diluted 2-fold to a final dosing concentration of 0.01 μM to 10 μM. For co-administration with other drugs, the final concentration of all drugs was 5 μM. After treating the cells for 48 hours, CCK-8 reagent was added at a 1:100 ratio. After incubation at 37°C for 1.5 hours, the absorbance was measured at 450 nm.
[0098] The calculation of survival rate is the same as in Example 1.
[0099] Cell photography and data statistics: same as in Example 3.
[0100] Extraction of cell protein: The protein extraction method is the same as that in Example 1.
[0101] 3. Experimental results Depend on Figure 15 As shown in the data, compared with the osimertinib monotherapy group, the survival rate of MIHA was higher in the SAM and osimertinib co-administration group, and there was no significant difference in the survival rate of MIHA in the Tio, GA, Sily, NAC and osimertinib co-administration group compared with the osimertinib monotherapy group.
[0102] like Figure 16 As shown in the figure, compared with the osimertinib single-administration group, the vacuoles in MIHA were greatly reduced in the SAM and osimertinib co-administration group.
[0103] like Figure 17 As shown in the figure, compared with the group treated with osimertinib alone, the expression levels of LAMP1 and LC3 proteins in the SAM and osimertinib co-treatment group were reduced, indicating that SAM alleviated the autophagy pathway activation caused by osimertinib.
[0104] These results indicate that SAM has a protective effect on osimertinib-induced liver injury.
[0105] Example 8 1. Animal experiments Twenty male C57BL / 6 mice, aged 6-8 weeks, were randomly divided into 4 groups: blank control group, SAM group, osimertinib (OSI) group, and OSI+SAM group, with 5 mice in each group.
[0106] 3. Experimental Design like Figure 18 As shown, the OSI dose was 50 mg / kg / day, administered by oral gavage; the SAM dose was 25 mg / kg / day, administered by intraperitoneal injection. Mice in each group were treated with the drugs according to the above groupings. Serum and liver tissue were collected 21 days later for pathological analysis.
[0107] 3. Experimental results like Figure 19 As shown, compared with the blank control group, the necrotic area in the liver tissue of the mice in the OSI group was significantly increased, and the necrotic area in the liver tissue of the mice in the SAM+OSI combination group was significantly decreased.
[0108] like Figure 20 As shown in the figure, compared with the blank control group, the concentrations of ALT and AST in the serum of mice in the OSI group were increased, and the concentrations of ALT and AST in the serum of mice in the SAM+OSI treatment group were decreased.
[0109] These results indicate that SAM can treat osimertinib-induced hepatotoxicity in vivo.
[0110] Example 9 1. Reagents and Materials The reagents and materials are the same as in Examples 1 and 7.
[0111] 2. Experimental Design Cellular administration: NCI-1975 cells were seeded at an appropriate density in 96-well plates and cultured overnight to allow attachment. Osimertinib was dissolved in dimethyl sulfoxide (DMSO) to a 16 mM stock solution. The drug solution was prepared and diluted 2-fold to a final dosing concentration of 0.013 μM. For the drug-drug combination group, the final SAM concentration was 5 μM. After 48 hours of treatment, CCK-8 reagent was added at a 1:100 ratio. After incubation at 37°C for 1.5 hours, absorbance was measured at 450 nm.
[0112] 3. Experimental results like Figure 21 As shown, the survival rate of cells in the SAM and osimertinib co-treated group remained unchanged compared with that in the osimertinib alone-treated group.
[0113] The above results indicate that SAM has no effect on the tumor-killing effect of osimertinib.
Claims
1. Use of an autophagy inhibitor in the preparation of a drug for preventing or treating liver toxicity caused by osimertinib, characterized in that: The liver toxic side effects include hepatocellular damage or death caused by osimertinib-induced activation of the hepatocellular autophagy pathway.
2. The use according to claim 1, characterized in that The manifestations of the liver toxic side effects include the generation of intracytoplasmic vacuoles in hepatocytes, and the vacuolar structures are autophagolysosome-like vacuoles.
3. The use according to claim 1, characterized in that The autophagy inhibitor is bafilomycin A1, hydroxychloroquine or S-adenosylmethionine.
4. An anti-tumor combination drug, characterized in that: The invention comprises a first preparation formed by osimertinib and a pharmaceutically acceptable carrier, and a second preparation formed by hydroxychloroquine or S-adenosylmethionine and a pharmaceutically acceptable carrier.
5. The anti-tumor combination drug according to claim 4, characterized in that: The effective dose of hydroxychloroquine in the medicine is 10-15 mg / kg; the effective dose of S-adenosylmethionine is 25-30 mg / kg.
6. The anti-tumor combination drug according to claim 4, characterized in that: The pharmaceutically acceptable carrier includes at least one of a filler, a disintegrant, a lubricant, and a binder.
7. The anti-tumor combination drug according to claim 4, characterized in that: The preparation form of the medicine is an oral preparation.
8. Use of the anti-tumor combination drug according to any one of claims 4 to 7 in the preparation of drugs for treating lung cancer.
9. The use according to claim 8, characterized in that The lung cancer is non-small cell lung cancer.
10. The use according to claim 9, characterized in that The lung cancer is lung adenocarcinoma.