A drug targeting the SQLE gene or protein for treating crizotinib-induced liver toxicity

Abstract

The present invention discloses a drug for treating crizotinib-induced liver toxicity by targeting the SQLE gene or protein, belonging to the field of pharmaceutical technology. The drug reverses crizotinib-induced liver toxicity by downregulating the expression of the SQLE gene or the accumulation of SQLE protein. By downregulating SQLE, the present invention can effectively reverse crizotinib-induced apoptosis of hepatocytes, revealing that the SQLE gene is a key gene for crizotinib-induced liver injury and providing a new preventive and therapeutic target for intervening in crizotinib-induced hepatotoxicity. The present invention proposes that SQLE can be degraded through the autophagy pathway, providing a new direction for currently searching for intervention strategies for drug-induced hepatotoxicity and to a certain extent solving the current situation of few clinically available intervention drugs and single mechanisms. The intervention drug reverses crizotinib-induced liver toxicity by downregulating SQLE, expanding the clinical application value of crizotinib.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly to the application of SQLE gene or SQLE protein as a target in the preparation of drugs for treating crizotinib-induced liver toxicity. Background Art

[0002] Crizotinib (PF-02341066) is an oral competitive tyrosine kinase inhibitor, and its targets include recombinant ALK, abnormally amplified c-MET, and recombinant ROS1. It was approved by the US FDA in 2011 for the treatment of locally advanced non-small cell lung cancer with EML4-ALK fusion gene mutation and advanced non-small cell lung cancer with positive ROS1 progression.

[0003] Although crizotinib shows good anti-tumor efficacy in clinical applications, its serious liver toxicity has attracted increasing attention. According to the FDA report, after taking crizotinib for treatment, the incidences of the elevation of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) by more than five times in patients are 11.2% and 5.7% respectively, and even 2 patients died due to severe liver toxicity. Currently, the intervention strategy for its liver toxicity is to stop or reduce the drug dosage. However, there are also clinical reports showing that a 62-year-old female patient showed obvious symptoms of acute liver failure 24 days after receiving crizotinib. Even after taking the drug withdrawal strategy and supplemented with adjuvant treatment, the patient still died at 40 days (van Geel RM, et al., Crizotinib-induced fatal fulminant liver failure. Lung Cancer, 2016.93: p.17-9.).

[0004] Therefore, it is an urgent need at present to provide new preventive and therapeutic targets for crizotinib-induced liver toxicity.

[0005] Squalene epoxidase (SQLE) catalyzes the epoxidation of the carbon-carbon double bond of squalene in the cholesterol synthesis pathway to produce 2,3-oxidosqualene, and is considered to be the rate-limiting enzyme of sterol biosynthesis. There are reports showing that SQLE can be used as a new and effective target for the treatment of non-alcoholic fatty liver disease. Its targeted therapy can bring significant benefits to non-alcoholic steatohepatitis (NASH), and at the same time, this protein can also be used as a serum biomarker for screening and assisting in the diagnosis of NASH. However, there is no literature report on the relationship between SQLE protein and crizotinib-induced liver toxicity, and further research is needed.

[0006] Autophagy activators can reduce the accumulation of abnormal functional proteins under drug action by activating the autophagy degradation pathway, thereby reducing the killing effect of drugs on cells. Some studies have reported that autophagy activators have a certain protective effect on hepatocyte apoptosis (Chen Y, et al. Dihydromyricetin protects against liver ischemia / reperfusion induced apoptosis via activation of FOXO3a-mediated autophagy. Oncotarget, 2016 Nov 22; 7(47): 76508-76522).

[0007] However, there is no report on the treatment of SQLE-related diseases or injuries with autophagy activators. Summary of the Invention

[0008] The object of the present invention is to explore genes / proteins related to crizotinib-induced liver toxicity, and use them as action targets for preventing and treating crizotinib liver toxicity, screening drugs for treating crizotinib liver toxicity side effects, solving the liver toxic side effects of crizotinib use, and expanding the clinical application value of crizotinib.

[0009] To achieve the above object, the present invention adopts the following technical solutions:

[0010] The present invention provides the use of the SQLE gene or SQLE protein as a target in the preparation of drugs for treating crizotinib liver toxicity, and the drugs down-regulate the expression of the SQLE gene or the accumulation of the SQLE protein. The nucleotide sequence of the SQLE gene is shown in SEQ ID No. 1, and the amino acid sequence of the SQLE protein is shown in SEQ ID No. 2.

[0011] The present invention studies and finds that crizotinib can inhibit the degradation of SQLE protein in hepatocytes, resulting in the accumulation of SQLE protein in hepatocytes, and both the action of crizotinib and overexpression of SQLE will aggravate the apoptosis of hepatocytes, suggesting that the accumulation of SQLE protein is the key cause of crizotinib-induced liver toxicity, and SQLE may be a potential target for preventing or treating crizotinib-induced liver toxicity. Therefore, inhibiting the expression of the SQLE gene / protein can be used as a means to intervene in crizotinib liver toxicity.

[0012] In the research of the present invention, RNA interference technology was used to down-regulate the expression of SQLE in hepatocytes, thereby reversing the apoptosis of hepatocytes induced by crizotinib. Specifically, after using siRNA, the apoptosis of hepatocytes induced by crizotinib was reduced, and the apoptosis-related protein cleaved-PARP was significantly down-regulated. Therefore, the present invention provides a new therapeutic target for intervening in the hepatotoxicity caused by crizotinib. Developing a corresponding pharmaceutical preparation targeting the SQLE gene to knockdown the expression of this gene, the drug achieves the purpose of treating the hepatotoxic side effects caused by crizotinib by down-regulating the expression of the SQLE gene.

[0013] Preferably, the drug contains siRNA targeting the SQLE gene. The siRNA targeting the SQLE gene can inhibit the expression of SQLE, thereby reversing the liver toxicity induced by crizotinib.

[0014] Specifically, the nucleotide sequence of the siRNA is: 5’-AACAUGAUAACCACCCGGCTT-3’.

[0015] The present invention also provides the application of the SQLE gene or SQLE protein as a target in screening drugs for treating crizotinib-induced liver toxicity.

[0016] Specifically, using a cell or animal model to screen drugs that promote the degradation of SQLE, and evaluating the activity of the drug to be tested by measuring the expression level of SQLE protein.

[0017] The research of the present invention found that co-administering an autophagy activator can significantly down-regulate the accumulation of SQLE caused by crizotinib and reverse the apoptosis of hepatocytes induced by crizotinib. Therefore, the present invention provides a new use of autophagy activators in intervening in the hepatotoxicity caused by crizotinib. The drug achieves the purpose of treating the hepatotoxic side effects caused by crizotinib by reducing the accumulation of SQLE protein.

[0018] Another object of the present invention is to provide the application of autophagy activators in the preparation of drugs for treating diseases or injuries caused by abnormal expression of SQLE protein.

[0019] The present invention has been proved by research that autophagy activators can significantly reduce the accumulation of SQLE protein in hepatocytes caused by crizotinib, indicating that the autophagy pathway can be used as a key pathway for regulating the degradation of SQLE protein. Therefore, autophagy activators can be used to treat diseases or injuries caused by abnormal expression of SQLE protein.

[0020] Furthermore, the disease or injury is a liver disease caused by the accumulation of SQLE protein.

[0021] Furthermore, the disease or injury is crizotinib-induced liver toxicity reaction. The autophagy activator can down-regulate the accumulation of SQLE protein caused by crizotinib by combining with crizotinib.

[0022] The autophagy activator includes but is not limited to: rapamycin, metformin or a pharmaceutically acceptable salt thereof.

[0023] Further, the drug also includes a pharmaceutically acceptable excipient.

[0024] The dosage form of the drug can be an oral solid preparation, an oral liquid preparation, an injection, a lyophilized powder injection, a large volume infusion, a patch, an ointment, a gel, a soft capsule or a suppository.

[0025] The present invention also provides an anti-tumor combined drug composition, including a first preparation with crizotinib as an active ingredient and a second preparation with an autophagy activator as an active ingredient.

[0026] The drug composition is used for the combined medication for treating non-small cell lung cancer, especially for preparing a drug for treating locally advanced non-small cell lung cancer with EML4-ALK fusion gene mutation or advanced non-small cell lung cancer with ROS1 positive progression.

[0027] The beneficial effects of the present invention are as follows:

[0028] (1) The present invention provides the application of SQLE gene or SQLE protein as a target in the preparation of a drug for treating crizotinib-induced liver toxicity. Specifically, the present invention provides siRNA targeting the SQLE gene or an autophagy activator targeting the SQLE protein as a drug for reversing crizotinib-induced liver toxicity. The drug reverses crizotinib-induced liver toxicity by down-regulating the expression of the SQLE gene or the accumulation of the SQLE protein.

[0029] (2) The present invention finds that down-regulating SQLE can effectively reverse crizotinib-induced apoptosis of hepatocytes, revealing that the SQLE gene is a key gene for crizotinib-induced liver injury and providing a new prevention and treatment target for intervening in crizotinib-induced liver toxicity. Clinically, siRNA molecules targeting SQLE can play an important role in the treatment and intervention of crizotinib-induced liver toxicity.

[0030] (3) The present invention first proposes that SQLE can be degraded through the autophagy pathway, providing a new direction for currently searching for intervention strategies for drug-induced liver toxicity and solving the current situation of few available intervention drugs and single mechanism in clinical practice to a certain extent. Description of the Drawings

[0031] Figure 1Effects of crizotinib and SQLE overexpression plasmid on SQLE protein and hepatic parenchymal cells HL-7702, where A shows the effects at different crizotinib concentrations, B shows the effects at different crizotinib action times, and C shows the effects of SQLE overexpression plasmid.

[0032] Figure 2 Effect of SQLE gene-specific siRNA on crizotinib-induced apoptosis of hepatic parenchymal cells HL-7702.

[0033] Figure 3 Effect of crizotinib on SQLE transcription in mouse liver and the effect of combined use of protein synthesis inhibitor actinomycin on SQLE protein level, where A is the transcriptional level and B is the protein level.

[0034] Figure 4 Effect of combined use of autophagy activators and inhibitors on SQLE protein expression level, where A is the combined use of chloroquine and B is the combined use of rapamycin.

[0035] Figure 5 Effect of combined use of autophagy activator metformin on SQLE protein expression level.

[0036] Figure 6 Effect of combined use of autophagy activator on crizotinib-induced apoptosis of hepatic parenchymal cells HL-7702, where A is the combined use of rapamycin and B is the combined use of metformin hydrochloride.

[0037] Figure 7 Effect of combined use of metformin hydrochloride on crizotinib-induced slow weight gain and increased liver weight / body weight ratio in mice, where A is weight gain and B is liver weight / body weight ratio.

[0038] Figure 8 Effect of combined use of metformin hydrochloride on crizotinib-induced liver injury in mice.

[0039] Figure 9 Effect of combined use of metformin hydrochloride on crizotinib-induced upregulation of transaminases in mice, where A is ALT and B is AST.

[0040] Figure 10 Effect of combined use of metformin hydrochloride on crizotinib-induced SQLE protein accumulation in mouse liver, where A is the western blot result and B is the quantitative analysis result.

[0041] Figure 11 Effect of combined use of metformin hydrochloride on SQLE protein accumulation detected by immunohistochemical staining of liver sections. Detailed implementation methods

[0042] The present invention will be further described below in conjunction with specific embodiments. The following embodiments 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 belong to the scope of the present invention.

[0043] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0044] The present invention will be further described below in conjunction with specific embodiments. The following embodiments 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 belong to the scope of the present invention.

[0045] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0046] C57BL / 6J mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.; sodium carboxymethyl cellulose (CMC-Na) was purchased from Shanghai Sinopharm Group; human normal liver parenchymal cell line HL-7702 was purchased from Guangzhou Jinio Biotechnology Co., Ltd.; SQLE antibody was purchased from Santa Cruz Biotechnology; β-Actin antibody was purchased from Hangzhou Daige Biotechnology Co., Ltd.; cleaved-PARP antibody was purchased from Hangzhou Huaan Biotechnology Co., Ltd.; chloroquine was purchased from Shanghai Taosu Biochemical Technology Co., Ltd.; rapamycin was purchased from Shanghai Taosu Biochemical Technology Co., Ltd.; siRNA was purchased from Shanghai Jima Pharmaceutical Co., Ltd., the negative control (NC) sense chain sequence was 5'-UUCUCCGAACGUGUCACGUTT-3'; the siSQLE sense chain was 5'-AACAUGAUAACCACCCGGCTT-3'; transfection reagent Purchased from Polyplus Transfection Company.

[0047] Crizotinib, CAS No. 877399-52-5, chemical name 3-[(R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-5-[1-(piperidin-4-yl)-1H-pyrazol-4-yl]pyridin-2-amine, molecular formula C 21 H 22 Cl2FN5O, molecular weight 450.34, purchased from Shanghai Taosu Biochemical Technology Co., Ltd. The structural formula is as follows:

[0048]

[0049] Metformin Hydrochloride, with a CAS number of 1115-70-4, a molecular formula of C4H 12 ClN5, a molecular weight of 165.62, purchased from Shanghai TaoSure BioScience Co., Ltd. The structural formula is as follows:

[0050]

[0051] Example 1

[0052] Human normal hepatic parenchymal cells HL-7702 were seeded in 12-well plates at a density of 80,000 cells / well, and different concentrations of crizotinib (0, 1.5, 3, and 4.5 μM) were applied for 24 h or 3 μM crizotinib was applied for different times (0, 6, 12, and 24 h). The cells were harvested, and proteins were extracted and quantified.

[0053] Human normal hepatic parenchymal cells HL-7702 were seeded in 12-well plates at a density of 150,000 cells / well. After overnight adherent stability, the SQLE overexpression plasmid (original vector: pCDNA3.0-3'FLAG, Gene ID of the SQLE coding sequence: 6713) and the negative control vector were introduced into HL-7702 cells through the jetPRIME transfection reagent. The cells were harvested 24 h later, and proteins were extracted and quantified.

[0054] Protein level detection was performed by western blot, and the results are as Figure 1 shown. Compared with the control group, with the increase of the action concentration and time, crizotinib significantly up-regulated the protein levels of cleaved-PARP and SQLE in the mouse liver, suggesting that crizotinib could induce cell apoptosis and SQLE protein accumulation ( Figure 1 A, B). After overexpressing SQLE in cells, the level of the apoptotic protein cleaved-PARP increased significantly ( Figure 1 C), suggesting that SQLE might be the key protein for crizotinib-induced cell apoptosis.

[0055] Example 2

[0056] Human normal liver parenchymal cells HL-7702 were seeded in 12-well plates at a density of 80,000 cells per well. After overnight adherence and stabilization, siRNA targeting SQLE (sense strand: 5’-AACAUGAUAACCACCCGGCTT-3’) and negative control NC (sense strand: 5’-UUCUCCGAACGUGUCACGUTT-3’) were transfected into HL-7702 cells using jetPRIME transfection reagent. After stable expression, cells were treated with 3 μM crizotinib for 24 h, then harvested, and proteins were extracted and quantified. Subsequently, protein levels were detected by western blot.

[0057] The results were as Figure 2 shown. After knocking down SQLE and then treating with crizotinib, the increase in SQLE protein level caused by the drug could be reversed, and at the same time, the level of apoptotic protein cleaved-PARP decreased, indicating that siRNA targeting SQLE could reduce cell apoptosis by reversing the SQLE protein accumulation caused by crizotinib.

[0058] Example 3

[0059] Twelve male C57BL / 6J mice were randomly divided into 2 groups, namely the control group and the crizotinib group, with 6 mice in each group, and administered by gavage. The dose of crizotinib was 100 mg / kg / day, and the control group was replaced with 0.4% CMC-Na. After continuous administration for 6 weeks, the liver was dissected, and part of the liver tissue was lysed and disrupted. After sufficient lysis of the lysate, the transcriptional level of SQLE in mouse liver tissue was examined.

[0060] Human normal liver parenchymal cells HL-7702 were seeded in 12-well plates at a density of 150,000 cells per well. After overnight adherence and stabilization, 3 μM crizotinib was administered at the same time. After 6 h, the medium was changed, and the protein synthesis inhibitor cycloheximide at a concentration of 10 μg / mL was added for different times (0, 0.5, 1, 2, 4, and 6 h). Cells were harvested, proteins were extracted and quantified, and then protein levels were detected by western blot.

[0061] The results were as Figure 3 shown. There was no significant difference in the transcriptional level of SQLE in mouse liver ( Figure 3 A). Under the action of the protein synthesis inhibitor cycloheximide, it was found that crizotinib could prolong the half-life of SQLE ( Figure 3 B), suggesting that crizotinib could inhibit the degradation of SQLE.

[0062] Example 4

[0063] Human normal liver parenchymal cells HL-7702 were seeded in 12-well plates at a density of 150,000 cells / well. After overnight adherence and stabilization, 6 groups were set up, namely the control group, the crizotinib group, the chloroquine group, the crizotinib + chloroquine group, the rapamycin group, and the crizotinib + rapamycin group. After 24 hours of treatment, the cells were harvested, proteins were extracted and quantified, and then protein levels were detected by western blot.

[0064] The results were as Figure 4 shown that the blockade of lysosomal function by chloroquine aggravated the crizotinib-induced SQLE protein accumulation ( Figure 4 A), while the autophagy activator rapamycin could down-regulate the crizotinib-induced SQLE protein accumulation ( Figure 4 B).

[0065] Example 5

[0066] Human normal liver parenchymal cells HL-7702 were seeded in 12-well plates at a density of 150,000 cells / well. After overnight adherence and stabilization, 4 groups were set up, namely the control group, the crizotinib group, the metformin hydrochloride group, and the crizotinib + metformin hydrochloride group. After 24 hours of treatment, the cells were harvested, proteins were extracted and quantified, and then protein levels were detected by western blot.

[0067] The results were as Figure 5 shown that metformin hydrochloride could down-regulate the crizotinib-induced SQLE protein accumulation.

[0068] As Figure 6 shown, the apoptosis of liver parenchymal cells induced by crizotinib was significantly reversed after the combination of the autophagy activator rapamycin ( Figure 6 A) and metformin hydrochloride ( Figure 6 B).

[0069] Example 6

[0070] I. Twenty male C57BL / 6J mice were randomly divided into 4 groups, namely the control group, the crizotinib group, the metformin hydrochloride group, and the crizotinib + metformin hydrochloride combination group, with 5 mice in each group. The drugs were administered by gavage. The dose of crizotinib was 100 mg / kg / day, and the dose of metformin hydrochloride was 200 mg / kg / day. The control group was replaced with 0.4% CMC-Na, and the drugs were administered continuously for 6 weeks.

[0071] Blood samples were collected by orbital blood sampling to detect the levels of the biochemical markers ALT and AST of liver injury in the serum.

[0072] The livers were dissected, weighed, and the organ coefficients of the mice were examined. The liver tissues were embedded, sectioned, and stained with HE.

[0073] The results were asFigure 7 As shown in Figure A, the weight gains of the control group, crizotinib group, metformin hydrochloride group, and combination group were 7.44 ± 0.33%, 4.24 ± 0.82%, 7.62 ± 0.38%, and 6.06 ± 0.59% respectively ( Figure 7 A).

[0074] The liver organ coefficients (LW / BW) were 4.30 ± 0.19%, 5.27 ± 0.20%, 3.97 ± 0.32%, and 4.63 ± 0.14% respectively ( Figure 7 B).

[0075] The above data indicate that the slow weight gain and up - regulation of organ coefficients in mice caused by crizotinib were significantly reversed after the combination of metformin hydrochloride.

[0076] The results are as Figure 8 shown. HE staining was performed on liver tissue sections. The immune cell infiltration and vacuolar degeneration damage caused by crizotinib alone were significantly improved after the combination of metformin hydrochloride.

[0077] The results are as Figure 9 shown. The liver function markers ALT of the control group, crizotinib group, metformin hydrochloride group, and combination group were 31.8 ± 2.08, 58.2 ± 9.79, 36.6 ± 7.97, and 33.12 ± 2.49 U / L respectively; AST were 113.16 ± 15.34, 173.64 ± 18.07, 127.92 ± 19.75, and 135.72 ± 16.37 U / L respectively, indicating that crizotinib can cause liver function damage in mice, and the liver function damage induced by crizotinib was significantly reversed after the combination of metformin hydrochloride.

[0078] Second, take partial liver tissues of mice in 4 groups, extract proteins and quantify them. Subsequently, the expression level of SQLE protein after drug administration was detected by western blot, and the western blot results were quantitatively analyzed using Image J software.

[0079] The results are as Figure 10 shown. Compared with the control group, crizotinib significantly up - regulated the SQLE protein level in the liver of mice, while the combination of metformin hydrochloride could reverse the SQLE protein accumulation caused by crizotinib.

[0080] Immunohistochemical staining was performed on liver sections to detect the change of SQLE protein level. The increase in SQLE level caused by crizotinib was significantly reversed after the combination of metformin hydrochloride. The results are shown in Figure 11 .

Claims

1. Application of a substance against SQLE a gene or SQLE protein in the preparation of a drug for treating crizotinib-induced liver toxicity, characterized in that The substance targeting SQLE the gene or SQLE protein is an siRNA targeting the SQLE gene or an autophagy activator. The nucleotide sequence of the siRNA is: 5’-AACAUGAUAACCACCCGGCTT- 3’. The autophagy activator is rapamycin, metformin or a pharmaceutically acceptable salt thereof. The drug downregulates SQLE gene expression or SQLE protein accumulation. The SQLE nucleotide sequence of the gene is as shown in SEQ ID No.1, and the amino acid sequence of the SQLE protein is as shown in SEQ ID No.

2. The manifestations of crizotinib-induced liver toxicity include: hepatocyte apoptosis, infiltration of immune cells in liver tissue, and vacuolar degeneration injury of hepatocytes.

2. The application according to claim 1, characterized in that The drug also includes pharmaceutically acceptable excipients.

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