6-(2-hydroxyethyl)benzo[d]thiazol-4-ol in reversing osimertinib resistance

Abstract

The application relates to application of 6-(2-hydroxyethyl)benzo[d]thiazole-4-ol combined with osimertinib in preparation of a drug for treating non-small cell lung cancer. A drug for preventing and / or treating human non-small cell lung cancer is provided, and active ingredients of the drug include 6-(2-hydroxyethyl)benzo[d]thiazole-4-ol (HBT) and osimertinib. The application provides that HBT is used for reversing drug resistance of osimertinib in treatment of non-small cell lung cancer, and can be combined with osimertinib, so that a good treatment effect of non-small cell lung cancer is obtained.

Description

TECHNICAL FIELD

[0001] The application relates to the field of medicine, and particularly relates to application of 6-(2-hydroxyethyl)benzo[d]thiazol-4-ol in reversing osimertinib resistance. BACKGROUND

[0002] Non-small cell lung cancer (NSCLC) is the most common malignant tumor, and its morbidity and mortality are high. More than 60% of NSCLC patients are in the middle or late stage or have metastasis when diagnosed, which brings challenges to the diagnosis and treatment of the patients. Traditional treatment methods include surgical resection, radiotherapy and chemotherapy, but the overall effect is still poor, and the five-year survival rate of the patients is still less than 20%.

[0003] Osimertinib (Osi) is a third-generation epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) drug, and is the first choice for patients with advanced NSCLC carrying EGFR mutations. Although Osi can significantly inhibit the occurrence and development of NSCLC in the early stage, most NSCLC patients develop drug resistance after one year of Osi treatment. The mechanism of Osi resistance can be divided into EGFR-dependent mechanism and EGFR-independent mechanism. At present, different treatment strategies have been developed for the above-mentioned mechanisms of Osi resistance, but the effect is still poor (Schmid S, Li JJN, Leighl NB. Mechanisms of osimertinib resistance and emerging treatment options[J]. Lung Cancer. 2020; 147: 123-129.). Therefore, it is urgent and necessary to find safe and effective drugs to overcome this difficult challenge.

[0004] Our research group previously isolated a benzothiazole derivative, 6-(2-hydroxyethyl)benzo[d]thiazol-4-ol (HBT), from the endophytic fungus Aspergillus sp. 1022LEF. Previous studies have shown that HBT has insecticidal effects (X.L. Yuan, D.L. Zhao, et al. Characterization of a New Insecticidal Benzothiazole Derivative from Aspergillus sp. 1022LEF against the Fall Armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae), J Agric Food Chem, 2024. 72(50):27939-27952). The structure of HBT was disclosed, and it was characterized by spectroscopic methods, including one- and two-dimensional nuclear magnetic resonance data, Hirsch data, and single-crystal X-ray diffraction analysis. Further transcriptomic and proteomic analyses showed that HBT disrupts basic biosynthetic processes by impeding DNA replication and protein synthesis, affecting mitochondria-mediated autophagy, and interfering with hormone synthesis, thereby leading to the death of Drosophila and achieving insecticidal effects.

[0005] Currently, there is no report on the anti-tumor effect of 6-(2-hydroxyethyl)benzo[d]thiazol-4-ol (HBT) and the overcoming of osimertinib resistance in NSCLC.

[0006] Therefore, the present application is proposed. SUMMARY

[0007] Based on this, the purpose of the present application is to provide a new use of 6-(2-hydroxyethyl)benzo[d]thiazol-4-ol (HBT), i.e. to provide a treatment strategy for NSCLC patients with osimertinib acquired resistance and application, so as to provide a new treatment method for NSCLC cells with osimertinib acquired resistance.

[0008] The technical solutions achieving the above-mentioned purposes include the following.

[0009] The first aspect of the present application provides the use of 6-(2-hydroxyethyl)benzo[d]thiazol-4-ol (HBT) in the preparation of a drug for reversing osimertinib resistance in non-small cell lung cancer.

[0010] The first aspect of the present application provides the use of 6-(2-hydroxyethyl)benzo[d]thiazol-4-ol (HBT) in the preparation of a drug for reversing osimertinib resistance in non-small cell lung cancer.

[0011] The third aspect of the present application provides a medicine for preventing and / or treating non-small cell lung cancer in human, wherein the active ingredients of the medicine comprise 6-(2-hydroxyethyl)benzo[d]thiazole-4-ol (HBT) and osimertinib, and the 6-(2-hydroxyethyl)benzo[d]thiazole-4-ol and the osimertinib are respectively independent administration units, or the 6-(2-hydroxyethyl)benzo[d]thiazole-4-ol (HBT) and the osimertinib jointly form a combined administration unit.

[0012] In some embodiments, the non-small cell lung cancer is osimertinib-resistant non-small cell lung cancer.

[0013] In some embodiments, the osimertinib resistance is osimertinib-acquired resistance.

[0014] The present application is proved by experiments that 6-(2-hydroxyethyl)benzo[d]thiazole-4-ol (HBT) can significantly inhibit the proliferation and clonal formation of human lung adenocarcinoma PC9 / OR (osimertinib resistance) cells. In vivo experiments prove that HBT can significantly inhibit the growth of subcutaneous transplanted tumors of PC9-OR cells. The present application provides 6-(2-hydroxyethyl)benzo[d]thiazole-4-ol (HBT) for reversing the drug resistance of osimertinib in the treatment of non-small cell lung cancer, which can be combined with osimertinib to obtain a good treatment effect for non-small cell lung cancer. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 For the construction of PC9-OR cells, the CCK-8 method is used to detect the IC50 of osimertinib on PC9 / PC9-OR cells, and it is proved that the PC9-OR cells are successfully constructed.

[0016] Figure 2 For the effect of HBT on the viability of PC9 / OR cells, wherein A is the chemical structural formula of HBT, B is the result of CCK-8, D is the live and dead staining structure of HBT, and E is the clonal formation experiment of HBT.

[0017] Figure 3 For the effect of HBT on the subcutaneous transplanted tumors of PC9-OR cells, wherein A is the growth curve of the tumor, B is the growth curve of the body weight of the mouse, C is the tumor weight statistical result and tumor picture of the mouse, and D is the result of H&E staining for detecting the necrosis of the tumor and immunohistochemical detection of the expression level of the proliferation index Ki67 of the tumor cells.

[0018] Figure 4This diagram illustrates the good safety profile of HBT in tumor-bearing mice. In the diagram, A shows the H&E staining of the major organs of the mice, B shows the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl transferase (γ-GTP) in the liver of the mice, and C shows the levels of uric acid (UREA), creatinine (Gr), and uric acid (UA) in the mice. Detailed Implementation

[0019] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0020] Unless otherwise specified, experimental methods in the following examples were performed under standard conditions, such as those described in the fourth edition of *Molecular Cloning: A Laboratory Manual*, edited by Green and Sambrook, published in 2013, or according to the manufacturer's recommendations. All commonly used chemical reagents used in the examples are commercially available products.

[0021] Unless otherwise defined, all technical and scientific terms used in this invention are consistent with those belonging to this invention.

[0022] The terms used herein are generally understood to have the same meaning as those skilled in the art. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0023] To facilitate understanding of this technology, some terms and phrases are defined below.

[0024] HBT: 6-(2-hydroxyethyl)benzo[d]thiazol-4-ol.

[0025] IC50: half maximal inhibitory concentration. In this invention, the half-maximal inhibitory concentration (IC50) of osimertinib on PC9 cells or PC9 / OR is calculated based on the inhibition rate of osimertinib gradient concentrations on PC9 cells or PC9 / OR.

[0026] Drug resistance is the phenomenon where pathogens (such as bacteria, viruses, fungi, and parasites) or tumor cells become less sensitive to drugs or even completely lose their effectiveness, leading to decreased or failed treatment outcomes. In this invention, osimertinib is indicated as having poor or ineffective treatment efficacy for non-small cell lung cancer.

[0027] Acquired drug resistance (ADR) refers to the phenomenon where pathogens (such as bacteria, viruses, fungi, or tumor cells) that were originally sensitive to antimicrobial drugs develop resistance to the drug through gene mutation or the acquisition of exogenous drug resistance genes after exposure, resulting in decreased or ineffective treatment. In this invention, osimertinib acquired resistance refers to the development of resistance to osimertinib in patients after a period of treatment.

[0028] In this invention, the cell viability of PC9 / OR cells treated with different concentrations of HBT for 48 h was determined using a CCK-8 assay. The results showed that HBT effectively inhibited cell viability in a dose-dependent manner, with an IC50 value of 7.31 μM. Simultaneously, treatment of PC9 / OR cells with 1.25, 2.5, and 5 μM HBT for 24 h, as confirmed by live / dead cell staining experiments, demonstrated that HBT inhibited PC9-OR cell proliferation, and the same results were observed in clonogenic assays. The construction of a subcutaneous xenograft model of PC9-OR cells confirmed that HBT at doses of 1.25 mg / kg and 2.5 mg / kg significantly inhibited the growth of subcutaneous xenografts of PC9-OR cells, with significantly higher efficacy than osimertinib.

[0029] The present invention will be further described in detail below with reference to specific embodiments.

[0030] Material

[0031] Materials: RPMI 1640 medium was purchased from Gibco, USA; fetal bovine serum was purchased from Ecosai Biotechnology, China; penicillin-streptomycin (C0222) and osimertinib (B1104) were purchased from APExBIO Biotechnology, USA; DCFH-DA (HY-D0940) was purchased from MCE Biotechnology, USA; CCK-8 kit (C0005) was purchased from TargetMol, USA; Calcein-AM / PI dye (KGA511) was purchased from KGI Biotechnology, China.

[0032] The preparation method and verification of 6-(2-hydroxyethyl)benzo[d]thiazol-4-ol (HBT) used in the embodiments of the present invention are in accordance with the reference: (XL Yuan, DL Zhao, et al. Characterization of a New Insecticidal Benzothiazole Derivative from Aspergillus sp. 1022LEF against the Fall Armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae), J AgricFood Chem, 2024. 72(50):27939-27952).

[0033] The structural formula of the prepared 6-(2-hydroxyethyl)benzo[d]thiazol-4-ol (HBT) is as follows, and it was used in the following experiments:

[0034] .

[0035] Example 1: Establishment and validation of an NSCLC osimertinib acquired resistance cell model

[0036] Cell culture: The human non-small cell lung cancer cell line PC9 (carrying an EGFR exon 19 deletion mutation) was purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. PC9 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum, 1% penicillin and streptomycin at 37°C in a 5% CO2 incubator.

[0037] Modeling: Using osimertinib-sensitive PC9 cells, an osimertinib-acquired resistance cell line PC9 / OR was established using a drug concentration escalation method.

[0038] PC9 cells in the logarithmic growth phase were treated with 0–5 μM osimertinib in incremental concentrations until the cells could grow stably in a culture medium containing 5 μM osimertinib, which were then known as PC9 / OR cells.

[0039] verify

[0040] Cell viability was assessed using the CCK-8 assay. Specifically, PC9 / OR cells in logarithmic growth phase were seeded in 96-well plates (5 × 10⁶ cells / wells). 3Cells / well were incubated for 24 hours. Different concentrations of osimertinib (0, 1.25, 2.5, 5, 10, 20 μM) were added afterward. The cells were treated at 37℃ and 5% CO2 for 48 hours, followed by incubation with 10% CCK-8 solution for another 1 hour. The absorbance at 450 nm was measured using a microplate reader, and the half-maximal inhibitory concentration (IC50) was calculated. Cell viability (%) = (experimental group absorbance - blank group absorbance) / (control group absorbance - blank group absorbance). The results are shown below. Figure 1 As shown.

[0041] Figure 1 Analysis of the CCK-8 assay results showed that osimertinib had IC50 values ​​of 0.4045 μM for PC9 and 8.073 μM for PC9 / OR.

[0042] Example 2: Determination of the effect of HBT on PC9 / OR cell viability

[0043] Cell viability was assessed using the CCK-8 assay. Specifically, PC9 / OR cells in logarithmic growth phase were seeded in 96-well plates (5 × 10⁶ cells / wells). 3 Cells / well were incubated for 24 hours. Different concentrations of HBT (0.625, 1.25, 2.5, 5, 10, 20 μM) were added, and the cells were treated at 37℃ and 5% CO2 for 48 hours. Then, 10% CCK-8 solution was added and the cells were incubated for another 1 hour. The absorbance at 450 nm was measured with a microplate reader, and the half maximal inhibitory concentration (IC50) was calculated. Cell viability (%) = (absorbance of experimental group - absorbance of blank group) / (absorbance of control group - absorbance of blank group). Figure 2 In B, the analysis of CCK-8 assay results showed that the IC50 value of HBT for PC9 / OR was 7.31 μM, and it exhibited a significant dose-dependent effect.

[0044] The effect of HBT on PC9-OR cell proliferation was detected using the Calcein-AM / PI staining method. Specifically, PC9 / OR cells in the logarithmic growth phase were seeded in 96-well plates (3 × 10⁻⁶ cells / wells). 5 Cells were cultured in wells (1 cell / well) for 24 hours, and then different concentrations of HBT (1.25, 2.5, 5.0 μM) were added. Cells were treated at 37°C and 5% CO2 for 48 hours. Calcein-AM and PI dye were then added, and the cells were incubated for 15 minutes. Cell viability was observed under a fluorescence microscope. PI-labeled (red fluorescence) cells were dead cells, and Calcein-AM-labeled (green fluorescence) cells were live cells. Figure 2As shown in D, HBT can significantly inhibit the proliferation of PC9-OR cells.

[0045] Cell viability was assessed using a plate colony formation assay. Specifically, PC9 / OR cells in logarithmic growth phase were seeded in 6-well plates (1×10⁶ cells / wells). 3 Cells were cultured at 100 cells / well for 24 hours until adherence. Different concentrations of HBT were then added, and the cells were treated at 37°C and 5% CO2 for 48 hours. The culture medium was then replaced with a drug-free medium, and the cells were cultured for another 7 days. Cells were fixed with 4% paraformaldehyde and stained with crystal violet. The results are shown below. Figure 2 As shown in E, HBT treatment of PC9 / OR cells for 48 h significantly inhibited cell colony formation.

[0046] Example 3: HBT assay for PC9 / OR cell subcutaneous xenografts

[0047] The effect of HBT on overcoming osimertinib resistance was evaluated using a PC9-OR cell subcutaneous xenograft model.

[0048] Specifically, logarithmic growth phase PC9 / OR cells were seeded into BALB / c-nu mice (5×10⁻⁶ cells / year). 6 (Number of cells / mouse) After tumor formation, tumor-bearing mice were randomly divided into four groups: solvent group (saline group), osimertinib 5 mg / kg group, HBT 1.25 mg / kg group, and HBT 2.5 mg / kg group, with 5 mice in each group. The mice were administered the medication intraperitoneally every other day, and the length and short diameter of the tumor and body weight were monitored. Eighteen days after administration, the mice were anesthetized, and the tumors were harvested, weighed, and photographed. Tumor volume (mm²) 3 = 0.5*a*a*b (a is the long diameter of the tumor, b is the short diameter of the tumor). H&E staining was used to detect the necrotic area of ​​the tumor, and immunohistochemistry was used to detect the expression level of the tumor cell proliferation index Ki67.

[0049] See the experimental results. Figure 3 . Figure 3 A and Figure 3 Both B-cell assays showed that HBT significantly inhibited the growth of subcutaneous xenografts of PC9-OR cells. Figure 3 The results of C confirmed that HBT had no significant effect on the body weight of mice at doses of 1.25 mg / kg and 2.5 mg / kg. Figure 3 Results showed that HBT at doses of 1.25 mg / kg and 2.5 mg / kg could increase the area of ​​tumor necrosis and inhibit the expression of the tumor cell proliferation index Ki67.

[0050] Safety testing of HBT: 18 days after administration, mice were tested for alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl transferase isoenzyme (γ-GTP), as well as uric acid (UREA), creatinine (Gr), and uric acid (UA).

[0051] HBT has good safety in tumor-bearing mice; please refer to [link / reference]. Figure 4 .in, Figure 4 A shows the results of H&E staining of the major organs of the mouse. Figure 4 B represents the detection levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl transferase isoenzyme (γ-GTP) in the liver of mice. Figure 4 C represents the levels of urea acid (UREA), creatinine (Gr), and uric acid (UA) in mice. The above results indicate that HBT at doses of 1.25 mg / kg and 2.5 mg / kg showed no significant toxicity to the major organs of mice, demonstrating good safety.

[0052] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. Use of 6-(2-hydroxyethyl)benzo[d]thiazol-4-ol (HBT) in the preparation of a drug for reversing osimertinib resistance of non-small cell lung cancer.

2. Use of 6-(2-hydroxyethyl)benzo[d]thiazol-4-ol combined with osimertinib in the preparation of a drug for treating non-small cell lung cancer.

3. The use according to claim 2, wherein, The non-small cell lung cancer is osimertinib-resistant non-small cell lung cancer.

4. Use according to claim 3, wherein, The osimertinib resistance is osimertinib-acquired resistance.

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