Use of linopter for treating lung squamous cell carcinoma

Abstract

The application discloses a pharmaceutical use of linopteridin and a medicine containing the linopteridin. The use is that the linopteridin is applied to preparing a TBK1 inhibitor, and the TBK1 inhibitor combined with an anti-PD-1 antibody is applied to preparing a medicine for treating lung squamous carcinoma. The medicine is a TBK1 inhibitor containing the linopteridin, and a medicine composition containing the linopteridin and the anti-PD-1 antibody. The linopteridin can directly combine and inhibit the TBK1 kinase activity, and reduce the TBK1 phosphorylation level. The linopteridin combined with the anti-PD-1 antibody can significantly inhibit the lung squamous carcinoma tumor growth and has good safety. The aspect provides a new drug administration approach for treating the lung squamous carcinoma.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more particularly to the pharmaceutical use of saurolophytin and pharmaceuticals containing saurolophytin. Background Technology

[0002] Lung cancer is one of the leading causes of cancer-related morbidity and mortality worldwide, with squamous cell carcinoma accounting for approximately 30% of cases. In recent years, immune checkpoint inhibitors (ICIs), represented by inhibitors of programmed death-1 (PD-1) and its ligand (PD-L1), have become the first-line standard treatment, providing some patients with durable clinical benefits and hope for survival. However, clinical data shows that in unselected squamous cell carcinoma patients, the objective response rate (ORR) of single-agent PD-1 inhibitors is typically only around 20%, meaning that approximately 80% of patients do not initially respond to treatment. Furthermore, some patients with initial responses gradually develop acquired resistance during treatment, leading to disease progression. This primary and acquired resistance is a key scientific issue currently limiting the efficacy of ICIs. Current intervention strategies for PD-1 / PD-L1 treatment resistance mainly include checkpoint combination therapy (such as combination with CTLA-4 inhibitors), chemotherapy, and targeted drugs. However, most clinical trials have been forced to be interrupted or terminated due to problems such as poor efficacy gains from combination therapy, increased toxicity such as immune adverse reactions, and acquired resistance.

[0003] Therefore, exploring effective sensitization strategies to overcome immunotherapy resistance has become a hot topic in current research on squamous cell carcinoma of the lung. TANK-binding kinase 1 (TBK1), a key kinase in the innate immune signaling pathway, not only participates in the regulation of tumor cell survival but also closely relates to the efficacy of immune checkpoint inhibitors by modulating the immunosuppressive state in the tumor microenvironment. As a key molecule connecting innate immunity and inflammatory responses, TBK1 has been shown to participate in regulating tumor-associated macrophage polarization and effector T cell function. However, existing TBK1 synthesis inhibitors are limited in their application in combination immunotherapy due to insufficient selectivity or toxicity issues. Traditional Chinese medicine (TCM) monomers, guided by TCM theory, are widely sourced, structurally diverse, and often possess multi-target regulation and good safety profiles, making them a potential treasure trove for discovering novel TBK1 inhibitors. Currently, there are no reports of TBK1 inhibitors derived from TCM being used in combination with anti-PD-1 antibodies for the treatment of squamous cell carcinoma of the lung.

[0004] Pectolinarigenin (PEC) is a natural flavonoid compound widely found in Guangxi's distinctive traditional medicinal herbs (Gui medicine), such as *Cirsium japonicum* (Zhuang medicine name: Yalinzi) and *Hedyotis diffusa* (Zhuang medicine name: Kehenmo). Its structure is as follows:Figure 1 As shown in Figure A. Existing studies have preliminarily confirmed its direct anti-tumor activity in lung cancer and liver cancer. PEC can affect multiple signaling pathways closely related to tumor growth and survival. Currently, there are no reports of using anchovy flavonoids as a TBK1 inhibitor in combination with anti-PD-1 antibodies for the treatment of squamous cell carcinoma of the lung. Summary of the Invention

[0005] The main objective of this invention is to overcome the shortcomings of the existing technology and provide a new pharmaceutical use for saural flavonoids in the treatment of squamous cell carcinoma of the lung.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides the application of saural flavin in the preparation of TBK1 inhibitors.

[0008] Secondly, the present invention provides the use of a TBK1 inhibitor in combination with an anti-PD-1 antibody in the preparation of a medicament for treating squamous cell carcinoma of the lung, wherein the TBK1 inhibitor comprises saurolophytin.

[0009] In one or more embodiments, the anti-PD-1 antibody is selected from one or more of nivolumab, pembrolizumab, toripalimab, sintilimab, camrelizumab, tislelizumab, or penaplimab.

[0010] Thirdly, the present invention provides a TBK1 inhibitor comprising saurolophytin.

[0011] The TBK1 inhibitor also contains pharmaceutically acceptable excipients.

[0012] Fourthly, the present invention provides a pharmaceutical composition for treating squamous cell carcinoma of the lung, comprising a TBK1 inhibitor and an anti-PD-1 antibody, wherein the TBK1 inhibitor comprises saurolophytin.

[0013] In one or more embodiments, the anti-PD-1 antibody is selected from one or more of nivolumab, pembrolizumab, toripalimab, sintilimab, camrelizumab, tislelizumab, or penaplimab.

[0014] In one or more embodiments, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

[0015] In one or more embodiments, the pharmaceutical composition may be generated as a pharmaceutical kit comprising a TBK1 inhibitor and an anti-PD-1 antibody. In this case, the TBK1 inhibitor is present in a dosage form different from the anti-PD-1 antibody, and the two are packaged separately (i.e., one dosage form contains the TBK1 inhibitor; the other dosage form contains the anti-PD-1 antibody).

[0016] The beneficial effects of this invention are as follows: This invention demonstrates through experiments that scallop flavonoids can directly bind to and inhibit TBK1 kinase activity, thereby reducing TBK1 phosphorylation levels; the combined administration of scallop flavonoids and anti-PD-1 antibodies can significantly inhibit the growth of lung squamous cell carcinoma with good safety, providing a new drug administration route for the treatment of lung squamous cell carcinoma. Attached Figure Description

[0017] Figure 1 To validate the in vitro target of scallopeflavin. A. Chemical structure of scallopeflavin; B. Results of in vitro kinase inhibition experiments with scallopeflavin, including IC50. 50 =0.6309 μM; C. SPR (surface plasmon resonance) experimental results of scutellarin, KD=2.542E-7.

[0018] Figure 2 Western blotting results showing the inhibition of TBK1 phosphorylation and PD-L1 expression in SK-MES-1 cells by piracetam flavonoids. A. Piracetam flavonoids inhibited the expression of p-TBK1 and PD-L1 in a dose-dependent manner; B. Western blotting protein expression statistics, *P<0.05, **P<0.01.

[0019] Figure 3 The results show the effects of scutellarin on the inhibition of SK-MES-1 cell viability at different time points. A. Cell viability results after 6 h of scutellarin administration; B. Cell viability results after 12 h of scutellarin administration; C. Cell viability results after 24 h of scutellarin administration. *P<0.05, **P<0.01, ***P<0.001.

[0020] Figure 4 Results of co-culture experiments of SK-MES-1 cells and Jurkat T cells with different concentrations of saurolophus. *P<0.05,**P<0.01.

[0021] Figure 5 The results are from animal experiments using safflower flavonoids combined with αPD-1 in mice. A. In vivo fluorescence imaging during treatment of a mouse orthotopic lung xenograft model; B. Representative orthotopic tumor images; C. Tumor fluorescence intensity line plot, *P<0.05, **P<0.01, ***P<0.001.

[0022] Figure 6 A. Line graph showing the in vivo safety evaluation results of scallop flavonoids in AE mice; B. Serum biochemical liver function index ALT results; C. Serum biochemical liver function index AST results. Detailed Implementation

[0023] This invention will demonstrate, through multiple experimental examples, the efficacy of saural flavin as a TBK1 inhibitor combined with anti-PD-1 antibody against squamous cell carcinoma of the lung, so as to prove that it can be used to prepare drugs for the treatment of squamous cell carcinoma of the lung.

[0024] Experimental materials and reagents:

[0025] Human lung squamous cell carcinoma SK-MES-1 cells and mouse lung squamous cell carcinoma KLN205 cells (purchased from Zhejiang Baidi Biotechnology Co., Ltd.) were cultured in DMEM medium containing 10% fetal bovine serum, 5% penicillin and streptomycin, and high glucose (all purchased from Thermo Fisher Scientific China); sauerkraut flavonoids (purchased from Shanghai Yuanye Biotechnology Co., Ltd.); purified TBK1 protein (purchased from Thermo Fisher Scientific China); antibodies against p-TBK1, TBK1, and Beta Actin (all purchased from Wuhan Sanying Biotechnology Co., Ltd.); 0.25% trypsin-EDTA digestion buffer, RIPA lysis buffer, and CCK8 assay kit (purchased from Beijing Solarbio Science & Technology Co., Ltd.); BCA protein concentration assay kit (purchased from Beyotime Biotechnology Co., Ltd., China); 10% SDS-PAGE one-step gel preparation kit, NC membrane, Tris-SDS-glycine electrophoresis powder, ice-free rapid transfer powder, and skim milk powder (purchased from Shanghai Yamei Biomedical Technology Co., Ltd.); Matrigel (purchased from Moji Biotechnology Co., Ltd.); ECL Plus super-sensitive luminescent liquid (purchased from Sizhengbo Technology Co., Ltd.)

[0026] Experimental equipment: CO2 cell incubator (Thermo Fisher Scientific China), clean bench (Shanghai Zhicheng Analytical Instruments Manufacturing Co., Ltd.), high-speed refrigerated centrifuge (Eppendorf China), fluorescence microscope (Leica Microsystems (Shanghai) Trading Co., Ltd.), microplate reader (Xiamen Jianfa High-Tech Co., Ltd.).

[0027] Data analysis: All experiments were repeated at least three times. Data are expressed as mean ± standard deviation. SPSS 23.0 was used for analysis. The t-test was used to compare differences between groups, and P < 0.05 was considered statistically significant.

[0028] Experiment Example 1: SPR and In Vitro Kinase Inhibition Experiment

[0029] SPR (Surface Plasmon Resonance) binding experiment: Recombinant human TBK1 protein was immobilized on the surface of a CM5 sensor chip via amine coupling. Unreacted active groups were blocked with ethanolamine, and a running buffer containing a fixed proportion of DMSO was used as the mobile phase for DMSO correction. Squid flavin was serially diluted to multiple concentration points using the same buffer and injected into the chip channels sequentially at a set flow rate. Response signals were recorded during the binding phase, and the chip was rinsed with running buffer during the dissociation phase. If necessary, a mild regeneration solution was used to regenerate the chip surface to restore the baseline. The control channel was set as a blank fixation or protein-free fixation channel to eliminate non-specific binding and bulk effects. After reference subtraction and baseline correction of the sensing curves for each concentration, a 1:1 Langmuir binding model or a steady-state binding model was used for fitting to obtain the binding constant KD and related kinetic parameters.

[0030] In vitro TBK1 kinase inhibition assay: An in vitro kinase reaction system was prepared, containing recombinant TBK1 kinase (5 μg / ml), substrate protein (0.1 mg / ml), ATP (20 μM), and reaction buffer. Squid flavonoids were prepared in the same solvent system and serially diluted to ensure a consistent final concentration of DMSO in all groups. Different concentrations of squid flavonoids were pre-incubated with TBK1 for 10 min, followed by the addition of substrate protein and ATP for 1 h. Then, ADP-Glo™ Reagent was added, and the reaction was terminated by reacting for 40 min. Finally, Kinase Detection Reagent was added, and the reaction was continued for 30 min to quantify the reaction product and generate a chemiluminescent signal. The inhibitor-free group served as a 100% activity control. The relative activity of TBK1 at each concentration was calculated, and inhibition rate-concentration curves were plotted. An IC50 was obtained by fitting the curve using a four-parameter logistic model. 50 Simultaneously, enzyme-free wells or positive controls with known TBK1 inhibitors were set up to verify the stability and specificity of the system.

[0031] In in vitro kinase inhibition experiments ( Figure 1 B), scallop flavin can effectively inhibit TBK1-mediated substrate phosphorylation, IC50 50 The value was 0.6309 μM. The binding kinetic parameters of the compound saurolophus flavin and TBK1 protein were measured by SPR technology, such as... Figure 1 As shown in Figure C, the binding affinity KD value is 2.542E-7 M, indicating strong binding ability. This result, along with SPR binding affinity data, confirms the potential of saurolophus flavonoids as a direct inhibitor of TBK1.

[0032] Experimental Example 2: Squid flavonoids inhibit phosphorylation expression of TBK1 in SK-MES-1 cells

[0033] The effect of different concentrations of safflower flavin solution on TBK1 phosphorylation levels in SK-MES-1 cells was detected using Western blotting. The specific steps are as follows:

[0034] SK-MES-1 cells were seeded at a depth of 25 cm. 2 In cell culture flasks, when the cell density reached 80%, 0, 0.313, 0.625, 1.25, 2.5, 5, 10, 20, 40, and 80 μM of scutellarin solution were added. After 6 h, total protein from the drug-treated cells was extracted using a mixed lysis buffer of RIPA and phosphatase / protease inhibitor (100:1). Protein quantification was performed using the BCA standard method. A 10% SDS-PAGE gel was prepared for electrophoresis, then transferred to an NC membrane. After blocking with 5% skim milk for 1 h, primary antibodies (p-TBK1 and TBK1 diluted 1:1000, Beta Actin diluted 1:10000) were added and incubated overnight at 4°C. After washing three times with TBST for 5 min each time, secondary antibody (anti-Rabbit IgG) was added and incubated at room temperature for 0.5 h. After washing three times with TBST for 5 min each time, ECL chemiluminescence solution was added, and the bands were exposed in an imaging system.

[0035] Immunoblotting results showed that after treating SK-MES-1 cells with 0, 2.5, 5, and 10 μM crocin solution for 6 h, the protein expression level of p-TBK1 decreased in a concentration-dependent manner, with significant differences compared to the control group (*P<0.05). Figure 2 As shown.

[0036] Experiment Example 3: Squid flavonoids inhibit the proliferation of SK-MES-1 cells.

[0037] The effect of different concentrations of safflower flavonoids on the proliferation of lung squamous cell carcinoma cells was detected using the CCK8 assay. The specific steps were as follows: Adherent SK-MES-1 cells were digested with 0.25% trypsin-EDTA digestion solution at a concentration of 8 × 10⁻⁶ cells / cells. 3 Cells were seeded at a density of [number] cells / well in 96-well plates, with three replicates per group. After 24 hours, once the cells had fully adhered, 0, 1.25, 2.5, 5, 10, 20, 40, and 80 μM of scutellarin solution were added to the cell culture medium. After culturing for 24 hours and 48 hours, 90 μL of fresh culture medium was added, followed by 10 μL of CCK8 solution, and the cells were cultured for another 1 hour. The absorbance at 450 nm was then measured using a microplate reader.

[0038] The results of CCK8 assay showed that saury flavonoids inhibited the proliferation of SK-MES-1 cells in a concentration- and time-dependent manner, with a statistically significant difference compared to the control group (P<0.05). Its IC50 value was [not specified in the original text]. 50The value was approximately 86.67 μM at 24 h, such as Figure 3 As shown.

[0039] Experiment Example 4: Willow crocin enhances T cell killing rate in in vitro co-culture

[0040] T-cell killing assay: SK-MES-1 cells were seeded at a density of 8000 cells / well in 96-well plates and allowed to adhere overnight. Tumor cells were pretreated with different concentrations of salicylic acid for 6 h, then the drug-containing medium was removed. Jurkat cells were activated at an effector-to-target ratio of 5:1 (50 ng / mL LPA + 1 μg / mL iomycin, activation for 24 h) and co-cultured for 24 h. After 24 h, the supernatant T cells were discarded, washed with PBS, and incubated with 10% CCK8 for 1 h. The absorbance at 450 nm was measured, and the T-cell killing rate was calculated.

[0041] T cell kill rate = )

[0042] like Figure 4 The results showed that in vitro co-culture experiments demonstrated that the combined use of saural flavonoids significantly enhanced the tumor killing rate of anti-PD-1 T cells.

[0043] Experimental Example 5: Squid flavonoids inhibit tumor growth in vivo.

[0044] C57 mouse orthotopic lung xenograft model: Using 6-8 week old C57 mice, a needle was quickly inserted into the pleural cavity about 4 mm deep at a 90-degree angle to the chest wall surface, along the upper edge of the 6th rib in the right lung of the mouse, between the 5th and 6th ribs, and injected with 2×10 5 KLN205 cells were used, and the tumor size in the model group was 100 mm after 5-7 days. 3 Mice were randomly divided into four groups: a model group, an anti-PD-1 monotherapy group, a scallop flavonoid monotherapy group, and a scallop flavonoid + anti-PD-1 combination group, with eight mice in each group. The scallop flavonoid group received an intraperitoneal injection of 20 mg / kg for two days daily; the anti-PD-1 group received an intraperitoneal injection of 200 μg / mouse for three days; and the model group received an equal volume of physiological saline. Mouse weight and tumor volume were measured regularly to record survival status. Fourteen days after administration, samples were collected from various organs, tumors, and whole blood to record tumor volume, tumor weight, and organ damage.

[0045] The results showed that 14 days after injection of scutellarin, tumor growth in mice was significantly inhibited, and tumor weight and volume were also suppressed, with significant differences compared to the model group (***P<0.001). Furthermore, the combination therapy group also showed statistically significant differences compared to the single-drug group (*P<0.05). In addition, there was no significant difference in mouse body weight, and serum liver function indicators AST and ALT were within the normal range (ALT: 10.06-96.47 U / L; AST: 36.31-235.48 U / L). Figure 5-6 As shown.

[0046] The applicant first explored the direct interaction between scallop flavonoids and TBK1, confirming a stable binding between scallop flavonoids and recombinant TBK1 protein through SPR experiments. Further, in vitro kinase inhibition experiments and Western blot analysis demonstrated that scallop flavonoids could dose-dependently inhibit TBK1 kinase activity and reduce p-TBK1 expression levels, indicating that scallop flavonoids possess the ability to inhibit the TBK1 signaling pathway at both the molecular and cellular levels. Secondly, the applicant evaluated the biological effects of scallop flavonoids on lung squamous cell carcinoma cells, detecting a certain inhibitory effect on LUSC cell proliferation. Finally, a combined treatment experiment of scallop flavonoids and anti-PD-1 antibody was conducted in a mouse model of lung squamous cell carcinoma. The results showed that the tumor volume and endpoint tumor weight in the combined group were significantly lower than those in the single-drug group, while no significant weight loss was observed, indicating that the combined regimen has a clear in vivo anti-tumor effect and good tolerability.

[0047] This invention addresses the clinical challenge of limited efficacy of PD-1 monoclonal antibodies in the treatment of squamous cell lung cancer. It constructs a combined treatment strategy of "natural small molecule targeting TBK1—enhancing anti-PD-1 efficacy," forming a complete chain of evidence from multiple levels, including molecular binding, enzyme activity inhibition, cellular function, and animal models. Existing technologies have not disclosed the application of anchovy flavonoids as a TBK1 inhibitor in combination with anti-PD-1 antibodies for the treatment of squamous cell lung cancer. This study is the first to clearly demonstrate its direct targeting of TBK1 and validate in vivo models that the combined treatment significantly enhances anti-tumor effects. It possesses clear technical efficacy support and feasibility, demonstrating innovation and application value in the field of natural product immunosensitization.

Claims

1. Application of sauropoiesisin in the preparation of TBK1 inhibitors.

2. The application of TBK1 inhibitors combined with anti-PD-1 antibodies in the preparation of drugs for treating squamous cell carcinoma of the lung, characterized in that, The TBK1 inhibitor contains saurolophytin.

3. The application according to claim 2, characterized in that, The anti-PD-1 antibody is selected from one or more of nivolumab, pembrolizumab, toripalimab, sintilimab, camrelizumab, tislelizumab, or penaplimab.

4. A TBK1 inhibitor, characterized in that, It contains saurolophytin.

5. The TBK1 inhibitor according to claim 4, characterized in that, It also includes pharmaceutically acceptable excipients.

6. A pharmaceutical composition for treating squamous cell carcinoma of the lung, characterized in that, It contains a TBK1 inhibitor and an anti-PD-1 antibody, wherein the TBK1 inhibitor contains saurolophytin.

7. The pharmaceutical composition according to claim 6, characterized in that, The anti-PD-1 antibody is selected from one or more of nivolumab, pembrolizumab, toripalimab, sintilimab, camrelizumab, tislelizumab, or penaplimab.

8. The pharmaceutical composition according to claim 6, characterized in that, It also includes pharmaceutically acceptable excipients.

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