Application of fluperphenazine in preparation of LYN inhibitor

Abstract

The invention relates to the technical field of medicines, in particular to application of fluperphenazine in preparation of an LYN inhibitor. The invention finds that the fluperphenazine is an LYN inhibitor for the first time, and can be used for preventing and treating LYN-mediated related diseases. Pharmacological experiments verify the inhibition effect of the fluoperphenazine on LYN, and the fluoperphenazine can be developed into drugs for preventing and treating LYN-mediated related diseases through further experiments.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the application of fluphenazine in the preparation of LYN inhibitors. Background Technology

[0002] LYN (LYN proto-oncogene) is an important member of the Src family of tyrosine kinases, playing a crucial regulatory role in intracellular signaling pathways and participating in various physiological processes such as cell proliferation, differentiation, apoptosis, and immune responses. Recent studies have shown that abnormal activation or high expression of LYN is closely related to the development and progression of various diseases, such as hematologic malignancies (e.g., chronic myeloid leukemia, acute myeloid leukemia, lymphoma), solid tumors (e.g., breast cancer, lung cancer, pancreatic cancer), and autoimmune diseases (e.g., rheumatoid arthritis, systemic lupus erythematosus).

[0003] In hematologic malignancies, LYN, as a key upstream kinase in the B-cell receptor (BCR) signaling pathway, can lead to persistent activation of the pathway by aberrant activation, promoting tumor cell survival, proliferation, and drug resistance. For example, in chronic myeloid leukemia (CML), LYN can enhance the kinase activity of the BCR-ABL fusion protein by phosphorylating it, while also activating downstream signaling pathways such as STAT5 and PI3K / AKT, leading to resistance of tumor cells to traditional targeted drugs (such as imatinib). In acute myeloid leukemia (AML), high LYN expression can promote the malignant proliferation of leukemia cells by regulating the differentiation and apoptosis of myeloid cells. In solid tumors, LYN can enhance the invasive and metastatic abilities of tumor cells and participate in the regulation of the tumor microenvironment by interacting with other signaling molecules (such as EGFR, HER2, and c-Met). In autoimmune diseases, LYN can influence the occurrence and development of inflammatory responses by regulating the activation and function of immune cells (such as B cells, T cells, and macrophages).

[0004] Therefore, developing highly effective and specific LYN inhibitors has become an important research direction for treating the aforementioned LYN-related diseases. Currently, some LYN inhibitors have entered preclinical or clinical research stages, but existing LYN inhibitors still have some shortcomings, such as low specificity (easily inhibiting other Src family kinases), significant toxic side effects, and the ease with which tumor cells develop drug resistance, which limits their widespread clinical application. Furthermore, the complex synthesis processes and high production costs of some LYN inhibitors also hinder their industrialization and promotion.

[0005] Fluphenazine (Flu) is a known phenothiazine antipsychotic drug with the molecular formula C64. 22 H 26 F3N3OS, with a molecular weight of 437.52, has the chemical structural formula shown in formula (Ⅰ):

[0006] (I).

[0007] In existing technologies, fluphenazine is mainly used to treat mental illnesses such as schizophrenia and bipolar disorder. Its mechanism of action is primarily through blocking dopamine D2 receptors in the central nervous system, regulating the transmission of dopaminergic neurotransmitters, and thereby improving mental symptoms. To date, there are no reports of fluphenazine possessing LYN inhibitory activity, or its use in the preparation of LYN inhibitors or the treatment of LYN-mediated diseases. Summary of the Invention

[0008] In view of this, the object of the present invention is to provide a new use for fluphenazine, namely, its application in the preparation of LYN inhibitor drugs.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0010] This invention provides the use of fluphenazine or its geometric isomers, pharmaceutically acceptable salts, esters, solvates, and hydrates in any of the following:

[0011] (i) Preparation of LYN inhibitors;

[0012] (ii) Prepare reagents or kits for inhibiting LYN or LYN-related signaling pathways;

[0013] (iii) Prepare pharmaceutical compositions for the prevention and / or treatment of LYN-mediated diseases;

[0014] The structural formula of the fluphenazine is shown in formula (Ⅰ):

[0015] (I).

[0016] In some specific embodiments of the present invention, the reagents or kits described above may be reagents or kits for scientific research purposes.

[0017] In some specific embodiments of the present invention, the above-mentioned application achieves the suppression of LYN-related signaling pathways by inhibiting LYN phosphorylation.

[0018] In some specific embodiments of the present invention, the LYN-related signaling pathways in the above applications include the B-cell receptor signaling pathway.

[0019] In some specific embodiments of the present invention, the fluphenazine described above is used as the active pharmaceutical ingredient.

[0020] In some specific embodiments of the present invention, the fluphenazine described above is used as the sole active pharmaceutical ingredient.

[0021] In some specific embodiments of the present invention, the pharmaceutical composition described above is a tablet, capsule, granule, drop, lyophilized product, granule, ointment, or injection.

[0022] In some specific embodiments of the present invention, the pharmaceutical composition described above further includes pharmaceutically acceptable excipients.

[0023] In some specific embodiments of the present invention, the excipients used above are carriers, solvents, emulsifiers, dispersants, wetting agents, binders, stabilizers, colorants, or fragrances.

[0024] In some specific embodiments of the present invention, the preparation method of the pharmaceutical composition described above includes: adding pharmaceutical excipients to fluphenazine to prepare tablets, capsules, granules, drops, lyophilized products, granules, ointments, or injections.

[0025] In some specific embodiments of the present invention, the mass fraction of fluphenazine in the pharmaceutical composition used above is at least 98%, and the pharmaceutical excipient is an excipient.

[0026] The present invention also provides the pharmaceutical composition for the above-described applications.

[0027] The present invention also provides the use of the above-described pharmaceutical composition in the preparation of medicaments for the prevention and / or treatment of LYN-mediated related diseases.

[0028] The present invention also provides the medicine described above for the application.

[0029] The present invention also provides methods for preventing and / or treating LYN-mediated diseases, including administering the above-described pharmaceutical composition or drug to humans or animals in need.

[0030] In some specific embodiments of the present invention, the LYN-mediated related diseases of the above-mentioned applications or methods include tumors, autoimmune diseases, respiratory diseases, metabolic and circulatory system-related diseases, or neurodegenerative diseases, and may be at least one of breast cancer, ovarian cancer, gastric cancer, colorectal cancer, lung cancer, pancreatic cancer, liver cancer, glioblastoma, chronic lymphocytic leukemia, acute lymphoblastic leukemia, multiple myeloma, rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, psoriasis, atopic dermatitis, asthma, chronic obstructive pulmonary disease, atherosclerosis, diabetic nephropathy, Alzheimer's disease, and Parkinson's disease.

[0031] In some specific embodiments of the present invention, the lung cancer described in the above application or method is non-small cell lung cancer.

[0032] This invention establishes in vivo and in vitro models of non-small cell lung cancer and, for the first time, selects fluphenazine as a therapeutic drug to study its effect on LYN, observing the inhibitory effect of fluphenazine on LYN, thereby providing a novel treatment strategy for LYN-related diseases.

[0033] The application of fluphenazine in the preparation of LYN inhibitors proposed in this invention is based on the multi-target pharmacological activity potential of fluphenazine and the important therapeutic value of LYN kinase. By exploring new indications for already marketed drugs, this invention provides a novel treatment strategy for LYN-related diseases (especially tumors and autoimmune diseases with high LYN expression or abnormal activation). This research direction not only aligns with the innovative concept of drug reuse but also provides new insights into solving the current challenges in LYN inhibitor development, possessing significant theoretical and clinical application value. Compared with existing technologies, this invention has the following beneficial effects:

[0034] (1) This invention first discovered that fluphenazine has LYN inhibitory activity and applied it to the preparation of LYN inhibitors, which expanded the clinical application scope of fluphenazine and provided a new direction for the secondary development of fluphenazine.

[0035] (2) Fluphenazine, as a marketed antipsychotic drug, has relatively clear pharmacokinetic and toxicological data, a long history of clinical application, and its safety and tolerability have been fully verified. Using it to prepare LYN inhibitors can greatly shorten the drug development cycle, reduce development costs, and accelerate the industrialization process. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0037] Figure 1 The results of network pharmacology methods for identifying targets for fluphenazine—intersection targets;

[0038] Figure 2 , Figure 3 Results of using network pharmacology methods to find targets for fluphenazine—molecular docking results;

[0039] Figure 4 Results of network pharmacology method for identifying targets of fluphenazine – RMSD results;

[0040] Figure 5 The CETSA test results of fluphenazine and LYN are shown.

[0041] Figure 6 The results of the DARTS experiment show the effects of fluphenazine and LYN;

[0042] Figure 7 The SPR test results of fluphenazine and LYN are shown.

[0043] Figure 8 Immunofluorescence results of fluphenazine on p-LYN(Y397) protein;

[0044] Figure 9 The image shows the Western blot results of fluphenazine on p-LYN (Y397) protein;

[0045] Figure 10 Schematic diagram of in vivo experiment;

[0046] Figure 11 Western blot results of the effects of fluphenazine on p-LYN(Y397) protein in subcutaneous xenograft tumor tissue of nude mice;

[0047] Figure 12 Immunohistochemical results of fluphenazine on p-LYN(Y397) protein in subcutaneous xenograft tumor tissue of nude mice;

[0048] Figure 13 The results of HE staining of the heart, liver, spleen, lungs and kidneys of nude mice with fluphenazine are shown. Detailed Implementation

[0049] This invention discloses the application of fluphenazine in the preparation of LYN inhibitor drugs. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0050] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0051] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0052] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0053] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and does not constitute a limitation on the scope of the invention. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0054] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0055] This invention provides a new use for fluphenazine, namely, its application in the preparation of LYN inhibitors.

[0056] In one implementation, the present invention provides the use of fluphenazine in the preparation of reagents or kits for inhibiting LYN or LYN-related signaling pathways, such as reagents or kits for screening in vitro kinases / epigenetic enzymes, inhibiting cell signaling pathways, constructing disease models, and preparing protein degradation probes.

[0057] In another implementation, the present invention provides the use of fluphenazine in the preparation of a pharmaceutical composition for treating LYN inhibitors, wherein the pharmaceutical composition comprises an inert, non-toxic, and pharmacologically suitable excipient.

[0058] In another implementation, the present invention provides a pharmaceutical composition for inhibiting LYN, specifically, the composition contains a therapeutically effective dose of fluphenazine active ingredient.

[0059] In some examples, the pharmaceutical composition also includes a pharmaceutically acceptable one or more inert, non-toxic, and pharmacologically suitable excipients.

[0060] In some examples, the excipient is a carrier, solvent, emulsifier, dispersant, wetting agent, binder, stabilizer, colorant, or fragrance.

[0061] In some examples, the pharmaceutical composition is a tablet, capsule, granule, drop, lyophilized product, granule, ointment, or injection.

[0062] In some examples, the preparation method of the pharmaceutical composition includes: adding pharmaceutical excipients to fluphenazine to form tablets, capsules, granules, drops, lyophilized products, granules, ointments, or injections.

[0063] In some examples, the LYN inhibitor pharmaceutical composition can be formulated into tablets, capsules, granules, drops, lyophilized products, ointments, or injections by adding a common pharmaceutical excipient to fluphenazine with a purity of 98% or higher.

[0064] In practice, the pharmaceutical compositions according to the invention can act systemically and / or locally, and for this purpose can be taken in a suitable manner, for example, via oral, parenteral, pulmonary, or nasal routes. The compositions according to the invention can be taken in a form suitable for these routes of administration.

[0065] Suitable for oral administration are administration forms that act and rapidly and / or in an improved manner according to the level of existing technology, and comprise pharmaceutical compositions according to the invention in crystalline and / or amorphous and / or dissolved forms, such as tablets (uncoated or coated tablets, which, for example, have a coating that resists gastric juices or delays dissolution or does not dissolve, upon release of the composition according to the invention), tablets that break rapidly in the mouth, or films, films / lyophilized products, capsules (e.g., hard or soft capsules), sugar-coated tablets, granules, pellets, powders, emulsions, suspensions, aerosols, or solutions.

[0066] Parenteral administration can avoid absorption steps (e.g., intravenous, intra-arterial, intracardiac, intraspinal, intralumbar, or intra-articular) or simultaneously include absorption (e.g., intramuscular, subcutaneous, intradermal, percutaneous, or intraperitoneal). Suitable forms of administration for parenteral administration are particularly for injection and infusion in the form of solutions, suspensions, emulsions, lyophilized products, or sterile powders.

[0067] Another suitable route of administration is, for example, a form of medication for inhalation, such as a powder inhaler or nebulizer, or a form of medication that can be taken nasally, such as drops, solution or spray.

[0068] In practice, the pharmaceutical composition according to the invention can be converted into the described oral form. This can be done in a manner known per se by mixing with inert, non-toxic, and pharmacologically suitable excipients. These excipients particularly include carriers (e.g., microcrystalline cellulose, lactose, mannitol, starch), solvents (e.g., liquid polyethylene glycol), emulsifiers and dispersants or wetting agents (e.g., sodium lauryl sulfate, polysorbate oleate, propylene glycol), binders (e.g., polyvinylpyrrolidone), synthetic and natural polymers (e.g., albumin), stabilizers (e.g., antioxidants such as ascorbic acid), colorants (e.g., inorganic pigments such as iron oxides), and masking fragrances and odors.

[0069] In practice, the effective dose of fluphenazine can be varied depending on the administration method, the patient's age and weight, the severity of the condition, and other relevant factors. The recommended dose for oral administration is 100-200 mg once or twice daily; the recommended dose for parenteral administration is 50-100 mg once daily.

[0070] Unless otherwise specified, all other raw materials, reagents, consumables, and instruments involved in this invention are commercially available products and can be purchased from the market. The materials involved in the embodiments are as follows.

[0071] 1. Laboratory animals

[0072] Eighteen female BALB / c athymic nude mice, aged 3–5 weeks and weighing 18–25 g, were provided by Shanghai Silex Laboratory Animal Co., Ltd. During the experiment, the housing temperature was 20 ± 3.2℃, and the relative humidity was 65%–75%, with free access to food and water.

[0073] 2. Medicines and reagents

[0074] 2.1 Medicines

[0075] Fluphenazine hydrochloride (purity > 98%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd., stored at -20℃, and prepared fresh each time it is needed.

[0076] 2.2 Reagents

[0077] Cell lines: A549 and H1299 cells, purchased from the Cell Bank / Stem Cell Bank of the Chinese Academy of Sciences, Shanghai, China; fetal bovine serum, produced by Gibco; RPMI-1640 medium, purchased from Procell; Matrigel, BD, USA; LYN (2796S), Phospho-LYN (Tyr397) (70926), CST, USA.

[0078] 2.3 Instruments

[0079] Electronic balance, Shanghai Ohaus Instruments Co., Ltd.; Vernier calipers (HAMILTION); Thermo Fisher Scientific, USA; Biosafety workbench, Suzhou Antai Air Technology Co., Ltd.; Inverted microscope, Olympus, Japan.

[0080] The verification scheme of this invention is as follows: the effects of fluphenazine on LYN are studied through in vitro experiments (molecular docking, CETSA, DARTS, SPR, Western blotting and immunofluorescence) and in vivo animal experiments, and the inhibitory effect of fluphenazine on LYN is observed, thereby providing experimental basis for the development of fluphenazine into an LYN inhibitor.

[0081] The present invention will be further illustrated below with reference to the embodiments.

[0082] Example

[0083] 1. Western blot

[0084] (1) Lyse cell samples or tumor tissues with Beyotime RIPA buffer and centrifuge to obtain the supernatant. Add 5× Loading Buffer and heat at 100°C for 7 minutes. Store protein samples at -80°C.

[0085] (2) Perform SDS-PAGE electrophoresis. Constant voltage mode, set the voltage to 140 V, 70 minutes.

[0086] (3) Linear transfer (wet transfer). Transfer membrane at a constant current of 320 mA for 1.5 hours.

[0087] (4) Immunological reaction and color development. Block the NC membrane with 5% BSA on a shaker at room temperature for 1 hour. After blocking, incubate overnight at 4°C with a 1:1000 solution of 1×TBST diluted specific primary antibody. After primary antibody incubation, wash the membrane for 5 minutes each time, 4 times. Then, incubate with a 1:50000 solution of secondary antibody on a shaker at room temperature for 1 hour. After secondary antibody incubation, wash the membrane for 5 minutes each time, 4 times. Finally, perform ECL chemiluminescence color development.

[0088] 2. Molecular docking experiments

[0089] (1) The 2D structure of the small molecule fluphenazine was drawn using ChemDraw 15.0 and saved as a cdx file. Chem3D 15.0 was loaded, and energy minimization was performed using the MM2 force field. The structure was then saved as a pdb file. AutoDockTool 1.5.6 was loaded, charges were added, atom types were assigned, and all rotatable bonds were set to flexible. The structure was then saved as a pdbqt file for molecular docking.

[0090] (2) Obtain the LYN crystal structure (ID: P07948) from AlphaFold Data Bank as a protein acceptor for molecular docking. In PyMOL 1.7, delete the water of crystallization and other small molecules from the crystal structure, add hydrogen atoms, and save. Load the AutoDockTool 1.5.6 program, add charges, assign atom types, and save as a pdbqt format as the molecular docking acceptor.

[0091] (3) Molecular docking was performed using AutoDock Vina 1.1.2. The ligand was set to flexible and the acceptor to rigid. The exhaustiveness search precision was set to 100, and other parameters were left as default.

[0092] 3. Molecular Dynamics (MD) Simulations

[0093] Molecular dynamics simulations of the protein-small molecule complex obtained through molecular docking were performed using the Amber18 software package. The protein was simulated using ff14SB force field parameters, while the small molecule ligand was simulated using GAFF universal force field parameters. The AM1-BCC atomic charges were calculated using the ANTECHAMBER module. The protein-small molecule complex was loaded into the tleap module, where hydrogen atoms and antagonistic ions were automatically added to neutralize the charge. The TIP3P dominant water model was selected, and periodic boundary conditions were set. The molecular dynamics simulation workflow consisted of four steps: energy minimization, heating, equilibrium, and production kinetics simulation. First, the heavy atoms of the protein (and small molecule) were constrained, and energy minimization was performed on the water molecules for 10,000 steps (including 5,000 steps of steepest descent and 5,000 steps of conjugate gradient). Then, the constraints were lifted, and energy optimization was performed on the entire system for 10,000 steps. After energy minimization, the system was slowly heated to 300 K over 50 ps. After heating, the system was equilibrated for 50 ps under the npt ensemble. Finally, molecular dynamics simulations of the system were performed in the npt ensemble for 100 ns with a time step of 2 fs. Trajectory data were saved every 20 ps and correlation analysis was performed using the CPPTRAJ module. The binding free energies of ligands and proteins were calculated using the MMPBSA.py module.

[0094] 4. Surface Plasmon Resonance (SPR) Experiment

[0095] (1) Immobilization of CM5 chips and solution preparation

[0096] At 25°C, buffer (10 mM PBS) was flowed over the chip surface. The CM5 chip was activated using EDC and NHS from the Amine Coupling Kit. Human LYN recombinant protein was diluted to 10 μg / mL with sodium acetate at pH = 4.0 and then coupled to the activated CM5 chip. The chip was then blocked with Ethanolamine-HCl. Calibration curves containing 2%–3% DMSO were prepared using DMSO-free buffer (10 mM PBS), with a total of 8 concentration gradients. Fluphenazine was dissolved in DMSO to 20 mM, and then serially diluted with buffer (10 mM PBS) to obtain solutions of different concentrations. These solutions were flowed over the surface of the protein-coupled chip, and the instrument detected the protein-small molecule binding and dissociation curves.

[0097] (2) Flu affinity determination

[0098] The curves were fitted using SPR analysis software to calculate the binding rate constant and dissociation rate constant, as well as the equilibrium dissociation constant, in order to assess the affinity of fluphenazine for recombinant human LYN protein.

[0099] 5. Cell thermal migration assay (CETSA)

[0100] (1) Cell freeze-thaw and lysis

[0101] Prepare two fully confluent 10 cm culture dishes containing cells. Wash twice with PBS, then add 1 mL of PBS (containing 1× protease / phosphatase inhibitor) to each dish. Scrape cells into cryovials using a cell scraper. Add an appropriate amount of liquid nitrogen to a capped foam box, gently tap the cryovials to mix the cells, and quickly freeze in liquid nitrogen for 30 seconds. Carefully remove the cryovials with forceps and allow them to thaw at room temperature. Once completely thawed, repeat the freeze-thaw cycle twice more to ensure complete cell lysis. Before each thawing, gently tap the cryovials to mix the sample. After the freeze-thaw cycle, transfer the liquid to EP tubes and centrifuge at 10,000 g for 15 minutes at 4°C to obtain the supernatant.

[0102] (2) Protein concentration determination

[0103] The concentration of supernatant protein was determined using a BCA kit.

[0104] (3) Drug incubation

[0105] Add fluphenazine to one supernatant to a final concentration of 20 μM, and add an equal amount of DMSO to the other supernatant. Mix well to ensure that the drug or DMSO is fully bound to the protein. Incubate at room temperature. After 2 hours, divide the proteins that have finished incubating with the drug or DMSO into 10 equal portions and place them on ice.

[0106] (4) Temperature gradient processing

[0107] Set the PCR instrument to gradient temperature mode, setting eight temperature points: 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, 75℃, and 80℃. Place the sample on the temperature plate the instant it reaches the specified temperature; do not place it during the temperature rise process. Heat for 3 minutes, then allow it to rest at room temperature for 3 minutes, and finally place it on ice. Repeat this process for all eight temperatures. Centrifuge at 10000 g for 30 minutes and collect the supernatant.

[0108] (5) Protein detection

[0109] Add an appropriate amount of 2×SDS loading buffer to the supernatant, vortex to mix, centrifuge, boil at 100℃ for 7 minutes, and collect the protein.

[0110] (6) Western blot analysis

[0111] Refer to Method 1 for detailed steps of protein blotting.

[0112] 6. Target Stability Analysis of Drug Affinity Response (DARTS) Experiment

[0113] (1) Add the above reagents in the following ratio to a 50 mL centrifuge tube: 233.76 mg NaCl, 40 μL Triton X-100, 5 mL 200 mM Tris-HCl (pH adjusted to 7.5 with HCl), 2 mL glycerol, and 3 mL deionized water. Mix well to obtain the lysis buffer. Add the above reagents in the following ratio to a centrifuge tube: 29.25 mg NaCl, 11.1 mg CaCl2, 5 mL 100 mM Tris-HCl (pH adjusted to 8.0 with HCl), and 5 mL deionized water. Mix well to obtain the reaction buffer.

[0114] (2) Cultivate a cell culture dish that has grown to 10 cm in length, wash twice with PBS, add 1 mL of lysis buffer, place on ice for half an hour to allow complete lysis, collect the cells, centrifuge, and divide the supernatant into 5 groups.

[0115] (3) The supernatant was incubated with DMSO, 2 μM, 20 μM and 200 μM fluphenazine at room temperature for 1 hour according to the pre-grouped groups.

[0116] (4) Hydrolyze the protein supernatant in (3) with 0.2% streptomycin in the reaction buffer at room temperature for 10 minutes.

[0117] (5) Add an appropriate amount of 2×SDS loading buffer to the EP tube in (4), mix well, boil at 100℃ for 7 minutes, and collect the sample.

[0118] (6) Western blot analysis

[0119] Refer to Method 1 for detailed steps of protein blotting.

[0120] 7. Immunofluorescence assay

[0121] (1) Digest A549 and H1299 cells, resuspend them and plate them in a six-well plate. When the cells adhere to the plate and grow to 50%~60%, add fluphenazine for 48 hours.

[0122] (2) After the fluphenazine treatment, the A549 and H1299 cells in the six-well plate were digested and centrifuged to obtain cell pellets, and the cells were resuspended in complete culture medium.

[0123] (3) Add 10 μL of PBS to the 12-well plate beforehand, cover with a climbing slide to avoid air bubbles, and then add 1 mL of complete culture medium. Take an appropriate amount of cell suspension and add it to the 12-well plate, controlling the density to reach 50%~60% on the second day.

[0124] (4) On the second day, aspirate the culture medium, slowly add 1 mL of PBS along the side wall, wash 2-3 times, discard the PBS, add 200 μL of 4% polyethanol fixative to the 12-well plate to fix the cells on the smear for 30 minutes, discard the fixative after fixation, add PBS and wash 2-3 times on a shaker.

[0125] (5) Add 200 μL of 0.5% Triton X-100 along the sidewall into a 12-well plate, incubate on a shaker for 20 minutes, and wash once with PBS to remove excess permeate.

[0126] (6) Block with 5% BSA on a shaker for 1 hour. After blocking, add 200 μL of primary antibody prepared in the appropriate proportion with 5% BSA according to the antibody instructions. Incubate overnight on a shaker at 4°C.

[0127] (7) Use a pipette to recover the primary antibody into a centrifuge tube, add 500 μL PBST to wash the cells on the slide three times, 3 minutes each time. Prepare the fluorescent secondary antibody in advance with fluorescent secondary antibody dilution buffer, add 200 μL of the pre-prepared fluorescent secondary antibody to each well, and incubate on a shaker in the dark for 1 hour.

[0128] (8) After incubation, the fluorescent secondary antibody was recovered. 500 μL of 1×TBST was added to each well to wash the cells three times for 3 minutes each time. After removing the excess PBS, 200 μL of pre-prepared DAPI (DAPI and PBS were diluted 1:1000) was added to each well and the cells were incubated on a shaker in the dark with aluminum foil for 3 minutes.

[0129] (9) Recover the nuclear staining solution DAPI, add 500 μL PBST to each well and wash three times, 3 minutes each time. Aspirate the PBST, add 10 μL of anti-fluorescence quencher to the slide in advance, remove the slide with a needle tip, place it face down on the slide, and take pictures for analysis using a confocal microscope.

[0130] 8. CDX nude mouse xenograft model

[0131] (1) Lung adenocarcinoma cells A549 were used as tumorigenic cells in this experiment. A549 cells with good growth were selected and cultured in 10cm dishes. The cells were passaged one day before subcutaneous tumorigenesis and inoculated into nude mice when they were in the logarithmic growth phase.

[0132] (2) After digesting the cells in the large dish, centrifuge, wash the cells twice with PBS, mix by pipetting, centrifuge again, resuspend the cells in 1 mL of serum-free culture medium, mix by pipetting, count the cells, and prepare a final concentration of 4 × 10⁻⁶. 7Pipe 3000 μL of cell suspension into a total tube containing cells / mL, add matrix gel at a 1:1 volume ratio, mix well by pipetting, and place on ice.

[0133] (3) Eighteen female nude mice aged 3-5 weeks were selected as experimental subjects in this experiment. Iodine was used to disinfect the injection site under the right armpit of the mice, and 100 μL of the mixture was injected subcutaneously.

[0134] (4) After the transplanted tumors grew, 18 nude mice were randomly divided into a blank control group (blank solvent), a low-dose fluphenazine group (Flu, 7.5 mg / kg), and a high-dose fluphenazine group (Flu, 15 mg / kg). The tumor volume was measured with calipers every two days, and the weight of the nude mice was measured with an electronic balance. The formula for calculating the tumor volume is: V = L × D 2 / 2 Calculate the tumor volume, where L is the longest diameter of the transplanted tumor and D is the shortest diameter of the transplanted tumor. The tumor volume should reach 80-100 mm. 3 Then the medication was administered.

[0135] (5) Every two days, the low-dose group and the high-dose group were injected intraperitoneally with fluphenazine at 7.5 mg / kg and 15 mg / kg, respectively, while the blank control group was injected with an equal amount of blank solvent. After 7 treatments, the nude mice were sacrificed and the tumors were removed.

[0136] (6) Take a photo.

[0137] 9. Tissue paraffin embedding and sectioning

[0138] (1) Fix the detached animal tumor in 4% polymethanol tissue fixative for 12-24 hours.

[0139] (2) Place the fixed tissue in running water and rinse for several hours to remove the fixative present in the tissue. The rinsing time should be adjusted according to the fixation time.

[0140] (3) Place the rinsed tissue in a tissue box and then place it in 75% ethanol for 2 hours, 85% ethanol for 2 hours, 90% ethanol for 2 hours, 95% ethanol for 1 hour, 100% ethanol for 30 minutes, and 100% ethanol for 30 minutes.

[0141] (4) Place the dehydrated tissue in a system of ethanol and xylene in a 1:1 ratio for 1 hour, in xylene No. 1 for 30 minutes, and in xylene No. 2 for 30 minutes.

[0142] (5) Place the tissue that has been processed above into the melted paraffin in the embedding machine and soak it for at least 3 hours. If necessary, soak it overnight.

[0143] (6) After soaking in paraffin, take out the corresponding tissue from the tissue box and place it into an embedding tool of appropriate size for embedding. After filling it with liquid wax, place it in a -20℃ freezing table to cool and solidify. Then take the wax block out of the embedding frame and trim the wax block.

[0144] (7) Before sectioning, the wax block can be placed on a freezing table to cool for a long time. Adjust the water bath temperature of the slide spreader to 40°C in advance. Place the trimmed wax block on the slide slicer, spread the slice flat on the water surface, carefully lift the tissue with a glass slide, and place it in a 60°C oven for baking. After baking, the white slides can be stored at room temperature.

[0145] 10. Immunohistochemistry of paraffin-embedded tissue sections

[0146] (1) Dewaxing and hydrating the white film. Place it in xylene No. 1 for 10 minutes, xylene No. 2 for 10 minutes, xylene No. 3 for 10 minutes, anhydrous ethanol for 5 minutes, 95% ethanol No. 1 for 5 minutes, 95% ethanol No. 2 for 5 minutes, 80% ethanol for 5 minutes, 75% ethanol for 5 minutes, and PBS for 5 minutes three times.

[0147] (2) Antigen retrieval. Add one packet of antigen retrieval solution to 1000 mL of ultrapure water, add hydrochloric acid to adjust the pH to about 6, put the dewaxed and hydrated white tablets into the antigen retrieval solution, place them in a 90℃ water bath for 30 minutes, remove them and let them cool to room temperature, and wash them with PBS for 5 minutes.

[0148] (3) Lay the white slide flat in a dark box and use an immunohistochemical pen to circle the tissue along the tissue edge. Add 40 μL to block endogenous peroxidase and react at room temperature for 10 minutes. Then wash with PBS three times, three minutes each time.

[0149] (4) Prepare the primary antibody (primary antibody) at an appropriate concentration using BSA according to the instructions. Add 40-60 μL of primary antibody per sample according to the tissue size. No primary antibody is added to the negative control group. Incubate overnight at 4°C. Then wash three times with PBS for three minutes each time.

[0150] (5) On the second day, the primary antibody was recovered, the reaction enhancement solution was added, and the dark box was placed in a constant temperature incubator at 37°C for 20 minutes.

[0151] (6) Add the enhanced enzyme-labeled goat anti-rabbit IgG polymer produced by Beijing Zhongshan Jinqiao Company, and place the dark box in a 37°C constant temperature incubator for 20 minutes. Then wash with PBS three times, three minutes each time.

[0152] (7) Prepare the DAB chromogenic solution in advance. Prepare the DAB chromogenic solution according to the volume ratio of reagent 2 to reagent 1 of 20:1. Store at 4℃ protected from light. Note that it should be prepared fresh each time. Add 40 μL of DAB chromogenic solution to each tissue sample and observe the color change of the tissue. Terminate the reaction with PBS when appropriate and record the reaction time. After the reaction is completed, terminate the color development with PBS.

[0153] (8) After the reaction is complete, stain in hematoxylin staining solution for 6 minutes, and then quickly rinse off the excess staining solution under running water. Differentiate in 1% hydrochloric acid-acetic acid solution for 3 seconds, and then quickly rinse off the excess staining solution under running water.

[0154] (9) Turn on the water bath and adjust the temperature to 55°C. Prepare 300 mL of clean water in advance and place it in the 55°C water bath. Then place the glass slide in the reaction dish containing clean water and invert it for 5 minutes.

[0155] (10) Place the slide in 75% ethanol for 2 seconds, 80% ethanol for 2 seconds, 90% ethanol for 2 seconds, and anhydrous ethanol for 2 seconds in sequence. After drying, add mounting solution to seal the slide.

[0156] 11. Statistical Methods

[0157] Statistical analysis and graphing of experimental data were performed using GraphPad Prism 10.0 software. Student's t-test was used for comparisons between two groups. Quantitative data were expressed as mean ± standard error (mean ± SD), and categorical data were analyzed using the chi-square test. A p-value < 0.05 was considered statistically significant. Each experiment was repeated at least three times.

[0158] Example 1: Identifying the target of fluphenazine using network pharmacology methods

[0159] To identify the direct target of fluphenazine, the SuperPred and Pharmmapper databases were first used to search for potential target proteins. Venny 2.1.0 software was used to obtain the intersection targets of the two databases, which were: Tyrosine-protein kinase Lyn, Aldose reductase, and Mineralocorticoid receptor. Literature review determined that Lyn is a likely target of fluphenazine. Figure 1 Preliminary molecular docking results show that fluphenazine can form stable hydrogen bonds and hydrophobic interactions with the active pocket of LYN kinase (key amino acid residues Leu374, Leu253, and Val261), with a binding energy of -9.2 kcal / mol, suggesting a strong binding potential between the two. Figure 2 , Figure 3The RMSD curve represents the variation in protein conformation. From... Figure 4 As can be seen from the data, the RMSD fluctuations of the protein were relatively smooth throughout the entire kinetic process, especially after 50 ns, when the RMSD stabilized around 0.4 nm. This result indicates that the binding of small molecules to the receptor protein does not lead to a sustained and significant change in its conformation. Based on the MMGBSA equation, the binding free energy of fluphenazine ligand to LYN protein was calculated (using the trajectory from 50 to 100 ns), and the binding free energy was -59.6 kcal / mol, indicating that the two have a very good binding interaction.

[0160] Example 2: CETSA experiment verified that LYN is a target of fluphenazine.

[0161] Based on the preliminary screening results of network pharmacology, LYN protein was selected for further validation using CETSA and Western blotting experiments. On a thermal cycler, the program was set to gradient temperature ramp, with eight temperature points established, followed by Western blotting experiments. Based on the experimental results, such as... Figure 5 As shown, fluphenazine was more stable than LYN in the DMSO group after incubation with LYN protein as the temperature increased.

[0162] Example 3: DARTS experiment verifies that LYN is a target of fluphenazine.

[0163] Based on the preliminary screening results of network pharmacology, the LYN protein was selected for further validation using DARTS and Western blotting experiments. After protein extraction, it was incubated with different concentrations of fluphenazine, followed by streptomycin lysis. The streptomycin concentrations were 0 and 1:500. The binding of the fluphenazine small molecule ligand to the target protein LYN stabilized the protein and enhanced its resistance to proteolytic enzyme activity. Figure 6 As shown, the target protein becomes more stable with increasing fluphenazine concentration.

[0164] Example 4: SPR experiment verifies that LYN is the target of fluphenazine.

[0165] The SPR results of fluphenazine and LYN protein are as follows: Figure 7 As shown, the dissociation equilibrium constant K between fluphenazine and LYN protein is... D =5.47×10 -6 M indicates that fluphenazine and LYN have a good affinity.

[0166] Example 5: Fluphenazine was able to inhibit the phosphorylation of LYN in in vitro experiments.

[0167] To evaluate the effect of fluphenazine on LYN protein, immunofluorescence and Western blotting experiments were performed. NSCLC cells were treated with different concentrations of fluphenazine for 48 h, and immunofluorescence experiments were conducted to detect the expression of p-LYN(Y397) and LYN. The results are shown below. Figure 8 The fluorescence of p-LYN (Y397) gradually decreased, showing a negative correlation with fluphenazine concentration, while the fluorescence intensity of LYN remained largely unaffected by fluphenazine. Western blot analysis also indicated that fluphenazine reduced LYN phosphorylation levels in A549 and H1299 cells in a dose-dependent manner, while the total LYN protein level remained unchanged. (See results below.) Figure 9 .

[0168] Example 6: In vivo animal experiments further confirmed that fluphenazine can inhibit the phosphorylation of LYN.

[0169] Next, a nude mouse xenograft model was established. Figure 10 After seven administrations, the tumor tissue was excised for immunohistochemical and Western blot experiments, such as... Figure 11 , Figure 12 As shown, fluphenazine also inhibited the phosphorylation level of p-LYN (Y397), and Figure 13 This indicates that fluphenazine has no significant toxicity to the heart, liver, spleen, lungs, and kidneys of mice.

[0170] Preparation Example 1

[0171] Take 20 g of fluphenazine monomer compound, add 280 g of pharmaceutical starch, mix them thoroughly, and make 1000 capsules, each weighing 0.3 g and containing 20 mg of fluphenazine.

[0172] Preparation Example 2

[0173] Take 100 g of fluphenazine monomer compound, add 200 g of pharmaceutical starch, mix them thoroughly, and make 1000 tablets, each weighing 0.3 g and containing 100 mg of fluphenazine.

[0174] Preparation Example 3

[0175] Take 20 g of fluphenazine monomer compound, add 100 mL of 1,2-propanediol, dissolve thoroughly, dilute with sterile water for injection to 1000 mL, mix well, and dispense into 1000 ampoules, each containing 1 mL of fluphenazine and 20 mg of fluphenazine.

[0176] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Use of fluphenazine or its isomers, pharmaceutically acceptable salts, esters, solvates, or hydrates in any of the following: (i) Preparation of LYN inhibitors; (ii) Prepare reagents or kits for inhibiting LYN or LYN-related signaling pathways; (iii) To prepare drugs for the prevention and / or treatment of LYN-mediated diseases; The structure of the fluphenazine is shown in formula (Ⅰ): （Ⅰ）。 2. The application as described in claim 1, characterized in that, Fluphenazine is the active pharmaceutical ingredient.

3. The application as described in claim 2, characterized in that, Fluphenazine is the sole active pharmaceutical ingredient.

4. The application as described in claim 1, characterized in that, The LYN-mediated diseases include at least one of the following: breast cancer, ovarian cancer, gastric cancer, colorectal cancer, lung cancer, pancreatic cancer, liver cancer, glioblastoma, chronic lymphocytic leukemia, acute lymphoblastic leukemia, multiple myeloma, rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, psoriasis, atopic dermatitis, asthma, chronic obstructive pulmonary disease, atherosclerosis, diabetic nephropathy, Alzheimer's disease, and Parkinson's disease.

5. The application as described in claim 4, characterized in that, The lung cancer in question is non-small cell lung cancer.

6. The application as described in claim 1, characterized in that, The drug is in the form of tablets, capsules, granules, drops, lyophilized products, granules, ointments, or injections.

7. The application as described in claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients.

8. The application as described in claim 7, characterized in that, The excipient is a carrier, solvent, emulsifier, dispersant, wetting agent, binder, stabilizer, colorant, or fragrance.

9. The application as described in claim 1, characterized in that, The preparation method of the drug includes: adding pharmaceutical excipients to fluphenazine to prepare tablets, capsules, granules, drops, lyophilized products, granules, ointments or injections.

10. The application as described in claim 9, characterized in that, The drug contains at least 98% fluphenazine by mass, and the pharmaceutical excipient is an excipient.

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