Use of triclabendazole in the preparation of an antitumour medicament or pharmaceutical composition

Abstract

The application discloses application of triclabendazole in preparation of an antitumor drug or a pharmaceutical composition, and belongs to the technical field of biological medicines. It is found for the first time that triclabendazole can target to induce degradation of PD-L1 protein in tumor cells, block a PD-1 / PD-L1 immune checkpoint signal path, reverse the immune inhibition state of T cells in a tumor microenvironment, restore the killing function of immune cells on tumor cells, and thus inhibit tumor growth. In vitro experiments show that triclabendazole reduces the PD-L1 protein level in breast cancer cells in a concentration-dependent manner. In vivo experiments show that triclabendazole can significantly inhibit the growth of a breast cancer transplanted tumor in mice, increase the proportion of tumor-infiltrating CD8 + T cells and granzyme B positive CD8 + T cells. In addition, triclabendazole and cisplatin in combination present an additive antitumor effect, triclabendazole can be orally administered, has good safety, and has a good clinical transformation prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to new pharmaceutical uses of the small molecule compound triclocarban, and particularly to the use of triclocarban in the preparation of drugs or pharmaceutical compositions for the treatment of malignant tumors. Background Technology

[0002] Malignant tumors are among the major diseases that seriously threaten human health, with solid tumors such as breast cancer and lung cancer consistently exhibiting high incidence and mortality rates. PD-L1 (programmed death-ligand 1) is a key immune checkpoint molecule, abnormally highly expressed on the surface of various tumor cells and on immune cells in the tumor microenvironment. When PD-L1 binds to the PD-1 receptor on the surface of T cells, it transmits inhibitory signals, leading to inhibited T cell proliferation, reduced cytokine secretion, and ultimately inducing T cell exhaustion, helping tumor cells effectively evade the body's immune surveillance. Clinical studies have shown that high expression of PD-L1 is closely related to poor prognosis and immunotherapy resistance in patients with breast cancer and other malignant tumors, and is an important driving factor in the formation of a tumor immunosuppressive microenvironment.

[0003] Currently, treatment strategies targeting PD-L1 primarily rely on monoclonal antibody drugs. However, these drugs suffer from drawbacks such as high production costs, potential immunogenicity, limited penetration through biological barriers, and typically restricted to injection administration. In recent years, strategies involving the direct induction of target protein degradation through small molecule compounds have emerged as a cutting-edge direction in anti-tumor drug development due to their potential advantages, including oral administration, easy permeation of biological membranes, regulation of protein homeostasis, relatively low production costs, and lower immunogenicity risk. However, despite some preclinical studies reporting small molecule compounds with PD-L1 degrading activity, no small molecule PD-L1 degrading agents have yet received regulatory approval for marketing, and this field remains in the exploratory stage. Furthermore, while the combined use of chemotherapy drugs and immune checkpoint inhibitors has received widespread attention, the optimal combination regimen, synergistic mechanisms, and safety profile still require further investigation.

[0004] Triclocarban (CAS No.: 101-20-2, chemical name: 1-(4-chlorophenyl)-3-(3,4-dichlorophenyl)urea) is a classic broad-spectrum antibacterial agent widely used as an antibacterial active ingredient in personal care products (such as soaps, shower gels, hand sanitizers, etc.) since the 1950s, and also in some topical disinfectants. Triclocarban's mechanism of action is mainly related to its inhibition of enoyl-ACP reductase (FabI) ​​in bacteria, thereby blocking bacterial fatty acid synthesis and exerting its antibacterial effect. To date, no reports have revealed a link between triclocarban and PD-L1 protein degradation, nor have there been any reports of its use in antitumor immunotherapy or clinical applications. Summary of the Invention

[0005] The purpose of this invention is to provide a novel pharmaceutical use for the small molecule compound triclocarban, addressing the technical problem of the lack of highly effective, orally administered small molecule degrading agents in existing PD-1 / PD-L1 immune checkpoint therapies. This invention is the first to discover that triclocarban can target and induce PD-L1 protein degradation, thereby achieving anti-tumor effects by remodeling the tumor immune microenvironment.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides the use of the compound of formula (I) in the preparation of an antitumor drug or an antitumor drug composition; Formula (I) The chemical name of the compound shown in formula (I) is triclocarban, and its CAS number is 101-20-2.

[0007] As an feasible approach, the antitumor drug or antitumor drug composition is used to treat PD-L1 positive malignant tumors.

[0008] As an feasible approach, the PD-L1 positive malignant tumors include, but are not limited to, breast cancer, non-small cell lung cancer, small cell lung cancer, gastric cancer, esophageal cancer, liver cancer, colorectal cancer, pancreatic cancer, renal cell carcinoma, bladder cancer, prostate cancer, ovarian cancer, cervical cancer, melanoma, head and neck squamous cell carcinoma, and thyroid cancer.

[0009] In a preferred embodiment of the present invention, the malignant tumor is breast cancer.

[0010] In the above-described applications provided by the present invention, the compound represented by formula (I) is used as the sole active ingredient; or, the compound represented by formula (I) is used in combination with at least one other antitumor active ingredient as an active ingredient.

[0011] In a preferred embodiment of the present invention, the compound shown in formula (I) inhibits tumor growth by degrading PD-L1 protein in tumor cells, blocking the PD-1 / PD-L1 immune checkpoint signaling pathway, reversing the immunosuppressive state of T cells in the tumor microenvironment, restoring the recognition and killing function of immune cells on tumor cells, and promoting immune cell infiltration. The term "degradation" refers to reducing the expression level of PD-L1 at the protein level.

[0012] In some embodiments of the present invention, the unit dose of triclocarban in the drug or pharmaceutical composition is from 1 mg to 500 mg.

[0013] In this invention, there are no particular restrictions on the route of administration of the drug or drug composition, including but not limited to oral administration, sublingual administration, rectal administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, pulmonary inhalation administration, mucosal absorption administration, topical administration, or transdermal absorption administration.

[0014] Preferably, the route of administration is oral administration or intraperitoneal injection.

[0015] In this invention, the pharmaceutical composition comprises a therapeutically effective amount of the compound represented by formula (I) and at least one pharmaceutically acceptable excipient.

[0016] Pharmaceutically acceptable excipients include, but are not limited to: solvents (such as water, ethanol, propylene glycol, polyethylene glycol), diluents (such as starch, dextrin, sucrose, lactose, mannitol, microcrystalline cellulose), excipients, disintegrants (such as crospovidone, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose), binders (such as hydroxypropyl methylcellulose, povidone, sodium carboxymethyl cellulose), lubricants (such as magnesium stearate, talc, micronized silica gel), surfactants (such as polysorbate, sodium lauryl sulfate), suspending agents, thickeners, preservatives (such as sodium benzoate, parabens), buffers (such as phosphate buffer, acetate buffer), isotonic adjusters (such as sodium chloride, glucose), pH adjusters, flavoring agents, and coloring agents.

[0017] In this invention, the dosage forms of the drug or drug composition include, but are not limited to, oral solid dosage forms (tablets, capsules, granules, powders, pills, drops), oral liquid dosage forms (oral solutions, oral suspensions, oral emulsions), injections (solution injections, suspension injections, emulsion injections, lyophilized powder injections), external preparations (ointments, creams, gels, patches, liniments, lotions), and sprays, aerosols, and powder sprays.

[0018] In a second aspect, the present invention provides the use of the compound of formula (I) in combination with a chemotherapeutic drug in the preparation of an antitumor drug composition; The chemotherapy drugs are selected from one or more of the following categories: (i) Platinum compounds, including but not limited to cisplatin, carboplatin, oxaliplatin, and nedaplatin; (ii) Alkylating agents, including but not limited to cyclophosphamide, ifosfamide, nitrogen mustard, chlorambucil, and melphalan; (iii) Antimetabolites, including but not limited to 5-fluorouracil, capecitabine, gemcitabine, methotrexate, pemetrexed, and cytarabine; (iv) Anthracyclines and their derivatives, including but not limited to doxorubicin, epirubicin, daunorubicin, idarubicin, and mitoxantrone; (v) Antimicrotubule inhibitors, including but not limited to paclitaxel, docetaxel, albumin-bound paclitaxel, vincristine, vinorelbine, and eribulin; (vi) Topoisomerase inhibitors, including but not limited to etoposide, teniposide, irinotecan, and topotecan; (vii) Targeted drugs, including but not limited to tyrosine kinase inhibitors, PARP inhibitors, CDK4 / 6 inhibitors, mTOR inhibitors, HDAC inhibitors, VEGF / VEGFR inhibitors, EGFR inhibitors, and HER2 inhibitors; (viii) Hormonal drugs, including but not limited to tamoxifen, letrozole, anastrozole, and fulvestrant.

[0019] In a preferred embodiment of the present invention, the chemotherapy drug is cisplatin.

[0020] In an embodiment of combined use, the mass ratio of the compound of formula (I) to the chemotherapeutic drug in the pharmaceutical composition is from 100:1 to 1:100.

[0021] Preferably, the mass ratio is 10:1 to 1:10.

[0022] More preferably, the mass ratio is 5:1 to 1:5.

[0023] More preferably, the mass ratio is 1:1.

[0024] In the implementation scheme of combined use, the compound shown in formula (I) and the chemotherapeutic drug can be administered simultaneously, separately, or sequentially.

[0025] "Simultaneous administration" refers to administering two active ingredients to the subject in the same pharmaceutical composition or as two separate pharmaceutical compositions at the same time point; "separate administration" refers to administering two active ingredients to the subject in two separate pharmaceutical compositions within the same or different time windows; "sequential administration" refers to administering one active ingredient to the subject first, and then administering the other active ingredient to the subject after a certain time interval.

[0026] Thirdly, the present invention provides an antitumor pharmaceutical composition comprising a therapeutically effective amount of the compound shown in formula (I) and at least one pharmaceutically acceptable excipient.

[0027] As one embodiment, the pharmaceutical composition further comprises at least one second active ingredient, the second active ingredient being selected from chemotherapy drugs, immunotherapy drugs, or targeted therapy drugs.

[0028] Preferably, the second active ingredient is a chemotherapy drug, more preferably cisplatin.

[0029] Fourthly, the present invention provides a method for treating malignant tumors, the method comprising administering to a subject in need a therapeutically effective amount of the compound represented by formula (I), or administering the pharmaceutical composition of the present invention.

[0030] Preferably, the method further includes administering a chemotherapy drug to the subject. The chemotherapy drug is preferably cisplatin.

[0031] The term "treatment" refers to reducing, delaying, inhibiting, or preventing the growth, metastasis, or recurrence of malignant tumors, including but not limited to reducing tumor volume, decreasing tumor burden, prolonging patient survival, and improving patient quality of life.

[0032] In this invention, the subject is a mammal, preferably a human.

[0033] The Triclocarban described in this invention is a known compound that can be purchased commercially (e.g., from compound suppliers such as Sigma-Aldrich, ChemDiv, and TargetMol) or prepared using known chemical synthesis methods in the art, such as by addition reaction of 4-chloroaniline and 3,4-dichlorophenyl isocyanate, or by other conventional urea compound synthesis methods in the art.

[0034] Based on the above technical solution, the present invention has the following beneficial effects: (I) This invention reveals for the first time a novel pharmaceutical application of Triclocarban as a PD-L1 protein degrader. Experiments have demonstrated that Triclocarban can induce PD-L1 protein degradation in a concentration-dependent manner in human breast cancer cells MCF-7 and mouse breast cancer cells 4T1, significantly reducing PD-L1 protein levels even at a concentration of 3 µM. This discovery provides a small molecule compound with definite degradative activity and oral applicability for the PD-1 / PD-L1 immune checkpoint pathway, offering a new strategy for developing non-antibody immune checkpoint inhibitors.

[0035] (II) This invention demonstrates, through a mouse 4T1 orthotopic xenograft model of breast cancer, that Triclocarban (5 mg / kg, intraperitoneal injection, once daily) can significantly inhibit tumor growth, with tumor volume and weight significantly reduced compared to the solvent control group, demonstrating its definite in vivo antitumor efficacy and verifying its potential for clinical development as an antitumor drug.

[0036] (III) This invention analyzed changes in the tumor immune microenvironment using flow cytometry and found that Triclocarban can significantly increase tumor-infiltrating CD8+. + The proportion of T cells was increased, while granzyme B positivity (Gzmb) was also upregulated. + The proportion of T cells was increased, reversing the immunosuppressive state of the tumor microenvironment and activating the body's anti-tumor immune response. These findings suggest that Triclocarban, by degrading PD-L1 protein, may synergistically enhance the clearance of tumors by immune cells while reversing the immunosuppressive microenvironment, providing a new drug candidate for tumor immunotherapy.

[0037] (iv) This invention reveals the synergistic effect of Triclocarban and the first-line chemotherapy drug cisplatin. In the 4T1 breast cancer model, the tumor inhibition effect of the Triclocarban and cisplatin combination therapy group was significantly better than that of either single drug group, indicating that the combination of Triclocarban and cisplatin can produce a better anti-tumor effect than either drug alone, and has the potential for clinical development of combination therapy.

[0038] (v) Compared with existing PD-1 / PD-L1 monoclonal antibody drugs, Triclocarban, as a small molecule compound, has significant advantages such as low production cost, no immunogenicity risk, oral administration, and good tissue penetration. It can significantly reduce treatment costs, improve patient compliance and drug accessibility, and has good social and economic benefits.

[0039] (vi) Triclocarban is a known compound that is widely used in personal care products. Its human safety data is relatively sufficient, which is expected to shorten the drug development cycle, reduce the risk of clinical development, and make it feasible to quickly advance to clinical trials. Attached Figure Description

[0040] Figure 1 Here is the chemical structural formula of the small molecule compound Triclocarban.

[0041] Figure 2 This is the result of Western blot analysis of the degradation efficiency of PD-L1 protein in human embryonic kidney HEK293T cells and mouse breast cancer 4T1 cells by Triclocarban and candidate compounds in Example 1 of the present invention; wherein, DMSO is the solvent control group, compounds 1-10 are candidate compounds, and Tubulin is the internal reference protein.

[0042] Figure 3 This is the result of Western blot analysis of the effect of different concentrations of Triclocarban on the PD-L1 protein level in human breast cancer MCF-7 cells and mouse breast cancer 4T1 cells in Example 2 of the present invention; the concentration gradient of Triclocarban was 0, 0.1, 0.3, 1, 3, 10 μM, and Tubulin was used as an internal reference protein.

[0043] Figure 4(a) is a schematic diagram of the animal experimental procedures for the solvent control group, the Triclocarban monotherapy group (5 mg / kg, intraperitoneal injection, once daily), the cisplatin monotherapy group (5 mg / kg, intraperitoneal injection, once every 3 days), and the Triclocarban and cisplatin combination group (Triclocarban 5 mg / kg intraperitoneal injection, once daily + cisplatin 5 mg / kg, intraperitoneal injection, once every 3 days). Figure 4(b) shows the in vivo antitumor pharmacodynamics of Triclocarban alone and in combination with cisplatin in a mouse 4T1 orthotopic xenograft model of breast cancer, with the tumor volume of each group of mice increasing with the number of days of administration. Figure 4(c) shows the statistical results of tumor weight after administration in each group of mice in the in vivo antitumor pharmacodynamic test of Triclocarban alone and in combination with cisplatin in a mouse 4T1 orthotopic xenograft model of breast cancer. Figure 4(d) shows the in vivo antitumor pharmacodynamics of triclocarban alone and in combination with cisplatin in a mouse 4T1 orthotopic breast cancer tumor model, as detected by flow cytometry in tumor tissues of mice in each group. + T cells at CD45 + CD3 + A statistical graph showing the percentage of T cells; Figure 4(e) shows the flow cytometry analysis of granzyme B positivity in tumor tissues of mice in a mouse 4T1 orthotopic breast cancer model, using triclocarban alone and in combination with cisplatin. + CD8 + T cells in CD8 + A statistical graph showing the percentage of T cells.

[0044] In the diagram, "+" indicates combined use; This indicates a statistically significant difference between the two groups (P<0.05). This indicates that P < 0.01; This indicates that P < 0.001; This means P < 0.0001. Detailed Implementation

[0045] To facilitate understanding of this invention, some terms used herein are defined and explained below. Unless otherwise expressly stated, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Terms not explicitly defined in this invention should be interpreted according to their ordinary meaning in the art.

[0046] In this document, the term "Triclocarban" refers to the compound with the chemical name 1-(4-chlorophenyl)-3-(3,4-dichlorophenyl)urea, also known as triclocarban, 3,4,4'-trichlorocarbanilide, or TCC, with CAS registry number 101-20-2 and chemical structural formula as shown in formula (I). This term includes any of its crystal forms, hydrates, and solvates.

[0047] In this article, the term "PD-L1" refers to Programmed Death-Ligand 1, also known as CD274 or B7-H1, a B7 family co-stimulatory molecule and a key immune checkpoint molecule expressed on the surface of various malignant tumor cells and immune cells in the tumor microenvironment. PD-L1 mediates immunosuppressive signals after binding to the PD-1 receptor on the surface of T cells.

[0048] In this article, the term "PD-1" refers to programmed death-1, also known as CD279, which is an immune checkpoint receptor expressed on the surface of immune cells such as T cells and B cells, and belongs to the CD28 family. After binding to its ligand PD-L1, PD-1 transmits an inhibitory signal, inhibiting the activation, proliferation, and effector function of T cells.

[0049] In this document, the term "degradation" refers to the reduction of expression levels of a target protein (such as PD-L1) at the protein level. In this invention, "degradation" specifically refers to the downregulation of PD-L1 protein levels in tumor cells induced by Triclocarban.

[0050] In this article, the term "anti-tumor" refers to inhibiting, blocking, delaying, reducing, or preventing the growth, proliferation, invasion, metastasis, or recurrence of tumors, including but not limited to reducing tumor volume, reducing tumor weight, reducing tumor burden, prolonging the survival of subjects, improving the quality of life of subjects, and enhancing the body's anti-tumor immune response.

[0051] In this article, the terms "tumor" and "malignant tumor" are used interchangeably. "Tumor" refers to any abnormal growth of tissue, whether benign or malignant; "malignant tumor" refers to newly formed malignant tissue with invasive and metastatic capabilities.

[0052] In this document, the term "subject" refers to an individual who receives the medicament or pharmaceutical composition described in this invention, said individual being a vertebrate, preferably a mammal, and more preferably a human. The subject may be a patient diagnosed with a malignant tumor, or an individual at risk of developing a tumor.

[0053] In this document, the term "therapeuticly effective amount" refers to an amount of active ingredient (such as Triclocarban) sufficient to produce the desired therapeutic effect in a subject. This amount varies depending on factors such as the type and severity of the disease being treated, the specific active ingredient used, the subject's age, weight, health status, sex, diet, route of administration, duration of treatment, and any concomitant medications. Those skilled in the art can determine the therapeutically effective amount for a specific subject based on routine experimental procedures.

[0054] In this document, the term "pharmaceutical composition" refers to a composition comprising the Triclocarban described herein and at least one pharmaceutically acceptable excipient. The composition optionally comprises one or more other active ingredients (such as chemotherapeutic agents, immunotherapeutic agents, or targeted therapies). The pharmaceutical composition is intended to be administered to a subject for preventative or therapeutic purposes.

[0055] In this document, the term "pharmaceuticalally acceptable excipient" refers to all ingredients other than the active ingredient that are routinely used in the pharmaceutical field, are compatible with the active ingredient, and are non-toxic to subjects, including but not limited to solvents, diluents, excipients, disintegrants, binders, lubricants, surfactants, suspending agents, thickeners, preservatives, buffers, isotonic adjusters, pH adjusters, flavoring agents, and coloring agents. The excipients should be "pharmaceutically acceptable," meaning that, to a reasonable extent of medical judgment, they are suitable for use in contact with tissues of humans and animals without excessive toxicity, irritation, allergic reactions, or other problems or complications, and have a reasonable benefit / risk ratio.

[0056] In this document, the term "combination dosing" refers to administering Triclocarban to a subject in the form of the same pharmaceutical composition as one or more other active ingredients (such as chemotherapy drugs), or in the form of separate pharmaceutical compositions at the same or staggered time points. Combination dosing includes, but is not limited to, simultaneous dosing, separate dosing, and sequential dosing. Simultaneous administration: Two or more active ingredients are administered in the same formulation or in two separate formulations at the same time point; Separate administration: Two or more active ingredients are administered in separate formulations within the same or different time windows; Sequential administration: One active ingredient is administered first, followed by another active ingredient after a certain interval.

[0057] In this article, the term "chemotherapeutic drugs" refers to drugs used for the chemotherapy of malignant tumors, including but not limited to platinum compounds (such as cisplatin, carboplatin, and oxaliplatin), alkylating agents, antimetabolites, anthracyclines and their derivatives, antimicrotubule agents, topoisomerase inhibitors, targeted drugs, and hormonal drugs.

[0058] In this document, the terms "comprising" and "including" are used in this invention. The terms "comprising," "including," "having," and any variations thereof are open-ended expressions, indicating that other elements may exist in addition to those listed. In contrast, the term "consisting of" is a closed-ended expression, indicating that the invention consists only of the listed elements.

[0059] In this document, the term “about” is used to modify a numerical value or range of values ​​to indicate a reasonable range of deviations from that value or range as understood by those skilled in the art.

[0060] In this document, the terms "optional" and "optionally" indicate that the event or situation they modify may or may not occur, including both the possibility of the event or situation occurring and the possibility of it not occurring.

[0061] The technical solution of the present invention will be described in more detail below with reference to specific embodiments and accompanying drawings. In implementing the present invention, except for the experimental steps, conditions, and methods described in detail below, all other techniques and common knowledge in the art are employed, and no additional special limitations are imposed.

[0062] It should be noted that the embodiments described herein are only some typical examples of the present invention, and not all implementation methods. The following description of exemplary embodiments is for illustrative purposes only and does not constitute any limitation on the present invention or its application scenarios.

[0063] In the demonstration and discussion of the various embodiments, the specific numerical values ​​and parameters involved are for illustrative purposes only and are not intended to be limiting. Therefore, other embodiments based on the concept of this invention can have their relevant numerical values ​​adjusted according to actual needs.

[0064] In addition, unless otherwise specified, the experimental materials used in the examples are all commercially available biochemical reagents, and the experimental operations also follow the standard procedures in this field.

[0065] Example 1: Screening and validation of Triclocarban's PD-L1 degradation efficiency 1.1 Experimental Materials Cell lines: human embryonic kidney HEK293T cells (ATCC number: CRL-3216) and mouse breast cancer 4T1 cells (ATCC number: CRL-2539) were both purchased from the American Center for Type Culture Collection.

[0066] Experimental drug: Triclocarban (CAS No.: 101-20-2, compound number: CE04-5558), purchased from ChemDiv compound library; candidate compounds 1-10 (numbered as compound 1 to compound 10 respectively), purchased from ChemDiv compound library.

[0067] Main reagents: DMEM high glucose medium (Gibco, catalog number: 11965092), fetal bovine serum (FBS, Gibco, catalog number: 10099141), RIPA lysis buffer (Beyotime, catalog number: P0013B), BCA protein quantification kit (Beyotime, catalog number: P0012), PD-L1 antibody (Cell Signaling Technology, catalog number: 13684), Tubulin antibody (Cell Signaling Technology, catalog number: 2144), HRP-labeled goat anti-rabbit secondary antibody (Cell Signaling Technology, catalog number: 7074), PVDF membrane (Millipore, catalog number: IPVH00010), ECL chemiluminescent substrate (Millipore, catalog number: WBKLS0100).

[0068] 1.2 Experimental Methods (1) Cell seeding: HEK293T cells and 4T1 cells were seeded into 12-well cell culture plates, with 2×10⁶ cells seeded into each well. 5 Cells were added to DMEM complete medium containing 10% FBS and cultured in a saturated humidity incubator at 37°C and 5% CO2 for 24 hours. When the cells grew to 60%-70% confluence, they were treated with drugs.

[0069] (2) Compound screening and treatment: A solvent control group (DMSO) and a drug treatment group were set up. Each drug group was added with a candidate compound (Triclocarban was candidate compound No. 4) at a final concentration of 10 μM, and the solvent control group was added with an equal volume of DMSO (final concentration ≤0.1%). Each group was set up with 3 replicates. Cell samples were collected 24 hours after drug treatment.

[0070] (3) Protein extraction: Aspirate the culture medium and wash the cells twice with pre-cooled PBS. Add 100 μL of RIPA lysis buffer (containing protease inhibitor) to each well, lyse on ice for 15 minutes, scrape the lysate with a cell scraper and transfer it to a 1.5 mL centrifuge tube. Centrifuge at 12,000 rpm for 10 minutes at 4°C and collect the supernatant.

[0071] (4) Protein quantification and sample preparation: The protein concentration of each sample was determined by the BCA method. All samples were adjusted to the same protein concentration using RIPA lysis buffer, 5× protein loading buffer was added (to make the final concentration 1×), mixed well, heated in a 95℃ metal bath for denaturation for 5 minutes, immediately cooled in an ice bath, and then briefly centrifuged before use.

[0072] (5) SDS-PAGE electrophoresis: Take an equal amount of protein sample (30 μg per well) and load it onto a 12% polyacrylamide separating gel for electrophoresis. First, electrophore at a constant voltage of 80 V until the bromophenol blue indicator enters the separating gel, and then electrophore at a constant voltage of 120 V until the bromophenol blue indicator migrates to the bottom of the gel.

[0073] (6) Transfer: The separated protein was transferred to a PVDF membrane (pre-activated with methanol) using the wet transfer method. The transfer conditions were 250 mA constant current and 120 minutes under ice bath conditions.

[0074] (7) Blocking and Antibody Incubation: After transfer, place the PVDF membrane in TBST blocking buffer containing 5% skim milk powder and block on a shaker at room temperature for 1 hour. Dilute the primary antibody (PD-L1 antibody 1:1000, Tubulin antibody 1:2000) with TBST and incubate overnight on a shaker at 4°C. The next day, wash the membrane three times with TBST for 10 minutes each time. Dilute the HRP-labeled secondary antibody (1:2000) with TBST and incubate on a shaker at room temperature for 1 hour. Wash the membrane three times with TBST for 10 minutes each time.

[0075] (8) Chemiluminescence detection: Mix the ECL chemiluminescence substrate according to the instructions, add it evenly to the surface of the PVDF membrane, expose and develop it in the chemiluminescence imaging system, and detect the gray values ​​of the protein bands of PD-L1 and Tubulin.

[0076] 1.3 Experimental Results like Figure 2 As shown, candidate compounds were screened for PD-L1 degradation activity at a concentration of 10 μM in HEK293T and 4T1 cells. The results showed that candidate compound 4 (Triclocarban) exhibited the highest degradation efficiency for PD-L1 protein in both cell types compared to the other candidate compounds, while no significant change in PD-L1 protein levels was observed in the solvent control group (DMSO). The expression levels of the internal control protein Tubulin were largely consistent across the treatment groups, indicating that each drug treatment had no significant non-specific effect on overall cellular protein expression.

[0077] The above results indicate that Triclocarban can significantly promote the degradation of PD-L1 protein in HEK293T and 4T1 cells, and its degradation efficiency for PD-L1 is superior to other candidate compounds screened at the same time, demonstrating good targeted degradation specificity and high efficiency.

[0078] Example 2: Concentration-dependent degradation of PD-L1 protein levels in breast cancer cells by Triclocarban 2.1 Experimental Materials Cell lines: human breast cancer MCF-7 cells (ATCC number: HTB-22) and mouse breast cancer 4T1 cells (ATCC number: CRL-2539) were both purchased from the American Center for Type Culture Collection.

[0079] Experimental drug: Triclocarban (CAS No.: 101-20-2), purchased from Sigma-Aldrich (Catalog No.: 105937).

[0080] Main reagents: DMEM high glucose medium (Gibco, catalog number: 11965092), RPMI-1640 medium (Gibco, catalog number: 11875093), fetal bovine serum (Gibco, catalog number: 10099141), RIPA lysis buffer (Beyotime, catalog number: P0013B), BCA protein quantification kit (Beyotime, catalog number: P0012), PD-L1 antibody (Cell Signaling Technology, catalog number: 13684), Tubulin antibody (Cell Signaling Technology, catalog number: 2144), HRP-labeled secondary antibody (Cell Signaling Technology, catalog number: 7074).

[0081] 2.2 Experimental Methods (1) Cell seeding: MCF-7 cells and 4T1 cells were seeded into 6-well cell culture plates, with 4 × 10⁶ cells seeded into each well. 5 MCF-7 cells were cultured in RPMI-1640 medium containing 10% FBS, and 4T1 cells were cultured in DMEM medium containing 10% FBS. Cells were incubated at 37°C with 5% CO2 for 24 hours. Drug treatment was initiated when cells reached 60%-70% confluence.

[0082] (2) Concentration gradient treatment: MCF-7 cells and 4T1 cells were treated with Triclocarban at concentrations of 0, 0.1, 0.3, 1, 3 and 10 μM, respectively, with 3 replicates per group. Cell protein lysates were collected 24 hours after drug treatment.

[0083] (3) Western blot detection: Protein extraction, quantification, SDS-PAGE electrophoresis, membrane transfer, antibody incubation, and chemiluminescence detection were performed according to the method described in Example 1 to analyze the changes in PD-L1 protein levels in cells after treatment with different concentrations of Triclocarban. Tubulin was used as an internal control.

[0084] 2.3 Experimental Results like Figure 3As shown, in MCF-7 cells and 4T1 cells, Triclocarban is capable of reducing PD-L1 protein levels in a concentration-dependent manner. As the treatment concentration of Triclocarban increases from 0.1 μM to 10 μM, the gray values of the PD-L1 protein bands in both types of cells show a gradual decreasing trend. A significant reduction in PD-L1 protein level can be observed at a concentration of 3 μM, and the degradation effect is the most significant at a concentration of 10 μM. The housekeeping protein Tubulin shows consistent expression levels in all concentration treatment groups, indicating that the degradation effect of Triclocarban on PD-L1 is targeted and specific.

[0085] The above results indicate that Triclocarban can induce PD-L1 protein degradation in breast cancer cells in a concentration-dependent manner, can significantly reduce PD-L1 protein level at a concentration of 3 μM, and has a clear concentration-effect relationship.

[0086] Example 3: In vivo anti-tumor pharmacodynamic evaluation of Triclocarban used alone and in combination with cisplatin in a mouse breast cancer 4T1 model 3.1. Experimental materials Experimental animals: SPF grade female BALB / c mice, 8-10 weeks old, weighing 18-22 g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. [Laboratory Animal Production License No.: SCXK (Beijing) 2021-0011]. All animal experimental operations were reviewed and approved by the Institutional Animal Care and Use Committee, and comply with relevant requirements for laboratory animal welfare and ethics. The mice are housed in an SPF grade animal room, with the temperature controlled at 22-26°C, relative humidity of 40%-70%, 12-hour light-dark alternating illumination, and free access to food and water.

[0087] Cell line: Mouse breast cancer 4T1 cells (ATCC No.: CRL-2539).

[0088] Experimental drugs: Triclocarban (Sigma-Aldrich, Cat. No.: 105937), Cisplatin (Sigma-Aldrich, Cat. No.: P4394), Matrigel (Corning, Cat. No.: 356234).

[0089] Main reagents: Type I collagenase (Gibco, catalog number: 17100019), Zombie Violet dye (BioLegend, catalog number: 423114), PerCP / Cyanine 5.5 anti-mouse CD45 antibody (BioLegend, catalog number: 103132), PE / Cyanine 7 anti-mouse CD3 antibody (BioLegend, catalog number: 100320), FITC anti-mouse CD8a antibody (BioLegend, catalog number: 100706), anti-mouse Gzmb antibody (BioLegend, catalog number: 515406).

[0090] 3.2 Experimental Methods (1) Cell preparation: 4T1 cells were resuscitated and passaged one week in advance. When the cells reached the 3rd generation (logarithmic growth phase), the culture medium was discarded, and the cells were washed twice with sterile PBS. Cells were digested with 0.25% trypsin-EDTA digestion solution, and digestion was terminated with DMEM medium containing 10% FBS. The cell suspension was collected. The cells were centrifuged at 1,000 rpm for 5 minutes at 4°C, the supernatant was discarded, and the cells were resuspended in sterile PBS and counted. The cell density was adjusted to 4 × 10⁶ cells / year. 6 Cells / mL, take 100 μL of cell suspension (containing 4 × 10⁶ cells / mL). 5 Mix 100 μL of matrix gel with 100 μL of cells in a 1.5 mL centrifuge tube and store on ice for later use.

[0091] (2) In situ tumor inoculation: Mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg). After the mice were completely anesthetized, the skin of the second mammary fat pad area of ​​the mice was disinfected with povidone-iodine. The cell suspension was drawn up with a 1 mL syringe (with a 26G needle) and slowly injected into the second mammary fat pad of the mice to form a local wheal. After the injection, the injection site was gently pressed with a sterile cotton swab for a moment to prevent leakage of the cell suspension. The mice were placed on a constant temperature heating pad and returned to their cages after they woke up.

[0092] (3) Tumor measurement and grouping: Starting from the 3rd day after inoculation, the long and short diameters of the tumor were measured daily using digital calipers, and the tumor volume was calculated according to the following formula: Tumor volume (mm) 3 = 1 / 2 × major axis × minor axis 2 When the average tumor volume reaches approximately 30-50 mm... 3 On day 5 post-inoculation, tumor-bearing mice were randomly divided into 4 groups of 8 mice each: Solvent control group (Vehicle): Intraperitoneal injection of an equal volume of solvent control (5% DMSO + 25% PEG400 + 5% Tween-80 + 65% PBS), once daily; Triclocarban monotherapy group: Triclocarban 5 mg / kg (dissolved in the above solvent) was administered intraperitoneally once daily; Cisplatin monotherapy group: cisplatin 5 mg / kg (dissolved in the above solvent) was administered intraperitoneally once every 3 days; The combination therapy group received intraperitoneal injection of Triclocarban 5 mg / kg once daily, plus cisplatin 5 mg / kg every 3 days.

[0093] The administration volume for each group was 10 μL / g body weight, and the administration was continued until the experimental endpoint.

[0094] (4) Observation and Recording: During the drug administration period, the long and short diameters of the tumor were measured every two days, and the weight of the mice was also measured. The mental state, activity level, food intake, and water intake of the mice were observed. When the average tumor volume in the solvent control group reached 1000 mm... 3 The experiment was terminated on the 27th day after vaccination.

[0095] (5) Tumor tissue collection: At the end of the experiment, mice were euthanized by cervical dislocation and disinfected by immersion in 75% ethanol for 3 minutes. The tumor tissue was completely dissected using ophthalmic scissors and forceps in a clean bench. The surface blood was washed away with pre-cooled PBS, and the surface moisture was blotted with absorbent paper before weighing the wet weight of the tumor. 150 mg of the tumor parenchyma was used for flow cytometry analysis. The remaining tumor tissue was flash-frozen in liquid nitrogen and then transferred to a -80°C freezer for storage.

[0096] (6) Tumor tissue digestion and single-cell suspension preparation: 150 mg of tumor parenchyma tissue was placed in a sterile culture dish and cut into approximately 1 mm pieces using ophthalmic scissors. 3 Cells were broken into small fragments and transferred to 6-well plates containing 2 mL of serum-free DMEM medium. 200 μL of 10× type I collagenase (final concentration 1×) was added to each well, and the cells were gently shaken to mix. The plates were then incubated at 37°C for 30 minutes, with gentle pipetting every 10 minutes to promote dispersion. After digestion, 1 mL of DMEM medium containing 10% FBS was added to each well to terminate the digestion. The digest was passed through a 40 μm cell strainer to a 15 mL centrifuge tube. The filtrate was collected and centrifuged at 3,000 rpm for 3 minutes at 4°C, and the supernatant was discarded. The cells were resuspended in 1 mL of PBS containing 0.5% BSA, centrifuged at 3,000 rpm for 3 minutes, and the supernatant was discarded. The washing process was repeated once.

[0097] (7) Flow cytometry staining: Staining of live and dead cells: Add 100 μL of Zombie Violet working solution (1:100 PBS dilution) to the cell pellet, gently pipette to resuspend the cells, and stain at room temperature in the dark for 30 minutes. After staining, add 1 mL of PBS, centrifuge at 3,000 rpm for 3 minutes, and discard the supernatant.

[0098] CD8 + T cell surface staining: Add 100 μL of T cell surface antibody mixture (PerCP / Cyanine 5.5 anti-mouse CD45, PE / Cyanine 7 anti-mouse CD3, FITC anti-mouse CD8a, all diluted 1:100 with PBS containing 0.5% BSA) to each tube, resuspend the cell pellet, and stain at room temperature in the dark for 30 minutes. After staining, add 1 mL of PBS, centrifuge at 3,000 rpm for 3 minutes, and discard the supernatant.

[0099] Gzmb + CD8 + T cell intracellular staining: For samples detecting granzyme B, after surface staining, fixation / permeabilization buffer was added according to the instructions, followed by resuspending the cells in 100 μL of permeabilization buffer containing anti-mouse Gzmb antibody (1:100 dilution). Staining was performed at room temperature in the dark for 30 minutes. After staining, 1 mL of permeabilization buffer was added for washing, followed by centrifugation at 3,000 rpm for 3 minutes, and the supernatant was discarded.

[0100] Washing and resuspending: Resuspend cells in 1 mL PBS, centrifuge at 3,000 rpm for 3 minutes, and discard the supernatant. Finally, resuspend the cell pellet in 200 μL PBS, pass it through a 40 μm cell filter into a flow cytometry tube, and store on ice in the dark for analysis.

[0101] (8) Flow cytometry detection: Detection was performed using a BD LSR Fortessa flow cytometer, with at least 1 × 10⁻⁶ samples collected for each sample. 5 CD45 + Cells. Data analysis was performed using FlowJo software, with the following gatekeeping strategy: lymphocyte populations were identified using FSC / SSC scatter plots → cell adhesion bodies were excluded using FSC-A / FSC-H → live and dead cells were identified using Zombie Violet negative markers → CD45... + CD3 + Double positive delineation of T cells → CD8 + Positive screening for CD8 + T cells. Analysis of CD8 + T cells account for 45% of CD45 + CD3 +The percentage of T cells, and Gzmb + Cells account for CD3 + CD8 + The percentage of T cells.

[0102] A schematic diagram of the grouping and drug administration process in animal experiments is shown in Figure 4(a).

[0103] 3.3 Experimental Results (1) Inhibitory effect of Triclocarban on the growth of 4T1 xenografts of breast cancer in mice As shown in Figure 4(b), compared with the solvent control group, the tumor volume growth in the Triclocarban monotherapy group (5 mg / kg) was significantly slowed down, and the difference in tumor volume between the two groups reached a statistically significant level from day 8 of administration. At the endpoint of the experiment, the mean tumor volume in the solvent control group was 1382.9 ± 199.9 mm. 3 The triclocarban monotherapy group had a mean diameter of 684.6 ± 237.1 mm. 3 The tumor growth inhibition rate (TGI) was 50.5%. The mean tumor volume in the cisplatin monotherapy group was 756.1 ± 118.0 mm. 3 The TGI was 45.3%. The mean tumor volume in the combination therapy group was 389.5 ± 71.2 mm. 3 The TGI was as high as 71.8%, which was significantly better than any single drug group.

[0104] As shown in Figure 4(c), the trends of tumor weight and volume at the experimental endpoint were consistent. The average tumor weight was 1.9 ± 0.3 g in the solvent control group, 0.9 ± 0.2 g in the triclocarban monotherapy group, 1.1 ± 0.2 g in the cisplatin monotherapy group, and 0.6 ± 0.2 g in the combination therapy group. All groups showed statistically significant differences in tumor weight compared to the solvent control group. P< 0.0001 The combination therapy group also showed statistically significant differences compared to each single-drug group. P<0.01 ).

[0105] (2) The regulatory effect of Triclocarban on the tumor immune microenvironment As shown in Figure 4(d), flow cytometry results showed that CD8+ in the tumor tissue of the solvent control group was significantly lower than that in the control group. + T cells account for 45% of CD45 + CD3 + The proportion of T cells was 6.5% ± 0.1%. This proportion significantly increased to 10.3% ± 3.3% in the Triclocarban monotherapy group. P<0.01 The cisplatin monotherapy group had a success rate of 12.3% ± 3.0%. P<0.001The combined medication group further increased to 17.5% ± 4.7% ( P<0.0001 vs solvent control group; P<0.01 vs Triclocarban monotherapy group ).

[0106] As shown in Figure 4(e), Gzmb in tumor tissue of the solvent control group + CD8 + T cells account for a significant portion of CD8. + The proportion of T cells was 41.5% ± 4.3%. This proportion increased to 48.5% ± 5.5% in the Triclocarban monotherapy group. P<0.05 In the cisplatin monotherapy group, the success rate was 48.4% ± 6.9%. P<0.05 The combined medication group further increased to 59.1% ± 4.0% ( P<0.0001 vs solvent pair Control group; P<0.001 vs Triclocarban monotherapy group ).

[0107] During the administration period, the mice in each group did not experience a significant decrease in body weight, and no obvious toxic reactions (such as diarrhea, piloerection, arched back, reduced activity, and other abnormal behaviors) were observed, indicating that Triclocarban was well tolerated at a dose of 5 mg / kg.

[0108] In summary, triclocarban (5 mg / kg, intraperitoneal injection, once daily) significantly inhibited tumor growth and reduced tumor volume and weight in a 4T1 breast cancer mouse model. Its antitumor mechanism is at least partially achieved through the following pathway: increasing tumor-invasive CD8... + The proportion of T cells was increased, while granzyme B-positive CD8 cells with direct cytotoxic activity were upregulated. + The proportion of T cells can be increased, thereby reversing the immunosuppressive state of the tumor microenvironment and restoring the T cell-mediated anti-tumor immune response.

[0109] When triclocarban is used in combination with the first-line chemotherapy drug cisplatin, it exhibits a synergistic effect superior to either drug alone in terms of tumor growth inhibition rate and immune microenvironment remodeling, suggesting that triclocarban can be used as a chemotherapy sensitizer in combination with standard chemotherapy regimens.

[0110] The above examples demonstrate that Triclocarban effectively blocks the PD-1 / PD-L1 immune checkpoint signaling pathway by targeting and degrading the PD-L1 protein, thereby exerting its anti-tumor effect by inhibiting tumor cell proliferation and activating anti-tumor immunity.

[0111] The embodiments described in this specification are only intended to help those skilled in the art better understand and implement the present invention. For those skilled in the art, appropriate adjustments, substitutions, or extensions can be made to these embodiments based on the content disclosed in this invention without requiring creative effort, and the basic principles of the invention can be applied to other similar scenarios. Therefore, the disclosed embodiments should not be considered as limiting the scope of protection of this invention. Any equivalent modifications, improvements, or alternatives made based on this invention without departing from its essential spirit should fall within the scope of protection of this invention.

Claims

1. The use of the compound shown in formula (I) in the preparation of antitumor drugs or antitumor drug compositions; Formula (I) The chemical name of the compound shown in formula (I) is triclocarban, and its CAS number is 101-20-2.

2. The application according to claim 1, characterized in that, The application is the preparation of drugs or drug compositions for the treatment of PD-L1 positive malignant tumors.

3. The application according to claim 2, characterized in that, The PD-L1 positive malignant tumors are selected from one or more of the following: breast cancer, non-small cell lung cancer, small cell lung cancer, gastric cancer, esophageal cancer, liver cancer, colorectal cancer, pancreatic cancer, renal cell carcinoma, bladder cancer, prostate cancer, ovarian cancer, cervical cancer, melanoma, head and neck squamous cell carcinoma, and thyroid cancer.

4. The application according to claim 1, characterized in that, The triclocarban content in a unit dosage form of the drug or drug composition is from 1 mg to 500 mg.

5. The application according to claim 1, characterized in that, The route of administration of the drug or drug composition is selected from one of the following: oral administration, sublingual administration, rectal administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, pulmonary inhalation administration, mucosal absorption administration, topical administration, or transdermal absorption administration.

6. The application according to claim 1, characterized in that, The drug or pharmaceutical composition comprises a therapeutically effective amount of the compound shown in formula (I) and at least one pharmaceutically acceptable excipient; the excipient is selected from one or more of solvents, diluents, excipients, disintegrants, binders, lubricants, surfactants, suspending agents, thickeners, preservatives, buffers, isotonic regulators, pH regulators, flavoring agents, and coloring agents.

7. The application according to claim 1, characterized in that, The dosage form of the drug or drug composition is selected from one of the following: tablets, capsules, granules, powders, pills, drops, oral solutions, oral suspensions, oral emulsions, injections, lyophilized powder injections, ointments, creams, gels, patches, liniments, lotions, sprays, aerosols, or powder sprays.

8. Use of the compound shown in formula (I) in combination with chemotherapeutic drugs in the preparation of antitumor drug compositions; the chemical name of the compound shown in formula (I) is triclocarban, and the CAS number is 101-20-2.

9. An antitumor drug composition, characterized in that, The pharmaceutical composition comprises a therapeutically effective amount of the compound represented by formula (I) and at least one pharmaceutically acceptable excipient; the chemical name of the compound represented by formula (I) is triclocarban, and the CAS number is 101-20-2; the pharmaceutical composition further comprises at least one second active ingredient selected from one or more chemotherapeutic drugs, immunotherapeutic drugs, or targeted therapies.

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