An antitumor pharmaceutical composition and its application

Abstract

This invention relates to the field of biomedical technology, and more particularly to an anti-tumor drug and its application; the pharmaceutical composition comprises quercetin or a pharmaceutically acceptable derivative thereof, and leucine. This pharmaceutical composition achieves anti-tumor activity against tumor cells and CD8+. + Bidirectional differential regulation of BCAT1 in T cells can simultaneously achieve "tumor suppression" and "immune activation".

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an antitumor pharmaceutical composition and its application. Background Technology

[0002] Colorectal cancer (CRC) is the third most common malignant tumor worldwide, and the second leading cause of death among malignant tumors. Although early-stage in situ colorectal cancer can be cured by surgical resection with a high 5-year survival rate, approximately 50% of patients will experience distant metastases. Lung metastases account for 10%-20% of metastatic cases, making it the second most common site of metastasis after liver metastases. Once lung metastases occur in colorectal cancer, the 5-year survival rate drops significantly, becoming a core bottleneck affecting patient prognosis.

[0003] Currently, clinical treatment options for colorectal cancer lung metastases are limited, primarily relying on systemic chemotherapy, targeted therapy, and immune checkpoint inhibitors. Immune checkpoint inhibitors (such as PD-1 monoclonal antibodies) have shown some efficacy in patients with microsatellite instability-high / mismatch repair deficient (MSI-H / dMMR) colorectal cancer lung metastases. However, the majority (95%) of patients with microsatellite stable / mismatch repair normal (MSS / pMMR) colorectal cancer respond extremely poorly to single-agent immune checkpoint inhibitor therapy, offering almost no clinical benefit. Furthermore, existing chemotherapy and targeted therapy regimens generally suffer from limited response rates, easy induction of drug resistance, and significant toxic side effects, failing to meet the clinical treatment needs for colorectal cancer lung metastases, especially MSS-type colorectal cancer lung metastases. Therefore, developing novel and effective treatment strategies has become a critical issue that urgently needs to be addressed.

[0004] In recent years, immunometabolic dysregulation in the tumor microenvironment (TME) has been identified as a key mechanism leading to the failure of antitumor immunity. In the immunosuppressive TME, tumor cells interact with CD8+... + T cells fiercely compete for key metabolic substrates such as glucose and amino acids, leading to cytotoxic CD8. + T cells experience metabolic exhaustion, significantly suppressing their core function of killing tumor cells. Branched-chain amino acid transaminase 1 (BCAT1), a key enzyme in branched-chain amino acid metabolism, is abnormally highly expressed in various malignant tumors and is closely related to tumor proliferation, invasion, metastasis, and resistance to EGFR inhibitors. Based on this, existing technologies have developed therapeutic strategies targeting BCAT1 in tumor cells, including small molecule inhibitors and antisense nucleic acids, aiming to directly inhibit the metabolic activity of tumor cells to block their growth and metastasis.

[0005] However, existing BCAT1 targeting technologies have significant drawbacks: the research perspective is entirely limited to tumor cells themselves, completely ignoring the role of BCAT1 in immune cells, especially CD8. + The potential role of quercetin in T cell function regulation cannot be restored by remodeling TME immune metabolism to restore anti-tumor immunity. Meanwhile, quercetin, as a widely studied natural flavonoid, has had numerous reports on its anti-inflammatory, antioxidant, and anti-tumor activities, including direct inhibition of tumor cell proliferation and apoptosis. Existing technologies (such as patents CN114040751A and EP2185533A2) disclose the application of quercetin or its derivatives in anti-tumor activity, but their mechanisms of action mainly focus on inducing tumor cell apoptosis, inhibiting the cell cycle, or inducing immunogenic cell death. They have not revealed the preventive and therapeutic effects of quercetin on distant metastasis of cancer cells (especially colorectal and lung metastases), nor have they linked it with CD8. + It has not established associations with specific metabolic targets within T cells (such as BCAT1) nor has it combined with branched-chain amino acids such as leucine to achieve synergistic regulation of immune metabolism.

[0006] Branched-chain amino acids (especially leucine) are essential for maintaining CD8. + Quercetin is an important nutrient for T cell effector function and activation of the mTORC1 pathway. However, no anti-tumor combination of quercetin and BCAT1, which regulates the dual metabolism of tumor cells and immune cells by targeting BCAT1, has been reported to date. Currently, there are no reports, either domestically or internationally, on "quercetin combined with leucine targeting CD8." + There are no reports on the technology of "T-cell BCAT1 prevention and treatment of colorectal cancer lung metastasis", and no studies have revealed the role of BCAT1 in CD8. + Mediating role in T cell metabolism and function regulation. Developing novel drug compositions with quercetin and leucine in combination and bidirectional regulation of BCAT1 as the core is of great significance for overcoming the treatment bottleneck of MSS-type colorectal cancer with lung metastases. Summary of the Invention

[0007] The technical problem to be solved by this invention is to provide an anti-tumor drug composition containing quercetin and leucine, which addresses the shortcomings of existing technologies and achieves effective targeting of tumor cells and CD8+. + Bidirectional differential regulation of BCAT1 on T cells can simultaneously achieve "tumor suppression" and "immune activation".

[0008] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0009] An antitumor pharmaceutical composition comprising quercetin or a pharmaceutically acceptable derivative thereof, and leucine.

[0010] As a preferred technical solution, the weight ratio of quercetin to leucine is 1:1 to 1:10.

[0011] As a further preferred technical solution, the weight ratio of quercetin to leucine is 1:1 to 1:5.

[0012] As an improved technical solution, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, forming a formulation suitable for oral or injectable administration.

[0013] The present invention relates to the use of a pharmaceutical composition in the preparation of a medicament for the treatment and / or prevention of cancer, wherein the cancer is a solid tumor; the cancer is colorectal cancer, lung cancer, breast cancer, or melanoma; preferably for the prevention or treatment of lung metastases from colorectal cancer.

[0014] This invention also protects the pharmaceutical composition in the preparation of drugs that regulate BCAA metabolism and enhance CD8 in the tumor microenvironment. + Use in drugs that enhance T-cell function.

[0015] The pharmaceutical composition works by: (1) upregulating BCAT1 in tumor cells, promoting BCAA degradation, and inhibiting tumor cell proliferation; (2) in CD8 + Inhibiting BCAT1 in T cells increases extracellular leucine utilization, activates the mTORC1 pathway, and promotes T cell effector differentiation; (3) reduces T cell exhaustion markers PD-1 and TIM-3, and increases the secretion of IFN-γ, granzyme B, and perforin.

[0016] After adopting the above technical solution, the beneficial effects of the present invention are:

[0017] This invention discloses for the first time BCAT1 in CD8 + Based on the key regulatory role of quercetin in T cells, a pharmaceutical composition containing quercetin and leucine was developed. This composition, on the one hand, upregulates BCAT1 expression in tumor cells, accelerates the breakdown of branched-chain amino acids (BCAAs), cuts off the "fuel" supply to tumor cells, and directly inhibits their proliferation and metastasis; on the other hand, it inhibits CD8... + The activity of BCAT1 in T cells is reduced, thus reducing the breakdown and consumption of extracellular leucine by T cells; this achieves a dual-targeting and synergistic tumor-suppressing effect.

[0018] Furthermore, by enhancing the bioavailability of leucine in the tumor microenvironment, CD8 is effectively activated. + The mTORC1 signaling pathway within T cells promotes T cell differentiation into effector phenotypes and significantly enhances their ability to secrete key cytotoxic molecules such as interferon-γ (IFN-γ), granzyme B, and perforin. Furthermore, this composition significantly reduces CD8+. +The expression of T cell surface exhaustion markers PD-1 and TIM-3 effectively reverses the T cell exhaustion state caused by metabolic competition, restoring and maintaining its long-term anti-tumor immune function. Especially for MSS-type colorectal cancer lung metastases, which are extremely difficult to treat clinically, this invention transforms "cold tumors" into "hot tumors" through immune metabolic remodeling, providing a novel and effective treatment strategy for patients with MSS-type colorectal cancer, which accounts for up to 95% of cases. Attached Figure Description

[0019] Figure 1 This is a diagram showing the inhibitory effect of quercetin on the growth and metastasis of colorectal cancer cells in vitro.

[0020] Figure 2 Quercetin promotes the increase of CD8 in peripheral blood + Figure 1 shows the effector differentiation of T cells and the results of quercetin's in vivo inhibition of experimental colorectal cancer lung metastasis.

[0021] Figure 3 Quercetin remodels the effector function of CD8+ T cells in the tumor microenvironment and alleviates their exhaustion state. (Results diagram)

[0022] Figure 4 Reprogramming CD8 for quercetin + A diagram showing the results of branched-chain amino acid metabolism in T cells;

[0023] Figure 5 The diagram shows the results of quercetin directly binding to and inhibiting BCAT1;

[0024] Figure 6 The diagram shows the results of quercetin and leucine synergistically enhancing anti-tumor immunity and inhibiting lung metastasis;

[0025] Figure 7 Representative H&E stained sections of multiple organs from healthy mice and colorectal cancer lung metastasis model mice after intraperitoneal injection of quercetin;

[0026] Figure 8 The results of flow cytometry analysis of the proportions of peripheral blood T cells, B cells and their differentiation subsets Tn, Tcm and Teff in healthy mice after intraperitoneal injection of quercetin.

[0027] Figure 9 To detect the presence of tumor-inducing molecules in peripheral blood of mice in the control and quercetin-treated groups by flow cytometry.

[0028] CD4 in lymph nodes and lung metastases + T cells, CD8 + T cells and B220 + A graph showing the proportion of B cells;

[0029] Figure 10The levels of CD4+ in peripheral blood, tumor draining lymph nodes, and lung metastases of mice in the control and quercetin-treated groups were detected by flow cytometry. + T cells, CD8 + A graph showing the results of T cells and the proportions of their Tn, Tcm, and Teff subsets;

[0030] Figure 11 Quercetin downregulates BCAT1 in MC38 cells, inhibits p-S6, and promotes CD8. + Graph showing the results of T cell-mediated tumor cell apoptosis;

[0031] Figure 12 GZMB levels in lung metastases of control and quercetin-treated mice were detected by flow cytometry. + CD8 + T cells, IFN-γ + CD8 + T cells and Perforin + CD8 + The graph shows the results of the T cell ratio. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0033] Example 1

[0034] An antitumor pharmaceutical composition comprising quercetin and leucine, mixed in a weight ratio of 1:1.

[0035] Prescription: Quercetin 50g, leucine 50g, microcrystalline cellulose 30g, sodium carboxymethyl starch 8g, appropriate amount of povidone K30 (prepared into a 5% aqueous solution as a binder) and magnesium stearate 1g, to make 1000 tablets in total;

[0036] Quercetin, leucine, microcrystalline cellulose, and sodium carboxymethyl starch were each passed through an 80-mesh sieve and thoroughly mixed. A suitable amount of 5% povidone K30 aqueous solution was added to prepare a soft mass, which was then passed through a 20-mesh sieve to form wet granules. The wet granules were dried in an oven at 50-60℃, controlling the moisture content to below 3%. The dried granules were then sized by passing through a 20-mesh sieve, and magnesium stearate was added and mixed thoroughly. Tableting was performed using a suitable die to obtain tablets containing 50mg of quercetin and 50mg of leucine per tablet.

[0037] Example 2

[0038] An antitumor pharmaceutical composition comprising quercetin and leucine, mixed in a weight ratio of 1:3.

[0039] Prescription: Quercetin 25g, leucine 75g, lactose 40g, sodium carboxymethyl starch 10g, appropriate amount of povidone K30 (prepared into a 5% aqueous solution as a binder) and magnesium stearate 1.5g, to make 1000 tablets;

[0040] Tablets were prepared according to the preparation method of Example 1, each containing 25 mg of quercetin and 75 mg of leucine.

[0041] Example 3

[0042] An antitumor pharmaceutical composition comprising quercetin and leucine, mixed in a weight ratio of 1:5.

[0043] Prescription: Quercetin 20g, Leucine 100g, Mannitol 30g, Hydroxypropyl-β-Cyclodextrin 100g, add water for injection to 2000 mL;

[0044] Preparation method (lyophilized powder for injection):

[0045] a. Under aseptic conditions, add the prescribed amounts of leucine, mannitol, and hydroxypropyl-β-cyclodextrin to approximately 80% of the total volume of water for injection and stir to dissolve;

[0046] b. Add quercetin, stir to ensure complete encapsulation and dissolution, and adjust the pH to 7.0–8.5 if necessary;

[0047] c. Add 0.1% (w / v) needle-grade activated carbon and stir at room temperature for 15 minutes;

[0048] d. Remove carbon and filter, then add water for injection to the full volume;

[0049] e. Fine filtration using a 0.22 μm microporous membrane, resulting in a clear filtrate.

[0050] f. Dispense into 2 mL portions per vial;

[0051] g. Half-stuffed, freeze-dried: pre-freezing: -45℃, 3 h; sublimation drying: -20℃~0℃, 20 h

[0052] Drying: 25℃, 4 h;

[0053] h. Vacuum stopper and capping to obtain the product. Each vial contains 20 mg of quercetin and 100 mg of leucine. Dissolve in normal saline or 5% glucose injection before intravenous infusion.

[0054] Example 4

[0055] An antitumor pharmaceutical composition comprising quercetin and leucine, mixed in a weight ratio of 1:8.

[0056] Prescription: Quercetin 12.5g, Leucine 100g, Mannitol 50g, Hydroxypropyl-β-cyclodextrin 37.5g, add water for injection to 1000mL;

[0057] Preparation method (lyophilized powder for injection): Following the lyophilized powder preparation method in Example 3, quercetin and leucine were mixed in a 1:8 ratio to obtain an injection containing 12.5 mg of quercetin and 100 mg of leucine per vial.

[0058] Example 5

[0059] An antitumor pharmaceutical composition comprising quercetin and leucine, mixed in a weight ratio of 1:10.

[0060] Prescription (for 1000 capsules): Quercetin 10g, Leucine 100g, Microcrystalline cellulose 25g, Pregelatinized starch 15g, Magnesium stearate 1.5g;

[0061] Preparation method (capsule form):

[0062] Quercetin, leucine, microcrystalline cellulose, and pregelatinized starch were each passed through an 80-mesh sieve and mixed evenly using an equal-volume incremental method. Then, magnesium stearate was added and the mixture was thoroughly mixed. Finally, the mixed powder was directly filled into hollow capsules of appropriate size to obtain capsules containing 10 mg of quercetin and 100 mg of leucine per capsule.

[0063] This study employed an integrated approach, combining in vitro functional experiments, metabolomics, in vivo metabolite models, and computational target prediction to elucidate the immunometabolic effects of quercetin. BCAT1 was identified as the direct physical target of quercetin, and its role in reprogramming BCAA metabolism to activate CD8 was elucidated. + The role of T cells in function. See below for details:

[0064] I. Experimental Materials and Methods

[0065] 1. Human tissue samples: Clinical samples were obtained from Hai'an Hospital Affiliated to Nantong University. Primary tumor specimens from nine colorectal cancer patients were analyzed using multiplex immunofluorescence staining. All participants signed written informed consent forms before enrollment. The study protocol was reviewed and approved by the Ethics Committee of Hai'an People's Hospital (Approval No.: 2025-L025).

[0066] 2. Cells: SW480 and MC38 cells were purchased from the Cell Bank of the Chinese Academy of Sciences. Cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) at 37°C and 5% CO2.

[0067] Cell viability assay (CCK-8): SW480 cells were inoculated at 1.2 × 10⁶ cells per well.4 8.5×10 3 and 7×10 3 MC38 cells were seeded at a density of 3 × 10⁶ cells per well in 96-well plates. 3 1.5×10 3 and 1.2×10 3 Cells were seeded at a density of [number] cells per well in 96-well plates for assays at 24h, 48h, and 72h, and incubated overnight. Cells were then treated with quercetin (MCE) at concentrations of 0, 12.5, 25, 50, 100, and 150 μM. At each specified time point, the DMEM medium containing 10% (v / v) fetal bovine serum (FBS) was replaced with 90 μL of serum-free DMEM medium, and 10 μL of CCK-8 reagent (APExBIO) was added to each well. Incubation was continued at 37°C and 5% (v / v) CO2 for 40 minutes; absorbance was measured using a microplate reader.

[0068] Scratch healing assay: SW480 cells were introduced at a rate of 1 × 10⁶ cells per well. 6 MC38 cells were seeded at a density of 2 × 10⁶ cells per well in 6-well plates. 5 Cells were seeded at a density of [number] cells per well and cultured overnight until fully confluent. Three parallel vertical lines were drawn in each well using a 200 μL pipette tip. The wells were then gently rinsed twice with 1X PBS (pH 7.4) to remove detached cells. The scratched areas were located under a microscope, and images were taken 0 h after scratching. Cells were then treated with quercetin (MCE) at concentrations of 0, 12.5, 25, 50, 100, and 150 μM. Images of each field of view were taken 24 h and 48 h after scratching. The scratch area was processed and analyzed using ImageJ software.

[0069] Apoptosis assay: SW480 cells were introduced at a density of 1 × 10⁶ cells per well. 6 MC38 cells were seeded at a density of 1.5 × 10⁶ cells per well in 6-well plates. 5 Cells were seeded at a density of [number] cells per well in 6-well plates and incubated overnight. Cells were treated with quercetin at concentrations of 0, 25, 50, 100, and 150 μM for 48 hours. After treatment, the supernatant was discarded, and the cells were washed once with 1×PBS (pre-chilled at 4°C). Cells were digested with 500 μL of EDTA-free trypsin (KeyGEN), neutralized, and collected by centrifugation at 1000 rpm for 5 minutes. The cell pellet was washed twice with 1×PBS (pre-chilled at 4°C) and resuspended in 500 μL of 1×Binding Buffer. Annexin V-FITC (2.5 μL) and PI (2.5 μL) (KeyGEN) were added to each sample, and the samples were incubated in the dark for 15 minutes. The samples were gently mixed and analyzed using flow cytometry.

[0070] Colony formation assay: Cells were seeded at a density of 600 cells per well in 6-well plates and then treated with quercetin at concentrations of 0, 25, 50, and 100 μM, respectively. The culture medium was changed every 3 days for a total of 14 days. After incubation, the cells were washed once with 1× PBS (pH 7.4) and fixed with 1 mL of 4% (w / v) paraformaldehyde (BOSTER) for 15 min. After fixation, the cells were washed three times with PBS and air-dried. They were then stained with 1 mL of crystal violet (Beyotime, 0.5%) in the dark for 20 min, washed three times with 1× PBS (pH 7.4), air-dried, and photographed.

[0071] 3. Animal tumor model and in vivo treatment: To establish a mouse model of colorectal cancer lung metastasis, MC38 cells (5 × 10⁻⁶) were administered via tail vein injection. 5 Mice were monitored twice daily, morning and evening. Approximately 18 days after injection, all animals were euthanized. To evaluate the effects of quercetin and leucine, mice with established colorectal cancer lung metastases were divided into three groups. The quercetin group received intraperitoneal injections of quercetin (50 mg / kg; MCE) every two days. The leucine group received leucine (50 mg / kg) via gavage according to the same protocol. The combination group received both drugs simultaneously, following the exact same dosing regimen.

[0072] Lymphocytes were isolated from mouse peripheral blood / tissue: Peripheral blood (100 μL per mouse) was collected from the orbital venous plexus and diluted 1:9 with 1X PBS (pH 7.4), then centrifuged at 450 × g for 15 min. The pellet was treated with ACK lysis buffer for 5 min to lyse red blood cells, washed with PBS, and resuspended in PBS containing 2% FBS. Inguinal lymph nodes (ILNs) were excised and washed in PBS. The tissue was then separated by mechanically grinding it on a 100 μm cell sieve using a syringe plunger. The resulting homogenate was filtered through a sieve, centrifuged at 300 × g for 7 min, and treated with ACK lysis buffer for 5 min to remove red blood cells. After washing with PBS, the cells were resuspended in PBS supplemented with 1% FBS. Lung metastases were fragmented into 2-3 mm pieces and placed in RPMI-1640 medium containing 10% (v / v) heat-inactivated FBS (ABW), 1% (v / v) penicillin-streptomycin (Beyotime), and 1% (w / v) type III collagenase (Worthington Biochemical). Enzymatic digestion was performed at 37°C for 30 minutes. The digested tissue was passed through a 100 μm cell sieve and centrifuged at 300 × g for 7 minutes. The pellet was resuspended in 40% Percoll and centrifuged at 800 × g for 15 minutes to separate lymphocytes. The interfacial layer containing lymphocyte-enriched cells was collected, treated with ACK lysis buffer for 5 minutes to remove red blood cells, washed with PBS, and finally resuspended in PBS supplemented with 2% (v / v) FBS. These procedures were performed according to the previously described method.

[0073] Flow cytometry and T-cell phenotype analysis: used for surface staining, cells were packed at 1 × 10⁶ cells per well. 6 Cells were seeded at a density of [number] cells per well in 96-well plates and incubated at 4°C for 30 minutes with antibodies labeled with fluorescent dyes against CD45, CD3, B220, CD8, and CD4 (diluted 1:200 in PBS containing 2% FBS). To assess the production of intracellular GZMB, perforin, and IFN-γ, cells were stimulated with CellActivation Cocktail (containing brevidin A, BioLegend) for 6 hours prior to fixation and permeabilization.

[0074] CD8 + Negative selection and in vitro culture of T cells: Lymphocytes were isolated from lung metastases and spleen under aseptic conditions using the aforementioned tissue processing methods. Then, mouse CD8+ cells were used... + The T-cell sorting kit (BEAVER) enriches CD8 cells through negative selection. + T cells. The purified cells were cultured on plates pre-coated with anti-CD3 (3 μg / mL) and anti-CD28 (1 μg / mL) antibodies. After 48 hours of culture, quercetin was added to the culture medium to a final concentration of 10 μM.

[0075] Histopathological analysis: The extent of lung metastasis was first assessed through gross morphological observation and lung weight / body weight ratio. Lung tissue containing metastatic lesions was fixed in formalin and embedded in paraffin. Serial sections were prepared from the paraffin-embedded lung tissue blocks using a Leica microtome, followed by hematoxylin and eosin (H&E) staining. Whole-slide imaging of the sections was performed using a KFBIO digital pathology slide scanner to capture the maximum cross-sectional area of ​​the lung for further analysis.

[0076] Multiplex immunofluorescence (mIHC) staining: Formalin-fixed, paraffin-embedded (FFPE) tissue sections (4 μm thick) from nine patients with or without metastatic colorectal cancer were dewaxed and hydrated. Sections were incubated in EDTA buffer (pH 9.0) at 99°C for 30 minutes for heat-induced antigen retrieval. After blocking, sections were incubated at 37°C for 60 minutes with primary antibodies: anti-CD8 (Zsbio), anti-CD4 (Zsbio), anti-BCAT1 (Abcam), anti-BCAT2 (Abcam), anti-PD-1 (Abcam), and anti-TCF-1 (CST). Subsequently, sections were incubated at 37°C for 20 minutes with HRP-conjugated secondary antibodies. Tyramine signal amplification (TSA) was performed using fluorescently labeled tyramine, following these steps: CD8 was treated with CF430 (Biotium, 96053), CD4 with AF488 (AAT, 11070), PD-1 with AF594 (AAT, 11082), TCF-1 with Cy3 (AAT, 11065), BCAT2 with Cy5 (AAT, 11066), and BCAT1 with XTSA690 (AKF, AXT6710000), incubated for 10 minutes at room temperature each time. Between each TSA cycle, the antibody was stripped by incubation at 99°C for 8 minutes in EDTA buffer at pH 9.0. Finally, cell nuclei were counterstained with DAPI.

[0077] Western blot: Total cellular proteins were extracted using lysis buffer suitable for both Western blotting and immunoprecipitation. 20 μg of protein from each sample was separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was then blocked with PBS containing 5% skim milk powder for 1 hour at room temperature. After blocking, the membrane was incubated overnight at 4°C with primary antibodies targeting specific proteins (including anti-BCAT1, anti-BCAT2, anti-BCKDH-α, anti-BCKDH-B, anti-DBT, and anti-β-actin). Following primary antibody incubation, the membrane was incubated with HRP-conjugated secondary antibody for 1 hour at room temperature. Protein detection was performed using enhanced chemiluminescence (ECL) substrates, and imaging was performed using a chemiluminescence imaging system.

[0078] Quantitative reverse transcription polymerase chain reaction (RT-qPCR): The RT-qPCR procedure is as follows. Total RNA was extracted from tumor cells or cultured cells using Trizol reagent (Invitrogen) according to the manufacturer's instructions. cDNA was synthesized from the extracted RNA using the HighCapacity RNA-to-cDNA kit (Applied Biosystems, Vazyme) according to the manufacturer's protocol. Real-time PCR was performed using SYBR Green Master Mix (Vazyme). Gene expression levels were determined based on the threshold cycle (Ct) value measured during the linear amplification phase. All data were normalized with GAPDH expression as an internal control and relative quantification was performed using the ΔΔCt method.

[0079] Molecular docking: The crystal structure of BCAT1 (PDBID: 7NWA) was preprocessed in PyMOL 3.0, including the removal of heteroatoms (such as solvent molecules, ions, and small molecules) and structural repair. The protein receptor and small molecule ligand (quercetin) were then prepared separately in AutoDock Tools 4 and saved as pdbqt files. Global docking of the processed receptor and ligand was performed using AutoDockVina 1.2.5. Default docking parameters were used: grid spacing = 0.375 Å, exhaustiveness = 8. The prepared pdbqt file was used as input, and docking simulations were run under specified settings. The docking results were sorted according to docking scores, and the conformation with the highest score was selected for further analysis. Protein-ligand interactions were analyzed using Discovery Studio Visualizer 2024, and structural visualizations were generated using PyMOL 3.0.

[0080] Molecular dynamics simulations: Molecular dynamics simulations were performed using the GROMACS software package. The protein receptor was described using the AMBER99SB-ILDN all-atom force field, while ligand parameters and topology were generated using the generic AMBER force field to ensure compatibility. The protein-ligand complex was placed in a cubic simulation box with a minimum distance of 1.0 nm between the solute and the box edge. The system was solvated using the SPC216 water model, with Na added. + or Cl -Ions neutralize the total net charge. Energy minimization is first performed using the steepest descent method to eliminate any spatial conflicts. The system then equilibrates in two consecutive phases: an isochoric ensemble and an isobaric ensemble. After equilibration, a 10 ns production MD simulation is performed using the CUDA toolkit with GPU acceleration. Structural stability and flexibility are assessed by calculating the root mean square deviation, root mean square fluctuations, and radius of gyration. Intermolecular interactions are evaluated using hydrogen bond analysis and center-of-mass distance analysis. Finally, a free energy landscape is constructed based on the relative Gibbs free energy, calculated according to the probability distributions of the first two principal components (PC1 and PC2).

[0081] Surface plasmon resonance (SPR) analysis: SPR analysis was performed using the Biacore system. All run buffers were 1×PBS-P+ (pH 7.4). For ligand immobilization, the CM5 sensor chip was installed according to the manufacturer's instructions. Channel 2 was activated using an equal volume mixture of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) at a flow rate of 10 μL / min. Recombinant human BCAT1 protein was then diluted to 50 μg / mL with sodium acetate buffer (pH 5.0) and immobilized onto the activated channel at the same flow rate, resulting in a final immobilization level of approximately 9655 response units (RU). The remaining active groups were blocked with ethanolamine-HCl (pH 8.5). Channel 1 underwent the same activation and blocking procedure, but was injected only with protein-free sodium acetate buffer as a reference surface for background subtraction. Solvent correction was performed using a calibration series of DMSO concentrations ranging from 4.5% to 5.8% prior to analyte binding assays to account for buffer effects. For kinetic analysis, quercetin was serially diluted in running buffer containing 5% DMSO and injected at a flow rate of 30 μL / min onto both the protein and reference surfaces for 150 seconds (binding phase), followed by buffer washing for dissociation. The sensing surface was regenerated with 10 mM glycine-HCl (pH 2.0) between each cycle. Binding kinetics were analyzed using Biacore Insight Evaluation Software. The sensor plot was double-referenced, and a globally fitted 1:1 Langmuir binding model was used to determine the binding rate constant (Ka), dissociation rate constant (Kd), and dissociation constant (KD).

[0082] Statistical Analysis: Flow cytometry data were analyzed using FlowJo 10.8.1 (BD Biosciences). Statistical analysis was performed using GraphPad Pism 10 and R (v4.1.2) in RStudio. Comparisons among multiple groups were performed using two-way ANOVA followed by Bonferroni post-hoc tests. Data are presented as mean ± standard error of the mean (SEM) of at least three independent experiments, with specific sample sizes specified in the legend.

[0083] II. Experimental Results and Analysis

[0084] 1. Inhibitory effect of quercetin on the growth and metastasis of colorectal cancer cells in vitro.

[0085] To evaluate the direct antitumor effect of quercetin, its in vitro effects on the viability, migration, apoptosis, and colony formation ability of colorectal cancer cells were first examined. Using CCK-8 and scratch assays, quercetin was found to inhibit the proliferation of human SW480 and mouse MC38 colorectal cancer cells in a dose- and time-dependent manner, and significantly inhibit their migration ability. Figure 1 AD). Further analysis using Annexin V-FITC / PI staining revealed that quercetin promoted early and late apoptosis in both cell lines in a dose-dependent manner. Figure 1 E, F). Notably, quercetin induced a significant increase in late apoptosis in SW480 cells, detectable even at a concentration of 25 μM. Furthermore, colony formation assays confirmed its potent inhibitory effect on long-term colony formation survival. Figure 1 G). In summary, these results indicate that quercetin has a direct growth-inhibiting and anti-metastatic effect on colorectal cancer cells in vitro.

[0086] in Figure 1 In the images, A) shows the viability of human SW480 and mouse MC38 colorectal cancer cells treated with different doses of quercetin using the CCK-8 assay (n=3). B) shows the quantitative analysis of the scratch healing / migration ability of SW480 and MC38 cells after quercetin treatment (n=3). C) shows representative images of SW480 cell scratch assays. D) shows representative images of MC38 cell scratch assays. E) shows flow cytometry contour plots illustrating apoptosis in SW480 and MC38 cells after quercetin treatment (Annexin VFITC / PI staining). F) shows the quantitative analysis of early and late apoptosis in E) (n=3). G) shows the clonogenic assay and corresponding quantitative analysis of quercetin-treated SW480 and MC38 cells (n=3).

[0087] 2. Quercetin promotes the increase of CD8+ in peripheral blood. + T cell effector differentiation

[0088] Inspired by its direct in vitro antitumor activity, the immunomodulatory potential of quercetin in vivo was then evaluated. Figure 2 A). Healthy mice received intraperitoneal injections of quercetin on days 1, 3, and 5. Figure 2 A). Serial orbital blood samples were collected on days 0, 2, 4, and 6, and comprehensive immunophenotypic analysis was performed by flow cytometry. Analysis showed dynamic and time-dependent changes in major lymphocyte subsets. By day 6, CD4... + and CD8 + The proportion of T cells was significantly increased in all cases. In contrast, the proportion of B cells briefly increased on day 2, then decreased on days 4 and 6. Figure 2 B, C; Supporting infographic S2A). Examination of T cell differentiation status shows naïve CD4. + T cells (Tn, CD62L) + CD44 - The proportion gradually decreased, reaching its lowest point on day 4, followed by a slight rebound on day 6. Conversely, CD4... + Central memory T cells (Tcm, CD62L) + CD44 + The proportion of CD4 increased significantly on day 4, while the effector CD4 + T cells (Teff, CD62L) - / lowCD44 + No significant changes were observed in CD8. + A similar trend was observed in T cells: a decrease in the initial subset, while CD8... + Tcm increased significantly on day 4. Notably, CD8... + The proportion of Teff increased significantly by day 4, and although it decreased slightly by day 6, it remained significantly higher than baseline. In summary, these data indicate that in vivo quercetin treatment induced systemic immune stimulation characterized by total T cell expansion and promoted effector differentiation, particularly in CD8 cells. + Within the T cell population ( Figure 2 D, E; Support Infographic S4).

[0089] in Figure 2 In the diagram, A) shows the timeline of drug administration and sampling. Healthy mice were intraperitoneally injected with quercetin on days 1, 3, and 5, and orbital blood was collected on days 0, 2, 4, and 6. B) shows the concentration of CD4+ in peripheral blood as detected by flow cytometry. + T cells, CD8 + Quantitative analysis of the change in T cell and B cell ratio over time (n=3). C) is a representative flow cytometry contour plot showing peripheral blood CD45. + CD4 in cell population + and CD8+ T cell population, D) is CD4. + and CD8 + Quantitative analysis of the time-dependent changes in Tn, Tcm, and Teff subsets within a T cell population (n=4). E) represents the CD4 subpopulation of the specified population. + (Tn, Tcm, Teff) and CD8 + Flow cytometry contour plots of the (Tn, Tcm, Teff) subsets. F) Schematic diagram of the experimental lung metastasis model and treatment regimen. Lung metastasis was established by tail vein injection of MC38 cells, followed by quercetin or control treatment. G) Body weight changes in mice in the control and quercetin-treated groups after tumor inoculation. H) Left: Lung weight / body weight ratio of mice in the control and quercetin-treated groups. Right: Statistical count of the number of metastatic nodules visible on the lung surface (n=6). I) Representative gross images of the lungs of mice in the control and quercetin-treated groups. J) Representative H&E-stained sections of lung tissue showing metastatic lesions. K) Representative H&E-stained sections of the liver, kidney, and spleen of mice in the control and quercetin-treated groups.

[0090] 3. Quercetin inhibits experimental colorectal cancer lung metastasis in vivo.

[0091] Based on these immunostimulatory effects observed in healthy mice, we subsequently established an experimental lung metastasis model of colorectal cancer to evaluate the therapeutic potential of quercetin against tumor dissemination. Figure 2 F). Lung metastasis was induced by tail vein injection of MC38 cells, followed by quercetin treatment or control treatment. Compared with the control group, mice treated with quercetin showed significantly reduced weight loss (F). Figure 2 G). Consistent with the reduction in metastatic burden, the lung weight / body weight ratio was significantly lower in the quercetin group, and the number of metastatic nodules visible on the lung surface was significantly reduced. Figure 2 H). Gross anatomical examination and subsequent H&E staining of lung tissue confirmed a significant reduction in metastatic lesions (H). Figure 2 I,J). Histological examination of other major organs did not show obvious signs of quercetin-induced toxicity, but a significant increase in spleen size was observed in the treatment group, suggesting a possible systemic immune response. Figure 2 J,K).

[0092] 4. Quercetin activates effector function and alleviates CD8. + T cell exhaustion

[0093] To investigate whether reduced metastasis is associated with enhanced antitumor immunity, we analyzed the immune cell phenotypes in peripheral blood, lung metastases, and tumor draining lymph nodes (TdLNs) using flow cytometry. Quercetin treatment significantly altered the systemic and local T cell patterns. At the systemic level, CD8+ in peripheral blood... + The proportion of T cells increased significantly ( Figure 3A; Supporting Infographic S3). A consistent shift from the initial phenotype to the effector phenotype was observed in both the tumor microenvironment and drainage sites. In TdLNs and lung metastases, quercetin treatment led to an initial CD8+ shift. + The proportion of T cells was significantly reduced, accompanied by CD8. + A significant increase in Teff. Despite the presence of CD4 in TdLNs and metastatic lesions. + The total proportion of T cells did not change significantly, but their composition changed markedly: naïve CD4+ + T cells decreased, while CD4 decreased. + Teff cells increased. Quercetin treatment also significantly enhanced CD8+ expression in lung metastases. + Effector functions of T cells ( Figure 3 BD; Support Infographic S4). Functional analysis shows that CD8 generates IFN-γ. + The proportion of T cells increased significantly, and CD8 cells expressing key cytotoxic mediators granzyme B (GZMB) and perforin were observed. + The proportion of T cells also increased significantly, collectively indicating a strong enhancement in its cytotoxic activity. Figure 3 E; Supports Infographic S6).

[0094] T cell exhaustion status was assessed using multiplex immunofluorescence staining. Quercetin treatment significantly reduced exhausted CD8+ cells in lung metastases. + The proportion of T cells, expressed as PD1 + CD8 + and TIM3 + CD8 + Decreased cell population frequency ( Figure 3 F, G). The reduction of these classic depletion markers indicates that quercetin not only enhances effector function but also alleviates CD8+ depletion in the tumor microenvironment. + The state of T cell depletion.

[0095] in Figure 3 A) CD4 counts in peripheral blood (n=4), tumor draining lymph nodes (TdLNs) (n=4), and lung metastases (n=5) from mice in the control group and quercetin-treated group. + T cells, CD8 + T cells and B220 + The proportion of B cells. B) represents CD4+ in lung metastases (n=5), peripheral blood (n=4), and TdLNs (n=4). + With CD8 + The proportions of Tn, Tcm, and Teff subsets within T cells. C) represents CD4+ in lung metastases, peripheral blood, and TdLNs. + (Tn, Tcm, Teff) and CD8 +Flow cytometry contour maps of T cell subsets (Tn, Tcm, Teff). D) represents CD8+ originating from lung metastases. + Representative flow cytometry contour plots of T cells expressing IFN-γ, GZMB, and perforin. E) shows IFN-γ in lung metastases. + GZMB + and perforin CD8 + Quantitative analysis of T cells. F) shows the detection of CD8+ cells in lung metastases using multiplex immunofluorescence. + TCF1 + CD8 + PD1 + and CD8 + TIM3 + Quantitative analysis of T cells (n=5). G) shows representative images of H&E staining and multiplex immunofluorescence from lung transfer sections of mice in the control and quercetin-treated groups.

[0096] 5. Quercetin reprogramming CD8 + Branched-chain amino acid metabolism in T cells

[0097] Given the observed CD8 + With T cell function activation, we hypothesized that quercetin may exert its effects by remodeling its metabolic state (a key determinant of T cell effector potential). Untargeted metabolomics analysis of lung metastases in tumor-bearing mice showed significant alterations in branched-chain amino acid (BCAA) metabolism after quercetin treatment. Figure 4 AC).

[0098] To directly examine whether quercetin regulates CD8 in the complex microenvironment of lung metastases. + T cell-associated BCAA metabolic pathways were first isolated from lung metastases in untreated tumor-bearing mice. + T cells, after in vitro activation, were treated with quercetin for transcriptional analysis. Figure 4 D). Transcriptional analysis showed that quercetin significantly downregulated the mRNA expression of key BCAA catabolic enzymes, particularly BCAT1 and BCAT2, as well as several other enzymes involved in this pathway. Figure 4 E). To validate these findings in vivo, CD8 was purified by magnetic sorting from the spleen of quercetin-treated mice. + T cells, protein levels were validated by Western blotting. Figure 4 F). Consistent with the transcriptional data, the protein levels of BCAT1, BCAT2, and other related metabolic enzymes were also significantly reduced. These results indicate that quercetin broadly inhibits CD8 by downregulating multiple key enzymes. + BCAA metabolism in T cells.

[0099] To investigate the clinical relevance of BCAA metabolism in T cells, we used multiplex immunofluorescence staining to analyze the primary tumor tissues of 9 colorectal cancer patients. Figure 4 H). Our results showed that patients with distant metastases had a significantly higher proportion of CD8+ in their primary tumors. + BCAT1 + T cells, and CD8 + BCAT1 + TCF1 + A significant increase in T cells. In contrast, CD8... + BCAT2 + or CD4 + BCAT1 + The proportion of T cells was not significantly correlated with metastatic status. Figure 4 I). These findings suggest that CD8 + BCAT1 expression is elevated in T cells, especially in TCF1. + Within this subgroup, it may be associated with colorectal cancer metastasis.

[0100] in Figure 4 In the diagram, A) is a bubble plot showing the non-targeted metabolomics analysis of lung metastases in the control group and quercetin-treated tumor-bearing mice. B) is a KEGG pathway enrichment analysis showing the metabolic pathways that were significantly altered in the lung metastases after quercetin treatment. C) is a schematic overview of the BCAA catabolic pathway. D) is a schematic diagram of the experimental procedure: isolation of CD8 from lung metastases / spleen of tumor-bearing mice. + T cells were activated in vitro using anti-CD3 / CD28 antibodies in the presence or absence of quercetin. E) In vitro quercetin-treated CD8 cells activated by quercetin, as determined by RT-qPCR. + mRNA expression levels of key BCAA-catabolizing enzymes (BCAT1, BCAT2, etc.) in T cells (n=4). F) is a representative Western blot image showing CD8 activation after quercetin treatment in vivo. + Protein expression of BCAA pathway-related enzymes in T cells. G) Quantitative analysis of Western blot results from group (F), normalized with β-actin as an internal reference. H) Representative multiplex immunofluorescence images of primary tumor tissues from colorectal cancer patients with or without distant metastasis. I) Quantitative analysis of immunofluorescence data from group (H), comparing CD8+ expression in primary tumors from patients with or without distant metastasis. + BCAT1 + T cells and CD8 + BCAT1 + TCF1 + The proportion of T cells and other subsets (n=9).

[0101] 6. Quercetin directly binds to and inhibits BCAT1.

[0102] To elucidate the molecular mechanism by which quercetin mediates BCAT1 inhibition, a computer simulation screening of potential BCAT1 binders was first performed using the HERB database. This analysis identified quercetin as a candidate ligand. Figure 5 A). Subsequent molecular docking simulations predicted a high binding affinity between quercetin and BCAT1. Figure 5 B). Detailed analysis of the binding interface revealed a stable complex interaction network, including hydrogen bonds, Pi-hydrogen bonds, electrostatic interactions (Pi-cationic), hydrophobic interactions (Pi-alkyl), and van der Waals forces. Specifically, hydrogen bonds are formed with residues Ser331, Tyr161, Val289, Arg119, and Thr333 on chain A of BCAT1, and Leu173 on chain B. Additional interactions include Pi-hydrogen bonds with Thr333 (chain A), Pi-cationic interactions with Lys222 (chain A), and Pi-alkyl interactions with Ala334 (chain A). These computational data strongly support that quercetin is a direct binder of BCAT1, providing a structural basis for its inhibitory effect. Figure 5 CD).

[0103] To verify this prediction from a biophysical perspective, we then employed surface plasmon resonance (SPR) analysis to assess the direct interaction between recombinant human BCAT1 protein and quercetin. Figure 5 (EF). SPR binding kinetics confirmed a strong and specific interaction. Quercetin exhibited rapid binding and slow dissociation with human BCAT1 protein, indicating high binding affinity and stable complex formation, consistent with molecular docking results, and providing direct biophysical evidence for quercetin's role as a BCAT1 binder.

[0104] in Figure 5 A) Screening quercetin for candidate ligands identified as BCAT1 using the HERB database. B) Molecular docking simulations predict a high binding affinity between quercetin and BCAT1. C)-D) Detailed views of the predicted binding interface between quercetin and BCAT1. E) Representative sensor map showing human BCAT1 protein immobilized on an SPR chip. A total of 9655 response units (RU) of protein were bound. F) The SPR binding sensor map and derived kinetic parameters (binding rate constant Ka 1.70e+03 (1 / Ms), dissociation rate constant Kd 6.98e-03 (1 / s), dissociation constant KD 4.11e-06 (M)) confirm the strong and specific interaction between quercetin and BCAT1.

[0105] 7. Quercetin and leucine synergistically enhance anti-tumor immunity and inhibit lung metastasis.

[0106] To investigate the functional interaction between quercetin and BCAA metabolism in vivo, three treatment cohorts were established: quercetin monotherapy, leucine monotherapy, and quercetin combined with leucine. Figure 6 A). Gross assessment of lung metastases showed a significant reduction in metastatic burden in both the quercetin monotherapy group and the combination therapy group. Figure 6 B). Consistent with this observation, compared with the untreated control group, the lung weight / body weight ratio was significantly reduced in all treatment groups, with the most significant reductions in the quercetin monotherapy and combination therapy groups. Figure 6 C). Importantly, the combination therapy resulted in the most significant reduction in the number of visible tumor nodules (C). Figure 6 D).

[0107] CD8 from lung metastases + Flow cytometry analysis of cytotoxic molecules in T cells showed that all three treatment groups exhibited GZMB. + A significant increase in the proportion of cells ( Figure 6 E). Both the quercetin monotherapy group and the quercetin combined with leucine group showed IFN-γ. + and Perforin + CD8 + The proportion of T cells increased significantly, with the most significant enhancement observed in the combination therapy group. Figure 6 F,G). It is worth noting that Perforin in the joint group... + CD8 + The frequency of T cells increased nearly 10-fold compared to the untreated control group.

[0108] in Figure 6 A) Schematic diagram of in vivo treatment strategy. Mice with established MC38 lung metastases received quercetin, leucine, or a combination thereof. B) Representative gross images of the lungs of mice in the untreated control group and each treatment group. C) Representative H&E-stained sections of lung tissue showing metastatic lesions. D) Statistics on the lung weight / body weight ratio (n=4) and the number of visible metastatic nodules on the lung surface in each treatment group. E) Cytotoxic CD8 from lung metastases. + Flow cytometry analysis of T cells. GZMB in the designated treatment group. + IFN-γ + and Perforin + CD8 + Quantitative analysis of representative flow cytometry contour plots of T cell proportions (n=3).

[0109] This study reveals a novel mechanism by which the binding of quercetin to BCAT1 enhances CD8 activation. +The cytotoxicity of T cells and the inhibition of metastatic tumor growth provide a theoretical basis for targeting BCAA metabolism in the treatment of advanced colorectal cancer. These results indicate that quercetin not only possesses independent anti-metastatic activity but also synergistically inhibits lung metastasis with leucine, supporting the therapeutic potential of co-targeting BCAA metabolism to enhance anti-tumor immunity and control metastasis.

[0110] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An antitumor pharmaceutical composition, characterized in that, The pharmaceutical composition comprises quercetin or a pharmaceutically acceptable derivative thereof, and leucine.

2. The antitumor pharmaceutical composition according to claim 1, characterized in that, The weight ratio of quercetin to leucine is 1:1 to 1:

10.

3. The antitumor pharmaceutical composition according to claim 2, characterized in that, The weight ratio of quercetin to leucine is 1:1 to 1:

5.

4. The antitumor pharmaceutical composition according to claim 1, characterized in that, The pharmaceutical composition further comprises a pharmaceutically acceptable carrier and is formulated as a preparation suitable for oral or injectable administration.

5. Use of a pharmaceutical composition according to any one of claims 1-3 in the preparation of a medicament for treating and / or preventing cancer.

6. The use according to claim 5, characterized in that, The cancer is a solid tumor; the cancer is colorectal cancer, lung cancer, breast cancer, or melanoma.

7. The use according to claim 6, characterized in that, Used for the prevention or treatment of lung metastases from colorectal cancer.

8. The pharmaceutical composition according to any one of claims 1-3, in the preparation of a drug that regulates BCAA metabolism and enhances CD8 in the tumor microenvironment. + Use in drugs that enhance T-cell function.

9. The use according to claim 8, characterized in that, The pharmaceutical composition works by: (1) upregulating BCAT1 in tumor cells, promoting BCAA degradation, and inhibiting tumor cell proliferation; (2) in CD8 + Inhibiting BCAT1 in T cells increases extracellular leucine utilization, activates the mTORC1 pathway, and promotes T cell effector differentiation; (3) reduces T cell exhaustion markers PD-1 and TIM-3, and increases the secretion of IFN-γ, granzyme B, and perforin.

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