Use of N-acetyl-L-phenylalanine in the preparation of a drug against colorectal cancer

Abstract

The application provides application of N-acetyl-L-phenylalanine in preparation of an anti-colorectal cancer drug, and belongs to the technical field of medicines.The application proves, for the first time, that N-acetyl-L-phenylalanine has significant anti-colorectal cancer activity and good safety through systematic in-vitro cell experiments, in-vivo pharmacodynamic experiments of animal models and comprehensive safety evaluation.The application provides a brand-new strategy for treatment of colorectal cancer.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of N-acetyl-L-phenylalanine in the preparation of anti-colorectal cancer drugs. Background Technology

[0002] Current treatments for colorectal cancer suffer from significant side effects and widespread drug resistance. While commonly used chemotherapy drugs (such as 5-fluorouracil, oxaliplatin, and irinotecan) have some efficacy in anti-tumor treatment, their toxic side effects are substantial, often causing myelosuppression, neurotoxicity, and gastrointestinal reactions, severely impacting patients' quality of life and treatment adherence. Platinum-based chemotherapy-induced neuropathy (CIPN) is a common dose-limiting toxicity factor. Simultaneously, drug resistance in colorectal cancer cells is becoming increasingly prominent; with each subsequent chemotherapy session, patients often develop resistance, leading to a significant decline in the effectiveness of subsequent treatments. While targeted therapies and immunotherapy have expanded clinical options, they are only effective for patients with specific molecular subtypes (e.g., KRAS G12C mutations account for approximately 2%-3% of colorectal cancer cases), limiting their applicability to a limited population. Furthermore, some targeted therapies also exhibit drug resistance and toxic side effects.

[0003] Therefore, new treatment strategies for colorectal cancer need to be developed. Summary of the Invention

[0004] In view of this, in order to overcome the shortcomings of the prior art, the present invention is proposed.

[0005] The first aspect of the present invention provides the use of N-acetyl-L-phenylalanine or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating colorectal cancer.

[0006] Furthermore, the pharmaceutically acceptable salts of the N-acetyl-L-phenylalanine include, but are not limited to, one or more of its alkali metal salts, alkaline earth metal salts, ammonium salts, and organic amine salts.

[0007] Furthermore, the alkali metal salt includes, but is not limited to, sodium salt, potassium salt, or lithium salt.

[0008] Furthermore, the alkaline earth metal salts include, but are not limited to, calcium or magnesium salts.

[0009] Furthermore, the ammonium salt includes a salt formed by ammonium ions and N-acetyl-L-phenylalanine.

[0010] Furthermore, the N-acetyl-L-phenylalanine or a pharmaceutically acceptable salt thereof exists in anhydrous form, hydrate, solvate, crystalline form, amorphous form, or mixtures thereof.

[0011] Furthermore, the colorectal cancer is selected from colorectal adenocarcinoma.

[0012] Furthermore, the drug is used to inhibit the proliferative activity of colorectal cancer cells.

[0013] Furthermore, the drug is used to promote apoptosis in colorectal cancer cells.

[0014] Furthermore, the drug is used to inhibit the migration of colorectal cancer cells.

[0015] Furthermore, the drug is used to inhibit the lateral migration of colorectal cancer cells.

[0016] Furthermore, the drug is used to inhibit the invasion of colorectal cancer cells.

[0017] Furthermore, the drug is used to inhibit the growth of colorectal cancer tumors.

[0018] Furthermore, the colorectal cancer cells were selected from RKO cells and MC38 cells.

[0019] Furthermore, the drug also includes pharmaceutically acceptable carriers and / or excipients.

[0020] A second aspect of the present invention provides a pharmaceutical composition for treating colorectal cancer, the pharmaceutical composition comprising N-acetyl-L-phenylalanine or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier and / or excipient.

[0021] Furthermore, the pharmaceutical composition may also include other drugs for treating colorectal cancer.

[0022] Furthermore, the other drugs for treating colorectal cancer include, but are not limited to, fluorouracil drugs (e.g., 5-fluorouracil), platinum drugs (e.g., oxaliplatin), irinotecan, raltitrexed, TAS-102, bevacizumab, and cetuximab.

[0023] Furthermore, the dosage forms of the pharmaceutical composition include, but are not limited to, oral formulations, injectable formulations, implantable formulations, or topical formulations. The oral formulations include, but are not limited to, tablets, coated tablets, dispersible tablets, sustained-release tablets, controlled-release tablets, capsules, soft capsules, granules, powders, pills, oral liquids, syrups, suspensions, and emulsions.

[0024] A third aspect of the present invention provides a method for treating colorectal cancer, the method comprising administering N-acetyl-L-phenylalanine or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition described in the second aspect of the present invention.

[0025] The advantages and beneficial effects of this invention are as follows: This invention provides the application of N-acetyl-L-phenylalanine in the preparation of anti-colorectal cancer drugs. Through systematic in vitro cell experiments, in vivo pharmacodynamic experiments in animal models, and comprehensive safety evaluations, this invention is the first to demonstrate that N-acetyl-L-phenylalanine possesses significant anti-colorectal cancer activity with good safety. This invention provides a novel strategy for the treatment of colorectal cancer. Attached Figure Description

[0026] Figure 1 The figure shows the experimental results of the inhibitory effect of N-acetyl-L-phenylalanine on colorectal cancer cells in vitro. Figure 1 In this figure, A represents the dynamic changes in cell proliferation activity of the human colorectal cancer cell line RKO after treatment with different concentrations of N-acetyl-L-phenylalanine (Ac-Phe). Figure 1 B in the figure represents the results of detecting the effect of Ac-Phe on the proliferation of mouse colorectal cancer cell line MC38 using the CCK-8 assay; Figure 1 C in the figure represents the apoptosis of RKO cells treated with 80 µM Ac-Phe for 24 hours, as detected by Annexin V-FITC / 7-AAD double staining flow cytometry. Figure 1 Figure D in the figure represents the results of evaluating the effect of 80 µMAc-Phe on the migration ability of RKO cells using the Transwell chamber method. Figure 1 E in the figure represents the results of the scratch healing experiment.

[0027] Figure 2 The figure shows the experimental results of N-acetyl-L-phenylalanine inhibiting the growth of subcutaneous colorectal cancer tumors. Figure 2 A in the diagram represents the tumor-bearing and drug administration process in C57BL / 6 mice; Figure 2 In the figure, B represents the size of a representative subcutaneous tumor in mice after treatment with solvent control group, Ac-Phe 10 mg / kg group, 50 mg / kg group, 200 mg / kg group and Bacteroides rich in oleate group; Figure 2 C in the graph represents the change in subcutaneous tumor volume over time in C57BL / 6 mice; Figure 2 In the figure, D represents the weight of subcutaneous tumors in C57BL / 6 mice; Figure 2 E in the diagram represents the tumor-bearing and drug administration process in BALB / c nude mice; Figure 2 F in the figure represents the subcutaneous tumor results of representative BALB / c nude mice after treatment with solvent control group and Ac-Phe group (50mg / kg); Figure 2 G in the graph represents the change in subcutaneous tumor volume over time in BALB / c nude mice; Figure 2 The figure shows the weight of subcutaneous tumors in BALB / c nude mice, represented by H.

[0028] Figure 3The results of the safety assessment of N-acetyl-L-phenylalanine are as follows: Figure 3 In this context, A represents the in vivo safety evaluation procedure for healthy C57BL / 6 mice; Figure 3 B in the figure represents the comparison of mouse weight changes before and after the experiment; Figure 3 In this figure, C represents the result of ELISA detection of IL-6 concentration in the serum of two groups of mice; Figure 3 In this context, D represents the serum IL-1β concentration in two groups of mice as determined by ELISA. Figure 3 E in the figure represents the immunohistochemical staining result of intestinal tissue. Detailed Implementation

[0029] The present invention will be further described below with reference to embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make equivalent modifications to the disclosed technical content to create equivalent embodiments. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the invention are all within the protection scope of the present invention.

[0030] Example 1: N-acetyl-L-phenylalanine has an inhibitory effect on colorectal cancer cells in vitro. 1. Experimental Materials 1.1 N-acetyl-L-phenylalanine

[0031] 1.2 Experimental cell lines

[0032] 1.3 Main Reagents

[0033] 1.4 Main Instruments and Equipment

[0034] 2. Experimental Methods 2.1 Cell Culture After resuscitating RKO and MC38 cells, respectively, they were seeded into corresponding culture media containing 10% fetal bovine serum and 1% penicillin-streptomycin, and cultured in incubators at 37°C, 5% CO2, and saturated humidity. When the cell confluence reached 80%-90%, the cells were digested with 0.25% trypsin-EDTA and passaged. Cells in the logarithmic growth phase were used for subsequent experiments.

[0035] 2.2 CCK-8 assay for cell proliferation activity Objective: To detect changes in the proliferation activity of RKO and MC38 cells at different time points after treatment with different concentrations of Ac-Phe.

[0036] Experimental Procedure: RKO and MC38 cells in logarithmic growth phase were digested with trypsin to prepare single-cell suspensions, and counted using a cell counter. Cell density was adjusted to 5 × 10³ cells / well, and 100 µL was seeded into each well of a 96-well plate. Sterile PBS was added to the edge wells to prevent edge effects. The 96-well plates were pre-cultured at 37°C with 5% CO2 for 24 hours to allow cell adhesion. The old culture medium was removed, and fresh culture medium containing different concentrations of Ac-Phe was added. Ac-Phe concentrations were set as follows: 0 µM (control group, containing an equal volume of DMSO, final DMSO concentration ≤0.1%), 5 µM, 20 µM, 80 µM, and 120 µM. Each concentration was used in 5 replicates. After adding the drug, the cells were cultured for another 5 days, and CCK-8 assays were performed at the corresponding time points of day 0, day 1, day 2, day 3, day 4, and day 5. For assay, add 10 µL of CCK-8 solution to each well, gently tap the plate to mix, and incubate for 1-4 hours (the specific incubation time depends on the cell type and color change; generally, RKO and MC38 cells are incubated for 2 hours). Measure the absorbance (OD) at 450 nm using a microplate reader. 450 (With time as the horizontal axis, OD) 450 The values ​​are used as the ordinate to plot the cell proliferation curve.

[0037] 2.3 Flow cytometry detection of apoptosis Objective: To detect the effect of Ac-Phe treatment on apoptosis in RKO cells.

[0038] Experimental procedure: RKO cells in the logarithmic growth phase were taken, digested with trypsin to prepare a single-cell suspension, and then subjected to 2×10⁻⁶ cells / cells. 5 Cells were seeded at a density of 2 cells / well in 6-well plates, with 2 mL of culture medium per well. Incubated at 37°C in a 5% CO2 incubator for 24 hours to allow cell adhesion. The old culture medium was removed, and fresh culture medium containing 0 µM (control group, equal volume of DMSO) and 80 µM Ac-Phe was added, respectively, and cultured for another 24 hours. Cell collection: The culture medium was carefully aspirated into centrifuge tubes (retaining suspended cells). Adhering cells were washed once with PBS, and trypsin without EDTA was added to digest the adherent cells. Digestion was stopped with the collected culture medium. Cells from the same treatment group were combined into centrifuge tubes. Centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were washed twice with pre-cooled PBS. Cells were resuspended in 1× Binding Buffer, and the cell density was adjusted to 1×10⁶ cells / well. 6 Cells / mL. Take 100 µL of cell suspension (containing 1×10⁶ cells / mL). 5Add 5 µL Annexin V-FITC and 5 µL 7-AAD staining solution to each flow cytometry tube, and mix gently. Incubate at room temperature in the dark for 15 minutes. Add 400 µL 1× Binding Buffer to each tube, mix gently, and analyze by flow cytometry within 1 hour. Flow cytometry analysis shows that Annexin V-FITC positive (green fluorescence) cells are early apoptotic cells, and 7-AAD positive (red fluorescence) cells are late apoptotic / necrotic cells. Calculate the total apoptotic cell ratio (early apoptosis + late apoptosis).

[0039] 2.4 Transwell chamber cell migration and invasion assay Objective: To investigate the effects of Ac-Phe treatment on the migration and invasion abilities of RKO cells.

[0040] 2.4.1 Transfer Experiment Experimental procedure: RKO cells in logarithmic growth phase were digested with trypsin, resuspended in serum-free MEM medium, counted, and the cell density was adjusted to 2 × 10⁶ cells / year. 5 Cells / mL. Add 600 µL of complete culture medium containing 10% FBS (as a chemokine) to each well of a 24-well plate, and place the Transwell chamber (8.0 μm pore size) into the 24-well plate. Take 200 µL of cell suspension (containing 4 × 10⁶ cells / mL). 4 Cells were added to the upper chamber of a Transwell plate. The experiment was divided into two groups: control group: serum-free medium containing an equal volume of DMSO (final DMSO concentration ≤0.1%) was added to the upper chamber; Ac-Phe group: serum-free medium containing 80 µM Ac-Phe was added to the upper chamber. Each group had 3 replicates. The 24-well plate was incubated at 37°C with 5% CO2 for 24 hours. The Transwell chambers were removed, and the cells and medium in the upper chamber were gently wiped away with a cotton swab. The chambers were gently washed once with PBS, and then fixed in methanol for 15 minutes. The chambers were removed, washed once with PBS, and stained with 0.1% crystal violet for 20 minutes. The chambers were washed three times with PBS and air-dried at room temperature. Five fields of view (200×) were randomly selected under an inverted microscope for photographing, and the number of cells that crossed the lower chamber membrane was counted. The average value was taken as the number of migrated cells.

[0041] 2.4.2 Invasion Experiment Experimental Procedure: Matrigel Coating: Remove Matrigel (Corning, catalog number 356234) from -20°C and thaw overnight at 4°C. Dilute Matrigel 1:8 with pre-chilled serum-free MEM medium (e.g., 50 µL Matrigel + 350 µL pre-chilled serum-free medium). Add 60 µL of the diluted Matrigel solution to each well in the upper chamber of the Transwell, gently agitate to evenly coat the membrane surface, and incubate at 37°C for 2 hours to allow the Matrigel to solidify. Aspirate any unsolidified liquid before use. Take RKO cells in the logarithmic growth phase, trypsinize them, resuspend them in serum-free MEM medium, count them, and adjust the cell density to 3 × 10⁻⁶ cells / well. 5 Cells / mL (more cells are needed in the invasion assay because some cells are blocked by Matrigel). Add 600 µL of complete culture medium containing 10% FBS to each well of a 24-well plate, and place the Matrigel-coated Transwell chambers into the 24-well plate. Take 200 µL of cell suspension (containing 6 × 10⁶ cells / mL). 4 Cells were added to the upper chamber of a Transwell plate. Experimental groups were the same as for the migration experiment: a control group (equal volume of DMSO) and an 80 µM Ac-Phe group, with three replicates per group. The 24-well plate was incubated at 37°C in a 5% CO2 incubator for 48 hours (invasion requires a longer time than migration). The Transwell chamber was removed, and uninvaded cells and Matrigel were gently wiped away with a cotton swab. Subsequent fixation, staining, photography, and counting procedures were the same as for the migration experiment (methanol fixation for 15 minutes → crystal violet staining for 20 minutes → PBS washing → microscopic photography and counting).

[0042] 2.5 Scratch Healing Test Objective: To investigate the effect of Ac-Phe treatment on the lateral migration ability of RKO cells.

[0043] Experimental procedure: RKO cells in the logarithmic growth phase were digested with trypsin to prepare a single-cell suspension, and then subjected to 5×10⁻⁶ cells / cells. 5Cells were seeded at a density of 1 cell / well in 6-well plates, with 2 mL of culture medium per well. The plates were incubated at 37°C in a 5% CO2 incubator until cell confluence reached 90%-100% (forming a monolayer). A scratch was then performed using a 200 µL sterile pipette tip to make a straight scratch perpendicular to the plate on the cell monolayer in each well. Cells were gently washed 2-3 times with PBS to remove cell debris, and the medium was replaced with serum-free culture medium containing different concentrations of Ac-Phe. Experimental groups included: control group (containing an equal volume of DMSO), 80 µM Ac-Phe group, and 120 µM Ac-Phe group. At 0, 12, 24, and 48 hours post-scratching, the healing process was observed and photographed under an inverted microscope (100×) to record the scratch healing status.

[0044] 3. Experimental Results Experimental results are as follows Figure 1 As shown.

[0045] Figure 1 In the figure, A represents the dynamic changes in cell proliferation activity of the human colorectal cancer cell line RKO after treatment with different concentrations of N-acetyl-L-phenylalanine (Ac-Phe). The CCK-8 assay was used to monitor the cell proliferation activity for 5 consecutive days. The results showed that the OD of the control group (0 µM) RKO cells was significantly lower. 450 The OD values ​​showed a steady upward trend over time, indicating normal cell proliferation; however, after treatment with 5 µM, 20 µM, 80 µM, and 120 µM Ac-Phe, the OD values ​​of cells in each concentration group showed a significant increase. 450 The values ​​were all lower than those of the control group at the same time point, and the inhibitory effect increased with increasing concentration and treatment time. Low concentration (5 µM) Ac-Phe showed a mild inhibitory effect from day 2, with a slight separation from the control group curve; the high concentration groups of 80 µM and 120 µM showed the most significant inhibitory effects, with OD values ​​increasing from day 2. 450 The concentration was significantly lower than that of the control group. By day 5, the cell viability of the 120 µM group had decreased to about 40% of that of the control group, showing clear concentration-dependent and time-dependent inhibitory characteristics.

[0046] Figure 1 B in the figure represents the results of CCK-8 assay on the effect of Ac-Phe on the proliferation of mouse colorectal cancer cell line MC38, showing an inhibitory pattern similar to that of RKO cells. During the observation period of 0-5 days, the OD of control MC38 cells was [missing data]. 450 The OD values ​​continued to rise, and the 5 µM, 20 µM, 80 µM, and 120 µM Ac-Phe treatment groups all showed varying degrees of cell proliferation inhibition. With increasing Ac-Phe concentration, the inhibitory effect gradually strengthened; the high concentration groups (80 µM, 120 µM) significantly inhibited MC38 cell proliferation from day 2 onwards, and by day 5, the OD values ​​of the high concentration groups were...450 The value was much lower than that of the control group. This result indicates that N-acetyl-L-phenylalanine has broad-spectrum anti-proliferative activity against colorectal cancer cells derived from both humans and mice.

[0047] Figure 1 In the figure, C represents the apoptosis of RKO cells treated with 80 µM Ac-Phe for 24 hours, detected by Annexin V-FITC / 7-AAD double staining flow cytometry. The results showed that the total proportion of Annexin V single-positive and Annexin V / 7-AAD double-positive cells in the control group RKO cells was low, indicating a low spontaneous apoptosis rate. In contrast, the proportions of early apoptotic cells (Annexin V single-positive) and late apoptotic / necrotic cells (double-positive) were significantly increased in the 80 µM Ac-Phe treatment group, with the total proportion of apoptotic cells significantly higher than that in the control group. These results demonstrate that N-acetyl-L-phenylalanine can effectively induce apoptosis in colorectal cancer cells, and apoptosis induction is one of the important mechanisms by which it exerts its anti-tumor effect.

[0048] Figure 1 Figure D in the diagram represents the results of evaluating the effect of 80 µM Ac-Phe on the migration ability of RKO cells using the Transwell assay. After 24 hours of culture, crystal violet staining showed that a large number of RKO cells in the control group penetrated the Transwell membrane, with a higher number of cells migrating to the lower chamber side; while in the Ac-Phe (80 µM) treatment group, the number of cells penetrating the chamber membrane was significantly less than that in the control group, indicating a significant inhibition of cell migration. An invasion assay (pre-coated with Matrigel) was also performed in this experiment, and the results showed that Ac-Phe also significantly reduced the number of cells penetrating the Matrigel and the chamber membrane, suggesting that N-acetyl-L-phenylalanine not only inhibits the migration ability of colorectal cancer cells but also effectively weakens their invasive ability, demonstrating potential value in inhibiting tumor metastasis.

[0049] Figure 1E in the figure represents the scratch healing assay results. The scratch healing assay was used to examine the effect of different concentrations of Ac-Phe (80 µM, 120 µM) on the lateral migration ability of RKO cells. Scratch healing was observed at 0, 12, 24, and 48 hours after scratching. The results showed that control group cells began migrating to the scratched area 12 hours after scratching, the scratch narrowed significantly at 24 hours, and the scratch was basically healed at 48 hours, with cells covering most of the scratched area. In contrast, the 80 µM Ac-Phe treatment group showed slower scratch healing at all time points than the control group, and the scratch was not completely healed at 48 hours. The 120 µM Ac-Phe treatment group showed a more significant inhibitory effect, with the scratch width remaining wide at 12, 24, and 48 hours, strongly hindering cell migration. These results indicate that N-acetyl-L-phenylalanine can inhibit the scratch healing ability of colorectal cancer cells in a concentration-dependent manner, i.e., inhibit the lateral migration ability of cells, and the inhibitory effect is more pronounced at a concentration of 120 µM.

[0050] Example 2: N-acetyl-L-phenylalanine can inhibit the growth of subcutaneous tumors in colorectal cancer. 1. Experimental Materials 1.1 Test compounds and strains

[0051] 1.2 Laboratory Animals

[0052] 1.3 Main Reagents

[0053] 1.4 Main Instruments and Equipment

[0054] 2. Experimental Methods 2.1 Cell Culture and Preparation of Tumor-Bearing Cells After resuscitation, MC38 cells (mouse colorectal cancer cell line) were cultured in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in a 5% CO2 incubator. When the cells reached the logarithmic growth phase and confluence reached 80%-90%, they were digested with 0.25% trypsin-EDTA and collected. The cells were washed twice with sterile PBS, resuspended, counted, and the cell density was adjusted to 5 × 10⁶ cells / year. 6 Quantity / mL, keep on ice for later use.

[0055] 2.2 Establishment and drug administration of C57BL / 6 mouse subcutaneous tumor-bearing model Animal strain: C57BL / 6 mice (immunely healthy).

[0056] Modeling procedure: Day 0: Each mouse was subcutaneously injected with 100 µL of MC38 cell suspension (containing 5 × 10⁻⁶ cells) into one side of the groin. 6 (cells). Day 7: When the subcutaneous tumor grows to a volume of 50-100 mm³, tumor-bearing mice are randomly divided into groups to begin drug administration. Tumor volume calculation formula: V = (major axis × minor axis²) / 2.

[0057] Experimental groups (n=5-8 per group): Solvent control group (Vehicle): Daily gavage with an equal volume of solvent (45% physiological saline + 5% Tween 80 + 40% PEG300 + 10% DMSO); Low-dose Ac-Phe group: Daily gavage with Ac-Phe 10 mg / kg; Medium-dose Ac-Phe group: Daily gavage with Ac-Phe 50 mg / kg; High-dose Ac-Phe group: Daily gavage with Ac-Phe 200 mg / kg; Bacteroides oleate-rich group: Daily gavage with bacterial solution 1×10 9 CFU / animal (Bacteroides oleiciplenus, HYCC532574, purchased from Yushao Biotechnology). Administration: Oral gavage at a fixed time daily for 28 consecutive days. The solvent control group received an equal volume of solvent, while the bacterial strain group received an equal volume of bacterial suspension.

[0058] Observation indicators: Measure the long and short diameters of the tumor every 2-3 days, calculate the tumor volume, and plot the tumor growth curve. Observe the general condition of the mice daily (activity, feeding, defecation, fur condition, etc.). On day 28, sacrifice the mice, dissect the subcutaneous tumor, weigh the tumor, and photograph it.

[0059] 2.3 Establishment and Drug Administration of BALB / c Nude Mouse Subcutaneous Tumor Model Animal strain: BALB / c nude mouse (immunodeficient, T cell absent).

[0060] Modeling procedure: Same as C57BL / 6 mice, on day 0, 5×10⁵ mmol / L was injected subcutaneously into the groin of each nude mouse. 6 One MC38 cell. Administration began on day 7 when the tumor volume reached 50-100 mm³. Continuous gavage was administered until day 28.

[0061] Experimental groups (n=5-8 per group): Solvent control group (ctrl): administered an equal volume of solvent by gavage daily; Ac-Phe group: administered Ac-Phe 50 mg / kg by gavage daily.

[0062] Observation indicators: Measure the long and short diameters of the tumor every 2-3 days, calculate the tumor volume, and plot the tumor growth curve. Observe the general condition of the mice daily (activity, feeding, defecation, fur condition, etc.). On day 28, sacrifice the mice, dissect the subcutaneous tumor, weigh the tumor, and photograph it.

[0063] 2.4 Data Processing and Statistics All data are expressed as mean ± standard deviation (Mean ± SD) or mean ± standard error (Mean ± SEM). Statistical analysis was performed using GraphPad Prism software. One-way ANOVA was used for comparisons among multiple groups, and independent samples t-tests were used for comparisons between two groups. A p-value < 0.05 was considered statistically significant.

[0064] 3. Experimental Results Experimental results are as follows Figure 2 As shown.

[0065] Figure 2 A in the diagram represents the tumor-bearing and drug administration process in C57BL / 6 mice. On day 0, each mouse received a subcutaneous injection of 5 × 10⁵ mg / L in the groin. 6 MC38 cells; on day 7, when the subcutaneous tumor volume reached 50-100 mm³, daily oral gavage administration began. The treatment groups included low-dose Ac-Phe (10 mg / kg), medium-dose (50 mg / kg), and high-dose (200 mg / kg) as well as Bacteroides oleiciplenus (1×10⁻⁶). 9 CFU / mouse), the solvent control group was given an equal volume of solvent (45% saline + 5% Tween 80 + 40% PEG300 + 10% DMSO); mice were administered the drug continuously until day 28, at which point they were sacrificed for tissue collection. This procedure was used to evaluate the therapeutic effects of Ac-Phe and Bacteroides oleate-rich on established colorectal tumors in immunocompetent mice.

[0066] Figure 2 Photograph B in the image shows subcutaneous tumors in representative mice from each group after sacrifice on day 28. The tumors are, in order: solvent control group, Ac-Phe 10 mg / kg group, 50 mg / kg group, 200 mg / kg group, and Bacteroides rich in oleate group (1×10⁻⁶). 9 (CFU). The images clearly show that the tumor volume in the solvent control group was larger, while the tumor volume in each drug administration group was significantly smaller than that in the control group; there was no significant difference in tumor volume between the Ac-Phe dosage groups and between the Ac-Phe group and the bacterial strain group, and no trend of further tumor shrinkage with increasing dosage was observed.

[0067] Figure 2In the figure, C represents a line graph showing the change in subcutaneous tumor volume over time in C57BL / 6 mice. The dynamic changes in tumor volume for each group of mice were plotted with time (day 7 to day 28) on the x-axis and tumor volume (mm³) on the y-axis. Before administration (day 7), the baseline tumor volume of each group was basically the same; after the start of administration, the tumor volume in the solvent control group increased rapidly, reaching a high level by day 28. In contrast, the tumor volumes in the Ac-Phe 10 mg / kg, 50 mg / kg, and 200 mg / kg groups, as well as the *Bacteroides oleate* group, were significantly smaller than those in the solvent control group, with statistically significant differences. Notably, the tumor volume curves of the three Ac-Phe dosage groups largely overlapped, showing no dose-dependent enhancement of inhibitory effect, and the inhibitory effects of the Ac-Phe group and the *Bacteroides oleate* group were also basically equivalent.

[0068] Figure 2 Figure D in the graph represents the subcutaneous tumor weight results in C57BL / 6 mice. The solvent control group had the highest average tumor weight; the tumor weights of all Ac-Phe dose groups and the Bacteroides oleate-rich group were significantly lower than those of the control group (P<0.05). Consistent with the trend of volume change, there was no significant difference in tumor weight among the low, medium, and high dose groups of Ac-Phe, showing no dose dependence, and the tumor-inhibiting effects of Ac-Phe and Bacteroides oleate-rich were also basically similar.

[0069] Figure 2 E in the diagram represents the tumor-bearing and drug administration process in BALB / c nude mice. On day 0, a subcutaneous injection of 5 × 10⁻⁶ mg / L... 6 MC38 cells were administered via gavage daily starting on day 7, but the nude mouse experiment only included a solvent control group and an Ac-Phe 50 mg / kg group, with mice sacrificed on day 28. This procedure was used to evaluate the antitumor effect of Ac-Phe in immunodeficient (T cell-deficient) mice to determine whether its effect depends on the adaptive immune system.

[0070] Figure 2 F in the figure shows a photograph of a subcutaneous tumor in a BALB / c nude mouse at the time of sacrifice. The photographs show the actual subcutaneous tumors of representative mice in the solvent control group and the Ac-Phe group (50 mg / kg) after sacrifice on day 28. Macroscopic observation reveals that the subcutaneous tumors in the solvent control group were larger; the tumors in the Ac-Phe group were significantly smaller than those in the control group, indicating that Ac-Phe can inhibit tumor growth even in immunodeficient nude mice.

[0071] Figure 2G in the graph represents a line graph showing the change in subcutaneous tumor volume in BALB / c nude mice over time. The graph plots time (day 7 to day 28) on the x-axis and tumor volume (mm³) on the y-axis, illustrating the dynamic changes in tumor volume between the two groups of nude mice. Before administration (day 7), the baseline tumor volumes in both groups were consistent. After administration, the tumor volume in the solvent control group increased rapidly and continuously, while the tumor volume in the Ac-Phe 50 mg / kg group increased more slowly. From the mid-to-late stages of administration, the tumor volume in the Ac-Phe 50 mg / kg group was significantly smaller than that in the control group, and the difference was statistically significant. This result indicates that Ac-Phe can still exert a tumor-suppressive effect in nude mice lacking T cells, suggesting that its anti-colorectal cancer effect is not entirely dependent on the adaptive immune system.

[0072] Figure 2 The figure shows the weight of subcutaneous tumors in BALB / c nude mice, where H represents the tumor weight. The solvent control group had a higher average tumor weight, while the Ac-Phe 50 mg / kg group had a significantly lower average tumor weight than the control group (P<0.05). This result further confirms that Ac-Phe gavage administration can effectively inhibit the growth of subcutaneous xenografts in nude mice, resulting in a significant reduction in tumor weight.

[0073] Example 3 Safety assessment of N-acetyl-L-phenylalanine 1. Experimental Materials 1.1 Test Compound

[0074] 1.2 Laboratory Animals

[0075] 1.3 Main Reagents

[0076] 1.4 Main Instruments and Equipment

[0077] 2. Experimental Methods 2.1 Animal grouping and administration Healthy C57BL / 6 mice were randomly divided into two groups (n=5-8 per group): The solvent control group (Vehicle) received an equal volume of solvent (45% saline + 5% Tween 80 + 40% PEG300 + 10% DMSO) via gavage daily; the Ac-Phe group received Ac-Phe 200 mg / kg (the highest dose used in the experiment) via gavage daily. Administration was performed orally at fixed times daily for 4 weeks. The general condition of the mice (activity, feeding, defecation, fur condition, etc.) was observed daily, and body weight was measured every 3 days. After 4 weeks, the mice were sacrificed, and serum and intestinal tissue were collected for subsequent testing.

[0078] 2.2 Weight monitoring Mice body weight was measured every 3 days during the drug administration period, and changes in body weight in each group were recorded. After the experiment, the rate of change in body weight of mice in each group was calculated, and the difference between the solvent control group and the Ac-Phe group was compared.

[0079] 2.3 Serum inflammatory factor detection Blood collection: Blood was collected from the orbital venous plexus before the mice were sacrificed. The blood was allowed to stand at room temperature for 30 minutes, then centrifuged at 3000 rpm for 10 minutes to separate the serum, which was then stored at -80℃ for later use.

[0080] IL-6 detection: The mouse IL-6 ELISA kit (BioLegend, catalog number 1210602) was used. The procedure was followed according to the instructions: serum samples and standards were added to a 96-well plate pre-coated with antibody, incubated, and then the detection antibody was added. After incubation with enzyme-labeled antibody, color development and termination of the reaction were performed. The OD value was measured at a wavelength of 450 nm, and the serum IL-6 concentration was calculated according to the standard curve.

[0081] IL-1β detection: The mouse IL-1β ELISA kit (BioLegend, catalog number 1210122) was used, and the operation steps were the same as above to calculate the serum IL-1β concentration.

[0082] 2.4 Immunohistochemical staining of intestinal tissue Sampling and fixation: After euthanizing the mice, the colon tissue was removed, the contents were gently rinsed with PBS, a colon segment of about 1 cm was cut off, and fixed in 4% paraformaldehyde for 24 hours.

[0083] Embedding and sectioning: The fixed tissue was dehydrated with graded ethanol, cleared with xylene, embedded in paraffin, and then prepared into continuous sections with a thickness of 4 μm using a microtome. The sections were attached to anti-detachment glass slides and baked at 60°C for 2 hours.

[0084] Immunohistochemical staining: Dewaxing and hydration: Sections were washed sequentially with xylene (10 min × 2 times), graded ethanol (100%, 95%, 80%, 70% each for 5 min), and distilled water. Antigen retrieval: Sections were placed in sodium citrate buffer (pH 6.0), microwaved to boiling, maintained for 15 min, and then allowed to cool naturally to room temperature. Blocking endogenous peroxidase: Incubation with 3% H2O2 for 10 min. Blocking nonspecific binding: Blocking with 5% goat serum at room temperature for 30 min. Primary antibody incubation: E-Cadherin antibody (CST, 3195S, 1:200 dilution), MUC2 antibody (Abcam, ab272941, 1:200 dilution), and ZO-1 antibody (Abcam, ab227228, 1:200 dilution) were added and incubated overnight at 4°C. Secondary antibody incubation: HRP-labeled goat anti-rabbit / mouse secondary antibody was added and incubated at room temperature for 30 min. DAB staining: Add DAB working solution and control the staining time under a microscope (approximately 1-2 minutes). Stop staining after a brown positive signal appears. Counterstaining and mounting: Counterstain cell nuclei with hematoxylin for 30 seconds, turn blue with tap water, dehydrate with graded ethanol, clear with xylene, and mount with neutral resin. Observation and photography: Observe the staining results under an optical microscope (200× or 400×) and compare the expression intensity and distribution of E-Cadherin, MUC2, and ZO-1 between the solvent control group and the Ac-Phe group.

[0085] 2.5 Data Processing and Statistics All data are expressed as mean ± standard deviation (Mean ± SD). Independent samples t-tests were used for comparisons between two groups. A p-value < 0.05 was considered statistically significant.

[0086] 3. Experimental Results Figure 3 In Figure A, the in vivo safety evaluation procedure for healthy C57BL / 6 mice is described. Mice were randomly assigned to a solvent control group and an Ac-Phe group (200 mg / kg). The mice were administered the drug daily by gavage for 4 weeks, during which their body weight and general condition were monitored. After 4 weeks, the mice were sacrificed, and serum samples were collected to detect the inflammatory factors IL-6 and IL-1β. Colonic tissue was also harvested for E-Cadherin, MUC2, and ZO-1 immunohistochemical staining. This procedure was used to evaluate the effects of long-term Ac-Phe gavage administration on the overall toxicity, inflammatory response, and intestinal barrier function in mice.

[0087] Figure 3B in the figure represents the comparison of mouse body weight changes before and after the experiment. The results showed that the body weight of mice in the solvent control group increased normally within 4 weeks; the body weight of mice in the Ac-Phe group (200 mg / kg) was basically the same as that of the control group, with no statistically significant difference between the two groups (P>0.05). These results indicate that continuous intragastric administration of Ac-Phe (200 mg / kg) for 4 weeks had no significant effect on the body weight of healthy C57BL / 6 mice, suggesting that this compound does not have significant systemic toxicity.

[0088] Figure 3 In the figure, C represents the serum IL-6 concentration in the two groups of mice as determined by ELISA. The serum IL-6 level in the solvent control group was approximately 124 ± 4.5 pg / mL, while that in the Ac-Phe group was approximately 125 ± 4.8 pg / mL. The levels were almost identical between the two groups, with no statistically significant difference (P>0.05). This result indicates that long-term gavage administration of Ac-Phe does not induce a systemic increase in IL-6, suggesting that it does not have a significant pro-inflammatory effect.

[0089] Figure 3 In the figure, D represents the serum IL-1β concentration in the two groups of mice as determined by ELISA. The serum IL-1β level in the solvent control group was 31 ± 4.3 pg / mL, and in the Ac-Phe group it was 29.7 ± 2.8 pg / mL, with no statistically significant difference between the two groups (P>0.05). This result further confirms that long-term administration of Ac-Phe does not cause an increase in the body's IL-1β level and does not have a significant inflammatory activating effect.

[0090] Figure 3 In the figure, "E" represents the immunohistochemical staining results of intestinal tissue. E-Cadherin is a marker protein for intercellular junctions, and its normal expression maintains the integrity of the intestinal epithelial structure. MUC2 is a mucin secreted by goblet cells and is a major component of the intestinal mucus layer. ZO-1 is a tight junction protein that maintains intestinal barrier function. Immunohistochemical staining results showed that in the solvent control group, E-Cadherin showed continuous positive staining on the intestinal epithelial cell membrane, MUC2 was abundantly expressed in goblet cells, and ZO-1 showed clear positive bands at intercellular junctions. In the Ac-Phe group, the expression sites, staining intensities, and distribution patterns of the above three proteins were basically consistent with those in the solvent control group, and there was no significant difference between the two groups. These results indicate that long-term gavage administration of Ac-Phe has no adverse effects on the structure and function of the intestinal barrier.

[0091] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. The use of N-acetyl-L-phenylalanine or a pharmaceutically acceptable salt thereof in the preparation of medicaments for the treatment of colorectal cancer.

2. The application according to claim 1, characterized in that, The colorectal cancer mentioned is selected from colorectal adenocarcinoma.

3. The application according to claim 1, characterized in that, The drug is used to inhibit the proliferative activity of colorectal cancer cells.

4. The application according to claim 1, characterized in that, The drug is used to promote apoptosis in colorectal cancer cells.

5. The application according to claim 1, characterized in that, The drug is used to inhibit the migration of colorectal cancer cells.

6. The application according to claim 5, characterized in that, The drug is used to inhibit the lateral migration of colorectal cancer cells.

7. The application according to claim 1, characterized in that, The drug is used to inhibit the invasion of colorectal cancer cells.

8. The application according to claim 1, characterized in that, The drug is used to inhibit the growth of colorectal cancer tumors.

9. The application according to any one of claims 3-7, characterized in that, The colorectal cancer cells were selected from RKO cells and MC38 cells.

10. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable carriers and / or excipients.

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