Use of N-lactoylphenylalanine for the preparation of a medicament for the treatment of tumors

Abstract

The application of N-lactoyl phenylalanine in the preparation of a drug for treating tumors belongs to the technical field of medicine. The structural formula of the N-lactoyl phenylalanine is. The drug plays a role by regulating T cell activation, gene profile changes related to effector function, promoting CD8 + T cell activation, and enhancing the secretion of killing effectors. N-lactoyl phenylalanine can be used in combination with an immune checkpoint inhibitor. The present application first provides a novel use of N-lactoyl phenylalanine (Lac-Phe) as an anti-tumor drug, and determines that the compound is one of the core functional molecules mediating the anti-tumor effect of exercise; and simultaneously first discloses the core action mechanism of Lac-Phe in enhancing CD8 + T cell activation, promoting effector secretion, and improving anti-tumor immune response, which can significantly optimize the efficacy of existing tumor immunotherapy, fill many gaps in the prior art, and have important scientific innovation value and high clinical conversion application prospect.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of N-lactylphenylalanine in the preparation of drugs for treating tumors. Background Technology

[0002] Malignant tumors pose a core challenge to global public health, and their clinical diagnosis, treatment, and prevention are key research areas in the biomedical field. In recent years, tumor immunotherapy has achieved landmark clinical breakthroughs, with mainstream regimens, represented by immune checkpoint inhibitors, fundamentally changing the clinical treatment landscape for various malignant tumors. However, existing immunotherapies still have core clinical limitations: insufficient intensity of the anti-tumor immune response and restricted activation of effector T cell function. This results in significant room for improvement in clinical efficacy and long-term patient survival benefits. There is an urgent need to develop novel, safe, and accessible therapeutic drugs that can enhance anti-tumor immune effects, as well as combination therapy regimens that can synergistically enhance existing treatment options.

[0003] Regular exercise can significantly reduce the risk of cancer and improve treatment outcomes and overall survival in cancer patients. Its core benefits have been confirmed by multiple studies and are related to exercise's role in reshaping the body's immune homeostasis and enhancing the body's CD8+. + T-cell anti-tumor immunity is closely related to exercise and has clear clinical translational potential. However, current research on the anti-tumor effects of exercise has not yet identified the core endogenous functional molecular mediators that mediate this effect, making it impossible to translate the anti-tumor health benefits of exercise into clinical drug intervention strategies that can be precisely implemented and quantified. This is a core technological bottleneck that urgently needs to be overcome in this field.

[0004] Previous studies have found that N-lactoyl-Phenylalanine (Lac-Phe) is an endogenous metabolite whose abundance is significantly upregulated in the body circulation after exercise. It can regulate appetite in mice and resist diet-induced obesity. These results have been published in Nature (2022, DOI:10.1038 / s41586-022-04828-5). However, whether this compound mediates the antitumor physiological benefits of exercise, whether it possesses antitumor activity and pharmaceutical applications, and its relationship with antitumor immunity and CD8+ remain unclear. + The correlation between T cell function regulation and the feasibility of combining it with other anti-tumor therapies are currently unclear, and there are no publicly reported related technical solutions, which cannot fill the aforementioned clinical and technical gaps. Summary of the Invention

[0005] This invention addresses the following two issues: 1. It fills the core technological gap in the existing technology regarding Lac-Phe in the field of anti-tumor therapy, clarifying the anti-tumor activity of this compound and providing its application in the preparation of anti-tumor drugs. This overcomes the core technological bottleneck of the existing technology, which has not yet clearly identified the core endogenous functional molecular mediators that mediate the anti-tumor effect of exercise, and cannot translate the anti-tumor health benefits of exercise into a precisely implementable clinical drug intervention strategy. 2. Addressing the core clinical limitations of existing tumor immunotherapy, such as limited activation of effector T cells and insufficient intensity of anti-tumor immune responses, this invention provides a novel therapeutic drug that can safely and efficiently enhance effector T cell function and improve the anti-tumor immune response, breaking through the efficacy bottleneck of existing immunotherapy and improving treatment outcomes and survival benefits for cancer patients.

[0006] This invention addresses the core functional molecules that mediate the antitumor effects of exercise in existing technologies, as well as the core clinical bottlenecks in tumor immunotherapy, such as limited activation and insufficient response of effector T cells. For the first time, it clarifies the antitumor activity and mechanism of action of the exercise-induced upregulated endogenous metabolite Lac-Phe, filling the gap in the application of this compound in the field of antitumor pharmaceuticals. At the same time, it provides a novel combination therapy strategy that can significantly improve the efficacy of existing tumor immunotherapy.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] The application of N-lactylphenylalanine in the preparation of drugs for treating tumors, wherein the structural formula of N-lactylphenylalanine is as follows: .

[0009] This invention constructs a tumor-bearing mouse exercise intervention model, conducts plasma non-targeted metabolomics differential analysis, systematically screens endogenous metabolites whose abundance is significantly and stably upregulated after exercise intervention, and identifies the core differentially expressed molecule Lac-Phe. At the same time, the model was validated to find that tumor growth in tumor-bearing mice was significantly inhibited after exercise intervention, clarifying the association between Lac-Phe and the anti-tumor effect of exercise.

[0010] Based on the above findings, this invention proposes for the first time a novel use of Lac-Phe in the preparation of antitumor drugs. Through in vivo drug intervention experiments in various mouse models and tumor models, the in vivo antitumor effect of Lac-Phe was systematically verified: in multiple independent tumor-bearing models, exogenous supplementation with Lac-Phe could stably and significantly inhibit tumor growth and prolong the survival of tumor-bearing animals, clarifying the broad-spectrum antitumor activity of this compound and providing sufficient pharmacodynamic support for its antitumor pharmaceutical application.

[0011] Furthermore, the drug regulates T cell activation, changes in the gene spectrum related to effector function, and promotes CD8 activation. +T cell activation enhances the secretion of cytotoxic effector factors to exert its effects.

[0012] The core of exercise-mediated anti-tumor effects relies on the body's immune system, especially CD8. + Based on the consensus in the T-cell field, this invention systematically elucidates the core mechanism by which Lac-Phe regulates anti-tumor immunity through a series of in vitro and in vivo experiments: via CD8... + T cell transcriptomic analysis revealed that Lac-Phe significantly regulates changes in the gene spectrum related to T cell activation and effector function; functional verification using flow cytometry and other methods confirmed that Lac-Phe significantly promotes CD8+. + Activation of T cells and increased secretion of cytotoxic effector factors fill the gap in our understanding of the compound's role in T cell function regulation.

[0013] Furthermore, N-lactylphenylalanine can be used in combination with immune checkpoint inhibitors.

[0014] To address the limitations of existing tumor immunotherapy, this study combines Lac-Phe with CD8... + The invention further constructed an in vivo model of combined therapy to systematically evaluate the synergistic effect of Lac-Phe in combination with existing anti-tumor immunotherapy methods, which significantly regulates the T-cell effector function. The experimental results confirmed that the combination of Lac-Phe and immune checkpoint inhibitors can significantly improve the anti-tumor efficacy, break through the response bottleneck of existing immunotherapy, and provide a new, safe and accessible synergistic combination therapy for clinical tumor treatment.

[0015] Compared to existing technologies, the core beneficial effects of this invention are: This invention provides a novel use for Lac-Phe as an anti-tumor drug for the first time, clarifying that this compound is one of the core functional molecules mediating the anti-tumor effect of exercise; simultaneously, it reveals for the first time that Lac-Phe enhances CD8... + The core mechanisms of action, including T cell activation, promotion of effector factor secretion, and enhancement of anti-tumor immune response, can significantly optimize the efficacy of existing tumor immunotherapy, fill multiple gaps in current technology, and possess both significant scientific innovation value and extremely high clinical translational application prospects.

[0016] This invention fills a core technological gap in the clinical translation of the antitumor effects of exercise, and for the first time clearly defines the antitumor pharmaceutical applications of Lac-Phe. Simultaneously, it overcomes the core clinical bottlenecks of existing tumor immunotherapy, effectively addressing industry pain points such as limited activation of effector T cell function and insufficient antitumor immune response, and proposing a safe and effective synergistic combination therapy. Attached Figure Description

[0017] Figure 1Anatomical diagrams of tumors in tumor-bearing mice in the exercise intervention group and the control group;

[0018] Figure 2 A statistical graph showing the tumor weight of tumor-bearing mice in the exercise intervention group and the control group;

[0019] Figure 3 Tumor growth curves of tumor-bearing mice in the exercise intervention group and the control group;

[0020] Figure 4 The survival curves of tumor-bearing mice in the exercise intervention group and the control group are shown.

[0021] Figure 5 Volcano plot of differentially expressed metabolites in plasma of tumor-bearing mice in the exercise group and control group;

[0022] Figure 6 List of the top 10 metabolites that increased after exercise intervention in tumor-bearing mice;

[0023] Figure 7 This is an anatomical diagram of the tumor after Lac-Phe intervention in a 4T1 orthotopic tumor-bearing model.

[0024] Figure 8 A statistical chart of tumor weight after Lac-Phe intervention in a 4T1 orthotopic tumor-bearing model;

[0025] Figure 9 Tumor growth curves after Lac-Phe intervention in the 4T1 in situ tumor-bearing model;

[0026] Figure 10 A comparison curve of survival after Lac-Phe intervention in the 4T1 orthotopic tumor-bearing model;

[0027] Figure 11 Anatomical diagram of tumors after Lac-Phe intervention in the MC38 subcutaneous tumorigenesis model;

[0028] Figure 12 A statistical chart of tumor weight after Lac-Phe intervention in the MC38 subcutaneous tumorigenesis model;

[0029] Figure 13 Tumor growth curves after Lac-Phe intervention in the MC38 subcutaneous tumorigenesis model;

[0030] Figure 14 A curve showing the survival of the MC38 subcutaneous tumorigenesis model after Lac-Phe intervention;

[0031] Figure 15 In vitro CD8 after Lac-Phe treatment + Heatmap of differentially expressed genes in the T cell transcriptome;

[0032] Figure 16In vitro CD8 after Lac-Phe treatment + T cell activation markers statistical graph;

[0033] Figure 17 In vitro CD8 after Lac-Phe treatment + Flow cytometry plot of T cell secreted factors;

[0034] Figure 18 CD8+ in vivo tumor-infiltrating cells after Lac-Phe treatment + T cell activation markers statistical graph;

[0035] Figure 19 CD8+ in vivo tumor-infiltrating cells after Lac-Phe treatment + Flow cytometry plot of T cell secreted factors;

[0036] Figure 20 Comparative images of tumor anatomy for each experimental group (solvent control, Lac-Phe monotherapy, immunotherapy monotherapy, and combination therapy);

[0037] Figure 21 A comparative chart of tumor weight statistics for each experimental group (solvent control, Lac-Phe monotherapy, immunotherapy monotherapy, and combination therapy group);

[0038] Figure 22 Comparison of tumor growth curves for each experimental group (solvent control, Lac-Phe monotherapy, immunotherapy monotherapy, and combination therapy group);

[0039] Figure 23 Survival curves for each experimental group (solvent control, Lac-Phe monotherapy, immunotherapy monotherapy, and combination therapy group). Detailed Implementation

[0040] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the technical solutions and core effects of the present invention, and are not intended to limit the scope of protection of the present invention. The embodiments provided below can serve as a guide for those skilled in the art to make further improvements, and do not constitute a limitation on the present invention in any way.

[0041] Unless otherwise specified, the experimental methods described in the following examples are conventional experimental methods in the art, performed according to the technical conditions and operating procedures described in publicly available literature, or the product instructions for the corresponding reagents and kits. Unless otherwise specified, the instruments and equipment used in the experiments are all conventional laboratory instruments and equipment. Unless otherwise specified, the reagents, cell lines, experimental animals, consumables, etc., used in the following examples can all be obtained through legitimate commercial channels.

[0042] Example 1: Construction of a tumor-bearing mouse exercise intervention model and screening of core differential metabolites

[0043] 1. Construction of a tumor-bearing mouse motor intervention model

[0044] (1) Treadmill adaptation training

[0045] Experimental mice were trained on an electric treadmill equipped with a tail-mounted electric shock grid. Before formal intervention, they underwent three acclimatization training sessions held every other day. The training parameters were: treadmill speed 5 m / min, incline 7.5°, and session duration 15 min, to eliminate interference from the treadmill environment. Control group mice were fed normally and required no intervention.

[0046] (2) Exercise pre-intervention

[0047] After the adaptive training, mice underwent formal treadmill training every two days. The training parameters were: treadmill speed 10 m / min, incline 15°, and a maximum training duration of 60 minutes, completed between 13:00 and 16:00 daily. The termination criterion for a single training session was the appearance of significant fatigue in the mouse, defined as the inability to resume running after five consecutive exposures to the tail electric shock grid; this training session was considered complete. Before tumor cell inoculation, mice completed a total of five training sessions (total duration 10 days) as a pre-intervention.

[0048] (3) Construction of tumor-bearing models and intervention after tumor formation

[0049] Log-phase MC38 colon cancer cells were selected, and the cell concentration was adjusted (1... Tumor modeling was achieved by subcutaneous inoculation of 10⁶ cells / mouse on the back of mice. After inoculation, mice continued to undergo the same treadmill training regimen as in the pre-intervention phase (once every 2 days, with the same parameters) until the experimental endpoint on day 18 after tumor formation.

[0050] (4) Indicator detection nodes and contents

[0051] Tumor growth monitoring: Every 2 days after tumor implantation is a fixed monitoring point. The long and short diameters of the tumor are measured using vernier calipers, the tumor volume is calculated, and a tumor growth curve is plotted.

[0052] Survival monitoring: The survival status of mice was recorded daily throughout the experiment, and the survival rate of mice was calculated.

[0053] Endpoint detection: On day 18 after tumor bearing, mice were euthanized and the tumor tissue was completely dissected, photographed, weighed, and the wet weight of the tumor was recorded.

[0054] 2. Screening of plasma core differential metabolites

[0055] (1) Collection of biological samples

[0056] On day 18 after tumor implantation, EDTA-anticoagulated whole blood samples were collected from mice via the orbital venous plexus. The supernatant plasma was separated by centrifugation at 4°C (5000 rpm, 5 min), aliquoted, and stored at -80°C for later use.

[0057] (2) Sample pretreatment

[0058] After thawing the plasma sample on ice, 50 μL of plasma was mixed with 300 μL of ice-cold acetonitrile / methanol mixture (1:4, v / v, containing internal standard), and vortexed for 3 min. The sample was centrifuged at 12,000 rpm for 10 min at 4 °C, and the supernatant was collected and incubated at -20 °C for 30 min, followed by another centrifugation at 12,000 rpm for 3 min for further clarification. Finally, 180 μL of the supernatant was transferred to an LC-MS vial for analysis.

[0059] (3) Non-targeted metabolomics detection

[0060] Plasma extracts were detected using reversed-phase liquid chromatography-mass spectrometry (RP-LC-MS), with data acquired in both positive and negative electrospray ionization (ESI) modes. Chromatographic separation was performed using a Waters ACQUITY Premier HSS T3 column. Mobile phase A consisted of water containing 0.1% formic acid, and mobile phase B consisted of acetonitrile containing 0.1% formic acid, eluted according to a pre-defined gradient program. During detection, the column temperature was controlled at 40℃, the flow rate at 0.4 mL / min, and the injection volume at 3 μL. Mass spectrometry acquisition employed a full-scan combined with data-dependent secondary mass spectrometry (MS / MS) mode, with a scan range of m / z 70–1000 and dynamic exclusion configured. Raw data underwent format conversion, peak extraction, correction, and comparative analysis. Subsequently, metabolite annotation and quality control filtering were performed using database analysis to complete downstream metabolomics analysis.

[0061] (4) Screening of differential metabolites

[0062] Unsupervised principal component analysis (PCA) and supervised orthogonal partial least squares discriminant analysis (OPLS-DA) were used to screen for significantly different metabolites between the exercise intervention group and the control group. The screening criteria were: independent samples t test P < 0.05 and |log2(fold difference)| > 1.

[0063] In this embodiment, the results of the exercise-induced anti-tumor mouse model are as follows: Figures 1-4 As shown in the figure, exercise intervention significantly inhibited tumor growth in mice, manifested as a slowdown in tumor volume growth rate, a decrease in tumor mass at the endpoint, and an overall downward shift in the tumor growth curve throughout the experimental period. Simultaneously, exercise intervention significantly improved mouse survival, indicating a good anti-tumor effect. Non-targeted metabolomics analysis was performed on mouse plasma collected at the experimental endpoint, and the results are shown in the figure. Figures 5-6As shown, Lac-Phe is one of the most significantly elevated plasma metabolites after exercise intervention, and the top 10 metabolites with the most significant upregulation were also screened.

[0064] Example 2: In vivo antitumor efficacy validation of Lac-Phe in multiple tumor models

[0065] This embodiment validates the in vivo antitumor activity of Lac-Phe using two independent tumor models. The unified dosing regimen was as follows: Lac-Phe prepared with sterile saline was administered intraperitoneally at a dose of 50 mg / kg, once every two days, starting 10 days before tumor cell inoculation. Modeling was initiated after five pre-injections, and the same dosing regimen was maintained until the experimental endpoint. The MC38 colon cancer model used male C57BL / 6 mice, with 1×10⁻⁶ mice subcutaneously inoculated into the right back. 6 MC38 cells in logarithmic growth phase; 4T1 mammary cancer orthotopic model using female BALB / c mice, with 5 × 10⁶ cells seeded into the fourth mammary fat pad. 5 4T1 cells in the logarithmic growth phase were used; tumor growth curves were measured every 2 days after modeling, mouse survival rates were calculated, mice were sacrificed at the experimental endpoint, tumor tissue was dissected, gross images were taken and weighed, and samples were prepared as needed for subsequent analysis.

[0066] In this embodiment, the intervention effects of Lac-Phe in the 4T1 orthotopic tumorigenesis mouse model and the MC38 subcutaneous tumorigenesis mouse model are as follows: Figures 7-10 and Figures 11-14 As shown in the figure. The results indicate that Lac-Phe intervention significantly inhibited tumor growth in mice in both tumor models, manifested as slowed tumor volume growth, reduced tumor quality at the experimental endpoint, and sustained inhibition of tumor growth curves throughout the observation period; at the same time, the survival time of mice in the Lac-Phe intervention group was significantly prolonged, suggesting that it has significant tumor-suppressing effects and survival benefits.

[0067] Example 3: Lac-Phe regulation of CD8 + In vitro validation of T cell effector function

[0068] In this embodiment, spleen tissue from C57BL / 6 mice was used to obtain high-purity primary CD8+ using an immunomagnetic bead negative sorting method. + T cells were divided into a blank control group and a Lac-Phe intervention group. Both groups were stimulated to activate the TCR using anti-CD3 / anti-CD28 coupled magnetic beads. The intervention group was co-cultured with a predetermined concentration of Lac-Phe during activation stimulation. Cell samples were collected after culture at a predetermined time point. Total RNA was extracted from a portion of the samples for RNA-seq transcriptome sequencing. Differential gene analysis was used to analyze Lac-Phe-induced CD8+. +Another part of the study involved alterations in the T cell transcription profile, followed by flow cytometry analysis to detect T cell activation markers and the secretion of cytotoxic effector factors. This systematically validated the effects of Lac-Phe on CD8+. + The regulatory role of T cell activation and effector function.

[0069] In this embodiment, the result is as follows: Figures 15-17 As shown. High-purity primary CD8 isolated in vitro. + After T cells were treated with Lac-Phe, it was found that Lac-Phe significantly regulated the gene expression profile related to T cell activation and effector function. Further functional experiments, including flow cytometry, confirmed that Lac-Phe significantly promoted CD8+ expression. + T cell activation and enhanced secretion of cytotoxic effector factors indicate that they play an important role in regulating T cell function.

[0070] Example 4: Lac-Phe regulation of CD8 + In vivo validation of T cell effector function

[0071] This embodiment is based on the MC38 subcutaneous colon cancer tumor-bearing model after Lac-Phe intervention in Example 2. Tumor tissue from mice excised at the experimental endpoint was collected and enzymatically digested using a commercially available tumor tissue digestion solution according to the product instructions. After red blood cell lysis, a single-cell suspension of tumor tissue was prepared. CD8+ in the tumor microenvironment was detected by flow cytometry. + The expression levels of T cell activation markers and the secretion levels of cytotoxic effector factors were verified in vivo under physiological conditions to enhance the effect of Lac-Phe on tumor-infiltrating CD8+. + The regulatory role of T cell activation and effector function.

[0072] In this embodiment, the result is as follows: Figures 18-19 As shown. In a Lac-Phe-treated tumor-bearing mouse model, tumor infiltration CD8 + The activation level of T cells was significantly increased, and the secretion of their related cytotoxic effector factors was also significantly increased, indicating that Lac-Phe can enhance CD8+ in the tumor microenvironment. + The anti-tumor effector function of T cells.

[0073] Example 5: Synergistic efficacy verification of Lac-Phe combined with immunotherapy

[0074] This embodiment uses the MC38 subcutaneous colon cancer tumor-bearing model to verify the synergistic anti-tumor effect of Lac-Phe combined with Anti-PD-1 immunotherapy. The experiment was set up with 4 parallel treatments: solvent control group, Lac-Phe monotherapy group, Anti-PD-1 monotherapy group, and Lac-Phe + Anti-PD-1 combination group. The Lac-Phe administration regimen was completely consistent with that in Example 2, with an intraperitoneal injection of 50 mg / kg every 2 days. Pre-administration was started 10 days before tumor cell inoculation, and the same regimen was maintained after modeling until the experimental endpoint. The Anti-PD-1 administration regimen was intraperitoneal injection on days 6, 9, 12, and 15 after tumor bearing, with 100 μg per mouse. The control group was injected intraperitoneally with the same dose of isotype-matched IgG at the same time points. The tumor growth curve was measured every 2 days after modeling, and the survival rate of mice was counted. At the end of the experiment, the mice were sacrificed, and the tumor tissue was dissected, photographed, and weighed to clarify the synergistic anti-tumor effect of the combination of the two.

[0075] In this embodiment, the result is as follows: Figures 20-23 As shown, both Lac-Phe intervention and PD-1 blockade alone can inhibit tumor growth to some extent, but the anti-tumor effect is further enhanced when the two are used in combination. Compared with the two single-factor intervention groups, the combination treatment group showed more significant tumor growth inhibition, lower experimental endpoint tumor quality, and higher mouse survival rate, indicating that Lac-Phe can effectively enhance the efficacy of PD-1 blockade therapy, and the combination regimen is superior to PD-1 intervention alone.

Claims

1. The application of N-lactylphenylalanine in the preparation of drugs for treating tumors, characterized in that: The structural formula of the N-lactylphenylalanine is as follows: .

2. The use of N-lactylphenylalanine according to claim 1 in the preparation of a medicament for treating tumors, characterized in that: The drug works by regulating T cell activation, changes in the gene spectrum related to effector function, and promoting CD8 activation. + T cell activation enhances the secretion of cytotoxic effector factors to exert its effects.

3. The use of N-lactylphenylalanine according to claim 1 in the preparation of a medicament for treating tumors, characterized in that: N-Lactylphenylalanine can be used in combination with immune checkpoint inhibitors.

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