A combination of ackermanii and an eet homolog and uses thereof

Abstract

The application discloses a kind of Akkermansia and EET homologs combined medicine and its purposes, it is related to biomedical technical field.The specific combined medicine of the application includes: A1) first effective component, the first effective component includes mucinophilic Akkermansia;And, A2) second effective component, the second effective component includes 14,15-epoxy eicosatrienoic acid (14,15-EET) or at least one of isomer, pharmaceutically acceptable salt, ester, prodrug of 14,15-epoxy eicosatrienoic acid.The combined medicine of the application shows excellent anti-inflammatory and anti-tumor effect in LPS-induced inflammation model and LLC subcutaneous tumor model, and the Bliss independent model is calculated to be synergistic index SI>1, indicating that it has good synergistic effect, can be used for the prevention, treatment and prognosis improvement of non-small cell lung cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a combination drug of Akkermansia and EET homologues and its use. Background Technology

[0002] Chronic inflammation is closely related to the development and progression of tumors. In the tumor microenvironment, neutrophil infiltration and the sustained release of pro-inflammatory factors (such as IL-1, IL-6, and TNF-α) can promote tumor growth, angiogenesis, and immunosuppression, thereby weakening the effects of conventional and immunotherapies. The lipid mediator 14,15-epoxyeicosatrienoic acid (14,15-EET) has been shown to possess clear anti-inflammatory and anti-platelet aggregation biological activities, demonstrating potential application value in regulating tumor-related inflammation.

[0003] However, in related technologies, traditional exogenous supplementation of 14,15-EET faces a significant "metabolic inactivation bottleneck" in vivo. The main reason is that exogenously supplemented 14,15-EET is readily hydrolyzed in vivo by soluble epoxide hydrolase (sEH, encoding gene Ephx2, protein EPHX2) into its diol metabolite (DHET / DiHETE), resulting in a short effective duration, unstable and poorly sustained anti-inflammatory effects, thus limiting its stable application in tumor-related inflammation regulation and anti-tumor therapy. On the other hand, Akkermania (… Akkermansia muciniphila Although Amu (Amycium muscarinii) has been reported to have immunomodulatory and anti-inflammatory potential as a gut symbiotic, current strategies mostly focus on using Amu alone or in combination with immune checkpoint inhibitors such as PD-1 / PD-L1. However, due to the heterogeneity of the tumor microenvironment and immune tolerance, some patients have difficulty obtaining stable anti-tumor effects.

[0004] Therefore, there is an urgent need to develop a combination drug that can effectively overcome the bottleneck of in vivo metabolism and inactivation of exogenous 14,15-EET, while having more stable and stronger anti-inflammatory and anti-tumor effects, in order to solve the problem that the efficacy of single agents or simple combination regimens in the treatment of tumors such as non-small cell lung cancer is unstable and the number of patients who benefit is limited. Summary of the Invention

[0005] The first aspect of the present invention is to provide a combination drug of Akkermansia and EET homologues, which can significantly increase the effective exposure time of 14,15-EET or its isomers in vivo, thereby stably enhancing its anti-inflammatory activity and synergistically inhibiting the progression of non-small cell lung cancer and reducing the local and systemic inflammatory burden of the tumor.

[0006] The second objective of this invention is to provide the application of the combination drug of the above-mentioned Akkermansia and EET homologues in the preparation of antitumor drugs.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A first aspect of the present invention provides a combined drug of Akkermansia and an EET homologue, comprising: A1) A first active ingredient, comprising *Akermansia mycotoxinophilus*; and, A2) A second active ingredient comprising at least one of 14,15-epoxyeicosatetrienoic acid (i.e., 14,15-EET) or an isomer of 14,15-epoxyeicosatetrienoic acid, a pharmaceutically acceptable salt, an ester, or a prodrug.

[0008] In some embodiments of the present invention, the *Ackermania* strain is purchased from the U.S. Culture Collection Center with accession number ATCC BAA-835.

[0009] In some embodiments of the present invention, the isomers of the 14,15-epoxyeicosatotrienoic acid include structural isomers and stereoisomers.

[0010] In some embodiments of the present invention, the structural isomer includes at least one of 5,6-epoxyeicosatotrienoic acid, 8,9-epoxyeicosatotrienoic acid, and 11,12-epoxyeicosatotrienoic acid.

[0011] In some embodiments of the present invention, the stereoisomer includes at least one of cis-trans isomers, enantiomers, and diastereomers.

[0012] In some embodiments of the invention, the pharmaceutically acceptable salt refers to the salt of 14,15-epoxyeicosatetrienoic acid or its isomers, as represented herein, which is non-toxic, biologically tolerable, or otherwise biologically suitable for administration to a subject. See also S.M. Berge et al., “Pharmaceutical Salts,” J. Pharm. Sci., 1977, 66, 1. 19. Preferred pharmaceutically acceptable salts are those that are pharmacologically effective and suitable for contact with the tissues of a subject without excessive toxicity, irritation, or allergic reactions.

[0013] In some embodiments of the present invention, the ratio of the first active ingredient to the second active ingredient is 10:10. 5 ~10 10 CFU: 0.01~5μg.

[0014] Preferably, the ratio of the first active ingredient to the second active ingredient is 10:1. 6 ~10 9 CFU: 0.01~2μg.

[0015] In some embodiments of the present invention, the combined drug is a single compound preparation containing the first active ingredient and the second active ingredient.

[0016] In some embodiments of the present invention, the combined drug is a combination of two separate formulations of the first active ingredient and the second active ingredient.

[0017] A second aspect of the invention provides the use of the combined drug as described in the first aspect in the preparation of an antitumor drug.

[0018] In some embodiments of the present invention, the tumor includes, but is not limited to, colon cancer, rectal cancer, colorectal cancer, lung cancer (such as non-small cell lung cancer), stomach cancer, liver cancer, pancreatic cancer, breast cancer, kidney cancer, fibrosarcoma, bile duct cancer, esophageal cancer, ovarian cancer, bladder cancer, malignant melanoma, or glioma.

[0019] In some embodiments of the present invention, it is also expected by those skilled in the art that the antitumor drug can partially or completely inhibit other solid tumors.

[0020] In some embodiments of the present invention, the tumor is non-small cell lung cancer.

[0021] In some embodiments of the present invention, the antitumor drug includes an anti-non-small cell lung cancer drug.

[0022] In some embodiments of the present invention, the antitumor drug further comprises a pharmaceutically acceptable carrier.

[0023] In this invention, a pharmaceutically acceptable carrier refers to a component in a pharmaceutical preparation or composition that is non-toxic to the subject, excluding the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, adjuvants, carriers, excipients, flow aids, sweeteners, diluents, preservatives, dyes / coloring agents, flavoring agents, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents, or emulsifiers. Pharmaceutically acceptable carriers known in the art can be used to formulate the pharmaceutical compositions of this invention.

[0024] In some embodiments of the present invention, the antitumor drug is administered via gastrointestinal or / and non-gastrointestinal routes.

[0025] In some embodiments of the present invention, the antitumor drug has at least one of the following functions: B1) Reduce the proportion of neutrophils in peripheral blood and / or the Pan-Immune Inflammation Value (PIV). B2) Reduce CD11b in tumor tissue + Ly6G + Neutrophil infiltration; B3) Downregulates the expression of Ephx2 gene and / or EPHX2 protein in tumor tissue.

[0026] The combined drug of Akkermansia and EET homologue of the present invention and its application have at least the following beneficial effects: This invention utilizes the combination of *Akermansia muciniphila* (… Akkermansia muciniphila The combination of live bacteria (Amu) and 14,15-epoxyeicosatrienoic acid (14,15-EET) or its homologues / isotypes solves the challenges of EET's short effective exposure time in vivo, difficulty in maintaining stable anti-inflammatory effects in tumor sites and further transforming them into sustained anti-tumor effects, and the difficulty of Amu forming a broader-spectrum and reproducible anti-inflammatory-immunomodulatory-anti-tumor synergistic effect in tumor immunotherapy.

[0027] This invention discovers that combining Amu with EET can utilize Amu to inhibit / downregulate the expression of soluble epoxide hydrolases (sEH, Ephx2 / EPHX2), reducing the rapid hydrolytic inactivation of EET in vivo, thereby significantly improving the effective exposure time and duration of action of EET. Compared with existing single-agent formulations or simple combination regimens, the combination therapy of this invention exhibits more stable and stronger anti-inflammatory and anti-tumor effects in LPS-induced inflammation models and LLC subcutaneous tumor models. The synergistic index SI > 1 calculated using the Bliss independent model indicates a good synergistic effect. In addition, the combination therapy of this invention can reduce the proportion of neutrophils in peripheral blood and the PIV inflammatory index, reduce local neutrophil infiltration in tumors, and alleviate the risk of inflammation and damage to multiple organs such as the heart, lungs, liver, and kidneys. It can provide a safe and effective combined intervention strategy for the prevention, treatment, and prognosis improvement of non-small cell lung cancer, and has a promising application prospect.

[0028] Other features and advantages of the present invention will be set forth in the following description. Attached Figure Description

[0029] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 The results of fluorescence signal detection in zebrafish under different treatment groups in this invention are shown, where A is a microscopic observation of fluorescence signal and B is a statistical graph of neutrophils. Figure 2These are the qPCR detection results of inflammation-related factors in zebrafish from different treatment groups in this invention, where A is... IL1 B is IL6 C is IL10 D is Tnf ; Figure 3 The results show the tumor volume correlation detection and statistical results of different treatment groups in the LLC subcutaneous tumor model. A is the actual image of the tumor on day 9, B is the tumor volume statistical result, C is the tumor size statistical result at different time points, and D is the tumor size statistical result on day 9. Figure 4 The results show the tumor inhibition rate and synergy index of different treatment groups in the LLC subcutaneous tumor model. A represents the tumor inhibition rate of different treatment groups, and B represents the synergy index. Figure 5 The results of peripheral blood neutrophil ratio and PIV inflammatory index in different treatment groups in the LLC subcutaneous tumor model are shown in Figure A, B, C, and D. Figure 6 The results of flow cytometry analysis of tumor tissues from different treatment groups in the LLC subcutaneous tumor model are shown in Figure A, where A is the flow cytometry plot and B is the statistical result. Figure 7 HE staining images of heart / lung / liver / kidney organs in different treatment groups in an LLC subcutaneous tumor model; Figure 8 The results of qPCR detection of pro-inflammatory factors in tumor tissues from different treatment groups in an LLC subcutaneous tumor model are shown, where A represents... IL1 B is IL6 C is IL10 D is Ly6g E is Tnf F is Ephx2 ; Figure 9 The images show the immunohistochemical staining and semi-quantitative scoring results of tumor tissues from different treatment groups in the LLC subcutaneous tumor model. In the image, A is the immunohistochemical staining image, and B is the statistical result of EPHX2 protein expression. Detailed Implementation

[0030] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0031] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0032] In the description of this invention, the reference term "and / or" includes all and any combination of one or more of the associated listed items. For example, A and / or B includes (A and B) and (A or B).

[0033] In this invention, all data were plotted and statistically analyzed using GraphPad Prism 9.3.1 software. For comparisons of three or more groups, one-way ANOVA was used. For pairwise comparisons between groups that did not show significant differences in one-way ANOVA and the model group, unpaired t-tests were used. Significance is indicated by *. p <0.05, **: p <0.01, ***: p <0.001, **** p <0.0001; not significant, marked as ns.

[0034] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0035] Example 1 This embodiment validates the presence of Akkermansia myxophila (based on the LPS zebrafish inflammation model). Akkermansia muciniphila The anti-inflammatory effects of Amu were demonstrated, and evidence was provided for the anti-inflammatory basis of its combined application in subsequent tumor models. The specific experiments are as follows.

[0036] 1. Experimental materials Akkermania: Akkermania myxophilus in this experiment ( Akkermansia muciniphila The strain (Amu) was lyophilized. Akkermansia muciniphila Purchased from the American Culture Collection Center, accession number ATCC BAA-835.

[0037] Culture medium: Brain Heart Infusion Broth (BHI) with 1% (m / v) mucin added.

[0038] Zebrafish: The Tg(lyz:DsRed) transgenic zebrafish strain was selected, which is a transgenic zebrafish strain that uses the promoter of the zebrafish lysozyme C (lyz) gene to drive the expression of the red fluorescent protein DsRed.

[0039] 2. Experimental Methods (1) Cultivation and acquisition of strains Take one lyophilized Amu culture, add 200 μL of BHI + 1% MUCIN medium to dissolve it, then take 200 μL and spread it on a blood agar plate and streak it; place it in an anaerobic tank and evacuate the gas control system, then incubate it at 37℃ under anaerobic conditions for 48 h.

[0040] Then, single colonies were picked and inoculated into 10 mL of BHI + 1% MUCIN medium and cultured anaerobically at 37℃ for 48 h to obtain seed culture. Finally, 500 mL of BHI + 1% MUCIN medium was taken, and the seed culture was inoculated at 1% (v / v) and cultured anaerobically at 37℃ for 48 h for later use.

[0041] (2) Construction and grouping of LPS zebrafish inflammation model Model establishment: Tg(lyz:DsRed) zebrafish strain was used for male-female breeding. After collecting fertilized eggs, they were incubated and fluorescence identification was performed during 24-48 h. Embryos carrying red fluorescence (visible lyz-positive neutrophils) were selected for further culture.

[0042] Administration and stimulation regimen: Amu treatment was initiated on day 4 (4 dpf) in zebrafish at a dose of 10. 6 CFU / 100 μL was applied once daily with simultaneous water changes until the end of the experiment; LPS stimulation was administered on day 6, with a final LPS concentration of 25 μg / mL; fluorescence imaging was performed on zebrafish in each group on day 7. A PBS control group and an LPS model group were also included.

[0043] Detection indicators: Zebrafish tissues from each group were homogenized, total RNA was extracted and reverse transcribed into cDNA, and qPCR was performed to detect the transcriptional levels of inflammation-related factors (IL1, IL6, IL10, and TnF). The anti-inflammatory effect of Amu in vivo was comprehensively evaluated by combining fluorescence signals and qPCR results. Neutrophil counts were also determined using bright-field and fluorescence imaging.

[0044] 3. Experimental Results The relevant results of fluorescence signal detection are as follows Figure 1 As shown, in the Tg(lyz:DsRed) zebrafish LPS inflammation model, LPS induced a significant increase in the fluorescence signal of lyz neutrophils; after Amu (LPS+Amu) treatment, the fluorescence / count of neutrophils decreased, and the inflammatory response was alleviated, indicating that the modeling was successful and that Amu administration helped to reduce inflammation.

[0045] qPCR test results as follows Figure 2 As shown in the figure. In the zebrafish LPS inflammation model, qPCR detection showed that LPS significantly upregulated inflammatory factors. IL1 , IL6 , IL10 and Tnf The expression of the above-mentioned inflammatory factors was reduced by Amu treatment (LPS+Amu), which reduced the transcription levels of the above-mentioned inflammatory factors, indicating that Amu has an anti-inflammatory effect in vivo.

[0046] Example 2 This embodiment investigates the tumor prevention and treatment effects of Akkermansia myxophilus combined with 14,15-EET based on mouse experiments. The specific experiments are as follows.

[0047] 1. Experimental Materials Experimental mice: Twenty 7-8 week old C57 mice in good condition were selected and purchased from the Department of Zoology, Central South University.

[0048] Experimental strain: Akkermansia myxophila ( Akkermansia muciniphila The strain (Amu) was lyophilized. Akkermansia muciniphila Purchased from the American Culture Collection Center, accession number ATCC BAA-835.

[0049] 14,15-EET solution: Prepare a working solution of 1–10 μg / mL using a 100 μg / mL stock solution of 14,15-EET (CAS No. 81276-03-1). Store the working solution under anaerobic conditions for later use in mouse experiments. Take precautions to prevent oxidation and degradation by protecting it from light, at low temperatures, and using inert gases.

[0050] 2. Experimental Methods (1) Cultivation and acquisition of strains Activation treatment: Take one lyophilized Amu culture, add 200 μL of BHI + 1% MUCIN medium to dissolve it, then take 200 μL and spread it on a blood agar plate and streak it; place it in an anaerobic tank and evacuate the gas control system, then incubate it at 37℃ under anaerobic conditions for 48 h.

[0051] Large-scale amplification: Single colonies were picked and inoculated into 10 mL of BHI + 1% MUCIN medium and cultured anaerobically at 37℃ for 48 h to obtain seed culture. Finally, 500 mL of BHI + 1% MUCIN medium was taken and inoculated with the seed culture at 1% (v / v) and cultured anaerobically at 37℃ for 48 h for later use.

[0052] Bacterial harvesting and washing / redissolving: After the culture was completed, the bacterial solution was collected and centrifuged at 6000 rpm for 10 min to obtain bacterial sludge; the bacterial sludge was washed twice with physiological saline and then redissolved for later use, and viable bacteria were counted for subsequent animal experiments via gavage.

[0053] (2) Laboratory animals and grouping Twenty 7–8 week old C57 mice in good condition were purchased from the Department of Zoology, Central South University. They were randomly divided into four groups of five mice each: PBS group, Amu group, 14,15-EET group, and Amu+14,15-EET group.

[0054] (3) Preparation of gut microbiota The group receiving intravenous infusion was prepared with Amu via gavage before tumor inoculation: the Amu dose was 10... 8 CFU / day, administered via gavage every other day, with treatment starting one week in advance to increase Amu abundance and efficacy in the gut.

[0055] (4) Establishment of a subcutaneous tumor model of lung cancer Once LLC cells reached the logarithmic growth phase, they were digested with trypsin, neutralized with culture medium, and collected by centrifugation. Residual serum was removed by washing twice with DPBS, and the cells were finally resuspended in DPBS. After cell counting, the cells were processed at a rate of 1×10⁻⁶. 6 A subcutaneous lung cancer tumor model was established by inoculating one cell per mouse into the right axillary subcutaneous tissue.

[0056] (5) Dosing regimen After tumor cells were inoculated and tumor formation began, the 14,15-EET and PBS groups were treated starting from day 1 after tumor formation. Both EET and PBS were administered every other day until the end of the experiment, with the first dose being doubled.

[0057] EET administration: The concentration of 14,15-EET working solution is 5 μg / mL, administered via intraperitoneal injection, with a dosing volume of 100 μl / day. Amu administration: Live Amu bacteria prepared according to the above culture were administered at 10... 8 CFU / day / 200μl continuous gavage; Combined group: Mice were simultaneously treated with Amu and 14,15-EET. Tumor-bearing mice were sacrificed two weeks later.

[0058] (6) Sample collection and testing After sacrifice, tumor tissue, organs such as heart, lungs, liver, and kidneys, and peripheral blood were collected for the following tests and analyses: ① Evaluation of antitumor efficacy: Tumor volume and final (day 9) tumor weight were recorded, and the inhibition rate was calculated. An independent-action model was used to calculate the ideal combined inhibition rate, and the synergy index (SI) was further calculated to assess the combined synergistic effect. The formulas for calculating the inhibition rate and the synergy index (SI) are as follows: The inhibition rate (I) is calculated using the following formula: I = (X control X treat ) / X control = 1 (X treat / X control ); Among them, X control This represents the mean value of the control group (e.g., the mean tumor volume / weight in the PBS or PD-1+NC group); X treat The mean value represents the treatment group (e.g., the mean tumor volume / weight of the Amu, EET, and Amu+EET groups); I represents the inhibition rate (0-1, with a larger value indicating stronger inhibition).

[0059] The formula for calculating the ideal bliss independence is as follows: I exp = I A + I B I A I B = 1 (1 I A (1) I B ); Among them, I exp The ideal combined inhibition rate (the combined inhibition rate predicted by the Bliss independent model); I A The inhibition rate of A alone (e.g., the inhibition rate of Amu alone compared to the control); I B The inhibition rate of B alone (e.g., the inhibition rate of EET alone compared to the control). The formula for calculating the Synergy Index (SI) is as follows: SI = I obs_comb / I exp ; Among them, I obs_comb The measured inhibition rate of the combination group (e.g., the inhibition rate of the Amu+EET group relative to the control); Iexp The ideal mixed inhibition rate is given by the formula above.

[0060] Judgment: SI>1 indicates cooperation; SI = 1 indicates addition / independence; SI<1 indicates antagonism.

[0061] ②Inflammation level assessment: The expression levels of local and systemic inflammatory factors in the tumor were assessed by flow cytometry or molecular detection, and the overall inflammatory status of the host was assessed by composite inflammatory indices such as PIV. ③ Histological safety assessment: Inflammation and damage were assessed in the heart, lungs, liver, and kidneys, and the effects of different treatments on the inflammatory burden of multiple organs were compared.

[0062] ④ Mechanism verification: Ephx2 qPCR detection and EPHX2 immunohistochemical analysis were performed on tumor tissues to corroborate the mechanism by which Amu inhibits / downregulates the sEH axis, thereby reducing EET hydrolysis and inactivation.

[0063] 3. Experimental Results Figure 3 For the tumor volume detection and statistical results of each group, in the LLC subcutaneous tumor model, the tumor volume growth was slowest and the final tumor weight was lowest after Amu+EET treatment; Amu alone was the second slowest, EET alone was the weakest, and PBS was the highest. By continuously measuring tumor volume and weighing at the endpoint, it is suggested that the combination of Amu and EET can more significantly inhibit tumor progression.

[0064] Figure 4 To statistically analyze the synergistic effect of antitumor efficacy, the inhibition rate was calculated based on tumor volume, and the ideal combined inhibition rate was extrapolated using the Bliss independent model. The measured inhibition rate of the Amu+EET group was higher than the Bliss predicted value, and the synergy index SI was >1 at multiple time points. This indicates that there is a synergistic effect between Amu and EET.

[0065] Figure 5 Statistical analysis of peripheral blood neutrophil percentage (Neu%) and PIV inflammatory index in each group showed that both the Amu group and the Amu+EET group reduced peripheral blood neutrophil percentage (Neu%) and PIV inflammatory index, with a more significant decrease in the combination group; simultaneously, PIV was significantly positively correlated with tumor volume / weight. Further, through group comparison and correlation analysis of inflammatory markers, the results showed that reducing systemic inflammatory burden was consistent with inhibiting tumor burden. Tumor tissue flow cytometry results are as follows... Figure 6 As shown, CD11b of the Amu group and the Amu+EET group is displayed. + Ly6G + Neutrophil infiltration was reduced in all groups, with the lowest reduction observed in the combination therapy group. Flow cytometry analysis and proportion statistics after tumor digestion indicated that the combination therapy was more effective in inhibiting neutrophil-related inflammatory responses within the tumor.

[0066] Figure 7 HE staining results for the heart, lungs, liver, and kidneys showed that inflammation and tissue damage were more pronounced in the PBS group, lessened in the EET group, further alleviated in the Amu group, and least severe in the Amu+EET group. Pathological comparisons of major organs suggest that combined use can enhance anti-tumor efficacy while reducing the risk of inflammation and damage in multiple organs.

[0067] Figure 8 The results of qPCR detection of pro-inflammatory factors in tumor tissue showed that the levels of pro-inflammatory factors such as IL1, IL6, and TnF in the Amu group and the Amu+EET group were significantly different. Ly6g Expression downregulation was more pronounced in the combination group; at the same time Ephx2 Amu and the combination therapy group showed a decrease. Tumor tissue transcriptional level analysis suggested that the combination therapy simultaneously suppressed local inflammation and downregulated the sEH axis. Tumor tissue IHC results are as follows: Figure 9 As shown, Amu and the combination of Amu and EET reduced EPHX2 protein expression (H-score decreased), with the lowest value observed in the combination group. Immunohistochemical staining and semi-quantitative scoring suggest that Amu combined with EET can downregulate the sEH / EPHX2 axis, providing histological evidence for the mechanism of "reducing EET hydrolysis and inactivation, enhancing anti-inflammatory effects, and converting them into anti-tumor effects".

[0068] The above results indicate that the Amu+EET combination group showed the most significant effects in inhibiting inflammatory responses, reducing inflammatory markers such as PIV, and inhibiting tumor progression. Compared with Amu or 14,15-EET alone, the combination showed more stable and stronger anti-inflammatory and anti-tumor effects. The synergy index SI>1 calculated according to the Bliss model indicates that the two have a synergistic effect. At the same time, the combination was accompanied by downregulation of the sEH (Ephx2 / EPHX2) axis, suggesting that the combination regimen helps to reduce the hydrolytic inactivation of 14,15-EET and improve the effective exposure and persistence of 14,15-EET in vivo, thereby more stably and strongly reducing the level of inflammation in the tumor and the host as a whole, while synergistically inhibiting the progression of non-small cell lung cancer.

[0069] In summary, this invention provides an Akkermania ( Akkermansia muciniphilaCombinations of live bacteria (Amu) with 14,15-epoxyeicosatotrienoic acid (14,15-EET) and its homologues and their use in tumor immunotherapy: By combining Amu with EET, the expression of soluble epoxide hydrolases (sEH, Ephx2 / EPHX2) can be inhibited / downregulated by Amu, thereby reducing the rapid hydrolytic inactivation of EET in vivo and significantly improving the effective exposure time and duration of action of EET. Compared with existing single-agent formulations or simple combination regimens, the combination drug regimen of this invention exhibits more stable and stronger anti-inflammatory and anti-tumor effects in LPS-induced inflammation models and LLC subcutaneous tumor models. The synergistic index SI > 1 calculated by the Bliss independent model indicates that it has a good synergistic effect. At the same time, the combination drug regimen of this invention can reduce the proportion of neutrophils in peripheral blood and the PIV inflammatory index, reduce local neutrophil infiltration in the tumor, and alleviate the risk of inflammation and damage to multiple organs such as the heart, lungs, liver, and kidneys. It provides a safe and effective combined intervention strategy for the prevention, treatment, and prognosis improvement of non-small cell lung cancer and has a promising application prospect.

[0070] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A combination drug of Akkermansia and an EET homologue, characterized in that, Composed of the first active ingredient and the second active ingredient composition: The first active ingredient is *Ackermania viride*, which was purchased from the American Culture Collection Center (ATCC) with accession number ATCC BAA-835. The second active ingredient is at least one of 14,15-epoxyeicosatotrienoic acid or a pharmaceutically acceptable salt thereof; The ratio of the first active ingredient to the second active ingredient is 10. 6 ~10 9 CFU: 0.01~2μg.

2. The combined drug of Akkermansia and EET homologue according to claim 1, characterized in that, The combined drug is a combination of two separate formulations of the first active ingredient and the second active ingredient.

3. The use of the combination drug of Akkermansia and EET homologue as described in claim 1 or 2 in the preparation of antitumor drugs; The anti-tumor drug is an anti-non-small cell lung cancer drug.

4. The application according to claim 3, characterized in that, The antitumor drug also includes a pharmaceutically acceptable carrier.

5. The application according to claim 3, characterized in that, The antitumor drug is administered via the gastrointestinal tract and / or non-gastrointestinal routes.

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