A tumor inhibiting preparation based on akkermansia muciniphila

Abstract

The application discloses a kind of based on inhibiting tumor preparation of mucinophilic akkermansia, belong to tumor medicine field, the present application will AKK and tumor cell are mixed according to certain proportion and are injected in mouse body, it is found that AKK can inhibit the growth of tumor cell, and AKK can survive at least 7 days in tumor cell, and the effect of living AKK is better than the effect of dead AKK, in the present application, by AKK is mixed in biological activity liquid, based on inhibiting tumor preparation of mucinophilic akkermansia is prepared, amino glucosamine base acid coupling agent can also be added in preparation inhibiting tumor preparation, amino glucosamine base acid coupling agent has amino glucosamine group coupled by succinic acid and 2-aminothiazole-5-carboxylic acid ester group.The present application is a kind of based on inhibiting tumor preparation of mucinophilic akkermansia, which has good inhibitory effect on tumor cells, good safety, and can be used for preparing anti-tumor drugs.

Description

Technical Field

[0001] This invention belongs to the field of tumor drugs, specifically relating to a tumor-inhibiting agent based on Akkermansia myxophilus. Background Technology

[0002] Akkermansia muciniphila (AKK) is an anaerobic bacterium that inhabits the human gut. As an important member of the human gut microbiota, AKK utilizes mucin in the gut as its sole carbon and nitrogen source, playing a crucial role in maintaining the integrity of the intestinal barrier. It can also colonize the intestines of other animals, including mice, and protect intestinal function.

[0003] These functions are mainly attributed to AKK's ability to promote the repair and regeneration of intestinal epithelial cells by consuming mucin, thereby enhancing the intestinal barrier function and preventing harmful substances from entering the body. Simultaneously, the decomposed mucin can serve as food for other probiotics. These combined effects can further enhance the host's immune system, strengthen the body's immunity, and prevent infections and inflammatory diseases.

[0004] AKK bacteria are closely related to human health. Numerous studies have shown that gut AKK directly affects metabolic diseases such as obesity and type 2 diabetes. In conclusion, AKK bacteria have broad application prospects in preventing various metabolic diseases. Summary of the Invention

[0005] The purpose of this invention is to provide a tumor-inhibiting agent based on Akkermansia myxophilus that has a good inhibitory effect on tumor cells, good safety profile, and can be used to prepare anti-tumor drugs.

[0006] The technical solution adopted by the present invention to achieve the above objectives is as follows:

[0007] This invention discloses a method for preparing an anti-tumor agent, comprising: mixing Akkermansia myxophilus and a bioactive liquid to prepare an anti-tumor agent; the content of Akkermansia myxophilus in the anti-tumor agent is (1-10)×10 8 CFU / mL. This invention investigated the inhibitory effect of Akkermansia myxophilus on tumors. However, the inhibitory effect of Akkermansia myxophilus requires a healthy immune system; otherwise, it cannot inhibit tumors. Live Akkermansia myxophilus is more effective than dead Akkermansia myxophilus, and the dosage of live Akkermansia myxophilus should not be too low, otherwise the inhibitory effect on tumors will be poor.

[0008] Preferably, the tumor-inhibiting agent further contains an excipient, including a glucosamine conjugate having an glucosamine group coupled with succinic acid and a 2-aminothiazol-5-carboxylic acid ester group. Furthermore, the present invention allows for the co-use of the glucosamine conjugate with Akkermansia myxophilus, which can further enhance the tumor-inhibiting effect. Insufficient amounts of the glucosamine conjugate will not improve the tumor-inhibiting effect.

[0009] More preferably, the content of excipients in the tumor-inhibiting preparation is 0.001-0.04 wt%.

[0010] More preferably, the content of glucosamine conjugate in the tumor inhibitor is 0.001-0.03 wt%.

[0011] Preferably, the glucosamine conjugate is prepared by reacting acetoxyglucosamine with 2-aminothiazol-5-carboxylic acid ester and then deacetylifying it; acetoxyglucosamine is prepared by reacting glucosamine with acetic anhydride to obtain acetoxyglucosamine, and then reacting it with succinic anhydride.

[0012] More preferably, the amount of glucosamine used is 10-30 wt% of acetic anhydride.

[0013] More preferably, the amount of 2-aminothiazole-5-carboxylic acid ester used is 10-30 wt% of acetoxyglucosamine.

[0014] More preferably, the amount of succinic anhydride used is 20-40 wt% of acetoxyglucosamine.

[0015] More preferably, the preparation of the glucosamine conjugate includes the preparation of acetoxyglucosamine and the preparation of the glucosamine conjugate.

[0016] More preferably, in the preparation of acetoxyglucosamine, glucosamine is mixed with acetic anhydride, concentrated sulfuric acid is added at 0-5°C, and the mixture is stirred at 20-40°C for 3-9 hours. After the reaction is completed, anhydrous ethanol is added at 0-5°C to precipitate the precipitate, and acetoxyglucosamine is obtained by separation. Acetoxyglucosamine and potassium carbonate are added to DCM and mixed, then succinic anhydride is added and the mixture is reacted at 20-40°C for 3-9 hours. After the reaction is completed, saturated sodium chloride solution is added, the pH is adjusted to 3-4, and the mixture is allowed to stand for separation. The organic phase is separated, dried with anhydrous sodium sulfate, and evaporated to dryness. The dispersed precipitate is added, and the precipitate is obtained by separation.

[0017] More preferably, in the preparation of acetoxyglucosamine, the amount of glucosamine used is 10-30 wt% of acetic anhydride.

[0018] More preferably, in the preparation of acetoxyglucosamine, the amount of concentrated sulfuric acid used is 60-100 wt% of glucosamine.

[0019] More preferably, in the preparation of acetoxyglucosamine, anhydrous ethanol is used in an appropriate amount, and saturated sodium chloride solution is used in an appropriate amount.

[0020] More preferably, in the preparation of acetoxyglucosamine, the amount of acetoxyglucosamine used is 10-30 wt% of DCM.

[0021] More preferably, in the preparation of acetoxyglucosamine, the amount of potassium carbonate used is 60-90 wt% of acetoxyglucosamine.

[0022] More preferably, in the preparation of acetoxyglucosamine, the amount of succinic anhydride used is 20-40 wt% of acetoxyglucosamine.

[0023] More preferably, in the preparation of acetoxyglucosamine, the dispersion precipitate includes DCM and diethyl ether, wherein the DCM and diethyl ether are mixed in a volume ratio of 1:5-10. The amount of dispersion precipitate used is 300-400 wt% of acetoxyglucosamine.

[0024] More preferably, in the preparation of the glucosamine conjugate, acetoxyglucosamine, DIPEA, and trimethylacetyl chloride are added to DMF and mixed, and reacted at 0-5°C for 10-30 min. Then, 2-aminothiazol-5-carboxylic acid ester is added, and the mixture is stirred at 30-50°C for 3-9 h. After the reaction is complete, saturated sodium chloride solution is added, the pH is adjusted to 3-4, a precipitate is formed, and the acetoxyglucosamine conjugate is obtained. Then, the acetoxyglucosamine conjugate, methanol, and sodium methoxide are mixed and stirred at 20-40°C for 2-6 h. After the treatment is complete, the pH is adjusted to 3-4, the mixture is rotary evaporated to dryness, a dispersion precipitate is added, a precipitate is formed, and the glucosamine conjugate is obtained.

[0025] More preferably, in the preparation of the glucosamine conjugate, the amount of acetoxyglucosamine used is 30-70 wt% of DMF.

[0026] More preferably, in the preparation of the glucosamine conjugate, the amount of DIPEA used is 20-30 wt% of acetoxyglucosamine.

[0027] More preferably, in the preparation of the glucosamine conjugate, the amount of trimethylacetyl chloride used is 20-30 wt% of acetoxyglucosamine.

[0028] More preferably, in the preparation of the glucosamine conjugate, the amount of 2-aminothiazole-5-carboxylic acid ester used is 10-30 wt% of acetoxyglucosamine.

[0029] More preferably, in the preparation of the glucosamine conjugate, the amount of methanol used is 400-800 wt% of the acetoxyglucosamine conjugate.

[0030] More preferably, in the preparation of the glucosamine conjugate, the amount of sodium methoxide used is 60-100 wt% of the acetoxyglucosamine conjugate.

[0031] More preferably, in the preparation of the glucosamine conjugate, the dispersion precipitate includes DCM and diethyl ether, wherein the DCM and diethyl ether are mixed in a volume ratio of 1:5-10. The amount of dispersion precipitate used is 300-400 wt% of the acetoxyglucosamine conjugate. A suitable amount of saturated sodium chloride solution is used.

[0032] Preferably, in the preparation of the tumor-inhibiting agent, Akkermansia myxophilus is dispersed in a bioactive liquid to obtain the tumor-inhibiting agent.

[0033] More preferably, in the preparation of the tumor-inhibiting agent, Akkermansia myxophilus includes live AKK and / or dead AKK.

[0034] More preferably, in the preparation of the tumor-inhibiting agent, the dead AKK is AKK that has undergone pasteurization.

[0035] More preferably, in the preparation of the tumor-inhibiting agent, the bioactive liquid is a sterile PBS solution or other pharmaceutical solvent or other medical solvent.

[0036] More preferably, in the preparation of the tumor-inhibiting agent, the content of Akkermansia myxophilus in the tumor-inhibiting agent is (1-10)×10⁻⁶. 8 CFU / mL.

[0037] Preferably, the excipient further includes DL-3-(3,4-dihydroxyphenyl)alanine, and the content of DL-3-(3,4-dihydroxyphenyl)alanine in the tumor-inhibiting agent is 0.001-0.01 wt%. In addition to using glucosamine conjugates, the excipient of the present invention can also use glucosamine conjugates and DL-3-(3,4-dihydroxyphenyl)alanine together. The combined use of glucosamine conjugates and DL-3-(3,4-dihydroxyphenyl)alanine can improve the tumor-inhibiting effect, but if only DL-3-(3,4-dihydroxyphenyl)alanine is used, the tumor-inhibiting effect cannot be improved.

[0038] This invention discloses the tumor-inhibiting agent prepared by the above-described preparation method.

[0039] This invention discloses a mouse model for inhibiting tumors, comprising: mixing the above-mentioned tumor-inhibiting agent and tumor cells and injecting them into mice to obtain a mouse model for inhibiting tumors.

[0040] Preferably, in the preparation of the mouse model, the tumor-inhibiting agent and colon cancer cells are mixed to obtain a mixture, and the mixture is inoculated subcutaneously into mice, with 1 million tumor cells inoculated at each inoculation site.

[0041] More preferably, the amount of tumor-inhibiting agent used is measured by the amount of Akkermansia myxophilus present therein, with an Akkermansia myxophilus to colon cancer cell ratio of 0.5-10:1.

[0042] This invention discloses an anti-tumor drug, comprising: the above-mentioned tumor-inhibiting agent.

[0043] Preferably, the antitumor drugs include anti-colon cancer drugs.

[0044] This invention utilizes a method of co-injecting AKK with tumor cells in a specific ratio into mice. It was found that AKK can inhibit tumor cell growth, and AKK can survive in tumor cells for at least 7 days. Furthermore, the effect of live AKK is superior to that of dead AKK. This invention prepares an anti-tumor agent based on *Ackermania mutans* by mixing AKK with a bioactive solution. In the preparation of this anti-tumor agent based on *Ackermania mutans*, glucosamine conjugates can be added as excipients. In the preparation of the glucosamine conjugate, glucosamine first reacts with acetic anhydride to protect the hydroxyl group, yielding acetoxyglucosamine. Then, the amino group on acetoxyglucosamine reacts with succinic anhydride to obtain acetoxyglucosamine amino acid. Acetoxyglucosamine amino acid reacts with 2-aminothiazol-5-carboxylic acid ester to generate the acetoxyglucosamine amino acid conjugate. Finally, the acetyl group is removed to obtain the glucosamine amino acid conjugate. Therefore, this invention has the following beneficial effects: good safety and good inhibitory effect on tumor cells. Therefore, the present invention is a tumor-inhibiting agent based on Akkermansia myxophilus that has a good inhibitory effect on tumor cells, good safety profile, and can be used to prepare anti-tumor drugs. Attached Figure Description

[0045] Figure 1 This is a schematic diagram of the delivery method.

[0046] Figure 2 This is a tumor growth curve.

[0047] Figure 3 This is a diagram showing the size of the tumor.

[0048] Figure 4 Image showing tumor weight in nude mice.

[0049] Figure 5 Image showing the weight of tumors in wild rats.

[0050] Figure 6 This is a graph showing the tumor cell inhibition rate. Detailed Implementation

[0051] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0052] The concepts involved in this application will first be described with reference to the accompanying drawings. It should be noted that the following descriptions of various concepts are only for the purpose of making the content of this application easier to understand and do not constitute a limitation on the scope of protection of this application; furthermore, the embodiments and features in the embodiments of this application can be combined with each other unless otherwise specified. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0053] The research content of this invention's implementation method is as follows:

[0054] Culture of *Ackermania myxophilus*: *Ackermania myxophilus*, accession number ATCC BAA-835, was purchased from Beijing Bowen Biotechnology Co., Ltd. *Ackermania myxophilus* was cultured in brain heart infusion (BHI) medium containing mucin under strictly anaerobic conditions. Colony formation units (CFU / mL) were determined by plate counting on BHI medium containing 1% agarose. For experimental use, the cultured *Ackermania myxophilus* was diluted with phosphate-buffered saline (PBS) to achieve a final concentration of 1 × 10⁻⁶ in 200 μL. 9 CFU was used to obtain a bacterial suspension. Pasteurization was performed by incubating the bacterial suspension at 70°C for 30 minutes.

[0055] Tumor cell culture: Mouse colorectal cancer cell lines MC38 and CT26 were used, purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. MC38 or CT26 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin. Cells were cultured at 37°C and 5% carbon dioxide. Before use, PCR testing confirmed the cells were free of mycoplasma contamination.

[0056] Specific pathogen-free (SPF) C57BL / 6 mice were purchased from the Animal Center of Hangzhou Medical College and acclimatized for one week before treatment. The C57BL / 6 mice were 4-6 weeks old and housed in an environment of 22-23°C with a 12-hour light-12-hour dark cycle, and had free access to water and regular food.

[0057] This study employed two delivery methods: 1) oral gavage, i.e., conventional gavage; and 2) direct subcutaneous injection after mixing with tumor cells, i.e., direct delivery. A schematic diagram of the delivery methods is shown below. Figure 1 As shown. AKK was dispersed in sterile PBS solution by oral gavage to obtain an AKK suspension. For the first delivery method, the drug was administered orally by gavage once every 3 days. Control group mice received the same volume of sterile PBS by gavage. For the second delivery method, the AKK suspension was mixed with tumor cells in a specific ratio to obtain a mixture, and finally 100 μL of the mixture was subcutaneously injected into mice. The concentration of the AKK suspension was 1 × 10⁻⁶. 9 One cell was suspended in 200 microliters of PBS.

[0058] Intratumoral injection of AKK: 1×10 6 MC38 cells were subcutaneously injected, followed by intratumoral injection of AKK suspension 5-7 days later. Tumor samples were collected at 24 and 48 hours post-injection, and the homogenates were diluted and seeded onto BHI plates containing gentamicin and kanamycin. The concentration of the AKK suspension was 5 × 10⁻⁶. 8 CFU was dissolved in 30 μL of PBS.

[0059] Intravenous injection of AKK: 1×10 6 MC38 cells were subcutaneously injected, and AKK suspension was introduced into mice via tail vein injection 7 days later. Blood and tumor samples were collected at 2, 6, 24, 48, and 76 hours after AKK suspension injection and cultured on BHI medium plates containing gentamicin and kanamycin. The concentration of AKK suspension included 10... 7 CFU dissolved in 100 μL of PBS, 10 8 CFU dissolved in 100 μL of PBS and 10 μL of water. 9 CFU was dissolved in 100 μL of PBS.

[0060] Subcutaneous inoculation of tumor cells / AKK mixture: AKK in an AKK suspension was mixed with tumor cells at a specific ratio to obtain 100 μL of the mixture, which was then subcutaneously injected into mice. The concentration of the AKK suspension was 1 × 10⁻⁶. 9Cells were suspended in 200 μL of PBS. The ratio of Akk cells to tumor cells was 10:1, 5:1, 1:1, 0.25:1, 0.1:1, and 0.05:1. Tumor growth was observed: tumor size was measured every 3 days after inoculation. Tumors were collected and weighed 25 days after inoculation and statistically analyzed. They were divided into two parts: 1) rapidly frozen at -80℃; 2) fixed in 10% formalin solution for 24–48 hours.

[0061] This study compared the effects of oral gavage and direct subcutaneous injection on tumor growth using colorectal cancer (CRC) cells in an syngeneic mouse model. In the oral gavage group, C57BL / 6 mice were administered 1×10⁻⁶ cells by gavage every 3 days. 9 AKK, for a period of 5 weeks. At the beginning of the third week, these mice were vaccinated with 1×10 6 MC38 tumor cells were collected and AKK was administered via gavage until the tumor was harvested. Using this method, this study observed that the AKK group showed approximately a 35% reduction in both tumor growth and tumor weight at the end of the experiment compared to the PBS control group.

[0062] In this study, AKK was administered orally to mice carrying tumors, and blood and tumor tissue were collected at different time points to isolate AKK. From blood samples, 763 and 113 colony-forming units (CFU / mL) were recovered at 2 and 6 hours after fresh administration, respectively. However, direct recovery of bacteria from tumors was unsuccessful. Therefore, an enrichment method was employed, namely, incubating tumor tissue homogenates in BHI medium for 5 days. The results showed that the tumors of these mice did indeed contain live AKK.

[0063] To further investigate the ability of bacteria to migrate from the bloodstream to tumor sites and establish colonization, this study injected tumor cells using a direct delivery method. When the tumor reached approximately 100 mm... 3 In this study, AKK was administered intravenously. Samples were then collected from blood and tumor at 2, 6, 24, and 48 hours post-injection. This study successfully isolated AKK directly from both blood and tumor. Specifically, bacteria were isolated from blood samples within 2 to 24 hours, but were undetectable after 48 hours. Notably, a greater quantity of AKK was isolated from tumor tissue at the corresponding time points compared to blood samples. In particular, even at 48 hours post-injection, over 1000 colony-forming units (CFU) were obtained per gram of tumor tissue, while no colonies formed in the blood samples at this time. No colonies formed by 76 hours were observed.

[0064] These results indicate that bacteria can reach tumor sites via the bloodstream and may survive within the tumor for a period of time. To further test the survival of AKK in tumors, this study introduced AKK via intratumoral injection at a dose of 5 × 10⁻⁶ per tumor. 8 AKK was isolated from the tumor at 24, 48, and 168 hours post-injection, with more than 10 AKK samples obtained at both time points. 7 The recovery rate of 1 colony-forming unit / gram of tumor was much higher than that of tail vein injection, indicating that AKK can survive in tumors for at least 7 days.

[0065] The above findings indicate that AKK can be administered directly to mice via injection. In this study, 1×10⁻⁶ AKK was injected subcutaneously. 6 MC38 cells were pre-mixed with an equal number of AKK cells at a 1:1 ratio. Within 3 weeks post-injection, tumor growth in the AKK group was significantly slower than in the PBS group. At tumor harvest, the tumor volume in the AKK group was only one-quarter that in the PBS group. Similarly, the tumor weight in the AKK group was reduced by more than four times. Clearly, the tumor inhibition rate achieved through this direct delivery method was significantly higher than that achieved through gavage. Furthermore, this study also replaced AKK with E. coli, but no inhibition was observed in the E. coli-treated group.

[0066] This study used a direct delivery method to test the interaction between different ratios of AKK and tumor cells. The results are as follows: Figure 2 and Figure 3 As shown, Figure 2 Tumor growth curves after different inoculation ratios are shown for groups A, B, C, D, E, F, and G. Figure 3Tumor sizes after different inoculation ratios are shown in Groups A, B, C, D, E, F, and G. × indicates tumor disappearance. Tumor sizes after different inoculation ratios show that all inoculation ratios, from 0.1:1 to 10:1, significantly reduced tumor growth. Compared to the PBS control group, this effect was directly proportional to the inoculation ratio. Notably, when the AKK:MC38 cell ratio was 5:1, 2 out of 6 tumors disappeared; while at a ratio of 10:1, 5 out of 6 tumors disappeared. According to the tumor growth curve, tumors initially grew for a period before decreasing, especially at an AKK:MC38 cell ratio of 10:1, indicating that AKK-mediated tumor suppression remains effective even when AKK is no longer present. Furthermore, this study conducted the same experiment using CT26 cells. By using different ratios of AKK and CT26 cells, it was demonstrated that the inhibitory effect of AKK and CT26 cells on CT26 cells was directly proportional to their ratio; that is, the higher the ratio of AKK to CT26 cells, the better the inhibitory effect. Moreover, the AKK-mediated tumor inhibition rate was higher in CT26 tumors than in MC38 tumors.

[0067] This study investigated immunodeficient nude mice and immunodeficient wild mice. Immunodeficient nude mice were athymic nude mice, and immunodeficient wild mice were normal mice. To more accurately quantify tumor burden, MC38 cells expressing luciferase were used. Results for immunodeficient nude mice are as follows: Figure 4 As shown, group A consisted of nude mice inoculated with MC38 cells and sterile PBS solution, while group B consisted of nude mice inoculated with MC38 cells and AKK suspension. The mean luciferase signals of groups A and B were almost identical, and there was no significant difference in tumor weight between the two groups. Results for immunodeficient wild mice are shown below. Figure 5 As shown, group A consisted of wild rats inoculated with MC38 cells and sterile PBS solution, while group B consisted of wild rats inoculated with MC38 cells and AKK suspension. The luciferase signal and tumor weight in group B were significantly lower than those in group A. This indicates that the host immune system plays a crucial role in AKK-mediated tumor suppression.

[0068] This study compared live AKK and dead AKK in a directly delivered mouse model. The results showed that live AKK was significantly superior to dead AKK in inhibiting tumor growth. Dead AKK was pasteurized AKK.

[0069] Hematoxylin and eosin (H&E) staining revealed apoptotic and necrotic areas in the dead AKK group, but these areas were less extensive than in the live AKK group. Conversely, the KI-67 signal was stronger in the dead AKK group than in the live AKK group. Immunohistochemical staining showed that dead AKK exhibited some tumor-suppressive activity compared to the PBS control group, but this effect was not as significant as that of live AKK. Quantitative analysis further confirmed this trend. Given the tumor-suppressive effect of dead AKK and tumor cells at a 1:1 ratio, this study subsequently tested whether increasing the ratio of dead AKK would enhance the tumor-suppressive effect as much as live AKK. However, even at a 10:1 ratio, the tumor-suppressive effect of dead AKK was not as significant as that of live AKK at a 1:1 ratio, which contrasts sharply with the ratio-dependent effect of live AKK. In summary, the direct delivery method has the following advantages: more effective tumor suppression, shorter experimental cycle, avoidance of cumbersome gavage procedures, and reduced animal stress.

[0070] Example 1: A tumor-inhibiting agent based on Akkermansia myxophilus.

[0071] Tumor-inhibiting agent: The tumor-inhibiting agent was obtained by dispersing *Ackermania myxophilus* in a bioactive solution. The *Ackermania myxophilus* was a dead AKK. The dead AKK was pasteurized AKK. The bioactive solution was sterile PBS. The concentration of *Ackermania myxophilus* in the tumor-inhibiting agent was 5 × 10⁻⁶. 8 CFU / mL.

[0072] Example 2: A tumor-inhibiting agent based on Akkermansia myxophilus

[0073] Tumor-inhibiting agent: The tumor-inhibiting agent was obtained by dispersing *Ackermania myxophilus* and an adjuvant in a bioactive solution. The *Ackermania myxophilus* was a dead AKK. The dead AKK was pasteurized AKK. The bioactive solution was sterile PBS. The concentration of *Ackermania myxophilus* in the tumor-inhibiting agent was 5 × 10⁻⁶. 8 CFU / mL. The excipient is a glucosamine conjugate, which is formed by the reaction of acetoxyglucosamine with 2-aminothiazol-5-carboxylic acid ester. The content of acetoxyglucosamine in the tumor inhibitor is 0.021 wt%.

[0074] Preparation of acetoxyglucosamine: Glucosamine and acetic anhydride were mixed, concentrated sulfuric acid was added at 0°C, and the mixture was stirred at 30°C for 6 hours. After the reaction was complete, anhydrous ethanol was added at 0°C, precipitating out acetoxyglucosamine. Acetoxyglucosamine and potassium carbonate were mixed in DCM, then succinic anhydride was added, and the mixture was reacted at 30°C for 6 hours. After the reaction was complete, saturated sodium chloride solution was added, the pH was adjusted to 3, and the mixture was allowed to stand for separation. The organic phase was separated, dried over anhydrous sodium sulfate, and evaporated to dryness. The dispersed precipitate was added, precipitating out acetoxyglucosamine. The amount of glucosamine used was 20 wt% of acetic anhydride, and the amount of concentrated sulfuric acid used was 80 wt% of glucosamine. Anhydrous ethanol was used in appropriate amounts. Saturated sodium chloride solution was used in appropriate amounts. The amount of acetoxyglucosamine used was 20 wt% of DCM, 80 wt% of potassium carbonate used, and 30 wt% of succinic anhydride used. The dispersion comprises DCM and diethyl ether, which are mixed in a volume ratio of 1:8. The amount of dispersion used is 350 wt% of acetoxyglucosamine.

[0075] Preparation of glucosamine conjugates: Acetoxyglucosamine, DIPEA, and trimethylacetyl chloride were mixed in DMF and reacted at 0°C for 20 min. Then, 2-aminothiazole-5-carboxylic acid ester was added, and the mixture was stirred at 40°C for 6 h. After the reaction was complete, saturated sodium chloride solution was added to adjust the pH to 3, and a precipitate was formed. The acetoxyglucosamine conjugates were then separated. The acetoxyglucosamine conjugates were then mixed with methanol and sodium methoxide and stirred at 30°C for 4 h. After treatment, the pH was adjusted to 3, and the mixture was rotary evaporated to dryness. The dispersion precipitate was added, and a precipitate was formed. The glucosamine conjugates were then separated. The amount of acetoxyglucosamine used was 50 wt% of DMF, 25 wt% of DIPEA, 25 wt% of trimethylacetyl chloride, and 20 wt% of 2-aminothiazole-5-carboxylic acid ester. The amount of methanol used is 600 wt% of the acetoxyglucosamine conjugate, and the amount of sodium methoxide used is 80 wt% of the acetoxyglucosamine conjugate. The dispersion precipitate consists of DCM and diethyl ether, which are mixed in a volume ratio of 1:8. The amount of dispersion precipitate used is 350 wt% of the acetoxyglucosamine conjugate. Saturated sodium chloride solution is used in appropriate amounts.

[0076] Example 3: A tumor-inhibiting agent based on Akkermansia myxophilus.

[0077] The difference between this embodiment and Example 2 lies in the preparation of the tumor-inhibiting agent.

[0078] Tumor-inhibiting agent: The tumor-inhibiting agent was obtained by dispersing *Ackermania myxophilus* and an adjuvant in a bioactive solution. The *Ackermania myxophilus* was a dead AKK. The dead AKK was pasteurized AKK. The bioactive solution was sterile PBS. The concentration of *Ackermania myxophilus* in the tumor-inhibiting agent was 5 × 10⁻⁶. 8 CFU / mL. The excipient is a glucosamine conjugate, which is formed by the reaction of acetoxyglucosamine with 2-aminothiazol-5-carboxylic acid ester. The content of acetoxyglucosamine in the tumor inhibitor is 0.009 wt%.

[0079] Example 4: A tumor-inhibiting agent based on Akkermansia myxophilus

[0080] The difference between this embodiment and Example 2 is that DL-3-(3,4-dihydroxyphenyl)alanine can also be added as an excipient.

[0081] Tumor-inhibiting agent: The tumor-inhibiting agent was obtained by dispersing *Ackermania myxophilus* and an adjuvant in a bioactive solution. The *Ackermania myxophilus* was a dead AKK. The dead AKK was pasteurized AKK. The bioactive solution was sterile PBS. The concentration of *Ackermania myxophilus* in the tumor-inhibiting agent was 5 × 10⁻⁶. 8 CFU / mL. The excipients are glucosamine conjugate and DL-3-(3,4-dihydroxyphenyl)alanine. The glucosamine conjugate is formed by the reaction of acetoxyglucosamine with 2-aminothiazol-5-carboxylic acid ester. The content of glucosamine conjugate in the tumor inhibitor is 0.021 wt%, and the content of DL-3-(3,4-dihydroxyphenyl)alanine in the tumor inhibitor is 0.007 wt%.

[0082] Example 5: A tumor-inhibiting agent based on Akkermansia myxophilus

[0083] Tumor-inhibiting agent: A *Ackermania myxophilus* was dispersed in a bioactive solution to obtain a tumor-inhibiting agent. The *Ackermania myxophilus* was a live AKK. The bioactive solution was sterile PBS. The concentration of *Ackermania myxophilus* in the tumor-inhibiting agent was 5 × 10⁻⁶. 8 CFU / mL.

[0084] Example 6: A tumor-inhibiting agent based on Akkermansia myxophilus

[0085] Tumor-inhibiting agent: Akkermansia myxophilus and excipients were dispersed in a bioactive solution to obtain the tumor-inhibiting agent. The Akkermansia myxophilus was live AKK. The bioactive solution was sterile PBS. The concentration of Akkermansia myxophilus in the tumor-inhibiting agent was 5 × 10⁻⁶. 8 CFU / mL. The excipient is a glucosamine conjugate, which is formed by the reaction of acetoxyglucosamine with 2-aminothiazol-5-carboxylic acid ester. The content of acetoxyglucosamine in the tumor inhibitor is 0.021 wt%.

[0086] Preparation of acetoxyglucosamine: Glucosamine and acetic anhydride were mixed, concentrated sulfuric acid was added at 0°C, and the mixture was stirred at 30°C for 6 hours. After the reaction was complete, anhydrous ethanol was added at 0°C, precipitating out acetoxyglucosamine. Acetoxyglucosamine and potassium carbonate were mixed in DCM, then succinic anhydride was added, and the mixture was reacted at 30°C for 6 hours. After the reaction was complete, saturated sodium chloride solution was added, the pH was adjusted to 3, and the mixture was allowed to stand for separation. The organic phase was separated, dried over anhydrous sodium sulfate, and evaporated to dryness. The dispersed precipitate was added, precipitating out acetoxyglucosamine. The amount of glucosamine used was 20 wt% of acetic anhydride, and the amount of concentrated sulfuric acid used was 80 wt% of glucosamine. Anhydrous ethanol was used in appropriate amounts. Saturated sodium chloride solution was used in appropriate amounts. The amount of acetoxyglucosamine used was 20 wt% of DCM, 80 wt% of potassium carbonate used, and 30 wt% of succinic anhydride used. The dispersion comprises DCM and diethyl ether, which are mixed in a volume ratio of 1:8. The amount of dispersion used is 350 wt% of acetoxyglucosamine.

[0087] Preparation of glucosamine conjugates: Acetoxyglucosamine, DIPEA, and trimethylacetyl chloride were mixed in DMF and reacted at 0°C for 20 min. Then, 2-aminothiazole-5-carboxylic acid ester was added, and the mixture was stirred at 40°C for 6 h. After the reaction was complete, saturated sodium chloride solution was added to adjust the pH to 3, and a precipitate was formed. The acetoxyglucosamine conjugates were then separated. The acetoxyglucosamine conjugates were then mixed with methanol and sodium methoxide and stirred at 30°C for 4 h. After treatment, the pH was adjusted to 3, and the mixture was rotary evaporated to dryness. The dispersion precipitate was added, and a precipitate was formed. The glucosamine conjugates were then separated. The amount of acetoxyglucosamine used was 50 wt% of DMF, 25 wt% of DIPEA, 25 wt% of trimethylacetyl chloride, and 20 wt% of 2-aminothiazole-5-carboxylic acid ester. The amount of methanol used is 600 wt% of the acetoxyglucosamine conjugate, and the amount of sodium methoxide used is 80 wt% of the acetoxyglucosamine conjugate. The dispersion precipitate consists of DCM and diethyl ether, which are mixed in a volume ratio of 1:8. The amount of dispersion precipitate used is 350 wt% of the acetoxyglucosamine conjugate. Saturated sodium chloride solution is used in appropriate amounts.

[0088] Example 7: A tumor-inhibiting agent based on Akkermansia myxophilus

[0089] The difference between this embodiment and Example 6 lies in the preparation of the tumor-inhibiting agent.

[0090] Tumor-inhibiting agent: Akkermansia myxophilus and excipients were dispersed in a bioactive solution to obtain the tumor-inhibiting agent. The Akkermansia myxophilus was live AKK. The bioactive solution was sterile PBS. The concentration of Akkermansia myxophilus in the tumor-inhibiting agent was 5 × 10⁻⁶. 8 CFU / mL. The excipient is a glucosamine conjugate, which is formed by the reaction of acetoxyglucosamine with 2-aminothiazol-5-carboxylic acid ester. The content of acetoxyglucosamine in the tumor inhibitor is 0.009 wt%.

[0091] Example 8: A tumor-inhibiting agent based on Akkermansia myxophilus

[0092] The difference between this embodiment and Example 5 is that DL-3-(3,4-dihydroxyphenyl)alanine can also be added as an excipient.

[0093] Tumor-inhibiting agent: Akkermansia myxophilus and excipients were dispersed in a bioactive solution to obtain the tumor-inhibiting agent. The Akkermansia myxophilus was live AKK. The bioactive solution was sterile PBS. The concentration of Akkermansia myxophilus in the tumor-inhibiting agent was 5 × 10⁻⁶. 8 CFU / mL. The excipients are glucosamine conjugate and DL-3-(3,4-dihydroxyphenyl)alanine. The glucosamine conjugate is formed by the reaction of acetoxyglucosamine with 2-aminothiazol-5-carboxylic acid ester. The content of glucosamine conjugate in the tumor inhibitor is 0.021 wt%, and the content of DL-3-(3,4-dihydroxyphenyl)alanine in the tumor inhibitor is 0.007 wt%.

[0094] Comparative Example 1: A tumor-inhibiting agent based on Akkermansia myxophilus

[0095] The difference between this embodiment and Example 6 is that the glucosamine conjugate in the excipient is replaced with acetoxyglucosamine.

[0096] Comparative Example 2: A tumor-inhibiting agent based on Akkermansia myxophilus

[0097] The difference between this embodiment and Example 6 is that the content of glucosamine conjugate in the tumor-inhibiting agent is 0.0005 wt%.

[0098] Comparative Example 3: A tumor-inhibiting agent based on Akkermansia myxophilus

[0099] The difference between this embodiment and Example 8 is that the tumor-inhibiting agent does not use glucosamine conjugate.

[0100] Example 9: A mouse model

[0101] Preparation of mouse model: A mixture was prepared by mixing the tumor inhibitor and mouse protocoloma cells. The mixture was then inoculated subcutaneously into mice, with 1 million tumor cells inoculated at each inoculation site. The tumor inhibitor was taken from Example 1, and the amount of tumor inhibitor used was measured based on the amount of dead AKK contained therein. The ratio of dead AKK to mouse protocoloma cells was 1:1.

[0102] Example 10: A mouse model

[0103] A tumor-inhibiting agent was mixed with murine protocoloma cells to obtain a mixture, which was then inoculated subcutaneously into mice, with 1 million tumor cells inoculated at each inoculation site. The tumor-inhibiting agent was taken from Example 2, and the amount of the tumor-inhibiting agent used was measured based on the amount of live AKK contained therein, with a ratio of live AKK to murine protocoloma cells of 1:1.

[0104] Experimental example:

[0105] The cytotoxicity of the acetoxyglucosamine and glucosamine conjugate prepared in Example 3 was tested using mouse fibroblast L929 cells as test cells and the MTT assay was employed. A blank control was used without the test sample. The amount of acetoxyglucosamine or glucosamine conjugate in the test sample was 0.03 wt%, and the culture medium was DMEM. The results showed that acetoxyglucosamine promoted cell proliferation of 99.3% and glucosamine conjugate promoted cell proliferation of 99.8%, indicating that neither acetoxyglucosamine nor glucosamine conjugate is cytotoxic.

[0106] This invention tested the tumor inhibition of the *Ackermania myxophilus*-based antitumor agents prepared in Examples 5-8 and Comparative Examples 1-3. MC38 cells were used as test cells and inoculated into mouse models. The test samples were the *Ackermania myxophilus*-based antitumor agents prepared in Examples 5-8 and Comparative Examples 1-3. The ratio of *Ackermania myxophilus* to MC38 cells in the antitumor agents was 0.5:1, and the number of MC38 cells was 1 million tumor cells. An equal volume of sterile PBS solution was injected as a blank control. The tumor cell inhibition rate was calculated based on the blank control. The results are as follows: Figure 6As shown, S5 is Example 5, S6 is Example 6, S7 is Example 7, S8 is Example 8, D1 is Comparative Example 1, D2 is Comparative Example 2, and D3 is Comparative Example 3. In this study, AKK was mixed with tumor cells in a certain proportion and injected into mice. It was found that AKK could inhibit the growth of tumor cells, and AKK could survive in tumor cells for at least 7 days. Furthermore, the effect of live AKK was superior to that of dead AKK. In this invention, an anti-tumor agent based on *Ackermania mutans* was prepared by mixing AKK in a bioactive liquid. In the preparation of the anti-tumor agent based on *Ackermania mutans*, glucosamine conjugates can also be added as excipients. In the preparation of glucosamine conjugates, glucosamine first reacts with acetic anhydride to protect the hydroxyl groups, obtaining acetoxyglucosamine. Then, the amino group on acetoxyglucosamine reacts with succinic anhydride to obtain acetoxyglucosamine amino acid. Acetoxyglucosamine amino acid reacts with 2-amino... Thiazole-5-carboxylic acid ester reacts to generate an acetoxyglucosamine conjugate, and finally, the acetyl group is removed to obtain the glucosamine conjugate. This invention reveals that when acetoxyglucosamine and AKK are used together to prepare an anti-tumor agent based on Akkermansia myxophilus, the resulting anti-tumor agent does not significantly enhance tumor inhibition. However, when the acetoxyglucosamine conjugate and AKK are used together to prepare an anti-tumor agent based on Akkermansia myxophilus, the resulting anti-tumor agent significantly enhances tumor inhibition. The amount of acetoxyglucosamine conjugate used needs to be within a certain range to be effective; if the amount is too low, the inhibitory effect cannot be enhanced. In addition to using the glucosamine conjugate, this invention can also add DL-3-(3,4-dihydroxyphenyl)alanine as an excipient. The combined use of DL-3-(3,4-dihydroxyphenyl)alanine and the glucosamine conjugate can further improve the anti-tumor effect of the anti-tumor agent.

[0107] The embodiments and / or implementation methods described above are merely preferred embodiments and / or implementation methods for implementing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any way. Any person skilled in the art can make some modifications or alterations to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as the technology or embodiments that are substantially the same as the present invention.

[0108] This document uses specific examples to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of this application. The above descriptions are only preferred embodiments of this application. It should be noted that due to the limitations of written expression, while there are objectively infinite specific structures, those skilled in the art can make several improvements, modifications, or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner. These improvements, modifications, changes, or combinations, or the direct application of the inventive concept and technical solution to other situations without modification, should all be considered within the scope of protection of this application.

Claims

1. A method for preparing a tumor-inhibiting agent, comprising: A tumor-inhibiting agent was prepared by mixing Akkermansia myxophilus and a bioactive liquid; the content of Akkermansia myxophilus in the tumor-inhibiting agent was (1-10)×10. 8 The tumor-inhibiting agent, CFU / mL, further contains excipients, including glucosamine conjugates. The preparation method of the glucosamine conjugates includes: in the preparation of acetoxyglucosamine, glucosamine is mixed with acetic anhydride, concentrated sulfuric acid is added at 0-5°C, and then the mixture is stirred at 20-40°C for 3-9 hours. After the reaction is complete, anhydrous ethanol is added at 0-5°C to precipitate the precipitate, and acetoxyglucosamine is obtained. Acetoxyglucosamine and potassium carbonate are mixed in DCM, then succinic anhydride is added, and the mixture is reacted at 20-40°C for 3-9 hours. After the reaction is complete, saturated sodium chloride solution is added, the pH is adjusted to 3-4, and the mixture is allowed to stand for phase separation. The organic phase is separated, dried over anhydrous sodium sulfate, and evaporated to dryness. The dispersed precipitate is added, and the precipitate is obtained. Acetoxyglucosamine is obtained. In the preparation of the glucosamine conjugates, acetoxyglucosamine is mixed with acetoxyglucosamine and potassium carbonate. Glucosamine, DIPEA, and trimethylacetyl chloride were mixed in DMF and reacted at 0-5°C for 10-30 min. Then, 2-aminothiazol-5-carboxylic acid ester was added, and the mixture was stirred at 30-50°C for 3-9 h. After the reaction was completed, saturated sodium chloride solution was added, and the pH was adjusted to 3-4. A precipitate was formed, and the acetoxyglucosamine conjugate was obtained. The acetoxyglucosamine conjugate, methanol, and sodium methoxide were then mixed and stirred at 20-40°C for 2-6 h. After the treatment, the pH was adjusted to 3-4, and the mixture was evaporated to dryness. A dispersion was added, and a precipitate was formed, and the glucosamine conjugate was obtained. The glucosamine conjugate content in the tumor inhibitor was 0.001-0.03 wt%. The tumor was colon cancer, the bioactive solution was sterile PBS, and the accession number of Akkermansia myxophilus was ATCC BAA-835.

2. The method for preparing a tumor-inhibiting agent according to claim 1, characterized in that: The amount of glucosamine used is 10-30 wt% of acetic anhydride.

3. The method for preparing a tumor-inhibiting agent according to claim 1, characterized in that: The amount of 2-aminothiazole-5-carboxylic acid ester used is 10-30 wt% of acetoxyglucosamine.

4. The method for preparing a tumor-inhibiting agent according to claim 1, characterized in that: The amount of succinic anhydride used is 20-40 wt% of acetoxyglucosamine.

5. The tumor-inhibiting agent prepared by any of the preparation methods described in claims 1-4.

6. An antitumor drug, comprising: The tumor-inhibiting agent according to claim 5, wherein the antitumor drug is an anti-colon cancer drug.

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