Tumor inhibition preparation based on Ackermania muciniphila

Abstract

The invention discloses a tumor inhibition preparation based on Ackermania muciniphila, and belongs to the field of tumor medicine.AKK and tumor cells are mixed according to a certain proportion and jointly injected into a mouse body, and it is found that AKK can inhibit growth of the tumor cells and can survive for at least 7 days in the tumor cells; in addition, the effect of AKK in a living body is better than that of AKK in a dead body, the AKK is mixed in a bioactive solution to prepare the tumor inhibition preparation based on Ackermania muciniphila, and an auxiliary agent glucosamino acid conjugate can be added in the preparation of the tumor inhibition preparation, so that the AKK-based tumor inhibition preparation can be used for preparing the tumor inhibition preparation based on Ackermania muciniphila and the AKK-based tumor inhibition preparation based on Ackermania muciniphila. The glucosamine acid conjugate has a glucosamine group and a 2-aminothiazole-5-carboxylic acid ester group, which are coupled by a succinic acid. The ackermansiella muciniphila-based tumor inhibition preparation provided by the invention has a good inhibition effect on tumor cells, is good in safety and can be used for preparing anti-tumor drugs.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of tumor drugs, and particularly relates to a tumor inhibiting preparation based on Akkermansia muciniphila. BACKGROUND

[0002] Akkermansia muciniphila (AKK for short) is an anaerobic bacterium that inhabits in the human intestinal tract. As an important member of the human intestinal microflora, AKK can utilize mucin in the intestinal tract as its sole carbon and nitrogen source, and plays an important role in maintaining the integrity of the intestinal barrier. Meanwhile, it can also colonize in the intestines of other animals including mice and protect the function of the intestines.

[0003] These functions are mainly attributed to the fact that AKK can 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. Meanwhile, the decomposed mucin can serve as food for other intestinal probiotics, and these combined effects can further enhance the host's immune system, increase the body's immunity, and prevent the occurrence of infectious and inflammatory diseases.

[0004] AKK is closely related to human health. A large number of studies have shown that intestinal AKK directly affects metabolic diseases such as obesity and type 2 diabetes. In summary, AKK bacteria have broad application prospects in preventing various metabolic diseases. SUMMARY

[0005] The application aims to provide a tumor inhibiting preparation based on Akkermansia muciniphila, which has good inhibitory effect on tumor cells, good safety, and can be used for preparing an anti-tumor drug.

[0006] The technical scheme adopted by the application to achieve the above-mentioned purpose is as follows: The application discloses a preparation method of a tumor inhibiting preparation, which comprises the following steps: mixing Akkermansia muciniphila and a bioactive liquid to prepare the tumor inhibiting preparation; and the content of the Akkermansia muciniphila in the tumor inhibiting preparation is (1-10) x 10 8 The application studies the inhibitory effect of Akkermansia muciniphila on tumors, but the effect of Akkermansia muciniphila needs a sound immune system, and otherwise, Akkermansia muciniphila cannot inhibit tumors. The living Akkermansia muciniphila has better effect than the dead Akkermansia muciniphila, and the use amount of the living Akkermansia muciniphila cannot be too small, otherwise the inhibitory effect on tumors is poor.

[0007] Preferably, the tumor inhibiting agent further comprises an auxiliary agent, the auxiliary agent comprising a glucosaminic acid conjugate having a glucosamine group conjugated with succinic acid and a 2-aminothiazole-5-carboxylate group. The present application can further improve the tumor inhibiting effect by using the glucosaminic acid conjugate in combination with Akkermansia muciniphila. The tumor inhibiting effect cannot be improved when the amount of the glucosaminic acid conjugate is too small.

[0008] More preferably, the content of the auxiliary agent in the tumor inhibiting agent is 0.001-0.04wt%.

[0009] More preferably, the content of the glucosaminic acid conjugate in the tumor inhibiting agent is 0.001-0.03wt%.

[0010] Preferably, the glucosaminic acid conjugate is prepared by reacting acetyloxyglucosaminic acid with 2-aminothiazole-5-carboxylate and then deacetylizing; the acetyloxyglucosaminic acid is prepared by reacting glucosamine with acetic anhydride to obtain acetyloxyglucosamine and then reacting with succinic anhydride.

[0011] More preferably, the amount of glucosamine used is 10-30wt% of the acetic anhydride.

[0012] More preferably, the amount of 2-aminothiazole-5-carboxylate used is 10-30wt% of the acetyloxyglucosaminic acid.

[0013] More preferably, the amount of succinic anhydride used is 20-40wt% of the acetyloxyglucosamine.

[0014] More preferably, the preparation of the glucosaminic acid conjugate comprises the preparation of acetyloxyglucosaminic acid and the preparation of the glucosaminic acid conjugate.

[0015] More preferably, in the preparation of the acetyloxyglucosaminic acid, glucosamine is mixed with acetic anhydride, concentrated sulfuric acid is added at 0-5℃, then stirred and reacted at 20-40℃ for 3-9h, after the reaction is completed, anhydrous ethanol is added at 0-5℃, the precipitate is separated to obtain acetyloxyglucosamine; acetyloxyglucosamine and potassium carbonate are mixed in DCM, then succinic anhydride is added, reacted at 20-40℃ for 3-9h, after the reaction is completed, saturated sodium chloride solution is added, the pH is adjusted to 3-4, and then separated by layering, the organic phase is separated, dried with anhydrous sodium sulfate, rotary evaporated to dryness, and then dispersed to precipitate, and the precipitate is separated to obtain acetyloxyglucosaminic acid.

[0016] More preferably, in the preparation of the acetyloxyglucosaminic acid, the amount of glucosamine used is 10-30wt% of the acetic anhydride.

[0017] Further more preferably, in the preparation of acetyloxyglucosamine acid, the amount of concentrated sulfuric acid used is 60-100wt% of the glucosamine.

[0018] Further more preferably, in the preparation of acetyloxyglucosamine acid, anhydrous ethanol is used in an appropriate amount. Saturated sodium chloride solution is used in an appropriate amount.

[0019] Further more preferably, in the preparation of acetyloxyglucosamine acid, the amount of acetyloxyglucosamine used is 10-30wt% of DCM.

[0020] Further more preferably, in the preparation of acetyloxyglucosamine acid, the amount of potassium carbonate used is 60-90wt% of acetyloxyglucosamine.

[0021] Further more preferably, in the preparation of acetyloxyglucosamine acid, the amount of succinic anhydride used is 20-40wt% of acetyloxyglucosamine.

[0022] Further more preferably, in the preparation of acetyloxyglucosamine acid, the dispersion precipitant includes DCM and diethyl ether, and the DCM and diethyl ether are mixed in a volume ratio of 1:5-10. The amount of dispersion precipitant used is 300-400wt% of acetyloxyglucosamine.

[0023] More preferably, in the preparation of glucosamine acid conjugate, acetyloxyglucosamine acid and DIPEA, trimethylacetyl chloride are mixed in DMF, reacted at 0-5℃ for 10-30min, then 2-aminothiazole-5-carboxylate is added, stirred and reacted at 30-50℃ for 3-9h, after the reaction is completed, saturated sodium chloride solution is added, the pH is adjusted to 3-4, the precipitate is precipitated and separated to obtain acetyloxyglucosamine acid conjugate; then acetyloxyglucosamine acid conjugate, methanol and sodium methoxide are mixed, stirred and treated at 20-40℃ for 2-6h, after the treatment is completed, the pH is adjusted to 3-4, rotary evaporation is performed to dryness, a dispersion precipitant is added, the precipitate is precipitated and separated to obtain glucosamine acid conjugate.

[0024] Further more preferably, in the preparation of glucosamine acid conjugate, the amount of acetyloxyglucosamine acid used is 30-70wt% of DMF.

[0025] Further more preferably, in the preparation of glucosamine acid conjugate, the amount of DIPEA used is 20-30wt% of acetyloxyglucosamine acid.

[0026] Further more preferably, in the preparation of glucosamine acid conjugate, the amount of trimethylacetyl chloride used is 20-30wt% of acetyloxyglucosamine acid.

[0027] More preferably, in the preparation of the glucosaminyl acid conjugate, the amount of 2-aminothiazole-5-carboxylic acid ester used is 10-30 wt% of the acetoxyglucosaminyl acid.

[0028] More preferably, in the preparation of the glucosaminyl acid conjugate, the amount of methanol used is 400-800 wt% of the acetoxyglucosaminyl acid conjugate.

[0029] More preferably, in the preparation of the glucosaminyl acid conjugate, the amount of sodium methoxide used is 60-100 wt% of the acetoxyglucosaminyl acid conjugate.

[0030] More preferably, in the preparation of the glucosaminyl acid conjugate, the dispersion eluent comprises DCM and diethyl ether, and the DCM and diethyl ether are mixed in a volume ratio of 1:5-10. The amount of the dispersion eluent used is 300-400 wt% of the acetoxyglucosaminyl acid conjugate. The saturated sodium chloride solution is used in an appropriate amount.

[0031] Preferably, in the preparation of the tumor inhibiting preparation, the mucinophilic Akkermansia bacteria are dispersed in a biologically active liquid to obtain the tumor inhibiting preparation.

[0032] More preferably, in the preparation of the tumor inhibiting preparation, the mucinophilic Akkermansia bacteria comprise live AKK and / or dead AKK.

[0033] More preferably, in the preparation of the tumor inhibiting preparation, the dead AKK is AKK subjected to pasteurization.

[0034] More preferably, in the preparation of the tumor inhibiting preparation, the biologically active liquid is a sterile PBS solution or other pharmaceutical solvent or other medical solvent.

[0035] More preferably, in the preparation of the tumor inhibiting preparation, the content of the mucinophilic Akkermansia bacteria in the tumor inhibiting preparation is (1-10) x 10 8 CFU / mL.

[0036] Preferably, the adjuvant further comprises DL-3-(3,4-dihydroxyphenyl)alanine, and the content of the DL-3-(3,4-dihydroxyphenyl)alanine in the tumor inhibiting preparation is 0.001-0.01 wt%. In addition to the use of the glucosaminyl acid conjugate in the adjuvant of the present application, the glucosaminyl acid conjugate and the DL-3-(3,4-dihydroxyphenyl)alanine can be used together. The use of the glucosaminyl acid conjugate and the DL-3-(3,4-dihydroxyphenyl)alanine together can improve the tumor inhibiting effect, but if only the DL-3-(3,4-dihydroxyphenyl)alanine is used, the tumor inhibiting effect cannot be improved.

[0037] This invention discloses the tumor-inhibiting agent prepared by the above-described preparation method.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] This invention discloses an anti-tumor drug, comprising: the above-mentioned tumor-inhibiting agent.

[0042] Preferably, the antitumor drugs include anti-colon cancer drugs.

[0043] 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 conjugate. Finally, the acetyl group is removed to obtain the glucosamine 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 inhibitory 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

[0044] Figure 1 This is a schematic diagram of the delivery method.

[0045] Figure 2 This is a tumor growth curve.

[0046] Figure 3 This is a diagram showing the size of the tumor.

[0047] Figure 4 Figure 4 is a graph of tumor weight for the nude mice.

[0048] Figure 5 Figure 5 is a graph of tumor weight for the wild-type mice.

[0049] Figure 6 Figure 6 is a graph of tumor cell inhibition rate. DETAILED DESCRIPTION

[0050] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative effort belong to the protection scope of the present application.

[0051] The concepts involved in the present application will be described below first with reference to the drawings. It should be noted that the following descriptions of the concepts are only for the purpose of making the content of the present application easier to understand, and do not represent the limitation on the protection scope of the present application; meanwhile, the embodiments in the present application and the features in the embodiments can be combined with each other without conflict. The present application will be described in detail below with reference to the drawings and in combination with the embodiments.

[0052] The research content of the embodiments of the present application is as follows: Culture of Akkermansia muciniphila: Akkermansia muciniphila with the preservation number of ATCC BAA-835 was purchased from Beijing Biozym Scientific Co., Ltd. Akkermansia muciniphila was cultured in a brain heart infusion (BHI) medium containing mucin under strict anaerobic conditions. Colony forming units (CFU / mL) were determined by performing plate colony counting on a BHI medium containing 1% agarose. For experimental use, the cultured Akkermansia muciniphila was diluted with a phosphate buffer solution (PBS) to achieve a final content of 1×10 9 CFU in 200 microliters of volume, to obtain a bacterial suspension. In pasteurization, the bacterial suspension was incubated at 70°C for 30 minutes.

[0053] Culture of tumor cells: The mouse colorectal cancer cell lines were MC38 and CT26, which were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. MC38 or CT26 was cultured in a DMEM medium added with 10% fetal bovine serum and 1% penicillin and streptomycin. The cells were cultured at 37°C under a condition of 5% carbon dioxide. Before use, the cells were confirmed to be free of mycoplasma contamination by PCR detection.

[0054] Specific pathogen free (SPF) C57BL / 6 mice were purchased from the Animal Center of Hangzhou Medical College and acclimated for one week prior to treatment. C57BL / 6 mice were 4-6 weeks old and housed in a 22-23 °C environment with a 12-hour light and 12-hour dark cycle and free access to water and regular food.

[0055] Two delivery methods were used in this study: 1) oral gavage, i.e. the conventional gavage method, and 2) direct mixing with tumor cells followed by subcutaneous injection, i.e. direct delivery method, as shown in the schematic diagram of delivery methods Figure 1 AKK for oral gavage was dispersed in sterile PBS solution to obtain AKK suspension. For the first delivery method, mice were administered once every 3 days by oral gavage. Control mice were gavaged with the same volume of sterile PBS. For the second delivery method, AKK suspension was mixed with tumor cells at a certain ratio to obtain a mixture, and finally 100 microliters of the mixture was injected subcutaneously into mice. The concentration of AKK suspension was 1 x 10 9 cells were suspended in 200 microliters of PBS.

[0056] Intratumoral injection of AKK: 1 x 10 6 MC38 cells were injected subcutaneously, and 5-7 days later, AKK suspension was administered by intratumoral injection. After injection, tumor samples were collected at 24 hours and 48 hours, and the tumor homogenate was diluted and spotted on BHI plates containing gentamicin and kanamycin. The concentration of AKK suspension was 5 x 10 8 CFU was dissolved in 30 microliters of PBS.

[0057] Intravenous injection of AKK: 1 x 10 6 MC38 cells were injected subcutaneously, and 7 days after injection, AKK suspension was introduced into mice by tail vein injection. At 2, 6, 24, 48, and 76 hours after AKK suspension injection, blood and tumor samples were collected and cultured in BHI medium plates containing gentamicin and kanamycin. The concentration of AKK suspension included 10 7 CFU was dissolved in 100 microliters of PBS, 10 8 CFU was dissolved in 100 microliters of PBS, and 10 9 CFU was dissolved in 100 microliters of PBS.

[0058] Subcutaneous inoculation of tumor cell / AKK mixture: AKK in AKK suspension was mixed with tumor cells at a certain ratio to obtain 100 microliters of mixture, and finally 100 microliters of mixture was injected subcutaneously into mice. The concentration of AKK suspension was 1 x 10 9Cells were suspended in 200 μΐ of PBS. The Akk and tumor cell ratios were: 10:1, 5:1, 1:1, 0.25:1, 0.1:1, and 0.05:1. Tumor growth was observed: the size of the tumors was measured every 3 days after inoculation. Tumors were collected: 25 days after inoculation, tumors were collected and weighed, and statistical analysis was performed. They were divided into 2 parts: 1) quick frozen -80°C; 2) fixed in 10% formalin solution for 24-48 hours.

[0059] This study compared the effects of oral gavage and direct subcutaneous injection on tumor growth using colorectal cancer (CRC) cells in a syngeneic mouse model. For the oral gavage group, this study gavaged C57BL / 6 mice with 1 x 10 9 AKK for 5 weeks. At the beginning of the third week, this study inoculated these mice with 1 x 10 6 MC38 tumor cells and continued the AKK gavage until the tumors were harvested. In this way, this study observed a reduction of approximately 35% in tumor growth and tumor weight at the end of the experiment in the AKK group compared to the PBS control group.

[0060] This study administered AKK to tumor-bearing mice by oral administration and collected blood and tumor tissue at different time points to isolate AKK. From the blood samples, this study recovered 763 and 113 colony-forming units CFU / mL at 2 and 6 hours after fresh administration, respectively. However, the direct recovery of bacteria from the tumor failed. Therefore, this study used an enrichment method, i.e., incubating tumor tissue homogenate in BHI medium for 5 days. The results showed that the tumors of these mice did contain live AKK.

[0061] To further investigate the ability of bacteria to transfer from the blood circulation system to the tumor site and establish colonization, this study injected tumor cells according to the direct delivery method. When the tumor reached approximately 100 mm 3 AKK via intravenous injection. Subsequently, at 2, 6, 24, and 48 hours after injection, this study collected samples from the blood and the tumor. This time, this study successfully isolated AKK directly from the blood and the tumor. Specifically, this study isolated bacteria from blood samples within 2 to 24 hours, but failed to detect any after 48 hours. Notably, this study isolated more AKK from tumor tissue at the corresponding time points compared to blood samples. In particular, this study obtained more than 1000 colony-forming units (CFU) from each gram of tumor tissue even 48 hours after injection, while no colonies were formed in blood samples at that time. No colonies were formed until 76 hours.

[0062] 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.

[0063] 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.

[0064] 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 size after different ratios of inoculation, where Group A, Group B, Group C, Group D, Group E, Group F, and Group G, x represents tumor disappearance; tumor size after different ratios of inoculation shows that all ratios of inoculation from 0.1:1 to 10:1 of AKK to MC38 cells significantly reduced tumor growth, and this effect was proportional to the ratio of inoculation compared to the PBS control group. Notably, when the ratio of AKK to MC38 cells was 5:1, 2 out of 6 tumors disappeared, and when the ratio of AKK to MC38 cells was 10:1, 5 out of 6 tumors disappeared. According to the tumor growth curve, the tumor initially grew for a period of time before it began to decrease, especially when the ratio of AKK to MC38 cells was 10:1, which indicates that AKK-mediated tumor inhibition is still effective even in the absence of AKK. In addition, the same experiment was conducted using CT26 cells by the researchers in this study, and it was proven that the inhibition of CT26 cells by AKK and CT26 cells was proportional to the ratio of their numbers, i.e., the greater the ratio of AKK to CT26 cells, the better the inhibition effect. Moreover, the AKK-mediated tumor inhibition rate was higher in CT26 tumors than in MC38 tumors.

[0065] The present study conducted experiments on both immunodeficient nude mice and non-immunodeficient wild mice. The immunodeficient nude mice were athymic nude mice, and the non-immunodeficient wild mice were normal mice. In order to more accurately quantify tumor burden, the researchers in this study used MC38 cells expressing luciferase. The results for immunodeficient nude mice are shown in Figure 4 , where Group A were nude mice inoculated with MC38 cells and sterile PBS solution, and Group B were nude mice inoculated with MC38 cells and AKK suspension. The average luciferase signal of Group A and Group B was almost the same, and there was no significant difference in tumor weight between the two groups. The results for non-immunodeficient wild mice are shown in Figure 5 , where Group A were wild mice inoculated with MC38 cells and sterile PBS solution, and Group B were wild mice inoculated with MC38 cells and AKK suspension. The luciferase signal and tumor weight of Group B were significantly lower than those of Group A. This indicates that the host immune system plays a key role in AKK-mediated tumor inhibition.

[0066] The present study compared live AKK and dead AKK in a direct delivery mouse model. The results showed that live AKK was significantly better than dead AKK in inhibiting tumor growth. Dead AKK was AKK treated with pasteurization.

[0067] H&E staining showed that there were areas of apoptosis and necrosis in the dead AKK group, but these areas were not as extensive as 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 a degree of tumor suppression 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 suppression effect of dead AKK and tumor cells at a 1:1 ratio, the study subsequently tested whether increasing the ratio of dead AKK could enhance the tumor suppression effect as with live AKK. However, even at a ratio of 10:1, the tumor suppression effect of dead AKK was not as significant as that of live AKK at a ratio of 1:1, which was in sharp contrast to the ratio-dependent effect of live AKK. In summary, the direct delivery method has the following advantages: more effective tumor suppression, shorter experimental period, avoidance of the cumbersome gavage procedure, and reduction of stress on animals.

[0068] Example 1: A mucinophilic Akkermansia-based tumor inhibiting preparation The tumor inhibiting preparation: the mucinophilic Akkermansia was dispersed in a bioactive liquid to obtain the tumor inhibiting preparation. The mucinophilic Akkermansia was dead AKK. The dead AKK was AKK treated by pasteurization. The bioactive liquid was a sterile PBS solution. The content of the mucinophilic Akkermansia in the tumor inhibiting preparation was 5 x 10 8 CFU / mL.

[0069] Example 2: A mucinophilic Akkermansia-based tumor inhibiting preparation The tumor inhibiting preparation: the mucinophilic Akkermansia and the adjuvant were dispersed in a bioactive liquid to obtain the tumor inhibiting preparation. The mucinophilic Akkermansia was dead AKK. The dead AKK was AKK treated by pasteurization. The bioactive liquid was a sterile PBS solution. The content of the mucinophilic Akkermansia in the tumor inhibiting preparation was 5 x 10 8 CFU / mL. The adjuvant was a glucosamine acid conjugate, which was obtained by the reaction of acetyloxyglucosamine acid with 2-aminothiazole-5-carboxylate, and the content of the acetyloxyglucosamine acid in the tumor inhibiting preparation was 0.021 wt%.

[0070] Preparation of acetyloxyglucosamine acid: glucosamine was mixed with acetic anhydride, concentrated sulfuric acid was added at 0℃, and then the reaction was stirred at 30℃ for 6h. After the reaction was completed, anhydrous ethanol was added at 0℃, the precipitate was separated, and acetyloxyglucosamine was obtained. Acetyloxyglucosamine and potassium carbonate were mixed in DCM, succinic anhydride was added, and the reaction was carried out at 30℃ for 6h. After the reaction was completed, saturated sodium chloride solution was added, the pH was adjusted to 3, and the organic phase was separated after standing and layering. The organic phase was dried over anhydrous sodium sulfate, rotary evaporated to dryness, and then a dispersion precipitant was added. The precipitate was separated to obtain acetyloxyglucosamine acid. The use amount of glucosamine was 20wt% of acetic anhydride, and the use amount of concentrated sulfuric acid was 80wt% of glucosamine. Anhydrous ethanol was used in an appropriate amount. Saturated sodium chloride solution was used in an appropriate amount. The use amount of acetyloxyglucosamine was 20wt% of DCM, the use amount of potassium carbonate was 80wt% of acetyloxyglucosamine, and the use amount of succinic anhydride was 30wt% of acetyloxyglucosamine. The dispersion precipitant included DCM and diethyl ether, and the volume ratio of DCM to diethyl ether in the dispersion precipitant was 1:8. The use amount of the dispersion precipitant was 350wt% of acetyloxyglucosamine.

[0071] Preparation of glucosamine acid conjugate: acetyloxyglucosamine acid and DIPEA, trimethylacetyl chloride were mixed in DMF, and the reaction was carried out at 0℃ for 20min. Then 2-aminothiazole-5-carboxylate was added, and the reaction was stirred at 40℃ for 6h. After the reaction was completed, saturated sodium chloride solution was added, the pH was adjusted to 3, and the precipitate was separated to obtain acetyloxyglucosamine acid conjugate. Then acetyloxyglucosamine acid conjugate, methanol, and sodium methoxide were mixed, and the mixture was stirred at 30℃ for 4h. After the treatment was completed, the pH was adjusted to 3, rotary evaporated to dryness, and then a dispersion precipitant was added. The precipitate was separated to obtain glucosamine acid conjugate. The use amount of acetyloxyglucosamine acid was 50wt% of DMF, the use amount of DIPEA was 25wt% of acetyloxyglucosamine acid, the use amount of trimethylacetyl chloride was 25wt% of acetyloxyglucosamine acid, and the use amount of 2-aminothiazole-5-carboxylate was 20wt% of acetyloxyglucosamine acid. The use amount of methanol was 600wt% of acetyloxyglucosamine acid conjugate, and the use amount of sodium methoxide was 80wt% of acetyloxyglucosamine acid conjugate. The dispersion precipitant included DCM and diethyl ether, and the volume ratio of DCM to diethyl ether in the dispersion precipitant was 1:8. The use amount of the dispersion precipitant was 350wt% of acetyloxyglucosamine acid conjugate. Saturated sodium chloride solution was used in an appropriate amount.

[0072] Example 3: A mucinophilic akkermansia-based tumor inhibiting preparation This example is different from example 2 in the preparation of the tumor inhibiting preparation.

[0073] The tumor inhibiting preparation is prepared by dispersing the mucinophilic Akkermansia and the adjuvant in a bioactive liquid. The mucinophilic Akkermansia is dead AKK. The dead AKK is AKK treated by pasteurization. The bioactive liquid is a sterile PBS solution. The content of the mucinophilic Akkermansia in the tumor inhibiting preparation is 5×10 8 CFU / mL. The adjuvant is a glucosamine acid conjugate, which is obtained by reacting acetylglucosamine acid with 2-aminothiazole-5-carboxylic acid ester. The content of the acetylglucosamine acid in the tumor inhibiting preparation is 0.009 wt%.

[0074] Example 4: A tumor inhibiting preparation based on mucinophilic Akkermansia The difference between this example and Example 2 is that DL-3-(3,4-dihydroxyphenyl)alanine can also be added as an adjuvant.

[0075] The tumor inhibiting preparation is prepared by dispersing the mucinophilic Akkermansia and the adjuvant in a bioactive liquid. The mucinophilic Akkermansia is dead AKK. The dead AKK is AKK treated by pasteurization. The bioactive liquid is a sterile PBS solution. The content of the mucinophilic Akkermansia in the tumor inhibiting preparation is 5×10 8 CFU / mL. The adjuvant is a glucosamine acid conjugate and DL-3-(3,4-dihydroxyphenyl)alanine, which is obtained by reacting acetylglucosamine acid with 2-aminothiazole-5-carboxylic acid ester. The content of the glucosamine acid conjugate in the tumor inhibiting preparation is 0.021 wt%, and the content of DL-3-(3,4-dihydroxyphenyl)alanine in the tumor inhibiting preparation is 0.007 wt%.

[0076] Example 5: A tumor inhibiting preparation based on mucinophilic Akkermansia The tumor inhibiting preparation is prepared by dispersing the mucinophilic Akkermansia and the adjuvant in a bioactive liquid. The mucinophilic Akkermansia is dead AKK. The dead AKK is AKK treated by pasteurization. The bioactive liquid is a sterile PBS solution. The content of the mucinophilic Akkermansia in the tumor inhibiting preparation is 5×10 8 CFU / mL.

[0077] Example 6: A tumor inhibiting preparation based on mucinophilic Akkermansia The tumor inhibiting preparation is prepared by dispersing the mucinophilic Akkermansia and the adjuvant in a bioactive liquid. The mucinophilic Akkermansia is dead AKK. The dead AKK is AKK treated by pasteurization. The bioactive liquid is a sterile PBS solution. The content of the mucinophilic Akkermansia in the tumor inhibiting preparation is 5×10 8CFU / mL. The adjuvant is an amino glucoside acid conjugate, which is prepared by reacting acetoxy amino glucoside acid with 2-aminothiazole-5-carboxylic acid ester, and the content of acetoxy amino glucoside acid in the tumor preparation is 0.021wt%.

[0078] Preparation of acetoxy amino glucoside acid: glucosamine is mixed with acetic anhydride, concentrated sulfuric acid is added at 0°C, and then the reaction is stirred at 30°C for 6h. After the reaction is completed, anhydrous ethanol is added at 0°C to precipitate the precipitate, and acetoxy amino glucoside is separated. Acetoxy amino glucoside and potassium carbonate are mixed in DCM, and then succinic anhydride is added. The reaction is carried out at 30°C for 6h. After the reaction is completed, saturated sodium chloride solution is added, the pH is adjusted to 3, and the layers are separated by standing. The organic phase is separated, dried with anhydrous sodium sulfate, and rotary evaporated to dryness. A dispersion precipitant is added, the precipitate is precipitated, and acetoxy amino glucoside acid is separated. The use amount of glucosamine is 20wt% of acetic anhydride, and the use amount of concentrated sulfuric acid is 80wt% of glucosamine. Anhydrous ethanol is used in an appropriate amount. Saturated sodium chloride solution is used in an appropriate amount. The use amount of acetoxy amino glucoside is 20wt% of DCM, the use amount of potassium carbonate is 80wt% of acetoxy amino glucoside, and the use amount of succinic anhydride is 30wt% of acetoxy amino glucoside. The dispersion precipitant includes DCM and diethyl ether, and the DCM and diethyl ether in the dispersion precipitant are mixed at a volume ratio of 1:8. The use amount of the dispersion precipitant is 350wt% of acetoxy amino glucoside.

[0079] Preparation of glucosamine acid conjugate: acetyloxy glucosamine acid and DIPEA, trimethylacetyl chloride were mixed in DMF, reacted at 0℃ for 20 min, then 2-aminothiazole-5-carboxylate was added, and the reaction was stirred at 40℃ for 6 h. After the reaction was completed, saturated sodium chloride solution was added, the pH was adjusted to 3, and the precipitate was separated to obtain acetyloxy glucosamine acid conjugate. Then the acetyloxy glucosamine acid conjugate, methanol and sodium methoxide were mixed and stirred at 30℃ for 4 h. After the treatment was completed, the pH was adjusted to 3, and the mixture was rotary evaporated to dryness. The precipitate was separated by dispersion to obtain glucosamine acid conjugate. The amount of acetyloxy glucosamine acid used was 50 wt% of DMF, the amount of DIPEA used was 25 wt% of acetyloxy glucosamine acid, the amount of trimethylacetyl chloride used was 25 wt% of acetyloxy glucosamine acid, and the amount of 2-aminothiazole-5-carboxylate used was 20 wt% of acetyloxy glucosamine acid. The amount of methanol used was 600 wt% of acetyloxy glucosamine acid conjugate, and the amount of sodium methoxide used was 80 wt% of acetyloxy glucosamine acid conjugate. The dispersion precipitating liquid included DCM and diethyl ether, and the volume ratio of DCM to diethyl ether in the dispersion precipitating liquid was 1:8. The amount of dispersion precipitating liquid used was 350 wt% of acetyloxy glucosamine acid conjugate. The saturated sodium chloride solution was used in an appropriate amount.

[0080] Example 7: A mucinophilic Akkermansia-based tumor inhibiting preparation This example is different from Example 6 in that the tumor inhibiting preparation is prepared.

[0081] Tumor inhibiting preparation: mucinophilic Akkermansia and adjuvant were dispersed in a biologically active liquid to obtain a tumor inhibiting preparation. The mucinophilic Akkermansia was live AKK. The biologically active liquid was a sterile PBS solution. The content of mucinophilic Akkermansia in the tumor inhibiting preparation was 5×10 8 CFU / mL. The adjuvant was a glucosamine acid conjugate, which was obtained by the reaction of acetyloxy glucosamine acid and 2-aminothiazole-5-carboxylate. The content of acetyloxy glucosamine acid in the tumor inhibiting preparation was 0.009 wt%.

[0082] Example 8: A mucinophilic Akkermansia-based tumor inhibiting preparation This example is different from Example 5 in that DL-3-(3,4-dihydroxyphenyl)alanine can also be added as an adjuvant.

[0083] Tumor inhibiting preparation: mucinophilic Akkermansia bacteria and adjuvant were dispersed in a bioactive liquid to obtain a tumor inhibiting preparation. The mucinophilic Akkermansia bacteria were live AKK. The bioactive liquid was a sterile PBS solution. The content of mucinophilic Akkermansia bacteria in the tumor inhibiting preparation was 5×10 8 CFU / mL. The adjuvant was an amino glucoside acid conjugate and DL-3-(3,4-dihydroxyphenyl)alanine, the amino glucoside acid conjugate was obtained by reacting acetoxy amino glucoside acid with 2-aminothiazole-5-carboxylic acid ester, the content of the amino glucoside acid conjugate in the tumor inhibiting preparation was 0.021 wt%, and the content of DL-3-(3,4-dihydroxyphenyl)alanine in the tumor inhibiting preparation was 0.007 wt%.

[0084] Comparative Example 1: a tumor inhibiting preparation based on mucinophilic Akkermansia bacteria This example is different from Example 6 in that the amino glucoside acid conjugate in the adjuvant is replaced by acetoxy amino glucoside acid.

[0085] Comparative Example 2: a tumor inhibiting preparation based on mucinophilic Akkermansia bacteria This example is different from Example 6 in that the content of the amino glucoside acid conjugate in the tumor inhibiting preparation is 0.0005 wt%.

[0086] Comparative Example 3: a tumor inhibiting preparation based on mucinophilic Akkermansia bacteria This example is different from Example 8 in that the amino glucoside acid conjugate is not used in the tumor inhibiting preparation.

[0087] Example 9: a mouse model Preparation of the mouse model: a mixture was obtained by mixing the tumor inhibiting preparation and mouse colon cancer cells, and the mixture was inoculated subcutaneously into mice, 1 million tumor cells per inoculation point. The tumor inhibiting preparation was taken from Example 1, and the use amount of the tumor inhibiting preparation was calculated based on the live AKK therein, and the number ratio of the live AKK to the mouse colon cancer cells was 1:1.

[0088] Example 10: a mouse model A mixture was obtained by mixing the tumor inhibiting preparation and mouse colon cancer cells, and the mixture was inoculated subcutaneously into mice, 1 million tumor cells per inoculation point. The tumor inhibiting preparation was taken from Example 2, and the use amount of the tumor inhibiting preparation was calculated based on the live AKK therein, and the number ratio of the live AKK to the mouse colon cancer cells was 1:1.

[0089] Test Example: The acetyloxyamino-glucosaminic acid and the amino-glucosaminic acid conjugate prepared in the embodiment 3 are subjected to cytotoxicity test, taking the mouse fibroblast L929 cell as the test cell, using MTT method for test, taking no test sample as blank control, the use amount of acetyloxyamino-glucosaminic acid or amino-glucosaminic acid conjugate in the test sample is 0.03wt%, the culture medium in the test is DMEM culture medium, after test, the cell proliferation rate of acetyloxyamino-glucosaminic acid to the test cell is 99.3%, the cell proliferation rate of amino-glucosaminic acid conjugate to the test cell is 99.8%, indicating that acetyloxyamino-glucosaminic acid and amino-glucosaminic acid conjugate have no cytotoxicity.

[0090] The tumor inhibition test is carried out on the tumor inhibition preparation based on mucinophilic Akkermansia prepared in the embodiments 5-8 and the comparative examples 1-3, taking MC38 cell as the test cell, inoculating in the mouse model for test, the test sample is the tumor inhibition preparation based on mucinophilic Akkermansia prepared in the embodiments 5-8 and the comparative examples 1-3, the number ratio of mucinophilic Akkermansia in the tumor inhibition preparation to MC38 cell in the test is 0.5:1, the number of MC38 cell is 1 million tumor cells, taking injection of the same volume of sterile PBS solution as blank control, taking the blank control as the benchmark to calculate the tumor cell inhibition rate, the result is as follows Figure 6S5 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, D3 is Comparative Example 3, and the present application uses AKK mixed with tumor cells in a certain ratio to be injected into mice in the study, it is found that AKK can inhibit the growth of tumor cells, and AKK can survive in tumor cells for at least 7 days, and the effect of living AKK is better than that of dead AKK. In the present application, AKK is mixed in a bioactive liquid to prepare a tumor inhibiting preparation based on mucinophilic Akkermansia. In the preparation of the tumor inhibiting preparation based on mucinophilic Akkermansia, glucosamine acid conjugate can also be added as an adjuvant. In the preparation of glucosamine acid conjugate, first, the hydroxyl group is protected by reacting glucosamine with acetic anhydride to obtain acetyloxy glucosamine. Then, the amino group on the acetyloxy glucosamine reacts with succinic anhydride to obtain acetyloxy glucosamine acid. Acetyloxy glucosamine acid reacts with 2-aminothiazole-5-carboxylate to form acetyloxy glucosamine acid conjugate. Finally, the acetyl group is removed to obtain glucosamine acid conjugate. The present application finds that when acetyloxy glucosamine acid is prepared together with AKK to obtain a tumor inhibiting preparation based on mucinophilic Akkermansia, the tumor inhibiting preparation obtained has no obvious improvement effect on tumor inhibition. However, when acetyloxy glucosamine acid conjugate is prepared together with AKK to obtain a tumor inhibiting preparation based on mucinophilic Akkermansia, the tumor inhibiting preparation obtained has a significant improvement effect on tumor inhibition. The use amount of acetyloxy glucosamine acid conjugate needs to be within a certain range to have an effect. If the use amount is too low, the inhibitory effect cannot be improved. After the use of glucosamine acid conjugate, DL-3-(3,4-dihydroxyphenyl) alanine can also be added as an adjuvant for common use. The common use of DL-3-(3,4-dihydroxyphenyl) alanine and glucosamine acid conjugate can further improve the tumor inhibiting effect of the tumor inhibiting preparation.

[0091] The above-described examples and / or embodiments are only used to illustrate the preferred embodiments and / or implementations of the present application, and do not limit the implementation of the present application in any form. Any person skilled in the art can make some changes or modifications without departing from the scope of the technical means disclosed in the present application, and other equivalent embodiments can be made, but should be considered as the same technology or embodiment as the present application.

[0092] The principles and implementation manners of the present application are described herein by using specific examples, and the above example descriptions are only used to help understand the method of the present application and its core idea. The above descriptions are only preferred embodiments of the present application, and it should be pointed out that, due to the limited nature of the language expression, there are objectively infinite specific structures, and for ordinary skilled persons in the technical field, some improvements, refinements or changes can be made without departing from the principles of the present application, and the above technical features can also be combined in an appropriate manner; these improvements, refinements, changes or combinations, or the direct application of the inventive concept and technical solution to other occasions without improvement, shall be regarded as the protection scope of the present 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 CFU / mL.

2. The method for preparing a tumor-inhibiting agent according to claim 1, characterized in that: The tumor-inhibiting agent also contains excipients, including glucosamine conjugates having glucosamine groups coupled with succinic acid and 2-aminothiazol-5-carboxylic acid ester groups.

3. The method for preparing a tumor-inhibiting agent according to claim 2, characterized in that: 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.

4. The method for preparing a tumor-inhibiting agent according to claim 3, characterized in that: The amount of glucosamine used is 10-30 wt% of acetic anhydride.

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

6. The method for preparing a tumor-inhibiting agent according to claim 3, characterized in that: The amount of succinic anhydride used is 20-40 wt% of acetoxyglucosamine.

7. The tumor-inhibiting agent prepared by any of the preparation methods described in claims 1-6.

8. A mouse model for inhibiting tumors, comprising: The tumor-inhibiting agent described in claim 7 was mixed with tumor cells and injected into mice to obtain a mouse model of tumor suppression.

9. An antitumor drug, comprising: The tumor-inhibiting agent according to claim 7.

10. An antitumor drug according to claim 9, characterized in that: The antitumor drugs include anti-colon cancer drugs.

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