Use of akkermansia muciniphila and related compositions for the preparation of an inhibitor of vascular calcification

Abstract

This invention discloses the application of *Ackermania myxophilus* and related compositions in the preparation of vascular calcification inhibitors. Specifically, it provides the application of *Ackermania myxophilus* and propionate in the preparation of vascular calcification inhibitors and drugs for inhibiting vascular calcification. Through vascular calcification intervention experiments, this invention demonstrates that *Ackermania myxophilus* can significantly alleviate the symptoms of vascular calcification, and the combination of *Ackermania myxophilus* and propionate further significantly alleviates the symptoms. Given the high correlation between vascular calcification and cardiovascular and cerebrovascular diseases, *Ackermania myxophilus* and its combination with propionate can also be used for the treatment and / or prevention of cardiovascular and cerebrovascular diseases. This invention provides new ideas and methods for the clinical treatment of vascular calcification and has significant clinical implications for its prevention and treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of Akkermansia myxophilus and related compositions in the preparation of vascular calcification inhibitors. Background Technology

[0002] Vascular calcification is a common pathological phenomenon characterized by ectopic calcium salt deposition in the blood vessel wall.

[0003] With the aging of the population, the prevalence of vascular calcification is increasing year by year. In the general population aged 45-75, the prevalence of aortic calcification and coronary artery calcification is approximately 63.1% and 46.7%, respectively. Factors that induce or accelerate vascular calcification, or factors that inhibit it, are often present during the initiation and formation of vascular calcification. Once formed, vascular calcification is difficult to reverse. It can lead to decreased vascular compliance, thickening of the arterial wall, narrowing of the lumen, plaque instability, and plaque rupture, resulting in a series of cardiovascular diseases and adverse cardiovascular and cerebrovascular events. Therefore, vascular calcification is considered one of the important causes of high morbidity and mortality of cardiovascular diseases.

[0004] Currently, there are no effective drugs for prevention and treatment in clinical applications. Therefore, the development of vascular calcification inhibitors is of great clinical significance for the prevention and treatment of vascular calcification. Summary of the Invention

[0005] The purpose of this invention is to provide the use of Akkermansia myxophilus and related compositions in the preparation of vascular calcification inhibitors.

[0006] This invention provides the application of Akkermansia myxophilus and propionate in the preparation of vascular calcification inhibitors.

[0007] In this application, the myxotrophic Akkermansia and propionate are packaged separately.

[0008] This invention also provides the use of Akkermansia myxophilus and propionate in the preparation of drugs that inhibit vascular calcification.

[0009] In this application, the myxotrophic Akkermansia and propionate are packaged separately.

[0010] This invention also provides the use of Akkermansia myxophilus and propionate in the preparation of medicaments for the treatment and / or prevention of cardiovascular and cerebrovascular diseases.

[0011] In this application, the myxotrophic Akkermansia and propionate are packaged separately.

[0012] The present invention also provides a vascular calcification inhibitor, the active ingredients of which are Akkermansia myxophilus and propionate.

[0013] In the vascular calcification inhibitor, Akkermansia mycotoxin and propionate are packaged separately.

[0014] The present invention also provides a drug whose active ingredients are Akkermansia myxophilus and propionate;

[0015] The drug is either (a) or (b) as follows:

[0016] (a) Drugs that inhibit vascular calcification;

[0017] (b) Medications used to treat and / or prevent cardiovascular and cerebrovascular diseases.

[0018] In the drug, Akkermansia myxophilus and propionate are packaged separately.

[0019] This invention also provides the application of *Akermansia myxophila* in the preparation of vascular calcification inhibitors.

[0020] This invention also provides the application of *Akermansia myxophila* in the preparation of drugs that inhibit vascular calcification.

[0021] The present invention also provides the use of Akkermansia myxophilus in the preparation of medicaments for the treatment and / or prevention of cardiovascular and cerebrovascular diseases.

[0022] The present invention also provides a vascular calcification inhibitor, the active ingredient of which is Akkermansia myxophila.

[0023] The present invention also provides a drug whose active ingredient is Akkermansia myxophilus;

[0024] The drug is either (a) or (b) as follows:

[0025] (a) Drugs that inhibit vascular calcification;

[0026] (b) Medications used to treat and / or prevent cardiovascular and cerebrovascular diseases.

[0027] Specifically, the *Ackermania* strain is ATCC number BAA-835.

[0028] The propionate mentioned above is sodium propionate and / or calcium propionate.

[0029] The propionate mentioned above is any one or a combination of the following substances: sodium propionate, potassium propionate, calcium propionate, and zinc propionate.

[0030] In the above applications, when preparing drugs, Akkermansia myxophilus and propionate can be used as one of the active ingredients or as the sole active ingredient as a whole (combination).

[0031] In the above applications, when preparing drugs, Akkermansia myxophilus and propionate can be used as one of the active ingredients or as the sole active ingredient as a whole (combination).

[0032] In the above applications, Akkermansia myxophilus can be used as one of the active ingredients or as the sole active ingredient when preparing drugs.

[0033] In the above applications, Akkermansia myxophilus can be used as one of the active ingredients or as the sole active ingredient when preparing drugs.

[0034] In the above applications, a carrier material may also be added during drug preparation.

[0035] In the aforementioned drugs, Akkermansia myxophilus and propionate, as a whole (combination), can be one of the active ingredients or the sole active ingredient.

[0036] In the aforementioned drugs, Akkermansia myxophilus and propionate can be used as one of the active ingredients or as the sole active ingredient, as a whole (combination).

[0037] In the aforementioned drugs, Akkermansia myxophilus can be one of the active ingredients, or it can be the sole active ingredient.

[0038] In the aforementioned drugs, Akkermansia myxophilus can be one of the active ingredients or the sole active ingredient.

[0039] The aforementioned drugs may also include carrier materials.

[0040] Carrier materials include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), and enteric-coated carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). These materials can be used to formulate various dosage forms, including but not limited to tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, and lozenges. These can be conventional formulations, sustained-release formulations, controlled-release formulations, and various microparticle delivery systems. To formulate unit-dose dosage forms into tablets, various carriers known in the art can be widely used. Examples of carriers include diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, and aluminum silicate; humectants and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, and polyvinylpyrrolidone; and disintegrants. Examples of carriers include dried starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid esters, sodium dodecyl sulfate, methylcellulose, and ethylcellulose; disintegration inhibitors include sucrose, tristearate, cocoa butter, and hydrogenated oil; absorption enhancers include quaternary ammonium salts and sodium dodecyl sulfate; and lubricants include talc, silica, corn starch, stearates, boric acid, liquid paraffin, and polyethylene glycol. Tablets can also be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer and multilayer tablets. Various carriers known in the art can be widely used to formulate unit-dose dosage forms into pills. Examples of carriers include diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, kaolin, and talc; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste, or flour paste; and disintegrants such as agar powder, dried starch, alginate, sodium dodecyl sulfonate, methylcellulose, and ethylcellulose. For preparing unit-dose dosage forms into suppositories, a wide variety of carriers known in the art can be used. Examples of carriers include polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, and semi-synthetic glycerides. For preparing unit-dose dosage forms into injectable formulations such as solutions, emulsions, lyophilized powders for injection, and suspensions, all diluents commonly used in the art can be used, such as water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxyethylene isostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters. In addition, colorants, preservatives, flavorings, tasters, sweeteners or other materials may be added to pharmaceutical preparations if necessary.

[0041] Specifically, when Akkermansia myxophilus is a powdered product, it is dissolved or suspended in a solvent to obtain Akkermansia myxophilus bacterial solution.

[0042] The solvent may specifically be (100ml): containing 0.5% porcine mucin, 4.5g brain and heart extract powder and 0.05% cysteine, with the remainder being deionized water.

[0043] Akkermansia myxophilus bacterial solution can be administered orally.

[0044] Akkermansia myxophilus bacterial solution can be administered via gavage.

[0045] The concentration of *Ackermania myxophilus* in the bacterial suspension was 5 × 10⁻⁶. 9 cfu / 200μL.

[0046] Specifically, when propionate is a powdered product, it is dissolved or suspended in a solvent to obtain a propionate solution.

[0047] The solvent may specifically be water.

[0048] Propionate solution can be administered orally.

[0049] The concentration of propionate in the propionate solution is 200 mmol / L.

[0050] 40×10 9 CFU (crystal fucoidotropic myxobin) is formulated with 100-500 mmol propionate.

[0051] 40×10 9 CFU (Chemical Oxygenin-Propionibacterium Fibrosum) formulation: 200-400 mmol propionate

[0052] 40×10 9 CFU (Chemical Fumin-Propionate) formulation of Akkermansia muciniphila: 246-328 mmol propionate.

[0053] This invention demonstrates through vascular calcification intervention experiments that *Ackermania myxophilus* can significantly alleviate the symptoms of vascular calcification, and the combined use of *Ackermania myxophilus* and propionate further significantly alleviates the symptoms (indicating a significant synergistic effect). Given the high correlation between vascular calcification and cardiovascular and cerebrovascular diseases, *Ackermania myxophilus* and its combination with propionate can also be used for the treatment and / or prevention of cardiovascular and cerebrovascular diseases. This invention provides new ideas and methods for the clinical treatment of vascular calcification and has significant clinical implications for its prevention and treatment. Attached Figure Description

[0054] Figure 1 Photographs taken using alizarin red staining to detect vascular calcification.

[0055] Figure 2 This is the result of a blood vessel calcium content test. Detailed Implementation

[0056] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the present invention in any way. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Unless otherwise specified, the materials, reagents, etc. used in the following embodiments are all commercially available. Unless otherwise specified, the quantitative experiments in the following embodiments are all performed in triplicate, and the results are averaged.

[0057] Akkermansia muciniphila (AkK): ATCC, BAA-835. AKK suspension is obtained by suspending Akkermansia muciniphila in a culture medium. 200 μL of AKK suspension contains 5 × 10⁻⁶ Akkermansia muciniphila. 9 CFU. Culture medium (100ml) formula: contains 0.5% porcine mucin, 4.5g brain heart extract powder and 0.05% cysteine, with the remainder being deionized water.

[0058] Vitamin D3: www.sigmaaldrich.cn, product code 47763; potency of 7.5 mg vitamin D3 is 300,000 IU. Nicotine: Shanghai PureOne Biotech Co., Ltd., product code P1006. Sodium propionate: www.sigmaaldrich.cn, product code P5436. Calcium propionate: www.sigmaaldrich.cn, product code 21230. Male SD rats: Guangdong Provincial Medical Laboratory Animal Center. Alizarin Red dye: Beyotime Biotechnology Co., Ltd., product code ST1078. Cell lysis buffer: Beyotime Biotechnology Co., Ltd., product code P0013. Calcium assay kit (methyl thymol blue microplate method): Beijing Regen Biotechnology Co., Ltd., product code TC1021. BCA protein quantification assay kit: www.sigmaaldrich.cn, product code 71285.

[0059] Implementation examples

[0060] I. Group Dosing

[0061] Seven-week-old male SD rats (weighing 170-180g) were randomly divided into seven groups.

[0062] The entire process took 42 days, from day 1 to day 42 of the experiment.

[0063] The intramuscular injection site is the groin area of ​​the hind leg.

[0064] Control group: On day 1 of the experiment, anhydrous ethanol (1 μl anhydrous ethanol / g body weight) was injected intramuscularly, and peanut oil was administered by gavage twice (1 μl peanut oil / g body weight each time; the time interval between the two gavages was 9 hours); on day 1 of the experiment, distilled water was available for free drinking; from day 2 to day 42 of the experiment, 200 mM sodium chloride solution was available for free drinking.

[0065] Model group (VDN): On day 1 of the experiment, vitamin D3 solution was administered intramuscularly (1 μl vitamin D3 solution / g body weight; the solvent of vitamin D3 solution was anhydrous ethanol; the dose of vitamin D3 was 7.5 mg / kg body weight), and nicotine solution was administered by gavage twice (1 μl nicotine solution / g body weight each time; the solvent of nicotine solution was peanut oil; the dose of nicotine administered by gavage was 25 mg / kg body weight each time; the time interval between the two gavages was 9 hours); on day 1 of the experiment, distilled water was available for free drinking; from day 2 to day 42 of the experiment, 200 mM sodium chloride aqueous solution was available for free drinking.

[0066] AKK strain group (VDN+AKK): On day 1 of the experiment, vitamin D3 solution (1 μl vitamin D3 solution / g body weight; the solvent of vitamin D3 solution was anhydrous ethanol; the dose of vitamin D3 was 7.5 mg / kg body weight) was injected intramuscularly, and nicotine solution (1 μl nicotine solution / g body weight each time; the solvent of nicotine solution was peanut oil; the dose of nicotine given each time was 25 mg / kg body weight; the time interval between the two gavages was 9 hours) was administered. From day 2 to day 9 of the experiment, each rat was given 200 μL of AKK bacterial solution by gavage daily. On day 1 of the experiment, distilled water was available for free drinking. From day 2 to day 42 of the experiment, 200 mM sodium chloride solution was available for free drinking.

[0067] Sodium propionate group (VDN+SP): On day 1 of the experiment, vitamin D3 solution was administered intramuscularly (1 μl vitamin D3 solution / g body weight; the solvent of vitamin D3 solution was anhydrous ethanol; the dose of vitamin D3 was 7.5 mg / kg body weight), and nicotine solution was administered by gavage twice (1 μl nicotine solution / g body weight each time; the solvent of nicotine solution was peanut oil; the dose of nicotine administered by gavage was 25 mg / kg body weight each time; the time interval between the two gavages was 9 hours); on day 1 of the experiment, distilled water was available for free drinking; from day 2 to day 42 of the experiment, 200 mM sodium propionate aqueous solution was available for free drinking daily.

[0068] Sodium propionate AKK combined group (VDN+SP-AKK): On day 1 of the experiment, vitamin D3 solution was injected intramuscularly (1 μl vitamin D3 solution / g body weight; the solvent of vitamin D3 solution was anhydrous ethanol; the dose of vitamin D3 was 7.5 mg / kg body weight), and nicotine solution was administered by gavage twice (1 μl nicotine solution / g body weight each time; the solvent of nicotine solution was peanut oil; the dose of nicotine administered by gavage was 25 mg / kg body weight each time; the time interval between the two gavages was 9 hours); from day 2 to day 9 of the experiment, each rat was administered 200 μL of AKK bacterial solution by gavage daily; on day 1 of the experiment, distilled water was available for free drinking; from day 2 to day 42 of the experiment, 200 mM sodium propionate aqueous solution was available for free drinking daily.

[0069] Calcium propionate group (VDN+CaP): On day 1 of the experiment, vitamin D3 solution was administered intramuscularly (1 μl vitamin D3 solution / g body weight; the solvent of vitamin D3 solution was anhydrous ethanol; the dose of vitamin D3 was 7.5 mg / kg body weight), and nicotine solution was administered by gavage twice (1 μl nicotine solution / g body weight each time; the solvent of nicotine solution was peanut oil; the dose of nicotine administered by gavage was 25 mg / kg body weight each time; the time interval between the two gavages was 9 hours); on day 1 of the experiment, distilled water was available for free drinking; from day 2 to day 42 of the experiment, 200 mM calcium propionate aqueous solution was available for free drinking daily.

[0070] Calcium propionate AKK combined group (VDN+CaP-AKK): On day 1 of the experiment, vitamin D3 solution was injected intramuscularly (1 μl vitamin D3 solution / g body weight; the solvent of vitamin D3 solution was anhydrous ethanol; the dose of vitamin D3 was 7.5 mg / kg body weight), and nicotine solution was administered by gavage twice (1 μl nicotine solution / g body weight each time; the solvent of nicotine solution was peanut oil; the dose of nicotine administered by gavage was 25 mg / kg body weight each time; the time interval between the two gavages was 9 hours); from day 2 to day 9 of the experiment, each rat was administered 200 μL of AKK bacterial solution by gavage daily; on day 1 of the experiment, distilled water was available for free drinking; from day 2 to day 42 of the experiment, 200 mM calcium propionate aqueous solution was available for free drinking daily.

[0071] Through continuous observation, during the experiment, each rat drank 30-40 ml of 200 mM sodium chloride solution (or 200 mM sodium propionate solution, or 200 mM calcium propionate solution) per day.

[0072] II. Detection of vascular calcification using alizarin red staining

[0073] After completing step one, the experimental animals (6 per group) were euthanized and the entire aorta (ascending aorta + aortic arch + thoracic segment of descending aorta + abdominal segment of descending aorta + common iliac artery) was removed.

[0074] Vascular staining: Each aorta was fixed in 4% paraformaldehyde solution for 24 hours, and then stained with alizarin red stain (containing 0.003% alizarin red and 1% KOH, with the remainder being water) for 30 hours.

[0075] See results Figure 1 Compared to the Control group, the VDN group rats showed a large amount of dark red calcium salt deposits on the aortic wall. Compared to the VDN group, the VDN+AKK group rats showed a significant reduction in dark red calcium salt deposits on the aortic wall. Compared to the VDN group, the VDN+SP group rats showed a significant reduction in dark red calcium salt deposits on the aortic wall. Compared to the VDN group, the VDN+SP-AKK group rats showed a significant reduction in dark red calcium salt deposits on the aortic wall. Compared to the VDN group, the VDN+CaP group rats showed a significant reduction in dark red calcium salt deposits on the aortic wall. Compared to the VDN group, the VDN+CaP-AKK group rats showed a significant reduction in dark red calcium salt deposits on the aortic wall. Compared to the VDN+SP group, the VDN+SP-AKK group rats showed a significant reduction in dark red calcium salt deposits on the aortic wall. Compared to the VDN+CaP group, the VDN+CaP-AKK group rats showed a significant reduction in dark red calcium salt deposits on the aortic wall.

[0076] III. Detection of Calcium Content in Blood Vessels

[0077] After completing step one, the experimental animals (6 per group) were euthanized and the entire aorta (ascending aorta + aortic arch + thoracic segment of descending aorta + abdominal segment of descending aorta + common iliac artery) was removed.

[0078] The entire aorta was placed in an EP tube, homogenized, and cell lysis buffer was added. The mixture was then sonicated and centrifuged at 3000g for 3 minutes at 4°C. The supernatant was collected. The total protein concentration in the supernatant was measured using a BCA protein quantification kit, in g / L. The molar concentration of calcium in the supernatant was measured using a calcium assay kit (in mmol / L), and then converted to calcium mass concentration (mg / L) based on the molecular weight of calcium. Vascular calcium content = calcium mass concentration divided by total protein concentration, in mg / g.

[0079] The results are shown in Table 1 and... Figure 2 Compared with the Control group, the vascular calcium content of rats in the VDN group increased by 331.1% (P < 0.0001). Compared with the VDN group, the vascular calcium content of rats in the VDN+AKK group decreased by 39.3% (P < 0.0001). Compared with the VDN group, the vascular calcium content of rats in the VDN+SP group decreased by 43.0% (P < 0.0001). Compared with the VDN group, the vascular calcium content of rats in the VDN+SP-AKK group decreased by 51.6% (P < 0.0001). Compared with the VDN group, the vascular calcium content of rats in the VDN+CaP group decreased by 33.9% (P < 0.0001). Compared with the VDN group, the vascular calcium content of rats in the VDN+CaP-AKK group decreased by 47.7% (P < 0.0001). These results indicate that administration of AKK bacterial solution alone can significantly reduce vascular calcium salt deposition; sodium propionate combined with AKK bacterial solution or calcium propionate combined with AKK bacterial solution can more significantly reduce vascular calcium salt deposition. The combined use of compound drugs and bacterial strains demonstrates a significant synergistic effect, effectively preventing and treating symptoms of vascular calcification.

[0080] Table 1 Results of vascular calcium content testing in rats of different groups

[0081] Control 42.1 VDN 181.5 VDN+AKK 110.2 VDN+SP 103.5 VDN+SP-AKK 87.9 VDN+CaP 119.9 VDN+CaP-AKK 95.0

[0082] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. Akkermansia myxophila ( Akkermansia muciniphila Application of ATCC BAA-835 and propionate in the preparation of vascular calcification inhibitors.

2. Application of Akkermansia myxophilus ATCC BAA-835 and propionate in the preparation of drugs that inhibit vascular calcification.

3. Application of Akkermansia myxophila ATCC BAA-835 in the preparation of vascular calcification inhibitors.

4. Application of Akkermansia myxophilus ATCC BAA-835 in the preparation of drugs that inhibit vascular calcification.

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