Application of ackermania muciniphila composition in preparation of medicine for treating Wilson's disease

Abstract

The invention discloses an application of an Ackermania muciniphila composition in preparation of a drug for treating Wilson's disease, and relates to the technical field of biological medicines, and the key points of the technical scheme are as follows: Ackermania muciniphila can regulate intestinal flora and micro-ecological environment of WD patients, improve copper expelling and reduce the biological activity and availability of copper absorption drugs, and can be used for preparing a drug for treating Wilson's disease. The drug toxicity is reduced, and adverse events are reduced; therefore, the effects of removing copper, reducing copper absorption medicine and reducing copper accumulation in the body of a WD patient are enhanced. The Ackermania muciniphila can be Ackermania muciniphila viable bacteria, Ackermania muciniphila thermal inactivation thalli or fragments thereof, culture supernatant freeze-dried powder and a composition of the Ackermania muciniphila viable bacteria, the Ackermania muciniphila thermal inactivation thalli or the fragments thereof and the culture supernatant freeze-dried powder. The copper accumulation level in the body of a WD patient is reduced from multiple dimensions, the clinical WD treatment efficiency is improved, meanwhile, the steady-state time of copper in the body of the patient is prolonged, and adverse drug events are reduced.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and more specifically, to the application of an Akkermansia muciniphila composition in preparing a medicine for treating Wilson's disease. Background Art

[0002] Wilson's Disease (WD), also known as hepatolenticular degeneration (HLD), is an autosomal recessive copper metabolism disorder. Due to APT7B gene mutation, WD patients suffer from copper transport disorders in the body, and excessive copper deposition in most organs and tissues is most obvious, especially in the liver, brain, cornea, and kidneys. The clinical manifestations of patients are progressive cirrhosis, mental symptoms, renal damage, corneal pigment rings, KF rings, and extrapyramidal symptoms.

[0003] At present, the treatment of copper accumulation mainly includes the use of zinc salts to reduce gastrointestinal copper absorption and the use of copper chelators, such as penicillamine, dimercaptodibutyric acid, sodium dimercaptopropane sulfonate, trientine, etc. However, WD patients must receive lifelong medication to remove copper. Long-term use of the above drugs can cause gastrointestinal symptoms such as nausea and vomiting, gastrointestinal bleeding, diarrhea, as well as rash, urticaria, fever, joint pain, decreased white blood cells or platelets, neutropenia, iron deficiency anemia, lymphadenopathy, immune disorders, drug-induced lupus, and other adverse events. In addition, abnormal copper metabolism also leads to changes in the intestinal microbial composition and biodiversity of WD patients, affecting the bioavailability, bioactivity and toxicity of drugs, thereby affecting the treatment effect of Wilson disease patients.

[0004] The human intestinal flora consists of more than 800 species and more than 7,000 strains. It is a huge ecosystem that participates in and maintains many physiological activities and functions of the body. The intestinal flora is affected by many factors such as the environment, lifestyle, eating habits, and drugs. Its structural and functional changes are closely related to the occurrence and development of metabolic diseases, inflammatory diseases, and tumors. At the same time, a sound intestinal flora structure includes the chelation function of heavy metal ions such as copper and iron in the intestine.

[0005] Akkermansia muciniphila (Akk bacteria) is a normal bacterium in the human intestine. In recent years, many medical studies have shown that Akk bacteria help maintain the stability of the human intestinal mucus layer, promote the integrity of the intestinal barrier, improve human metabolic status, alcoholic / non-alcoholic liver disease, blood sugar and insulin resistance, tumor microenvironment, regulate intestinal immune response, inhibit inflammation, and regulate the structure and function of other microbial groups in the intestine through cross-feeding. CN111603488A and CN104918626A disclose the use of a composition, drug combination and medicament of a strain of Akk bacteria or its fragments in the treatment of metabolic syndromes such as obesity, diabetes, dyslipidemia, etc. in subjects, and the strain number of the Akk bacteria is ATCC BAA-835. CN117965391A and CN117946949A disclose 2 strains of Akk bacteria and their use in lowering cholesterol, inhibiting fat synthesis, and improving diarrhea symptoms, and the strain numbers of the Akk bacteria are Amuci-1 and Amuci-2. CN117448243A discloses a strain of Akk bacteria and its application in the preparation of health products with functions such as immune enhancement, and the strain number of the Akk bacteria is Akk007. CN117050914A discloses a strain of Akk bacteria and its application in the preparation of products for improving GLP-1 secretion, bidirectionally regulating the glucose and lipid metabolism of subjects, and improving the pancreatic function of subjects, and the strain number of the Akk bacteria is FMT-AKK015. The above documents respectively study the application of different strains of Akkermansia muciniphila. So far, there is still no application of Akkermansia muciniphila in the preparation of drugs for treating Wilson's disease. Summary of the invention

[0006] The purpose of the present invention is to provide an application of an Akkermansia muciniphila composition in the preparation of a drug for treating Wilson's disease, thereby reducing the copper accumulation level in WD patients from multiple dimensions, improving the clinical treatment efficiency of WD, and at the same time prolonging the copper steady-state time in the patient's body and reducing the occurrence of adverse drug events.

[0007] The above technical purpose of the present invention is achieved through the following technical scheme: use of the Akkermansia muciniphila composition in the preparation of a drug for treating Wilson's disease, wherein the Akkermansia muciniphila composition comprises Akkermansia muciniphila and a copper-removing drug.

[0008] The present invention also provides a combination mode of an Akkermansia muciniphila composition: the Akkermansia muciniphila composition comprises Akkermansia muciniphila, a copper-removing drug and a drug for reducing copper absorption.

[0009] The present invention is further configured as follows: the Akkermansia muciniphila in the above two combinations is one or more of live Akkermansia muciniphila, heat-inactivated Akkermansia muciniphila bacteria or fragments thereof, and culture supernatant freeze-dried powder;

[0010] Copper removal drugs include penicillamine, trientine, dimercaptosuccinic acid or sodium dimercaptopropane sulfonate;

[0011] Drugs that reduce copper absorption are zinc sulfate, zinc acetate, zinc gluconate, or zinc glycinate.

[0012] The present invention is further configured as follows: the amount of live Akkermansia muciniphila is 1×10 2 cfu-1×10 15 cfu.

[0013] The present invention is further configured as follows: the optimal amount of live Akkermansia muciniphila is 1×10 8 cfu-1×10 9 cfu.

[0014] The present invention is further configured as follows: the amount of heat-inactivated Akkermansia muciniphila cells is 1×10 2 cell-1×10 15 cell.

[0015] The present invention is further configured as follows: the optimal amount of heat-killed Akkermansia muciniphila is 1×10 8 cell-1×10 11 cell.

[0016] The present invention is further configured as follows: the above two drugs reduce and maintain the concentrations of alanine aminotransferase, aspartate aminotransferase and total bilirubin, liver copper and urine copper in the subject.

[0017] The present invention is further configured as follows: the above two drugs increase and maintain the serum ceruloplasmin level of the subject.

[0018] The present invention is further configured as follows: the above two drugs reduce the incidence of nausea and vomiting, gastrointestinal bleeding, diarrhea, rash, urticaria, fever, joint pain, lymphadenopathy, immune disorders and other adverse events in the subjects.

[0019] In summary, the present invention has the following beneficial effects: The present invention discloses the use of an Akkermansia muciniphila composition in the preparation of a drug for treating Wilson's disease. Among them, Akkermansia muciniphila can improve the intestinal immune and metabolic dysfunction of Wilson's disease, restore the intestinal ecological imbalance of Wilson's disease, and enhance the therapeutic effect of copper-removing drugs or drugs that reduce copper absorption. The combination of Akkermansia muciniphila and copper-removing drugs / drugs that reduce copper absorption involved in the embodiments of the present invention enhances the intestinal copper chelating capacity of Wilson's disease subjects compared with taking copper-removing drugs or drugs that reduce copper absorption alone. It is proved that the combination helps to restore the intestinal microecological imbalance of Wilson's disease subjects by regulating the intestinal microbiota, while increasing the efficacy of the drug and reducing the occurrence of adverse events. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is the pathological staining diagram in Example 1 of the present invention;

[0021] Figure 2 is the biochemical index diagram in Example 1 of the present invention;

[0022] Figure 3 is a schematic diagram of the diversity of intestinal flora in Example 1 of the present invention;

[0023] Figure 4 It is the pathological staining diagram in Example 2 of the present invention;

[0024] Figure 5 is the biochemical index diagram in Example 2 of the present invention;

[0025] Figure 6 Schematic diagram of the diversity of intestinal flora in Example 2 of the present invention. DETAILED DESCRIPTION

[0026] The following is combined with Figure 1-6 The present invention is described in further detail.

[0027] Example 1

[0028] In one embodiment of the present invention, an 8-week treatment of a Wilson's disease subject is performed with live Akkermansia muciniphila in combination with the copper-displacing drug penicillamine.

[0029] Experimental methods

[0030] 1. Animal Model

[0031] In this experiment, male 12-week-old SPF ATP7B knockout mice were used to construct a Wilson disease model. Compared with wild-type healthy mice (healthy control group), the mice had significantly increased alanine aminotransferase and liver copper concentration, significantly decreased serum ceruloplasmin, and swollen hepatocytes accompanied by inflammatory infiltration, indicating that the model was successfully constructed.

[0032] 2. Cultivation of Akkermansia muciniphila and Preparation of Bacterial Suspension

[0033] Culture medium: modified BHI culture medium, formula: peptone 10.0 g / L, dehydrated calf brain extract powder 12.5 g / L, dehydrated bull heart extract powder 5.0 g / L, sodium chloride 5.0 g / L, glucose 2.0 g / L, disodium hydrogen phosphate 2.5 g / L.

[0034] Culture conditions: 37℃ anaerobic culture for 48h.

[0035] Preparation of bacterial suspension: The cultured bacterial suspension was centrifuged at 8000×g for 10 min at 4°C. The obtained bacterial bodies were reselected with physiological saline and quantified.

[0036] 3. Experimental groups and treatment plans

[0037] Group 1: Healthy control group (Control), 10 wild-type healthy mice aged 12 weeks, were given the same dose of saline as drug treatment as a placebo.

[0038] Group 2: Disease control group (Model), 10 12-week-old Wilson disease model mice, administered with the same dose of saline as the placebo as the drug treatment.

[0039] The third group: penicillamine treatment group (DPA), 10 12-week-old Wilson disease model mice, administered with 0.01 mg / g body weight penicillamine once a day.

[0040] Group 4: Akkermansia muciniphila combined with penicillamine treatment group (Akk+DPA), 10 12-week-old Wilson disease model mice, administered with live Akkermansia muciniphila (3×10 9 cfu) and penicillamine (0.01 mg / g body weight) once a day.

[0041] The experiment lasted for 8 weeks. At the end of the experiment, all mice were killed and blood, intestinal contents, and liver tissue samples were collected.

[0042] 4 Detection methods

[0043] Alanine aminotransferase (ALT) levels were determined by dry biochemical analyzer, equipment model FUJIDRI-CHEM 4000ie. Ceruloplasmin levels were detected by ELISA, and the kit used was purchased from Shanghai Fankewei. Liver copper concentration was detected by inductively coupled plasma mass spectrometry, equipment model Thermo ICP-MS system. Intestinal microbiota identification results were obtained by 16srRNA sequencing analysis. Sequencing primers were designed as: 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3') in the V3-V4 region. Equipment model Illumina NovaSeq. Liver tissue pathology test results were obtained by microscopic observation of liver tissue after hematoxylin & eosin staining.

[0044] 5 Statistical methods

[0045] All the data obtained were first tested for normal distribution. One-way ANOVA was used to compare the differences between any two groups for factors that were in line with the normal distribution, and non-parametric tests were used to analyze the differences between any two groups (U test) or more than two groups (Kruskal–Wallis test) for factors that were not in line with the normal distribution.

[0046] Experimental Results

[0047] like Figure 1 As shown in the figure, the results of hematoxylin & eosin staining showed that the disease control group (Model) showed obvious hepatocyte swelling and inflammatory infiltration compared with the healthy control group (Control). In the penicillamine treatment group (DPA) and the Akkermansia muciniphila combined with penicillamine treatment group (Akk+DPA), liver damage was significantly alleviated. Compared with the two groups, the Akk+DPA group showed better protection against liver damage—no inflammatory cells appeared in the field of view.

[0048] like Figure 2 As shown in the results, the penicillamine treatment group (DPA) and the Akkermansia muciniphila combined with penicillamine treatment group (Akk+DPA) significantly reduced liver function abnormalities caused by Wilson's disease, including increased alanine aminotransferase (ALT), decreased ceruloplasmin, and increased liver copper levels. Among them, the effect of the Akkermansia muciniphila combined with penicillamine treatment group (Akk+DPA) was the most significant.

[0049] like Figure 3 As shown, there were different degrees of differences in the diversity of intestinal flora in each group of mice, and the diversity characterization indicators of the Akkermansia muciniphila combined with penicillamine treatment group (Akk+DPA) were closest to those of the healthy control group (Control), indicating that the treatment involving Akkermansia muciniphila can promote the recovery of intestinal flora.

[0050] In summary, compared with traditional treatment, Akkermansia muciniphila combined with penicillamine treatment can more effectively promote the recovery of liver function in Wilson disease subjects, alleviate liver damage, reduce liver copper deposition, and promote the recovery of intestinal microecological imbalance in subjects.

[0051] Example 2

[0052] In another embodiment of the present invention, a 12-week treatment of subjects with Wilson's disease is performed with Akkermansia muciniphila (pasteurized), the copper-removing drug dimercaptosuccinic acid, and the absorption-reducing drug zinc gluconate.

[0053] Experimental methods:

[0054] 1. Animal Model

[0055] In this experiment, male 16-week-old SPF-grade ATP7B knockout mice were used to construct a Wilson disease model. Compared with wild-type healthy mice (healthy control group), the mice had significantly increased alanine aminotransferase, aspartate aminotransferase, total bilirubin, and liver copper concentrations, significantly decreased serum ceruloplasmin, and swollen hepatocytes accompanied by inflammatory infiltration, indicating that the model was successfully constructed.

[0056] 2. Cultivation of Akkermansia muciniphila and Preparation of Pasteurized Suspension

[0057] Culture medium: modified BHI culture medium, formula: peptone 10.0 g / L, dehydrated calf brain extract powder 12.5 g / L, dehydrated bull heart extract powder 5.0 g / L, sodium chloride 5.0 g / L, glucose 2.0 g / L, disodium hydrogen phosphate 2.5 g / L.

[0058] Culture conditions: 37℃ anaerobic culture for 48h.

[0059] Preparation of bacterial suspension: The cultured bacterial suspension was centrifuged at 8000×g for 10 min at 4°C. The obtained bacterial bodies were reselected with physiological saline and quantified.

[0060] Pasteurization: Place the bacterial suspension in an 80°C water bath for 15 minutes to obtain heat-killed bacteria.

[0061] 3. Experimental groups and treatment plans

[0062] Group 1: Healthy control group (Control), 10 wild-type healthy mice aged 16 weeks, were given the same dose of saline as drug treatment as a placebo.

[0063] Group 2: Disease control group (Model), 10 16-week-old Wilson disease model mice, were given the same dose of normal saline as the placebo as the drug treatment.

[0064] The third group: DMSA+ZnGlu treatment group, 10 16-week-old Wilson disease model mice, were administered 0.03 mg / g body weight of DMSA and 5 ug / g body weight of zinc gluconate twice a day.

[0065] Group 4: Akkermansia muciniphila combined with dimercaptosuccinic acid and zinc gluconate treatment group (Akk+DMSA+ZnGlu group): 10 16-week-old Wilson disease model mice were given 0.03 mg / g body weight dimercaptosuccinic acid and 5 μg / g body weight zinc gluconate twice a day; 3×10 9 cfu live Akkermansia muciniphila, once a day.

[0066] The experiment lasted for 12 weeks. At the end of the experiment, all mice were killed and blood, intestinal contents, and liver tissue samples were collected.

[0067] 4 Detection methods

[0068] Alanine aminotransferase (ALT) levels were determined by dry biochemical analyzer, equipment model FUJIDRI-CHEM 4000ie. Ceruloplasmin levels were detected by ELISA, and the kit used was purchased from Shanghai Fankewei. Liver copper and urine copper concentrations were detected by inductively coupled plasma mass spectrometry, equipment model Thermo ICP-MS system. Intestinal microbiota identification results were obtained by 16s rRNA sequencing analysis. Sequencing primers were designed as: V3-V4 region 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'). Equipment model Illumina NovaSeq. Liver tissue pathology test results were obtained by microscopic observation of liver tissue after hematoxylin & eosin staining.

[0069] 5 Statistical methods

[0070] All the data obtained were first tested for normal distribution. One-way ANOVA was used to compare the differences between any two groups for factors that were in line with the normal distribution, and non-parametric tests were used to analyze the differences between any two groups (U test) or more than two groups (Kruskal–Wallis test) for factors that were not in line with the normal distribution.

[0071] Experimental Results

[0072] like Figure 4As shown in the figure, the results of hematoxylin & eosin staining showed that the disease control group (Model) showed obvious hepatocyte swelling and inflammatory infiltration compared with the healthy control group (Control). In the dimercaptosuccinic acid combined with zinc gluconate treatment group (DMSA+ZnGlu) and the Akkermansia muciniphila combined with dimercaptosuccinic acid and zinc gluconate treatment group (Akk+DMSA+ZnGlu), liver damage was significantly reduced. Compared with the two groups, the Akk+DPA group showed better protection against liver damage—fewer inflammatory cells appeared in the field of view.

[0073] like Figure 5 As shown, the dimercaptosuccinic acid combined with zinc gluconate treatment group (DMSA+ZnGlu) and the Akkermansia muciniphila combined with dimercaptosuccinic acid and zinc gluconate treatment group (Akk+DMSA+ZnGlu) significantly reduced liver function abnormalities caused by Wilson's disease, including increased alanine aminotransferase (ALT), decreased ceruloplasmin, and increased liver copper levels. Among them, the Akkermansia muciniphila combined with dimercaptosuccinic acid and zinc gluconate treatment group (Akk+DMSA+ZnGlu) had the most significant effect.

[0074] like Figure 6 As shown, there were different degrees of differences in the diversity of intestinal flora in each group of mice, and the diversity characterization indicators of the Akkermansia muciniphila combined with dimercaptosuccinic acid and zinc gluconate treatment group (Akk+DMSA+ZnGlu) were closest to those of the healthy control group (Control), indicating that the treatment involving Akkermansia muciniphila can promote the recovery of intestinal flora.

[0075] In summary, compared with traditional treatment, Akkermansia muciniphila combined with penicillamine treatment can more effectively promote the recovery of liver function in Wilson disease subjects, alleviate liver damage, reduce liver copper deposition, and promote the recovery of intestinal microecological imbalance in subjects.

[0076] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. Use of a muciniphilic Akkermansia composition in the preparation of a drug for treating Wilson's disease, characterized in that: The Akkermansia muciniphila composition comprises Akkermansia muciniphila and a copper-removing drug; The Akkermansia muciniphila is one or more of live Akkermansia muciniphila bacteria, heat-killed Akkermansia muciniphila bacteria or fragments thereof, and culture supernatant freeze-dried powder; The copper-removing drug is penicillamine, trientine, dimercaptosuccinic acid or sodium dimercaptopropane sulfonate.

2. Use of the Akkermansia muciniphila composition according to claim 1 in the preparation of a drug for treating Wilson's disease, characterized in that: Another combination of the Akkermansia muciniphila composition is as follows: comprising Akkermansia muciniphila, a copper-removing drug and a drug for reducing copper absorption; The Akkermansia muciniphila is one or more of live Akkermansia muciniphila bacteria, heat-inactivated Akkermansia muciniphila bacteria or fragments thereof, and freeze-dried culture supernatant powder; The copper-removing drug is penicillamine, trientine, dimercaptosuccinic acid or sodium dimercaptopropane sulfonate; The copper absorption reducing drug is zinc sulfate, zinc acetate, zinc gluconate or zinc glycinate.

3. Use of the Akkermansia muciniphila composition according to claim 1 or 2 in the preparation of a drug for treating Wilson's disease, characterized in that: The amount of live Akkermansia muciniphila used is 1×10 2 cfu-1×10 15 cfu.

4. Use of the Akkermansia muciniphila composition according to claim 1 or 2 in the preparation of a drug for treating Wilson's disease, characterized in that: The optimal amount of live Akkermansia muciniphila is 1×10 8 cfu-1×10 9 cfu.

5. Use of the Akkermansia muciniphila composition according to claim 1 or 2 in the preparation of a drug for treating Wilson's disease, characterized in that: The amount of heat-killed Akkermansia muciniphila cells is 1×10 2 cell-1×10 15 cell.

6. Use of the Akkermansia muciniphila composition according to claim 1 or 2 in the preparation of a drug for treating Wilson's disease, characterized in that: The optimal amount of heat-killed Akkermansia muciniphila is 1×10 8 cell-1×10 11 cell.

7. Use of the Akkermansia muciniphila composition according to any one of claims 1 or 2 in the preparation of a drug for treating Wilson's disease, characterized in that: The drug reduces and maintains the concentrations of alanine aminotransferase, aspartate aminotransferase, glutamyl transpeptidase and total bilirubin, liver copper and urine copper in the subject.

8. Use of the Akkermansia muciniphila composition according to any one of claims 1 or 2 in the preparation of a drug for treating Wilson's disease, characterized in that: The drug increases and maintains the serum ceruloplasmin level in the subject.

9. Use of the Akkermansia muciniphila composition according to any one of claims 1 or 2 in the preparation of a drug for treating Wilson's disease, characterized in that: The drug reduces the incidence of nausea and vomiting, gastrointestinal bleeding, diarrhea, rash, urticaria, fever, joint pain, lymphadenopathy, immune disorders and other adverse events in subjects.

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