Application of Akkermansia muciniphila in the relief and treatment of schizophrenia

Abstract

The present invention provides the use of Akkermansia muciniphila in alleviating and / or treating schizophrenia. The inventors unexpectedly found that Akkermansia muciniphila, especially Akkermansia muciniphila AM06 with a deposit number of CGMCC No. 22793 and Akkermansia muciniphila AM02 with a deposit number of CGMCC No. 22794, can effectively improve cognitive dysfunction in schizophrenia, and when used in combination with antipsychotic drugs, can effectively inhibit weight gain caused by antipsychotic drugs.

Description

Technical Field

[0001] The present invention relates to an application of Akkermansia muciniphila, and in particular to an application of Akkermansia muciniphila in alleviating and / or treating schizophrenia. Background Art

[0002] Schizophrenia is a common and serious mental illness with unknown etiology. It usually starts in young adults and often causes disorders in perception, thinking, emotion and behavior, but generally without consciousness and intellectual impairment. The course of the disease is often prolonged, accounting for more than half of psychiatric inpatients, and about half of the patients eventually develop mental disability, which brings a serious burden to society, patients and their families.

[0003] In the DSM-5 diagnostic classification and criteria for mental disorders released in the United States in 2013, schizophrenia and other diseases were classified as spectrum disorders for the first time, called schizophrenia spectrum and other psychotic disorders, including schizotypal (personality) disorder, delusional disorder, brief psychotic disorder, schizophreniform disorder, schizophrenia, schizoaffective disorder (bipolar / depressive type), substance / drug-induced psychotic disorder, psychotic disorder due to other medical illness, catatonia, catatonia associated with other mental disorders, catatonia due to other medical illness, unspecified catatonia, other specified schizophrenia spectrum and other psychotic disorders, unspecified schizophrenia spectrum and other psychotic disorders. These psychotic disorders often require the use of antipsychotic drugs for treatment.

[0004] The first generation of antipsychotic drugs (typical antipsychotic drugs) refers to antipsychotic drugs that mainly act on central D2 receptors, including: ① phenothiazines such as chlorpromazine, thioridazine, perphenazine, fluphenazine and its long-acting agents, trifluoperazine, etc.; ② thioxanthenes such as clopenthixol and its long-acting agents, trifluoperazine and its long-acting agents, telden, etc.; ③ butyrophenones such as haloperidol and its long-acting agents, penfluridol, etc.; ④ benzamides such as sulpiride, etc. Phenothiazines are further divided into high-potency drugs such as perphenazine and trifluoperazine; low-potency drugs such as chlorpromazine and thioridazine (potency classification applies to first-generation drugs). This type of drug has been widely used in the clinical treatment of various mental illnesses since the 1950s, mainly treating various psychotic symptoms.

[0005] Many years after the first generation of antipsychotics came on the market, new second generation antipsychotics emerged, including clozapine, risperidone, olanzapine, quetiapine, ziprasidone and aripiprazole, etc. Compared with phenothiazines and other drugs, they have a higher 5-hydroxytryptamine (5-HT) 2 receptor blocking effect, called dopamine (DA)-5-hydroxytryptamine receptor antagonists (SDAs), and their effects on the mesolimbic system are more selective than those on the striatal system.

[0006] The most notable side effects of current treatments for psychiatric disorders are metabolic abnormalities, such as weight gain and extrapyramidal effects. The most challenging aspect in treating patients with schizophrenia is metabolic syndrome, which is more difficult to treat or reverse than other side effects caused by current therapies, especially in the long term, and this side effect complicates the condition. There is an urgent need to develop drugs for psychiatric disorders with improved safety. Summary of the invention

[0007] In order to overcome the above defects in the prior art, the present invention provides a use of Akkermansia muciniphila in alleviating and / or treating schizophrenia. The inventors unexpectedly found that Akkermansia muciniphila, especially Akkermansia muciniphila AM06 with a deposit number of CGMCC No. 22793 and Akkermansia muciniphila AM02 with a deposit number of CGMCC No. 22794, can effectively improve cognitive dysfunction in schizophrenia, and when used in combination with antipsychotic drugs, can effectively inhibit weight gain caused by antipsychotic drugs.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

[0009] The present invention first provides use of Akkermansia muciniphila in preparing a drug for alleviating and / or treating schizophrenia, wherein the Akkermansia muciniphila is at least one of Akkermansia muciniphila AM06 with a deposit number of CGMCC No. 22793 or Akkermansia muciniphila AM02 with a deposit number of CGMCC No. 22794.

[0010] In some embodiments, the Akkermansia muciniphila is one, two or more of live bacteria, inactivated bacteria with intact morphology and structure, inactivated bacteria with incomplete morphology and structure, bacterial lysate, and bacterial culture supernatant.

[0011] In some embodiments, the dosage form of the drug includes pills, tablets, granules, capsules, oral liquids or tube feeding preparations. The drug is administered to humans or animals.

[0012] The present invention also provides a pharmaceutical composition, which includes Akkermansia muciniphila and other antipsychotic drugs;

[0013] The other antipsychotic drugs include at least one of phenothiazine antipsychotics, thioxanthene antipsychotics, butyrophenone antipsychotics, benzamide antipsychotics, and dopamine-5-hydroxytryptamine receptor antagonists.

[0014] Preferably, the Akkermansia muciniphila is at least one of Akkermansia muciniphila AM06 with a deposit number of CGMCC No. 22793, or Akkermansia muciniphila AM02 with a deposit number of CGMCC No. 22794, or Akkermansia muciniphila standard strain ATCC BAA-835.

[0015] Preferably, the antipsychotic drug is clozapine, that is, the pharmaceutical composition is a combination of at least one of Akkermansia muciniphila AM06 with a deposit number of CGMCC No. 22793, or Akkermansia muciniphila AM02 with a deposit number of CGMCC No. 22794, or Akkermansia muciniphila standard strain ATCC BAA-835 and clozapine.

[0016] The present invention also provides use of at least one of Akkermansia muciniphila AM06 with a deposit number of CGMCC No. 22793, Akkermansia muciniphila AM02 with a deposit number of CGMCC No. 22794, or Akkermansia muciniphila standard strain ATCC BAA-835 in the preparation of a drug for alleviating or reducing weight gain caused by taking antipsychotic drugs.

[0017] In some embodiments, the antipsychotic drug is clozapine.

[0018] Beneficial effects of the present invention:

[0019] The present inventors unexpectedly discovered that Akkermansia muciniphila, especially Akkermansia muciniphila AM06 with a deposit number of CGMCC No. 22793 and Akkermansia muciniphila AM02 with a deposit number of CGMCC No. 22794, can effectively improve cognitive dysfunction in schizophrenia. When used in combination with antipsychotic drugs such as clozapine, they not only significantly increase the efficacy of antipsychotic drugs, but also inhibit weight gain caused by long-term use of antipsychotic drugs, thereby improving the quality of life of schizophrenia patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a colony characteristic diagram of Akkermansia muciniphila AM02 cultured using the method of Example 1;

[0021] Figure 2 This is a colony characteristic diagram of Akkermansia muciniphila AM06 cultured using the method of Example 1;

[0022] Figure 3 This is a microscopic observation picture of Akkermansia muciniphila AM02 cultured by the method of Example 1 after Gram staining;

[0023] Figure 4 This is a microscopic observation picture of Akkermansia muciniphila AM06 cultured by the method of Example 1 after Gram staining.

[0024] Strain deposit information

[0025] 1) AM02:

[0026] Classification name: Akkermansia muciniphila, preservation unit: General Microbiology Center of China Microbiological Culture Collection Administration, preservation address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing Road, preservation date: June 28, 2021, preservation number: CGMCC No.22794.

[0027] 2) AM06:

[0028] Classification name: Akkermansia muciniphila, preservation unit: General Microbiology Center of China Microbiological Culture Collection Administration, preservation address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing Road, preservation date: June 28, 2021, preservation number: CGMCC No.22793.

[0029] In the present application, "more than one kind" means three or more. DETAILED DESCRIPTION

[0030] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0031] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0032] Example 1 Cultivation of Akkermansia muciniphila

[0033] The Akkermansia muciniphila strain was streaked onto the GlcNAc-heme BHA plate and cultured anaerobically for 3 days. The colony morphology, staining properties, size, club shape, and distribution were observed.

[0034] Colony characteristics: After 3 days of culture on the above medium, Akkermansia muciniphila showed round, convex, neatly edged, opaque, white, and unevenly sized colonies, see Figure 1 and Figure 2 .

[0035] Microscopic appearance: Akkermansia muciniphila is a Gram-negative bacterium that is oval and arranged singly or in chains. Figure 3 and Figure 4 .

[0036] A single colony was selected and inoculated into 10 mL of BHI broth supplemented with GlcNAc for fermentation culture for 48 hours (temperature was 37°C). After the culture was completed, the bacterial solution was inoculated into 1 L of BHI broth supplemented with GlcNAc and continued to be expanded for 24 hours. The obtained bacterial solution was centrifuged and precipitated at a speed of 16000×g for 30 minutes, and the supernatant was removed and the precipitate was collected to obtain Akkermansia muciniphila bacterial sludge. Akkermansia muciniphila AM02, AM06 and BAA-835 were cultured separately.

[0037] Example 2 Preparation of live bacterial solution, inactivated bacterial solution and lysate of Akkermansia muciniphila

[0038] According to the fermentation culture method of Example 1, the corresponding AKK bacteria AM02, AKK bacteria AM06 and AKK bacteria ATCC BAA-835 were prepared.

[0039] (1) The bacterial strain was streaked onto a GlcNAc-heme BHA plate and cultured anaerobically at 37°C for 3 days.

[0040] (2) Enrichment: A single colony was selected from step (1) and inoculated into BHI broth supplemented with GlcNAc for enrichment culture. The culture time was 48 hours at a temperature of 37° C. The obtained bacterial solution was stored for later use.

[0041] (3) AKK bacteria live solution: After counting the live bacteria in the bacterial solution prepared in step (2), dilute it with physiological saline to 10 9 CFU / mL, save for future use.

[0042] (4) Inactivated bacterial solution of Akkermansia muciniphila: Take an appropriate amount of bacterial sludge, add physiological saline at a ratio of bacterial sludge: physiological saline (m:v) = 1:10, and stir at 1000 rpm with a stirrer for 5-20 minutes to evenly disperse the bacteria. Take 100 ml of the dispersed bacterial solution and place it in a sterile three-necked round-bottom flask (try to ensure that the bacterial solution does not adhere to the inner wall of the flask). Place the three-necked round-bottom flask on the heating plate of a magnetic stirrer, put in a sterile stirring bar, and insert a temperature electrode. Set the speed to 300-500 rpm and the temperature to 70°C. Heat for 30 minutes to obtain inactivated bacteria of Akkermansia muciniphila.

[0043] (5) AKK bacterial lysis solution: AKK bacterial lysis solution was prepared by ultrasonic disruption. Specific steps: 10 95 mL of live bacterial liquid of AKK bacteria with a CFU / mL was lysed by ultrasonic instrument for 30 minutes, with On for 10 seconds and Off for 10 seconds. The efficiency of bacterial lysis in this system reached 99%. After lysis, it was centrifuged at 6000 rpm, 10 minutes, and 4°C, and filtered with a 0.22 μm filter head. The filtrate was collected to prepare 10 9 CFU / mL of AKK bacterial lysate, save for later use.

[0044] Example 3 Pharmacological efficacy experiment of Akkermansia muciniphila in treating dizocillin (MK801)-induced schizophrenia model

[0045] (1) Experimental design and methods

[0046] Animal modeling and drug administration:

[0047] SPF grade SD rats, female, 2 months old, 100. The 100 SD rats were randomly divided into a blank group, a model group, a positive drug group (clozapine 1 mg / kg), an AM02 live bacteria group (10 9 CFU / each), AM02 inactivated bacteria group (10 9 cell / ), AM06 live bacteria group (10 9 CFU / each), AM06 inactivated bacteria group (10 9 cell / cell), AM06 bacterial lysate group (1 mL / cell), AM06 culture supernatant group (1 mL / cell), AM06 live bacteria (10 9 CFU / rat) and clozapine (1 mg / kg), 10 rats in each group. All rats except the blank group were intraperitoneally injected with MK801 (0.6 mg / kg) once a day for 21 consecutive days. At the same time as modeling, each rat was gavaged with the corresponding drug once a day for 21 consecutive days.

[0048] Detection indicators:

[0049] Evaluation of the degree of ataxia and stereotyped behavior in rats: 30 minutes after the last administration, the degree of ataxia and stereotyped behavior of rats were evaluated. The degree of ataxia was evaluated using a 4-level scoring method with reference to the Hoffman scoring standard, and the evaluation was performed once every 10 minutes; stereotyped behavior was evaluated with reference to the Sams-Dodd scoring standard. A double-blind method was used to perform a 5-level scoring, and the average score was taken for statistics; the higher the score, the more severe the symptoms.

[0050] Morris water maze test: On the 17th day after drug administration, rats were trained in the water maze, with 4 water entry points set in different quadrants, and the time required for the rats to climb onto the platform was recorded for 4 consecutive days. The platform was removed on the day when drug administration ended, and the swimming time of the rats in the target quadrant was recorded, and the swimming speed was calculated. This was repeated 4 times and the average value was taken.

[0051] (2) Experimental results

[0052] The results of each test index are shown in Table 1. Compared with the blank group, the ataxia and stereotypy scores of the model group increased significantly (p < 0.01), and the swimming time and swimming speed in the target quadrant decreased significantly (p < 0.01). All the groups treated with Akkermansia muciniphila could significantly reduce the ataxia and stereotypy scores to varying degrees (p < 0.05 or p < 0.01), and significantly increase the swimming time and swimming speed in the target quadrant (p < 0.05 or p < 0.01). Compared with the positive drug group and the AM06 live bacteria alone group, the AM06 live bacteria + positive drug combination group could more significantly improve the ataxia and stereotypy of the animals (p < 0.05), and significantly increase the swimming time in the target quadrant (p < 0.05). That is, the combination group has a synergistic effect relative to the single action group.

[0053] More than 85% of schizophrenia patients may have cognitive dysfunction in the early stage of the disease. Improving cognitive dysfunction in schizophrenia patients can improve the quality of life of patients and restore their social function, which is of great significance for the treatment of the disease. The above results show that the live bacteria, inactivated bacteria, bacterial lysate, and culture supernatant of AM02 and AM06 can significantly improve the cognitive dysfunction of schizophrenia model animals, and when Akkermansia muciniphila is used in combination with clozapine, it significantly increases the improvement effect of clozapine on cognitive function, resulting in a synergistic effect.

[0054] Table 1 Various test indicators (mean ± SD, n = 10)

[0055]

[0056]

[0057] Note: * indicates that the difference is significant compared with the model group, p < 0.05; ** indicates that the difference is extremely significant compared with the model group, p < 0.01;

[0058] a Indicates that compared with the positive drug group, the difference is significant p < 0.05.

[0059] b Compared with the group treated with AM06 live bacteria alone, the difference was significant (p < 0.05).

[0060] Example 4 Experimental study on the efficacy of Akkermansia muciniphila in alleviating weight gain induced by the psychotic drug clozapine (1) Experimental design and methods

[0061] Animal modeling and drug administration:

[0062] 50 SPF SD rats, female, 2 months old. 50 SD rats were randomly divided into blank group, model group, AM02 live bacteria group (10 9 CFU / each), AM06 live bacteria group (10 9 CFU / each), BAA-835 live bacteria group (10 9 CFU / rat), 10 rats in each group. Rats in all groups except the blank group were given clozapine by gavage at 5 mg / kg on the first day of the experiment, once a day, and clozapine was given at 25 mg / kg from the fifth day, once a day, and continued to be administered until the eighth week to induce a metabolic disorder model. At the same time as the modeling, each rat was given the corresponding drug by gavage, once a day, for 8 weeks.

[0063] Detection indicators:

[0064] The body weights of the animals were measured at 0, 4, 6, and 8 weeks of the experiment.

[0065] (2) Experimental results

[0066] Table 2 Body weight of rats in each group (mean ± SD, n = 10)

[0067]

[0068] Note: * Indicates that the difference is significant compared with the model group, p<0.05; ** Indicates that compared with the model group, the difference is significant p < 0.01;

[0069] # Compared with the BAA-835 live bacteria group, the difference was significant (p < 0.05). ## It indicates that compared with the BAA-835 live bacteria group, the difference is extremely significant, p < 0.01.

[0070] As shown in Table 2, compared with the blank group, the body weight of the model animals increased significantly from the 4th week (p < 0.01). Each group of Akkermansia muciniphila administration could significantly reduce the weight gain induced by clozapine (p < 0.05 or p < 0.01), and the effect of AM02 and AM06 in inhibiting the weight gain induced by clozapine was significantly better than that of BAA-835 (p < 0.05).

[0071] The most obvious side effects of current treatments for mental disorders are metabolic abnormalities, such as weight gain, which can affect patients' medication compliance. These results suggest that Akkermansia muciniphila can effectively inhibit weight gain caused by long-term use of antipsychotics such as clozapine.

[0072] The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of Akkermansia muciniphila in the preparation of a drug for alleviating and / or treating schizophrenia, characterized in that: The Akkermansia muciniphila is at least one of Akkermansia muciniphila AM06 with a deposit number of CGMCC No. 22793 or Akkermansia muciniphila AM02 with a deposit number of CGMCC No. 22794.

2. The use according to claim 1, characterized in that: The Akkermansia muciniphila is one, two or more of live bacteria, inactivated bacteria with complete morphological structure, inactivated bacteria with incomplete morphological structure, bacterial lysate, and bacterial culture supernatant.

3. The use according to claim 1 or 2, characterized in that: The dosage form of the drug includes pills, tablets, granules, capsules, oral liquids or tube feeding preparations.

4. A pharmaceutical composition, characterized in that These include Akkermansia muciniphila and other antipsychotics; The other antipsychotic drugs include at least one of phenothiazine antipsychotics, thioxanthene antipsychotics, butyrophenone antipsychotics, benzamide antipsychotics, and dopamine-5-hydroxytryptamine receptor antagonists; The Akkermansia muciniphila is at least one of Akkermansia muciniphila AM06 with a deposit number of CGMCC No. 22793 or Akkermansia muciniphila AM02 with a deposit number of CGMCC No. 22794; The pharmaceutical composition is used for alleviating and / or treating schizophrenia.

5. The pharmaceutical composition according to claim 4, characterized in that The antipsychotic drug is clozapine, that is, the pharmaceutical composition is a combination of at least one of Akkermansia muciniphila AM06 with a preservation number of CGMCC No. 22793 or Akkermansia muciniphila AM02 with a preservation number of CGMCC No. 22794 and clozapine.

Images