Probiotic Compositions, Formulations and Uses for Treating REM Sleep Behavior Disorder

Abstract

The present invention provides a probiotic composition for treating REM sleep behavior disorder, which is prepared from Bacillus licheniformis, Bifidobacterium longum, Lactobacillus acidophilus, Lactobacillus faecalis, and a pharmaceutically acceptable excipient. The present invention can improve REM sleep behavior disorder symptoms in patients. By improving RBD symptoms in idiopathic RBD and PD patients, the dose of levodopa in PD patients can be reduced.

Description

[Technical field]

[0001] The present invention relates to a probiotic composition, and in particular to a probiotic composition for treating REM sleep behavior disorder and a preparation prepared using said probiotic composition. [Background technology]

[0002] REM sleep behavior disorder (RBD) is a type of sleep disorder that occurs when a patient enters the REM sleep stage, and the muscles are not fully relaxed, resulting in screaming, talking, or physical movements associated with dreams. The most common behaviors are hitting, kicking, talking, screaming, and getting up in bed, waving their arms, grinding their teeth, laughing, singing, making phone calls, falling out of bed, etc. These patients generally need to be treated with drugs. Currently, effective treatments include drugs such as clonazepam and melatonin, but long-term use of clonazepam can cause a decline in the patient's cognitive ability, and it is also prone to develop dependence and drug resistance, and has significant side effects.

[0003] Patients with idiopathic RBD may develop neurodegenerative diseases such as Parkinson's disease (PD), dementia with Lewy bodies, and multiple system atrophy after a few years. RBD is a common non-motor symptom of Parkinson's disease, and is seen in more than half of Parkinson's disease patients. Currently, there are mainly the following types of drugs for treating Parkinson's disease: levodopa preparations, dopamine receptor agonists, monoamine oxidase inhibitors, and COMT inhibitors, which mainly improve the level of dopamine transmitters in the neuronal synaptic gap, and Benzoxal hydrochloride, amantadine, etc., which adjust the action of acetylcholine or other transmitters such as NMDA. These drugs only suppress motor symptoms and cannot delay the progression of the disease. In addition, as the disease progresses and long-term drug treatment continues, the amount and type of drugs administered to patients gradually increases, but the therapeutic effect decreases, and motor complications such as "motor waves," "dyskinesia," and "switch phenomenon" appear. In addition, while Parkinson's disease medications are effective in improving the motor symptoms of Parkinson's disease, they do not simultaneously improve RBD symptoms, and some medications may worsen the frequency of RBD attacks. Summary of the Invention [Problem to be solved by the invention]

[0004] The main technical problem that the present invention seeks to solve is to provide a probiotic composition for treating REM sleep behavior disorder.

[0005] Another technical problem that the present invention aims to solve is to provide a probiotic preparation prepared using the above-mentioned probiotic composition.

[0006] Another technical problem that the present invention seeks to solve is to provide new uses for the above-mentioned probiotic compositions and preparations. [Means for solving the problem]

[0007] To achieve the above objectives, the present invention adopts the following technical solutions: According to a first aspect of an embodiment of the present invention, there is provided a probiotic composition for treating REM sleep behaviour disorder, comprising Bacillus licheniformis, Bifidobacterium longum, Lactobacillus acidophilus, Lactobacillus faecalis and an art-acceptable excipient.

[0008] Preferably, the probiotic composition has a viable cell count of Bacillus licheniformis of 10.0×10 8 CFU / g or more, and the viable count of Bifidobacterium longum was 4.0×10 8 CFU / g or more, and the viable count of Lactobacillus acidophilus is 1.0×10 8 CFU / g or more, and the viable count of Lactobacillus faecalis is 0.5 × 10 8 CFU / g or more.

[0009] The above preferred range is a range in which the viable cell count of Bacillus licheniformis in the probiotic composition is 1×10 7 CFU / g or more, and the viable count of Bifidobacterium longum is l×l0 7 CFU / g or more, and the viable count of Lactobacillus acidophilus is 1×l0 7 CFU / g or more, and the live bacteria count of Faecalis is l x l0 7 The range can be extended to CFU / g or more, and the inventors can achieve the technical effects of the present invention by conducting clinical trials using probiotic preparations within this range.

[0010] Preferably, the viable cell count of the probiotic composition can be further controlled within the following range, but is not limited to the following range, and a good synergistic effect may be obtained between the strains. The viable cell count of Bacillus licheniformis is 10.0×10 8 ~10.0×10 10 CFU / g, and the viable cell count of the Bifidobacterium longum was 4.0 × 10 8 ~4.0×10 10 CFU / g, and the viable cell count of Lactobacillus acidophilus was 1.0×10 8~1.0×10 10 CFU / g, and the viable cell count of the faecalis bacteria was 0.5 × 10 8 ~0.5×10 10 CFU / g.

[0011] It has been confirmed through clinical trials that the above probiotic composition has good therapeutic effects on the RBD symptoms of idiopathic RBD patients and Parkinson's disease patients, and the therapeutic effects are stable and long-lasting. At the same time, it also has good improving effects on the motor symptoms of Parkinson's disease patients, and can play an important protective role in the treatment of Parkinson's disease patients, and can effectively reduce the dosage of levodopa required by patients.

[0012] A second aspect of the present invention provides a probiotic formulation for treating REM sleep behavior disorder, which is prepared using the above probiotic composition and art-acceptable excipients, which can be used to prepare and obtain different probiotic dosage forms, including but not limited to solvents, emulsifiers, disintegrants, solubilizers, antioxidants, pH adjusters, osmotic pressure adjusters, antibacterial agents, diluents, wetting agents, adhesives, and film-forming agents.

[0013] According to a third aspect of an embodiment of the present invention, there is provided a method for preparing a probiotic formulation for treating REM sleep behavior disorder, comprising the steps of:

[0014] Step (1): Prepare dried powders of Bacillus licheniformis, Bifidobacterium longum, Lactobacillus acidophilus, and Lactobacillus faecalis. do, Prepare each bacterial powder.

[0015] Step (2): Each of the dried powders obtained in step (1) is mixed with art-acceptable excipients to produce the final dosage form.

[0016] Here, the excipient described in step (2) above can be selected from solid preparations such as starch, dextrin, lactose, microcrystalline cellulose, or inorganic salts according to the dosage form to be prepared. The wetting agent and adhesive can be selected from starch paste and rubber adhesive. The disintegrant can be selected from dry starch, sodium carboxymethyl starch, and emergency disintegrant, and the film coating can be selected from enteric agents.

[0017] The probiotic composition described above is used in the preparation of a probiotic formulation for REM sleep behavior disorder.

[0018] The probiotic formulations described above are used in the preparation of drugs, dietary supplements and / or foods for treating REM sleep behavior disorder.

[0019] The probiotic preparation prepared by the above method is used in the preparation of a drug, health food and / or food for treating REM sleep behavior disorder.

[0020] According to a fourth aspect of an embodiment of the present invention, a food composition or supplement for treating REM sleep behavior disorder is provided.

[0021] The probiotic composition is prepared from Bacillus licheniformis, Bifidobacterium longum, Lactobacillus acidophilus, Lactobacillus faecalis and a pharmaceutically acceptable excipient.

[0022] In the probiotic composition, the viable cell count of Bacillus licheniformis was 1×10 7 CFU / g or more.

[0023] In the probiotic composition, the viable cell count of Bifidobacterium longum was 1×10 7 CFU / g or more.

[0024] In the probiotic composition, the viable cell count of Lactobacillus acidophilus was 1×10 7 CFU / g or more.

[0025] In the probiotic composition, the viable cell count of Lactobacillus faecalis is 1×10 7 CFU / g or more.

[0026] The food composition or supplement described above is used in the preparation of a dietary supplement and / or food for treating REM sleep behavior disorder in Parkinson's disease.

[0027] The above food types include, but are not limited to, biscuits, dairy products, substitute foods, meat products, sauces, ice cream, fermented products, juices, rice wine, confectionery, canned foods, pickles, seasonings, soy products, chocolates, bean paste, tea products, and puffed foods, etc. The food may be a food additive.

[0028] The types of the health food supplements include, but are not limited to, medicinal liquor, capsules, tablets, soluble granules, tea products, oral liquids, granules, fermented products, honey ointments, dews, powders, substitute foods, etc. The health food may further include health food additives.

[0029] The Bacillus licheniformis (BL), Bifidobacterium longum (BL), Lactobacillus acidophilus (LA) and Lactobacillus faecalis (EF) used in the present invention are all known to those skilled in the art. The freeze-dried powder of the above-mentioned species of bacteria of the present invention can be purchased commercially, and the freeze-dried powder meets the above-mentioned live bacteria requirements of the present invention, and can be prepared, mixed and dispensed according to a method known in the art. In addition, different demands such as powders, granules, tablets and capsules can be prepared by combining the probiotic composition with an acceptable excipient based on known pharmaceutical, health food and food preparation methods. The probiotic live bacteria used in the present invention can also be obtained by culturing using a culture method known in the art, and the preparation method and the compounding and extruding method of the probiotics are not limited to the contents disclosed in the present invention, and any known conventional method can be used to realize the present invention.

[0030] The above culture methods include, but are not limited to, liquid fermentation, solid fermentation and semi-solid fermentation.

[0031] The above types of liquid fermentation include, but are not limited to, batch fermentation, continuous fermentation and supplemented batch fermentation. The media used in the liquid fermentation include, but are not limited to, LB medium, AAM-AS liquid medium, MRS medium, PTYG medium, PY medium, YPD medium and BSM medium, and their improved, modified and optimized types.

[0032] The above types of solid fermentation methods include, but are not limited to, natural rich solid fermentation, microbial mixed solid enrichment fermentation, microbial mixed solid limited fermentation and single-cell pure fermentation. The media used in the solid fermentation methods include, but are not limited to, LB solid medium, YEB solid medium, Sabouraud agar medium, R2A agar medium, YPD solid medium, BSM solid medium, and their improved, modified and optimized types.

[0033] The types of semi-solid fermentation methods include, but are not limited to, intermittent fermentation, and the media used in the semi-solid fermentation methods include, but are not limited to, LB medium, YEB medium, Sabouraud agar medium, R2A agar medium, YPD medium, and their improved, modified, and optimized types.

[0034] The present invention recommends the following usage for patients with REM sleep behavior disorder. The probiotic capsule of the present invention is 250 mg per capsule, and the number of live bacteria in the capsule only needs to meet the above requirements of the present invention. The weight of the freeze-dried probiotic powder contained in the capsule is related to the preparation method of the dried powder and may vary slightly. The usage method is orally, 2 capsules each time, 3 times a day.

[0035] The present invention recommends the following method of use for Parkinson's disease patients. The probiotic capsule of the present invention is 250 mg per capsule, and the viable bacteria count in the capsule only needs to meet the above requirements of the present invention. The weight of the freeze-dried probiotic powder contained in the capsule is related to the preparation method and may vary slightly. The method of use is orally, 2 capsules per time, 3 times a day. Effect of the Invention

[0036] In the present invention, the probiotic composition and the components of the probiotic preparation prepared using the probiotic composition exert a synergistic effect, and clinical trials have confirmed that the probiotic composition can effectively treat and improve the RBD symptoms of patients, and that the therapeutic effect of the RBD symptoms is long-lasting and stable. The present invention has a therapeutic effect on the RBD symptoms of PD patients and can effectively improve the motor symptoms of PD patients, solving the problem that conventional drugs cannot simultaneously improve and treat the motor symptoms and non-motor symptoms of PD patients. The probiotic preparation provided by the present invention has no side effects, does not cause drug dependency and drug resistance, and can effectively reduce the dose of levodopa in PD patients, avoiding the patients from having early drug resistance to levodopa, and has a relatively good protective effect on patients. The present invention has good clinical application prospects and market prospects. [Brief description of the drawings]

[0037] [Figure 1] FIG. 13 shows changes in RBD-HK scores in three groups of RBD+PD patients. [Diagram 2] Figure 1 shows the change in baseline UPDRS-III scores after 12 and 24 weeks of treatment in three groups of RBD+PD patients. [Diagram 3] This is a comparison graph of the motor symptom improvement rates in three groups of RBD+PD patients treated for 12 weeks. [Figure 4] This is a comparison graph of the motor symptom improvement rates in three groups of RBD+PD patients treated for 24 weeks. [Diagram 5] FIG. 1 shows the trend of changes in levodopa dose equivalent in three groups of RBD+PD patients. [Figure 6] This is a comparison diagram of RBD-HK scores before and after iRBD treatment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] The inventors have found through clinical trials that the combination of Bacillus licheniformis, Bifidobacterium longum, Lactobacillus acidophilus and Lactobacillus faecalis has excellent therapeutic effects on the symptoms of RBD in patients with idiopathic RBD and patients with Parkinson's disease, has excellent durability and stability of therapeutic effects, and has a protective effect against the progression of the disease history of patients with Parkinson's disease. The present invention does not set specific requirements for the viable cell count of the four strains, as long as they meet the local production standards for probiotic products and can be established in the intestines of patients. The lower limit of the viable cell count of each strain described in the specific embodiments of the present invention is a recommended preferred method.

[0039] A specific embodiment is as follows. The raw materials for the freeze-dried powder of bifidobacteria used in the embodiments of the present invention can all be purchased commercially, and the specific sources are Jingyue Biotechnology Co., Ltd., Shannekang Biotechnology (Suzhou) Co., Ltd., and Beijing Beina Chuanglian Biotechnology Research Institute.

[0040] The freeze-dried probiotic powder can also be prepared by conventional techniques, and the specific preparation method is as follows. (1) A fungal species is inoculated into a seed medium to obtain an inoculum product. (2) The inoculum product of step (1) is fermented in a fermentation medium to obtain a fermentation product without changing the pH during the fermentation period. (3) The fermentation product and protective agent obtained in step (2) are freeze-dried to obtain a freeze-dried bacterial powder.

[0041] The above mentioned strains of bacteria are probiotics of Bacillus licheniformis, Bifidobacterium longum, Lactobacillus acidophilus or Lactobacillus faecalis.

[0042] The above-mentioned protective agent is a component known in the art, and may be selected from skim milk powder, isolactose, Vc-Na, starch, monosodium glutamate, and the like.

[0043] The cultivation of the above-mentioned species is a common technique, and reference can be made to "Effect of Biotin on the Growth and Lactic Acid Formation of Bacillus licheniformis" (Bioengineering Journal, January 1998), "Optimization of Bifidobacteria Enrichment Medium" (Food Industry Technology, No. 4, 2004), "Optimization of Lactobacillus acidophilus Enrichment Medium" (Food Industry Technology, No. 6, 2002) and "Study on Lactobacillus acidophilus and Fecal Streptococcus Healthy Yogurt Fermenting Agent" (Bulletin of Anhui Agricultural Technology Normal College, No. 1, 2000).

[0044] The above fermentation culture is a conventional technology, and can refer to the conventional techniques "Optimization of fermentation conditions for Bacillus licheniformis 10236" (Hebei Agricultural Science, May 2019), "Optimization of liquid fermentation conditions for bifidobacteria and measurement of growth curve" (Formulations and Technology, November 2012, Vol. 9, No. 31), "Development of Lactobacillus acidophilus freeze-drying protection agent and direct injection type composite fermentation agent" (Modern Food Science and Technology, 2019, 35(09):248-257.) and "Response surface methodology for optimizing Lactobacillus faecalis foreskin fermentation medium" (Food Industry Science and Technology, No. 21, 2019).

[0045] The preparation method of the freeze-dried bacterial powder is a common technique, and reference can be made to known techniques such as CN110367543A, CN110720638A and CN102356912B.

[0046] The probiotic composition, preparation method and colony standard of the present invention are all in accordance with Part 3 of the 2010 edition of the Chinese Pharmacopoeia and the 2000 edition of the China Regulations for Biological Products.

[0047] The CFU / g of the present invention is the number of colonies contained in the detection sample per gram, and CFU is the colony forming unit. The CFU / g in the following examples generally means "equal to" or "more than". In actual use, the number of live bacteria in general products is "more than" in order to ensure that the number of live bacteria within the quality assurance period is within the effective range and the survival rate of the bacterial strain in the intestine after the patient takes it.

[0048] The embodiment of the present invention described below is a probiotic capsule prepared using a three-dimensional motion mixer (Jiangsu Huangbao Group Co., Ltd. SBH system) and a capsule filling machine (Beijing Guowei and Jiao Medical Equipment Co., Ltd.). Those skilled in the art can also prepare the probiotic composition of the present invention into powder, granules, tablets, etc. according to the needs of use. The excipients used in each dosage form can be determined and prepared based on common knowledge in the technical field. The dosage form, excipients, and preparation method do not affect the final technical effect of the present invention, and the types of excipients, the amount of excipients used, the compounding relationship between excipients, and the preparation method are not used to limit the protection scope of the present invention. Of course, depending on the actual situation, one may not choose to prepare oneself, but may choose a tri-combined live bacteria preparation (e.g., a tri-combined preparation of live bacteria of Bifidobacterium longum, Lactobacillus acidophilus and Lactobacillus faecalis) that meets the above-mentioned viable bacteria count of the present invention, and the Bacillus licheniformis live bacteria capsule that meets the above-mentioned viable bacteria count of the present invention may be used in combination to achieve the above-mentioned technical effects of the present invention.

[0049] Example 1 Preparation of the probiotic formulation of the present invention (1) Bacillus licheniformis live bacteria, Bifidobacterium longum, and freeze-dried powders of Lactobacillus acidophilus and Lactobacillus faecalis are mixed with excipients to obtain a homogeneous mixture. In this embodiment, the excipients are pregelled starch and lactose. (2) The mixture obtained in step (1) is dispensed into capsules to prepare capsules, which are enteric-coated capsules, each of which contains 250 mg of the mixed bacteria powder.

[0050] Each capsule contains 10 x 10 live bacteria of Bacillus licheniformis. 8 CFU / g or more, Bifidobacterium longum is 4.0×10 8 CFU / g or more, Lactobacillus acidophilus is 1.0×10 8 CFU / g or more, and 0.5 × 10 8 CFU / g or more.

[0051] When the above viable cell count is met, the excipient is the remaining amount, and the mass ratio of pregelled starch to lactose is 1:1.

[0052] Example 2 Preparation of the probiotic formulation of the present invention (1) Mix live Bacillus licheniformis bacteria, Bifidobacterium longum, Lactobacillus acidophilus and Lactobacillus faecalis lyophilized powders and excipients to obtain a homogeneous mixture. In this embodiment, the excipients are pregelled starch and lactose. (2) The mixture obtained in step (1) is dispensed into capsules to prepare capsules, each of which is an enteric-coated capsule, and contains 250 mg of the mixed bacteria powder.

[0053] Each capsule contains 10 x 10 live bacteria of Bacillus licheniformis. 9 CFU / g or more, Bifidobacterium longum is 4.0×10 9 CFU / g or more, Lactobacillus acidophilus is 1.0×109 CFU / g or more, and Lactobacillus faecalis is 0.5×10 9 CFU / g or more.

[0054] When the above viable cell count was met, the excipient was the remaining amount, and the mass ratio of pregelatinized starch to lactose was 1:1.

[0055] Example 3 Preparation of the probiotic formulation of the present invention (1) Mix live Bacillus licheniformis bacteria, Bifidobacterium longum, Lactobacillus acidophilus and Lactobacillus faecalis lyophilized powders and excipients to obtain a homogeneous mixture. In this embodiment, the excipients are pregelled starch and lactose. (2) The mixture obtained in step (1) is dispensed into capsules to prepare capsules, each of which is an enteric-coated capsule, and contains 250 mg of the mixed bacteria powder.

[0056] Each capsule contains 10 x 10 live bacteria of Bacillus licheniformis. 10 CFU / g or more, Bifidobacterium longum is 4.0×10 10 CFU / g or more, Lactobacillus acidophilus is 1.0×10 10 CFU / g or more, and 0.5 × 10 10 CFU / g or more.

[0057] When the above viable cell count was met, the excipient was the remaining amount, and the mass ratio of pregelatinized starch to lactose was 1:1.

[0058] Example 4 Preparation of the probiotic formulation of the present invention (1) Mix live Bacillus licheniformis bacteria, Bifidobacterium longum, Lactobacillus acidophilus and Lactobacillus faecalis lyophilized powders and excipients to obtain a homogeneous mixture. In this embodiment, the excipients are pregelled starch and lactose. (2) The mixture obtained in step (1) is dispensed into capsules to prepare capsules, which are enteric-coated capsules, each of which contains 250 mg of the mixed bacteria powder.

[0059] Each capsule contains 1 x 10 live bacteria of Bacillus licheniformis. 7 CFU / g or more, Bifidobacterium longum is 1×10 7 CFU / g or more, Lactobacillus acidophilus 1×10 7 CFU / g or more, and faecalis bacteria is 1×10 7 CFU / g or more.

[0060] When the above viable cell count was met, the excipient was the remaining amount, and the mass ratio of pregelatinized starch to lactose was 1:1.

[0061] Efficacy Test of the Invention 1 - Parkinson's Disease Cohort Study 1. Materials and Methods 1) Research Subjects 1.Selection criteria Subjects who meet all of the following selection criteria are eligible to participate in this study. 1) Ages 40-80, both male and female

[0062] 2) Primary Parkinson's disease patients meet the 2015 Clinical Diagnostic Criteria for Parkinson's Disease MDS, including modified Hoehn-Yahr stage ≤3, no dementia, and Mini-Mental State Examination (MMSE) score >24.

[0063] 3) The motor and RBD symptoms of Parkinson's disease were stable for one month prior to entering the group, and all medications used in the month prior to entering the group were not adjusted, and no clonazepam, melatonin or other benzodiazepine drugs were used in the month prior to entering the group.

[0064] 4) They had not been treated with intestinal probiotics or prebiotics (including lactulose) or antibiotics in the 2 months prior to group entry, and if they had been used, a 2-month elution phase must have been performed.

[0065] Understand and agree to follow the study plan, agree to be included in the group, and sign the informed consent.

[0066] 1. Exclusion criteria Subjects who meet any of the following criteria are ineligible to participate in this study. 1) Superimposed parkinsonian and secondary parkinsonian syndromes such as multiple system atrophy, progressive supranuclear palsy, corticobasal degeneration, dementia with Lewy bodies, vascular parkinsonian syndromes, postencephalitic parkinsonian syndromes, or any other non-primary parkinsonian disease. 2) had taken probiotics, prebiotics (including lactulose), or antibiotics within 2 months prior to entering the group; 3) Alzheimer's disease, malignant tumor, spinal cord lesion, epilepsy, obstructive sleep apnea syndrome, autonomic nervous system disease (urinary retention, urinary incontinence, orthostatic hypotension, blood pressure drop of 30 / 15 mmHg or more 5 minutes after standing upright), etc. Within 3 months, new onset of cerebrovascular disease or serious sequelae of cerebrovascular disease affecting the evaluator. 4) History of gastrointestinal tumors and inflammatory bowel disease, and other acute or chronic gastrointestinal inflammation (including acute attacks of cholecystitis) within 3 months. 5) Serious cardiovascular disease (e.g., congestive heart failure with an American Heart Association rating of III-IV, or a history of myocardial infarction within the past 6 months) 6) Severe liver or kidney dysfunction, alanine transaminase, glutamine aminotransferase and total bilirubin higher than 1.5 times the upper limit of normal, serum creatinine higher than 1.5 times the upper limit of normal. 7) Pregnant and lactating women, or women of childbearing age between 40 and 60 who are HCG positive 8) Subjects known to have allergies to the test drug or related products 9) Those with a history of drug abuse or alcohol dependence 10) Subjects who have participated in other clinical trials within 3 months prior to entering the group 11) Patients who refuse to join the group, are unable to cooperate with the researchers, or are deemed by the researchers to be unsuitable for joining the group.

[0067] 1. Randomized Design: This is a prospective and open-ended cohort study. Patients were further stratified according to whether they had RBD, and divided into RBD+PD patients and RBD-PD patients, and patients in each stratum were further randomly divided into test group 1, test group 2, and control group.

[0068] RBD+ criteria (Stiasny-Kolster K, Mayer G, Schafer S, et al. The REM sleep behavior disorder screening questionnaire-a new diagnostic instrument[J]. Mov Disord, 2007,22(16):2386-2393. Shen SS, Shen Y, Xiong KP, Chen J, Mao CJ, Huang JY, Li J, Han F, Liu CF. Validation study of REM sleep behavior disorder questionnaire-HongKong(RBDQ-HK) in east China. SleeP Med. 2014 Aug;15(8):952-8.doi:10.1016 / j.sleep.2014.03.020.): RBD screening scale (RBDSQ) RBDSQ>5 points, and REM sleep behavior disorder scale (Rapid eye movement sleep behavior disorder questionnaire Hong Kong) of the Chinese University of Hong Kong Those who do not meet the above criteria are RBD-HK (Range-Blind-Kong, RBD-HK) score ≥ 17 points and are diagnosed with RBD by multiple-conductor sleep monitoring.

[0069] Dropout, discontinuation, and withdrawal procedures: Patients who could not continue to participate in the study or could not be contacted despite repeated follow-ups were dropped out. Patients who developed any inflammation during the observation period and needed to be treated with antibiotics were not considered to have discontinued the study and their scale scores were not used during the study. Patients who developed acute cardiocerebrovascular or other serious adverse events were dropped out.

[0070] 2. Test method 1) Intervention sample: Test group 1 (intervention group) uses the probiotic preparation obtained according to Example 1 of the present invention. Test group 2 (intervention group) uses 1.0 × 10 Bifidobacterium longum per capsule. 7 CFU, Lactobacillus acidophilus 1.0×10 7 CFU, faecalis 1.0 × 107 Probiotic capsules containing CFU (purchased from Shanghai Shangyao Xinyi Pharmaceutical Factory Co., Ltd., National Pharmaceutical Preparation Standard S10950032) were used. The control group was maintained on the same treatment drug as before, without adding any probiotics or prebiotics.

[0071] 2) Test method In test group 1, in addition to the usual PD treatment, 2 tablets of the formulation of Example 1 are administered three times a day. In test group 2, in addition to the usual PD treatment, 4 tablets of the formulation of test group 2 are administered twice a day according to the instructions. The blank control group is treated with only the usual PD medication.

[0072] All daily doses of anti-PD therapeutic drugs are converted to levodopa dose equivalents using the following formula: 100 mg levodopa = 100 mg piribedil = 1 mg praxos = 5 mg ropinirole = 10 mg selegiline = 100 mg amantadine = 133 mg carbidopa = 1 mg rasagiline. Entacarbone doses are calculated by increasing the dose equivalent of levodopa by 33%.

[0073] For RBD+PD patients, no additional sleep interventions such as clonazepam or other benzodiazepines or melatonin will be administered from the time of group entry until the end of the observation period.

[0074] 3) Evaluation: All patients will have electrocardiograms taken at the time of entering the group and at 12 weeks, and blood tests, liver and kidney function tests, and electrolytes tests. The RBD screening scale will be used to screen for REM sleep behavior disorder, and the RBD and its severity will be evaluated using the RBD-HK scale from the Chinese University of Hong Kong. The third part of the Unified Parkinson's Disease Rating Scale (UPDRS) UPDRS-III will be used as the severity evaluation index for motor symptoms, and the difference between the score at 12 weeks or 24 weeks and the score at 0 weeks will be statistically calculated. A difference of >3 will be weighted, a difference <-3 will be considered as improvement, and a difference of -3≦3 will be considered as stable. At 0 weeks, 12 weeks, and 24 weeks after entering the group, the UPDRS-III score and RBD-HK score will be completed, and the name and dosage of the patient's usual anti-PD medication will be recorded.

[0075] 4) Data statistics: Data are statistically analyzed using SPSS26.0. Quantitative data are expressed as mean ± standard difference (x±s), and quantitative data are expressed as percentages. The 12-week data are the main statistical indicators. Paired t-tests are used for intragroup comparisons of quantitative data, analysis of variance is used for comparisons between the three groups, analysis of variance is used for comparisons of the three groups at different time points, and chi-square test or Fisher exact test is used for quantitative data. P<0.05 is considered to be statistically significant.

[0076] 3. Test Results and Analysis General Patient Information This study was approved by the Ethics Committee of Beijing Friendship Hospital Affiliated to Capital Medical University.

[0077] Patients were recruited from the outpatient and ward-visiting neurology department of Beijing Friendship Hospital affiliated to Capital Medical University between May 2018 and December 2019, and all patients signed informed consent.

[0078] We collected 62 patients with primary Parkinson's disease who met the conditions for entering the group, 120 in the test group, 220 in the test group, 22 in the control group, and 2 in the control group who dropped out. Finally, 60 patients entered the group. Among them, 48 RBD+ and 12 RBD- were randomly distributed into three groups, with 16 RBD+PD patients and 4 RBD-PD patients in each group. Of these, 35 were male, with a median age of 66 years, and there was no significant difference in the distribution of age (P<0.05, analysis of variance) and gender (P<0.05, chi-square test) among the three groups. There were no obvious abnormalities in electrocardiograms, blood tests, liver and kidney function, electrolytes, etc. at the time of entering the group and at 12 weeks, and no patients were discontinued from observation early due to the above reasons. No patients were discontinued from observation due to serious adverse events. The HY staging of the three groups of patients was 2.10±0.68, 2.15+0.65 and 1.75+0.60, respectively, and the differences were not statistically significant (P=0.110>0.05, analysis of variance).

[0079] 1) Comparison of RBD effects 1. 12-week follow-up results The RBD-HK scores of all 16 RBD+PD patients in each group when they entered the group were 38.75±10.89 (experimental group 1), 33.19±12.50 (experimental group 2), and 30.56±12.79 (control group), respectively, and there was no statistically significant difference between the three groups (P=0.178>0.05, dispersion separation). After 12 weeks of treatment, the RBD-HK scores of experimental group 1 and experimental group 2 compared with those of this group at week 0, the difference was statistically significant (P<0.001 (experimental group 1), P<0.05 (experimental group 2)), and the scores before and after the control group were relatively insignificant (P>0.05). When the RBD-HK scores of the three groups were compared to the baseline difference, the comparison difference between the two groups was statistically significant (P<0.05). Among them, when comparing the RBD-HK scores of study group 1 and the control group (P<0.001), study group 2 and the control group (P=0.025), and study group 2 and study group 1 (P=0.035) with the baseline difference, the differences were statistically significant (P<0.05, analysis of variance) (Table 1).

[0080] Table 1 RBD-HK scores of RBD+PD patients in group 3 at 12 weeks JPEG0007671778000001.jpg42170Compared to before treatment in this group, *P<0.05, **P<0.01, ***P<0.001. Compared with the control group after treatment, #P<0.05, ##P<0.01, ###P<0.001.

[0081] 2. RBD outcomes at 24-week follow-up There were 14, 11 and 14 cases in the three groups, respectively, and they completed the 24-week follow-up (causes of dropout: some patients did not reach the 24-week follow-up period, and some patients could not be followed up due to the current 2019-2020 COVID-19). When comparing the RBD-HK scores between the three groups with the baseline difference, the differences were statistically significant (P<0.05). Among them, when comparing the RBD-HK scores between test group 1 and the control group (P<0.001), test group 2 and the control group (P=0.021), and test group 1 and test group 2 (P=0.048) with the baseline difference, the differences were all statistically significant (P<0.05, analysis of variance). Specifically, they are shown in Table 2 and Figure 1.

[0082] Table 2 RBD-HK scores of RBD+PD patients in group 3 at 24 weeks JPEG0007671778000002.jpg49170Compared to before treatment in this group, *P<0.05, **P<0.01, ***P<0.001. Compared with the control group, #P<0.05, ##P<0.01, ###P<0.001.

[0083] Summary: The results suggest that over time, the RBD-HK score of test group 1 (the group of the present invention) continues to decrease, that is, the patient's RBD status continues to improve. For test group 2 (the group of three bacteria), after 12 weeks of treatment, the patient's RBD status improved from when they entered the group, but the status did not further improve with continued treatment. The RBD-HK score of the control group had no statistical difference at 0 weeks, 12 weeks, and 24 weeks, suggesting that anti-Parkinson's disease motor symptom drugs have no obvious effect on the patient's RBD status. Furthermore, as shown in Figure 1, the RBDHK score of test group 1 continued to decrease, with a clear and stable tendency to decrease. Test group 2 also decreased, but a tendency to increase was evident in the later period, and although both groups have a certain therapeutic effect, the persistence and stability of the therapeutic effect of test group 1 was superior to that of test group 2. In summary, for RBD symptoms in PD patients, the therapeutic effect of test group 2 is superior to that of the control group, and the therapeutic effect of test group 1 of the present invention is superior to that of test group 2 and even superior to that of the control. In addition, the therapeutic effect of the present invention is continuously improved with the extension of the treatment time.

[0084] 2) Statistics on motor symptoms 1. Comparison of UPDRS-III scores Explanation: This score scale requires patients to come to the hospital and have a face-to-face evaluation with a doctor. Some patients missed the evaluation time because they could not arrive at the hospital within the stipulated time, some patients did not reach the 24-week follow-up time, and some patients could not be followed up due to the current 2019-2020 COVID-19 pandemic. Therefore, the number of follow-up patients has decreased in part.

[0085] Among the three groups of patients, 19, 16, and 18 patients completed the 12-week UPDRS follow-up, respectively, and the differences between the baseline UPDRS-III scores of the three groups were not statistically significant (P=0.05, analysis of variance). At 12 weeks of treatment, the UPDRS-III scores were compared with those at week 0, and the differences were statistically significant within the study group 1 (P=0.001) and study group 2 (P=0.029), while the differences were not statistically significant within the control group (P=0.111) (paired t-test).

[0086] After 12 weeks of treatment, the UPDRS-III scores were compared with the score difference at 0 weeks, and the differences of the three groups were -5.08 ± 5.82 (test group 1), -4.84 ± 8.01 (test group 2), and 1.92 ± 4.84 (control group), respectively, and the differences were statistically significant (P < 0.001). Among them, the differences in UPDRS-III scores between test group 1 and the control group (P = 0.004), and between test group 2 and the control group (P = 0.009) were compared, and the differences were statistically significant (P < 0.01). The differences in UPDRS-III scores between test group 2 and test group 1 were compared, and the differences were not statistically significant (P = 1.000 > 0.05) (analysis of variance), but the score difference of test group 1 was more improved than that of test group 2 after 12 weeks of treatment. (Table 3)

[0087] Table 3. UPDRSIII scores of 3 groups of PD patients treated for 12 weeks JPEG0007671778000003.jpg40170Compared to before treatment in this group, *P<0.05, **P<0.01, ***P<0.001. Compared with the control group after treatment, #P<0.05, ##P<0.01, ###P<0.001.

[0088] The number of patients who completed the 24-week follow-up of UPDRS scores in each group was 15, 7, and 8, respectively. By statistical analysis of these patients, the UPDRS-III scores at 24 weeks of treatment were compared with those at week 0, and the difference was statistically significant (P = 0.020) when comparing within the study group 1, but there was no statistical difference between study group 2 (P = 0.907) and the control group (P = 0.546). When comparing the UPDRS-III scores at 24 weeks with the difference at baseline, the differences among the three groups were not statistically significant (P = 0.09 > 0.05). Among them, when comparing the changes in UPDRS-III scores between study group 1 and the control group (P = 0.126), study group 2 and the control group (P = 1.000), and study group 1 and study group 2 (P = 0.435), all the differences were not statistically significant (P > 0.05) (analysis of variance). However, the difference in UPDRS-III scores after 24 weeks of treatment showed a more obvious improvement trend in study group 1 (-4.9 ± 7.20) than study group 2 (-0.29 ± 6.18) and the control group (1.38 ± 6.13), as shown in Table 4 and Figure 2.

[0089] Table 4. UPDRSIII scores of PD patients in the three groups at 24 weeks JPEG0007671778000004.jpg46170

[0090] Compared with the group before treatment, *P<0.05, **P<0.01, ***P<0.001. Compared with the control group after treatment, #P<0.05, ##P<0.01, ###P<0.001.

[0091] 2. Statistics on improvement rate According to the literature, the difference in UPDRS-III baseline score between after treatment and when entering the group is defined as stable disease ≦|±3| points, a loss of 3 points or more is improvement, and a gain of 3 points or more is aggravation of disease. After 12 weeks of treatment, the improvement rates of the three groups were compared, and the difference was statistically significant (P=0.008<0.01). Among them, the treatment effect after 12 weeks of treatment was compared, and the improvement rates of test group 1 and the control group (P=0.004), test group 2 and the control group (P=0.013) were compared, and the difference was statistically significant (P<0.05), while the improvement rates of test group 1 and test group 2 (P=0.691) were compared, and the difference was not statistically significant (P>0.05, analysis of variance). (Table 5)

[0092] Table 5. Comparison of the effects of 3 treatment methods after 12 weeks of treatment JPEG0007671778000005.jpg51170Compared to before treatment in this group, *P<0.05, **P<0.01, ***P<0.001. Compared with the control group at the same time after treatment, #P<0.05, ##P<0.01, ###P<0.001.

[0093] After 24 weeks of treatment, the improvement rates of test group 1 and the control group (P=0.015) were compared, and the difference was statistically significant (P<0.05); the improvement rates of test group 1 and test group 2 (P=0.198), and test group 2 and the control group (P=0.814) were compared, and the differences were not statistically significant (P>0.05, analysis of variance); the improvement rates of the three groups were compared, and the differences were not statistically significant (P=0.062>0.05, analysis of variance). However, the improvement rate of test group 1 was 66.7%, which was obviously higher than that of test group 2 (28.6%). As shown in Table 6 and Figure 3.

[0094] Table 6 Comparison of the effects of 3 treatment methods after 24 weeks of treatment JPEG0007671778000006.jpg52170

[0095] Compared with the group before treatment, *P<0.05, **P<0.01, ***P<0.001. Compared with the control group at the same time after treatment, #P<0.05, ##P<0.01, ###P<0.001.

[0096] The above results show that when patients were given probiotic treatment, the motor symptoms of the patients in test group 1 and test group 2 could be obviously improved, and test group 1 had a stronger improving effect. With the extension of the treatment time, the treatment of motor symptoms in test group 1 was effective, and the proportion of patients whose symptoms were improved also gradually increased, rising from 52.6% at 12 weeks to 66.7% at 24 weeks. However, the improvement rate of test group 2 (three bacteria group) decreased from 56.3% at 12 weeks to 28.6% at 24 weeks, and the improvement rate of the control group was basically stable at 11.1% to 12.5%. At 24 weeks, the improvement rate of test group 1 was obviously higher than that of the control group and test group 2. This indicates that the test group 1 of the present invention has a stronger tendency to improve the therapeutic effect on motor symptoms than test group 2, and the continuity and stability of the improvement tendency are better than test group 2. (Table 6, Figure 4)

[0097] Summary: Probiotic treatment can improve the motor symptoms of patients in both test group 1 and test group 2 of the present invention. Test group 1 of the present invention is superior to test group 2 in terms of UPDRS-III scoring and overall improvement rate. In addition, with the extension of treatment time, its improving effect on motor symptoms is gradually enhanced, and the overall improvement rate gradually increases, and at 24 weeks, 66.7% of patients have obvious improvement in motor symptoms, which is much higher than test group 2 and the control group. More importantly, test group 2 of the present invention has a stronger, more sustained and more stable improvement trend for motor symptoms.

[0098] 3) Comparison of levodopa dose equivalents The statistical sample of patients in this group was the same as the statistical sample of patients with motor symptoms.

[0099] There was no statistically significant difference in the levodopa dose equivalent at entry into the three groups (P=0.227). Comparing the difference between the levodopa dose equivalent at 12 weeks of treatment and baseline, the difference between the three groups was statistically significant (P=0.004,<0.05). Among them, the daily levodopa dose equivalent in test group 1 and test group 2 was lower at 12 weeks than at entry into the group, with the reduction in dosage being -22.37±45.56mg / d and -8.59±42.50n1g / d, respectively. Among them, the reduction in levodopa dose equivalent in test group 1 was obvious compared to the control group, and the difference was statistically significant (P=0.005)(P<0.05). However, when comparing the changes in levodopa dose equivalent between study group 2 and the control group (P = 0.051) and study group 1 and study group 2 (P = 1.000), the differences were not statistically significant (P > 0.05) (analysis of variance), as shown in Table 7.

[0100] Table 7. Comparison of levodopa dose equivalents in 3 groups of PD patients at 12 weeks JPEG0007671778000007.jpg47170Compared to before treatment, *P<0.05, **P<0.01, ***P<0.001. Compared with the control group after treatment, #P<0.05, ##P<0.01, ###P<0.001.

[0101] After 24 weeks of treatment, the changes in levodopa dose equivalents in the three groups were compared, and the differences were not statistically significant (P=0.22>0.05). Among them, the changes in levodopa dose equivalents in test group 1 and the control group (P=0.331), test group 2 and the control group (P=0.491), and test group 1 and test group 2 (P=1.000) were compared, and the differences were not statistically significant (P>0.05) (ANOVA). The results are shown in Table 8.

[0102] Table 8. Dose equivalent of levodopa in 3 groups of PD patients at 24 weeks JPEG0007671778000008.jpg54170Compared to before treatment, *P<0.05, **P<0.01, ***P<0.001. Compared with the control group after treatment, #P<0.05, ##P<0.01, ###P<0.001.

[0103] The number of patients who simultaneously completed the records of UPDRSIII score and levodopa dose equivalent at 12 and 24 weeks was 15 in study group 1, 7 in study group 2, and 8 in the control group. The changes in levodopa dose equivalent between the three groups are shown in Figure 5.

[0104] Summary: In the present invention, after treatment in test group 1, the severity of RBD of the patients was obviously improved, and the therapeutic effect was obviously higher than that of test group 2. In addition, the improving effect gradually increased with the extension of time. At the same time that RBD was improved, the motor symptoms of the patients were obviously improved, and the improvement rate tended to be obviously improved. The daily levodopa dose equivalent was obviously reduced compared to when entering the group, suggesting that the improvement of the motor symptoms was due to probiotics, not the increase in levodopa dose equivalent. Test group 1 tends to have a better therapeutic effect than test group 2.

[0105] The above clinical trial used the probiotic preparation prepared in Example 1, and the inventors were able to reach the same test conclusion by conducting tests using the probiotic preparation prepared in Example 4. Theoretically, as long as the viable cell count of the probiotic is equal to or greater than the above CFU / g, the technical effect of the present invention can be achieved, and considering the loss rate of viable cells during the storage and transportation of the probiotic, it is also feasible even if the viable cell count is slightly lower than that specified in the present invention.

[0106] Efficacy study of the present invention 2 - Cohort study of idiopathic RBD (iRBD) 1. Materials and Methods 1) Research Subjects 1.Selection criteria Subjects who meet all of the following selection criteria are eligible to participate in this study. 1) Ages 40-85, regardless of gender 2) RBD First Screening Scale (RBDSQ) RBDSQ>5 points and the Chinese University of Hong Kong REM Sleep Behavior Disorder Scale (RBD-HK) RBD-HK score ≥17 points. 3) Diagnostic criteria for iRBD: A physician from the Sleep Disorders Center in the Department of Neurology determined the diagnosis based on polysomnography (PSG) that the patient had a history of dream deduction behavior, and video polysomnography showed increased electromyographic activity or behavioral abnormalities during REM phase.

[0107] 2. Exclusion criteria 1) History of serious psychiatric illness or substance abuse, Hamilton Depression Scale score ≥ 17 and / or Anxiety Scale score ≥ 14, or treatment with oral antidepressants or antipsychotics, or treatment with oral clonazepam or other benzodiazepines and / or melatonin within the past 4 weeks.

[0108] 2) Having other central nervous system diseases including cerebrovascular disease, brain tumor, inflammation, genetic metabolic disease, brain trauma, and clear central nervous system degenerative diseases (Parkinson's disease, multiple myelopathy, dementia with Lewy bodies, etc.).

[0109] 3. Dropout, discontinuation and withdrawal plan: Patients who cannot continue to participate in this study or cannot be contacted after multiple follow-ups will be dropped out. Patients who develop any inflammation during the observation period and need to be treated with antibiotics will not be counted as participants who have discontinued the study. Patients who develop acute cardiocerebrovascular or other serious adverse events will be dropped out.

[0110] 2) Test method 1. Intervention method: The probiotic preparation obtained in Example 4 of the present invention was administered 2 tablets per day, 3 times a day, for 12 consecutive weeks.

[0111] 2. Evaluation: All patients will undergo electrocardiograms at the time of group entry and at 12 weeks, blood tests, liver and kidney function, and electrolytes tests. RBD and its severity will be evaluated using the REM Sleep Behavior Disorder Scale (RBDSQ) screening and the RBD-HK scale from the Chinese University of Hong Kong.

[0112] 3. Data statistics: Data were statistically analyzed using SPSS26.0. Metric data were expressed as mean ± standard difference (x±s), and the data from 12 weeks was used as the main statistical indicator. Paired t-test was used, and P<0.05 was considered to indicate statistical significance of the difference.

[0113] II. Test results and analysis This study was approved by the Ethics Committee of Beijing Friendship Hospital Affiliated to Capital Medical University.

[0114] Patients who visited the neurology outpatient clinic and ward of Beijing Friendship Hospital affiliated to Capital Medical University from January 2019 to January 2021 were recruited, and all patients signed informed consent.

[0115] A total of 6 iRBD patients who met the inclusion criteria and did not meet the exclusion criteria were recruited, including 5 men and 1 woman, with an average age of 64.7 ± 2.7 years. The RBDSQ scores of all patients exceeded 5 points. The RBDHK score before intervention was 41.50 ± 15.67 points. After 12 weeks of treatment, 5 patients' RBD status was obviously improved, and 1 patient remained stable, with an RBDHK score of 26.17 ± 9.99 points, which was statistically different from before intervention (P = 0.038, P < 0.05) (Figure 6).

[0116] 3. Summary After the present invention was used to treat idiopathic RBD, the RBD status of most patients (5 / 6 cases) was significantly improved compared to before treatment, and the seizure status of one case was stable, suggesting that the present invention can significantly improve the symptoms of idiopathic RBD. Idiopathic RBD is a prodromal expression of neurodegenerative diseases such as Parkinson's disease, and the present invention can improve idiopathic RBD seizures and is expected to delay the risk of iRBD patients developing neurodegenerative diseases such as PD or Lewy body dementia.

Claims

1. Use of a probiotic composition in the manufacture of a medicament for treating REM sleep behavior disorder in Parkinson's disease, comprising: The drug for treating REM sleep behavior disorder of Parkinson's disease improves motor symptoms in Parkinson's disease patients, The probiotic composition is prepared from Bacillus licheniformis, Bifidobacterium longum, Lactobacillus acidophilus, Lactobacillus faecalis and an acceptable excipient; The viable cell count of the Bacillus licheniformis was 10.0 x 10 ８ CFU / g or more, The viable cell count of the Bifidobacterium longum was 4.0 x 10 ８ CFU / g or more, The viable cell count of the Lactobacillus acidophilus is 1.0 x 10 ８ CFU / g or more, The viable cell count of the faecalis bacteria was 0.5×10 ８ CFU / g or more; 2. The use according to claim 1 ,

2. The excipients are pregelled starch and lactose.

2. The use according to claim 1 .

3. The mass ratio of the pregelled starch to the lactose is 1:

1.

3. The use according to claim 2.

4. The probiotic composition is administered in addition to conventional treatment for Parkinson's disease. The use according to any one of claims 1 to 3.

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