A synbiotic formulation for assisting in the treatment of drug-refractory epilepsy and uses thereof

Abstract

This invention belongs to the field of biomedicine, specifically relating to a synbiotic preparation for adjuvant treatment of drug-resistant epilepsy and its application. The synbiotic provided by this invention consists of probiotics and prebiotics. The probiotics include Bifidobacterium, Lactobacillus, and Enterococcus; the prebiotic components include fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, inulin, resistant dextrin, and konjac flour. This invention is the first to use the NHS3 seizure severity score as a primary efficacy indicator, filling a long-neglected dimension of seizure severity that has led to the unrecognized nature of many clinically significant partial responses.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a synbiotic preparation for adjuvant treatment of drug-resistant epilepsy and its application. Background Technology

[0002] Traditional antiepileptic drugs (such as carbamazepine, sodium valproate, and levetiracetam) have shown clear clinical efficacy in controlling seizures, but they suffer from drawbacks including single-target action (primarily acting on ion channels or GABA receptors) and a high risk of various adverse reactions (such as liver damage, hematological abnormalities, cognitive decline, behavioral disorders, osteoporosis, and teratogenic risks to the fetus). Compared to chemical drugs, synbiotics (a combination of probiotics and prebiotics) offer potential advantages in adjunctive treatment of epilepsy, including regulating gut microbiota, high safety, multi-target action, and fewer adverse reactions. Therefore, synbiotics hold promise as a beneficial supplement to traditional antiepileptic drugs, providing patients with a safer and more comprehensive adjunctive treatment option.

[0003] However, synbiotics face challenges such as low levels of clinical evidence, unclear specific effective strains and mechanisms of action, and individual variability in treatment efficacy. Therefore, conducting large-scale randomized controlled trials and identifying the optimal strain combination and pathway of action are crucial for developing synbiotic adjuvant antiepileptic agents. It is known in existing technologies that specific combinations of prebiotics can regulate the gut microbiota. For example, related studies have shown that the combination of fructooligosaccharides, xylooligosaccharides, resistant dextrin, and polydextrose can significantly promote an increase in Bifidobacterium abundance. Furthermore, the "Chinese Expert Consensus on the Clinical Application of Microecological Regulators" recommends the use of fructooligosaccharides and resistant starch to promote probiotic colonization in the gut, and recommends the use of inulin to improve glucose metabolism and increase plasma butyrate levels to exert anti-inflammatory effects. For example, publication number CN119286730A, titled "Streptococcus thermophilus AK223-6 and its application in the preparation of products for regulating the gut, improving memory, and improving epilepsy," discloses a strain of Streptococcus thermophilus AK223-6 and its postbiotic product. During fermentation, fructooligosaccharides were used as a prebiotic. This postbiotic not only increases beneficial bacteria (Bifidobacteria and Lactobacillus) and promotes human digestion and absorption, but also improves memory, alleviates epilepsy, and reduces the frequency of seizures. However, the specific mechanisms by which existing synbiotics treat epilepsy are unclear, and there is a lack of systematic reviews on the severity of epileptic seizures. In particular, the existing technology lacks evidence of synbiotics improving the clinical severity of epilepsy.

[0004] In conclusion, it is necessary to propose a novel synbiotic formulation that can effectively assist in the treatment of epilepsy in order to meet the application needs in this field. Summary of the Invention

[0005] The purpose of this invention is to provide a synbiotic preparation for adjuvant treatment of drug-resistant epilepsy and its application, which partially solves or alleviates the above-mentioned deficiencies in the prior art. The specific technical solution adopted by this invention is as follows.

[0006] The application of a probiotic and prebiotic composition in the preparation of a drug for adjuvant treatment of refractory epilepsy, wherein the probiotics include Bifidobacterium, Lactobacillus, and Enterococcus; and the prebiotic components include fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, inulin, resistant dextrin, and konjac flour. Furthermore, the prebiotic ingredients include 1-5g of fructooligosaccharides, 1-5g of galactooligosaccharides, 1-3g of xylooligosaccharides, 1-5g of inulin, 5-10g of resistant dextrin, and 1-5g of konjac powder.

[0007] As a preferred embodiment, the prebiotic ingredients include 3g of fructooligosaccharides, 5g of galactooligosaccharides, 1g of xylooligosaccharides, 2g of inulin, 7.5g of resistant dextrin, and 1.5g of konjac powder.

[0008] Furthermore, the prebiotic is used to promote the colonization of the probiotics in the intestines of patients with drug-resistant epilepsy.

[0009] Furthermore, the prebiotic is used to promote the colonization of Bifidobacteria in the intestines of patients with drug-resistant epilepsy.

[0010] Furthermore, the probiotic and prebiotic composition is used to improve the clinical severity of epileptic seizures.

[0011] Furthermore, the probiotic and prebiotic composition is used to alter the gut microbiota structure in patients with drug-resistant epilepsy.

[0012] Another aspect of the present invention provides a synbiotic formulation.

[0013] A synbiotic preparation for relieving drug-resistant epilepsy, the synbiotic preparation comprising probiotics and a prebiotic composition; wherein the probiotics include Bifidobacterium, Lactobacillus, and Enterococcus; and wherein the prebiotic components include fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, inulin, resistant dextrin, and konjac flour.

[0014] As a preferred embodiment, the prebiotic ingredients include 3g of fructooligosaccharides, 5g of galactooligosaccharides, 1g of xylooligosaccharides, 2g of inulin, 7.5g of resistant dextrin, and 1.5g of konjac powder.

[0015] Furthermore, the synbiotic preparation is an oral formulation.

[0016] Another aspect of the present invention provides an evaluation method.

[0017] An assessment method for evaluating the degree of remission of drug-resistant epilepsy by a synbiotic preparation, wherein the synbiotic preparation comprises probiotics and a prebiotic composition; the probiotics include Bifidobacterium, Lactobacillus, and Enterococcus; the prebiotic components include fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, inulin, resistant dextrin, and konjac flour; the assessment method involves performing an NHS3 score on patients after taking the synbiotic preparation.

[0018] Furthermore, the evaluation method includes setting up a drug administration group (prebiotic preparation group), control group 1 (probiotic group) and control group 2 (placebo group).

[0019] Furthermore, the assessment method includes using the NHS3 score to assess the severity of epilepsy in patients in the treatment group, control group 1, and control group 2 after the treatment period (end of week 12) and observation period (end of week 24).

[0020] Beneficial technical effects: (1) This invention is the first to use the NHS3 seizure severity score as the main efficacy indicator of seizure severity in patients with refractory epilepsy, filling the gap in the dimension of seizure severity that has been neglected for a long time, resulting in the failure to identify a large number of clinically significant partial responses.

[0021] (2) This invention is the first to simultaneously set up a synbiotic group, a probiotic-only group, and a placebo group in the same controlled trial (RCT) design. Through parallel comparison of the three groups, it was confirmed that only the synbiotic group showed significant changes in the microbial community structure, while no changes were observed in the probiotic-only group. This finding reveals the unique value of the synbiotic strategy (the synergistic effect of prebiotics on probiotic colonization) in solving the colonization bottleneck.

[0022] (3) This invention, through a large-sample, multicenter, double-blind randomized controlled trial, found that after treatment with synbiotics to promote Bifidobacterium colonization, the severity of epilepsy in patients was significantly reduced compared to the control group (poor colonization group). This clinical population result reveals the importance of adding colonization promoters (prebiotics) to probiotic treatment.

[0023] (4) In view of the current situation that existing treatments for DRE patients have limited efficacy and new drug development bottlenecks, this invention provides a safe, convenient and easy-to-promote adjuvant treatment plan, filling the gap in clinical treatment. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. The elements or parts in the drawings are not necessarily drawn to scale. Obviously, the drawings described below are some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0025] Figure 1 The NHS3 score for the severity of epileptic seizures provided in one embodiment of the present invention; Figure 2 This invention provides an analysis of differential species and KEGG functional enrichment of gut microbiota among different test groups after a 12-week treatment period, as part of one embodiment of the invention (A represents the analysis of relative average abundance differences of the same species, B represents the KEGG functional enrichment analysis of groups A and B, C represents the KEGG functional enrichment analysis of groups A and C, and D represents the KEGG functional enrichment analysis of groups B and C; * represents p<0.05; ** represents p<0.01; *** represents p<0.001). Figure 3 The following is a graph showing the results of alpha gene diversity at different stages in three experimental groups, provided for one embodiment of the present invention: (a) Group A - Synbiotic group; (b) Group B - Probiotic-only group; (c) Group C - Placebo group; Figure 4 The following is a graph (PCoA) showing the results of β-gene diversity at different stages of three experimental groups provided in one embodiment of the present invention: (A) Group A - Synbiotic group; (B) Group B - Probiotic-only group; (C) Group C - Placebo group; (DF) Comparison of diversity of the three experimental groups at the baseline period, treatment period and observation period. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0027] In this document, "and / or" includes any and all combinations of one or more of the listed related items.

[0028] In this article, "multiple" means two or more, that is, it includes two, three, four, five, etc.

[0029] As used in this specification, the term "about" typically means + / -5% of the value, more typically + / -4% of the value, more typically + / -3% of the value, more typically + / -2% of the value, even more typically + / -1% of the value, and even more typically + / -0.5% of the value.

[0030] In this specification, certain embodiments may be disclosed in a range-bound format. It should be understood that this "range-bound" description is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of a range should be considered as having specifically disclosed all possible subranges and the individual numerical values ​​within those ranges. For example, a description of the range 1-6 should be considered as having specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and the individual numbers within those ranges, such as 1, 2, 3, 4, 5, and 6. This rule applies regardless of the breadth of the range.

[0031] Definition of noun: The synbiotic described in this invention is a combination of probiotics and prebiotics.

[0032] The drug-resistant epilepsy (DRE) patients described in this invention have the following characteristics: 1) Patients with DRE who have not achieved seizure-free status after two or more standard ASMs treatments, especially those with high seizure severity and limited response to existing treatments; 2) Patients who are unsuitable for or do not accept surgical treatment, including those with unclear intracranial lesions, high surgical risks, or those who refuse surgery; 3) Epilepsy patients with evidence of gut microbiota dysbiosis, such as those with a history of antibiotic use, gastrointestinal symptoms, or abnormal gut microbiota testing; 4) Patients who wish to seek safe and low-side-effect adjunctive treatments in addition to conventional drug therapy.

[0033] Experimental materials: Probiotics (trade name: Bifidobacterium; dosage form: capsule; strength: 210mg / capsule; administration method: oral), prebiotics (dosage form: powder strip; strength: 20g / strip; administration method: oral), placebo 1 (capsule, matched with probiotics, administration method: oral) and placebo 2 (powder strip, matched with prebiotics, administration method: oral).

[0034] Prebiotic formula (20g / bag): The preferred formula is: 3g fructooligosaccharides, 5g galactooligosaccharides, 1g xylooligosaccharides, 2g inulin, 7.5g resistant dextrin, and 1.5g konjac powder.

[0035] Placebo 1: Pregelatinized starch, lactose, and purified water.

[0036] Placebo 2: 19.99 g maltodextrin powder and 0.01 g galactooligosaccharide 70 powder.

[0037] Probiotics: Bifidobacterium longum, Lactobacillus acidophilus, Enterococcus faecalis.

[0038] 2. Experimental Methods: This document provides an example design for a large-scale randomized controlled trial (RCT) to verify that synbiotic treatment promotes Bifidobacterium colonization and thus reduces the severity of seizures in patients with drug-resistant epilepsy. The RCT includes the following steps: patient recruitment, randomization, double-blinding, controlled trial, visitation, assessment, and data analysis. Details are as follows.

[0039] (1) Recruitment: A total of 248 adult patients with refractory epilepsy were recruited and informed consent was obtained. All patients or their families were informed that they should not take antibiotics or other medications at will, and that they should be informed in a timely manner if there are any special circumstances: vital signs, epileptic seizures, laboratory assessment, MMSE and MoCA scale assessment, routine electroencephalogram, electrocardiogram, and cranial MRI.

[0040] (2) Randomization: In accordance with the requirements of this trial, the subjects were divided into three groups, A, B and C, which did not differ at baseline (demographics, vital signs and epilepsy conditions such as frequency, severity and medication). They were given three treatments, namely synbiotic (Group A), probiotic alone (Group B) and placebo (Group C).

[0041] Table 1 Notes: Medication: Used / Not Used; Marital Status: Single / Married / Divorced; Education: Primary School / Junior High School / High School / University / Postgraduate.

[0042] (3) Blinding method: Use SAS9.4 software to generate blind bottoms.

[0043] Blinding includes: first unblinding blinding (specifying only group A, group B, and group C for each case) and second unblinding blinding (clarifying which experimental or control group group A, group B, and group C specifically represent).

[0044] Following the blinding procedure described above, the medications were packaged and blinded. Each subject (including the experimental and control groups) was assigned a medication bag (containing probiotics or placebo) and a medication box (containing prebiotics or placebo), containing all medications to be administered during the treatment period. The medication bags and boxes were labeled with serial numbers, and all medications inside the bags and boxes were also labeled with the same serial number, matching the subject's randomization number. The probiotic and placebo bottles were labeled with the dosage instructions (4 capsules twice daily), while the prebiotic and placebo medication boxes were labeled with the dosage instructions (1 sachet twice daily). An emergency letter was also prepared for each case, with the subject's randomization number on the envelope and a sealed letter inside indicating the case's group, for use in case of emergency unblinding. All emergency envelopes for each subject were properly stored during the trial and submitted to the clinical research unit after the study concluded.

[0045] Three copies of the blinded study plan were sealed and kept separately by an independent statistician, the clinical research unit in charge, and the National Clinical Trial Institution of the Clinical Trial Research Center, which is also the clinical lead research center. Blinding was maintained throughout the entire process, from the generation of random numbers, the numbering of the study drug, the enrollment of subjects, the recording and evaluation of study results, monitoring, data management, and statistical analysis.

[0046] (4) Controlled trial: A three-group control strategy of synbiotic, probiotic and placebo was used. The grouping strategy is sufficient to show that, based on the existing results, the colonization of Bifidobacterium can alleviate the condition of epilepsy, and the failure of colonization may lose some clinical effect.

[0047] (5) Follow-up: The trial was divided into three phases: baseline, treatment (12 weeks), and observation period (24 weeks). At baseline, it was ensured that there were no differences in clinical phenotypes or gut microbiota metagenomics among the three experimental groups. After 12 weeks of treatment, patients' seizure diaries, serum and stool samples, and scale data were collected periodically. The same procedure was followed after the observation period.

[0048] (6) Testing and evaluation: Serum and stool samples were submitted from the subjects at three different stages, and the severity of seizures at each stage was assessed by the laboratory personnel. Clinical scales, laboratory liver and kidney function tests, and adverse reaction records indicated that the three interventions were safe.

[0049] (7) Data analysis: Statistical analysis was conducted on the frequency of clinical attacks and severity scores to identify differences between groups during the stages. The actual changes in gut microbiota and corresponding metabolic changes were obtained through the detection and analysis of fecal metagenomics and their metabolites.

[0050] 3. Experimental Groups: A total of 248 patients with drug-resistant epilepsy (DRE) who underwent adjuvant therapy were randomly divided into three groups: group A was the experimental group, and groups B and C were the control groups. 1) Group A: Synbiotic group (probiotics + prebiotics), dosage: DRE patients take 4 probiotic capsules and 1 prebiotic powder orally twice a day, morning and evening.

[0051] 2) Group B: Probiotics only group (probiotics + placebo 2), dosage: DRE patients take 4 probiotic capsules and 1 placebo 2 orally twice a day, morning and evening.

[0052] 3) Group C: Placebo group (placebo 1 + placebo 2), dosage: DRE patients take one placebo 1 and one placebo 2 orally twice a day, morning and evening.

[0053] Example 1 1. NHS 3 rating This invention uses the National Hospital Seizure Severity Scale-3 (NHS3) score as the core efficacy indicator to systematically evaluate the treatment effects on epilepsy patients in three groups: A, B, and C. The scoring criteria include a range of severity-related evaluation indicators such as generalized tonic-clonic seizures, falls, injuries, urinary and fecal incontinence, loss of consciousness, recovery time, and automatisms.

[0054] After the treatment period (end of week 12) and observation period (end of week 24), the severity of epilepsy was assessed using the NHS3 score, with higher scores indicating greater severity. At the end of treatment, NHS3 scores compared to baseline were compared using the Wilcoxon test for significance between groups. The results showed that group A was significantly lower than groups B and C (P<0.05), and group B was significantly lower than group C (P<0.05).

[0055] Table 2. Tests of NHS3 total score at different time points Baseline-time point NHS3 scores were calculated at different time points, where V3 represents week 12 of the treatment period and V6 represents week 24 of the observation period.

[0056] Figure 1 shows the comparison of clinical severity scores among the three groups, specifically the changes in clinical severity score (NHS3) between the treatment and observation periods. The differences between groups were analyzed based on the GEE test, corrected for by intervention group and follow-up time, and pairwise within groups were determined using the Wilcoxon paired test. Results showed significant differences in total NHS3 scores between groups at both the end of treatment and the end of observation (A>B>C, P<0.05). This indicates that group A showed a significant improvement in clinical severity score compared to groups B and C.

[0057] 2. Analysis of differences in bacterial community abundance 2.1 Differential species analysis of gut microbiota and KEGG functional enrichment analysis After 12 weeks of treatment, feces were collected from three groups of DRE patients to perform differential species analysis of gut microbiota and KEGG functional enrichment analysis.

[0058] Experimental results: see Figure 2 In the analysis of gut microbiota differences among the test groups after the 12-week treatment period for AD, only the treatment group (synbiotic group) showed an abundance of Bifidobacterium spp. Bifidobacterium The gut microbiota, mainly composed of bacteria, underwent significant changes, while the control group showed no significant changes. Figure 2 A shows the top 10 bacterial communities with significant differences among the three groups at time point V3; Figure 2 Figure 2B shows the functional annotations of the bacterial communities in groups A and B based on KEGG enrichment analysis; Figure 2C shows the functional annotations of the bacterial communities in groups A and C based on KEGG enrichment analysis. Figure 2 D shows the functional annotations of the B and C bacterial communities based on KEGG enrichment analysis.

[0059] The above experimental results demonstrate that the combination of probiotics and prebiotics is significantly superior to the probiotic-only group and the placebo group in promoting the colonization of Bifidobacteria and enriching the core dominant bacteria, and promotes the reduction of NHS3 clinical severity scores, thereby improving the clinical phenotype of patients with drug-resistant epilepsy.

[0060] 2.2 α and β diversity See Figure 3 The treatment period and the observation period each lasted 3 months, for a total of 6 months. The data collection nodes at the end of each month were named the six stages V1-V6, with V0 being the baseline period, V3 being the end of the treatment period, and V6 being the end of the observation period. Figure 3 The alpha diversity analysis showed that only treatment group A showed a significant change in gene diversity, which decreased after treatment and rebounded after drug withdrawal, while control groups B and C showed no significant changes.

[0061] See Figure 4β diversity analysis showed that β diversity was significantly increased in the treatment group during the administration period, and the gut microbiota community structure was significantly separated from the baseline and control group; while after the drug was discontinued and the observation period began, β diversity in the treatment group began to decrease.

[0062] Changes in α and β diversity indicate that the combination of probiotics and prebiotics can induce targeted remodeling of the gut microbiota structure in DRE patients. Combined with the aforementioned reduction in NHS3 clinical severity scores, these changes in microbiota diversity and community structure are consistent with the clinical phenotype improvement in the synbiotic group, suggesting that gut microbiota remodeling may be one of the relevant mechanisms by which synbiotic intervention improves the clinical manifestations of DRE patients.

[0063] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0064] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. Use of a probiotic and prebiotic composition for the manufacture of a medicament for the adjunctive treatment of drug-resistant epilepsy, characterised in that, The probiotics include Bifidobacterium, Lactobacillus, and Enterococcus; the prebiotic components include fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, inulin, resistant dextrin, and konjac flour.

2. The application as described in claim 1, characterized in that, The prebiotic ingredients include 3g of fructooligosaccharides, 5g of galactooligosaccharides, 1g of xylooligosaccharides, 2g of inulin, 7.5g of resistant dextrin, and 1.5g of konjac powder.

3. The application as described in claim 1, characterized in that, The prebiotic is used to promote the colonization of the probiotics in the intestines of patients with drug-resistant epilepsy.

4. The application as described in claim 3, characterized in that, The prebiotic is used to promote the colonization of Bifidobacteria in the intestines of patients with drug-resistant epilepsy.

5. The application as described in claim 1, characterized in that, The probiotic and prebiotic composition is used to improve the clinical severity of epileptic seizures.

6. The application as described in claim 1, characterized in that, The probiotic and prebiotic composition is used to alter the gut microbiota structure in patients with drug-resistant epilepsy.

7. A synbiotic preparation for relieving drug-resistant epilepsy, characterized in that, The synbiotic preparation includes probiotics and a prebiotic composition; the probiotics include Bifidobacterium, Lactobacillus, and Enterococcus; the prebiotic components include fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, inulin, resistant dextrin, and konjac flour.

8. The synbiotic formulation as described in claim 7, characterized in that, The prebiotic ingredients include 3g of fructooligosaccharides, 5g of galactooligosaccharides, 1g of xylooligosaccharides, 2g of inulin, 7.5g of resistant dextrin, and 1.5g of konjac powder.

9. The synbiotic formulation as described in claim 7, characterized in that, The aforementioned synbiotic preparation is an oral formulation.

10. A method for assessing the degree of remission of drug-resistant epilepsy by a synbiotic preparation, characterized in that, The synbiotic preparation includes a combination of probiotics and prebiotics; the probiotics include Bifidobacterium, Lactobacillus, and Enterococcus; the prebiotic components include fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, inulin, resistant dextrin, and konjac flour; the assessment method is to perform NHS3 scoring on patients after taking the synbiotic preparation.

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