Phytobacterium plantarum for solving symptoms of irritable bowel syndrome

Abstract

The present disclosure provides a method of preventing, reducing, or ameliorating the symptoms of IBS in a subject in need thereof with Phytobacterium plantarum (L.plantarum). Also provided are Phytobacterium plantarum for use in the prevention, alleviation or amelioration of IBS symptoms. Also provided are compositions comprising Phytobacterium plantarum for use in the prevention, alleviation or amelioration of IBS symptoms.

Description

[0001] Field

[0002] The present disclosure generally relates to probiotics for improving symptoms of irritable bowel syndrome.

[0003] Background

[0004] Irritable bowel syndrome (IBS) is a disorder that is generally diagnosed in the absence of any specific pathogenic disease and is generally characterized by abdominal pain or discomfort and abnormal bowel frequency or stool form. IBS is further classified according to the predominant bowel habit pattern, such as diarrhea-predominant IBS (IBS-D), constipation-predominant IBS (IBS-C), and IBS with mixed bowel habits (IBS-M). A meta-analysis estimated the global prevalence of IBS to be 9.2% and 3.8% based on the Rome III and Rome IV criteria, respectively. A global study conducted by the Rome Foundation in 33 countries reported that the incidence of IBS in most countries was between 3% and 5%. IBS-D is the most common subtype, and 31.5% of IBS patients reported IBS-D, meeting the Rome IV diagnostic criteria. The pathophysiology of IBS has not been fully elucidated; however, there is evidence that changes in the gut microbiota are associated with IBS. Observational studies have shown that compared with controls, patients with IBS-D have significantly impaired health-related quality of life (QoL), more absenteeism, and a significant decrease in overall work productivity.

[0005] Management of IBS symptoms can include pharmacological, psychological, and complementary approaches. There is no single management strategy that is generally adopted for specific IBS subtypes. Recent studies have shown that agents that restore the gut microbiota may help manage IBS symptoms.

[0006] Probiotics are defined as live microorganisms that, when administered in sufficient amounts, confer health benefits on the host. For example, when administered in sufficient amounts, they can inhibit the growth of pathogenic bacteria or confer health benefits. Several probiotic strains, particularly Lactobacilli, have recently been reclassified into 25 genera, and the genus Bifidobacterium has been proposed for patients with IBS. Several mechanisms have been hypothesized to explain the effects of probiotics on gut health, including normalization of the gut microbiota, prevention of overgrowth of pathogenic bacteria, modulation of visceral afferent pathways, and enhancement of gut barrier function. However, significant heterogeneity between studies, as well as limitations in study quality, design, and efficacy, make it problematic to draw definitive conclusions. Over the past few decades, several clinical trials have been conducted to evaluate the efficacy and safety of probiotics, as is evident from many publications. However, previous reviews have shown that the results of IBS probiotic studies are conflicting due to different IBS subtypes and the use of different combinations of different probiotic strains at different doses in the studies, which may obscure the beneficial effects of individual strains within the species.

[0007] Lactiplantibacillus plantarum (recently reclassified from Lactobacillus plantarum) is a species that is frequently isolated from the healthy human gastrointestinal tract and has been studied in GI clinical trials including those for IBS. L. plantarum is a Gram-positive, catalase-negative bacterium and is a member of the large group of lactic acid bacteria. The name of this species is derived from its prevalence in naturally fermented plants, which were the primary food source long before meat and dairy became major food sources. L. plantarum differs from many other Lactobacillus species in that it has a relatively large genome, has a prominent ability to ferment a variety of different carbohydrates, has a high growth requirement for manganese, and has high tolerance to low pH, often dominating in spontaneously lactic acid-fermented foods with a pH below 4.0. L. plantarum has good acid and bile salt tolerance and has the ability to adhere to the human intestinal mucosa. It can promote gut integrity or gut motility, alter the composition of the gut microbiota, inhibit the colonization or growth of pathogenic microorganisms, and may be able to modulate immune function, thus helping to manage various diseases.

[0008] Overview

[0009] The present disclosure provides methods for preventing, alleviating or improving IBS symptoms in a subject in need thereof using Lactiplantibacillus plantarum. Also provided is the use of Lactiplantibacillus plantarum for preventing, alleviating or improving IBS symptoms. Also provided is the use of a composition comprising Lactiplantibacillus plantarum for preventing, alleviating or improving IBS symptoms. The present disclosure provides uses, methods and compositions for improving the quality of life of IBS subjects.

[0010] It has been found that the methods, uses and compositions of the present disclosure alleviate the symptoms of IBS, especially IBS-D.

[0011] Brief Description of the Drawings

[0012] Figure 1 Shows the mean ± SD of the total score of the IBS Symptom Severity Scale (IBS-SSS).

[0013] Figure 2 Shows the APS-NRS scores during the intervention.

[0014] Figure 3 Shows the percentage of responders based on the IBS-SSS.

[0015] Figure 4 Shows the percentage of responders based on stool consistency.

[0016] Figure 5 Shows the total IBS-QoL score during the intervention.

[0017] Figure 6 Shows the PSS scores during the intervention.

[0018] Detailed Description

[0019] The present disclosure provides methods for preventing, alleviating or improving IBS-D symptoms in a subject in need thereof using Lactiplantibacillus plantarum. Also provided is Lactiplantibacillus plantarum for preventing, alleviating or improving IBS-D symptoms. Also provided is a composition comprising Lactiplantibacillus plantarum for preventing, alleviating or improving IBS-D symptoms.

[0020] The preferred probiotic used herein is Lactiplantibacillus plantarum. More preferably, the probiotic comprises the Lactiplantibacillus plantarum strain Lpla33 available from Chr. Hansen A / S TM (CLEPIUS TM ). More preferably, the probiotic comprises the Lactiplantibacillus plantarum strain with accession number DSM 34687 deposited on June 28, 2023.

[0021] The growth of various Lactobacillus species to form cell cultures, cell pastes, and spore preparations is generally known in the art. Additionally, various Lactiplantibacillus plantarum products are commercially available, such as UALp-05 TM / Lpla33 TM .

[0022] As used herein, the term "composition" can be construed as, but is not limited to, a nutritional composition, a nutraceutical composition, a nutritional supplement, or a drug.

[0023] As used herein, the term "therapeutically effective amount" or "effective amount" refers to the amount of an active ingredient that elicits a biological or pharmacological response in a subject, which includes at least partially alleviating, preventing, or mitigating the symptoms of the condition being treated.

[0024] The term "irritable bowel syndrome" (IBS) refers to the occurrence of one or more of a group of symptoms, particularly recurrent abdominal pain and changes in bowel habits, which may be diarrhea, constipation, or both. IBS is part of a group of disorders called functional gastrointestinal disorders (more recently called gut-brain interaction disorders). These disorders are all characterized by chronic or recurrent gastrointestinal symptoms for which no structural or biochemical cause can be found.

[0025] According to an embodiment, the Lactiplantibacillus plantarum of the present disclosure can be administered in solid, semi-solid, or liquid oral dosage forms. In one embodiment of the present disclosure, the pharmaceutical preparation can be in the form of an emulsion, a solution, a suspension, a syrup, an elixir, a tablet, a capsule, a pill, a granule, and a suppository. In another embodiment of the present disclosure, the pharmaceutical preparation can be in the form of a water-dispersible granule (WG), a suspension concentrate (SC), a wettable powder (WP), an emulsifiable concentrate (EC), a granule, a gel, a suspo-emulsion (SE), a capsule suspension, and a mixed preparation of a suspension (ZC), etc., and preferably a water-dispersible granule (WG), a suspo-emulsion (SE), and a mixed preparation of a capsule suspension and a suspension (ZC). In one embodiment, the Lactiplantibacillus plantarum of the present disclosure can be dried. The drying can include spray drying, fluidized bed drying, or freeze drying. In one embodiment, the Lactiplantibacillus plantarum of the present disclosure is in an oral dosage form of a sachet powder or granule, a liquid, a solution, a suspension, an emulsion, or a syrup.

[0026] In another embodiment of the present disclosure, the Lactiplantibacillus plantarum composition may include at least one pharmaceutically acceptable excipient selected from the group consisting of fillers, binders, diluents, thickeners, solvents, coating agents, dispersants, preservatives, sweeteners, flavoring agents, defoaming agents, and stabilizers. Pharmaceutically acceptable fillers may be selected from the group consisting of lactose, microcrystalline cellulose, starch, pregelatinized starch, calcium phosphate, calcium sulfate, calcium carbonate, mannitol, sorbitol, xylitol, sucrose, maltose, fructose, glucose, maltodextrin, and the like. Pharmaceutically acceptable binders may be selected from the group consisting of starch, natural sugars, corn sweeteners, natural and synthetic gums, cellulose derivatives, gelatin, povidone, polyethylene glycol, waxes, sodium alginate, alcohols, water, and the like. Pharmaceutically acceptable diluents may be selected from the group consisting of calcium carbonate, calcium hydrogen phosphate, tricalcium phosphate, calcium sulfate, microcrystalline cellulose, microcrystalline siliconized cellulose, powdered cellulose, dextrate, dextrose, fructose, lactitol, anhydrous lactose, lactose monohydrate, lactose dihydrate, lactose trihydrate, mannitol, sorbitol, starch, pregelatinized starch, sucrose, talc, xylitol, maltose, maltodextrin, maltitol, and the like. Pharmaceutically acceptable sweeteners may be selected from the group consisting of alitame, acesulfame potassium, aspartame, D-tryptophan, dextrose, erythritol, fructose, galactose, glycerol, glycyrrhizin, glucose, isomaltitol, xylitol, xylose, lactitol, lactose, levulose, maltitol, maltodextrin, maltol, maltose, mannitol, corn syrup, neohesperidin dihydrochalcone, neotame, saccharin, cyclamate, sorbitol, sucralose, sucrose, tagatose, thaumatin, trehalose, and the like. Pharmaceutically acceptable flavoring agents may be selected from the group consisting of natural flavor oils, anethole, acetic acid, ascorbic acid, phosphoric acid, fumaric acid, lactic acid, lemon, linalool, malic acid, menthol, eucalyptol, citrus, citric acid, cinnamone, tartaric acid, thymol, vanilla, strawberry, and the like. Pharmaceutically acceptable preservatives may be selected from the group consisting of parabens, phenol, chlorocresol, alkyl p-hydroxybenzoates, benzoic acid and its salts, boric acid and its salts, citric acid and its salts, sorbic acid and its salts, neutral preservatives, mercury preservatives, quaternary compounds, and the like.

[0027] In one embodiment, the composition of the present invention may include not less than about 1 billion, about 2 billion, about 3 billion, about 4 billion, about 5 billion, about 6 billion, about 7 billion, about 8 billion, about 9 billion, about 10 billion cells (colony forming units or CFU) of Lactiplantibacillus plantarum.

[0028] In one embodiment, the daily dose of Lactiplantibacillus plantarum is not less than about 1 billion, about 2 billion, about 3 billion, about 4 billion, about 5 billion, about 6 billion, about 7 billion, about 8 billion, about 9 billion, about 10 billion cells (colony forming units or CFU).

[0029] DSM 34687 is the preferred Lactiplantibacillus plantarum for use herein. The microorganism can be deposited with a depository institution having the status of an international depository authority under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure: DSMZ - Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Inhoffenstr. 7B, 38124 Braunschweig, Germany. The applicant requests that, in accordance with the current regulations of the industrial property offices of the Contracting States to the Budapest Treaty, samples of the deposited microorganism can only be made available to experts before the grant of a patent right. Examples

[0030] Example 1

[0031] A randomized, double - blind, placebo - controlled, multi - center study was conducted in adults aged 18 to 70 years to evaluate the safety and efficacy of Lactiplantibacillus plantarum Lpla33 TM in patients with diarrhea - predominant irritable bowel syndrome (IBS). Approximately 450 potential participants were screened, and taking into account a 20% screening failure rate, up to 360 participants were recruited. The recruited participants were then randomly assigned to 3 groups in a 1:1:1 ratio (Lactiplantibacillus plantarum Lpla33 TM [not less than (NLT) 1 billion CFU / day], Lactiplantibacillus plantarum Lpla33 TM [NLT 10 billion CFU / day] and placebo).

[0032] The EMA and USFDA have provided detailed guidelines for evaluating drugs for the treatment of IBS to achieve consistency in study design and methods. The standards set by the regulatory agencies also apply to probiotics, which are increasingly considered a promising option for IBS management. The regulatory agencies have emphasized that the primary goal of IBS management is to relieve the most bothersome symptoms of the disease; therefore, the primary efficacy endpoint of IBS trials should be the improvement in the severity of these IBS symptoms. We have adopted this as the primary objective of this study. We included the IBS Severity Scoring System (IBS-SSS) (a widely validated patient-reported composite outcome assessment tool) for its assessment, while the Abdominal Pain Severity - Numerical Rating Scale (APS-NRS) was used to determine the severity of abdominal pain / discomfort. In addition, responder definitions for different outcome measures have been recommended, which are the secondary endpoints of this study. According to the USFDA's recommendations, a two-week screening period was included in this study to confirm the diagnosis. Several studies have shown that patients with IBS have a lower health-related quality of life, especially in the diarrhea-predominant subtype, for which the quality of life of study participants needs to be measured. Therefore, we included the IBS Quality of Life (IBS-QoL) and Perceived Stress Scale (PSS) in the secondary objectives of the study.

[0033] At the first visit to the center, the study design was fully explained to eligible study participants in an easy-to-understand language, after which a formally signed and dated informed consent form from the study participants and a consent form for microbiome analysis (using stool specimens) were obtained. The medical history and past / current medications were recorded. After recording the demographic details (age, gender, and ethnicity) and anthropometrics (height, weight, and BMI), a clinical examination was performed, along with the measurement of vital signs (blood pressure and pulse rate), body temperature, and SpO2 levels. All results were recorded in the source document (SD). For women of childbearing age, a urine pregnancy test was performed. Participants were asked to self-administer the APS-NRS and the Bristol Stool Form Scale (BSS) separately before screening, with recall periods of the last three months and one month, respectively. The investigator evaluated the participants for the full inclusion and exclusion criteria. Participants who met the inclusion and exclusion criteria were considered for the run-in period. A stool collection kit and electronic diary login credentials were provided to all eligible participants. Participants were provided with instructions to submit a morning stool sample on Day 0 and to fill in the electronic diary every day. The fasting blood glucose (FBG), hemoglobin (Hb), and thyroid-stimulating hormone (TSH) of the participants were evaluated by an accredited laboratory. Placebo and rescue medications (RM) were distributed to participants in the placebo run-in period. Participant diaries (for diet recall) were distributed, and clear instructions for filling in these diaries were provided by the investigator or CRC.

[0034] Participants took placebo daily before lunch for two weeks. They filled out an electronic diary for APS-NRS on Day 7 and Day 14 after the screening visit, with a recall period of 7 days, and their compliance was checked by the Clinical Research Coordinator (CRC) or any other designated person. After screening, for the next 14 days, participants filled out the BSS self-administered using an electronic diary every evening. Compliance was checked by the CRC or any other designated person.

[0035] Day 0

[0036] The placebo and diet diary were reconciled, and compliance was determined. The RM was reconciled and its use (if any) was recorded. After measuring body weight, a clinical examination was conducted, followed by the assessment and recording of vital signs (blood pressure and pulse rate), body temperature, and SpO2 level. Dietary recall was performed using the diet diary to determine the average calorie intake (% protein, % fat, % carbohydrate, and % fiber intake) from any 3 days (including weekends) of the screening visit. Participants filled out the IBS-SSS, IBS-QoL, and PSS. Participants were re-evaluated according to the randomization criteria, and eligible participants were randomly assigned. A urine pregnancy test was performed for women of childbearing age. All randomized participants filled out the APS-NRS and BSS (electronic diary) with a recall period of 7 days at Week 1, Week 2, Week 3, and Week 4. The CRC regularly checked their compliance. The study product (Investigational Product (IP) or placebo) and RM were assigned according to the randomization chart and protocol instructions. Participants filled out the diet diary (for dietary recall) every 14 days to record the 24-hour dietary intake for two working days and one weekend. The first stool sample of the day (Day 0) was collected from participants who consented to microbiome analysis.

[0037] Day 28(+ / -2)

[0038] Verify the study medication and dietary recall forms. The investigator or CRC determined the compliance with the study product. The investigator monitored and reported any AEs or SAEs that occurred. Verify the RM and record its use (if any). Conduct urine pregnancy tests on all women of childbearing age. Record concomitant medications (if any) in the SD. After measuring body weight, evaluate the clinical examination and vital signs (blood pressure and pulse rate), body temperature, and SpO2 levels and record them on the SD. Use a dietary diary for dietary recall to estimate the average calorie intake (% protein, % fat, % carbohydrate, and % fiber intake) for any 3 days (including weekends) during each 14-day period in the past 28 days. The participants self-administered the IBS-SSS, IBS-QoL, and PSS questionnaires. The participants self-administered the APS-NRS and BSS (electronic diary) at weeks 5, 6, 7, and 8 with a recall period of 7 days. The CRC regularly checked their compliance. The study product (IP or placebo) and RM were assigned according to the randomization chart and instructions on the protocol were provided. Stool collection kits were distributed and the participants were given instructions to bring a morning stool sample on day 56. Dietary diaries (for dietary recall) were distributed and the investigator or CRC provided clear instructions for filling out these diaries.

[0039] Day 56(+ / -2)

[0040] Verify the study medication and dietary diary. The investigator or CRC determined the compliance with the study product. The investigator monitored and reported any AEs or SAEs that occurred. Verify the RM and record its use (if any). Conduct urine pregnancy tests on all women of childbearing age. Record concomitant medications (if any) in the SD. After measuring body weight, conduct a clinical examination while measuring the vital signs (blood pressure and pulse rate), body temperature, and SpO2 levels. All results were recorded on the SD. The participants were required to self-administer the APS-NRS, IBS-SSS, BSS, IBS-QoL, and PSS and instructions for completing the above questionnaires were provided. Use a dietary diary for dietary recall to estimate the average calorie intake (% protein, % fat, % carbohydrate, and % fiber intake) for any 3 days (including weekends) during each 14-day period in the past 28 days. Collect the first stool sample of the day (day 56) from the participants for microbiome analysis.

[0041] Chr. Hansen (Wausau) provided the investigational products used in the study. The production of the products complied with all required regulations and the CRO provided a sufficient quantity of the products to the clinical research center. The following table provides the detailed information on the investigational products. To ensure the same total capsule weight, the content of microcrystalline cellulose varied slightly among the groups. Microcrystalline cellulose is a commonly used excipient. Chemically, it is an inert substance that does not degrade during digestion and has no significant absorption. Therefore, the slight differences in quantity among the groups will not affect the study results.

[0042] Detailed Information of the Test Products

[0043]

[0044] In the random visits, eligible participants were evenly distributed among three groups - the low-dose probiotic group, the high-dose probiotic group, and the control group, as described below. The study products were provided to the participants according to the randomization chart.

[0045] Group Interventions, Protocols, and Intervention Durations

[0046]

[0047]

[0048] In this study, participants were provided with Lactiplantibacillus plantarum Lpla33 capsules containing 1 billion or 10 billion colony-forming units (CFU) per capsule or placebo. In a published study, a dose of 10 billion CFU / capsule of Lactiplantibacillus plantarum was administered to 108 participants, and it was reported to be safe and effective after 4 weeks of ingestion. The study reported one single adverse event (transient vertigo) and no changes in blood parameters were detected. In another clinical trial, Lactiplantibacillus plantarum was safe at a dose of 10 billion CFU / day for 4 weeks. According to a meta-analysis of IBS studies, a daily dose of 10 billion CFU / day was considered a midpoint range. There are other references regarding the safety profile of Lactiplantibacillus plantarum. Current studies on Lactiplantibacillus plantarum Lpla33 include a lower dose of 1 billion CFU / day for 8 weeks (considered a low dose in probiotic IBS studies) and a higher dose of 10 billion CFU / day (considered a midpoint dose in probiotic IBS studies). Each probiotic dose group was evaluated separately compared to the placebo group.

[0049] Irritable Bowel Syndrome - Symptom Severity

[0050] Study participants were required to answer each question on a 100-point visual analog scale of the IBS Symptom Severity Scale. The IBS-SSS score ranges from 0 to 500, and a higher score indicates more severe symptoms. If necessary, participants could be classified as having mild (75 - 175), moderate (175 - 300), or severe (>300) IBS. A reduction of 95 points is associated with a clinically meaningful improvement.

[0051] Assessment method: The IBS-SSS questionnaire is available on the electronic diary. Participants filled out the questionnaire themselves using the electronic diary on Day 0, Day 28, and Day 56, with a recall period of 10 days. According to the set randomization criteria, participants with an IBS-SSS total score > 175 on Day 0 were randomly grouped. Compared with the placebo, the score at the Day 56 visit was evaluated against the baseline score.

[0052] Except on Day 56, the change in IBS-SSS from baseline was evaluated on Day 28 compared with the placebo.

[0053] Assessment method: Under the supervision of the investigator or CRC, study participants filled out the questionnaire themselves using the electronic diary at baseline (Day 0) and the Day 28 visit, with a recall period of 10 days. Compared with the placebo, the score at the Day 28 visit was evaluated against the baseline score.

[0054] The severity of abdominal pain was evaluated using the 11-point APS NRS, where 10 represents the most severe pain and 0 represents no pain, as specified: "How much abdominal pain did you feel last week, on a scale of 0 (none) to 10 (very severe)." The APS-NRS score categories include 0 - none, 1 to 3 - mild, 4 to 6 - moderate, 7 to 9 - severe, and 10 = very severe. The NRS has been well psychometrically tested in IBS patients, showing appropriate changes in response to treatment and being closely related to the severity of overall GI symptoms. The FDA also recommends using it as a provisional endpoint for assessing treatment benefits in IBS clinical trials. A change in abdominal pain improvement of at least 30% compared with the baseline was considered a clinically significant change.

[0055] At the screening visit, participants were asked to rate the severity of abdominal pain in the past 3 months before the screening visit using the APS-NRS. Login credentials for the electronic diary (including the APS-NRS) were provided to eligible participants. After screening, individuals were asked to rate the intensity of abdominal pain in the past 7 days every weekend (using the electronic diary). The average of 2 weeks was considered the final pain score and was used to evaluate participants according to the set randomization criteria. If a participant met the randomization criteria, he / she was instructed to continuously rate the APS-NRS at the end of Week 1, Week 2, Week 3, Week 4, Week 5, Week 6, Week 7, and Week 8 (end of the study visit), with a recall period of 7 days. For the Day 28 visit, the average of Weeks 1 to 4 was considered; similarly, the score for Day 56 was the average of Weeks 5 to 8. The APS-NRS scores from baseline to the end of Day 28 and Day 56 were compared to evaluate the efficacy of the IP compared with the placebo. The window period for self-administered APS-NRS was +1 day.

[0056] Note: At least three responses between two consecutive visits were considered mandatory.

[0057] Most IBS patients (77.7%) had both loose / watery stools and hard / lumpy stools, and they fluctuated on average three times per month between these two extremes. These frequent fluctuations in stool consistency occurred naturally across all IBS subtypes and were not due to the use of laxatives or antidiarrheal medications. IBS presents as abdominal pain or discomfort that is relieved by defecation or is associated with a change in the frequency of stools (increase or decrease) or the appearance of stools (loose or hard) at the time of onset. Stool consistency is evaluated using the Bristol Stool Scale, a validated ordinal scale of stool types that ranges from 1 to 7 and includes types 1-2 and 6-7, which are combined with other symptoms of constipation and diarrhea, respectively. Types 3 to 5 are generally considered the most common stool forms and are typical in cross-sectional surveys of healthy adults.

[0058] Assessment method: During the screening visit, participants were asked to rate their stool consistency for the past 1 month before screening using the BSS. They rated their stool consistency as follows: 1 = separate hard lumps, like nuts, difficult to pass; 2 = sausage-shaped, but lumpy; 3 = sausage-shaped, but with cracks on the surface; 4 = like a sausage or snake, smooth and soft; 5 = soft blobs with clear edges; 6 = fluffy blobs with ragged edges, mushy stools; and 7 = watery, no solid pieces, entirely liquid. Eligible participants were provided with login credentials for an electronic diary (which provided the BSS). After screening, individuals were asked to rate the stool consistency of their abnormal bowel activity every evening for the next 14 days (lead-in period). Then, on Day 0, participants were evaluated according to the set randomization criteria, and if he / she met the randomization criteria, they were enrolled in the study.

[0059] On Day 0, participants were instructed to rate their stool consistency daily using the BSS in the electronic diary each week. Weekly BSS scores were evaluated to assess changes in stool consistency toward normal, but not the frequency of stools, as the former is related to colonic transit time and is considered a better indicator of bowel function. At the end of Day 28 and Day 56, the percentage of participants who had transitioned to normal stool consistency compared with their baseline (Day 0) BSS scores was determined to evaluate the efficacy of IP compared with placebo. The window period for self-administered BSS was +1 day. Note: At least three responses were mandatory between two consecutive visits.

[0060] In this study, responders were defined as study participants whose overall symptom severity on the IBS-SSS improved by 95 points when compared from baseline (Day 0) to Day 28 and Day 56.

[0061] The USFDA guidelines recommend evaluating the response to interventions for IBS based on weekly responders (defined as the number of days with at least one stool of Bristol type 6 or 7 per week decreased by 50% compared to baseline). In this study, we also used this definition to evaluate the response to interventions.

[0062] Although IBS is not life-threatening, it is associated with a reduced life expectancy and has a significant impact on an individual's life. Previous studies have confirmed that patients with IBS have a higher prevalence of depression and a lower quality of life than their healthy peers. In this study, the IBS-related quality of life (QoL) was assessed by the IBS-QoL, a 34-item questionnaire rated on a 5-point scale (range: 34 - 170), with higher scores indicating a worse quality of life. It measures 8 domains relevant to patients with IBS: restlessness, activity interference, body image, health concerns, food avoidance, social responses, sexuality, and relationship problems. The IBS-QOL has high internal consistency and high reproducibility. The IBS-QoL is sensitive to treatment response in patients with IBS. Each item is rated on a 5-point Likert scale (Items 1, 2, 4, 8 - 10, 12, 13, 16, 25 - 29, 34 are rated as "1 - not at all; 2 - slightly; 3 - moderately; 4 - quite a bit; 5 - very severely" & Items 3, 5 - 7, 11, 14, 15, 17 - 24, 30 - 33 are rated as "1 - not at all; 2 - slightly; 3 - moderately; 4 - quite a bit; 5 - a lot"). There is a 30-day recall period for filling out this questionnaire. Individual responses to the 34 items are summed and converted to a total score of 0 - 100, with higher scores indicating an increase in IBS-specific QoL. An increase of 10 points or more is considered a clinically significant improvement.

[0063] Assessment method: Study participants self-administered a digital questionnaire at baseline, day 28, and day 56 visits.

[0064] There is an increasing recognition that bidirectional gut-brain axis dysregulation is a conceptual model for the pathophysiology of IBS. It involves functional abnormalities in the enteric nervous system, the autonomic nervous system, and / or the central nervous system. Studies have shown that stress can lead to overactivity or underactivity of the hypothalamic-pituitary-adrenal axis as well as the autonomic, metabolic, and immune systems. It can also alter gut-brain interactions and ultimately affect different physiological functions of the gastrointestinal tract. On this basis, this study included the Perceived Stress Scale (PSS), a widely used psychological tool for measuring the degree to which an individual's life situation is assessed as being stressful. The PSS items were specifically designed to understand how unpredictable, uncontrollable, and overloaded the individuals being evaluated felt their daily lives were affected by stress. The reliability and reproducibility of this tool have been validated in various clinical contexts. The PSS consists of 10 questions regarding the stress levels experienced in the past 30 days, and the responses are recorded on a 4-point Likert scale (0 = never, 1 = almost never, 2 = sometimes, 3 = often, 4 = always). The PSS score is obtained by reversing the responses to four positively worded items (items 4, 5, 7, and 8) (e.g., 0 = 4, 1 = 3, 2 = 2, 3 = 1, and 4 = 0) and then summing all the scale items. Higher scores indicate higher stress levels, and the scores range from 0 to 40.

[0065] Assessment method: The study participants self-administered the digital PSS at baseline (day 0), day 28, and day 56.

[0066] Results

[0067] The baseline mean total IBS-SSS scores of the three study groups were comparable. After 56 days of intervention, the high-dose Lactiplantibacillus plantarum (10 billion CFU) group reported the largest decrease in the total score from the baseline value (-51.76%), followed by the low-dose Lactiplantibacillus plantarum (1 billion CFU) group (-40.66%). The placebo group reported the least decrease in the mean total IBS-SSS score from the baseline (-19.05%), less than half of the magnitude observed in the low-dose and high-dose probiotic groups. A statistically significant decrease in the IBS-SSS scores from the baseline was observed in all groups (p < 0.05). In addition, the effects of the two Lactiplantibacillus plantarum doses on the total IBS-SSS score were statistically significant compared with the placebo (p < 0.05); however, the effect size of the high-dose Lactiplantibacillus plantarum was 1.5 times higher than that of the low-dose group, which was statistically significant (p = 0.0298).

[0068] Summary of the total IBS-SSS scores and changes from the baseline on day 56

[0069] Note:

[0070] [1] Calculate the p-value using ANCOVA with treatment as the factor and baseline as the covariate.

[0071] [2] Calculate the p-value using a paired t-test.

[0072] [3] Calculate the p-value using ANCOVA, adjusted with Dunnett's test, with treatment as the factor and baseline as the covariate, compared to the placebo.

[0073] [4] Calculate the p-value using a two-sample t-test between 1 billion CFU and 10 billion CFU.

[0074] Abbreviations: N = number of participants; SD = standard deviation

[0075] At day 28, the mean total IBS-SSS score of the three study groups was significantly decreased compared to the baseline score ( Figure 1 ). The decrease in the high-dose Lactiplantibacillus plantarum group was the highest compared to the baseline score, followed by the low-dose group. The placebo group reported the least decrease in the total IBS-SSS score compared to the baseline, but it was statistically significant. The difference between the three groups was also statistically significant. The effect size of the low-dose group was half of that of the high-dose group; however, the difference was not statistically significant (p = 0.0956).

[0076] Summary of the total IBS-SSS score and change from baseline at day 28

[0077] Note:

[0078] [1] Calculate the p-value using ANCOVA with treatment as the factor and baseline as the covariate.

[0079] [2] Calculate the p-value using a paired t-test.

[0080] [3] Calculate the p-value using ANCOVA, adjusted with Dunnett's test, with treatment as the factor and baseline as the covariate, compared to the placebo.

[0081] [4] Calculate the p-value using a two-sample t-test between 1 billion CFU and 10 billion CFU.

[0082] Abbreviations: CI = confidence interval, N = number of participants SD = standard deviation.

[0083] The mean baseline APS-NRS scores of the three groups were similar and ranged from 5.00 to 9.50.

[0084] At day 56, the mean APS-NRS scores of all three groups were decreased compared to the baseline, and the decrease was statistically significant. See Figure 2It was reported that the highest reduction (-33.33%) was observed in the high-dose Lactiplantibacillus plantarum group compared to the baseline, and the lowest (-13.17%) was in the placebo group. Statistically significant differences were observed among the three groups. The effect size (-1.44) in the high-dose group was significantly greater than that in the low-dose Lpla33 group (-0.89) (p = 0.0057).

[0085] After 28 days of intervention, the average baseline score in the low-dose group decreased by 12.18%, which was similar to the decrease (-12.83%) observed in the high-dose Lpla33 group (p = 0.7089). The placebo group had the least decrease in the average APS-NRS score (-8.54%). In all three groups, the decrease in the average APS-NRS score compared to the baseline was statistically significant; however, the differences among the three groups were not significant.

[0086] Summary of APS-NRS scores

[0087]

[0088] Abbreviations: CI = confidence interval, N = number of participants, SD = standard deviation

[0089] Changes in APS-NRS scores

[0090]

[0091] Note:

[0092] [1] The p-value was calculated using ANCOVA with treatment as the factor and baseline as the covariate.

[0093] [2] The p-value was calculated using a paired t-test.

[0094] [3] The p-value was calculated using ANCOVA, adjusted using the Dunnett test, with treatment as the factor and baseline as the covariate, compared to the placebo.

[0095] [4] The p-value was calculated using a two-sample t-test between 1 billion CFU and 10 billion CFU.

[0096] Abbreviations: CI = confidence interval, N = number of participants, SD = standard deviation

[0097] For this study, responders were defined as study participants with a reduction of 95 points in the overall symptom severity of IBS-SSS on day 56 compared to baseline (day 0). The following table shows the number and percentage of responders on day 28 and day 56. The largest number of responders was observed in the high-dose Lactiplantibacillus plantarum group on day 28 and day 56. At the end of the study, 72.63% of the participants responded to the higher dose of probiotics, while 59.62% of the participants in the low-dose Lactiplantibacillus plantarum group responded. The difference between the groups was statistically significant. In contrast, only 26.26% of the participants in the placebo group responded to the intervention. On day 28 and day 56, the three groups differed significantly in their response to the intervention (p<0.05)( Figure 3 ).

[0098] Percentage of responders based on improvement in total IBS-SSS score

[0099]

[0100] Note: [1] The p-values for the three groups were calculated using the chi-square test.

[0101] [2] The p-value was calculated using the chi-square test between 1 billion CFU and 10 billion CFU.

[0102] [3] The p-value was calculated using the chi-square test between 1 billion CFU and the placebo.

[0103] [4] The p-value was calculated using the chi-square test between 10 billion CFU and the placebo.

[0104] Abbreviation: N = number of participants

[0105] Based on stool consistency, weekly responders were defined as having at least one day with a consistency of type 6 or 7 in the stool per week, with a 50% reduction compared to baseline. According to this definition, compared to the placebo group (day 28: 17.17% and day 56: 26.26%), the high-dose Lactiplantibacillus plantarum group reported the most responders on day 28 (48.42%) and day 56 (88.42%), followed by the low-dose Lactiplantibacillus plantarum group (day 28: 30.77% and day 56: 62.50%) (p<0.05). The three groups differed significantly in weekly stool consistency responders. In addition, a significant difference was observed between the two Lactiplantibacillus plantarum groups. See Figure 4 .

[0106] Percentage of responders based on stool consistency (BSS type 6 and 7)

[0107]

[0108] Note: [1] The p-values for the three groups were calculated using the chi-square test.

[0109] [2] The p-value was calculated using the chi-square test between 1 billion CFU and 10 billion CFU.

[0110] [3] The p-value was calculated using the chi-square test between 1 billion CFU and placebo.

[0111] [4] The p-value was calculated using the chi-square test between 10 billion CFU and placebo.

[0112] Abbreviation: N = number of participants

[0113] The mean baseline total IBS-QoL scores of the three groups were similar ( Figure 5 ). On day 56, a statistically significant improvement (increase) in the mean total IBS-QoL score compared to the baseline score was observed in all three groups. The between-group comparison of the mean total IBS-QoL score on day 56 also showed a statistically significant difference. The high-dose Lactiplantibacillus plantarum group and the low-dose Lactiplantibacillus plantarum group reported greater improvements compared to placebo (p < 0.05). The effect size of the high-dose Lactiplantibacillus plantarum group (24.5741) was significantly greater than that of the low-dose group (13.40) (p < 0.05).

[0114] On day 28, a statistically significant improvement in the mean total IBS-QoL score of the three groups compared to the baseline score was observed (p < 0.05). Greater improvements were observed in the high-dose (14.09) Lactiplantibacillus plantarum group and the low-dose (5.40) Lactiplantibacillus plantarum group compared to placebo (p < 0.05). In addition, the difference between the two Lactiplantibacillus plantarum dose groups was statistically significant (p = 0.0009).

[0115] Summary of the total IBS Quality of Life (QoL) score

[0116]

[0117] Abbreviation: CI = confidence interval, SD = standard deviation, N = number of participants

[0118] Change in the total IBS Quality of Life score

[0119]

[0120] Note:

[0121] [1] The p-value was calculated using ANCOVA with treatment as the factor and baseline as the covariate.

[0122] [2] The p-value was calculated using the paired t-test.

[0123] [3] The p-value was calculated using ANCOVA and adjusted using Dunnett's test, with treatment as the factor and baseline as the covariate, compared to the placebo.

[0124] [4] The p-value was calculated using a two-sample t-test between Lpla33 - 1 billion CFU and Lpla33 - 10 billion CFU.

[0125] Abbreviations: CI = confidence interval, N = number of participants, SD = standard deviation

[0126] These three groups had similar baseline mean PSS scores ( Figure 6 ). Compared to the baseline, a statistically significant decrease in the mean PSS score was observed in both Lactiplantibacillus groups. In contrast, the decrease in the placebo group was not significant at day 28 or day 56 (p > 0.05). Compared to the placebo, both doses of Lactiplantibacillus showed a significant decrease in the PSS score (p < 0.05). The effect size (-5.24) in the high-dose Lactiplantibacillus group was greater than that in the low-dose group (-3.55); however, the difference between the two dose groups was not statistically significant (p = 0.1708).

[0127] Summary of PSS scores

[0128]

[0129] Change in PSS scores

[0130]

[0131] Note:

[0132] [1] The p-value was calculated using ANCOVA with treatment as the factor and baseline as the covariate.

[0133] [2] The p-value was calculated using a paired t-test.

[0134] [3] The p-value was calculated using ANCOVA and adjusted using Dunnett's test, with treatment as the factor and baseline as the covariate, compared to the placebo.

[0135] [4] The p-value was calculated using a two-sample t-test between Lpla33 - 1 billion CFU and Lpla33 - 10 billion CFU.

[0136] Abbreviations: CI = confidence interval, N = number of participants, SD = standard deviation

[0137] This randomized placebo-controlled parallel-group trial was conducted to investigate the efficacy of supplementing two different doses of Lactiplantibacillus plantarum [10 billion CFU / day and 100 billion CFU / day] for 56 days on IBS symptoms compared with placebo. The trial achieved its primary and secondary objectives and demonstrated that at the end of day 56, both the high-dose and low-dose Lactiplantibacillus plantarum significantly reduced the IBS symptom severity score, more than twice the extent observed in the placebo group. The positive effects in both treatment groups were also confirmed at day 28, as the symptom severity score was reduced to a statistically significant extent compared with baseline and the placebo group. The reduction in the IBS-SSS score in both probiotic groups exceeded the previously reported criterion for a clinically meaningful response, i.e., a reduction of 95 points. In addition, the high-dose Lactiplantibacillus plantarum showed a greater effect size in reducing the IBS symptom severity compared with the low-dose Lactiplantibacillus plantarum.

[0138] Similarly, supplementing Lactiplantibacillus plantarum at days 28 and 56 also significantly alleviated abdominal pain compared with baseline levels and the placebo group. In addition, the higher dose of Lactiplantibacillus plantarum showed a greater effect size in reducing the APS-NRS score. The USFDA has defined an improvement in abdominal pain of at least 30% compared with baseline as clinically meaningful, which was achieved by administering Lactiplantibacillus plantarum at a dose of 100 billion CFU / day.

[0139] Responder analysis is one of the secondary outcomes of IBS trials recommended by the USFDA. Responder analysis was conducted using two different criteria. First, the IBS-SSS score improved by 95 points compared with baseline, and second, the number of days with at least one stool of consistency type 6 or 7 per week was reduced by 50% compared with baseline. At the end of the study, according to the symptom severity criterion, 72.63% of the participants in the high-dose Lactiplantibacillus plantarum group responded, followed by 59.62% in the low-dose group and 26.26% in the placebo group. According to the stool consistency criterion, after day 56, the percentages of responders in the high-dose Lactiplantibacillus plantarum group (88%) and the low-dose Lactiplantibacillus plantarum group (63%) were significantly higher than that in the placebo group (26%).

[0140] It has been determined that IBS significantly affects the quality of life of affected individuals, so it is prudent to evaluate the effectiveness of the investigational product in improving quality of life. The IBS QoL is a validated questionnaire specifically designed to evaluate the quality of life of patients with IBS. In this study, the QoL of the participants was evaluated, and a statistically significant improvement in scores was observed in both Lactiplantibacillus plantarum groups at Day 28 and Day 56 compared to the baseline and placebo groups. The IBS-QoL scores in both groups were significantly improved to a clinically meaningful level (i.e., 10 points or more). In addition, the effects of the two Lactiplantibacillus plantarum doses on stress-related symptoms were evaluated using the Perceived Stress Scale, and both doses were observed to be effective in alleviating stress in the participants.

[0141] The results showed that compared with the placebo, the Lactiplantibacillus plantarum doses of 1 billion CFU / day or 10 billion CFU / day significantly improved both the primary and secondary study endpoints, and a greater effect size was observed with the 10 billion CFU / day dose. This study was well-powered and incorporated important recommendations put forward by the USFDA and other experts for randomized controlled trials of IBS.

Claims

1. Use of Lactiplantibacillus plantarum DSM 34687 (Lpla33 TM ) for alleviating or preventing IBS-D symptoms in a subject.

2. The use according to claim 1, wherein the dosage of Lactiplantibacillus plantarum is at least about 1 billion CFU per day.

3. The use according to claim 1, wherein the dosage of Lactiplantibacillus plantarum is at least about 10 billion CFU per day.

4. The use according to claims 1 - 3, wherein the Lactiplantibacillus plantarum is administered for at least 28 days.

5. The use according to claims 1 - 3, wherein the Lactiplantibacillus plantarum is administered for at least 56 days.

6. A composition comprising Lactiplantibacillus plantarum DSM for alleviating or preventing IBS - D symptoms 34687 (Lpla33 TM ) composition 7. The composition according to claim 6, wherein the composition comprises at least about 1 billion CFU of Lactiplantibacillus plantarum.

8. Use of Lactiplantibacillus plantarum DSM 34687 (Lpla33 TM ) for improving the quality of life (IBS-QoL) of subjects suffering from IBS-D.

9. The use according to claim 8, wherein the dosage of Lactiplantibacillus plantarum is at least about 1 billion CFU per day.

10. The use according to claim 8 or 9, wherein the dosage of Lactiplantibacillus plantarum is at least about 10 billion CFU per day.

11. The use according to claims 8 - 10, wherein the Lactiplantibacillus plantarum is administered for at least 28 days.

12. The use according to claims 8 - 11, wherein the Lactiplantibacillus plantarum is administered for at least 56 days.

13. A method for alleviating or preventing IBS-D symptoms in a subject, the method comprising administering to the subject an effective amount of Lactiplantibacillus plantarum DSM 34687 (Lpla33 TM ).

14. The method according to claim 1, wherein the dosage of Lactiplantibacillus plantarum is at least about 1 billion CFU per day.

15. The method according to claim 1, wherein the dosage of Lactiplantibacillus plantarum is at least about 10 billion CFU per day.

16. The method according to claims 1 - 3, wherein the Lactiplantibacillus plantarum is administered for at least 28 days, preferably at least 56 days.

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