WO2024089260A1

WO2024089260A1 - Lactiplantibacillus plantarum for use in addressing the symptoms of irritable bowel syndrome

Info

Publication number: WO2024089260A1
Application number: PCT/EP2023/080102
Authority: WO
Inventors: Christopher Martoni
Original assignee: Chr. Hansen A/S
Priority date: 2022-10-28
Filing date: 2023-10-27
Publication date: 2024-05-02
Also published as: KR20250095634A; AU2023366340A1; CN120282792A

Abstract

The present disclosure provides a method of preventing, reducing, or ameliorating the symptoms of IBS, in a subject in need thereof, with L. plantarum. Further provided is L. plantarum for use in preventing, reducing, or ameliorating the symptoms of IBS. Further provided is a composition comprising L. plantarum for use in preventing, reducing, or ameliorating the symptoms of IBS.

Description

LACTIPLANTIBACILLUS PLANTARUM FOR USE IN ADDRESSING THE SYMPTOMS OF IRRITABLE BOWEL SYNDROME

FIELD

The present disclosure generally relates to probiotics for ameliorating symptoms of irritable bowel syndrome.

BACKGROUND

Irritable Bowel Syndrome (IBS), a condition usually diagnosed in the absence of any specific causative disease, is generally characterized by abdominal pain or discomfort and abnormalities in stool frequency or form. IBS is classified further by 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 9.2% and 3.8% pooled global prevalence of IBS based on Rome III and Rome IV criteria, respectively. A global study conducted by the ROME foundation across 33 countries reported IBS rates between 3% and 5% in most countries. The IBS-D, the most common subtype, has been reported by 31.5% of people with IBS, meeting the Rome IV diagnostic criteria. The pathophysiology of IBS has not been fully elucidated; however, evidence suggests that alterations in the gut microbiota have been associated with IBS. Observational studies indicated that patients with IBS-D experienced significant impairment of health-related quality of life (QoL), more work absenteeism, and significant overall work productivity loss compared to controls.

Management for IBS symptoms can include pharmacological, psychological, and complementary approaches. There is currently no single management strategy for a particular IBS subtype that has been universally adopted. Recent studies suggest that agents that restore gut microflora may help manage IBS symptoms.

Probiotics are defined as live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. For example, they may suppress the growth of pathogenic bacteria or confer health benefits when administered in adequate amounts. Several probiotic strains, particularly the lactobacilli, recently reclassified into 25 genera, and the Bifidobacterium genera, have been proposed for use in IBS patients. Several mechanisms have been hypothesized to explain the effect of probiotics on gut health, including normalizing intestinal flora, preventing the overgrowth of pathogenic bacteria, modulating visceral afferent pathways, and enhancing intestinal barrier function. However, significant heterogeneity between studies and study quality, design, and power limitations have made drawing definitive conclusions problematic. Over the past few decades, several clinical trials have been conducted to evaluate probiotics' efficacy and safety, as evident from many publications. However, previous reviews have identified that results from probiotic studies in IBS are conflicting because of different IBS subtypes and the use of different combinations of probiotic strains in varied doses across studies, which might have obscured the beneficial effects of individual strains within that species.

Lactobacillus plantarum (recently reclassified as Lactiplantibacillus plantarum) is a frequently isolated species from the healthy human GI tract that has been studied in numerous GI clinical studies, including for IBS. L. plantarum is a Gram-positive, catalase-negative bacterium that is a member of the broad classification of lactic acid bacteria. The name of the species originated from its common occurrence in spontaneously fermented plants, which were major food sources long before meat and milk became dominant. L. plantarum differs from many other lactobacilli species in that L. plantarum has a relatively large genome, possesses a striking ability to ferment many different carbohydrates, has a high growth requirement for manganese, and has a high tolerance to low pH, frequently predominating in spontaneously lactic-acid-fermented foods where the pH is below 4.0. L. plantarum has good acid and bile salt tolerance and the capability to adhere to the human intestinal mucosa. It can promote intestinal integrity or motility, alter the composition of the intestinal flora, inhibit the colonization or growth of pathogenic microbes, and is potentially capable of modulating immune function, thereby helping manage several disorders.

SUMMARY

The present disclosure provides a method of preventing, reducing, or ameliorating the symptoms of IBS, in a subject in need thereof, with L. plantarum. Further provided is L. plantarum for use in preventing, reducing, or ameliorating the symptoms of IBS. Further provided is a composition comprising L. plantarum for use in preventing, reducing, or ameliorating the symptoms of IBS. The present disclosure provides uses, methods, and compositions for improving the quality of life of a subject with IBS.

The methods, uses and compositions of the present disclosure have been found to relieve the symptoms of IBS, especially IBS-D.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the mean ± SD IBS-Symptom Severity Scale (IBS-SSS) total score.

Figure 2 shows the APS-NRS scores over the intervention period.

Figure 3 shows the percentage of responders based on IBS-SSS.

Figure 4 shows the percentage of responders based on stool consistency.

Figure 5 shows the IBS-QoL total score over intervention period.

Figure 6 shows the PSS score over intervention period.

DETAILED DESCRIPTION

The present disclosure provides a method of preventing, reducing, or ameliorating the symptoms of IBS-D, in a subject in need thereof, with L. plantarum. Further provided is L. plantarum for use in preventing, reducing, or ameliorating the symptoms of IBS-D. Further provided is a composition comprising L. plantarum for use in preventing, reducing, or ameliorating the symptoms of IBS-D.

A preferred probiotic for use herein is Lactiplantibacillus plantarum. More preferably, said probiotic includes a Lactiplantibacillus plantarum strain available from Chr. Hansen A/S as Lpla33™ (CLEPIUS™). More preferably, said probiotic includes the Lactiplantibacillus plantarum strain with the accession number DSM 34687 deposited on June 28, 2023. The growth of various lactobacilli species to form cell cultures, cell pastes, and spore preparations is generally well-known within the art. In addition, various Lactiplantibacillus plantarum products are available from commercial sources such as UALp-05™/ Lpla33™.

The term “composition”, as used herein can be construed as but not limited to a nutritional composition, a nutraceutical composition, a nutritional supplement or a pharmaceutical drug.

The term “therapeutically effective amount” or “effective amount” as used herein means that amount of active ingredient that elicits the biological or medicinal response in a subject which includes at least partial reduction, prevention, or alleviation of the symptoms of the condition being treated.

The term "irritable bowel syndrome" (IBS) is defined as one or more of a group of symptoms that occur together, notably repeated abdominal pain and altered bowel habits, which may be diarrhea, constipation, or both. IBS is part of a spectrum of disorders called functional gastrointestinal disorders (more recently termed disorders of gut-brain interaction). These disorders are all characterized by chronic or recurrent gastrointestinal symptoms for which no structural or biochemical cause can be found.

According to an embodiment, the Lactiplantibacillus plantarum of the present disclosure may be administered in solid, semi-solid, or liquid oral dosage form. In an embodiment of the present disclosure, the pharmaceutical formulation can be in the form of emulsions, solutions, suspensions, syrups, elixirs tablets, capsules, pills, granules, and suppository. In another embodiment of the present disclosure, the pharmaceutical formulation can be in the form of water dispersible granules (WG), suspension concentrates (SC), wettable powders (WP), emulsifiable concentrates (EC), granules, gel, suspo emulsions (SE), mixed formulation of capsule suspension and suspension concentrates (ZC) and the like and preferably, water dispersible granules (WG), suspo emulsions (SE) and mixed formulation of capsule suspension and suspension concentrates (ZC). In an embodiment, the Lactiplantibacillus plantarum of the present disclosure can be dried. Drying can include spray drying, fluid bed drying, or freeze-drying. In an embodiment, the Lactiplantibacillus plantarum of the present disclosure is in an orally administered dosage form of powder or granule for sachet, liquid, solution, suspension, emulsion or syrup.

In another embodiment of the present disclosure, the Lactiplantibacillus plantarum composition can include at least one pharmaceutically acceptable excipient selected from the group consisting of fillers, binders, diluents, thickening agents, solvents, coating agents, dispersing agents, preservatives, sweeteners, flavoring agents, antifoaming agent and stabilizers. Pharmaceutically acceptable filler may be selected from the group comprising lactose, microcrystalline cellulose, starch, pre-gelatinized starch, calcium phosphate, calcium sulfate, calcium carbonate, mannitol, sorbitol, xylitol, sucrose, maltose, fructose, dextrose, maltodextrin, and the like. Pharmaceutically acceptable binder may be selected from the group comprising starches, 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 comprising calcium carbonate, calcium phosphate dibasic, calcium phosphate tribasic, calcium sulfate, microcrystalline cellulose, microcrystalline silicified cellulose, powdered cellulose, dextrates, dextrose, fructose, lactitol, lactose anhydrous, lactose monohydrate, lactose dihydrate, lactose trihydrate, mannitol sorbitol, starch, pregelatinized starch, sucrose, talc, xylitol, maltose maltodextrin, maltitol, and the like. Pharmaceutically acceptable sweetener may be selected from the group comprising alitame, acesulfame potassium, aspartame, D-tryptophan, dextrose, erythritol, fructose, galactose, glycerol, glycyrrhizin, glucose, isomalt, xylitol, xylose, lactitol, lactose, levulose, maltitol, maltodextrin, maltol, maltose, mannitol, corn syrup, neohesperidin dihydrochalcone, neotame, saccharin, siclamate, sorbitol, sucralose, sucrose, tagatose, taumatin, trehalose, and the like. Pharmaceutically acceptable flavoring agent may be selected from the group comprising natural flavoring oils, anethole, acetic acid, ascorbic acid, phosphoric acid, fumaric acid, lactic acid, lemon, linalool, malic acid, menthol, eucalyptol, orange, citric acid, cinnamone, tartaric acid, thymol, vanilla, strawberry, and the like. Pharmaceutically acceptable preservative may be selected from the group comprising parabens, phenol, chlorocresol, parahydroxy benzoic acid alkyl esters, benzoic acid and salts thereof, boric acid and salts thereof, citric acid and salts thereof, sorbic acid and salts thereof, neutral preservatives, mercurial preservatives, quaternary compounds, and the like.

In an embodiment, the composition of the present invention can 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 count of cells (colony forming units or CFU) of Lactiplantibacillus plantarum.

In an 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 count of cells (colony forming units or CFU).

DSM 34687 is a preferred L. plantarum for use herein. Microorganisms may be deposited at a Depositary institution having acquired the status of international depositary authority under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure: Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures Inhoffenstr. 7B, 38124 Braunschweig, Germany. The applicant requests that a sample of deposited microorganisms may only be made available to an expert, subject to available provisions governed by Industrial Property Offices of States Party to the Budapest Treaty, until the date on which the patent is granted.

EXAMPLES

EXAMPLE 1

A randomized, double-blind, placebo-controlled multi-center study was undertaken in adults ranging from 18 to 70 years old for evaluating the safety and efficacy of L. plantarum Lpla33™ in individuals with diarrhea- predominant IBS. Approximately 450 potential participants were to be screened, and considering a screening failure rate of 20%, up to 360 participants were to be recruited. The recruited participants were then randomized in a ratio of 1 : 1: 1 within 3 groups (L. plantarum Lpla33™ [Not less than (NLT) 1 billion CFU/day], L. plantarum Lpla33™ [NLT 10 billion CFU/day], and placebo).

EMA and USFDA have provided detailed guidelines for evaluating drugs to treat IBS to bring uniformity in the study designs and methodology. The standards set by the regulatory authorities stand valid for probiotics also, which have been increasingly considered a promising management option for IBS. The regulatory authorities have emphasized that the primary objective of IBS management is to alleviate the most bothersome symptoms of the disease; therefore, the primary efficacy endpoint in the IBS trial should be an improvement in the severity of these IBS symptoms. Abiding by this recommendation, we have included the same as the primary objective of the present study. We have included IBS-Severity Scoring System (IBS-SSS), a widely validated comprehensive patient-reported outcome assessment tool for its assessment, and Abdominal Pain Severity- Numeric Rating Scale (APS-NRS) for determining the severity of abdominal pain/discomfort. Also, responder definitions for different outcome measurements have been recommended, which were included as secondary endpoints in the present study. A screening period of two weeks to confirm the diagnosis was incorporated into the study, as suggested by the USFDA. Several studies have demonstrated a lower health-related quality of life in IBS patients, notably among the diarrhea-predominant subtype, that necessitates measuring the study participants' quality of life. Therefore, we included IBS-Quality of Life (IBS-QoL) and Perceived Stress Scale (PSS) in the secondary objectives of the study.

On the first visit to the site, the study design was thoroughly explained to the suitable research participants in a language well understood, following which research participants duly signed and dated informed consent was obtained along with the consent for the microbiome analysis (using the stool specimen). The medical history and prior/ongoing medicine usage were recorded. After recording the demographic details (age, gender, &: ethnicity) and anthropometries (height, weight, &: BMI), the clinical examination was performed along with the measurement of vitals (blood pressure and pulse rate), body temperature, and SpO2 levels. All the findings were recorded on the source document (SD). In the case of females of childbearing potential, a urine pregnancy test was performed. Participants were asked to self-administer the APS-NRS and Bristol stool scale (BSS) with a recall period of the last three months and one month, respectively, prior to screening. Participants were assessed for complete inclusion and exclusion criteria by an investigator. Participants who complied with the inclusion and exclusion criteria were considered for the run-in phase. Stool collection kits and log-in credentials for an e-diary were provided to all the eligible participants. Instructions were provided to the participants for submitting morning stool samples on day 0 and regarding the daily filling of the e-diary. Participants were assessed for fasting blood glucose (FBG), hemoglobin (Hb), and thyroid-stimulating hormone (TSH) from an accredited laboratory. Placebo and rescue medication (RM) were dispensed to the participants for the placebo run-in phase. Participant diaries (for dietary recall) were dispensed, and the investigator or CRC provided clear instructions for filling up the same.

Participants consumed the placebo daily before lunch for 2 weeks. They filled e-diary for APS-NRS on the 7^th and 14^th-day post-screening visit with a recall period of 7 days, and the compliance of the same was checked by the clinical research coordinator (CRC) or any other designated personnel. Post- screening, BSS was selfadministered by the participant daily in the evening using an e-diary for the next 14 days. Compliance was checked by the CRC or any other designated personnel.

DAY O

Placebo and diet diary were reconciled, and compliance was ascertained. RM was reconciled, and its usage (if any) was recorded. After measuring weight, the clinical examination was performed, followed by vitals (blood pressure and pulse rate), body temperature, and SpO2 levels were assessed and recorded. The dietary recall was done using the diet diary to determine the average intake of calories (% protein, % fat, % carbohydrates, and % fiber intake) of any 3 days (inclusive of a weekend day) from the screening visit. Participants filled out the IBS-SSS, IBS-QoL, and PSS. Participants were assessed again as per the randomization criteria, and the eligible participants were randomized. In the case of females of childbearing potential, a urine pregnancy test was performed. All the randomized participants were administered the APS-NRS and BSS (e-diary) on weeks 1, 2, 3, and 4 with a recall period of 7 days. The compliance of the same was checked by the CRC periodically. Study products (Investigational Product(s) (IP) or placebo) along with RM were dispensed as per the randomization chart with instructions regarding the regimen. The diet diary (for dietary recall) was filled by the participant every 14 days to record 24-h dietary intake for two weekdays and one weekend day. The first stool sample of the day (on day 0) was collected by the participants who consented to microbiome analysis.

DAY 28 (+/-2) Study medication and diet recall chart were reconciled. Compliance was ascertained for the study products by the investigator or CRC. Any AEs or SAEs experienced were monitored and reported by the investigator. RM was reconciled, and its usage (if any) was recorded. A urine pregnancy test was performed for females of childbearing potential. Concomitant medication usage (if any) was recorded in the SD. After measuring weight, the clinical examination along with vitals (blood pressure and pulse rate), body temperature, and SpO2 levels was assessed and recorded on the SD. The dietary recall was done using the diet diary to approximate the average intake of calories (% protein, % fat, % carbohydrates, and % fiber intake) of any 3 days [inclusive of a weekend day] during every 14 days in the last 28 days. Participants self-administered the IBS-SSS, IBS-QoL, and PSS questionnaires. The participants self- administered the APS-NRS and BSS (e-diary) on weeks 5, 6, 7, and 8, with a recall period of 7 days. The compliance of the same was checked by the CRC periodically. Study products (IP(s) or placebo) and RM were dispensed as per the randomization chart, and instructions regarding the regimen were provided. Stool collection kits were dispensed, and instructions were provided to the participants to bring the morning stool samples on day 56. The diet diary (for dietary recall) was dispensed, and the investigator or CRC provided clear instructions for filling up the same.

DAY 56 (+/-2)

Study medication and diet dairy were reconciled. Compliance was ascertained for the study products by the investigator or CRC. Any AEs or SAEs experienced were monitored and reported by the investigator. RM was reconciled, and its usage (if any) was recorded. A urine pregnancy test was performed for females of childbearing potential. Concomitant medication usage (if any) was recorded in the SD. After measuring weight, the clinical examination was performed along with the measurement of vitals (blood pressure and pulse rate), body temperature, and SpO2 levels. All the findings were recorded on the SD. Participants were instructed to self-administer the APS-NRS, IBS-SSS, BSS, IBS-QoL, and PSS, and instructions were provided to complete the mentioned questionnaires. The dietary recall was done using the diet diary to approximate the average intake of calories (% protein, % fat, % carbohydrates, and % fiber intake) (of any 3 days [inclusive of a weekend day] during every 14 days in the last 28 days). The first stool sample of the day (on day 56) was collected by the participants for microbiome analysis.

Chr. Hansen (Wausau) supplied the investigational products used in the study. The products were manufactured in compliance with all required regulations, and sufficient quantities of the product were supplied to the clinical study sites by the CRO. The details of the investigational products are provided in the table below. In order to ensure an identical overall capsule weight, the included amount of microcrystalline cellulose differed slightly between groups. Microcrystalline cellulose is a commonly used excipient. Chemically, it is an inert substance, is not degraded during digestion, and has no appreciable absorption. Therefore, the slight difference in its quantity between groups would not affect study outcomes.

Details of the investigational products

On the randomization visit, the eligible participants were equally distributed across the three groups - the low- dose probiotic group, the high-dose probiotic group, and the control group, as described below. The participants were provided with the study products as per the randomization chart.

Group wise intervention, regimen, and intervention duration

In the present study, participants were provided with L. plantarum Lpla33 capsules containing either 1 or 10 billion colony-forming units (CFU) per capsule or placebo. In a published study, L. plantarum at a dose of 10 billion CFU/capsule was administered to 108 participants and was reported to be safe and efficacious for consumption over 4 weeks. The study reported a single adverse event (transient vertigo) and detected no change in blood parameters. In a separate clinical trial, L. plantarum was shown to be safe at a dose of 10 billion CFU/day for 4 weeks. A daily dose of 10 billion CFU/day has been considered a mid-point range according to meta-analyses of IBS studies. Additional references are also available regarding the safety profile of L. plantarum. The current study with L. plantarum Lpla33 included a lower dose of 1 billion CFU/day (considered a low dose among probiotic IBS studies) and a higher dose of 10 billion CFU/ day (considered a mid-point dose among probiotic IBS studies) for a period of 8-weeks. Each probiotic dosage group was assessed individually in comparison to the placebo.

Irritable Bowel Syndrome-Symptom Severity

Research participants were asked to respond to each question on a 100-point visual analog scale of the IBS- Symptom Severity Scale. Scores on the IBS-SSS can range from 0 to 500, with higher scores indicating more severe symptoms. Participants, if required, can be categorized as having mild (75-175), moderate (175-300), or severe (>300) IBS. A decrease of 95 points has been associated with a clinically meaningful improvement. Method of assessment: The IBS-SSS questionnaire was available on the e-diary. The participant selfadministered the questionnaire on days 0, 28, and 56 using the e-diary with a recall period of 10 days. As per the set randomization criteria, participants with IBS-SSS total scores > 175 on day 0 were randomized. Scores were evaluated on the day 56 visits from baseline compared to placebo.

In addition to day 56, change in IBS-SSS on day 28 from baseline was assessed compared with placebo.

Method of assessment: _The study participants self- administered the questionnaire using the e-diary on baseline (day 0) and day 28 visits under the supervision of an investigator or CRC, with a recall period of 10 days. The scores were evaluated on the day 28 visit from baseline compared to the placebo.

Abdominal pain severity was assessed via the 11-point APS-NRS, with 10 representing the most severe pain and 0 representing no pain, with the following directions: “How much abdominal pain have you felt in the last week, on a scale from 0 (none) to 10 (very severe). ’’APS-NRS score ratings included 0 - None, 1 to 3 - Mild, 4 to 6 - Moderate, 7 to 9 - Severe, and 10 = very severe. The NRS has been well tested psychometrically in IBS patients, showed an appropriate change in response to treatment, and correlated well to the overall GI- symptom severity. The FDA has also recommended it as a provisional endpoint for evaluating treatment benefits in IBS clinical trials. Improvement in abdominal pain of at least 30% compared to baseline has been proposed as a clinically meaningful change.

On the screening visit, the participants were asked to rate the abdominal pain severity using APS-NRS over the past 3 months prior to the screening visit. Eligible participants were provided with log-in credentials for an e- diary, including APS-NRS. Post-screening, the individual was asked to rate the intensity of abdominal pain (using e-diary) over the past 7 days at the end of each week. The average of the 2-weeks was considered the final pain score and was used for assessing participants as per set randomization criteria. If the participant fulfilled the randomization criteria, he/she was instructed to rate APS-NRS consecutively at the end of weeks 1, 2, 3, 4, 5, 6, 7, and 8 (End of the study visit) with a recall period of seven days. For the day 28 visit, the average score for weeks 1 - 4 was considered; similarly, for day 56, the score was averaged for weeks 5 - 8. The APS-NRS score was compared from baseline to the end of days 28 and 56 to assess the efficacy of IP versus placebo. The window period for the self-administration of APS-NRS was + 1 day.

Note: Minimum of three responses between two consecutive visits was considered mandatory.

Most IBS patients (77.7%) have both loose/watery stools and hard/lumpy stools, and they fluctuate between these extremes an average of 3 times per month. These frequent stool consistency fluctuations occur naturally in all IBS subtypes and are not due to taking laxatives or antidiarrheal medications. IBS presents with abdominal pain or discomfort that is relieved by defecation or is associated at its onset with a change in stool frequency (either an increase or decrease) or a change in the appearance of the stool (either loose or hard). Stool consistency was assessed via the Bristol Stool Scale, a validated ordinal scale of stool types ranging from 1 through 7, with types 1-2 and 6-7, in conjunction with other symptoms indicative of constipation and diarrhea, respectively. Types 3-5 are generally considered to be the most normal stool form and are the modal stool forms in cross-sectional surveys of healthy adults.

Method of assessment: _During the screening visit, the participants were asked to rate their stool consistency over the past 1 month before screening using BSS. They rated the stool consistency as follows: 1 = separate hard lumps like nuts, difficult to pass; 2 = sausage shaped but lumpy; 3 = like a sausage but with cracks on the surface; 4=like a sausage or snake, smooth and soft; 5 = soft blobs with clear-cut edges; 6 = fluffy pieces with ragged edges, a mushy stool; and 7 = watery, no solid pieces, entirely liquid. Eligible participants were provided log-in credentials for an e-diary where BSS was available. Post-screening, the individual was asked to rate the stool consistency of their abnormal bowel movements daily in the evening for the next 14 days (run-in period). The participants were then assessed as per the set randomization criteria on day 0, and if he/she fulfilled the randomization criteria, they were enrolled in the study.

On day 0, the participants were instructed to rate their stool consistency/day every week using BSS on e-diary. Weekly BSS scores were assessed to evaluate change in stool consistency to normal, but not stool frequency, as the former correlates with colonic transit time and is thought to be a better indicator of bowel function. At the end of days 28 and 56, the percentage of participants transitioning to normal stool consistency compared to baseline (day 0) BSS scores were identified for evaluating the efficacy of IP compared with placebo. The window period for the self-administration of BSS was + 1 day. Note: Minimum of three responses between two consecutive visits is mandatory.

In the present study, a responder was defined as a research participant whose overall symptom severity on the IBS-SSS improved by 95 points when comparisons were made from baseline (day 0) to day 28 and day 56.

USFDA guidelines recommend evaluating the intervention response for IBS in terms of a weekly responder, defined as a decrease of at least 50% in the number of days per week with at least one stool that has a consistency of Type 6 or 7 compared with baseline. In the present study, we have also utilized this definition to evaluate response to intervention.

Although IBS is not life-threatening, it is associated with decreased life expectancy and considerably impacts individuals’ lives. Research has previously confirmed that patients with IBS have a higher prevalence of depression and lower quality of life than their healthy peers. In the present study, IBS-related quality of life (QoL) was assessed via the IBS-QoL, a 34-item questionnaire with each item rated on a 5-point scale (range: 34-170), with increasing scores indicating the deteriorating quality of life. It measures 8 domains found relevant to patients with IBS: dysphoria, interference with activity, body image, health worry, food avoidance, social reaction, sexual and relationship issues. The IBS-QOL has high internal consistency and high reproducibility. IBS-QoL is sensitive to treatment response in patients with IBS. Each of the items are rated on a 1 - 5 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 - Extremely” & 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 and 5 - A great deal”. The questionnaire was administered with a 30-day recall period. The individual responses to the 34 items were summed and transformed to a 0-100 scale for a total score, with increasing scores indicating improved IBS specific QoL. An increase of 10 points or more is considered a clinically meaningful improvement.

Method of assessment: The questionnaire was self- administered digitally by the study participants on the baseline, day 28, and day 56 visits.

Disturbance of the bidirectional brain-gut axis has been increasingly recognized as a conceptual model of IBS pathophysiology. It comprises abnormal functioning of the enteric, autonomic, and/or central nervous systems. Research indicates that stress can result in overactivity or underactivity along the hypothalamic- pituitary-adrenal axis and of the autonomic nervous, metabolic, and immune systems. It can also alter braingut interactions, ultimately affecting the gastrointestinal tract's different physiological functions. On this basis, the present study included Perceived Stress Scale (PSS), a widely used psychological instrument that measures the degree to which situations in one’s life are appraised as stressful. PSS items are specifically designed to understand how unpredictable, uncontrollable, and overloaded the assessed individuals find their daily lives influenced by stress. The tool has been validated for reliability and reproducibility in various clinical implications. PSS includes 10 questions about levels of stress experienced in the last 30 days with responses being captured on a Likert scale of 0 - 4 (0 = Never 1 = Almost Never 2 = Sometimes 3 = Fairly Often 4 = Very Often). PSS scores are obtained by reversing responses (e.g., 0 = 4, 1 = 3, 2 = 2, 3 = 1 & 4 = 0) to the four positively stated items (items 4, 5, 7, &: 8) and then summing across all scale items. Higher scores indicate increased levels of stress, and the scores range from 0 - 40.

Method of assessment: PSS was self- administered digitally by the research participants on baseline (day 0), day 28, and 56.

RESULTS The baseline mean IBS-SSS total score was comparable across the three study groups. After 56 days of intervention, the high-dose L. plantarum (10 billion CFU) group reported the highest reduction (-51.76%) in total scores from the baseline values, followed by the low-dose L. plantarum (1 billion CFU) group (-40.66%). The placebo group reported the lowest reduction (-19.05%) in the mean IBS-SSS total score from the baseline, which was less than half the magnitude observed in the low-dose and high-dose probiotic groups. It was observed that the IBS-SSS score reduction from baseline in all the groups was statistically significant (p<0.05).

Also, the effect of the two L. plantarum doses on IBS-SSS total score was statistically significant (p<0.05) compared to placebo; however, the effect size of the high-dose L. plantarum was 1.5-fold higher than the low- dose group which was statistically significant (p=0.0298).

Summary for IBS-SSS total score and change from baseline on day 56

Notes:

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

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

[3] p-value was calculated using ANCOVA using Dunnett t-test adjustment with treatment as factor and baseline as covariate vs. Placebo.

[4] p-value was calculated using two-sample t-tests between 1 billion CFU and 10 billion CFU.

Abbreviation: N = number of participants; SD = standard deviation

On day 28, there was a statistically significant reduction in the mean total IBS-SSS score from the baseline scores across the three study groups (figure 1). The magnitude of the reduction from the baseline score was highest in the high-dose L. plantarum group, followed by the low-dose group. The placebo group reported the lowest but statistically significant decrease in the total IBS-SSS score from baseline. The difference between the three groups was also statistically significant. The effect size of the low-dose group was half of that observed in the high-dose group; however, it was not statistically significant (p=0.0956).

Summary of IBS-SSS total score and change from baseline on day 28

Notes:

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

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

The mean baseline APS-NRS score across the three groups was similar and ranged from 5.00 to 9.50.

On day 56, the mean APS-NRS score was reduced from baseline in all three groups, and the reduction was statistically significant. See Figure 2. The highest reduction from baseline was reported in the high-dose L. plantarum group (-33.33%) and the lowest in the placebo group (-13.17%). A statistically significant difference was observed between the three groups. The effect size was significantly larger in the high-dose group (-1.44) than in the low-dose Lpla33 group (-0.89) (p=0.0057).

After 28 days of intervention, the mean baseline score was reduced by 12.18% in the low-dose group, similar to the reduction observed (-12.83%) in the high-dose Lpla33 group (p=0.7089). The placebo group had the lowest decrease in the mean APS-NRS score (-8.54%). The reduction in the mean APS-NRS score from baseline in all three groups was statistically significant; however, the difference between the three groups was not significant.

Summary of APS-NRS score

Abbreviation: CI = confidence interval, N = number of participants, SD = standard deviation Change in APS-NRS score

For the present study, a responder was defined as a research participant whose overall symptom severity on the IBS-SSS reduced by 95 points on day 56 compared to baseline (day 0). The table below shows the number and percentage of responders on days 28 and 56. The maximum number of responders was observed in the high-dose L. plantarum group on day 28 and day 56. At the end of the study, 72.63% of participants responded to the higher dose of probiotics, while 59.62% responded in the low-dose L. plantarum group. The difference between groups was statistically significant. In comparison, only 26.26% of participants in the placebo group experienced a response to the intervention. The three groups differed significantly in terms of response to intervention (p<0.05) on both day 28 and day 56 (figure 3).

Percentage of responders based on IBS-SSS total score improvement

Notes: [1] p-value was calculated using the Chi-Square test across the three groups.

[2] p-value was calculated using the Chi-Square test between 1 billion CFU and 10 billion CFU

[3] p-value was calculated using the Chi-Square test between 1 billion CFU and Placebo.

[4] p-value was calculated using the Chi-Square test between 10 billion CFU and Placebo.

Abbreviation: N=number of participants

Based on stool consistency, a weekly responder was defined as a decrease of at least 50% in the number of days per week with at least one stool with a consistency of Type 6 or 7 compared with baseline. According to this definition, the high-dose L. plantarum group reported the most responders on day 28 (48.42%) and day 56 (88.42%), followed by the low-dose L. plantarum group (day 28: 30.77% and day 56: 62.50%) compared to the placebo group (day 28: 17.17% and day 56: 26.26%) (p<0.05). The three groups differed significantly regarding weekly stool consistency responders. Also, a significant difference was noted between the two L. plantarum groups. See Figure 4.

Percentage of responders based on stool consistency (BSS type 6 & 7)

The mean baseline IBS-QoL total score was similar across the three groups (figure 5). On day 56, a statistically significant improvement (increase) in the mean IBS-QoL total scores from the baseline scores was observed in all three groups. Between-group comparison of the mean IBS-QoL total score at day 56 also showed a statistically significant difference. A greater improvement was reported in the high-dose and low-dose L. plantarum group compared to the placebo (p<0.05). The effect size was significantly larger in the high-dose L. plantarum group (24.5741) than in the low-dose group (13.40) (p<0.05).

On day 28, there was a statistically significant improvement in the mean IBS-QoL total scores from the baseline scores in all three groups (p<0.05). A greater improvement was observed in the high dose (14.09) and low dose (5.40) L. plantarum groups compared to placebo (p<0.05). Also, the difference between the two L. plantarum dose groups was statistically significant (p=0.0009).

Summary for IBS-Quality of Life (QoL) total score

Abbreviation: CI = confidence interval, SD = Standard deviation, N = number of participants Change for IBS-Quality of Life total score

Notes: [1] p-value was calculated using ANCOVA with treatment as a factor and baseline as a covariate.

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

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

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

The three groups had similar mean PSS scores at baseline (figure 6). Compared to the baseline, a statistically significant reduction in the mean PSS score was observed in both L. plantarum groups. In contrast, the reduction in the placebo group was not significant on day 28 or day 56 (p>0.05). Both doses of L. plantarum demonstrated a significant reduction in PSS scores as compared to placebo (p<0.05). The effect size was larger in the high-dose L. plantarum group (-5.24) than in the low-dose group (-3.55); however, the difference between the two dose groups was not statistically significant (p=0.1708).

Summary of PSS Score

Change in PSS Score

Notes: 1] p-value was calculated using ANCOVA with treatment as a factor and baseline as a covariate.

2] p-value was calculated using paired t-test. 3] p-value was calculated using ANCOVA using Dunnett t-test adjustment with treatment as factor and Baseline as covariate vs. Placebo. 4] p-value was calculated using two-sample t-tests between Lpla33 -1 billion CFU and Lpla33 -10 billion CFU.

Abbreviation: CI = confidence interval, N = number of participants, SD = standard deviation The present randomized placebo-controlled parallel-group trial was conducted to investigate the efficacy of 56 days of supplementation of two different doses of L. plantarum [NLT 1 billion CFU/day and 10 billion CFU/day] on IBS symptoms compared to placebo. The trial achieved its primary and secondary objectives and demonstrated that high and low doses of L. plantarum significantly reduced the IBS symptom severity score by more than twice the magnitude observed in the placebo group at the end of day 56. The positive effect in the two treatment groups was also demonstrated at day 28 as the symptom severity score was reduced to a statistically significant extent compared to baseline and the placebo group. The reduction in IBS-SSS scores in both probiotic groups surpassed the previously reported criteria of clinical meaningful response, i.e., a decrease of 95-points. Further, the higher dose L. plantarum demonstrated a greater effect size in reducing IBS symptom severity as compared to the lower dose L. plantarum.

Likewise, abdominal pain was also significantly reduced on day 28 and day 56 with L. plantarum supplementation compared to baseline levels and the placebo group. Additionally, the higher dose L. plantarum demonstrated a greater effect size in reducing the APS-NRS score. USFDA has defined an improvement in abdominal pain of at least 30% compared to baseline as clinically meaningful, which was achieved by L. plantarum when administered at a dose of 10 billion CFU/day.

Responder analysis is one of the secondary outcomes recommended by the USFDA for IBS trials. The responder analysis was performed using two different criteria, firstly, a 95-point improvement in the IBS-SSS score from baseline, and secondly, a decrease of at least 50% in the number of days per week with at least one stool with a consistency of Type 6 or 7 compared with baseline. In the high-dose L. plantarum group, 72.63% of participants responded as per symptom severity criteria, followed by the low-dose group with 59.62% and the placebo group with 26.26% at the end of the study. Based on stool consistency criteria, the percentage of responders in the high-dose L. plantarum group (88%) and low-dose L. plantarum group (63%) was significantly greater than the placebo (26%) after Day 56.

As it is well-established that IBS significantly impacts the quality of life of the affected individuals, it is prudent to assess the effectiveness of an investigational product in improving quality of life. IBS-QoL is a validated questionnaire explicitly designed to evaluate the quality of life of the IBS-affected population. In this study, QoL of the participants was evaluated and a statistically significant improvement was observed in the scores on days 28 and 56 in the two L. plantarum groups compared to baseline and the placebo group. Both groups significantly improved the IBS-QoL score to a level considered clinically meaningful (i.e., 10 points or more). Additionally, the effect of the two L. plantarum doses on stress-related symptoms was assessed using the Perceived Stress Scale and it was observed that the two doses were effective in relieving stress in the participants.

The results demonstrate that L. plantarum, at a dose of 1 or 10 billion CFU/day, produced a significant improvement in the primary and secondary study endpoints as compared to placebo, with a greater effect size observed with the 10 billion CFU/day dose. The study was adequately powered and incorporated important recommendations proposed by the USFDA and other experts for IBS randomized controlled trials.

Claims

1. L. plantarum DSM 34687 (Lpla33™) for use in the reduction or prevention of the symptoms of IBS- D in a subject.

2. The use of claim 1 wherein the dosage of L. plantarum is at least about 1 billion CFU per day.

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

4. The use of claim 1-3 wherein the L. plantarum is administered for at least 28 days.

5. The use of claim 1-3 wherein the L. plantarum is administered for at least 56 days.

6. A composition comprising L. plantarum DSM 34687 (Lpla33™) for use in the reduction or prevention of the symptoms of IBS-D.

7. The composition of claim 6, wherein said composition comprises at least about 1 billion CFU of the L. plantarum.

8. L. plantarum DSM 34687 (Lpla33™) for use in improving the quality of life (IBS-QoL) of a subject suffering from IBS-D.

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

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

11. The use of claim 8-10 wherein the L. plantarum is administered for at least 28 days.

12. The use of claim 8-11 wherein the L. plantarum is administered for at least 56 days.

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

14. The method of claim 1 wherein the dosage of L. plantarum is at least about 1 billion CFU per day.

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

16. The method of claim 1-3 wherein the L. plantarum is administered for at least 28 days, preferably at least 56 days.