Proof-of-Concept "Stress & Anxiety Dampening Effects of Lpc-37"
Phase: NA
Conditions: Healthy, Stress, Psychological
Sponsor: Daacro

 
 
 
 
 
 
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Proof-of-concept “Stress & anxiety dampening effects of  
Lpc-37” 
 
NH-03862 Sisu (PR) 
 
 
Protocol Number:  
Du01 – 2017, Version F3.0, 14th March 2018 
Principal Investigator: 
Juliane Hellhammer, Ph.D. 
Max-Planck-Straße 22 
54296 Trier,  
Germany 
Sponsor: 
 
 
DuPont Nutrition and Health 
Danisco Sweeteners Oy 
 
 
 
 
Sokeritehtaantie 20 
 
 
 
 
 
02460 Kantvik, 
 
 
 
 
Finland  
 
 
 
 
 

 
 
 
 
 
 
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1 
 
PROTOCOL SUMMARY 
1.1 
SYNOPSIS  
Title: 
Proof-of-concept “Stress & anxiety dampening effects of Lpc-37” 
 
 
Study Description: 
The purpose of this clinical trial is to determine whether a single strain of 
bacteria derived from the species Lactobacillus paracasei (Lpc-37) can 
modulate stress experienced by healthy male and female participants 
exposed to the Trier Social Stress Test (TSST). This randomized, double-
blind, placebo-controlled, parallel-groups clinical trial is statistically 
powered to detect a significant modulation of the increase in heart rate 
(HR), as the primary outcome, in response to the TSST. Self-reporting 
inventories related to stress, anxiety, subjective feelings and sleep will be 
completed by participants to identify the potential impact of probiotic 
supplementation on other psychological and physiological outcomes. 
Designed as a proof-of-concept study, the results of this study will serve as 
an indication that the chosen study design is suitable to investigate stress-
related effects of probiotics. 
 
 
Objectives: 
 
Primary Objective:   
 
The primary objective of this trial is to evaluate the efficacy of Lactobacillus 
paracasei Lpc-37 (Lpc-37) on HR before, during and after the TSST. 
 
 
 
Secondary Objectives: 
 
The secondary objectives of this trial are to evaluate: 
 
1. The effect of Lpc-37 on state anxiety as measured with the State- 
Trait-Anxiety-Inventory (STAI) before and after treatment. 
2. The effect of Lpc-37 on state anxiety as measured with the STAI 
before and after the TSST. 
3. The effect of Lpc-37 on the Cortisol Awakening Response (CAR) 
before and after treatment.  
4. The effect of Lpc-37 on salivary cortisol before and after the TSST. 
5. The effect of Lpc-37 on perceived stress as measured with the 
Perceived Stress Scale (PSS) before and after treatment. 
6. The effect of Lpc-37 on depression, anxiety and stress as measured 
with the Depression-Anxiety-Stress Scale (DASS) before and after 
treatment. 
7. The effect of Lpc-37 on anxiety as measured with the Beck Anxiety 
Inventory (BAI) before and after treatment. 
 

 
 
 
 
 
 
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8. The effect of Lpc-37 on perceived stress, anxiety, insecurity and 
exhaustion as measured by Visual Analog Scales (VAS) before and 
after treatment. 
9. The effect of Lpc-37 on perceived stress, anxiety, insecurity and 
exhaustion as measured by VAS before, during and after the TSST. 
10. The effect of Lpc-37 on sleep quality, health, wellbeing and mood 
as measured by a daily online diary over a period of 7 weeks. 
11. The effect of Lpc-37 on systolic and diastolic blood pressure (BP) 
before and after treatment. 
12. The effect of Lpc-37 on systolic and diastolic BP before and after the 
TSST. 
13. The effect of Lpc-37 on salivary alpha-amylase (sAA) before and 
after the TSST. 
 
 
  
Endpoints: 
Primary Endpoint: 
 
The primary endpoint is change in HR in response to the TSST. The efficacy 
is defined as a reduced increase in HR in response to the TSST in the verum 
group compared to the placebo group. 
 
Secondary Endpoints:  
 
Endpoints related to parameters assessed before (Visit 2) and after the 
intervention (Visit 3). The difference of:  
• 
STAI-state scores (Form X1) 
• 
PSS scores 
• 
DASS depression scores 
• 
DASS anxiety scores 
• 
DASS stress scores 
• 
BAI scores 
• 
VAS stress perception scores 
• 
VAS anxiety scores 
• 
VAS insecurity scores 
• 
VAS exhaustion scores 
• 
Systolic BP  
• 
Diastolic BP 
 
Efficacy is defined as a larger reduction of the listed scores, as well as 
systolic and diastolic BP, after 5 weeks of daily Lpc-37 intake in the verum 
group compared to the placebo group.  
 
Endpoints related to cortisol and the CAR assessed before (Visit 2) and 
after the intervention (Visit 3). The difference of: 
• 
Area under the curve with respect to ground (AUCG) 
• 
Area under the curve with respect to increase (AUCI)  
• 
Cortisol at the time of awakening 

 
 
 
 
 
 
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• 
Cortisol at 8 pm 
 
Efficacy is defined as a larger number of participants showing a 
normalization of the CAR after 5 weeks of daily Lpc-37 intake in the verum 
group compared to the placebo group.  
 
Endpoints related to parameters assessed before and after the TSST. The 
difference of:  
• 
STAI-state scores (Form X1) 
• 
Systolic BP 
• 
Diastolic BP 
 
Efficacy is defined as a reduced increase of STAI-state (X1) scores, systolic 
and diastolic BP in the verum group compared to the placebo group.  
 
Endpoints related to parameters assessed before, during and after TSST. 
The change of: 
• 
VAS stress perception scores 
• 
VAS anxiety scores 
• 
VAS insecurity scores 
• 
VAS exhaustion scores 
• 
Salivary cortisol 
• 
sAA 
 
Efficacy is defined as a reduced increase of the VAS stress perception, 
anxiety, insecurity and exhaustion scores. For salivary cortisol and sAA, 
efficacy is defined as subgroup-specific normalization of the response, i.e. a 
higher response for chronically high stressed participants and a lower 
response for chronically low stressed participants in the verum group 
compared to the placebo group. 
 
The changes over time from before (Visit 1) and after the intervention 
(Visit 3) as assessed with a daily online diary in:  
• 
Sleep duration  
• 
Sleep related recovery scores 
• 
Sleep disruptions (binary) 
• 
Reported number of sleep disruptions 
• 
Perceived health status scores 
• 
Mood scale scores  
• 
Perceived productivity scores 
 
Efficacy is defined as an increase of sleep duration, sleep related recovery 
scores, perceived health status scores, perceived productivity scores, mood 
scores and a decrease of number of sleep disruptions over the course of the 
intervention in the verum group compared to the placebo group. 
 
 

 
 
 
 
 
 
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Safety:  
 
The safety objectives of this clinical trial are to evaluate if the vital signs 
(Body Mass Index (BMI), BP, HR, and the incidence and intensity of adverse 
events (AEs) are comparable between the verum group and the placebo 
group.  
 
Study Population: 
The study population included healthy male and female adults (18-45 years 
inclusive). A total of 120 healthy participants were recruited and 
randomized into one of two study groups (verum or placebo) after 
stratifying for chronic stress (below or above age-related median score of 
the Screening Scale of Chronic Stress subscale of the Trier Inventory for 
Chronic Stress (TICS) and sex (male and female).  
 
For the stratification based on chronic stress, participants with a chronic 
stress score of ≤13 were stratified into the low chronic stress subgroups and 
participants with a chronic stress score of ≥14 were stratified into the high 
chronic stress subgroups.  
 
Group 1: Lactobacillus paracasei Lpc-37 at 1.75 x 1010 colony forming units 
(CFU) per day  
 
Group 2: Placebo  
 
Participants took one capsule of their assigned investigational product every 
morning for 5 weeks.   
 
Inclusion Criteria: 
 
- 
Voluntary, written, informed consent to participate in the study 
- 
Male or female aged between 18-45 years (inclusive) 
- 
BMI between 18.5 – 29.9 kg/m2  
- 
Medical examination at baseline indicates they are healthy in the 
opinion of the Principal Investigator  
- 
Ability of the participant (in the Principal Investigator’s opinion) to 
comprehend the full nature and purpose of the study including 
possible risks and side effects  
- 
Agreement to comply with the protocol and study restrictions  
- 
Available for all study visits  
- 
Females of child-bearing potential required to provide a negative 
urine pregnancy test and to use contraceptives 
- 
Easy access to internet 
 
Exclusion Criteria: 
 

 
 
 
 
 
 
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- 
Self-reported diagnosis of one or more DSM-IV axis 1 disorder(s), 
including but not limited to current major depression, anxiety 
disorder, bipolar spectrum disorder or schizophrenia 
- 
Have a significant acute or chronic coexisting illness (cardiovascular, 
gastrointestinal (inflammatory bowel syndrome, inflammatory 
bowel disease), immunological, metabolic, neurodevelopmental or 
any condition which contraindicates, in the Principal Investigator’s 
judgement, entry to the study  
- 
Currently taking (from day of screening onwards) or have previously 
taken (last 4 weeks prior to screening) psychoactive medication 
(anxiolytics, sedatives, hypnotics, anti-psychotics, anti-depressants, 
anti-convulsants, centrally acting corticosteroids, opioid pain 
relievers)  
- 
Currently taking (from day of screening onwards) medication or 
dietary supplements that the Principal Investigator believes would 
interfere with the objectives of the study, pose a safety risk or 
confound the interpretation of the study results (e.g. melatonin, 
omega-3 dietary supplements, non-steroidal anti-inflammatory 
drugs (NSAIDS), over-the-counter (OTC) sleep medication (not 
categorized as sedatives, hypnotics or anti-depressants), anti-
coagulants, 
proton 
pump 
inhibitors, 
anti-histamines, 
pseudoephedrine, cortisone, beta-blockers)  
- 
Recent (within last 4 weeks prior to screening) or ongoing antibiotic 
therapy during the intervention period  
- 
Daily consumption of concentrated sources of probiotics and/or 
prebiotics within 2 weeks of screening and throughout the 
intervention period other than the provided study products (e.g., 
probiotic/prebiotic tablets, capsules, drops or powders)  
- 
Pregnant or lactating female, or pregnancy planned during 
intervention period  
- 
Not fluent in German 
- 
Have self-reported dyslexia  
- 
History of alcohol, drug, or medication abuse  
- 
Self-declared illicit drug users (including cannabis and cocaine) for 
3 weeks prior to screening and during the intervention period  
- 
Contraindication to any substance in the investigational products  
- 
Hypertension (systolic ≥ 140 mmHg, diastolic ≥ 90 mmHg) 
- 
Known hyper- or hypothyroidism unless treated and under control 
(stable for more than 3 months) 
- 
Persons having previously participated in the TSST 
- 
Smoking > 5 cigarettes/day 
- 
Employee of the sponsor or contract research organization 
- 
Participation in another study with any investigational product 
within 60 days of screening and during the intervention period  
- 
Principal Investigator believes that the participant may be 
uncooperative and/or noncompliant and should therefore not 
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- 
Participant under administrative or legal supervision  
 
Sample size: 
 
The estimated sample size was computed for a repeated measurement 
ANOVA with  
• 
2 groups 
• 
7 measurements (HR during the TSST) 
• 
a small effect size of f = 0.1 
• 
α-error probability = 0.05 
• 
power (1 – β-error probability) = 0.85 
and resulted in a group size of 56 participants each. In order to account for 
protocol deviations, the estimated sample size was rounded up to a total 
sample size of 60 participants per study group. 
 
Statistical analyses: 
 
For all endpoints, analyses will be performed for the Intention-to-Treat (ITT) 
and Per Protocol (PP) population separately. Subgroup analyses will be 
performed for the different strata, i.e. female participants, male 
participants, high stressed participants and low stressed participants. 
 
Efficacy analyses are planned as linear mixed model (lmm) analyses and 
repeated measures analyses of variance (RM ANOVA) or alternatively as t-
tests, Friedman analyses of variance, Wilcoxon Signed-Rank test, Kruskal 
Wallis test, and Mann Whitney U tests in case of violation of model 
assumptions. Two-sided hypothesis testing (α=0.05) will be performed. 
 
Mixed models will be built up gradually, first testing how many time 
polynomials should be included, then testing possible covariates, and lastly 
adding the effect of study group and time × group interaction terms (e.g. 
time1 × group, time2 × group). Models will be built including time and 
intercept as random factors. In case of convergence difficulties, time will be 
dropped from the random effects. Fixed effects include time, group, time × 
group interaction terms and covariates. Categorical variables are effect-
coded, time will be centered. To assess main effects and interactions of 
variables with more than one coefficient (time, time x group interaction), 
Type II F-tests will be conducted using Satterthwaite's degrees of freedom 
method. 
 
If the assumptions of a statistical analysis are violated in the efficacy 
analyses despite efforts of transformation, alternative tests will be used. If 
the assumptions for mixed models are violated, RM ANOVAs will be used. If 
the assumptions for RM ANOVAs are violated, one-way ANOVAs will be 
performed. If the assumptions for parametric testing are violated, non-
parametric tests will be calculated. Time effects will then be evaluated by 
Wilcoxon Signed-Rank tests or Friedman ANOVAs, group effects by Mann 

 
 
 
 
 
 
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Whitney U tests, and time × group interactions by Mann Whitney U tests on 
difference values between measurements (e.g. group difference in the 
difference value between HR sitting before the TSST minus HR standing 
before the TSST). 
 
Phase: 
2 
 
 
Description of 
Sites/Facilities Enrolling 
Participants: 
The sponsor contracted daacro GmbH & Co. KG (contract research 
organization) and their site located at the Science Park of Trier, Germany 
(Max-Planck-Straße 22, 54296 Trier, Germany) to perform the clinical trial. 
This was a single-site clinical trial.  
 
 
 
Description of Study 
Intervention: 
 
The investigational products are: 
 
Group 1: Lactobacillus paracasei Lpc-37 at 1.75 x 1010 CFU per day 
 
Ingredients: Lactobacillus paracasei Lpc-37, microcrystalline cellulose, 
magnesium stearate, silicon dioxide  
Batch No.: 1103180371 
 
Group 2: Placebo  
 
Ingredients: Microcrystalline cellulose, magnesium stearate, silicon dioxide  
Batch No.: 1103180369 
 
Posology: 
 
The study products will be provided in capsules. The study products will be 
consumed once daily for 5 weeks, 30 minutes before breakfast or their first 
meal of the day, with a glass of plain (non-sparkling) water. 
 
 
Study Duration: 
 
5 weeks 
 
date of First Participant First Visit: April 10, 2018  
date of Last Participant Last Visit: October 9, 2018   
 
Participant Duration: 
 
The study visits are defined as; Screening Visit (Visit 1), Baseline Visit (Visit 
2), and after 5 weeks of intervention with either verum or placebo, the Post-
Treatment Visit (Visit 3).  
The investigation steps and duration of each study visit is outlined in 
Schema 1.2.  
 
 
 

 
 
 
 
 
 
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1.2 
SCHEMA 
 
Overall Study Design 
Study day 
No. of visit at 
study site 
Investigation steps 
Duration of visit 
 
Visit 0 
Telephone screening: Interested participants receive 
information via phone and a first check of 
inclusion/exclusion criteria is performed. Eligible 
participants are scheduled for a screening visit at the 
study site. 
approx. 20 min. 
1 
Visit 1 
Screening visit: At Visit 1, participants will be informed 
about the study procedure extensively. Eligibility 
criteria will be checked, and participants sign an 
informed consent. The TICS questionnaire will be filled 
in for stratification and participants receive a random 
number. Enrolled participants receive a training on how 
to handle the daily online diary entry and on how to 
collect saliva samples at home during the 2 days before 
Visit 2. A saliva collection kit will be handed out.    
approx. 75 min. 
 
2 - 15 
 
 
Run-in: During the two-week run-in period participants 
are not allowed to consume products containing 
concentrated 
sources 
of 
probiotics/prebiotics. 
Participants fill in the daily online diary in the mornings. 
Saliva samples will be collected during the 2 days 
before Visit 2.  
 
16 
Visit 2 
Baseline visit: Participants return their saliva samples. 
Weight, HR and BP will be measured. Baseline 
questionnaires (STAI-Trait, STAI-State, VAS stress, 
anxiety, insecurity and exhaustion, BAI, DASS, PSS) will 
be assessed. The investigational product will be handed 
out and intake will be explained. A second saliva 
collection kit will be handed out for post-treatment 
assessment during 2 days prior to Visit 3. 
approx. 45 min. 
17-51 
 
Intervention: Participants take 1 capsule of the 
investigational product and fill in the online diary daily 
in the morning. Saliva samples will be collected during 
the 2 days before Visit 3. 
 
51 
Visit 3 
After 5 weeks of intervention intake participants return 
to the study site. Again, saliva samples will be collected. 
Overall compliance with the study protocol will be 
checked and AEs will be assessed. Weight, HR and BP 
will be measured. Again, questionnaires (STAI-State, 
VAS stress, anxiety, insecurity and exhaustion, BAI, 
approx. 125 min. 

 
 
 
 
 
 
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DASS, PSS) will be assessed. The TSST will be conducted. 
6 saliva samples for the assessment of cortisol and sAA 
will be collected before and after the TSST. A 
continuous HR measurement will take place before, 
during and after the TSST. BP will be assessed before 
and after the TSST. STAI state anxiety will be assessed 
before and after the TSST, while VAS stress, anxiety, 
insecurity and exhaustion will be assessed before, 
during and after the TSST. Participants receive their 
400€ study compensation. 
Abbreviations: V, visit; TICS, Trier Inventory for Chronic Stress; HR, heart rate; BP, blood pressure; VAS, 
visual analog scale; BAI, beck anxiety inventory; DASS, depression-anxiety-stress scale; PSS, perceived 
stress scale; AE, adverse event; TSST, Trier Social Stress Test; sAA, salivary alpha amylase.  
 
 
 

 
 
 
 
 
 
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1.3 
SCHEDULE OF ACTIVITIES (SOA) 
  
Visit 
V0 
V1 
Run-in 
CAR pre 
V2 
Treatment 
CAR post 
V3 (TSST) 
Study day (treatment day) 
 
1 
2-15 
14-15 
16 
17-51 
49-50 
51 
Telephone screening 
× 
 
 
 
 
 
 
 
Study inclusion, TICS 
 
× 
 
 
 
 
 
 
Vital signs (BP, pulse, weight, 
height, BMI) 
 
× 
 
 
× 
 
 
× 
STAI-Trait 
 
 
 
 
× 
 
 
 
General psychometric assessment 
(STAI-State, VAS stress, anxiety, 
insecurity and exhaustion, DASS, 
BAI, PSS) 
 
 
 
 
× 
 
 
× 
Daily online diary 
 
 
x 
 
× 
× 
 
 
Study treatment dispense 
 
 
 
 
× 
 
 
 
Intake of investigational product 
 
 
 
 
 
× 
 
 
Compliance check 
 
 
 
 
× 
 
 
× 
TSST-protocol including 
questionnaires VAS, STAI-State, 
continuous HR, BP pre-/post- TSST, 
saliva samples (cortisol, AA)  
 
 
 
 
 
 
 
× 
CAR profile 
 
 
 
× 
 
 
× 
 
Abbreviations: TICS, Trier Inventory for Chronic Stress; BP, blood pressure; BMI, body mass index; VAS, 
visual analog scale; DASS, depression-anxiety-stress scale; BAI, beck anxiety inventory; PSS, perceived 
stress scale; TSST, Trier Social Stress Test; HR, heart rate; AA, alpha-amylase; CAR, cortisol awakening 
response