Effects of Simethicone and Multilac Baby on Fecal Calprotectin Levels Infantile Colic
Phase: NA
Conditions: Infantile Colic
Sponsor: The President Stanisław Wojciechowski State University of Applied Sciences in Kalisz

Study Protocol - Effects of Simethicone and the Multi-strain 
Synbiotic Multilac Baby on Fecal Calprotectin in Colicky Babies 
Document Date: December 07, 2020 
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Cover page 
 
 

Study Protocol - Effects of Simethicone and the Multi-strain 
Synbiotic Multilac Baby on Fecal Calprotectin in Colicky Babies 
Document Date: December 07, 2020 
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1. General Information 
Infantile colic or extensive crying is a major burden for newborns, their parents and 
healthcare providers [Landgren and Hallström, 2011]. In most of the cases infantile 
colic will disappear after the first three to five months of life [Sung, 2018]. 
Nevertheless, due to its stressful nature for parents, infant colic is among the leading 
causes to consult a health care professional during early infancy [Lucassen, 2010]. 
The parental burden associated with infantile colic finally results in enormous 
pressure on pediatricians to prescribe at least some kind of remedy. Diagnosis of 
infantile colic can be based on the Wessel’s criteria, defined by crying and 
restlessness for more than three hours a day for more than three days per week for 
more than three weeks [Wessel et al., 1954]. Depending on diagnostic details, 
occurrence rates between 3 to 40% of all infants have been found [Lucassen et 
al.2001, Helseth and Begnum, 2002]. 
2. Background Information 
The etiology of infant colic remains unclear [Sarasu et al., 2018], with a variety of 
potential causes (gastrointestinal, hormonal, neurodevelopmental, and psychosocial) 
discussed. This has resulted in a broad range of therapeutic approaches aiming to 
address the problem [Lucassen, 2010, Mai et al, 2018]. 
Simethicone [Simethicone], a mixture of dimethicone and SiO2, is a rather old product 
that, it is claimed, acts as a topical barrier for protecting the gut mucosa against 
irritants. It is not absorbed and is virtually non-toxic. While its use in diagnostic 
procedures is well-established, the therapeutic effects in a number of 
gastroenterological indications are contradictory [Meier and Streuwald, 2007]. In 
infantile colic, a number of smaller simethicone trials [Metcalf et al., 1994, Sethi and 
Sethi, 188, Danielsson and Hwang, 1985] have been published, but evidence did not 
reach the threshold of significance. Despite the absence of evidence for beneficial 
effects, simethicone is widely used for the treatment of infantile colic in some 
countries. 
There is growing evidence that the gut microbiotas of colicky infants significantly 
differ from those of non-colicky babies [Savino et al., 2004, Savino et al., 2005, 
Savino et al., 2009]. In the gut microbiota of colicky babies a lower level of 
commensal bacteria like lactobacilli and bifidobacteria and higher numbers of 
proteobacteria have been found. Among these proteobacteria were Escherichia and 
Klebsiella bacteria [Savino et al., 2011], which are well known for their gas-producing 
properties, as well as the potential production of inflammatory lipopolysaccharides 
(LPS). In addition, it has been described that the gut microbiota of colicky infants 
exhibits a slower bacterial colonization, a reduced microbiota diversity and a lower 
microbiota stability [De Werth et al., 2013]. These findings have triggered a number 
of studies investigating the effect of supplementation of the gut microbiota of colicky 

Study Protocol - Effects of Simethicone and the Multi-strain 
Synbiotic Multilac Baby on Fecal Calprotectin in Colicky Babies 
Document Date: December 07, 2020 
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babies with products containing probiotic bacteria without a prebiotic component 
(probiotics), or with (synbiotics). 
Although infantile colic is in most cases a self-limiting condition, pediatricians are 
writing a prescription for most of the affected infants. Results from surveys, 
performed among German and Polish pediatricians, revealed that in these two 
countries simethicone and pro-/synbiotics were the most frequently prescribed 
products for infantile colic [Sommermeyer et al., 2020]. In a recently published study 
it was found that the multi-strain synbiotic, Multilac Baby, antagonizes the in-vitro 
growth of the pathogenic bacteria Escherichia coli EPEC, Shigella sonnei, 
Salmonella typhimurium, Klebsiella pneumoniae and Clostridioides difficile [Piatek et 
al. 2020]. A clinical study evaluating effects of simethicone and Multilac Baby 
demonstrated that the synbiotic significantly improved the crying behavior of colicky 
babies when compared with the effects of simethicone [Piatek et al., 2020, accepted 
for publication in Beneficial Microbes]. 
Findings of a disturbed gut-microbiota in colicky babies have triggered interest in 
investigating gut-inflammation in these babies. An established marker for the level of 
gut inflammation marker is calprotectin [Stríz & Trebichavský, 2004]. Calprotectin is a 
24 kDa dimer of calcium binding proteins S100A8 and S100A9 [Pthirana et al., 2018]. 
The complex accounts for up to 60% of the soluble protein content of the neutrophil 
cytosol. The complex is resistant to enzymatic degradation, and can be easily 
measured in feces. Inflammatory processes result in an influx of neutrophils into the 
bowel lumen [Walsham & Sherwood, 2016] Since calprotectin comprises as much as 
60% of the soluble protein content of the cytosol of neutrophils, it can serve as a 
marker for the level of intestinal inflammation Measurement of fecal calprotectin has 
been shown to be strongly correlated with 111-indium-labelled leucocytes - 
considered the gold standard measurement of intestinal inflammation [Costa et al., 
2003; Gisbert & McNicholl, 2009]. So far, there is only one study published which has 
characterized the effects of the probiotic L. reuteri on the level of fecal calprotectin in 
colicky babies [Savino et al., 2018]. Based on the findings of this study it was 
concluded that probiotics have the potential to improve gut-inflammation in babies 
with infantile colic. 
3. Objectives/Purpose 
The objective of the study is to compare the effects of simethicone and the multi-
strain synbiotic Multilac Baby on calprotectin levels in colicky and non-colicky babies. 
 
 

Study Protocol - Effects of Simethicone and the Multi-strain 
Synbiotic Multilac Baby on Fecal Calprotectin in Colicky Babies 
Document Date: December 07, 2020 
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4. Study Design 
In a first step, the crying behavior of newborns shall be assessed by employing a 
parental daily diary for three weeks. Diagnosis of infantile colic will be based on the 
Wessel criteria (Rule of Three) [Wessel et al., 1954]: extensive evening crying for at 
least three hours per day, during at least three days per week, during the last three 
weeks. 
After the diagnosis has been performed (Day 1), calprotectin shall be determined in 
non-colicky babies (Arm 1, n=30-40) and colicky babies (Arms 2&3, each n=50-60). 
Based on the results from this steps it will be possible to determine if average 
calprotectin levels of colicky babies are different compared to those of non-colicky 
babies.  
Colicky babies will then randomly assigned to a treatment with simethicone (Arm 2, 
n=50-60) and treatment with Multilac Baby (Arm 3, n=50-60). Treatment duration will 
be four weeks (until Day 28). Crying behavior will be assessed with a parental daily 
diary during the last three weeks before the end of treatment. At the end of the 
treatment (Day 28), calprotectin level in feces will be performed by using a point of 
care test (Buehlmann Quantum Blue Calprotectin). 
 
 

Study Protocol - Effects of Simethicone and the Multi-strain 
Synbiotic Multilac Baby on Fecal Calprotectin in Colicky Babies 
Document Date: December 07, 2020 
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5. Selection and Exclusion of Subjects 
Work stream 
 
Inclusion criteria 
The study is aiming to recruit babies aged 3 to 6 weeks of aged. 
Exclusion criteria 
- 
Organic causes for crying 
- 
Previous treatment with antibiotics 
- 
Previous treatment with probiotic 
- 
Previous treatment with synbiotic 
 
 

Study Protocol - Effects of Simethicone and the Multi-strain 
Synbiotic Multilac Baby on Fecal Calprotectin in Colicky Babies 
Document Date: December 07, 2020 
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6. Treatment of Subjects 
Calculation 
https://www.sealedenvelope.com/power/binary-superiority/ 
Settings 
Significance level (alpha) 
5% 
Power (1-beta) 
80% 
Percentage 'success'  
in control group 
50% 
Percentage 'success'  
in experimental group 
75% 
Sample size required per group 
55 
Total sample size required 
110 
 
Randomization 
A two-treatment equal allocation randomization scheme will be used. Product will be 
allocated to patient based on the randomization scheme by the pediatrician on entry 
into the study. 
https://www.sealedenvelope.com/simple-randomiser/v1/lists 
Settings 
Treatment groups 
Group A, Group B 
Block sizes 
2,4,6,8,10 
List length 
100 
 
 
 

Study Protocol - Effects of Simethicone and the Multi-strain 
Synbiotic Multilac Baby on Fecal Calprotectin in Colicky Babies 
Document Date: December 07, 2020 
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Randomization process 
 
 
 

Study Protocol - Effects of Simethicone and the Multi-strain 
Synbiotic Multilac Baby on Fecal Calprotectin in Colicky Babies 
Document Date: December 07, 2020 
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Control Group – Non-colicky Babies (Arm 1) 
It is planned to include 30-40 babies to this arm to determine the calprotectin levels in 
non-colicky babies. 
Simethicone (Arm 2) 
Treatment for four weeks with simethicone (Espumisan®, 100 mg/ml, Berlin-Chemie / 
Menarini Polska Sp z o.o., Warsaw, Poland). Simethicone administered 3-6 times per 
day with each treatment comprising 6 drops of the 100 mg/ml emulsion. It is planned 
to include 50-60 patients to this arm. 
Multilac Baby (Arm 3) 
Treated for four weeks with one stick pack of a multi-strain synbiotic (Multilac® Baby, 
Vivatrex GmbH, Aachen, Germany) per day. Each stick pack of Multilac® Baby 
contains a total of 109 colony forming units (CFU) with equal CFU amounts of the 
following probiotic bacteria: L. acidophilus LA-14, L. casei R0215; L. paracasei Lpc-3; 
L. plantarum Lp-115; L. rhamnosus GG, L. salivarius Ls-33, B. lactis Bl-04, B. bifidum 
R0071, B. longum R0175 and 1.43 g of the prebiotic fructooligosaccharides. It is 
planned to include 50-60 patients to this arm. 
7. Assessment of Efficacy 
Crying behavior 
Crying behavior will be assessed by using a parental diary (24-hour Parental Daily 
Report). Primary outcome measures are (i) “crying days last 3 weeks” and (ii) 
“average of daily evening crying duration last 3 weeks” and (iii) change of fecal 
calprotectin level between (Day 1 and Day 28). 
Gut calprotectin level 
Calprotectin will be measured in feces samples taken before start of treatment (Day 
1) and at the end of treatment (Day 28) with Simethicone and Multilac Baby. 
8. Assessment of Safety 
Safety will be assessed by using a parental diary (24-hour Parental Daily Report). 
9. Adverse Events 
Adverse Events will be reported by the standard procedure established to report 
adverse events of pharmaceutical products by physicians. 
10. 
Discontinuation of the Study 
The study will be discontinued in case of: 

Study Protocol - Effects of Simethicone and the Multi-strain 
Synbiotic Multilac Baby on Fecal Calprotectin in Colicky Babies 
Document Date: December 07, 2020 
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- 
occurrence of adverse events, 
- 
failure to recruit patients. 
11. 
Statistics 
The statistical analyses will be conducted with GraphPad Prism software version 8.2 
(GraphPad Software, Dan Diego, California, USA) or MedCalc Statistical Software 
version 19.2.1 (MedCalc Software Ltd., Ostend, Belgium). The Student t-test will be 
used to compare mean values of continuous variables approximating a normal 
distribution. For non-normally distributed variables, the Mann-Whitney U test will be 
used. The X2 test or Fisher exact test will be used, as appropriate, to compare 
percentages. The MedCalc Statistical Software version 19.2.1 will be used to 
calculate the relative risk (RR) and number needed to treat (NNT) [Altman, 1998], all 
with a 95% confidence interval (CI). The difference between the treatment groups will 
be considered significant when the p-value will be less than 0.05, when the 95% CI 
for RR will not include 1.0, or when the 95% CI for mean difference will not include 
1.0, or when the 95% CI for mean difference will not include 0. One-way ANOVA 
followed by Dunnett’s multiple comparisons test as well as Cochran-Armitage Chi-
squared test for trend will be performed using GraphPad Prism software. All 
statistical tests will be two-tailed and will be performed at the 5% level of significance. 
All analyses will be conducted on an intention-to-treat basis, including all patients in 
the groups to which they will be allocated. 
12. 
Quality Control and Assurance 
According to local GCP SOPs. 
13. 
Ethics 
Study will have to be approved by the Ethics Committee of The President Stanisław 
Wojciechowski State University of Applied Sciences in Kalisz. 
Informed consent of parents of colicky babies will be collected from parents in writing 
before enrolling of the babies into the study. 
 
 

Study Protocol - Effects of Simethicone and the Multi-strain 
Synbiotic Multilac Baby on Fecal Calprotectin in Colicky Babies 
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14. 
Data handling and Recordkeeping 
Data will be handled by study investigators monitored and supervised by the principle 
investigator of the study (Prof. Dr. Hanna Kraus). Records will be kept at the office of 
the principle investigator applying all local regulations of data protection provisions. 
 
15. 
Publication Policy 
It is planned to publish the study results in a peer-reviewed scientific/medical journal. 
 
 

Study Protocol - Effects of Simethicone and the Multi-strain 
Synbiotic Multilac Baby on Fecal Calprotectin in Colicky Babies 
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16. 
Project Timetable/Flowchart 
Start of study after approval by the ethics committee and establishment of the 
calprotectin measurements. Start of the study is expected in 12/2020. 
Patient enrollment and study progress: 
 
 
 

Study Protocol - Effects of Simethicone and the Multi-strain 
Synbiotic Multilac Baby on Fecal Calprotectin in Colicky Babies 
Document Date: December 07, 2020 
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17. 
References 
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642–647. doi:10.1016/s1590-8658(03)00381-5 
Danielsson B, Hwang CP. ”Treatment of infantile colic with surface active substance 
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De Weerth C, Fuentes S, et al. ”Crying in infants. On the possible role of intestinal 
microbiota in the development of colic.” Gut Microbes Vol. 4, No. 5, 2013, pp. 416-21. 
Helseth S, Begnum S. ”A comprehensive definition of infant colic: parents' and 
nurses' perspectives.” Journal of Clinical Nursing Vol. 11, No. 5, 2002, pp. 672-80. 
Landgren K, Hallström I. ”Parents′ experience of living with a baby with infantile colic 
– a phenomenological hermeneutic study.” Scandinavian Journal of Caring Sciences 
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Lucassen P, Assendelft W, et al. ”Systematic review of the occurrence of infantile 
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Pathirana GW, Chubb P, Gillett MJ, Vasikaran SD. “Faecal Calprotectin“.Clinical 
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Synbiotic Multilac Baby on Fecal Calprotectin in Colicky Babies 
Document Date: December 07, 2020 
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Piatek J, Krauss H, et al. “In-Vitro Growth Inhibition of Bacterial Pathogens by 
Probiotics and a Synbiotic: Product Composition Matters.“ International Journal of 
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Synbiotic Multilac Baby on Fecal Calprotectin in Colicky Babies 
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Walsham NE, Sherwood RA. “Fecal calprotectin in inflammatory bowel disease“. 
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18. 
Supplements/Appendices 
Randomization Letter 
Parental Letter Multilac Baby 
Parental Letter Espumisan 
Patient Assessment Sheet Day 1 EN/PL 
24 Hours Parental Diary Form EN/PL