The study was designed as a mono-centre, parallel-treatment, multi-dose, double-blind study in subjects with functional constipation. The trial incorporated a planned, adaptive interim analysis to adjust for sample size and to evaluate the different doses with regard to the main study objective. The first part of the study was performed with four study arms: two arms with a daily dose of 300 mL (active and placebo) and two arms with a daily dose of 500 mL (active and placebo). Following the interim analysis, the lower-volume/lower-dose arms were terminated, and the final sample size (for the 500 mL dose) was adjusted. Results for the lower dose are not presented.
The study was approved by the Ethics Committee (Office for Health and Social Affairs, Berlin, Germany) and by the Competent Authority (Federal Institute for Drugs and Medical Devices, Bonn, Germany). It was conducted in compliance with the Declaration of Helsinki as well as the German Pharmaceuticals Act, the principles of ICH-GCP, and the German GCP-V. The study was registered in the European Clinical Trials Database (EudraCT) as EudraCT No 2012-005130-11. Participants gave written informed consent prior to the study.
A total of 106 otherwise healthy subjects with functional constipation were enrolled to the study site in Berlin, Germany. Subjects aged 18–70 with functional constipation according to ROME III criteria having two-to-four bowel movements per week during the preceding months were included [4, 16]. They were asked to adhere to their former diet and physical activity and had to be used to consuming at least 300 mL of water (incl. tea). Women of childbearing potential had to agree to use contraception methods. The exclusion criteria were as follows: acute gastritis and enteritis, bleeding tendency and risk of rupture in the intestinal tract, disorders in motility and secretion in the digestive tract, acute or chronic disease of the gastrointestinal tract, irritable bowel syndrome, abdominal pain, abdominal surgery within the last 6 months prior to study start, known pelvic floor dysfunction, susceptibility to development of kidney stones, hyperresorptive hypercalciuria with urinary stones, urinary infections with E. coli, renal insufficiency, acute or chronic kidney or urinary tract disease, alkalosis, severe respiratory disease, cardiovascular system insufficiency, acute inflammatory diseases, dehydrated conditions, restricted fluid tolerance, acute or chronic neurological or psychiatric illness, weight loss of ≥3 kg within the last 3 months prior to the study, clinically relevant excursions of laboratory parameters, BMI > 35 kg/m2, thyroid dysfunction, known sensitivity to the ingredients of the product, use of any preparations that could affect the gastrointestinal tract during the last 2 weeks and during the study (except rescue medication, a bisacodyl suppository that could be used in case of no bowel movements for 4 days; max. four suppositories were permitted during the entire study), use of sympathomimetics and cardiac glycosides, supplementation of magnesium, vitamins or other minerals during the study, intake of mineral water other than the study product during the study, pregnancy or nursing, drug, alcohol or medication abuse, participation in another clinical trial during the last 30 days prior to study start, relationship with or dependence on the sponsor or the investigator, problems with complying with or following the protocol due to language difficulties, or commitment to an institution by virtue of an order issued either by the judicial or the administrative authorities.
Furthermore, subjects had to fulfil the following randomisation criteria during the run-in period before obtaining the investigational product (assessed per subject diary): two-to-four bowel movements per week and intake of at least 300 mL water (incl. tea) daily.
During the study period of 6 weeks, the subjects consumed their daily dose of Donat Mg natural mineral water or placebo in two portions: prior to breakfast and in the evening before dinner. Donat Mg natural mineral water is derived from a spring in Rogaska Slatina, Slovenia. It is enriched by minerals from dissolving rocks 280-600 m under ground providing Donat Mg with 13 g/L of dissolved mineral substances. Both the mineral water and the placebo were produced and bottled by Droga Kolinska, d.d (Slovenia). Main ingredients of Donat Mg natural mineral water are sodium (1600 mg/L), magnesium (1000 mg/L), calcium (370 mg/L), sulphate (2000 mg/L), and hydrogen carbonate (7600 mg/L). The placebo was sparkling water derived from another spring in Rogaska Slatina with a low content of minerals (<1 mg/L sodium, 30 mg/L magnesium, 73 mg/L calcium, 17 mg/L sulphate, 390 mg/L hydrogen carbonate) and a quantity of CO2 (3.5 g/L) comparable to the active product.
The clinical phase of the study included a run-in period of 10 ± 2 days and an intervention period of 6 weeks ± 3 days. A total of five visits were performed: a screening visit, a baseline visit (after the run-in period), a telephone visit 7 ± 3 days after baseline, a control visit 21 ± 3 days after baseline and the final visit 42 ± 3 days after baseline.
Compliance was checked by counting returned unused investigational product and assessing the trial duration. The accepted compliance rate was defined as 75–125 % of the correct quantity of investigational product and max. 3 days deviation from the 6-week study period.
The primary outcome was the subject-rated change in the number of complete spontaneous bowel movements (CSBMs) per week between placebo and active over the course of the study. A CSBM was defined as a bowel movement with sensation of complete evacuation and with no laxative/enema in the 24 h preceding the bowel movement. In addition, the number of overall bowel movements (BM), the number of spontaneous bowel movements (SBM, bowel movements with no laxative/enema in the 24 h preceding the bowel movement) and the number of complete bowel movements (CBM, defined as bowel movements with a sensation of complete evacuation) were assessed. Subjects were asked to record their stool consistency using Bristol Stool Form Scale (BSFS) questionnaire .
Subjects had to document their daily bowel movements, the respective stool form, the sensation of complete evacuation following defecation (yes/no) and whether they had used any rescue medication in a daily diary. In addition, subjects had to fill in the questionnaires Gastrointestinal Symptoms Rating Scale (GSRS) , the Short Form 12 Health Survey Questionnaire (SF-12)  and the short form of the international physical activity questionnaire (IPAQ-SF) at each visit post screening . Eating and drinking habits were recorded in a diary on 3 days of each week (to calculate the mean answer to the items regarding intake of certain foods/day, the following score was used: none = 1, once = 2, twice or three times = 3, more than three times = 4; the intake of liquids was recorded in mL). The efficacy of the investigational products was evaluated by the participants and the investigator independently at the end of the study by means of a global-scaled evaluation with “very good”, “good”, “moderate” or “poor”.
Biochemical parameters such as liver function and lipid parameters as well as blood pressure and heart rate were assessed at screening and at the end of the intervention.
Adaptive design and statistics
The adaptive interim analysis was scheduled to take place when data from 50 % of originally planned number of subjects were available . The analysis was done by an independent statistician using the full analysis set (FAS). An Independent Data Monitoring Committee (IDMC) was in place to provide appropriate recommendations to the trial sponsor while all staff involved in the conduct of the trial remained blind to the results of the interim analysis. Two sets of one-sided confirmatory hypotheses were planned to be tested at the interim and final analysis for the primary efficacy variable dCSBM in each of the dose groups (i.e. 300 mL each verum or placebo; 500 mL each verum or placebo) (Table 1).
The trial results were analysed according to a two-stage adaptive group sequential design with one interim analysis using O’Brien and Fleming stopping boundaries . Each hypothesis was tested (at the interim and final analysis) with the nonparametric one-sided Mann–Whitney U test. The inverse-normal method was used to combine the p values for each of the two sets of one-sided confirmatory hypotheses .
The overall experiment-wise significance level [i.e. the family-wise error rate (FWER)] was set to = 0.025 (one-sided). Adjustment of the significance level was performed to control the overall type I error = 0.025 (one-sided). In the interim analysis, the null hypotheses could not be rejected for any of the dose levels. The IDMC recommended stopping the low-volume-dose groups and to adjust the sample size for the higher dose. The final analysis of the primary endpoint was only performed for the 500-mL dose with the test statistic T2 critical value to reject the null hypothesis of 1.977. The secondary outcome parameters were tested in the exploratory sense using the nonparametric procedures Mann–Whitney U test, Wilcoxon test and Chi-square test. Changes in clinical parameters over time were analysed by using exploratory ANOVA. The analysis was performed using the FAS and at least for the primary outcome in addition using the valid case analysis set (VCAS).
Sample size estimation
For the sample size estimation before the interim analysis, the calculation was based on the assumption of an effect size of 0.7 for the lower dose and 1.0 for the higher dose. The sample size re-estimation for the study continuation with the higher dose was calculated based on the target conditional power using the procedure suggested by Chang . The initial estimates and the observed treatment difference and standard deviation at the interim analysis were considered. The conditional power was set to 80 %. Critical value α2 for the final analysis was set to 0.0240.