Humic Acids in Patients with Diarrhoea-Predominant Irritable Bowel Syndrome: Results from A Randomised Controlled Trial

Structured Summary Irritable bowel syndrome (IBS) is the most common functional gastrointestinal disorder. There is no definitive treatment for IBS and all medications implemented in its treatment have concerns of tolerability and safety associated with their use. The WH67®-R-2012 study was a randomised, double-blind, placebo-controlled clinical trial comparing the effect of humic acids versus placebo in patients with diarrhoea-predominant IBS (IBS-D). The primary endpoint of the study was an improvement of IBS symptoms measured with the irritable bowel syndrome severity scoring system (IBS-SSS). Secondary endpoints were improvements in quality of life (IBS-QOL), the Bristol Stool Form Scale (BSFS), and in the short form 36 (SF-36). Forty-six patients were recruited to participate in the study. No adverse events were reported and no patient discontinued the study. There was no main effect for treatment (F=1.56, p=0.221), centre (F=1.71, p=0.201), or sex (F=2.47, p=0.127). We defined a response to treatment as an improvement of at least 50 points on the IBS-SSS. 57.9% of patients responded to treatment with humic acids and 36.8% of patients responded to treatment with placebo (χ 2 =1.69, p=0.194). In patients receiving humic acids, women were more likely to be responders than men (71.4% vs. 20.0%, χ 2 =4.00, p=0.045). This was the first study testing the effect of humic acids as a potential treatment for IBS-D. No adverse events were reported, reflecting the very good tolerability of humic acids. If proven efficacious in a larger study, humic acids could represent a novel treatment option, which is much-needed for IBS-D. Humic Acids in Patients with Diarrhoea-Predominant Irritable Bowel Syndrome: Results from A Randomised Controlled Trial.


Introduction
Irritable bowel syndrome (IBS) is the most common functional gastrointestinal disorder [1]. Exact prevalence rates vary depending on diagnostic criteria [2]. A recent study found a prevalence rate of 16.6% in a German sample diagnosed according to Rome III criteria [3]. Hallmarks of IBS are visceral hypersensitivity and an altered bowel habit [1]. This results in chronic gastrointestinal symptoms including pain, discomfort, bloating, and stool irregularities such as constipation and diarrhoea [2]. The discomfort level caused by these symptoms leads people affected to seek medical attention and negatively impacts on their quality of life [2,4,5]. IBS can be classified into a diarrhoea-predominant (IBS-D), constipationpredominant (IBS-C), and alternating form (IBS-A) according to the predominant stool pattern, [1] with IBS-D being the most prevalent form [8]. IBS-D may also be the subtype with the most detrimental effect on quality of life [6]. IBS is the result of a disturbed gut-brain axis and studies have found evidence for dysbiosis in IBS [7]. Other proposed disease mechanisms include autonomic dysfunction, altered signalling to and processing in the central nervous system, altered immune function, and altered endocrine markers including CRF, cortisol, and glucagon-like peptide 1 [1]. Risk for developing IBS is higher after travellers' diarrhoea infection, [3] in women [8] and in younger people. Comorbidities are common in people with IBS. Examples of common somatic comorbidities are pain syndromes, overactive bladder, and migraine [9]. Common psychiatric comorbidities include depression and anxiety.
To avoid unnecessary cost and suffering incurred by IBS, speedy and accurate diagnosis are crucial. Direct costs per IBS patient per year are 791 euros in Germany, with indirect costs peaking at 995 euros per patient per year [10]. People suffering from IBS are more likely to undergo abdominal surgery, especially appendectomies, cholecystectomies, or hysterectomies [11]. Currently, there is no definitive treatment for IBS [12]. Given the differences in symptomatology, it also seems unlikely that one single treatment will be able to cure all forms of IBS. Medications used in the treatment of IBS often act symptomatically and include antispasmodics and antidepressants, especially tricyclic antidepressants [12][13][14]. For IBS-D, use of antidiarrheal agents like loperamide is common. Antibiotics [12] and probiotics [15][16][17][18] have also been used and proved effective in some instances. All these medications have concerns of tolerability and safety associated with their use and for most substances there is only limited evidence supporting their use [19].
Many patients request natural remedies from their gastroenterologist [20] and many IBS patients use complementary and alternative medicine [21,22]. Humic acids are such natural remedies that have been used since ancient times [20]. At present, they are mostly used in veterinary medicine to treat intestinal diseases [23,24]. Humic acids are complex organic substances mainly present in soil, but also in ground water [20,25,26]. They are generated through humification, a process of decomposition of organic material by microbial transformation [20,27]. Humic substances consist of polycyclic phenolic compounds [26].
Furthermore, humic acids have been shown to have an impact on the human microbiome, increasing most bacterial groups while leaving the individual microbial profile unchanged and microbial diversity intact [20]. Antibacterial properties of humic acids have been ascribed to quinones present in humic acids, while phenolic and carboxylic groups are probably responsible for antiinflammatory and antioxidant properties [25]. In general, it is assumed that humic acids act locally [24], which is supported by a recent study in adult pigs [23]. In Germany, only one standardised and registered humic acids product is available [20]. According to the summary of product characteristics, Activomin ® can be used to improve the subjective feeling of well-being in gastrointestinal disorders [37]. There are no known side effects of the product, and humic acids in general have a low oral toxicity [24].

Methods
The WH67 ® -R-2012 study was a randomised, double-blind, placebo-controlled clinical trial comparing the effect of humic acids (Activomin ® , WH Pharmawerk Weinböhla GmbH, Weinböhla, Germany) versus placebo in patients with IBS-D. The study was registered on the German Clinical Trials Register (DRKS00005183) and was approved by the Ethics Committee of the State Chamber of Physicians of Saxony in Germany (EK-BR-37/13-1). All patients provided their written informed consent to participate in the study.

Patients
Patients were eligible to participate in the study if they were diagnosed with IBS-D according to Rome III criteria, were at least

Questionnaires
Symptom severity was assessed using the irritable bowel severity scoring system (IBS-SSS) [38,39]. It consists of five questions rated on a visual analogue scale ranging from 0 to 100, yielding a maximum total score of 500. Patients can be classified into mild (75-<175), moderate (175-<300), and severe (>300) IBS. A change of 50 on the IBS-SSS predicts a significant improvement [39]. Quality of life (QOL) was assessed using the IBS-QOL measure [40]. It consists of 34 items specific to IBS and offers high internal consistency and high reproducibility. To assess the effect of humic acids on stool form, we used the Bristol Stool Form Scale (BSFS). It provides qualitative information on stool form, classifying it into seven different categories. It can be used to detect changes in gastrointestinal function and is reflective of intestinal transit time [41]. vitality, and general health perceptions [42]. Furthermore, medication intake, number of bowel movements, abdominal pain, and discomfort were assessed using a patient diary.

Procedures
Patients were screened two weeks before the first assessment.
During this screening, inclusion and exclusion criteria, demographic data, medical history including current medications, and scores

Clinical Endpoints
The primary endpoint of the study was an improvement of IBS symptoms measured with the IBS-SSS. Secondary endpoints were improvements in the IBS-QOL, BSFS, and in the SF-36. Further secondary endpoints were adverse events, the number of patients who discontinued the study, and the use of rescue medication (bisacodyl, acetaminophen, loperamide). As an exploratory endpoint, response to treatment was defined as a 50-point improvement in the IBS-SSS.

Statistical Analyses
Prior to the study, a power analysis was performed using an alpha of 0.05 and a power of 1beta=0.80, assuming a clinically relevant difference of 50 points 38  Forty-six patients were recruited to participate in the study.
Eight patients reported BSFS values of 1-3 at the first laboratory visit, reflecting solid stool forms. Since this is unexpected for IBS-D, these patients were excluded from further analysis, leaving 38 evaluable patients. The patient flow is depicted in Figure 1 and baseline demographic and disease characteristics are shown in     More patients in the humic acids group responded to treatment than in the placebo group; however, this difference was not statistically significant (57.9% vs. 36.8%, χ 2 =1.69, p=0.194).

Discussion
This randomised double blind placebo controlled clinical trial failed to demonstrate a benefit for the treatment of IBS-D using humic acids over placebo. Unexpectedly, we found an interaction between treatment and centre, which showed contrary treatment responses in the two major centres. Hence, we decided to conduct a responder analysis using a 50-point-decrease on the IBS-SSS as clinically meaningful response. Women suffering from IBS-D were more likely to respond to treatment with the humic acid preparation Activomin ® than men and more women responded to treatment with humic acids than with placebo. No adverse events were reported, reflecting the very good tolerability of humic acids which has also been observed elsewhere [20,27] and which is reflected in their low oral toxicity [24].
Humic acids have long been used in veterinary medicine to treat gastrointestinal diseases [43,44]. However, to our knowledge, this was the first study testing the effect of humic acids as a potential treatment for IBS-D in humans. In two prior studies, a prebiotic-probiotic compound consisting of leonardite, a source of humic acids, and several strains of commensal bacteria, was able to alleviate symptoms of IBS [45,46]. In this compound, leonardite is used as a prebiotic for the bacteria contained in the product [47]. In the light of findings that humic acids can increase the concentration of colonic microbiota, [24] a prebiotic effect is one possible mechanism through which humic acids might improve IBS symptoms. It has been suggested that prebiotics could be a useful future direction for the development of therapies for IBS, [21] as dysbiosis was also found to be present in IBS [7]. Another potential mechanism of action for humic acids is a general protection of the intestinal mucosa, [27] which could be provided by humic acids coating the intestinal wall [48]. Lastly, since low-grade inflammation has been suggested as one disease mechanism of IBS, [49] the antiinflammatory effect of humic acids could be beneficial [31][32][33][34][35][36] Women were more likely to respond to treatment than men.
This higher propensity of women to react to treatment with humic acids could also provide an explanation for the interaction between treatment and centre, which was observed in the ANCOVA. The centre where the decrease in IBS-SSS values was greater in the group that received humic acids had a higher proportion of women (84.2% vs. 63.2%). Greater response to treatment in women has also been observed with serotonergic agents, [53] although this finding has recently been challenged by a new study [54]. The response to placebo was high in the present study. A high response rate to placebo can generally be observed in IBS [55,56].
One study found placebo and a probiotic to equally lower IBS-SSS scores to the extent of around 50 points [57]. Even open-label administration of placebo can produce significant improvements in IBS-SSS compared to no medication when the same level of physician interaction is provided [58]. The results of this study are limited by its small sample size. Subsequently the sample included only a very low number of men; this was also due to the lower prevalence of IBS among men [8]. should also focus on potential differential effects of humic acids on women and men.