Prof. G. R. Corazza, Clinica Medica I, IRCCS Policlinico San Matteo, Università di Pavia, Piazzale Golgi 5, 27100 Pavia, Italy. E-mail: firstname.lastname@example.org
Background : Butyrate exerts anti-inflammatory effects in experimental colitis and on Crohn's disease lamina propria mononuclear cells in vitro.
Aim : To explore the efficacy and safety of oral butyrate in Crohn's disease.
Methods : Thirteen patients with mild–moderate ileocolonic Crohn's disease received 4 g/day butyrate as enteric-coated tablets for 8 weeks. Full colonoscopy and ileoscopy were performed before and after treatment. Endoscopical and histological score, laboratory data, Crohn's disease activity index and mucosal interleukin (IL)-1β, IL-6, IL-12, interferon-γ, tumour necrosis factor-α and nuclear factor-kappa B (NF-κB) were assessed before and after treatment.
Results : One patient withdrew from the study, and three patients did not experience clinical improvement. Among the nine patients (69%) who responded to treatment, seven (53%) achieved remission and two had a partial response. Endoscopical and histological score significantly improved after treatment at ileocaecal level (P < 0.05). Leucocyte blood count, erythrocyte sedimentation rate and mucosal levels of NF-κB and IL-1β significantly decreased after treatment (P < 0.05).
Conclusions : Oral butyrate is safe and well tolerated, and may be effective in inducing clinical improvement/remission in Crohn's disease. These data indicate the need for a large investigation to extend the present findings, and suggest that butyrate may exert its action through downregulation of NF-κB and IL-1β.
Butyrate, a short-chain fatty acid (SCFA) produced by bacterial fermentation of dietary fibres, plays a crucial physiological role in maintaining the health and integrity of the intestinal mucosa by regulating the balance between epithelial cell proliferation, differentiation and apoptosis.1 Apart from its well-known growth-preventing and apoptosis-inducing roles in neoplastic colonocytes,2 butyrate has been shown to reduce inflammation in experimental colitis,3 and to exert in vitro trophic and anti-inflammatory effects in normal colonocytes.4 On this basis, it has been topically administered to patients with distal ulcerative colitis with overall favourable results.4–8
Despite these observations, the mechanisms underlying the role of butyrate in preventing intestinal inflammation have yet to be clarified. Although its therapeutic benefit in ulcerative colitis has been attributed to the restoration of energy metabolism in colonocytes,9 recent observations do not support this mechanism, at least regarding the primary nature of the deficiency.10 Conversely, other studies have demonstrated that butyrate exerts immunomodulatory effects by downregulating T-helper cell (Th1) responses,11, 12 by modulating antigen presentation associated molecules,13 and by inhibiting vascular cell adhesion molecule-1-mediated leucocyte adhesion to endothelial cells.14
Recently, the demonstration in vitro that butyrate decreases proinflammatory cytokine expression by lamina propria mononuclear cells of Crohn's disease patients via inhibition of nuclear factor-kappa B (NF-κB) activation and inhibitor protein-kappa B (IκB) degradation has provided a rationale for its use in the treatment of Crohn's disease.15 However, to date, no in vivo study has been performed to address the therapeutic action of butyrate in this condition. In the present uncontrolled pilot study, we explored for the first time the efficacy of oral butyrate in inducing remission or an improvement of intestinal inflammation in mildly to moderately active Crohn's disease. Moreover, to better elucidate the immunological mechanisms underlying the actions of butyrate, we investigated the levels of NF-κB and proinflammatory cytokines interleukin (IL)-1β, IL-6, IL-12, interferon (IFN)-γ and tumour necrosis factor (TNF)-α in the intestinal lesions of Crohn's disease patients before and after butyrate administration.
Patients and Methods
Thirty-five Crohn's disease patients were screened for this study. Of them, 13 were enrolled in the study and 22 were excluded. The conditions for exclusion were: (i) complications (stenosis, strictures, fistulas or abscesses), (ii) patients experiencing first attack of the disease, (iii) involvement that was only ileal or colonic or extending more than the hepatic flexure, and (iv) previous resective surgery. In addition, subjects treated with oral or topical steroids, antibiotics or azathioprine in the 3 months before entry into the study, or who had ever received ciclosporin, methotrexate or infliximab were also excluded. The 13 patients (mean age 40.4 years, range 24–79) invited to participate had a Crohn's disease activity index (CDAI) of between 220 and 320, signifying mild to moderate disease. Diagnosis of Crohn's disease was ascertained according to the usual clinical criteria,16 and the site and extent of the disease were confirmed by endoscopy, histology and enteroclysis in all patients.
Treatment and assessment
Crohn's disease patients received 2 g butyrate twice a day for 8 weeks as enteric-coated tablets designed to release the active constituent in the terminal ileum and colon (Promefarm s.r.l., Milan, Italy). In vitro tests showed that coating resists disintegration at pH 1–6 and dissolves at pH > 7. All patients had had a relapse during maintenance treatment with oral mesalazine 2.4 g/day, and this treatment was continued at the same dose throughout the study. No other medication was allowed, and oral or topical steroids, antibiotics or azathioprine were discontinued at least 3 months earlier.
Full colonoscopy and ileoscopy were performed immediately before the start of the therapy and 8 weeks later, with collection of multiple size-appropriate intestinal biopsy specimens. Before treatment, samples were taken from three different locations, namely terminal ileum, caecum and ascending colon, and after treatment they were collected from the same areas as before treatment. Some biopsy samples were processed according to standard methods for traditional histology, others were homogenized for the determination of NF-κB and cytokine levels. Both endoscopical and histological investigations were carried out by physicians and pathologists unaware of the patient's treatment. The endoscopical and histological scoring system was graded from 0 to 3 as previously described.17 Laboratory data including leucocyte blood count, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), orosomucoid and fibrinogen were obtained within 1 week of entry into the investigation and after 8 weeks of treatment.
Disease activity was assessed by CDAI. Patients with scores below 150 were classified as being in remission, whereas those with scores over 450 had severe disease.16 CDAI was determined in the week before the start of therapy, after 4 and 8 weeks of treatment, and 2 weeks after the end of therapy. Clinical response, defined as a decrease in CDAI by at least 70 points, or complete remission, defined as a CDAI < 150 points, were the primary endpoints of the study. All the clinical features of patients are reported in Table 1.
Table 1. Clinical data for patients with Crohn's disease
After 4 weeks of treatment, patients were evaluated clinically, and in the case of clinical worsening of the disease they were immediately withdrawn from the study and then, considered as treatment failures. Only patients who experienced clinical response or remission were followed up for further 2 weeks after the end of treatment, as suggested by the European Medicine Agency guidelines.18
Each patient who took part in the study was enrolled after appropriate local Ethics Committee approval and informed consent was obtained in all cases.
Cytokines were determined on mucosal biopsy samples collected from terminal ileum. Two mucosal biopsy specimens were added to 0.3 mL of Tris–HCl 20 mmol/L pH 7.4, containing 1 μmol/L phenylmethylsulphonyl fluoride, 10 μmol/L leupeptin, 10 μmol/L pepstatin and 1 μmol/L aprotinin and homogenized using a hand-held Potter-Elvehjem tissue grinder (Phoenix Equipment Inc., Rochester, NY, USA) and by sonication (5 W, 80% output, 1 min and 50 s, altering 10 s sonication and 10 s pause) with a sonicator (Vibracell; Sonic and Materials Inc., Danbury, CT, USA). The amount of protein in mucosal homogenates was determined by the Coomassie protein assay (Bio-Rad Laboratories, Hemel Hempstead, UK), and 40 μL of each lysate sample was used for each assay. IL-1β, IL-6, IL-12, IFN-γ, TNF-α and NF-κB levels were measured by ELISA (enzyme-linked immunosorbent assay) using the human IL-1β ELISA kit, the human IL-6 ELISA kit, the human IL-12 ELISA kit, the human IFN-γ ELISA kit, the human TNF-α ELISA kit (all from Chemicon, Temecula, CA, USA), and the NF-κB activation ELISA kit (Oxford Biomedical Research, Oxford, MI, USA), respectively, according to the manufacturers’ instructions, and data were expressed as pg/mg protein. The NF-κB activation ELISA kit employed an oligonucleotide containing the DNA-binding NF-κB consensus sequence. NF-κB present in the samples bound specifically to the oligonucleotide coated on the plate, and DNA-associated NF-κB was selectively recognized by the primary antibody (p50 and p105 specific). Secondary antibody–alkaline phosphatase conjugate and chemiluminescent substrate were applied and plates were read in a chemiluminometer (Ciba-Corning Magic Lite Analyser; LabX, Midland, ON, Canada).
Data were analysed in the GraphPad Prism statistical PC program (GraphPad Software, San Diego, CA, USA) by means of the non-parametric Mann–Whitney U-test. Correlations were studied by Spearman's rank correlation test. A level of P < 0.05 was considered statistically significant.
There was one dropout because of clinical worsening at week 3. Table 1 shows that of the 12 Crohn's disease patients who concluded the study three patients did not experience clinical improvement after treatment, and nine patients (69%) responded to treatment – seven of them (53%) with complete response and two with partial response. The median pre-treatment CDAI (252, range 223–306) decreased significantly (P < 0.0001) to 162 (range 76–247) after 4 weeks and to 140 (93–263) after 8 weeks. The median CDAI value remained unchanged, 2 weeks after the end of therapy (143, range 92–240). Figure 1 shows the CDAI score assessed before and after 4 and 8 weeks of treatment and 2 weeks after the end of treatment in the 12 Crohn's disease patients who completed the study.
Administration of butyrate was well tolerated in all patients and no adverse side-effects were observed. Compliance was evaluated on the basis of the number of tablets returned by the patients at the end of the study. As no patient returned >2% of the tablets, compliance was thus considered excellent.
In Table 2, the results of the laboratory investigations show that both leucocyte blood counts and ESR decreased significantly after 8 weeks of treatment in comparison with pre-treatment findings. However, no significant difference was found in the serum levels of orosomucoid, fibrinogen and CRP before and after treatment. In particular, the lack of a change in CRP may be a result of the only modestly raised concentrations prior to treatment, which is consistent with the mild–moderate disease activity in our uncomplicated patients. However, it is noteworthy that CRP concentrations were found to be significantly (rs = 0.61, P = 0.0251) correlated with CDAI scores before treatment. Both endoscopical and histological score significantly improved after treatment at the level of the terminal ileum and caecum, but not the ascending colon (Table 2). Among the proinflammatory cytokines that were measured in intestinal biopsies, IL-1β turned out to be significantly decreased after treatment. A favourable downward trend was observed for IL-6 and IL-12 levels, but this did not reach statistical significance. Neither IFN-γ nor TNF-α levels changed before and after therapy. The NF-κB was significantly lower after treatment when compared with pre-treatment mucosal levels (Table 2). No correlation was found between the mucosal levels of all the cytokines determined or NF-κB and CDAI.
Table 2. Laboratory, pathological and immunological findings for the 12 Crohn's disease patients who concluded the study at entry and after 8-week treatment
In the present study, we demonstrated, for the first time that butyrate administered orally at a dose of 4 g/day for 8 weeks is effective in inducing clinical improvement and/or remission, and in downregulating ileocaecal inflammation in mildly to moderately active Crohn's disease.
It has been suggested that chronic intestinal inflammation may result from either the inability of colonocytes to oxidize SCFA (in ulcerative colitis)9 or from decreased luminal concentrations of SCFA (in diversion colitis).19 Therefore, the use of SCFA has been proposed in gut inflammation to provide a readily available energy source for colonocytes, so as to favour the healing processes of the colonic mucosa.1 Several controlled and uncontrolled trials in the past few years have explored the efficacy of topical butyrate in the treatment of distal ulcerative colitis,4–8 but just recently the demonstration that butyrate exerts anti-inflammatory effects in vitro on intestinal lamina propria mononuclear cells from Crohn's disease patients has raised the possibility that butyrate may be used in the treatment of Crohn's disease.15
Our data showed that oral administration of butyrate for 8 weeks induces a clinical improvement in 69% of Crohn's disease patients, together with a significant decrease of CDAI, leucocyte blood count and ESR. It is noteworthy that remission was achieved in 53% of patients and still persisted 2 weeks after the end of therapy. Both endoscopical and histological examinations showed a significant improvement of mucosal inflammation in the terminal ileum and caecum after treatment, but not in the ascending colon, thus suggesting that the active substance was released mainly at the ileocaecal level.
As reported in a previous trial on ulcerative colitis,20 the oral administration of butyrate at a daily dose of 4 g, which corresponds to 35–60% of the normal daily intracolonic production of butyrate,21 was found to be safe in our patients, and no side effects were observed. Since all patients had had a relapse during maintenance treatment with oral mesalazine at the dose of 2.4 g/day, this treatment was maintained constant throughout the study.
To better define the mechanisms whereby butyrate suppresses inflammation in Crohn's disease, we investigated, before and after treatment, the mucosal levels of NF-κB and proinflammatory cytokines, both shown to be negatively regulated by butyrate in the colonic mucosa.12, 22–24 The transcription factor NF-κB, which plays a central role in inflammatory responses and is involved in the transcriptional regulation of many proinflammatory cytokine genes, has been proposed as a good target for the treatment of inflammatory bowel disease.25 Recently, several reports have described the ability of butyrate to interfere with the activity of NF-κB in the colon.15, 22, 23 Our in vivo study showed a significant reduction of NF-κB and IL-1β after butyrate administration in Crohn's disease patients, thus confirming the anti-inflammatory effects of butyrate reported in vitro on Crohn's disease lamina propria mononuclear cells by Segain et al.15 Moreover, we observed downregulation of IL-6 and IL-12 mucosal levels, although this did not reach statistical significance. However, we cannot exclude the possibility that this may depend on a type II statistical error because of the small sample of patients enrolled in our monocentric study. On the contrary, no difference was found in TNF-α and IFN-γ levels before and after butyrate treatment.
Although data emerging from the present uncontrolled pilot study do not allow definite conclusions to be drawn, because of the low statistical power of the protocol and the absence of a treatment control arm, they do provide encouragement to further investigate, in controlled trials on larger series of patients, the efficacy of butyrate in Crohn's disease, in order to ascertain whether this agent may effectively represent an alternative therapeutic strategy in this condition or may increase the response rate in a combined regimen.
We gratefully acknowledge the contribution made to the study by Promefarm s.r.l. that provided all the drugs used and monitored the conduct of the study. However, the analysis and conclusions are those of the clinical authors and have not been modified by the company.