The breakdown of glycosaminoglycans is an important consequence of inflammation at mucosal surfaces, and inhibition of metalloprotease activity may be effective in treating chronic inflammation.
The breakdown of glycosaminoglycans is an important consequence of inflammation at mucosal surfaces, and inhibition of metalloprotease activity may be effective in treating chronic inflammation.
To report an alternative approach, using the nutriceutical agent N-acetyl glucosamine (GlcNAc), an amino-sugar directly incorporated into glycosaminoglycans and glycoproteins, as a substrate for tissue repair mechanisms.
GlcNAc (total daily dose 3–6 g) was administered orally as adjunct therapy to 12 children with severe treatment-resistant inflammatory bowel disease (10 Crohn’s disease, 2 ulcerative colitis). Seven of these children suffered from symptomatic strictures. In addition, similar doses were administered rectally as sole therapy in nine children with distal ulcerative colitis or proctitis resistant to steroids and antibiotics. Where pre- and post-treatment biopsies were available (nine cases), histochemical assessment of epithelial and matrix glycosaminoglycans and GlcNAc residues was made.
Eight of the children given oral GlcNAc showed clear improvement, while four required resection. Of the children with symptomatic Crohn’s stricture, only 3 of 7 have required surgery over a mean follow-up of > 2.5 years, and endoscopic or radiological improvement was detected in the others. Rectal administration induced remission in two cases, clear improvement in three and no effect in two. In all cases biopsied there was evidence of histological improvement, and a significant increase in epithelial and lamina propria glycosaminoglycans and intracellular GlcNAc.
GlcNAc shows promise as an inexpensive and nontoxic treatment in chronic inflammatory bowel disease, with a mode of action which is distinct from conventional treatments. It may have the potential to be helpful in stricturing disease. However, controlled trials and an assessment of enteric-release preparations are required to confirm its efficacy and establish indications for use.
Remodelling of the extracellular matrix is an inevitable consequence of inflammation. The central role of fibroblasts in determining the outcome of either tissue restoration or scarring is now recognized to be of central importance.1 In both Crohn’s disease and ulcerative colitis there is particularly widespread breakdown of sulphated glycosaminoglycans (GAGs), related to the infiltration of activated macrophages.2 This action is not specific to inflammatory bowel disease, and is not only inducible in vitro in cell monolayers or whole organ culture,3, 4 but also occurs within the lung in neonatal respiratory distress syndrome.5 In all these cases the release of tumour necrosis factor-α (TNF-α) appears to play an important role.6–9 The molecular basis of TNF-associated matrix breakdown is by its induced expression of matrix-degrading metalloproteases (MMPs),10 and TNF blockade abrogates MMP-mediated GAG degradation and tissue damage in vitro.8 This may contribute to the efficacy of anti-TNF therapy in inflammatory bowel disease.11
Breakdown of sulphated GAGs is not only a common inflammatory response, but will also lead inevitably to a range of predictable physiological consequences including vascular leak, microthrombosis and alterations in cellular proliferation.12, 13 The initial response by fibroblasts is the production of a provisional matrix dominated by hyaluronan and dermatan sulphate, both of which may have pro-inflammatory as well as pro-fibrotic effects.14–16 If primary repair is not then possible, fibroblasts alter their matrix secretion pattern in response to macrophage cytokines to produce collagens, resulting in scarring.1, 13, 17
It is likely that inhibition of matrix degradation may be of benefit in a variety of conditions. Therapeutic inhibition of MMPs by their natural antagonists, tissue inhibitors of metalloproteases (TIMPs), has indeed been shown to prevent tissue damage in experimental allergic encephalomyelitis, despite vigorous ongoing inflammation.18 Active inflammatory bowel disease is characterized by the relative excess of mucosal MMPs over TIMPs,19 and thus the therapeutic use of TIMPs would appear to be an attractive option for limiting matrix degradation. These inhibitors are now the subject of intense research activity, but are likely to be expensive and unavailable for some years.
We report a pilot study of an alternative approach to the treatment of matrix degradation in inflammatory bowel disease. Instead of inhibiting MMP action, we attempted to augment tissue repair mechanisms by the administration of a metabolic fuel for fibroblast and epithelial GAG synthesis, N-acetyl glucosamine (GlcNAc; Figure 1). This naturally occurring amino sugar, which is derived from crab shell chitin, is both inexpensive and without known toxic effects. It is avidly taken up in vitro, in preference even to glucosamine, by inflammatory bowel disease tissue.20 In addition to its role in the biosynthesis of GAGs, it may potentially augment epithelial defences by contributing to mucin biosynthesis.21 In addition, there is intriguing recent evidence showing that GlcNAc also acts intracellularly as an antagonist of O-phosphorylation and may thus regulate many inflammatory pathways.22 Nutriceuticals such as GlcNAc have been demonstrated to have therapeutic effects in arthritis,23, 24 but there have been no previous reports of their use in intestinal inflammation.
Although GlcNAc is without known adverse effects, and is available without physician prescription in North America, it has not been used in paediatric inflammatory disease. We therefore erred on the side of caution, selecting children with treatment-resistant disease, and initially using low doses. GlcNAc powder, derived by enzymatic degradation of crab shell chitin, was supplied under licence by Glucogenics Pharmaceuticals, Toronto, Canada and was reconstituted locally by dissolving it water. Its therapeutic administration to children with chronic inflammatory bowel disease resistant to treatment was approved by the Local Research Ethics Committee. Informed consent was obtained from all parents and children, and pre- and post-treatment biopsies were taken when possible, in accordance with our standard clinical practice.
GlcNAc therapy was considered for two main groups:
1. Single-agent rectal administration for children with distal colitis or inflammation of the rectal stump after colectomy for ulcerative colitis, with a view to obtaining histological evidence of efficacy. The patients were selected on the basis of intractable symptoms that had failed to respond to rectal steroids and at least one other therapy.
2. Oral administration for children with extensive or severe inflammatory bowel disease that was resistant to conventional therapy. Because of our theoretical consideration that GlcNAc therapy might retard the further development of fibrosis, this group included several children with severely symptomatic strictures who would ordinarily have been referred urgently for surgical assessment. Because of the inclusion criteria of disease severity and chronicity, many of these children continued their regular treatments during the period of GlcNAc administration.
GlcNAc powder was reconstituted in water to a concentration of 1–1.5 g in 10 mL. Total daily doses of 3–6 g were given in three divided doses for both oral and rectal administration. Because of the known extensive first-pass clearance by the liver, the orally administered GlcNAc was held against the buccal mucosa for 1–2 min before swallowing. GlcNAc enemas were administered by the children or their parents using a Jacques catheter and were well-tolerated by all but one child (not shown) who discontinued therapy immediately because of pain on catheter insertion, and had previously refused rectal steroids for similar reasons.
We treated nine children with rectal inflammation resistant to steroids and other therapies using rectally administered GlcNAc (1.5–2.0 g in 10 mL water twice daily). For eight of these, children with therapy-resistant proctitis or distal colitis, GlcNAc was administered rectally as single therapy (clinical details are given in Table 1). In five cases (four ulcerative colitis, one Crohn’s) the rectum had been defunctioned following colectomy, and thus the primary pathology was a combination of inflammatory bowel disease and diversion colitis. Two other cases were of distal ulcerative colitis (one with ileo-anal pouchitis) and there was one case of ulcerative proctitis. The ninth case received oral and rectal GlcNAc for severe Crohn’s colitis with anal stricture (Table 2, patient 12), in addition to other treatments.
The patients had previously been treated unsuccessfully with other therapies including topical steroids (9/9), oral or topical aminosalicylates (7/9), antibiotics (7/9), topical ciclosporin (3/9), long-chain fatty acids (1/9) and oral interferon-α (1/9). Proctoscopic biopsies before and after 6 weeks of treatment were obtained in six cases (four rectal stump inflammation, one distal ulcerative colitis and one pouchitis).
Clinical details of the 12 patients treated with oral GlcNAc are shown in Table 2. All suffered from inflammatory bowel disease that had proved resistant to conventional therapy. Nine children suffered from severe Crohn’s disease affecting the small intestine, maximal in the terminal ileum in three—in the duodenum in one and throughout the entire small intestine in the other two (cases 1–9). Five children suffered from severe colitis (two ulcerative colitis—cases 10 and 11, three Crohn’s disease—cases 1, 9 and 12). Cases 1 and 9 had severe Crohn’s disease affecting the small bowel and colon. All remained uncontrolled despite extensive medical therapy including enteral nutrition (11/12), aminosalicylates (10/12), corticosteroids (11/12) and azathioprine (9/12). Three had previously undergone surgical resections for strictures, one of whom (case 2) had two strictures resected and several stricturoplasties performed two years before.
In eight patients there was evidence of symptomatic stricture, seven of the small intestine (cases 1–6, 8) and one anal (case 12). Those with small bowel strictures had symptoms of pain severe enough to limit their activity and nutrition, and with current radiological or endoscopic evidence to confirm a critically narrow stricture. In two cases there were multiple strictures throughout the small intestine (cases 2 and 5); in one case the child had previously undergone laparotomy and multiple stricturoplasty, and had been noted at operation to have several further strictures. All these children would have been referred in normal circumstances for either surgical resection or stricturoplasty. GlcNAc therapy was commenced in all children in 1996 or 1997, and thus a 2–3-year follow-up may be reported.
All of the five children with colitis had been considered for colectomy on the basis of failed medical therapy before the commencement of GlcNAc therapy. In all cases apart from case 11, who had been commenced on azathioprine 1 month previously, there had been no other medications introduced in the 3 months before GlcNAc.
To determine whether the mucosal responses were specific for GlcNAc therapy, or merely a nonspecific reflection of reduced inflammation, we studied the matrix response (see below) in six children (ages 11.6–17.3, two Crohn’s disease, four ulcerative colitis) whose colitis had been treated with corticosteroids and aminosalicylates. All had shown clinical and histological improvement.
Histochemistry for sulphated GAGs, using cationic colloidal gold (1/100 Biocell International, Cardiff, UK) at pH 1.2 was performed as previously reported.2, 3, 5 We have confirmed this technique by enzyme digestion studies to be specific for epithelial and endothelial heparan sulphate, and matrix chondroitin and dermatan sulphates within the mucosal tissues.2, 5 This stain does not recognize sialic acids. Intracellular and matrix-bound GlcNAc residues were detected in situ using biotinylated lectin wheat germ agglutinin (WGA [Vector Laboratories Inc., Burlingame, USA, 20 μg/mL]), following blockade of endogenous peroxidase by incubation in 3% hydrogen peroxide solution.25 This stain will detect protein-bound GlcNAc and sialic acid residues. Staining was developed, following incubation with streptavidin-horseradish peroxidase (DAKO, Denmark, 1:200), with diaminobenzidine (Sigma, St. Louis, USA). In order to minimize the variation between staining runs, the pre- and post-treatment biopsies for each case were stained together and for identical times.
The density of sulphated GAGs and WGA staining was assessed separately in the available pre- and post-treatment biopsies by point densitometry (Cambridge Instruments) at high magnification (× 400), using a 12.5 μm diameter window at wavelength 530 nm. Separate assessment was made of epithelial and matrix staining density, taking the mean of at least 10 separate measurements in each compartment. Mean values were derived for the entire groups before and after treatment, and statistical comparisons made by matched pair t-test.
No adverse side-effects of treatment were noted in any patient, apart from mild stinging on rectal insertion in one. One child in whom therapy was planned was unable to tolerate rectal catheter insertion for GlcNAc or steroids and is not further reported. All those given oral therapy found the suspension palatable, and in several cases pleasant.
The overall clinical response is shown in Table 1 and in case 12, Table 2. Of seven children treated with GlcNAc enema as single therapy, five showed clear improvement and two did not respond. Four of the five responders had ulcerative colitis with probable diversion colitis of the rectal stump, the fifth pouchitis. The two nonresponders suffered from Crohn’s disease of the rectal stump and ulcerative proctitis, and have since remained resistant to all therapies. Two children with ano-rectal strictures (Table 1 case 8, Table 2 case 12), in whom rectal GlcNAc was added to their pre-existing treatment showed a definite improvement in their stricture-related symptoms, the former able to discontinue his regular surgical dilatations and remaining in full remission after 2 years, the latter (who also received oral GlcNAc) showing improvement in his stricture but ultimately requiring subtotal colectomy for intractable Crohn’s colitis and growth failure.
Of the five responders to GlcNAc monotherapy, one patient had a clinical response within 3 days of starting treatment with complete resolution of bleeding and discharge, two were asymptomatic or with minimal symptoms after 4 weeks and two responded more slowly, with clear improvement noted by 3 months. Of note, all the four patients who were in clinical remission when GlcNAc supply was temporarily interrupted had a recurrence of rectal bleeding and mucous discharge within 2–10 days of ending the treatment, improving when it restarted.
The histological response is shown in Figures 2 and 3. Pre- and post-treatment rectal biopsies were taken in six cases, five of whom showed histological improvement, although none returned to complete normality. In all cases there was a reduction in the inflammatory infiltrate, with particular improvement in epithelial morphology and goblet cell density.
Histochemical staining for sulphated GAGs (Figures 2 and 3) showed significantly increased density within the matrix of the lamina propria (mean OD (optical density) post-treatment 167.0 ± s.e. 29.2 units vs. pre-treatment 81.3 ± 13.3, P=0.02) and nonsignificant change in the epithelium (141.2 ± 31.0 vs. 72.5 ± 13.1, P=0.06). By contrast the enhancement of WGA lectin staining for terminal GlcNAc residues and sialic acids was more striking in the epithelium (60.1 ± 16.9 vs. 30.4 ± 4.8, P < 0.01) than the matrix (68.2 ± 8.2 vs. 38.7 ± 8.4, P < 0.05).
Eight of the 12 children treated showed clear clinical improvement when GlcNAc was added to their current treatment, while four failed to respond (Table 2, cases 1, 8, 9 and 12). These cases were resistant to all therapies used and required surgery. One other case (case 7) showed a marked improvement in his duodenal stricturing disease but relapsed when he discontinued GlcNAc because of a problem with supply and required surgery. The nonresponders were notably those with severe Crohn’s terminal ileitis and colitis, who showed minimal or unsustained response to any treatment and eventually underwent subtotal colectomy. By contrast, the patients with predominantly upper small intestinal disease showed marked improvement. This clinical response was mirrored, in the one patient whose duodenal mucosa was rebiopsied, by enhanced matrix restoration in the epithelium and lamina propria together with restoration of epithelial morphology—this response was clearly greater than in the two cases where post-treatment ileal biopsies were obtained (Figures 2 and 3 and below).
By contrast to the children with severe active ileo-colitis, those with critical small bowel strictures did better, whether in the upper small bowel or the terminal ileum (cases 2–8). So far only three of these seven have required surgery after a mean follow-up period of over 2.5 years, and the others are free from stricture-related symptoms. Of the three children who did come to surgery, two did so within 3 months of starting GlcNAc therapy, one opting for surgery after 2 weeks (case 8), and the other (case 7) as reported above. The third case showed marked clinical improvement over > 2 years, before defaulting on all medications and relapsing, requiring resection of an ileal stricture 3 years after commencing GlcNAc (case 5). Endoscopic or radiological assessment has demonstrated improvement, although of variable degree, in all cases. In the case who had previously undergone laparotomy and stricturoplasty, there was substantial improvement of contrast radiolography to a normal appearance (Figure 3), which has been maintained for 3.5 years. In the other cases there was radiological and/or endoscopic improvement without the same complete resolution.
In addition to clinical improvement in the majority of patients, there was evidence at the mucosal level of enhanced expression of sulphated GAGs, particularly within the extracellular matrix, and of intracellular GlcNAc residues, particularly within the epithelium (Figures 1 and 2). Enhanced goblet cell mucus production was a notable feature in several of the specimens.
Taking together, all cases where pre- and post-treatment biopsies were available (i.e. rectal and/or oral), there was significant increase in matrix sulphated GAGs (mean OD post treatment 171.8 ± s.e. 19.6 units vs. pre-treatment 98.6 ± 13.1, P < 0.01), and epithelial GAGs (142.8 ± 20.4 vs. 79.7 ± 9.9 units, P < 0.05). In addition, the density of WGA staining for GlcNAc residues was increased within both the lamina propria matrix (71.1 ± 7.3 vs. 40.1 ± 5.6 units, P < 0.01) and the epithelium (57.4 ± 5.5 vs. 31.9 ± 3.2 units, P < 0.01). The enhancement of matrix composition was more striking in those areas where the therapy could be applied more directly (i.e. the upper small bowel and the rectum) than in the terminal ileum (Figure 2), suggestive of first-pass hepatic metabolism.
By contrast, only 1/6 patients treated with corticosteroids showed a similar enhancement of either GAG or WGA staining, despite significant histological improvement in all.
In this open-label Phase I study of GlcNAc therapy in paediatric inflammatory bowel disease, we have found preliminary evidence to suggest clinical efficacy when administered either as monotherapy for rectal inflammation or as adjunct therapy when administered orally for treatment-resistant inflammatory bowel disease. However we recognize limitations with the study design: this was an open-label study, limited by ethical considerations to a population of children with severe or treatment-resistant disease. Properly placebo-controlled studies are clearly necessary to confirm its efficacy and determine indications for use of GlcNAc in inflammatory bowel disease, as is now occurring in arthritis.23, 24, 26 With these reservations, we found that, in all cases where pre- and post-treatment biopsies were obtained, there was an enhancement of matrix and epithelial expression of sulphated glycosaminoglycans, as well as increased intracellular density of monomeric GlcNAc residues, to suggest that the clinical response was associated with matrix restoration and improvement in epithelial morphology. In addition, a possible response in established strictures was noted in several patients.
Chitin, the polymeric form of GlcNAc, is the major structural component of crustacean shells, and is thus the second most plentiful natural polymer after cellulose. It is readily soluble in water, with a pleasant taste, and cheap to manufacture.23 GlcNAc has been used orally as a human nutritional supplement in the USA for many years without reported adverse effects. Large-dose intravenous administrations have demonstrated no toxicity or alteration of glucose or insulin concentrations.27 In man, glucosamine and other amino sugars are formed from glucose and glutamine through a series of biochemical reactions (Figure 1). The rate-limiting initial step is catalysed by glucosamine synthetase which transfers an amide group from glutamine to fructose-6-phosphate to form glucosamine-6-phosphate. The transacetylase which catalyses the formation of N-acetyl glucosamine-6-phosphate plays a key role in the availability of all amino sugars. Both these enzymes may be critical regulators of the repair process in inflammatory bowel disease. Mucosal glucosamine synthetase activity is not up-regulated in active disease, and histological remission and healing of ulceration only occur when local concentrations increase.28, 29 As GlcNAc is preferentially taken up over glucosamine in vitro in inflammatory bowel disease biopsies, acetylation of glucosamine may be another rate-limiting step in the response to mucosal inflammation.
Two important pathways through which carbohydrates may aid epithelial defences in inflammation have been demonstrated. Firstly there is strong evidence of abnormal mucin glycosylation in inflammatory bowel disease,21, 25 and it was notable in several patients that mucin production was increased in response to rectal GlcNAc therapy (see Figure 3D). Future studies should address the specific composition of these induced mucins. Secondly, a role for heparan sulphate proteoglycans in aiding epithelial restitution by binding growth factors has been postulated.30 The histology and histochemistry of those children showing the greatest clinical response suggests that restoration of the epithelial barrier may be important in the action of GlcNAc in inflammatory bowel disease.
A more direct anti-inflammatory mechanism is suggested by evidence of an action of GlcNAc as a competitive antagonist of phosphorylation at serine and threonine residues following O-linked attachment (O-GlcNAcylation).22 GlcNAc thus reduces the production of superoxide anion and leucocyte elastase from stimulated neutrophils in vitro31 and has notably prevented enteropathy following gluten challenge of coeliac disease biopsies.32 More specific studies of phosphorylation will therefore be required to determine whether therapeutic O-GlcNAcylation occurs in vivo with this treatment.
We found a clinically unusual response in children with established strictures, although we recognize that this was not a controlled study. There is evidence however, that both GAGs and GlcNAc may retard the progression of peritoneal fibrosis in chronic peritoneal dialysis,33, 34 and post-operative adhesions may be prevented by hyaluronate-based membranes.35 It is an intriguing possibility that the fibrotic response may be more responsive to restoration of substrate supply than is currently recognized. However, these are clearly preliminary results, and formal placebo-controlled clinical trials are now required. If therapeutic efficacy is confirmed, nutriceutical therapy with N-acetyl glucosamine may be a safe and inexpensive addition to current treatment regimens in inflammatory bowel disease. However, the reduced efficacy we found in active ileo-colonic disease, compared to sites exposed to higher concentrations, suggests that formulations allowing targeted local release will be necessary for optimal efficacy.
We thank the children and their families for taking part in the study. We are grateful to Drs Mike Thomson and David Casson for endoscopic assistance and to Professor Hudson Freeze for useful advice and discussion. Robert Heuschkel was supported by the Crohn’s in Childhood Research Appeal. This work was undertaken by Dr Murch and colleagues with the Royal Free Hampstead NHS Trust who received a portion of its funding from the NHS Executive; the views expressed are those of the authors and not necessarily those of the Trust or the NHS Executive.
Silvia Salvatore collated all data and performed histochemical and lectin staining; Robert Heuschkel, Steve Tomlin and Professor John Walker-Smith coordinated the clinical aspects of the study; Sue Davies performed histological analysis; Siân Edwards performed radiological studies; Ian French performed in vitro analysis, safety and toxicity studies on N-acetyl glucosamine prior to patient administration; Simon Murch was lead investigator, supervised the clinical studies and laboratory investigation, and wrote the manuscript.
Ian French is Scientific Director of Glucogenics Pharmaceuticals, who supplied the N-acetyl glucosamine for the study. However all analysis and data presentation was performed by the other authors, for whom no conflict of interest exists.