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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Background

In Crohn's disease (CD), knockdown of Smad7, an inhibitor of Transforming Growth Factor (TGF)-β1 activity, with a specific antisense oligonucleotide (GED0301) seems to be safe and tolerable and associates with TGF-β1-mediated suppression of inflammatory pathways.

Aim

Since TGF-β1 has pro-fibrogenic effects in many organs, we evaluated whether GED0301 treatment associates with the formation of small bowel strictures.

Methods

Fifteen patients with active, inflammatory CD, receiving oral GED0301 once daily for 7 days, were monitored for the formation of small bowel strictures by Small Intestine Contrast Ultrasonography (SICUS). Serum basic fibroblast growth factor (bFGF) and human chitinase 3-like 1 (also known as YKL-40), two markers of CD-related intestinal strictures, and matrix metalloproteinases (MMP) and tissue inhibitor 1 of MMPs (TIMP1) were analysed at day 0 and day 180 by ELISA. Crohn's disease activity index (CDAI) changes were also monitored.

Results

Fourteen patients completed the 6-month study; the remaining underwent intestinal resection for a severe relapse not responsive to medical treatment. No patient developed small bowel stricture and none experienced obstructive symptoms during the study period. GED0301 treatment induced no significant change in the circulating levels of bFGF, YKL-40, MMPs and TIMP1. Seven of 12 patients who reached clinical remission following GED0301 treatment maintained a CDAI < 150 at day 180.

Conclusion

Short-term treatment of patients with Crohn's disease using GED0301 is not associated with the development of small bowel stricture, thus reinforcing the concept that this drug is safe at least at early time points.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Crohn's disease (CD) is a chronic inflammatory disorder that primarily affects small intestine and colon and causes tissue damage.[1, 2] The aetiology of CD is unknown, but a large body of evidence suggests that the pathological process is driven by excessive immune response, which is direct against components of the luminal flora and sustained by defects in counter-regulatory mechanisms.[1, 2] One such mechanism involves the suppressive cytokine Transforming Growth Factor (TGF)-β1.[3, 4] Indeed, in CD tissue, TGF-β1 activity is inhibited by high Smad7, an intracellular protein that binds to the TGF-β1 receptor and prevents TGF-β1-driven signalling.[5, 6] The functional relevance of these findings is supported by the demonstration that inhibition of Smad7 in CD mucosal cells with a specific antisense oligonucleotide restores TGF-β1 activity thereby down-regulating the production of inflammatory cytokines.[5] Consistently, studies in mice have shown that induction of experimental CD-like colitis is associated with enhanced expression of Smad7 and reduced TGF-β1 activity, and oral administration of Smad7 antisense oligonucleotide to mice attenuates intestinal inflammation.[7] To further explore the role of Smad7 in gut inflammation, we developed a Smad7 antisense oligonucleotide-containing pharmaceutical compound, termed GED0301. GED0301 is an oral gastro-resistant formulation with a pH-dependent, delayed-release of the oligonucleotide in the terminal ileum and right colon. We performed a phase 1 clinical trial and showed that administration of GED0301 to active, steroid-dependent/resistant CD patients was safe and well tolerated.[8] A clinical benefit of GED0301 was also documented in all patients, thus suggesting that Smad7 could represent a valid target for therapeutic interventions in CD.

The natural history of CD patients can be characterised by the development of local complications and/or extraintestinal manifestations.[9] For example, nearly 30% of patients develop single or multiple strictures, which lead to partial or complete obstruction of the lumen and often require surgical resection.[10] Strictures are characterised by intestinal smooth muscle cell hyperplasia and hypertrophy and fibrosis due to excessive extracellular matrix production, including collagen. The exact mechanism(s) by which chronic inflammation promotes intestinal fibrogenesis and factors involved in this process are not fully understood. Since TGF-β1 regulates collagen expression and extracellular matrix dynamics and has profibrotic effects in many tissues,[11, 12] it is conceivable that inhibition of Smad7 with GED0301 could paradoxically favour the development of strictures.

This study was designed to assess prospectively the development of small bowel strictures in CD patients receiving 1-week treatment with GED0301 using small intestine contrast ultrasonography (SICUS).

Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Patients

This study was an extension of a phase 1, open-label, dose-escalation study (EudraCT No.: 2009-012465-66) aimed at evaluating the safety and tolerability of GED0301 in 15 active CD patients who had an inflammatory phenotype and were dependent and/or resistant to steroids. Nine of 15 patients had received previous bowel surgery due to the development of strictures associated with obstructive symptoms (N = 6) or enteric fistulae (N = 3). Patients were allocated to receive 40 mg (N = 5 patients, cohort 1), 80 mg (N = 5 patients, cohort 2) or 160 mg (N = 5 patients, cohort 3) GED0301. The drug was given orally once a day for 7 consecutive days.[8]

Crohn's disease activity index (CDAI) was evaluated at day 0 and day 180. Clinical response was defined as decrease from baseline CDAI score of >70 points, while remission was defined as CDAI score <150.[13]

Small intestine contrast ultrasonography

Small intestine contrast ultrasonography was performed 2–4 weeks before starting the treatment and at day 180 according to previously published methods.[14-16] Patients were given oral contrast solution consisting of polyethylene glycol, which distends the intestinal lumen thus allowing a more accurate characterisation of the bowel wall. Examinations were performed every 20 minutes until oral contrast laid out the terminal ileum by a single gastroenterologist with a longstanding expertise (more than 5000 SICUS examinations).

Small intestine contrast ultrasonography criteria for the presence of small bowel CD lesions and complications were: (i) increased bowel wall thickness (BWT) (>3 mm); (ii) small bowel dilation, defined as a lumen diameter >25 mm; (iii) bowel stricture defined as lumen diameter <10 mm, measured at the level of maximally distended loop, with or without dilation; (iv) fistulas defined as hypoechoic tract with or without hyperechoic content; (v) abscess identified as roundish anechoic lesions, with an irregular wall, often presenting internal echoes and posterior echo enhancement; (vi) mesenteric adipose tissue alterations and lymph nodes. At the end of ultrasound investigation the following measurements were reported on a standardised form: (i) lesion extent; (ii) maximum BWT measured; (iii) minimum lumen diameter measured; (iv) presence and number of strictures; (v) presence and number of bowel dilation; (vi) presence of abscess; (vii) presence of fistula; (viii) presence of mesenteric adipose tissue alterations.

Blood sampling and enzyme-linked immunosorbent assay

Peripheral venous blood samples, collected from each patient at day 0 and 6 months (day 180) after the end of GED0301 treatment were centrifuged for 15 min at 1000g, and serum samples were stored at −20°C until tested. All serum samples were analysed at the same time point for human basic fibroblast growth factor (bFGF), human chitinase 3-like 1 (CHI3L1, also known as YKL-40), human tissue inhibitor of metalloproteinases 1 (TIMP-1), human Pro-Matrix Metalloproteinase 1 (Pro-MMP-1), human active and pro-Matrix Metalloproteinase 3 (total MMP-3), human active and pro-Matrix Metalloproteinase 9 (total MMP-9) by ELISA according to the manufacturer's instructions (R&D Systems, Minneapolis, MN, USA).

Statistical analysis

Values were expressed as median and range in all figures. Data were analysed using the Wilcoxon matched pairs in the GraphPad Prism statistical PC program (GraphPad Software, San Diego, CA, USA). A P < 0.05 was considered statistically significant.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Clinical data

Demographic and clinical data of patients were described elsewhere as this was an extension of a phase 1 clinical trial performed to assess the safety and tolerability of GED0301.[8] Briefly, study population included 15 patients (11 male, 73%) with active, steroid-dependent and/or -resistant CD and a median age of 37 years (range: 24–45 years). The mean CD duration was 4 years (range: 1–29 years). All patients had an inflammatory behaviour of CD and lesions were confined to the terminal ileum. At enrolment, the median CDAI score of all patients was 287 (range: 221–400). As previously reported, at the end of the treatment (day 8) all 15 patients experienced a clinical response, as defined by a decrease in CDAI score >70 points. Clinical remission was documented in 12/15 (80%) of patients.

Fourteen of 15 patients completed the study protocol. The remaining patient, who was treated with 160 mg GED0301, underwent an ileocolonic resection 3 months after stopping GED0301 treatment because of a severe relapse of the disease, which was resistant to medical therapy. Histopathology of the surgical specimen confirmed the presence of severe inflammatory lesions with no evidence of fibrostenosis. No patient experienced obstructive symptoms during the study period. At day 180, the median CDAI score was 144 (range: 65–250). Seven of 12 patients (58.3%) maintained clinical remission without any need for additional therapy, while the remaining seven had a clinically active disease as defined by a CDAI higher than 150 (Figure 1). Two of these seven patients were treated with anti-TNF-α, one patient with corticosteroids and azathioprine and the remaining with high doses of corticosteroids.

image

Figure 1. Crohn's disease activity index (CDAI) values of Crohn's disease patients before and 180 days after GED0301 treatment. CDAI values are shown for each patient enrolled in the three separate cohorts. Fourteen of 15 patients who entered the trial completed the study protocol, as one patient of cohort 3 underwent intestinal resection for severe relapse of the disease. Dotted line indicates the CDAI value discriminating patients with active disease (>150) from those with inactive disease (<150).

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Evaluation of CD-related lesions and complications by SICUS

Small intestine contrast ultrasonography can accurately assess the presence, size and number of small bowel lesions in CD patients.[17] Since SICUS is also non-invasive, radiation-free and inexpensive, we monitored the appearance of strictures in GED0301-treated patients using this technique. Baseline evaluation confirmed that all patients had lesions confined to the distal ileum. In particular, median extent of involved distal ileum was 15 cm (range: 5–50 cm), median BWT was 7 mm (range: 3.5–17 mm) and median lumen diameter was 7 mm (range: 2–13 mm) (Figure 2). No patient had colonic lesions. Six of 15 patients had mesenteric adipose tissue alterations. No patient had fistulas and/or abscesses.

image

Figure 2. Effect of GED0301 on CD-related lesions by SICUS. Each point in the graphs indicates the value of small bowel wall thickness (a), lumen diameter (b) and lesion extent (c) in a single patient as assessed by SICUS at baseline and 180 days after stopping treatment; horizontal bars indicate the median values. In panels (d) and (e), white arrows indicate bowel wall thickness and black arrowheads indicate lumen diameter of neo-terminal ileum of a single patient at day 0 and day 180. In panel (e) at the same level bowel wall thickness (white arrows) and lumen diameter (black arrowheads) show no changes at day 180.

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No significant differences in terms of small bowel thickness, lumen diameter and lesion extent was seen 6 months after the end treatment as compared with parameters at baseline [Figure 2 panel (a)–(c)]. In particular, at day 180, median extent of diseased distal ileum was 13.5 cm (range: 5–45 cm), median BWT was 7.5 mm (range: 3.5–14 mm) and median lumen diameter was 6 mm (range: 2–13 mm). SICUS confirmed the presence of mesenteric adipose tissue alteration in six of 14 patients and no patient developed abscesses or fistulas.

GED0301 does not alter the circulating levels of YKL-40 and bFGF

YKL-40, a growth factor released by activated neutrophils and activated macrophages, stimulates connective tissue cells.[18] Patients with CD, particularly those with strictures, have high serum levels of YKL-40, and this factor has been considered as a useful marker of stricture formation.[19, 20] Another marker of tissue repair and remodelling, whose levels are increased in the blood of CD patients, is bFGF.[21] Therefore, we next examined whether GED0301 treatment associated with changes in the circulating levels of these two proteins. At day 180, the median level of YKL-40 was slightly but not significantly increased as compared with day 0 (Figure 3a). Similarly, GED0301 treatment did not significantly alter the median levels of bFGF (Figure 3b).

image

Figure 3. GED0301 treatment does not affect serum levels of bFGF and YKL-40. Each point in the graphs indicates the serum level of YKL-40 (a) and bFGF (b) in each patient as measured by ELISA at day 0 and day 180. Horizontal bars represent the median value.

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Effects of GED0301 on MMP and TIMP production

An excessive synthesis and deposition of extracellular matrix (ECM) components by intestinal miofibroblasts and inhibition of ECM degradation due to imbalance between activity of MMPs and TIMPs are thought to play a major role in the fibrogenic process.[22]

Therefore, we quantified the serum levels of MMPs and TIMPs by ELISAs before (day 0) and 6 months (day 180) after the end of GED0301 treatment. No significant change in the serum concentrations of pro-MMP1, MMP-3, MMP-9 and TIMP-1 was seen following GED0301 therapy (Figure 4).

image

Figure 4. The serum concentrations of MMP-1, MMP-3, MMP-9 and TIMP-1 are not affected by GED0301 treatment. Each point in the graphs indicates the serum level of MMP-1 (a), MMP-3 (b), MMP-9 (c) and TIMP-1 (d) in each patient as measured by ELISA at day 0 and day 180. Horizontal bars represent the median value.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

A considerable amount of work has been recently performed in our laboratory to support the view that CD-related inflammation is marked by defective activity of TGF-β1, due to high intracellular levels of Smad7, an inhibitor of TGF-β1 signalling.[5, 6] These findings are supported by the demonstration that inhibition of Smad7 with a specific antisense oligonucleotide, termed GED0301, attenuates experimental colitis in mice.[7] Moreover, we have recently performed a phase 1 trial and shown that oral administration of GED0301 in CD patients is safe and tolerable and associates with clinical benefit.[8] This work was an extension of the phase 1 trial, aimed at assessing whether restoring TGF-β1 signalling with GED0301 leads to the development of small bowel strictures. The most relevant finding of our study is the observation that 7 day-treatment with GED0301 promotes neither the formation of strictures nor the appearance of obstructive symptoms. Moreover, GED0301 treatment does not significantly alter the serum levels of YKL40 and bFGF, two useful markers of stricture formation in CD.[19-21]TGFβ-1 is also crucial in the regulation of synthesis and breakdown of extracellular matrix proteins, given that it down regulates MMP expression and enhances expression of TIMP-1.[22, 23] Changes in the serum concentration of MMPs, reflecting the state of their activity, have been demonstrated in many pathological processes, including CD.[21] GED0301 treatment associated with no significant change in the serum concentrations of MMP1, MMP3, MMP9 and TIMP1. We restricted our analysis to these MMPs and TIMP1 because previous studies have shown that serum and mucosal expression and activity of these proteins, which are tightly controlled by TGFβ-1, are deregulated in CD.[23-26] Taken together our data indicate that, at least at early time points, a short-term treatment with GED0301 does not increase the risk of strictures. This message is strengthened by the prospective collection of data and morphological assessment of strictures with description of intestinal wall and lumen diameter using the SICUS, which has a very high sensitivity and specificity for detecting local CD complications.[27] It has been also reported that SICUS is comparable to computed tomography (CT) enterography and magnetic resonance enterography for the diagnosis of intestinal strictures[28] and to small bowel follow-through and CT for small bowel evaluation.[29] These observations together with the fact that SICUS avoids radiation support the use of this procedure for monitoring the formation of small bowel strictures in CD patients. Nonetheless, we think it is fair to point out some limitations of the study. The number of tested patients was very small as the study was primarily designed to evaluate the safety and tolerability of the drug. The nonblinded design of the trial and the lack of a placebo-controlled group represent further limits of the work. Therefore, data of the present work should be confirmed by an ad-hoc randomised controlled study performed in a larger number of patients. As the time-frame of therapy was limited to 1 week, it would also be relevant to ascertain the risk of strictures in patients receiving long-term treatment with GED0301.

TGF-β1 is a pleiotropic cytokine, which regulates the activity of multiple cell types involved in the regulation of both inflammatory and reparative processes.[4, 30, 31] TGF-β1 delivers mostly negative signals to immune cells, thereby acting as a powerful mucosal immunosuppressor, but it is also chemotactic for fibroblasts, stimulates fibroblast proliferation and increases the synthesis of a number of extracellular matrix proteins including collagens.[11] Therefore, transient TGF-β1 activity helps healing and regeneration of damaged tissues while persistent TGF-β1 function can promote excessive collagen deposition and promotes fibrosis.[12, 32] Based upon these observations one could hypothesise that enhancing TGF-β1 activity with GED0301 can inevitably lead to the formation of strictures. Interestingly, however, our results indicate that CD patients treated with GED0301 do not develop strictures. We do not know the reason for this apparent paradox, even though it is noteworthy that intestinal fibrogenesis is a very complex process caused by multiple, rather than single, cytokines/factors. In this context, it is noteworthy that treatment of CD patients with anti-TNF-α does not promote the development of strictures, despite TNF-α blockade enhances TGF-β1 activity.[33] Another possibility is that, in the inflamed gut of CD patients, Smad7 is mostly up-regulated by immune cells rather than stromal cells; so inhibition of Smad7 would promote resolution of the inflammation without affecting fibrogenesis process. It is also reasonable to believe that orally administered GED0301 is taken-up prevalently by epithelial cells, with the down-stream effect of restoring epithelial barrier integrity and leaving unaltered the function of stromal cells. Studies in experimental models of intestinal fibrosis suggest that chronic mucosal inflammation is the driving force for abundant collagen deposition and fibrogenic ECM matrix changes, and that abrogation of intestinal inflammation prevents extensive fibrosis.[34] It is thus conceivable that GED0301 acting on the CD inflammatory lesions of the gut wall can stop and/or revert, instead of promoting, the evolution of the stenotic process.

In conclusion, data of the present work indicate that short-term treatment of patients with active CD with GED0301 associates neither with the formation of strictures nor with the appearance of obstructive symptoms. This observation together with the demonstration that GED0301 is safe and tolerable, reduces the fraction of circulating effector T cells with gut-homing properties and decreases CDAI values to support the necessity of randomised, placebo-controlled, studies aimed at evaluating the efficacy of the drug in CD and ascertain whether GED0301 can find a place in the therapeutic armamentarium of CD patients.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Declaration of personal interests: Giovanni Monteleone has received research funding from Giuliani SpA, Milan, Italy and filed a patent related to the treatment of inflammatory bowel diseases with Smad7 antisense oligonucleotides, while the remaining authors have no conflict of interest. Declaration of funding interests: The study was funded in part by Giuliani SpA, Milan, Italy, and in part by a grant (GM) from the Broad Medical Research Foundation (IBD-0301R).

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References