Project Description

INTRODUCTION
Inflammatory Bowel Disease (IBD)
Epidemiology
Etiology and Pathophysiology
Treatment Options
Nutrition and overcoming malabsorption in IBD
Figure 1

The INFLATREAT project
Objectives
Central hypotheses
Specific aims
Project Outline
Figure 2
Results
Figure 3
Figure 4
Project Outcome
Dissemination and Exploitation

INTRODUCTION

Inflammatory Bowel Disease (IBD)
Inflammatory Bowel Disease (IBD) is a heterogeneous group of diseases comprising ulcerative colitis (UC) and Crohn’s disease (CD). CD is defined as a transmural granulomatous inflammation that may involve any segment of the gastrointestinal (GI) tract but most commonly affects the colon and the ileum. UC is limited to the large intestine or colon affecting only the mucosal layer.

Epidemiology
The disease is mainly prevalent in developed countries with a high occurrence of both CD and UC in North America and Northern Europe. However the incidence is rising internationally and 2.8 million people are currently diagnosed with IBD world-wide. The European Federation of Crohn’s and Ulcerative Colitis Associations (E.F.C.C.A) estimate that over one million Europeans are afflicted with either UC or CD. IBD is a lifelong disease usually starting in early adulthood and it is a cause of significant morbidity and economic cost in the European Union. Currently, the global market for pharmaceutical approaches to IBD is estimated at c. €20 billion and this is set to continue to rise.

Etiology and Pathophysiology
Although there are many hypotheses regarding the etiology of IBD, neither the etiology nor the pathophysiology is well understood. The abnormally exacerbated immune response to otherwise innocuous stimuli is thought to play a key role in the pathophysiology of IBD resulting in upregulation of pro-inflammatory mediators that trigger a complicated cascade of cytokine and chemokine activation leading to chronic inflammation. Pro-inflammatory cytokines stimulate the genetic expression of endothelial cell adhesion molecules (ECAMs) through the generation of reactive oxygen and nitrogen species resulting in the recruitment and extravasation of leukocytes into the bowel wall tissue. The inability of IBD patients to effectively regulate the activation of this inflammatory cascade leads to chronic inflammation. Decreased antioxidant defences and the increase in free radicals and other reactive oxygen and nitrogen species inherent to the inflammatory process result in increased oxidative stress. Under normal conditions, cells can handle minor oxidative stress due to the presence of protective enzymatic antioxidants (superoxide dismutase, glutathione peroxidase and others) and non-enzymatic antioxidants (α-tocopherol, vitamin C and others). However, the increased release of reactive oxygen and nitrogen species in IBD overloads the cellular antioxidant systems leading to cellular damage and apoptosis.

Treatment Options
At present there is no cure available for IBD, although technically UC can be ‘cured’ by surgical removal of the colon. A large majority of patients require chronic medical treatment, and a significant number require surgery due to complications including small bowel obstruction, fistula, abscess, ulceration or perforation. Patients are not only burdened by symptoms of IBD (e.g. diarrhea, abdominal pain and fatigue) but also by the side-effects of the medication. Conventional treatments in IBD aim at down-regulating the exacerbated immune response with the ultimate aim of suppressing inflammation and maintaining remission. Current therapies include aminosalicylates (5-ASA), corticosteroids, immunomodulators, antibiotics and biologic therapies. Although these therapies are partially effective unfortunately they have potentially serious side effects that limit their widespread prophylactic use. Alternative medicines including naturally occurring antioxidants/anti-inflammatory compounds are currently being investigated for the management of IBD and may be effective in reducing inflammation and would be safe to use on a long-term basis. Natural compounds, such as dietary fiber, Omega-3 polyunsaturated fatty acids or prebiotic and / or probiotics, were shown to exert anti-inflammatory activity in recent studies performed in humans and were well tolerated.

Nutrition and overcoming malabsorption in IBD
The inflammation inherent in IBD often results in malabsorption and nutrient deficiency, especially with respect to the fat-soluble vitamins A, D, E, and K and other nutrients which help limit inflammatory responses. Other deficiencies in vitamin B-12, folic acid and vitamin C, iron and trace minerals, such as zinc, selenium and copper have been well documented. The coordinating partner in this EUREKA project, Yasoo Health Ltd, has developed proprietary technology for formulating lipophilic compounds into dispersible micellar-like vehicles to enhance their absorption (Figure 1). Yasoo is already using this technology to overcome malabsorption of lipophilic compounds in one of their clinically tested, commercially available, products, AquADEKS®, for the adjuvant treatment of cystic fibrosis.

Figure 1

The INFLATREAT project

Objectives
Naturally occurring antioxidant/anti-inflammatory compounds have the ability to reduce oxidative stress and could therefore have a potential use in the management and adjunctive therapy of IBD. The aim of this EUREKA project is to evaluate selected naturally occurring lipophilic antioxidants and their modified derivatives for their ability to ameliorate inflammation associated with IBD. The ultimate goal is to develop a novel formulation of naturally occurring antioxidant / anti-inflammatory compounds for the management and adjunctive therapy of IBD.

Central hypotheses

The inability of IBD patients to effectively regulate the activation of the immune response contributes to the amplification of pro-inflammatory mediators such as TNF-α and IFN-γ leading to chronic inflammation.
A critical pathway in the pathophysiology of IBD is the production of reactive oxygen and nitrogen species which increase expression of Endothelial Cell Adhesion Molecules (ECAMs) such as mucosal addressin cell adhesion molecule-1 (MAdCAM-1) or vascular cell adhesion molecule-1 (VCAM-1).
Specific naturally occurring antioxidants / anti-inflammatory compounds alone or in combination may be effective in decreasing the production and release of pro-inflammatory mediators including ECAMs and cytokines and increase the expression of anti-inflammatory cytokines such as IL-10.
Pro-inflammatory cytokines stimulate the genetic expression of ECAMs which in turn directs the tissue infiltration by leukocytes, which induce and sustain gut inflammation. Decreased expression of pro-inflammatory cytokines and ECAMs will decrease extravasation of leukocytes into intestinal tissue resulting in improved clinical outcomes.

Specific aims
One of the specific aims of this project is to evaluate naturally occurring lipophilic antioxidants and their modified derivatives for their ability to:

decrease ECAM expression in IBD related cell culture models
suppress pro-inflammatory cytokine-mediated responses in human cells
increase anti-inflammatory cytokine-mediated responses in human cells
reduce inflammation and improve clinical outcomes in an experimental mouse model of colitis.

The project aims to develop a formulation of active compounds that will enhance the systemic absorption of the selected naturally occurring antioxidants/anti-inflammatory compounds and increase their uptake at the site of inflammation by using proprietary technology, developed by Yasoo (Figure 1).

Project Outline
An outline of the project structure is shown in Figure 2. Naturally occurring lipophilic antioxidants and their modified derivatives were selected to be evaluated for their ability to suppress the expression of VCAM-1 or MAdCAM-1 in various cells including immortomouse colonic endothelial (CE) cells, human microvascular endothelial cells (HMEC-1) and human umbilical vein endothelial cells (HUV-EC-C) (Figure 3). In addition the selected compounds were assayed for their ability to prevent the production and release of pro- and anti- inflammatory cytokines including IL-1b, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, GMCSF, IFN-γ and TNF-α in peripheral blood mononuclear cells (PBMCs) isolated from healthy blood donors. The anti-inflammatory properties of the most efficacious compounds from the in vitro assays were then evaluated in the dextran-sulfate sodium (DSS) mouse model of colitis (Figure 4). The DSS-model is widely used as a model for acute inflammatory bowel disease and it is considered a good indicator of clinical effectiveness.

Results
The anti-inflammatory actions of the selected compounds were assayed for their ability to prevent the production and release of pro- and anti- inflammatory cytokines in peripheral blood mononuclear cells (PBMCs) isolated from healthy blood donors. The results indicated that several of our test compounds possessed strong anti-inflammatory action as they prevented the production and release of the following pro-inflammatory cytokines: IL-2, IL-6, IL-8, IFN-γ and TNF-α from human PBMC cells. Some of the compounds tested, also had the ability to increase the expression of the anti-inflammatory cytokine IL-10 which is essential in immuno-regulation of the intestinal tract in IBD. Several of the selected compounds were also found to be effective in suppressing MAdCAM-1 and VCAM cytokine-induced adhesion molecule expression in endothelial cells in vitro. The compounds from the in vitro assays found to have the most efficaceous in vitro effects in both the VCAM and the cytokine assays were evaluated in an IBD animal model for their antioxidant / anti-inflammatory properties. The chosen compounds either individually or in combination proved to be protective against colitis causing a delay in the onset of the disease. A synergistic effect was also achieved with certain combinations of compounds resulting in a consistent improvement in delaying the onset of the disease. The results of the project gave conclusive evidence that some of the natural compounds evaluated have strong anti‐inflammatory properties in vitro in human models (Figure 3) and were effective in mitigating the disease parameters of IBD in a mouse model (Figure 4).

Figure 2 (click to enlarge)

Figure 3 (click to enlarge)

Figure 4 (click to enlarge)

Project Outcome
EUREKA’s support has been invaluable in enabling us, through the cell and animal work performed by the three participants in this project, to identify the most efficacious anti-inflammatory compounds with the potential to ameliorate the symptoms of IBD. The compounds are now being developed as a prototype formulation aiming to reduce inflammation, relapses and complications of IBD. The exact product composition and formulation is undergoing further optimization by Yasoo Health. Pilot clinical trials in IBD patients are planned to assess the efficacy of the final formulations in the adjuvant treatment of IBD. The prototype will be developed into a novel, scientifically validated and commercially successful product for the management and adjuvant treatment of patients with IBD.

Dissemination and Exploitation
This EUREKA project (E!3930-INFLATREAT) was selected as one of the 24 best practice projects in a recent survey performed for the European Seventh Framework Programme entitled “Use and Diffuse” (www.useanddiffuse.eu) investigating the relationship between outcomes for participants and the dissemination and exploitation methods used by the project. Over two hundred EC funded R&D projects were surveyed. Further work is now being carried out by the Use and Diffuse programme to elucidate a methodology of good practice from these projects’ experiences towards the development of a Use & Diffuse Handbook to be published later during 2009.

Some of the results of this project will be submitted for publication shortly; a manuscript is currently in preparation entitled “α-, δ- and γ- Tocopherols reduce Inflammatory Angiogenesis in Human Microvascular Endothelial Cells”.

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