Final Report Summary - FXR-IBD (Industry-Academia exchange to further FXR-based therapeutic intervention and non-invasive diagnosis in Inflammatory Bowel Disease)
INDUSTRY-ACADEMIA EXCHANGE TO FURTHER FXR-BASED THERAPEUTIC INTERVENTION AND NON-INVASIVE DIAGNOSIS IN INFLAMMATORY BOWEL DISEASE
Website: fxr-ibd.eu
Inflammatory Bowel Disease (IBD) covers a group of disorders that are characterized by chronic intestinal inflammation. Complications can be severe and in rare cases, IBD might even lead to mortality. IBD affects approximately 0.2% of the human population with a higher prevalence in Europe. The main types of IBD are Crohn’s Disease (CD) and Ulcerative Colitis (UC). IBD appears to result from a deregulated mucosal immune system combined with a compromised intestinal epithelial barrier function in genetically predisposed individuals.
Current treatment options of patients with IBD are mainly aimed at suppressing the immune response, i.e. treatment of the symptoms rather than targeting the root cause of the affliction. However, although reasonably effective, treatment failures and significant side effects such as bone demineralization, growth retardation, diabetes, and hypertension often occur. This clearly stresses the need for novel treatment options in the treatment of IBD.
Another problem is that no specific, non-invasive test exists to monitor IBD disease activity. Currently, patients have to undergo endoscopy, which is a burden for the patient, but may also present complications, such as bleedings and infections.
The above illustrates that there is an urgent need for further multi-disciplinary research, in order to develop novel and improved IBD diagnostic and treatment options.
The present training project proposal brought together skills, experiences and (fundamental, translational, and commercial) knowledge both from industry and academia, in the context of an innovative research project on novel diagnostic and therapeutic approaches for IBD and provides further education and training to a large group of researchers. In this study we aimed to:
1. Develop a novel treatment for IBD, using the recently described anti-inflammatory effects of the bile acid sensor Farnesoid X receptor (FXR). We aimed to develop agonists capable of selectively activating the FXR as potential pharmaceutical compounds.
2. Develop a non-invasive diagnostic tool using well-defined changes in the gut microbiome of people affected with IBD as a marker to diagnose and monitor disease progression or remission in stool samples.
The project was divided into 6 Work Packages (WP).
Work Package 1. Project Management.
In the first month of the project, we organized the kick-off meeting in Utrecht (29th of January 2014). In addition, we developed the FXR-IBD.eu website. We had subsequent 2-day meetings at Enterome (25-26th Sept. 2014), Perugia (May 2015), Utrecht (midterm review Nov. 2016), Paris (Sept. 2016), Perugia (Jan 2018) and Utrecht (Closing meeting, April 2018). In addition, we organized an open symposium on Microbiota in Inflammatory Bowel Disease: innovation in diagnostic and therapeutic tools', in order to update members of the consortium and other interested scientists in this fast developing field . Another mini-symposium was organised on the topic of: Bile acid signalling and the interaction with the microbiome, in which the latest research on this topic was discussed .
Work Package 2. Investigate the molecular mechanisms of FXR-anti-inflammatory actions.
Previously, the UMC Utrecht groups have shown that activation of the bile acid sensor FXR by the semi-synthetic agonist (OCA, obeticholic acid) inhibits intestinal inflammation in mice1. We aimed to investigate the molecular mechanisms of FXR anti-inflammatory actions. The anti-cholestatic and metabolic functions of FXR are attributed to classical ligand-dependent gene transactivation. However, the exact mechanism driving the anti-inflammatory effects of ligand-driven FXR activation remained poorly described. We found that FXR repressed pro-inflammatory genes via tethering to NF-KB, a key transcription factor involved in regulating the expression of pro-inflammatory cytokines. We reasoned that we could potentially separate the FXR anti-inflammatory actions from its metabolic actions on bile acid, glucose lipid and amino acid metabolism, since the metabolic actions are the result of direct FXR transactivation mechanisms. Based on this notion, high-throughput screening platforms were designed to screen for FXR ligands that only have anti-inflammatory/transrepression of NF-kB activity (WP4). In addition, we investigated the differential mechanism of the 4 encoded FXR isoforms. Since two of the isoforms are predominantly present in the intestine and the other two predominantly in the liver, we reasoned that selective targeting of the isoforms would be a way to tissue-specifically activate FXR. We indeed find that different isoforms regulate different genes that result in different functional consequences. We are currently exploring how the different isoforms could be selective targeted, to understand whether isoform-selectivity could be used to separate different FXR actions.
Work Package 3. Development of a non-invasive tool to assay disease remission.
Using alterations in number and type of bacteria residing in the stool samples of patients, Enterome aims to develop a microbiome fingerprint using whole genome metagenomics profiling on stool samples. This fingerprint will be translated into a non-invasive diagnostic tool. By the start of the project, Enterome had already finished the analysis of its internal study named “CrohnOmeter”, into which they recruited 99 patients with a total of 950 samples collected by Q4 2014. Analysis of 439 samples and subgroups has been performed and had led to the identification of two patentable discoveries, the first one (dep. 19/09/2014) was on a gene named CDENT29 which was assembled when Enterome created a gene specific catalogue for Crohn Disease and the second patent is a novel marker of disease activity. UMCU has collected over 100 new stool samples from Crohn Disease patients that were biobanked by Enterome and is used to validate the findings in a separate cohort.
In order to elucidate how the specific bacterial species affect host gut health, we combined efforts to investigate factors known to be associated with improved/aggravating IBD symptoms in order to better understand the function of differentially abundant bacteria in IBD. Therefore, Enterome and the UMCU have collaborated on identifying bacterial components that are associated with smoking in Crohn Disease patients, since smoking is associated with more severe intestinal inflammation. Although the study is limited in terms of sample numbers, some significant signal was picked up and a joint publication has been published in Inflammatory Bowel disease Journal (Opstelten et al. 2016).
Inside the CrohnOmeter study, Enterome has detected several MetaGenomic Species (MGS) correlated to the severity of the disease. Among these MGS, the 6063-3, related with Bifidobacterium longum, has drawn our attention due to its good correlation with the activity level of IBD, and the importance of this genus for bile acid metabolism. Based on this fact and also to contribute to the development of a non-invasive tool to evaluate the disease state (WP3 Aim), we have developed a procedure of bacterial selection and enrichment based on specific genetic signatures. Optimization is still ongoing to get sufficient amounts of sorted bacteria, but then it will allow for the first time to correlate metagenomics data analysis with real strains and bacterial species. Furthermore, it will allow to specifically characterize the full genome of the bacteria associated with disease severity in ChronOmeter study.
We have also generated screening methods for identification of bacterial components/genes modifying bile acid synthesis and FXR activity. Reporter assays were developed in order to measure bacterium-induced production of FXR ligands. In addition, an assay was developed to measure the interaction between FXR and NF-kB upon induction by different bacterial lysates. A publication is underway addressing the validation of these assays and the potential of B. dorei and E. limosum to induce FXR.
Work Package 4. Development of anti-inflammatory ligands.
An automated high-throughput reporter-based screen has been set-up to separate tethering versus direct transactivation by FXR and used for compound screening. A small library of 1200 compounds of FDA-approved drugs was tested to validate the robustness of the screening method and it revealed one compound, Mometasone furoate, that reduced anti-inflammatory activity via FXR with no or limited effects on the metabolic function of FXR. With these results, for the first time it has been shown that separation of metabolic and anti-inflammatory functions of FXR in vitro is possible (Bijsmans et al 2015). In addition, using the same screening method, UMCU has screened a library of novel compounds generated by TES Pharma for their ability to separate FXR functions, and also here we have found some interesting compounds that are currently tested in additional assays. These interesting compounds are bile acid derivatives characterized by an extended side chain that binds the non-canonical FXR S2 accessory ligand pocket, whereas the main scaffold binds the canonical S1 ligand pocket. To understand the genome-wide consequences of different compounds that were selected during these studies, intestinal organoids were incubated with different compounds and RNA has been isolated. Comparative RNA-sequencing data is currently being analyzed. This way, we hope to understand at the level of metabolic and inflammatory genes whether differential regulation by these compounds can be observed. These data will enable us to predict which compounds should be prioritized for in vivo validation in mice to selectively inhibit gut inflammation (WP5). These studies will be proceeded after the closure of the FXR-IBD project. We were invited to write a review for the prestigious Journal Pharmacology and Therapeutics. UMCU and TES Pharma/UNI Perugia have collaboratively reviewed the literature on the relevance of developing FXR selective ligands from a human disease perspective (Massafra et al 2018).
Work Package 5. In vivo validation.
In this work package, animal studies were conducted with novel FXR agonists to investigate whether intestinal inflammation symptoms are decreased and whether they indeed act only on inflammation and not on metabolic actions of FXR. So far, we have tested one new compound in a dose finding study in vivo. 0, 10, 30 and 50 ug/kg of the compound was given for 7 days per oral gavage to mice (n=5 per group). We have established that this compound is very potent and highly selective for FXR. Unfortunately, this compound is not suitable as an FXR-selective compound to decrease the inflammatory response, as it affects both metabolic and anti-inflammatory functions of FXR.
Workpackage 6 Dissemination of results.
We have disseminated our results via publications, symposia, conferences etc. More can be found at our website: www.FXR-IBD.eu.
Website: fxr-ibd.eu
Inflammatory Bowel Disease (IBD) covers a group of disorders that are characterized by chronic intestinal inflammation. Complications can be severe and in rare cases, IBD might even lead to mortality. IBD affects approximately 0.2% of the human population with a higher prevalence in Europe. The main types of IBD are Crohn’s Disease (CD) and Ulcerative Colitis (UC). IBD appears to result from a deregulated mucosal immune system combined with a compromised intestinal epithelial barrier function in genetically predisposed individuals.
Current treatment options of patients with IBD are mainly aimed at suppressing the immune response, i.e. treatment of the symptoms rather than targeting the root cause of the affliction. However, although reasonably effective, treatment failures and significant side effects such as bone demineralization, growth retardation, diabetes, and hypertension often occur. This clearly stresses the need for novel treatment options in the treatment of IBD.
Another problem is that no specific, non-invasive test exists to monitor IBD disease activity. Currently, patients have to undergo endoscopy, which is a burden for the patient, but may also present complications, such as bleedings and infections.
The above illustrates that there is an urgent need for further multi-disciplinary research, in order to develop novel and improved IBD diagnostic and treatment options.
The present training project proposal brought together skills, experiences and (fundamental, translational, and commercial) knowledge both from industry and academia, in the context of an innovative research project on novel diagnostic and therapeutic approaches for IBD and provides further education and training to a large group of researchers. In this study we aimed to:
1. Develop a novel treatment for IBD, using the recently described anti-inflammatory effects of the bile acid sensor Farnesoid X receptor (FXR). We aimed to develop agonists capable of selectively activating the FXR as potential pharmaceutical compounds.
2. Develop a non-invasive diagnostic tool using well-defined changes in the gut microbiome of people affected with IBD as a marker to diagnose and monitor disease progression or remission in stool samples.
The project was divided into 6 Work Packages (WP).
Work Package 1. Project Management.
In the first month of the project, we organized the kick-off meeting in Utrecht (29th of January 2014). In addition, we developed the FXR-IBD.eu website. We had subsequent 2-day meetings at Enterome (25-26th Sept. 2014), Perugia (May 2015), Utrecht (midterm review Nov. 2016), Paris (Sept. 2016), Perugia (Jan 2018) and Utrecht (Closing meeting, April 2018). In addition, we organized an open symposium on Microbiota in Inflammatory Bowel Disease: innovation in diagnostic and therapeutic tools', in order to update members of the consortium and other interested scientists in this fast developing field . Another mini-symposium was organised on the topic of: Bile acid signalling and the interaction with the microbiome, in which the latest research on this topic was discussed .
Work Package 2. Investigate the molecular mechanisms of FXR-anti-inflammatory actions.
Previously, the UMC Utrecht groups have shown that activation of the bile acid sensor FXR by the semi-synthetic agonist (OCA, obeticholic acid) inhibits intestinal inflammation in mice1. We aimed to investigate the molecular mechanisms of FXR anti-inflammatory actions. The anti-cholestatic and metabolic functions of FXR are attributed to classical ligand-dependent gene transactivation. However, the exact mechanism driving the anti-inflammatory effects of ligand-driven FXR activation remained poorly described. We found that FXR repressed pro-inflammatory genes via tethering to NF-KB, a key transcription factor involved in regulating the expression of pro-inflammatory cytokines. We reasoned that we could potentially separate the FXR anti-inflammatory actions from its metabolic actions on bile acid, glucose lipid and amino acid metabolism, since the metabolic actions are the result of direct FXR transactivation mechanisms. Based on this notion, high-throughput screening platforms were designed to screen for FXR ligands that only have anti-inflammatory/transrepression of NF-kB activity (WP4). In addition, we investigated the differential mechanism of the 4 encoded FXR isoforms. Since two of the isoforms are predominantly present in the intestine and the other two predominantly in the liver, we reasoned that selective targeting of the isoforms would be a way to tissue-specifically activate FXR. We indeed find that different isoforms regulate different genes that result in different functional consequences. We are currently exploring how the different isoforms could be selective targeted, to understand whether isoform-selectivity could be used to separate different FXR actions.
Work Package 3. Development of a non-invasive tool to assay disease remission.
Using alterations in number and type of bacteria residing in the stool samples of patients, Enterome aims to develop a microbiome fingerprint using whole genome metagenomics profiling on stool samples. This fingerprint will be translated into a non-invasive diagnostic tool. By the start of the project, Enterome had already finished the analysis of its internal study named “CrohnOmeter”, into which they recruited 99 patients with a total of 950 samples collected by Q4 2014. Analysis of 439 samples and subgroups has been performed and had led to the identification of two patentable discoveries, the first one (dep. 19/09/2014) was on a gene named CDENT29 which was assembled when Enterome created a gene specific catalogue for Crohn Disease and the second patent is a novel marker of disease activity. UMCU has collected over 100 new stool samples from Crohn Disease patients that were biobanked by Enterome and is used to validate the findings in a separate cohort.
In order to elucidate how the specific bacterial species affect host gut health, we combined efforts to investigate factors known to be associated with improved/aggravating IBD symptoms in order to better understand the function of differentially abundant bacteria in IBD. Therefore, Enterome and the UMCU have collaborated on identifying bacterial components that are associated with smoking in Crohn Disease patients, since smoking is associated with more severe intestinal inflammation. Although the study is limited in terms of sample numbers, some significant signal was picked up and a joint publication has been published in Inflammatory Bowel disease Journal (Opstelten et al. 2016).
Inside the CrohnOmeter study, Enterome has detected several MetaGenomic Species (MGS) correlated to the severity of the disease. Among these MGS, the 6063-3, related with Bifidobacterium longum, has drawn our attention due to its good correlation with the activity level of IBD, and the importance of this genus for bile acid metabolism. Based on this fact and also to contribute to the development of a non-invasive tool to evaluate the disease state (WP3 Aim), we have developed a procedure of bacterial selection and enrichment based on specific genetic signatures. Optimization is still ongoing to get sufficient amounts of sorted bacteria, but then it will allow for the first time to correlate metagenomics data analysis with real strains and bacterial species. Furthermore, it will allow to specifically characterize the full genome of the bacteria associated with disease severity in ChronOmeter study.
We have also generated screening methods for identification of bacterial components/genes modifying bile acid synthesis and FXR activity. Reporter assays were developed in order to measure bacterium-induced production of FXR ligands. In addition, an assay was developed to measure the interaction between FXR and NF-kB upon induction by different bacterial lysates. A publication is underway addressing the validation of these assays and the potential of B. dorei and E. limosum to induce FXR.
Work Package 4. Development of anti-inflammatory ligands.
An automated high-throughput reporter-based screen has been set-up to separate tethering versus direct transactivation by FXR and used for compound screening. A small library of 1200 compounds of FDA-approved drugs was tested to validate the robustness of the screening method and it revealed one compound, Mometasone furoate, that reduced anti-inflammatory activity via FXR with no or limited effects on the metabolic function of FXR. With these results, for the first time it has been shown that separation of metabolic and anti-inflammatory functions of FXR in vitro is possible (Bijsmans et al 2015). In addition, using the same screening method, UMCU has screened a library of novel compounds generated by TES Pharma for their ability to separate FXR functions, and also here we have found some interesting compounds that are currently tested in additional assays. These interesting compounds are bile acid derivatives characterized by an extended side chain that binds the non-canonical FXR S2 accessory ligand pocket, whereas the main scaffold binds the canonical S1 ligand pocket. To understand the genome-wide consequences of different compounds that were selected during these studies, intestinal organoids were incubated with different compounds and RNA has been isolated. Comparative RNA-sequencing data is currently being analyzed. This way, we hope to understand at the level of metabolic and inflammatory genes whether differential regulation by these compounds can be observed. These data will enable us to predict which compounds should be prioritized for in vivo validation in mice to selectively inhibit gut inflammation (WP5). These studies will be proceeded after the closure of the FXR-IBD project. We were invited to write a review for the prestigious Journal Pharmacology and Therapeutics. UMCU and TES Pharma/UNI Perugia have collaboratively reviewed the literature on the relevance of developing FXR selective ligands from a human disease perspective (Massafra et al 2018).
Work Package 5. In vivo validation.
In this work package, animal studies were conducted with novel FXR agonists to investigate whether intestinal inflammation symptoms are decreased and whether they indeed act only on inflammation and not on metabolic actions of FXR. So far, we have tested one new compound in a dose finding study in vivo. 0, 10, 30 and 50 ug/kg of the compound was given for 7 days per oral gavage to mice (n=5 per group). We have established that this compound is very potent and highly selective for FXR. Unfortunately, this compound is not suitable as an FXR-selective compound to decrease the inflammatory response, as it affects both metabolic and anti-inflammatory functions of FXR.
Workpackage 6 Dissemination of results.
We have disseminated our results via publications, symposia, conferences etc. More can be found at our website: www.FXR-IBD.eu.