Community Research and Development Information Service - CORDIS

Periodic Report Summary 2 - FIBRO-TARGETS (Targeting cardiac fibrosis for heart failure treatment)

Project Context and Objectives:
In Europe, 5% of hospital admissions in adults are due to heart failure (HF) – a larger proportion caused by myocardial infarction. In addition, ageing of the EU population will make incidence and prevalence of HF rise inexorably, unless effective preventive therapy is applied. So far antihypertensive therapy has had some success in preventing HF. Nevertheless, no therapy targeting pathophysiological mechanisms in HF such as fibrosis, inflammation or myocyte growth, is available for clinical use. At last, beyond the impact on myocardial interstitial fibrosis (MIF), nearly 45% of all deaths in the developed world can be attributed to some type of chronic fibro-proliferative disease.
With this scenario, finding new targets, biomarkers and/or promising molecules with anti-fibrotic capabilities, would have a great impact in the society in a mid-term.
In this sense, FIBRO-TARGETS is based on the hypothesis that the intervention on novel fibrosis-related targets involved in the processes of fibroblast differentiation to myofibroblasts, on the collagen synthesis over degradation balance and/or on collagen maturation may allow for interstitial repair, thus providing a new strategy for the prevention and treatment of MIF.
Since the beginning of the program, the setting of several experiment models (for in vitro read-outs and in vivo in small and large animal) relevant to one or several studied signaling pathways has allowed to further or newly characterize the capacity of candidate biotargets to modulate collagen production and cross-linking.

According to the anti- or pro- fibrotic properties of the studied biotargets, molecules (hits) were identified as potentially able to interfere with biotarget biological activity. The proposed agonist and antagonists have been selected within large libraries of molecules for most of the biotargets. When conventional structure activity relationship (SAR) analysis could not be used because of the unknown crystalline structure of OGN a different approach was taken to find homologues of OGN and use amino-acid sequence alignment and 3D protein structure overlay to identify conserved peptidic motifs that may be important for the OGN activities. Additional suitable cell-based screening assays have been developed to determine the efficacy parameters for the potential hits for several biotargets from each WP.

The therapeutic potential of some of the most promising hits are being tested in vitro, and will be challenged in vivo in small and big animal during the upcoming period.

Meanwhile, to validate therapeutic relevance of the hits identified for the studied biotargets, a common database of controls and patients susceptible of developing fibrosis or having fibrosis has been set. The merged clinical data of 1,200 individuals are associated with biosamples that will allow the biomarker profiling of selected individuals for the biotargets resulting from the analysis and discoveries made experimentally. An optimization of the biosamples use has required the development and/or validation of multiplexing bioassays for selected/identified targets. A first set of samples from patients and matched controls will be assayed soon. The resulting stratification of the patient according to signal-specific BM profile and diagnostic algorithms will represent a clinical tool for faster decision making based on surrogate endpoints, but also for more advance application such as theranostic, companion, and complementary BM tests. But before that, experimental and clinical assessment of the selected biotargets will provide strong evidence to set the ground for new anti-fibrotic therapeutic strategies.

Project Results:
Up to now the setting of several experiment models relevant to one or several studied signaling pathways allowed testing the capacity of number of molecules to modulate collagen production and cross-linking (CCL). Powerful read-outs (including cell-based assays (including SMAD2-4xSBE iHCF)) have been set to both characterize the anti- or pro-fibrotic properties of candidates biotargets and to screen molecules interfering with their effects. Depending on the unraveled anti- or pro-fibrotic properties, agonist and antagonists have been selected among large libraries of molecules (480 natural compound library and Prestwick Chemical Library) or identified based on target-based strategies.

The main findings are that:

- cardiotrophin-1 (CT-1), NGAL, Gal-3 and osteoglycin (OGN) display pro-fibrotic activity that can be blocked by newly identified interfering molecules. Dose response, specificity, toxicity have been done for several hits.

- apelin displays anti-fibrotic activity and apelin receptor agonists have been shown to inhibit TGF-beta-induced fibrosis).

- proteomic approaches led to the identification of novel mineralocorticoid receptor downstream targets that will be further explored as candidates biotargets.

- using functional high-throughput miRNA library screens >2000 miRNAs have been tested to clarify their mechanistic role in MIF and the capacity to block cardiac fibrosis of >480 natural compounds has been tested. A number of interesting powerful candidates (both for miRNAs and natural compounds) have been identified and their anti-fibrotic activities are currently tested in vivo to pave the way for their future clinical use.

Hits for each studied signaling pathways have been identified and their anti-fibrotic properties have been partly or extensively characterized in vitro. The potential hits have been entered in an in vitro assays pipeline where several have been evaluated for their efficacy, toxicity and ADME characteristics. In order to identify possible cross-modulators, all most promising hits from one WP have been supplied to partners from other WPs. Upcoming period will allow their evaluation in vivo and would end up in a set of best performing candidates (hits) that would prevent MIF or block its progression.

- Protocols and procedures needed for conducting the evaluation of selected agents in small and large animal models were established. Small animal testing has been conducted. For pig experiments, protocols for myocardial infarction as well as induction of left ventricular hypertrophy were established and all animals of the control groups were successfully finished up to the final follow up examinations. Serial in vivo PET-MRI imaging was established and refined for the purposes of the project. In control AMI animals, the presence of severely ischemic but viable myocardium at the anteroseptal location after MI was confirmed. A novel model for inducing cardiac hypertrophy in pigs was developed, and imaging and molecular biomarkers showed gradual development of cardiac fibrosis.

Using human clinical data and associated biosamples, the clinical relevance of those molecules have been evaluated.

In patients:

- increased CT-1 and down-regulated miR-122 and miR-19b levels were associated with myocardial fibrosis and collagen production.
- OGN plays an important role in MIF during various forms of HF and that in patients with a history of myocardial infarction OGN levels are higher and correlate with left ventricular dimensions. Convergence of important mechanistic pathways in cardiac fibroblasts, namely CT-1 and SPARC has been characterized.
- a common database regrouping >12,000 individuals susceptible of developing fibrosis or having fibrosis has been set. The comorbidities and phenotypic subgroups have been selected based on the FIBRO-TARGETS data dictionary. The associated biosamples will allow the measurement of candidate biotargets. A first set of 604 relevant cases matched by sex and age to 2 controls has been assigned to one selected comorbidity and associated biosamples should be assayed during the up coming third period.

Potential Impact:
For the WP1

We expect to identify and validate new specific targets involved in the development of myocardial interstitial fibrosis in chronic HF patients and to develop, at a pre-clinical stage, potential inhibitors of these pathways. Taking into account the high and increasing prevalence of HF and the fact that myocardial fibrosis is present even in those HF patients treated according to the clinical guidelines, and that it contributes to cardiac dysfunction and affect the clinical outcome, the development of novel safe and effective anti-fibrotic therapies could, in the long term, improve life expectancy and quality of HF patients. Moreover, HF constitutes a major economic burden for the European Health Systems, and these therapeutic agents might also have a favorable impact on this issue.

For the WP2

We expect to have extensively elucidated the roles of OGN and SPARC in the heart during cardiac diseases associated with excessive fibrosis. We aim to unravel their signalling pathways and to show that these findings are relevant to human disease. Finally we aim to have tools with which we can manipulate these pathways in small animal models of disease to improve fibrosis and disease outcome.

For the WP3

The identification of novel drugs to combat interstitial cardiac fibrosis has a huge importance. Currently miRNA therapeutic drugs are tested in human clinical trials in HCV infection (N Engl J Med. 2013 May 2;368(18):1685-94) and miRNA inhibitors are also tested in first forms of tissue fibrosis, e.g. in kidney fibrosis patients ( Thus, miRNA modulation in humans with the aim to inhibit cardiac fibrosis is a realistic goal and has great chances as innovative translational anti-fibrotic strategies.

For the WP4

- Identify the role of these novel biotargets in the consequences of mineralocorticoid receptor activation
- Identify potential therapeutic opportunities in interfering with these novel biotargets

For the WP5

In particular WP5 will try to find new molecule candidates that show certain performance thresholds in reverting or stopping the fibrotic processes. WP5 is applying a dual (target-based in silico-in vitro and traditional in vitro library screening) screening approaches, to find candidates with promising efficacy, toxicity and basic ADME parameters.

For the WP6

The use of small and large animal models for myocardial infarction will ensure accurate evaluation of potential targets and treatment options that are developed during the FIBRO-TARGETS project. Particularly the large animal models, which include modern imaging technologies, are an important step for translation to the clinic. The comprehensive analysis comprising imaging methods and molecular and transcriptional analyses helps extracting as much information as possible from animal experiments.

For the WP7

In the frame of the WP7, analysis of biosamples selected from 12,922 subjects (HF and controls) of the merged cohort will help defining BM profile and diagnostic algorithms allowing faster decision making based on surrogate endpoints, but also for more advance application such as theranostic, companion, and complementary BM tests and stratifying patients according to their biomarker profiles. This BM-based tool will be useful for the pharma industry developing drugs in HF domain. For clinical applications, the BM-based stratification aims to improve patients’ management and quality of life using optimized targeted therapy avoiding side effects. It should be noted that several large diagnostic companies have expressed interests in HF biomarkers qualified by Firalis.

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