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Responsabile: dott.ssa Sara De Iudicibus
Nonostante l'introduzione di farmaci biologici, i glucocorticoidi sono ancora utilizzati come trattamento standard nelle malattie infiammatorie croniche intestinali (MICI): la risposta clinica a questi farmaci è però molto variabile. Recentemente, piccole sequenze di microRNA si sono dimostrate importanti regolatori della trascrizione genica, e potrebbero avere un ruolo nella modulazione degli effetti dei glucocorticoidi. L’obiettivo del progetto è l’identificazione di biomarkers utili alla personalizzazione della terapia steroidea in pazienti pediatrici con MICI, attraverso l’integrazione di risultati di farmacogenomica (microRNA e mRNA) e di farmacodinamica (test di sensibilità in vitro). Tali analisi, effettuate su prelievi dei pazienti ottenuti in tempi programmati durante il trattamento con glucocorticoidi, permetteranno inoltre di studiare i meccanismi molecolari coinvolti nel fenomeno di resistenza a questi farmaci.
Background
The incidence of inflammatory bowel disease (IBD) is increasing in recent years, in particular in children and adolescent, and it is currently estimated that 20%-30% of patients experience onset of their symptoms under 20 years of age.
To date, a curative pharmacological therapy for IBD does not exist and the therapeutic approach is mainly aimed at the treatment and control of the inflammation, through drugs capable of inducing and maintaining remission. In spite of the introduction in therapy of highly effective biological agents, glucocorticoids (GCs) are still employed to induce remission in moderate to severe IBD, but considerable inter-individual differences in their efficacy and side effects have been reported. Given the high incidence of suboptimal response, associated with a significant number of side effects, that are particularly severe in paediatric patients, the identification of subjects that are most likely to respond poorly to GCs is extremely important. However, the mechanisms of steroid resistance and/or dependence are scarcely understood and there is presently no means to predict the response in advance. Genes involved in GC pharmacodynamic (genes of the GC receptor heterocomplex), and associated with the inflammatory pathway (genes encoding for inflammatory mediators) could be considered important candidates for an epigenetic approach involving microRNA (miRNA). miRNAs are small non-coding RNA molecules that suppress gene expression at post-transcriptional level, and are fine-tuning regulators of diverse biological processes, including the development and function of the immune system, apoptosis, metabolism and inflammation. Emerging data have implicated the deregulated expression of certain miRNA networks in the pathogenesis of autoimmune and inflammatory diseases, such as IBD.
Aims
The proposed research will develop new strategies for therapy personalization, based on clinical pharmacology assays, and in particular pharmacodynamic and pharmacogenomic assessments.
Study Design
Fifty paediatric patients with IBD, treated at the Paediatric Clinic of IRCCS Burlo Garofolo will be enrolled in a prospective study. Clinical activity, inclusive of clinical and inflammatory index evaluation, will be assessed by "Pediatric Crohn's Disease Activity Index" (PCDAI) for patients with Crohn's Disease, and "Ulcerative Colitis Activity Index" (UCAI) for patients with Ulcerative Colitis. Side effects will be recorded at each clinical evaluation. Peripheral blood will be obtained from these patients at three different times, at diagnosis before treatment, after 30 days of 1 to 2 mg/kg/day of prednisone and 12 months after diagnosis: the pharmacodynamic (GC sensitivity on primary cultures of patients' leukocytes) and pharmacogenetic (candidate DNA and mRNA and miRNA profiles) assays will be performed for each patient at these three time-points.
Summary Preliminary Data
Several recent studies of our laboratory on pharmacogenetic of IBD, have demonstrated that the BclI polymorphism in GC receptor gene (NR3C1), and the Leu155His polymorphism in NALP1 gene (involved in IL- 1beta activation), are associated with the GC clinical response in paediatric IBD patients.
Moreover, preliminary experiments in our lab have revealed a relation between the GC clinical response of paediatric patients affected by chronic inflammatory diseases and the in vitro sensitivity of their leukocytes to these agents.
Materials and methods
In vitro response to GCs will be evaluated by means of 3H thymidine incorporation assay performed on mononuclear cells isolated from peripheral blood. In addition, mRNA and miRNA expression during GC treatment will be evaluated and associated with in vitro sensitivity and patients' clinical response.
The expression of the selected candidate genes (GC receptor heterocomplex genes and genes associated with the inflammatory pathway) will be evaluated in lymphocytes obtained at diagnosis, after 30 days and 12 months, using TaqMan gene expression assays.
The miRNA patients profiles at each time-points will be generated by using an up-to-date real time based agnostic miRNA expression assay (e.g. TaqMan Array microRNA cards).
Main Expected Results and Impact
This study will develop an innovative set of pharmacological tools, integrating pharmacodynamics and pharmacogenomics, to be applied at the optimization of GC therapy in paediatric IBD patients, in order to increase therapy efficacy and to reduce adverse events.
Significance and Relevance for National Health System (SSN)
Quality of life is severely affected in IBD, due to the chronic character of the disease that implies frequent hospitalizations and aggressive therapies, with a significant risk of side effects and a considerable impact on health care costs. This project could provide new pharmacological markers useful to adjust treatment a priori, with an important impact on improved cure rates, avoiding inadequate regimens, and this is particularly important in paediatric patients, and reducing overall heath-care cost.
