RESEARCH

The coordinated research program focuses on translational pharmacology in pediatrics, with particular attention to therapy personalization strategies. We aim to contribute to the understanding of molecular and cellular mechanisms that influence drug response in pediatric patients. The established collaboration with the Scientific Institute for Research, Hospitalization and Healthcare (IRCCS) Maternal and Child Institute Burlo Garofolo in Trieste constitutes a cornerstone of our research activity. This collaboration facilitates the development of translational projects with concrete relevance for optimizing pediatric therapeutic protocols.

With a combination of pharmacokinetic, pharmacodynamic, and pharmacogenomic approaches and in vitro models, the group studies the role and interconnection of these characteristics and their association with patient response to therapy.

Development of patient-specific in vitro models for therapy personalization

Molecular hypotheses generated based on observations made in patients are verified in vitro to obtain information on the cellular and molecular mechanisms underlying relevant clinical phenomena and to further improve clinical practice.

Currently, these in vitro experiments focus on developing innovative pharmacological models based on induced pluripotent stem cells (iPSCs) and intestinal organoids.

iPSCs are obtained by reprogramming patients' somatic cells into an embryo-like state through the forced expression of selected genes; these cells maintain the donors' genetic heritage, thus allowing the generation of personalized models. The pluripotency of iPSCs, which makes it possible to differentiate these cells into all tissues of the human organism through appropriate culture conditions, allows obtaining cell lines that are otherwise not easily accessible from human subjects, including pancreatic ones. Advances in iPSC technology create new possibilities for modeling diseases and controlling adverse drug effects in a personalized way. In particular, the focal point of our current research in this field is based on the generation of the exocrine pancreas, both two-dimensional and three-dimensional, from iPSCs, in order to study drug-induced pancreatitis. Intestinal organoids, on the other hand, are generated from intestinal biopsies from which it is possible to isolate the crypts where adult stem cells are located. Through the cultivation of adult stem cells, it is possible to generate 3D intestinal structures that preserve the patient's genetic and epigenetic background. Currently, several projects on intestinal organoids from pediatric patients with IBD are ongoing, both to identify new pharmacological therapies for the treatment of these diseases and to investigate the mechanisms of action of drugs currently used in clinical practice.

Immunofluorescence performed on exocrine pancreatic organoids derived from iPSCs. A) Nuclei stained in blue with DAPI; B) Pancreatic amylase stained in red; C) Cytokeratin-19 stained in green; D) Merge.

Pharmacogenetics and pharmacoepigenetics in pediatric inflammatory bowel diseases

IBD is characterized by chronic idiopathic intestinal inflammation and includes two main disorders: Crohn's disease and ulcerative colitis. The peak onset of IBD is in people aged between 15 and 30 years; however, disease onset in pediatric patients is frequent, and increasing incidence rates globally have been demonstrated in both developed and developing countries, constituting a true epidemic in children. Currently, there is no curative therapy for these diseases, but many immunosuppressive drugs are currently employed.

Glucocorticoids are administered to induce remission, while the thiopurine antimetabolites azathioprine and mercaptopurine are effective in maintaining remission.

For glucocorticoids, the efficacy of these drugs is highly variable, and side effects, particularly severe in pediatric patients, are common and often unpredictable: understanding the complex gene regulation mediated by glucocorticoids could shed light on the causes of this variability. In this context, epigenetic characteristics, such as non-coding RNA expression profiles or DNA methylation profiles, represent a field of research of interest to us.

For azathioprine, current research considers the identification of new mechanisms of action and new cellular targets such as the intestinal epithelium. New pathways involved in thiopurine efficacy, including autophagy and the STING pathway, are under study.

Pharmacogenomic variables considered in IBD include single nucleotide polymorphisms in genes involved in mercaptopurine biotransformation such as TPMT, or in pharmacodynamics including PACSIN2 and ATG16L1.

Among the most promising and innovative pharmacodynamic markers under study are extracellular vesicles from which we isolate and quantify candidate miRNAs associated with the response to thiopurines.

The integration of various pharmacological measurements (pharmacogenetic, pharmacokinetic, and pharmacodynamic) with their response to therapy aims to provide physicians with tools to best adapt treatment to individual patient characteristics.

Pharmacological approaches for personalizing therapy of nephrotic syndrome (NS)

NS is a childhood kidney disease caused by impaired glomerular function that typically presents in the first decade of life. Glucocorticoids are considered the first-line therapy for idiopathic childhood NS and can induce remission in 90-95% of patients. Glucocorticoid responsiveness at diagnosis is of great prognostic importance regarding kidney function, which is generally well preserved in glucocorticoid-sensitive NS. Cases of glucocorticoid-resistant NS, primary and secondary, are observed in 5-10% and 1-3% of children, respectively, and these patients are subject to progressive disease and kidney failure. Relapses are also common in children; pediatric patients with recurrent disease are at risk of serious complications.

Many efforts have been made to predict the response to glucocorticoids in children with NS to avoid unnecessary drug therapies and side effects, but with conflicting results.

The main objective of this research is to evaluate the role of pharmacogenomic characteristics involved in glucocorticoid pharmacodynamics in response to steroid therapy in pediatric patients with NS treated with a standardized protocol. These characteristics include epigenetic profiles, particularly the methylation of the NLRP3 gene promoter and the expression of lncRNAs GAS5 and GAS5-AS1. These analyses will lead to the identification of useful genetic markers that allow predicting the efficacy of steroid therapy a priori.

Therapeutic personalization in juvenile idiopathic arthritis (JIA): molecular markers and response to methotrexate (MTX)

For children with JIA who do not respond to the antimetabolite MTX, the delay in identifying the optimal treatment at an early stage of the disease can lead to irreversible joint damage.

In this context, the primary objective is to identify useful predictive markers to predict the effectiveness of MTX, so as to promptly direct JIA patients with lower likelihood of response to the drug towards more aggressive therapies with biological drugs.

For this purpose, in vitro studies aimed at investigating the mechanism of action of MTX will allow identifying new efficacy markers, including, for example, single nucleotide polymorphisms (SNPs) in candidate genes. Additionally, recent evidence suggests a possible role of long non-coding RNAs (lncRNAs) in the mechanism of action of the drug. In particular, lincRNA-p21 is known for its interaction with p53 and its ability to modulate the inflammatory response through the regulation of NF-κB. In vitro studies will allow deepening the mechanism underlying the action of this lncRNA both in immortalized cell lines representative of the pathology and in peripheral blood mononuclear cells (PBMCs) of patients. Subsequently, the validation of these markers will occur in patients through correlation with the clinical response to MTX, evaluated through disease activity indices, such as the Juvenile Arthritis Disease Activity Score (JADAS), the American College of Rheumatology Pediatric (ACRped), and the achievement of clinical remission. In this way, new predictive markers of response to MTX will be identified, which will allow optimizing the therapeutic pathway in patients with JIA and improving disease management.

Schematization of the mechanisms of action of MTX; Inhibition of de novo synthesis of purines and pyrimidines through inhibition of dihydrofolate reductase (DHFR) and thymidylate synthase (TYMS); induction of lincRNA-p21 and its binding to the mRNA of the p65 subunit of NF-κB, following activation of DNA-dependent protein kinase (DNA-PK) and p53; increased adenosine release by inhibition of AICAR transformylase (ATIC), resulting in activation of adenosine A2A receptors (ADORA2A), responsible for the anti-inflammatory effect.

Circulating neuronal extracellular vesicles: predictive biomarkers of ketamine adverse effects

Ketamine is a dissociative anesthetic, a non-competitive antagonist of the N-methyl-D-aspartate (NMDA) receptor that blocks the excitatory effect of glutamate. It thus reduces NMDA receptor-mediated synaptic excitation in the central nervous system, resulting in anesthetic and analgesic action. Ketamine is a drug present in the therapeutic arsenal of pediatricians dealing with procedural analgosedation of children outside the operating room. Like all drugs, ketamine can induce side effects. The most frequently recorded side effects during procedural sedation outside the operating room, after ketamine administration, are vomiting and post-sedation agitation. The aim of the study is to evaluate the presence of miRNAs isolated from plasma extracellular vesicles originating from the central nervous system, which may be associated with the development of vomiting or post-sedation agitation. In addition, the study also involves the use of personalized neuronal models derived from the differentiation of patient-specific iPSCs; these models will be used to further investigate the role of miRNAs in predisposing patients to the development of ketamine-induced post-sedation agitation and vomiting.

Neuronal extracellular vesicles isolated from patient plasma. A) Transmission electron microscopy: overview and enlarged detail. Reference scale = 100 nm. B) Representative image and enlargement of a neuronal vesicle via ONI (Nanoimager) with quantification of tetraspanin distribution (doi: 10.1002/cpt.3420).

Thalidomide-induced peripheral neuropathy

Thalidomide is a drug with immunomodulatory and antiangiogenic properties, used in the treatment of erythema nodosum leprosum, multiple myeloma, and inflammatory bowel diseases (IBD) in patients refractory to conventional therapies. However, prolonged use of the drug is often limited by the onset of peripheral neuropathy (TiPN), the main adverse drug reaction and one of the most common causes of treatment discontinuation.

Given the still unclear pathogenesis of TiPN, the study uses iPSCs derived from pediatric patients with IBD, stratified based on the presence or absence of TiPN, to generate sensory neurons, useful for studying the molecular and cellular mechanisms underlying this adverse drug reaction. These cellular models will be used to further investigate the role of ADAMTS2 and CELSR2, two genes identified as downregulated in preliminary transcriptomic studies conducted by the research group on PBMCs from patients with IBD and on the SHSY-5Y cell line treated with the drug.

Safety and efficacy of innovative therapies for pediatric patients with Aicardi-Goutières syndrome using patient-specific models

Aicardi-Goutières syndrome (AGS) is a rare genetic disease, classified as a primary immunodeficiency with onset in the neonatal period and early childhood. Clinically, patients with AGS present with immune manifestations, neurodevelopmental degeneration, and progressive disorders resulting in poor life expectancy. AGS has a highly heterogeneous phenotype and is characterized by an abnormal accumulation of endogenous nucleic acids that trigger an aberrant interferon (IFN)-mediated immune response (resulting in a destructive interferonopathy). To date, pharmacological treatments are partially effective and mainly supportive. The aim of the study is to generate neural stem cells (NSCs) and neurons from patient-specific iPSCs for in vitro screening of the efficacy and safety of conventional and potentially innovative therapies for AGS.