The research group belongs to the Department of Life Sciences and the Department of Medical, Surgical and Health Sciences.
The coordinated research program focuses on both basic and translational aspects of the pharmacology of immunosuppressants and antileukemic drugs. The research interests of the team are focused on the identification and study of the molecular and cellular characteristics that can be used to personalize immunosuppressive and antileukemic therapy in pediatric patients. 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 patients' response to therapy.
At the center of the investigations is the personalization of therapy for pediatric inflammatory bowel diseases, acute lymphoblastic leukemia, nephrotic syndrome and juvenile idiopathic arthritis, with particular attention to the drugs used in these diseases.

Pharmacological approaches for the personalization of the therapy of pediatric inflammatory bowel diseases (MICI)
IBDs are characterized by chronic idiopathic inflammation of the intestine and include two main disorders: Crohn's disease and ulcerative rectocolitis. The onset peak of MICI is in people aged between 15 and 30; however, the onset of the disease in pediatric patients is frequent and an increase in the global incidence rates (especially Crohn's disease) have been demonstrated in both developed and developing countries, constituting a real epidemic in children. At present, there is no curative therapy for these diseases, but many immunosuppressive drugs are currently employed.
Monoclonal antibodies directed to TNFα (mainly infliximab and adalimumab) and other cytokines are effective for inducing remission and for maintenance therapy in refractory forms of the disease. Glucocorticoids (GCs) are administered to induce remission, although their use is limited by particularly severe adverse effects in pediatric patients. The thiopurine antimetabolites azathioprine (AZA) and mercaptopurine (MP) are effective for maintaining remission. In cases that do not respond to these therapies, thalidomide has recently been proposed as an effective agent.
Biological therapy with TNF-alpha antagonists such as infliximab has significantly improved the clinical management of pediatric patients with severe IBD: studies are underway to monitor treatment with infliximab and adalimumab with innovative pharmacokinetic and pharmacodynamic approaches to further optimize and rationalize the TNF-alpha antagonist therapy in children who need these expensive drugs. The information obtained from these translational studies will be extremely useful for optimizing therapy, improving cure rates and reducing side effects.
For glucocorticoids, the efficacy of these drugs is very variable and the side effects, particularly serious in pediatric patients, are common and often unpredictable: an understanding of 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 new and promising field of research. For azathioprine, current research considers the concentration of thiopurine metabolites in children with IBD and associates it with polymorphisms in determinants of thiopurine biotransformation candidates, such as thiopurine-S-methyl transferase (TPMT), inosine-triphosphate-pyrophosphatase (ITPA) and glutathione-S-transferase (GST). Particular attention is paid to the developmental aspects of thiopurine biotransformation during patient growth.

For thalidomide, through high-throughput sequencing approaches and analysis of candidate genes, the ongoing studies aim to identify genetic and epigenetic determinants associated with both efficacy but also with known side effects, including neuropathy.

Recently the study of interindividual variability in the response to immunosuppressive drugs in IBD has been developed thanks to the development of innovative patient-specific models of intestinal tissue, namely organoids.

Pharmacological approaches for the personalization of acute lymphoblastic leukemia (ALL) therapy

Risk-adjusted polychemotherapy protocols successfully treat 85% of patients with ALL, the most frequent hematological malignancy of infancy. In addition to this important success rate, LA treatment is complicated by interindividual variations, currently unpredictable, in the clinical response. In fact, among patients who fall back, 50-70% have a fatal outcome; in addition, most patients develop severe or even lethal adverse chemotherapy-related effects. A prospective multicenter study, mainly focused on methotrexate antimetabolites and mercaptopurine used in the ALL chemotherapy protocol is underway; this study is conducted on patients enrolled in the AIEOP-BFM protocol (Italian Association of Hematology and Pediatric Oncology - Berlin Frankfurt Munchen) ALL 2009 and intends to integrate different pharmacological measurements (pharmacogenetics, pharmacokinetics and pharmacodynamics) with their response to therapy, with the aim of provide doctors with the tools to best adapt the treatment to the individual characteristics of the patients. The pharmacogenomic variables considered are single nucleotide polymorphisms in the genes involved in the biotransformation of mercaptopurine (TPMT, ITPA, PACSIN2), in the SLCO1B1 gene involved in the excretion of methotrexate and in the deletions of GST-M1 and GST-T1, important for the detoxification of different drugs. The variants of interest are also examined retrospectively in patients enrolled in the previous AIEOP-BFM ALL 2000 protocol whose DNA and clinical data are already available. Pharmacokinetic measurements include the clearance of methotrexate during the consolidation phase and the evaluation of the main active metabolites of MP (thioguanine nucleotides and methylmercaptopurine methylated nucleotide) as well as the enzymatic activity of TPMT and ITPA, monitored during the consolidation and therapy of maintenance. Pharmacodynamic parameters are provided by the in vitro MTT test on blasts at the diagnosis that measures the cytotoxic effects of antileukemic drugs. Statistical analyzes evaluate the association between the in vitro pharmacological parameters of the patients and the in vivo response, to verify the prognostic role on the clinical outcome.

Pharmacological approaches for the personalization of nephrotic syndrome (SN) therapy

SN is a childhood kidney disease caused by impaired glomerular function that typically occurs in the first decade of life. Glucocorticoids are considered the first-line therapy of idiopathic infantile SN and are capable of inducing remission in 90-95% of patients. The reactivity of glucocorticoids to diagnosis is of great prognostic importance as regards renal function, which is generally well preserved in SNs sensitive to these drugs. Cases of primary and secondary glucocorticoid-resistant SNs 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 relapsing disease are at risk of serious complications.
Much effort has been made to predict glucocorticoid response in children with SN to avoid unnecessary drug therapies and side effects, but with conflicting results.
The main objective of this research is the evaluation of the role of the pharmacogenomic characteristics involved in the pharmacokinetics and pharmacodynamics of glucocorticoids in the response to steroid therapy in pediatric patients with SN treated with a standardized protocol. These characteristics include genetic polymorphisms and epigenetic profiles, in particular DNA methylation. These analyzes will lead to the identification of useful genetic markers, which allow to predict the a priori efficacy and steroid therapy. The 

secondary actives are to verify the possible relationships between genetic polymorphisms and glucocorticoid response in vitro (mononuclear cell proliferation test).

Pharmacological approaches for the personalization of juvenile idiopathic arthritis (AIG) therapy

For children with AIG who do not respond to the methotrexate antimetabolite, the delay in identifying the optimal treatment at an early stage of the disease can lead to long-term joint damage.
The primary objective is to identify the pharmacogenetic and pharmacokinetic markers useful in predicting the efficacy of methotrexate in patients with JIA. In fact, the identification of patients who could respond to methotrexate before treatment would be very useful for the clinician and our study should support the development of multilocus pharmacogenetic profiles to predict the response to methotrexate in these patients. Genotyping should be performed at diagnosis and patients with a genotype predisposing to response may be treated with methotrexate, given the high probability of response to this treatment. This study provides a rationale for reserving biological drugs to patients who are unlikely to benefit from less expensive but still effective treatments such as methotrexate. On the contrary, patients with variants associated with the lack of efficacy of methotrexate should be transferred more quickly to more aggressive treatment (i.e., methotrexate + biologists or biologists alone).
The study was carried out within an international consortium involving Italian (Burlo and Meyer pediatric hospitals) and US (coordinated by the Cincinnati hospital) centers. The clinical response to methotrexate is assessed as disease activity values ​​(JADAS and ACRped values ​​and changes) and as stable clinical remission over a 6-month period. The most relevant SNPs for each gene considered are tested on the patients' DNA. Pharmacokinetic characteristics such as the concentration of active drug metabolites, polyglutamate methotrexate and the activity of the enzyme inosine-triphosphate-pyrophosphatase are measured in the erythrocytes of the patients.
Studies have recently been launched regarding the development of peptide biosensors to quantify the activity of tyrosine kinases constitutively active in certain subtypes of acute lymphoblastic leukemia, in particular those that have the BCR-ABL translocation or other translocations that lead to a profile transcriptomic similar to BCR-ABL leukemias (BCR-ABL like leukemia); Furthermore, a study has been launched regarding the personalization of the myeloablative regimen with busulfan in leukemic patients in whom chemotherapy is unsuccessful or in which the disease relapses and who undergo bone marrow transplantation.

In vitro mechanistic studies on the pharmacokinetics, pharmacodynamics and pharmacogenomics of immunosuppressive and anti-leukemic drugs

The molecular hypotheses generated on the basis of the observations made in patients are verified in vitro, to obtain information on the cellular and molecular mechanisms underlying the relevant clinical phenomena and to further improve clinical practice.
Currently, these in vitro experiments are focused on:
- identification of long non-coding RNA as potential markers involved in the molecular mechanism of glucocorticoids, thus providing a new assessment of their involvement in the phenomenon of steroid resistance; an important purpose of this research is to provide a tool that could allow the optimization of steroid therapy in patients treated with these drugs;
- development of a biotechnological model to study the effects of thiopurins in vitro and the contribution of genetic determinants in the modulation of the cytotoxic effects and of the biotransformation of antimetabolites, with particular attention to the events that occur in intestinal, hepatic and pancreatic tissues: this model could therefore be used to validate the role of genetic factors (also emerging from whole genome studies) on the sensitivity of patients to thiopurins and to evaluate the effects of treatment adjustments;
- development of innovative pharmacological models based on induced pluripotent stem cells (iPSC): these are stem cells obtained by reprogramming the somatic cells in an embryo-like state through the forced expression of selected genes. The pluripotency of iPSC allows to obtain cell lines otherwise not easily accessible by human subjects, since they can be differentiated into various types of cells, growing iPSC under appropriate conditions. Advances in iPSC technology create new possibilities for modeling diseases and controlling adverse effects of drugs in a more personalized way. Pioneering studies have been conducted on liver, heart and nerve cells. However, studies based on iPSC technology, exocrine pancreas and drug-induced pancreatitis are currently limited and this is the focal point of our current research in this area.