Thiopurine metabolites variations during co-treatment with aminosalicylates for inflammatory bowel disease:Effect of N-acetyl transferase polymorphisms

AIM: To evaluate variation of the concentration of thiopurine metabolites after 5-aminosalicylate(5-ASA) interruption and the role of genetic polymorphisms of N-acetyl transferase(NAT) 1 and 2. METHODS: Concentrations of thioguanine nucleotides(TGN) and methymercaptopurine nucleotides(MMPN), metabolites of thiopurines, were measured by high performance liquid chromatography in 12 young patients(3 females and 9 males, median age 16 years) with inflammatory bowel disease(6 Crohn's disease and 6 ulcerative colitis) treated with thiopurines(7 mercaptopurine and 5 azathioprine) and 5-ASA. Blood samples were collected one month before and one month after the interruption of 5-ASA. DNA was extracted and genotyping of NAT1, NAT2, inosine triphosphate pyrophosphatase(ITPA) and thiopurine methyl transferase(TPMT) genes was performed using PCR assays. RESULTS: Median TGN concentration before 5-ASA interruption was 270 pmol/8 x 108 erythrocytes(range: 145-750); after the interruption of the aminosalicylate, a 35% reduction in TGN mean concentrations(absolutemean reduction 109 pmol/8 × 108 erythrocytes) was observed(median 221 pmol/8 × 108 erythrocytes, range: 96-427, P value linear mixed effects model 0.0011). Demographic and clinical covariates were not related to thiopurine metabolites concentrations. All patients were wild-type for the most relevant ITPA and TPMT variants. For NAT1 genotyping, 7 subjects presented an allele combination corresponding to fast enzymatic activity and 5 to slow activity. NAT1 genotypes corresponding to fast enzymatic activity were associated with reduced TGN concentration(P value linear mixed effects model 0.033), putatively because of increased 5-ASA inactivation and consequent reduced inhibition of thiopurine metabolism. The effect of NAT1 status on TGN seems to be persistent even after one month since the interruption of the aminosalicylate. No effect of NAT1 genotypes was shown on MMPN concentrations. NAT2 genotyping revealed that 6 patients presented a genotype corresponding to fast enzymatic activity and 6 to slow activity; NAT2 genotypes were not related to thiopurine metabolites concentration in this study. CONCLUSION: NAT1 genotype affects TGN levels in patients treated with thiopurines and aminosalicylates and could therefore influence the toxicity and efficacy of these drugs; however the number of patients evaluated is limited and this has to be considered a pilot study.

Induced pluripotent stem cells for therapy personalization in pediatric patients:Focus on drug-induced adverse events

Adverse drug reactions(ADRs)are major clinical problems,particularly in special populations such as pediatric patients.Indeed,ADRs may be caused by a plethora of different drugs leading,in some cases,to hospitalization,disability or even death.In addition,pediatric patients may respond differently to drugs with respect to adults and may be prone to developing different kinds of ADRs,leading,in some cases,to more severe consequences.To improve the comprehension,and thus the prevention,of ADRs,the set-up of sensitive and personalized assays is urgently needed.Important progress is represented by the possibility of setting up groundbreaking patient-specific assays.This goal has been powerfully achieved using induced pluripotent stem cells(iPSCs).Due to their genetic and physiological species-specific differences and their ability to be differentiated ideally into all tissues of the human body,this model may be accurate in predicting drug toxicity,especially when this toxicity is related to individual genetic differences.This review is an up-to-date summary of the employment of iPSCs as a model to study ADRs,with particular attention to drugs used in the pediatric field.We especially focused on the intestinal,hepatic,pancreatic,renal,cardiac,and neuronal levels,also discussing progress in organoids creation.The latter are three-dimensional in vitro culture systems derived from pluripotent or adult stem cells simulating the architecture and functionality of native organs such as the intestine,liver,pancreas,kidney,heart,and brain.Based on the existing knowledge,these models are powerful and promising tools in multiple clinical applications including toxicity screening,disease modeling,personalized and regenerative medicine.

Pharmacogenetics of azathioprine in inflammatory bowel disease: A role for glutathione-S-transferase?

Azathioprine is a purine antimetabolite drug commonly used to treat inflammatory bowel disease(IBD).In vivo it is active after reaction with reduced glutathione(GSH)and conversion to mercaptopurine.Although this reaction may occur spontaneously,the presence of isoforms M and A of the enzyme glutathione-S-transferase(GST)may increase its speed.Indeed,in pediatric patients with IBD,deletion of GST-M1,which determines reduced enzymatic activity,was recently associated with reduced sensitivity to azathioprine and reduced production of azathioprine active metabolites.In addition to increase the activation of azathioprine to mercaptopurine,GSTs may contribute to azathioprine effects even by modulating GSH consumption,oxidative stress and apoptosis.Therefore,genetic polymorphisms in genes for GSTs may be useful to predict response to azathioprine even if more in vitro and clinical validation studies are needed.reserved.

Role of tristetraprolin phosphorylation in paediatric patients with inflammatory bowel disease

BACKGROUND Intestinal inflammation and epithelial injury are the leading actors of inflammatory bowel disease(IBD),causing an excessive pro-inflammatory cytokines expression.Tristetraprolin(TTP),an mRNA binding protein,plays a role in regulating the inflammatory factors,recognizing specific sequences on the 3’untranslated region of cytokine mRNAs.TTP activity depends on its phosphorylation state:the unphosphorylated TTP degrades pro-inflammatory cytokine mRNAs;on the contrary,the phosphorylated TTP fails to destabilize mRNAs furthering their expression.The phospho-TTP forms a complex with the chaperone protein 14-3-3.This binding could be one of the factors that promote intestinal inflammation as a cause of disease progression.AIM To assess if TTP phosphorylation has a role in paediatric IBD.METHODS The study was carried out on a cohort of paediatric IBD patients.For each patient enrolled,a specimen of inflamed and non-inflamed colonic mucosa was collected.Furthermore,the experiments were conducted on macrophages differentiated from blood samples of the same patients.Macrophages from healthy donors’blood were used as controls.Co-immunoprecipitation assay and immunoblotting analyses were performed to observe the formation of the phospho-TTP/14-3-3 complex.In the same samples TNF-αexpression was also evaluated as major factor of the pro-inflammatory activity.RESULTS In this work we studied indirectly the phosphorylation of TTP through the binding with the chaperone protein 14-3-3.In inflamed and non-inflamed colon mucosa of IBD paediatric patients immunoblot assay demonstrated a higher expression of the TTP in inflamed samples respect to the non-inflamed;the coimmunoprecipitated 14-3-3 protein showed the same trend of expression.In the TNF-αgene expression analysis higher levels of the cytokine in inflamed tissues compared to controls were evident.The same experiments were conducted on macrophages from IBD paediatric patients and healthy controls.The immunoblot results demonstrated a high expression of both TTP and co-immunoprecipitated 14-4-3 protein in IBD-derived macrophages in comparison to healthy donors.TNF-αprotein levels from macrophages lysates showed the same trend of expression in favour of IBD paediatric patients compared to healthy controls.CONCLUSION In this work,for the first time,we describe a relation between phospho-TTP/14-3-3 complex and IBD.Indeed,a higher expression of TTP/14-3-3 was recorded in IBD samples in comparison to controls.

Induced pluripotent stem cells as an innovative model to study drug induced pancreatitis

Drug-induced pancreatitis is a gastrointestinal adverse effect concerning about 2%of drugs.The majority of cases are mild to moderate but severe episodes can also occur,leading to hospitalization or even death.Unfortunately,the mechanisms of this adverse reaction are still not clear,hindering its prevention,and the majority of data available of this potentially life-threatening adverse effect are limited to case reports leading to a probable underestimation of this event.In particular,in this editorial,special attention is given to thiopurine-induced pancreatitis(TIP),an idiosyncratic adverse reaction affecting around 5%of inflammatory bowel disease(IBD)patients taking thiopurines as immunosuppressants,with a higher incidence in the pediatric population.Validated biomarkers are not available to assist clinicians in the prevention of TIP,also because of the inaccessibility of the pancreatic tissue,which limits the possibility to perform dedicated cellular and molecular studies.In this regard,induced pluripotent stem cells(iPSCs)and the exocrine pancreatic differentiated counterpart could be a great tool to investigate the cellular and molecular mechanisms underlying the development of this undesirable event.This particular type of stem cells is obtained by reprogramming adult cells,including fibroblasts and leukocytes,with a set of transcription factors known as the Yamanaka’s factors.Maintaining unaltered the donors’genetic heritage,iPSCs represent an innovative model to study the mechanisms of adverse drug reactions in individual patients’tissues not easily obtainable from human probands.Indeed,iPSCs can differentiate under adequate stimuli into almost any somatic lineage,opening a new world of opportunities for researchers.Several works are already available in the literature studying liver,central nervous system and cardiac cells derived from iPSCs and adverse drug effects.However,to our knowledge no studies have been performed on exocrine pancreas differentiated from iPSCs and drug-induced pancreatitis,so far.Hence,in this editorial we focus specifically on the description of the study of the mechanisms of TIP by using IBD patient-specific iPSCs and exocrine pancreatic differentiated cells as innovative in vitro models.

Biomarkers for gastrointestinal adverse events related to thiopurine therapy

Thiopurines are immunomodulators used in the treatment of acute lymphoblastic leukemia and inflammatory bowel diseases.Adverse reactions to these agents are one of the main causes of treatment discontinuation or interruption.Myelosuppression is the most frequent adverse effect;however,approximately 5%-20%of patients develop gastrointestinal toxicity.The identification of biomarkers able to prevent and/or monitor these adverse reactions would be useful for clinicians for the proactive management of long-term thiopurine therapy.In this editorial,we discuss evidence supporting the use of PACSIN2,RAC1,and ITPA genes,in addition to TPMT and NUDT15,as possible biomarkers for thiopurine-related gastrointestinal toxicity.

Patient-derived organoids for therapy personalization in inflammatory bowel diseases

Inflammatory bowel diseases(IBDs)are chronic inflammatory disorders of the intestinal tract that have emerged as a growing problem in industrialized countries.Knowledge of IBD pathogenesis is still incomplete,and the most widely-accepted interpretation considers genetic factors,environmental stimuli,uncontrolled immune responses and altered intestinal microbiota composition as determinants of IBD,leading to dysfunction of the intestinal epithelial functions.In vitro models commonly used to study the intestinal barrier do not fully reflect the proper intestinal architecture.An important innovation is represented by organoids,3D in vitro cell structures derived from stem cells that can self-organize into functional organ-specific structures.Organoids may be generated from induced pluripotent stem cells or adult intestinal stem cells of IBD patients and therefore retain their genetic and transcriptomic profile.These models are powerful pharmacological tools to better understand IBD pathogenesis,to study the mechanisms of action on the epithelial barrier of drugs already used in the treatment of IBD,and to evaluate novel target-directed molecules which could improve therapeutic strategies.The aim of this review is to illustrate the potential use of organoids for therapy personalization by focusing on the most significant advances in IBD research achieved through the use of adult stem cells-derived intestinal organoids.

Pharmacogenetics of thiopurines

Polychemotherapeutic protocols for the treatment of pediatric acute lymphoblastic leukemia(ALL)always include thiopurines.Specific approaches vary in terms of drugs,dosages and combinations.Such therapeutic schemes,including risk-adapted intensity,have been extremely successful for children with ALL who have reached an outstanding 5-year survival of greater than 90%in developed countries.Innovative drugs such as the proteasome inhibitor bortezomib and the bi-specific T cell engager blinatumomab are available to further improve therapeutic outcomes.Nevertheless,daily oral thiopurines remain the backbone maintenance or continuation therapy.Pharmacogenetics allows the personalization of thiopurine therapy in pediatric ALL and clinical guidelines to tailor therapy on the basis of genetic variants in TPMT and NUDT15 genes are already available.Other genes of interest,such as ITPA and PACSIN2,have been implicated in interindividual variability in thiopurines efficacy and adverse effects and need additional research to be implemented in clinical protocols.In this review we will discuss current literature and clinical guidelines available to implement pharmacogenetics for tailoring therapy with thiopurines in pediatric ALL.