Pharmacokinetics and pharmacodynamics of thiopurines in an in\ua0vitro model of human hepatocytes: Insights from an innovative mass spectrometry assay

AIM: To apply an innovative LC-MS/MS method to quantify thiopurine metabolites in human hepatocytes and to associate them to cytotoxicity. METHODS: Immortalized human hepatocytes (IHH cells) were treated for 48 and 96 h, with 1.4 7 10-4 M azathioprine and 1.1 7 10-3 M mercaptopurine, concentrations corresponding to the IC50 values calculated after 96 h exposure in previous cytotoxicity analysis. After treatments, cells were collected for LC-MS/MS analysis to quantify 11 thiopurine metabolites with different level of phosphorylation and viable cells were counted by trypan blue exclusion assay to determine thiopurines in vitro effect on cell growth and survival. Statistical significance was determined by analysis of variance (ANOVA). RESULTS: Azathioprine and mercaptopurine had a significant time-dependent cytotoxic effect (p-value ANOVA = 0.012), with a viable cell count compared to controls of 55.5% and 67.5% respectively after 48 h and 23.7% and 36.1% after 96 h; no significant difference could be observed between the two drugs. Quantification of thiopurine metabolites evidenced that the most abundant metabolite was TIMP, representing 57.1% and 40.3% of total metabolites after 48 and 96 h. Total thiopurine metabolites absolute concentrations decreased over time: total mean content decreased from 469.9 pmol/million cells to 83.6 pmol/million cells (p-value ANOVA = 0.0070). However, considering the relative amount of thiopurine metabolites, TGMP content significantly increased from 11.4% cells to 26.4% (p-value ANOVA = 0.017). A significant association between thiopurine effects and viable cell counts could be detected only for MeTIMP: lower MeTIMP concentrations were associated with lower cell survival (p-value ANOVA = 0.011). Moreover, the ratio between MeTIMP and TGMP metabolites directly correlated with cell survival (p-value ANOVA = 0.037). CONCLUSION: Detailed quantification of thiopurine metabolites in a human hepatocytes model provided useful insights on the association between thioguanine and methyl-thioinosine nucleotides with cell viability

Multicentric Case-Control Study on Azathioprine Dose and Pharmacokinetics in Early-onset Pediatric Inflammatory Bowel Disease

BACKGROUND: Early-onset inflammatory bowel disease (IBD) is generally aggressive, with a high probability of complications and need of surgery. Despite the introduction of highly effective biological drugs, treatment with azathioprine continues to be important even for early-onset IBD; however, in these patients azathioprine response seems to be reduced. This study evaluated azathioprine doses, metabolite concentrations, and their associations with patients' age in children with IBD treated at 6 tertiary pediatric referral centers. METHODS: Azathioprine doses, metabolites, and clinical effects were assessed after at least 3 months of therapy in 17 early-onset (age 12 and <18 yrs, controls) patients with IBD. Azathioprine dose was titrated on therapeutic efficacy (response and adverse effects). Azathioprine metabolites and thiopurine methyltransferase activity were determined by high-performance liquid chromatography with ultra violet-vis detection (HPLC-UV) methods. RESULTS: Frequency of patients in remission was similar among early-onset and control groups, respectively (82% and 84%, P value = 0.72). Early-onset patients required higher doses of azathioprine (median 2.7 versus 2.0 mg\ub7kg\ub7d, P value = 1.1 7 10). Different doses resulted in comparable azathioprine active thioguanine nucleotide metabolite concentrations (median 263 versus 366 pmol/8 7 10 erythrocytes, P value = 0.41) and methylmercaptopurine nucleotide concentrations (median 1455 versus 1532 pmol/8 7 10 erythrocytes, P value = 0.60). Lower ratios between thioguanine nucleotide metabolites and azathioprine doses were found in early-onset patients (median 98 versus 184 pmol/8 7 10 erythrocytes\ub7mg\ub7kg\ub7d, P value = 0.017). Interestingly, early-onset patients presented also higher thiopurine methyltransferase activity (median 476 versus 350 nmol methylmercaptopurine/mg hemoglobin/h, P-value = 0.046). CONCLUSIONS: This study demonstrated that patients with early-onset IBD present increased inactivating azathioprine metabolism, likely because of elevated activity of the enzyme thiopurine methyltransferase

5-Aminoimidazole-4-carboxamide ribonucleotide-transformylase and inosine-triphosphate-pyrophosphatase genes variants predict remission rate during methotrexate therapy in patients with juvenile idiopathic arthritis

For children with juvenile idiopathic arthritis (JIA) who fail to respond to methotrexate, the delay in identifying the optimal treatment at an early stage of disease can lead to long-term joint damage. Recent studies indicate that relevant variants to predict methotrexate response in JIA are those in 5-aminoimidazole-4-carboxamide ribonucleotide-transformylase (ATIC), inosine-triphosphate-pyrophosphatase (ITPA) and solute-liquid-carrier-19A1 genes. The purpose of the study was, therefore, to explore the role of these candidate genetic factors on methotrexate response in an Italian cohort of children with JIA. Clinical response to methotrexate was evaluated as clinical remission stable for a 6-month period, as ACRPed score and as change in Juvenile Arthritis Disease score. The most relevant SNPs for each gene considered were assayed on patients' DNA. ITPA activity was measured in patients' erythrocytes. Sixty-nine patients with JIA were analyzed: 52.2 % responded to therapy (ACRPed70 score), while 37.7 % reached clinical remission stable for 6 months. ATIC rs2372536 GG genotype was associated with improved clinical remission (adjusted p value = 0.0090). For ITPA, rs1127354 A variant was associated with reduced clinical remission: (adjusted p value = 0.028); this association was present even for patients with wild-type ITPA and low ITPA activity. These preliminary results indicate that genotyping of ATIC rs2372536 and ITPA rs1127354 variants or measuring ITPA activity could be useful to predict methotrexate response in children with JIA after validation by further prospective studies on a larger patient cohort

Glucocorticoid pharmacogenetics in pediatric idiopathic nephrotic syndrome

Idiopathic nephrotic syndrome represents the most common type of primary glomerular disease in children: glucocorticoids (GCs) are the first-line therapy, even if considerable interindividual differences in thepir efficacy and side effects have been reported. Immunosuppressive and anti-inflammatory effects of these drugs are mainly due to the GC-mediated transcription regulation of pro- and anti-inflammatory genes. This mechanism of action is the result of a complex multistep pathway that involves the glucocorticoid receptor and several other proteins, encoded by polymorphic genes. Aim of this review is to highlight the current knowledge on genetic variants that could affect GC response, particularly focusing on children with idiopathic nephrotic syndrome

Role of Pharmacogenetics in Hematopoietic Stem Cell Transplantation Outcome in Children

Hematopoietic stem cell transplantation (HSCT) is an established therapeutic procedure for several congenital and acquired disorders, both malignant and nonmalignant. Despite the great improvements in HSCT clinical practices over the last few decades, complications, such as graft vs. host disease (GVHD) and sinusoidal obstructive syndrome (SOS), are still largely unpredictable and remain the major causes of morbidity and mortality. Both donor and patient genetic background might influence the success of bone marrow transplantation and could at least partially explain the inter-individual variability in HSCT outcome. This review summarizes some of the recent studies on candidate gene polymorphisms in HSCT, with particular reference to pediatric cohorts. The interest is especially focused on pharmacogenetic variants affecting myeloablative and immunosuppressive drugs, although genetic traits involved in SOS susceptibility and transplant-related mortality are also reviewed

Epratuzumab and Blinatumomab as Therapeutic Antibodies for Treatment of Pediatric Acute Lymphoblastic Leukemia: Current Status and Future Perspectives

More than 85% of children affected by acute lymphoblastic leukemia (ALL) are successfully treated; however relapse remains a remarkable clinical concern, with 50-60% of relapsing patients facing a fatal outcome. Management of relapsed patients includes standardized intensive risk-adapted regimens based on conventional drugs, and hematopoietic stem cells transplantation for patients with unfavourable features. Biological drugs, in particular the monoclonal antibody epratuzumab and the bi-functional recombinant single chain peptide blinatumomab, have been recently recognized as novel potential agents to be integrated in salvage ALL therapy to further improve rescue outcome

Family Business and Entrepreneurship: Competencies and Organizational Behavior

Family-owned enterprises go through various stages of growth and development over time once the second and subsequent generations enter the business. They present a unique set of challenges and problems, mostly due to generational tran-sition, role confusion, different vision of the business, qualification and skills. This quantitative study aims at increasing the understanding of which competencies are relevant to family business success and how they significantly influence both organizational behavior and family dynamics. Findings highlight the importance of family business as a unique context to study organizational behavior and con-firmed how family enterprises are influenced by family culture and kinship ties

Exploring the influence of working environments' restorative quality on organisational citizenship behaviours

The perceived potential for environmental restoration while at work can represent a job resource, given that restoration alleviates stress, which can free resources for both performance and extra-role activities. This study explores the association between perceived restorativeness (PR) and organisational citizenship behaviours (OCB – helpfulness, loyalty and voice). Further, this study tests the moderating effect of organisational justice (OJ – procedural, distributive and interactional) on the association between PR and OCB because a lack of OJ can be conceived as a job demand (following the job demands–resources model). Employees (n = 146) from different organisations responded to a questionnaire. Regression analysis with 1,000 bootstrap samples indicated a moderating effect of OJ (for low levels and the mean value of interactional justice – an OJ sub-dimension) on the PR and OCB (voice) relationship. Generally, the restorative quality of the work environment may play a pivotal part in promoting extra-role behaviou

Short-Range Recordings of Shallow Underwater Explosions with Short-Period and Broadband Seismometers in the Bay of Hyères, France

International audienceABSTRACT This study presents the analysis of the seismic records of eight underwater explosions triggered by the French Navy Mine Warfare Office in December 2018 in the Bay of Hyères, southeast France. The explosions (charges of 80–680 kg TNT-equivalent) were recorded by 17 temporary stations composed of medium-to-broadband velocimeters and accelerometers and of microelectromechanical systems accelerometers with sampling rates of 250 or 500 samples per second, installed at less than 10 km from the sources. The relatively high sampling rates used in this study allow us to observe, at the same time, seismic, hydroacoustic, and acoustic signals at the shore. This experiment offers a unique opportunity to investigate the impact at the shore, and in the water, of relatively large-charge weight detonations as well as of the amplification effects of the sedimentary cover. We notably see that the hydroacoustic signal is, at the rocky sites, the most energetic wavepacket recorded, and that its observation seems to be conditioned by the bathymetry and the seabed sedimentary cover. We also show that the hydroacoustic phase can be advantageously used to precisely estimate the location and origin time of the explosions. Finally, analyses of the spectrum and site response along the shore and at a bell tower on the Porquerolles Island provide interesting insights on the mitigation of the possible nuisance caused at the shore by the disposal of unexploded historical ordnance

Mathematical Competence Scale (MCS) for Primary School: The Psychometric Properties and the Validation of an Instrument to Enhance the Sustainability of Talents Development through the Numeracy Skills Assessment

The adequate development of the numeracy skills is a target of the fourth of the Sustainable Development Goals and is considered the basis for a financial literacy: both are competences needed for successful social and professional inclusion. Building on these goals, we carried out a unidimensional Mathematical Competence Scale (MCS) for primary school. The aim of this study was to present the psychometric properties and the validation process of MCS, designed basing on Item Response Theory. The final version of the scale, which measures different domains of mathematical knowledge (Data Analysis and Relationships, Geometry, Dimensions and Measurements, Numbers and Calculations), was validated on the entire population of 2935 fourth graders in Ticino Canton, Switzerland. The results reveal the high level of correlation between the six mathematical dimensions and confirm the assumption of a latent &#8220;mathematical construct&#8221;. However, even the multidimensional model could be considered a good model because it fitted the data significantly better than the one-dimensional model. In particular, the differences of the deviance between the two models are significant (&#967;2 (20) = 642.66, p &lt; 0.001). Moreover, findings show a significant gender effect and a positive correlation between students&#8217; actual school performance during the same academic year and MCS scores. MCS allows a reading of the learning and teaching process in the perspective of the psychology of sustainability and sustainable development and helps a teacher to sustain student talent through the development of numeracy skills; in fact, the scale is intended both as an assessment tool and an innovative approach for shaping the development of curriculum, and therefore has potential to serve as a bridge between empirical research, classroom practice and a positive (school and professional) career development