Spectrophotometric Screening for Potential Inhibitors of Cytosolic Glutathione S-Transferases.

Glutathione S-transferases (GSTs) are metabolic enzymes responsible for the elimination of endogenous or exogenous electrophilic compounds by glutathione (GSH) conjugation. In addition, GSTs are regulators of mitogen-activated protein kinases (MAPKs) involved in apoptotic pathways. Overexpression of GSTs is correlated with decreased therapeutic efficacy among patients undergoing chemotherapy with electrophilic alkylating agents. Using GST inhibitors may be a potential solution to reverse this tendency and augment treatment potency. Achieving this goal requires the discovery of such compounds, with an accurate, quick, and easy enzyme assay. A spectrophotometric protocol using 1-chloro-2,4-dinitrobenzene (CDNB) as the substrate is the most employed method in the literature. However, already described GST inhibition experiments do not provide a protocol detailing each stage of an optimal inhibition assay, such as the measurement of the Michaelis-Menten constant (Km) for CDNB or indication of the employed enzyme concentration, crucial parameters to assess the inhibition potency of a tested compound. Hence, with this protocol, we describe each step of an optimized spectrophotometric GST enzyme assay, to screen libraries of potential inhibitors. We explain the calculation of both the half-maximal inhibitory concentration (IC50) and the constant of inhibition (Ki)-two characteristics used to measure the potency of an enzyme inhibitor. The method described can be implemented using a pool of GSTs extracted from cells or pure recombinant human GSTs, namely GST alpha 1 (GSTA1), GST mu 1 (GSTM1) or GST pi 1 (GSTP1). However, this protocol cannot be applied to GST theta 1 (GSTT1), as CDNB is not a substrate for this isoform. This method was used to test the inhibition potency of curcumin using GSTs from equine liver. Curcumin is a molecule exhibiting anti-cancer properties and showed affinity towards GST isoforms after in silico docking predictions. We demonstrated that curcumin is a potent competitive GST inhibitor, with an IC50 of 31.6 ± 3.6 µM and a Ki of 23.2 ± 3.2 µM. Curcumin has potential to be combined with electrophilic chemotherapy medication to improve its efficacy.

Role of advanced nurse practitioners in the care pathway for children diagnosed with leukemia.

PURPOSE: The development of advanced nursing practices (ANP) can meet challenges presented by evolving needs of health care. We aimed at describing the approach taken to implement ANP in a pediatric oncology-hematology unit. METHOD: The Participatory, Evidence-informed, Patient-centered Process for Advanced practice was considered as a conceptual framework for the development, implementation and evaluation of the nursing role. Successive steps were taken for identifying patients and family needs, defining the health care priorities, and establishing a new health care model. RESULTS: The seven steps of the PEPPA framework were carried out over a year and contributed to the identification of the tasks and the role of the advanced nurse practitioner in the patient care pathway. The implementation of ANP was gradually achieved with the development of new evidenced-based health care procedures. The subsequent approval of a contract specification for advanced nurse practitioners facilitated the onset of a specific consultation, which facilitated a holistic approach. CONCLUSION: The impact of the implementation can be appreciated at different levels: i) the patient and his or her family, with improvement of communication and continuity of care; ii) the interdisciplinary team, with development of a dynamic and greater motivation of the health care providers; and iii) the profession, with a greater recognition of the competencies and conceptualization of the new role. An assessment of the process, the structure and the results should be carried out to validate the role of the advanced nurse practitioner throughout the long journey of a child with leukemia.

Association of busulfan exposure with survival and toxicity after haemopoietic cell transplantation in children and young adults: a multicentre, retrospective cohort analysis.

BACKGROUND: Intravenous busulfan combined with therapeutic drug monitoring to guide dosing improves outcomes after allogeneic haemopoietic cell transplantation (HCT). The best method to estimate busulfan exposure and optimum exposure in children or young adults remains unclear. We therefore assessed three approaches to estimate intravenous busulfan exposure (expressed as cumulative area under the curve [AUC]) and associated busulfan AUC with clinical outcomes in children or young adults undergoing allogeneic HCT. METHODS: In this retrospective analysis, patients from 15 centres in the Netherlands, USA, Canada, Switzerland, UK, Italy, Germany, and Australia who received a busulfan-based conditioning regimen between March 18, 2001, and Feb 12, 2015, were included. Cumulative AUC was calculated by numerical integration using non-linear mixed effect modelling (AUCNONMEM), non-compartmental analysis (AUC from 0 to infinity [AUC0-∞] and to the next dose [AUC0-τ]), and by individual centres using various approaches (AUCcentre). The main outcome of interest was event-free survival. Other outcomes of interest were graft failure or relapse, or both; transplantation-related mortality; acute toxicity (veno-occlusive disease or acute graft versus-host disease [GvHD]); chronic GvHD; overall survival; and chronic-GvHD-free event-free survival. We used propensity-score-adjusted Cox proportional hazard models, Weibull models, and Fine-Gray competing risk regressions for statistical analyses. FINDINGS: 790 patients were enrolled, 674 of whom were included: 274 (41%) with malignant and 400 (59%) with non-malignant disease. Median age was 4·5 years (IQR 1·4-10·7). The median busulfan AUCNONMEM was 74·4 mg × h/L (95% CI 31·1-104·6), which correlated with the standardised method AUC0-∞ (r2=0·74), but the latter correlated poorly with AUCcentre (r2=0·35). Estimated 2-year event-free survival was 69·7% (95% CI 66·2-73·0). Event-free survival at 2 years was 77·0% (95% CI 72·1-82·9) in the 257 patients with an optimum intravenous busulfan AUC of 78-101 mg × h/L compared with 66·1% (60·9-71·4) in the 235 patients at the low historical target of 58-86 mg × h/L and 49·5% (29·2-66·0) in the 44 patients with a high (>101 mg × h/L) busulfan AUC (p=0·011). Compared with the low AUC group, graft failure or relapse occurred less frequently in the optimum AUC group (hazard ratio [HR] 0·57, 95% CI 0·39-0·84; p=0·0041). Acute toxicity (HR 1·69, 1·12-2·57; p=0·013) and transplantation-related mortality (2·99, 1·82-4·92; p<0·0001) were significantly higher in the high AUC group (>101 mg × h/L) than in the low AUC group (<78 mg × h/L), independent of indication; no difference was noted between AUC groups for chronic GvHD (<78 mg × h/L vs ≥78 mg × h/L, HR 1·30, 95% CI 0·73-2·33; p=0·37). INTERPRETATION: Improved clinical outcomes are likely to be achieved by targeting the busulfan AUC to 78-101 mg × h/L using a new validated pharmacokinetic model for all indications. FUNDING: Research Allocation Program and the UCSF Helen Friller Family Comprehensive Cancer Center and the Mt Zion Health Fund of the University of California, San Francisco.

Systems medicine, personalized health and therapy.

The 7th Santorini Conference was held in Santorini, Greece, and brought together 200 participants from 40 countries in several continents, including Europe, USA but also Japan, Korea, Brazil and South Africa. The attendees had the opportunity to: listen to 60 oral presentations; participate in two lunch symposia; look at 103 posters, which were divided in two groups ('systems medicine and environment' and 'pharmacogenomics and cancer') and attend a dedicated exhibition with six companies. The meeting was organized by the Institut National de la Santé et de la Recherche Médicale (INSERM) U1122; IGE-PCV and by 'Biologie Prospective' with the collaboration of the European Society of Pharmacogenomics and Theranostics (ESPT), under the auspices of international organizations (e.g., International Federation of Clinical Chemistry and Laboratory medicine [IFCC], European Federation of Clinical Chemistry and Laboratory Medicine [EFLM], European Diagnostic Manufacturers Association [EDMA], Federation of European Pharmacological Societies [EPHAR], European Science Foundation [ESF]). The 3 days of the conference stimulated intensive discussions on systems biology and the influence of omics technologies on personalized health. Sixty speakers were invited or selected from early abstracts and gave presentations on the following topics: From systems biology to systems medicine/pharmacology; Omics/translating pharmacogenomics/proteomic biomarkers/metabolomics; Human nutrition and health/personalized medicine. We are summarizing here the main topics and presentations, according to the successive sessions.