New drugs are needed for the treatment of relapsed acute lymphoblastic leukemia and preclinical evaluation of the MEK inhibitor, selumetinib, has shown that this drug has excellent activity in those leukemias with RAS pathway mutations. The proapoptotic protein, BIM is pivotal in the induction of cell death by both selumetinib and glucocorticoids, suggesting the potential for synergy. Thus, combination indices for dexamethasone and selumetinib were determined in RAS pathway-mutated acute lymphoblastic leukemia primagraft cells in vitro and were indicative of strong synergism (combination index <0.2; n=5). Associated pharmacodynamic assays were consistent with the hypothesis that the drug combination enhanced BIM upregulation over that achieved by a single drug alone. Dosing of dexamethasone and selumetinib singly and in combination in mice engrafted with primary-derived RAS pathway-mutated leukemia cells resulted in a marked reduction in spleen size which was significantly greater with the drug combination. Assessment of the central nervous system leukemia burden showed a significant reduction in the drug-treated mice, with no detectable leukemia in those treated with the drug combination. These data suggest that a selumetinib-dexamethasone combination may be highly effective in RAS pathway-mutated acute lymphoblastic leukemia. An international phase I/II clinical trial of dexamethasone and selumetinib (Seludex trial) is underway in children with multiply relapsed/refractory disease.

Overall, pharmacogenetic factors had only a minor influence on cyclophosphamide or anthracycline-based adjuvant therapy of breast cancer.

Medial prefrontal cortex (mPFC) and orbitofrontal cortex (OFC) play critical roles in cognition and behavioural control. Glutamatergic, GABAergic, and monoaminergic dysfunction in the prefrontal cortex has been hypothesised to underlie symptoms in neuropsychiatric disorders. Here we characterised electrically-evoked field potentials in the mPFC and OFC. Electrical stimulation evoked field potentials in layer V/VI of the mPFC and layer V of the OFC. The earliest component (approximately 2 ms latency) was insensitive to glutamate receptor blockade and was presumed to be presynaptic. Later components were blocked by 6,7-dinitroquinoxaline-2,3-dione (DNQX (20 μM) and were assumed to reflect monosynaptic (latency 4-6 ms) and polysynaptic activity (latency 6-40 ms) mediated by glutamate via AMPA/kainate receptor. In the mPFC, but not the OFC, the monosynaptic component was also partly blocked by 2-amino-5-phosphonopentanoic acid (AP-5 (50-100 μM) indicating the involvement of NMDA receptors. Bicuculline (3-10 μM) enhanced the monosynaptic component suggesting electrically-evoked and/or glutamate induced GABA release inhibits the monosynaptic component via GABA A receptor activation. There were complex effects of bicuculline on polysynaptic components. In the mPFC both the mono-and polysynaptic components were attenuated by 5-HT (10-100 μM) and NA (30 and 60 μM) and the monosynaptic component was attenuated by DA (100 μM). In the OFC the mono-and polysynaptic components were also attenuated by 5-HT (100 μM), NA (10-100 μM) but DA (10-100 μM) had no effect. We propose that these pharmacologically characterised electrically-evoked field potentials in the mPFC and OFC are useful models for the study of prefrontal cortical physiology and pathophysiology.

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No abstract

Dexamethasone is a key component in the treatment of childhood acute lymphoblastic leukaemia (ALL). Despite playing a key role in the improved survival of ALL over several decades, intensification of dexamethasone therapy has also contributed to the increased toxicity associated with treatment, which is now seen to be at unacceptable levels given the favourable disease prognosis. Therefore the focus for treatment is now shifting towards reducing toxicity whilst maintaining current survival rates. As approximately 50% of patients were successfully treated on less intensive protocols of the 1980s, it has been questioned whether therapy intensification is necessary in all patients. Furthermore, there remains a subset of children who are still not cured of their disease. New strategies are therefore needed to identify patients who could benefit from dose reduction or intensification. However, adjusting a potentially life threatening therapy is a challenging task, particularly given the heterogeneous nature of ALL. This review focuses on the potential for patient stratification based on our current knowledge of dexamethasone pharmacokinetics, pharmacogenetics and the action of dexamethasone at the cellular level. A carefully designed, combined approach is needed if we are to achieve the aim of improved personalization of dexamethasone therapy for future patients.

No abstract

Introduction: Glucocorticoids (GC) have been at the forefront of acute lymphoblastic leukemia (ALL) treatment for a number of decades. However, there is heterogeneity of response, both in terms of GC-related toxicity and leukemia cell sensitivity. Contributing factors include marked pharmacokinetic variability observed in children (Yang et al. JCO, 2008; Jackson et al.AACR annual meeting abstract CT115, 2016) and a several hundred fold range of GC cellular response. Cellular resistance can be caused by deletion of the glucocorticoid receptor (GR) but is more commonly downstream of the GR. One neglected upstream parameter relates to GC accumulation, which may be an important factor in ALL GC response, given the evidence for drug transporters in ALL cells. Therefore, this study aimed to determine whether variation in intracellular dexamethasone (dex) levels is a determinant of dex sensitivity in an ALL setting. Methods: A number of cell lines including PreB697, GC resistant PreB697 sub-lines and REH cells, along with primagraft material from 9 patients and 6 primary patient samples (5 presentation and 1 relapse) were studied. The relative sensitivity of cells to dex was assessed using Alamar Blue drug sensitivity assay. Two methods were developed to assess intracellular dex accumulation; a liquid-chromatography mass spectrometry (LC/MS) method and a flow cytometry method, using dex conjugated to the fluorochrome FITC analysed on a FACSCalibur flow cytometry machine. GR status of the cells was confirmed by western blotting. Results: Dex GI50 values (concentration giving 50% growth inhibition) ranged from 37nM in PreB697 cells to >1000nM in GC resistant sub-lines and REH cells. Dex GI50 values in patient ALL cells ranged from 2 to >1000nM. Dex resistant cells were defined as having a dex GI50 of >500nM. The mean GI50 of the dex sensitive cells was 3.8nM. Western blotting suggested wildtype GR status in all samples, with R3D11 and REH serving as hemizygous deleted and GR negative controls, respectively. The mean dex accumulation was measured in cells using an LC/MS assay developed from a fully validated assay measuring plasma dex concentrations (Jackson et al. NCRI annual meeting abstract BACR9, 2014). Dex was stable in RF10 media for at least 8 hours and there was no matrix effect of RF10 media on dex chromatograms compared to dex in plasma. An incubation concentration of 500nM was chosen as this is the observed median value of dex cell exposure clinically. Dex concentrations were quantifiable in cell numbers of 1 x 106after incubation with 500nM dex, allowing measurement of patient samples where limited numbers of cells are available. Dex accumulation in cell lines after incubation with 500nM dex for 4h was 2.1 and 1.8 pmol/million cells in PreB697 and dex resistant sub-lines, respectively (range for resistant subclones 1.2 - 2.1pmol/million cells). There was greater variability in patient cells with a 40-fold range seen, but dex accumulation was not significantly different between sensitive (mean, 1.0 pmol/million; range, 0.1-2.3) and resistant cells (1.4 pmol/million; range, 0.4-4.4) (unpaired students t-test, p=0.17). To assess intra-leukemia heterogeneity in terms of dex accumulation, a flow based assay was established using dex-FITC. Incubation conditions of 500nM dex-FITC at 37°C for 45 minutes were optimal. Dex-FITC accumulation did not differ significantly between sensitive and resistant cells; mean fluorescence intensity of 4.2 (range 1.5-5.9) versus 4.1 (range 2.0 - 9.1) in sensitive and resistant cells, respectively (p=0.97, unpaired students t test). Dex-FITC accumulation appeared uniform within the ALL samples examined. Conclusions: These data suggest that variations in dex accumulation are unlikely to play a role in dex resistance in ALL, at least in vitro. Advancement of the flow-based dex accumulation assay to include leukaemia-associated immunophenotype markers will allow measurement in dex-resistant MRD in vivo. Given that 35% of patients do not achieve plasma concentrations of 200nM dex (Jackson et al. AACR annual meeting abstract CT115, 2016), a combined approach incorporating pharmacokinetic assessments, drug accumulation and cellular response in ALL cells, may allow a comprehensive understanding of dex pharmacology in order to optimise its clinical utility. Disclosures No relevant conflicts of interest to declare.