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E.C. Matheson et al.
was determined using the median-effect principle and was com- pared to the effect achieved with a combination of the two drugs to derive a combination index (CI) value.
Pharmacokinetic analyses
Plasma pharmacokinetics of selumetinib and dexamethasone were determined using non-compartmental analysis in female CD1 mice after oral dosing. Plasma concentrations of both drugs were measured by liquid chromatographic mass spectrometry (API4000 LCMS/MS, Applied Biosystems, CA, USA), attached to a Perkin Elmer chromatography system (Perkin Elmer Ltd, Beckonsfield, UK) and calibrated using standards prepared in blank mouse plasma. In both cases separation was performed using a Gemini 3μ C18 110A column, (50x3 mm), fitted with a 4x2 mm C18 cartridge (Phenomenex, Macclesfield, UK.
In vivo experiments
All experiments were performed under the UK Home Office
NCL-PLL60/4552. Drug efficacy studies were performed as previ- ously described.6 Briefly, primagraft cells were injected intrafemorally and mice were monitored for engraftment every 3- 4 weeks by tail vein bleed. Blood red cells were lysed and ana- lyzed by flow cytometry on a BD FACSCanto II, using anti- human CD10, CD34 and CD19 and anti-mouse CD45 antibodies. Human leukemia cells were gated and their number expressed as a percentage of the total number of nucleated cells. Once the level
of human leukemia cells reached >1% of total cells, mice were randomized into groups to receive control vehicle (0.5% hydroxy- propyl methylcellulose + 0.1% polysorbate 80) or drug treatment (6 mice per group) with dexamethasone, selumetinib or both, administered by oral gavage. Selumetinib was dosed at 25 mg/kg bid, while the dexamethasone dose varied in each study. Tumor burden was monitored weekly by flow cytometry. Pharmacodynamic studies were performed in highly engrafted mice which were dosed for 72 h. Spleens were removed following euthanasia and assessed by flow cytometry to confirm an engraft- ment of >85%. Cells were lysed and analyzed by western blotting for levels of p-ERK, ERK2, BIM, MCL1 and a-tubulin, as described above.
Additional details of the study methods are provided in the Online Supplementary Material.
Results
The combination of selumetinib and dexamethasone show synergy in vitro in RAS pathway-mutated acute lymphoblastic leukemia and is associated with enhanced induction of BIM
To investigate possible synergism, the R3F9 cell line and primagraft ALL cells, with and without Ras pathway mutations (n=8), were treated with dexamethasone,
AB
CD
Figure 1. The combination of selumetinib and dexamethasone shows synergy in vitro in RAS pathway-mutated acute lymphoblastic leukemia and is associated with enhanced levels of BIM. (A) Viability curves of Ras pathway-mutant acute lymphoblastic leukemia (ALL) cells (L829R) with individual drugs and the selume- tinib/dexamethasone drug combination. (B) Histogram of combination indices for the selumetinib/dexamethasone combination in wildtype and Ras pathway-mutant ALL cells; mutated genes are shown in brackets. (C) Western analyses of ALL cells (L829R) treated with control vehicle (CV) or GI50 values of selumetinib (10 μM) and dexamethasone (10 μM), singly and in combination. (D) A representative median effect curve (data shown are from L897) after simultaneous drug dosing and with each drug added 24 h prior to the partner drug, followed by a further 72 h incubation. CV: control vehicle; Sel: selumetinib; Dex: dexamethasone; CI: combination index.
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haematologica | 2019; 104(9)