FIGURE 6 from Novel Spirocyclic Dimer, SpiD3, Targets Chronic Lymphocytic Leukemia Survival Pathways with Potent Preclinical Effects

SpiD3 prodrug (SpiD3AP) displays antileukemic activity in Eµ-TCL1 mice. A, Synthesis of SpiD3AP. Reagents and conditions (a) dimethyl amine (2 mol/L in MeOH), MeOH: DCM (2: 1), 0°C. B, Spleen-derived malignant B cells from terminally diseased Eµ-TCL1 mice (n = 7) were stimulated ex vivo with 1X PMA/Ionomycin and treated with increasing concentrations of SpiD3 or SpiD3AP for 48 hours. Mitochondrial activity was assessed via MTS assay and normalized to the stimulated vehicle. Error bars and IC50 values are shown as mean ± SEM. C, In vitro microsomal stability studies comparing the stability of SpiD3 and SpiD3AP. Diclofenac (2 µmol/L) was used as a positive control in the metabolic stability study. T1/2: half-life, CLint: intrinsic clearance. D, Pharmacokinetic (PK) profile of SpiD3AP administered intravenously at 10 mg/kg body weight. Pharmacokinetic parameters include Clₒbs: clearance observed, T1/2: half-life, C0: initial concentration, AUClast: area under the curve last, MRTInfₒbs: mean residence time observed, Vssₒbs: steady state volume of distribution. Results are represented as mean ± SEM (n = 3 mice). E–G, Diseased Eµ-TCL1 mice (median age = 10. 2 months) were randomized to receive 10 mg/kg SpiD3AP or vehicle equivalent (VEH) via intravenous injection for 3 consecutive days. Equal numbers of male and female mice were used per treatment arm (n = 6 mice/arm). At study end (∼3 hours after the last intravenous injection), mice were sacrificed for tissue harvest. Flow cytometry evaluation of disease burden in blood (E) and spleen (F). Error bars are shown as mean ± SEM. G, Representative immunoblot analysis of MYC expression in spleen-derived malignant B cells from the treated mice. GAPDH served as the loading control. Asterisks indicate significance versus VEH. *, P