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

SpiD3 prodrug (SpiD3_AP) displays antileukemic activity in Eµ-TCL1 mice. A, Synthesis of SpiD3_AP. 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 SpiD3_AP 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 SpiD3_AP. Diclofenac (2 µmol/L) was used as a positive control in the metabolic stability study. T_1/2: half-life, CL_int: intrinsic clearance. D, Pharmacokinetic (PK) profile of SpiD3_AP administered intravenously at 10 mg/kg body weight. Pharmacokinetic parameters include Cl_obs: clearance observed, T_1/2: half-life, C₀: initial concentration, AUC_last: area under the curve last, MRT_Inf_obs: mean residence time observed, V_ss_obs: 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 SpiD3_AP 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 < 0.05.