ARQ 531

Cell experiment:

Biochemical inhibition assay is measured using full length BTK constructs of wild type or C481S mutant. Profiling on 236 kinases identifies 45 kinases with >50% inhibition at 200 nM concentration of ARQ 531. Subsequently, the potency of this ATP competitive inhibitor is determined on such kinases at the physiological 1 mM ATP concentration cells are treated with increasing concentrations of inhibitors in SUDHL-4 for 2 hours, following stimulation with either anti-IgM or growth factors cells are lysed for Western blot analysis[1].

Animal experiment:

Mice[1]Six week old female CB-17 SCID mice (1-2 weeks) are used. Mice are housed in sterile micro isolator cages, five mice per cage and receive food and water ad libitum. Female SCID mice are implanted subcutaneously with 8x106 TMD8 cells in 0.2 mL HBSS with 50% standard concentration BD matrigel in the upper right flank area. Mice are monitored and staged on day 14 (post injection of tumor cells) when size reaches approximately 400 mg. Oral daily dosing with ARQ 531 at 100 mg/kg, vinblastine or vehicle began on stage day. Tumor measurements and body weights are collected three times a week. In vivo Target and pathway inhibition is studied in mouse TMD8 xenograft model. Percent inhibition relative to the vehicle control is determined using densitometry analysis and the intensity of actin band is used as a loading control and the percentage of vehicle group is designated as 100%. DBA1/J mice are immunized with collagen to develop the arthritis, following the onset of arthritis, mice are randomized into treatment groups. Treatment is initiated by oral dosing of ARQ 531 at 25, 50 and 75 mg/kg and continued daily through arthritis day 14. Clinical scores are assessed for each of the paws on study arthritis days 1-15. Dexamethasone at 3 mg/kg is used as a control[1].

References:

[1]. S Eathiraj, et al. Targeting Ibrutinib-Resistant BTK-C481S Mutation with ARQ 531, a Reversible Non-Covalent Inhibitor of BTK. Clinical Lymphoma Myeloma & Leukemia, 2016, 16: S47-S48.