Mirdametinib

MEK:0.33 nM (cell free)

After 1 week of oral administration of PD0325901 (20–25 mg/kg/day) in mice, no tumor growth was detected in mice inoculated with PTC cells bearing a BRAF mutation. For PTC with the RET/PTC1 rearrangement, the average tumor volume of the orthotopic tumor was reduced by 58% as compared with controls [2].

The 50% growth inhibition (GI50) by PD0325901 was 11 nmol/L for the PTC cells with the RET/PTC1 rearrangement and 6.3 nmol/L for PTC cells with a BRAF mutation, with both concentrations readily achievable in serum [2]. PD0325901 significantly increases aggregate cohesion, stiffness, and viscosity but only when tumor cells have access to high concentrations of fibronectin. Treatment also results in reorganization of actin from cortical into stress fibers, in both 2D and 3D culture [3]. On day 7 post-treatment (D14), the strongest effect in the cultures treated with a combination of ALK5 inhibitor SB431542 (2 μM) and MEK inhibitor PD0325901 (0.5 μM), which resulted in ~45 large ALP+ colonies with characteristic hESC-like morphology, of which over 24 colonies were TRA-1-81+, and about 6–10 colonies stained positive for SSEA4 and NANOG, a mature pluripotency factor that is not ectopically introduced [4].

DMSO:12.1 mg/mL (25 mM),H2O:Insoluble

A cell death detection enzyme-linked immunosorbent assay was used per the manufacturer's instructions. Briefly, 4 × 10^4 cells were plated in 24-well plates in triplicate the day before treatment. PTC cells were treated with 0.1 μmol/L PD0325901 for 96 hours. Cells treated with 1 μmol/L staurosporine served as positive controls for apoptosis. At the end of treatment, cells were lysed using the lysis buffer provided in the kit for 30 minutes at room temperature and then centrifuged in 24-well plates. Lysates (20 μL of supernatant) were transferred to streptavidin-coated wells and incubated for 2 hours at room temperature with two antibodies (biotin-labeled anti-histone antibody and peroxidase-conjugated anti-DNA antibody). After the wells were washed three times, the samples were incubated with peroxidase substrate (ABTS) and the amount of colored product was determined spectrophotometrically at 405 nm. The background was measured at 490 nm [2].

Athymic Ncr-nu/nu mice were obtained from the National Cancer Institute at ages 6 to 8 weeks and housed for at least 1 week after arrival. Mice (10–14 per group) were anesthetized s.c. with a cocktail (100 μL/10 g body weight of 10 mg/mL ketamine and 1 mg/mL xylazine). K2 and TPC-1 cells stably infected with a retrovirus expressing luciferase (5 × 105 cells in 5 μL RPMI1640 medium) were inoculated into the thyroid gland, and the mice were monitored weekly for tumor growth by Xenogen (IVIS 200 imaging system) using Living Image 3.0 software. One week after inoculation, PD0325901 was dissolved in 80 mmol/L citric buffer (pH 7) by sonication and given to mice daily by oral gavage (20–25 mg/kg) for 3 weeks (5 consecutive days/week). Mice were sacrificed only due to tumor burden or loss of 20% of body weight. Tumor sizes were measured with calipers and tumor volume (V) was calculated by the formula (V = length × width × depth). Control mice were given 80 mmol/L citric buffer (pH 7) alone. All in vivo experiments were done at least twice [2].