Entinostat (MS-275)

Rat liver enzyme is diluted 1:6 with HDAC buffer. Recombinant human HDACs are diluted 1:4 in HDAC buffer. For standard HDAC assays, 60 μL of HDAC buffer is mixed with 10 μL of diluted enzyme solution at 30 °C. The HDAC reaction is started by adding 30 μL substrate solution in HDAC buffer followed by 30 min of incubation at 30 °C. The reaction is stopped by adding 100 μL trypsin solutions (10 mg/ml trypsin in 50 mM Tris-HCl [pH 8.0], 100 mM NaCl, 2 μM TSA). After a 20 min incubation period at 30 °C, the release of AMC is monitored by measuring the fluorescence at 460 nm (λ_ex = 390 nm). Fluorescence intensity is calibrated using free AMC. For standard time course experiments, 20 pmol of substrate is used in the initial 100 μL HDAC reaction. K_m and V_max values are determined by measuring the fluorescence AMC generated by enzymatic cleavage of 2–50 pmol of substrate. The experimental data are analyzed using a Hanes plot. The AMC signals are recorded against a blank with buffer and substrate but without the enzyme.

A2780, Calu-3, HL-60, K562, St-4, HT-29, KB-3-1, Capan-1, 4-1St and HCT-15 cells

~ 10 μM

3 days

Cancer cells (5 × 10³) are seeded into each well of 96-well plates and cultured with graded concentrations of MS-275 for 3 days. The cells are stained with 0.1 mg/mL neutral red for 1 hour in a CO₂-incubator, and, after aspiration of the medium, OD540 of the neutral red solubilized with 50 μL of ethanol and 150 μL of 0.1 M Na₂HPO₄ is measured. The IC50 value is determined by plotting growth inhibition of the cells against the logarithm of the drug concentration.

A2780, HT-29, HTC-15, KB-3-1, 4-1St, St-4, Capan-1 and Calu-3 cells are injected subcutaneously into the flank of nude mice.

12.3, 24.5 and 49 mg/kg

Administered orally once daily 5 days per week for 4 weeks