HDAC6-IN-1

Cell experiment:

A375, A2780s, SKBR3, HepG2, HeLa, HCT116, A549, and SKOV-3 cells are cultured in DMEM. RPMI8226, K562, H460, HT29, and Ramos cells are cultured in RPMI-1640 medium. MV4-11 cells are cultured in IMDM. All media contains 10% fetal bovine serum (FBS), 100 units/mL Penicillin, and 100 μg/mL Streptomycin. Cells are incubated at 37 °C in a humidified atmosphere of 5% CO2. Cells in logarithmic phase are seeded into 96-well culture plates at densities of 3000-5000 cells per well and subsequently treated with various concentrations of compounds (e.g., SKLB-23bb;10, 100, 1000, and 10000 nM) for 72 h in final volumes of 200 μL. Upon end point, 20 μL of MTT (5 mg/mL) is added to each well, and the cells are incubated for an additional 1-3 h. After carefully removal of the medium, the precipitates are dissolved in 150 μL of DMSO via mechanically shaking, and then absorbance values at a wavelength of 570 nm are taken on a spectrophotometer. IC50 values are calculated using percentage of growth versus untreated control[1].

Animal experiment:

Mice[1]For the MV4-11 and Ramos xenograft models, MV4-11 and Ramos cells (107 cells in 100 μL of serum-free IMDM) are injected subcutaneously into the right flanks of 5- to 6-week-old female NOD/SCID mice. For the HCT116 xenograft, HCT116 cells (107 cells in 100 μL of serum-free DMEM) are injected subcutaneously into the right flanks of 5- to 6-week-old female Balb/c nude mice. When the size of the formed xenografts reach 100-150 mm3, the mice are randomly divided (6 mice per group in MV4-11 model, 8 mice per group in Ramos model, and 7 mice per group in HCT116 model) into control group and treated groups. The mice in the experimental groups receive intravenous (iv) injection (50 mg/kg) or oral administration (25 mg/kg) of SKLB-23bb every 2 days. The mice in the vehicle group receive iv injection or oral administration of equal amount of physiological saline containing 5% ethanol and 5% Cremophor EL. Those in the SAHA or LBH-589 or ACY-1215 groups (positive controls) receive ip injection (50 mg/kg for SAHA and 10 mg/kg for LBH-589, dissolved in physiological saline containing 10% DMSO and 45% PEG400 to a concentration of 10 mg/mL) or oral administration (100 mg/kg for SAHA and 40 mg/kg for ACY-1215, dissolved in the same way described above) every 2 days. Tumor burden is measured every 2 days by a caliper. Tumor volume (TV) is calculated. The day that treatment started is defined as day 0. At the end of the experiment, mice are sacrificed and tumors are collected and weighed[1]. Rats[1]SKLB-23bb is administered to SD rats ntravenously (iv) at 12 mg/kg body weight and orally at 12 mg/kg body weight. Blood samples are taken, and the plasma is analyzed for concentration of SKLB-23bb using an LC-MS/MS system[1].

References:

[1]. Yang Z, et al. Discovery of Selective Histone Deacetylase 6 Inhibitors Using the Quinazoline as the Cap for the Treatment of Cancer. J Med Chem. 2016 Feb 25;59(4):1455-70.