The effect of Ricolinostat with or without PS-341 on the viability of MM cell lines, patient MM cells, and PBMCs is assessed by measuring MTT dye absorbance. PBMCs from healthy donors are isolated and stimulated with 2.5 μg/mL of phytohemagglutinin (PHA) for 48 hours in the presence of increasing concentrations of Ricolinostat (ACY-1215). DNA synthesis is measured by tritiated thymidine uptake. CD4⁺ T cells are purified from human blood with the Rosette Sep negative-selection kit. Cells are stimulated by CD3/CD28 Dynabeads for 7 days in the presence of compounds. Cell viability is assessed using alamarBlue. MM cells (2-3×10⁴ cells/well) are incubated in 96-well culture plates with medium and different concentrations of Ricolinostat (ACY-1215), PS-341, and/or recombinant IL-6 (10 ng/mL) or IGF-1 (50 ng/mL) for 24 hours at 37°C, and tritiated thymidine incorporation is measured^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Mice^[1]
To evaluate the in vivo anti-MM activity of Ricolinostat, male SCID mice are inoculated subcutaneously with 5×10⁶ MM.1S cells in 100 μL of serum-free RPMI 1640 medium. When tumors are measurable, mice are treated IP with Ricolinostat 50 mg/kg dissolved in 10% DMSO in 5% dextrose in water consecutively for 5 days a week for 3 weeks; PS-341 0.5 mg/kg dissolved in 0.9% saline solution biweekly (IV) for 3 consecutive weeks; or combination with the same dosing regimen used for the individual agents. The control group receive the carrier alone at the same schedule as the combination group. Tumor size is measured every other day in 2 dimensions using calipers, and tumor volume is calculated with the formula: V=0.5(a×b²) where a is the long diameter of the tumor and b is the short diameter of the tumor. Mice are killed when the tumor reaches 2 cm³ or is ulcerated. Survival and tumor growth are evaluated from the first day of treatment until death.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Santo L, et al. Preclinical activity, pharmacodynamic, and pharmacokinetic properties of a selective HDAC6 inhibitor, ACY-1215, in combination with PS-341 in multiple myeloma. Blood. 2012 Mar 15;119(11):2579-89.  [Content Brief]