Carfilzomib (PR-171)

Beige-nude-XID mice

L-Phenylalaninamide, (S)--[[2-(4-morpholinyl)acetyl]amino]benzenebutanoyl-L-leucyl-N-[(1S)-3-methyl-1-[[(2R)-2-methyl-2-oxiranyl]carbonyl]butyl]-

100 nM

2.0 mg/kg

5 nM [1], 5 nM [1], 5 nM [1], 5 nM [1], 5 nM [1], 5 nM [1]

Carfilzomib moderately reduces tumor growth in an in vivo xenograft model. Carfilzomib effectively decreases multiple myeloma cell viability following continual or transient treatment mimicking. Carfilzomib increases trabecular bone volume, decreases bone resorption and enhances bone formation in non-tumor bearing mice. [3]

Enzyme-linked immunosorbent assay for subunit profiling of carfilzomib, ANBL-6 cells (2, 106/well) are plated in 96-well plates and treated with Carfilzomib doses from 0.001 to 10 uM for 1 hour. Cells are then lysed (20 mM Tris-HCl, 0.5 mM EDTA), and cleared lysates are transferred to polymerase chain reaction (PCR) plates. A standard curve is generated using untreated ANBL-6 cell lysates starting at a concentration of 6 ug protein/uL. The active site probe [biotin-(CH2)4-Leu-Leu-Leu-epoxyketone, 20 uM] is added and incubated at room temperature for 1 hour. Cell lysates are then denatured by adding 1% sodium dodecyl sulfate (SDS) and heating to 100C, followed by mixing with 20 uL per well streptavidin-sepharose high-performance beads in a 96-well multiscreen DV plate and incubated for 1 hour. These beads are then washed with enzyme-linked immunosorbent assay (ELISA) buffer (PBS, 1% bovine serum albumin, and 0.1% Tween-20), and incubated overnight at 4C on a plate shaker with antibodies to proteasome subunits. Antibodies used included mouse monoclonal anti-beta1, anti-beta2, anti-beta1i, and anti-beta5i, goat polyclonal anti-beta2i, and rabbit polyclonal anti-beta5 (affinity-purified antiserum against KLH-CWIRVSSDNVADLHDKYS peptide). The beads are washed and incubated for 2 hours with horseradish peroxidase-conjugated secondary goat antirabbit, goat antimouse or rabbit antigoat antibodies. After washing, the beads are developed using the supersignal ELISA picochemiluminescence substrate. Luminescent detection is performed. Raw luminescence is converted to ug/mL by comparison with the standard curve and expressed as the % inhibition relative to vehicle control. Curve fits are generated using the following nonsigmoidal dose-response equation: Y = Bottom + (Top-Bottom)/(1 + 10 ((LogEC50 X), HillSlope)), where X is the logarithm of concentration, Y is the % inhibition, and EC50 is the dose showing 50% effect.

719, 91

DMSO 50 mg/mL, Water <1 mg/mL, Ethanol <1 mg/mL