Proteasome (IC50 = 5 nM)

Carfilzomib induces intrinsic and extrinsic apoptotic signaling pathways and activates c-Jun-N-terminal kinase (JNK), which in turn inhibits proliferation in a range of cell lines and patient-derived neoplastic cells, including multiple myeloma. When compared to bortezomib, carfilzomib exhibits greater anti-MM activity, overcomes resistance to both bortezomib and other agents, and works in concert with dexamethasone (Dex). At doses of 10 nM, carfilzomib exhibits over 80% inhibition of ChT-L activity in the β5 subunit, indicating preferential in vitro inhibitory potency. Preferential binding specificity for the β5 constitutive 20S proteasome and the β5i immunoproteasome subunits is caused by brief exposure to low-dose carfilzomib. After 8 hours, measuring caspase activity in ANBL-6 cells pulsed with carfilzomib reveals significant increases in caspase-8, caspase-9, and caspase-3 activity, resulting in 3.2-, 3.9-, and 6.9-fold increases, respectively, over control cells. The mitochondrial membrane integrity is reduced to 41% (Q1 + Q2) in carfilzomib pulse-treated cells, while it is 75% in vehicle-treated control cells.[1] Carfilzomib has also demonstrated preclinical efficaciousness against solid and hematological malignancies in another study. [2] Carfilzomib directly prevents the formation of osteoclasts and the resorption of bone.[3]

Carfilzomib moderately reduces tumor growth in an in vivo xenograft model. Carfilzomib successfully reduces the viability of multiple myeloma cells after either continuous or brief treatment mimicking. In mice without tumors, carfilzomib improves bone formation, reduces bone resorption, and increases the volume of trabecular bone.[3]

ANBL-6 cells (plated at 2 × 10⁶/well) are subjected to a 1-hour treatment with Carfilzomib at doses ranging from 0.001 to 10 μM. The next step involves lysing the cells (20 mM Tris-HCl, 0.5 mM EDTA), and the cleared lysates are then put onto PCR plates. Untreated ANBL-6 cell lysates are used to create a standard curve, with a concentration of 6 μg protein/μL. After adding the active site probe (biotin-(CH2)4-Leu-Leu-Leu-epoxyketone; 20 μM), the mixture is incubated for an hour at room temperature. After heating cell lysates to 100°C and adding 1% sodium dodecyl sulfate (SDS), the mixture is mixed with 20 μL of streptavidin-sepharose high-performance beads per well in a 96-well multiscreen DV plate, and the mixture is incubated for an hour. After washing the beads in a solution containing PBS, 1% bovine serum albumin, and 0.1% Tween-20, the beads are incubated with antibodies against proteasome subunits for an entire night at 4°C on a plate shaker. Goat polyclonal anti-β2i, rabbit polyclonal anti-β5 (affinity-purified antiserum against KLH-CWIRVSSDNVADLHDKYS peptide), and mouse monoclonal anti-β1, anti-β2, anti-β1i, and anti-β5i were among the antibodies used. Goat antirabbit, goat antimouse, or rabbit antigoat secondary antibodies conjugated with horseradish peroxidase are applied to the beads, followed by a 2-hour incubation period. The supersignal ELISA picochemiluminescence substrate is used to develop the beads after they have been cleaned. One carries out luminescent detection. The raw luminescence is expressed as the percentage inhibition compared to the vehicle control and converted to μg/mL by comparing it with the standard curve. The following nonsigmoidal dose-response equation is used to create curve fits: Y = Bottom + (Top-Bottom)/(1 + 10̂((LogEC50 − X) × HillSlope)), where EC50 is the dose that exhibits a 50% effect, X is the logarithm of concentration, and Y is the percentage of inhibition.

WST-1 is used to assess how the proteasome inhibitor Carfilzomib affects the growth of cells. The calculation of the inhibition of proliferation is based on parallel control cells that are given the vehicle alone. XLfit 4 software is used to interpolate the median inhibitory concentration (IC50) using a linear spline function. The following formula is used to determine the degree of resistance (DOR): DOR = IC50(resistant cells)/IC50(sensitive cells). After being pulsed with 100 nM carfilzomib, ANBL-6 cells are cleaned and suspended in PBS containing 5 μg/mL of JC-1, an enzyme that accumulates in mitochondria in a potential-dependent manner. Using a FacScan, the mitochondrial membrane potential-dependent color shift from 525 to 590 nm is examined. CellQuest software is used to analyze the data.

Beige-nude-XID mice are used in animal research. After pelleting 10×10⁶ Granta514 cells and twice washing them in 1X PBS, the cells are subcutaneously injected into the right flank. Following the appearance of tumors, carfilzomib-vorinostat is administered to five to six mice, and the growth or regression of the tumors is tracked throughout treatment. In DMSO and 10% sulfobutylether betacyclodextrin at a pH of 10 mM citrate buffer, stock vorinostat and carfilzomib are dissolved, respectively. Before injection, they are diluted and kept in small aliquots at -80°C for storage.

[1]. Blood . 2007 Nov 1;110(9):3281-90.

[2]. Curr Cancer Drug Targets . 2011 Mar;11(3):285-95.

[3]. Leukemia . 2013 Feb;27(2):430-40.

[4]. Mol Cancer Ther . 2011 Sep;10(9):1686-97.

C40H57N5O7

719.91

719.43

C, 66.73; H, 7.98; N, 9.73; O, 15.56

868540-17-4

Carfilzomib-d8;1537187-53-3

white solid powder

CC(C)C[C@@H](C(=O)[C@]1(CO1)C)NC(=O)[C@H](CC2=CC=CC=C2)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC3=CC=CC=C3)NC(=O)CN4CCOCC4

BLMPQMFVWMYDKT-NZTKNTHTSA-N

InChI=1S/C40H57N5O7/c1-27(2)22-32(36(47)40(5)26-52-40)42-39(50)34(24-30-14-10-7-11-15-30)44-38(49)33(23-28(3)4)43-37(48)31(17-16-29-12-8-6-9-13-29)41-35(46)25-45-18-20-51-21-19-45/h6-15,27-28,31-34H,16-26H2,1-5H3,(H,41,46)(H,42,50)(H,43,48)(H,44,49)/t31-,32-,33-,34-,40+/m0/s1

(2S)-4-methyl-N-[(2S)-1-[[(2S)-4-methyl-1-[(2R)-2-methyloxiran-2-yl]-1-oxopentan-2-yl]amino]-1-oxo-3-phenylpropan-2-yl]-2-[[(2S)-2-[(2-morpholin-4-ylacetyl)amino]-4-phenylbutanoyl]amino]pentanamide

PR-171; PR 171; PR171; Carflizomib; brand name: Kyprolis

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)