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Purity: ≥98%
Carfilzomib (formerly also known as PR-171; trade name: Kyprolis) is a novel, potent, and irreversible proteasome inhibitor that may have anti-tumor effects. It has a minimal or no impact on the PGPH and T-L activities, but it inhibits proteasome in ANBL-6 cells with an IC50 of less than 5 nM and showed preferential in vitro inhibitory potency against the ChT-L activity in the β5 subunit. A moderate level of antitumor activity was demonstrated by carfilzomib in an in vivo xenograft model. The FDA has approved it for use as an anti-cancer drug.
Targets |
Proteasome (IC50 = 5 nM)
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ln Vitro |
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]
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ln Vivo |
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]
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Enzyme Assay |
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.
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Cell Assay |
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.
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Animal Protocol |
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.
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References |
Molecular Formula |
C40H57N5O7
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Molecular Weight |
719.91
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Exact Mass |
719.43
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Elemental Analysis |
C, 66.73; H, 7.98; N, 9.73; O, 15.56
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CAS # |
868540-17-4
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Related CAS # |
Carfilzomib-d8;1537187-53-3
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Appearance |
white solid powder
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SMILES |
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
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InChi Key |
BLMPQMFVWMYDKT-NZTKNTHTSA-N
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InChi Code |
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
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Chemical Name |
(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
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Synonyms |
PR-171; PR 171; PR171; Carflizomib; brand name: Kyprolis
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HS Tariff Code |
2934.99.9001
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Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: This product is not stable in solution, please use freshly prepared working solution for optimal results. |
Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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Solubility (In Vitro) |
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Solubility (In Vivo) |
Solubility in Formulation 1: 2.5 mg/mL (3.47 mM) = in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.5 mg/mL (3.47 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.= View More
Solubility in Formulation 3: 2.5 mg/mL (3.47 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. Solubility in Formulation 4: 2% DMSO+castor oil: 10 mg/mL |
Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
1 mM | 1.3891 mL | 6.9453 mL | 13.8906 mL | |
5 mM | 0.2778 mL | 1.3891 mL | 2.7781 mL | |
10 mM | 0.1389 mL | 0.6945 mL | 1.3891 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration: mg/mL;
Method for preparing DMSO stock solution: mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation::Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O,mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
NCT Number | Recruitment | interventions | Conditions | Sponsor/Collaborators | Start Date | Phases |
NCT02970747 | Active Recruiting |
Drug: Carfilzomib | Multiple Myeloma in Relapse | iOMEDICO AG | October 25, 2016 | |
NCT02512926 | Active Recruiting |
Drug: Carfilzomib Drug: Etoposide |
Relapsed Leukemia Refractory Leukemia/td> | Stanford University | February 16, 2016 | Phase 1 |
NCT02899052 | Recruiting | Drug: Carfilzomib Drug: Venetoclax |
Multiple Myeloma | AbbVie | January 19, 2017 | Phase 2 |
NCT03795597 | Recruiting | Drug: Carfilzomib Drug: Busulfan IV |
Multiple Myeloma | Loyola University | May 22, 2019 | Phase 1 Phase 2 |
NCT04176718 | Recruiting | Drug: Carfilzomib Drug: Daratumumab |
Multiple Myeloma | Andrew Yee, MD | May 18, 2020 | Phase 2 |