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.

Absorption, Distribution and Excretion
Cmax, single IV dose of 27 mg/m^2 = 4232 ng/mL; AUC, single IV dose of 27 mg/m^2 = 379 ng•hr/mL; Carfilzomib does not accumulation in the systemic. At doses between 20 and 36 mg/m2, there was a dose-dependent increase in exposure.
Vd, steady state, 20 mg/m^2 = 28 L
Systemic clearance = 151 - 263 L/hour. As this value exceeds hepatic blood flow, it suggests that carfilozmib is cleared extrahepatically.

---

Metabolism / Metabolites
Carfilzomib was rapidly and extensively metabolized by the liver. The predominant metabolites were the peptide fragments and the diol of carfilzomib which suggests that the main metabolic pathways are peptidase cleavage and epoxide hydrolysis. The cytochrome P450 enzyme system is minimally involved in the metabolism of carfilzomib. All metabolites are inactive.

---

Biological Half-Life
Following intravenous administration of doses ≥ 15 mg/m^2, carfilzomib was rapidly cleared from the systemic circulation with a half-life of ≤ 1 hour on Day 1 of Cycle 1.

Hepatotoxicity
In large clinical trials of carfilzomib, elevations in serum aminotransferase levels were common, occurring in 8% to 13% of patients. However, values greater than 5 times the upper limit of normal (ULN) were uncommon, occurring in 1% to 2% of recipients. In several studies there were reports of clinically apparent liver injury including acute liver failure in patients receiving carfilzomib; however, in most instances multiple concomitant medications were being taken (such as lenalidomide) and the specific role of carfilzomib in causing the liver injury was not always clear. The onset of injury was typically during the first cycle of therapy. The clinical features and pattern of injury in clinically apparent cases of liver injury due to carfilzomib have not been described in the published literature. Hepatotoxicity is listed as a warning in the product label for carfilzomib and monitoring of serum enzymes during treatment is recommended.
Likelihood score: D (Possible cause of clinically apparent liver injury).

---

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the clinical use of carfilzomib during breastfeeding. Because carfilzomib is 97% bound to plasma proteins, the amount in milk is likely to be low. The manufacturer recommends that breastfeeding be discontinued during carfilzomib therapy and for 2 weeks after the last dose.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

---

Protein Binding
Over the concentration range of 0.4 - 4 micromolar, carfilzomib was 97% protein bound.

[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.

Carfilzomib is a synthetic tetrapeptide consisting of morpholin-4-acetyl, L-2-amino-4-phenylbutanoyl, L-leucyl and L-phenylalanyl residues joined in sequence with the C-terminus connected to the amino group of (2S)-2-amino-4-methyl-1-[(2R)-2-methyloxiran-2-yl]-1-oxopentan-1-one via an amide linkage. Used for the treatment of patients with multiple myeloma It has a role as an antineoplastic agent and a proteasome inhibitor. It is a tetrapeptide, a member of morpholines and an epoxide.
Carfilzomib is an injectable antineoplastic agent (IV only). Chemically, it is a modified tetrapeptidyl epoxide and an analog of epoxomicin. It is also a selective proteasome inhibitor. FDA approved carfilzomib in July 2012 for the treatment of adults with relapsed or refractory multiple myeloma as monotherapy or combination therapy.
Carfilzomib is a Proteasome Inhibitor. The mechanism of action of carfilzomib is as a Proteasome Inhibitor.
Carfilzomib is an irreversible proteasome inhibitor and antineoplastic agent that is used in treatment of refractory multiple myeloma. Carfilzomib is associated with a low rate of serum enzyme elevations during treatment and has been implicated to rare instances of clinically apparent, acute liver injury some of which have been fatal.
Carfilzomib is an epoxomicin derivate with potential antineoplastic activity. Carfilzomib irreversibly binds to and inhibits the chymotrypsin-like activity of the 20S catalytic core subunit of the proteasome, a protease complex responsible for degrading a large variety of cellular proteins. Inhibition of proteasome-mediated proteolysis results in an accumulation of polyubiquinated proteins, which may lead to cell cycle arrest, induction of apoptosis, and inhibition of tumor growth.

---

Drug Indication
Carfilzomib is indicated for the treatment of adult patients with relapsed or refractory multiple myeloma who have received one to three lines of therapy in combination with lenalidomide and dexamethasone; or dexamethasone; or daratumumab and dexamethasone; or daratumumab and hyaluronidase-fihj and dexamethasone; or isatuximab and dexamethasone. It is also indicated as a single agent for the treatment of patients with relapsed or refractory multiple myeloma who have received one or more lines of therapy.
FDA Label
Kyprolis in combination with daratumumab and dexamethasone, with lenalidomide and dexamethasone, or with dexamethasone alone is indicated for the treatment of adult patients with multiple myeloma who have received at least one prior therapy.
Treatment of acute lymphoblastic leukaemia
Treatment of Multiple Myeloma

---

Mechanism of Action
Carfilzomib is made up of four modified peptides and acts as a proteasome inhibitor. Carfilzomib irreversibly and selectively binds to N-terminal threonine-containing active sites of the 20S proteasome, the proteolytic core particle within the 26S proteasome. This 20S core has 3 catalytic active sites: the chymotrypsin, trypsin, and caspase-like sites. Inhibition of the chymotrypsin-like site by carfilzomib (β5 and β5i subunits) is the most effective target in decreasing cellular proliferation, ultimately resulting in cell cycle arrest and apoptosis of cancerous cells. At higher doses, carfilzomib will inhibit the trypsin-and capase-like sites.

---

Pharmacodynamics
Intravenous carfilzomib administration resulted in suppression of proteasome chymotrypsin-like activity when measured in blood 1 hour after the first dose. On Day 1 of Cycle 1, proteasome inhibition in peripheral blood mononuclear cells (PBMCs) ranged from 79% to 89% at 15 mg/m2, and from 82% to 83% at 20 mg/m2. In addition, carfilzomib administration resulted in inhibition of the LMP2 and MECL1 subunits of the immunoproteasome ranging from 26% to 32% and 41% to 49%, respectively, at 20 mg/m2. Proteasome inhibition was maintained for ≥ 48 hours following the first dose of carfilzomib for each week of dosing. Resistance against carfilzomib has been observed and although the mechanism has not been confirmed, it is thought that up-regulation of P-glycoprotein may be a contributing factor. Furthermore, studies suggest that carfilzomib is more potent than bortezomib.

C40H57N5O7

719.91

719.425

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

868540-17-4

Carfilzomib-d8;1537187-53-3

11556711

White solid powder

1.2±0.1 g/cm3

975.6±65.0 °C at 760 mmHg

204-208°C

543.8±34.3 °C

0.0±0.3 mmHg at 25°C

1.551

6.71

4

8

20

52

1180

5

C([C@@]1(OC1)C)(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)CN1CCOCC1)CCC1C=CC=CC=1)CC1C=CC=CC=1

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

Note: This product is not stable in solution, please use freshly prepared working solution for optimal results.

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

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.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 4: 2% DMSO+castor oil: 10 mg/mL

---

 (Please use freshly prepared in vivo formulations for optimal results.)