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]

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The proteasome has emerged as an important target for cancer therapy with the approval of bortezomib, a first-in-class, reversible proteasome inhibitor, for relapsed/refractory multiple myeloma (MM). However, many patients have disease that does not respond to bortezomib, whereas others develop resistance, suggesting the need for other inhibitors with enhanced activity. We therefore evaluated a novel, irreversible, epoxomicin-related proteasome inhibitor, Carfilzomib. In models of MM, this agent potently bound and specifically inhibited the chymotrypsin-like proteasome and immunoproteasome activities, resulting in accumulation of ubiquitinated substrates. Carfilzomib induced a dose- and time-dependent inhibition of proliferation, ultimately leading to apoptosis. Programmed cell death was associated with activation of c-Jun-N-terminal kinase, mitochondrial membrane depolarization, release of cytochrome c, and activation of both intrinsic and extrinsic caspase pathways. This agent also inhibited proliferation and activated apoptosis in patient-derived MM cells and neoplastic cells from patients with other hematologic malignancies. Importantly, carfilzomib showed increased efficacy compared with bortezomib and was active against bortezomib-resistant MM cell lines and samples from patients with clinical bortezomib resistance. Carfilzomib also overcame resistance to other conventional agents and acted synergistically with dexamethasone to enhance cell death. Taken together, these data provide a rationale for the clinical evaluation of carfilzomib in MM. [1]

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Continuous or physiologic transient administration of Carfilzomib or oprozomib is cytotoxic to human MM cells in vitro Under 48 h of continual drug incubation, carfilzomib and oprozomib exerted a cytotoxic effect on a panel of 10 human MM cell lines similar to bortezomib. In agreement with previous reports, the IC50 was approximately 2 nM for bortezomib, 3 nM for carfilzomib and 25 nM for oprozomib (Figure 1a). However, pharmacokinetic data indicate that in vivo exposure to drug is approximately 4 h following oral delivery of oprozomib and approximately 1 h with intravenous administration of carfilzomib or bortezomib. To more accurately replicate this physiological situation in vitro, cells were transiently treated with oprozomib for 4 h and with carfilzomib or bortezomib for 1 h followed by an additional 48 h culture in drug-free media. Myeloma cell lines remained susceptible to proteasome inhibition under short treatment conditions (Figure 1b), although increased doses were required to achieve similar efficacy (8 nM bortezomib, 6 nM carfilzomib and 50 nM oprozomib). Effective transient doses were still well below the maximum serum levels (Cmax) attained in patients (bortezomib: 0.162 μM (1.3 mg/m2 intravenous); carfilzomib: 0.95 μM (20 mg/m2 intravenous); oprozomib: 3.8 μM (30 mg per os)). The decrease in MM viability by carfilzomib and oprozomib was attributed to both inhibition of proliferation and apoptosis induction (data not shown), consistent with previous reports examining these PIs.
Oprozomib and carfilzomib inhibit OC differentiation and function in vitro. Carfilzomib and oprozomib promote osteogenic differentiation and mineralization in vitro [3].

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Interactions between the proteasome inhibitor Carfilzomib and the histone deacetylase (HDAC) inhibitors vorinostat and SNDX-275 were examined in mantle cell lymphoma (MCL) cells in vitro and in vivo. Coadministration of very low, marginally toxic carfilzomib concentrations (e.g., 3-4 nmol/L) with minimally lethal vorinostat or SNDX-275 concentrations induced sharp increases in mitochondrial injury and apoptosis in multiple MCL cell lines and primary MCL cells. Enhanced lethality was associated with c-jun-NH,-kinase (JNK) 1/2 activation, increased DNA damage (induction of λH2A.X), and ERK1/2 and AKT1/2 inactivation. Coadministration of carfilzomib and histone deacetylase inhibitors (HDACI) induced a marked increase in reactive oxygen species (ROS) generation and G(2)-M arrest. Significantly, the free radical scavenger tetrakis(4-benzoic acid) porphyrin (TBAP) blocked carfilzomib/HDACI-mediated ROS generation, λH2A.X formation, JNK1/2 activation, and lethality. Genetic (short hairpin RNA) knockdown of JNK1/2 significantly attenuated carfilzomib/HDACI-induced apoptosis, but did not prevent ROS generation or DNA damage. Carfilzomib/HDACI regimens were also active against bortezomib-resistant MCL cells [4].

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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Epoxyketone-based PIs exert bone anabolic effects on non-tumor bearing mice [3]
In vitro evidence suggests that PIs exert cell-autonomous effects on both OCs and OBs. To examine their effects on non-myelomatous bone, PIs were administered to non-tumor bearing immunocompetent C57Bl/6 mice for two weeks. Similar to bortezomib, treatment with Carfilzomib or oprozomib increased trabecular bone parameters (Figures 5a and b). All three PIs comparably inhibited OC function as measured by decreased serum levels of collagen breakdown products (carboxy-terminal telopeptide collagen crosslinks) resulting from bone resorption (Figure 5c). Furthermore, all drugs significantly increased OB activity as measured by increased serum levels of N-terminal propeptide of type I procollagen, a marker of bone formation, compared with controls (Figure 5d). Notably, carfilzomib exerted an increase in N-terminal propeptide of type I procollagen that was significantly greater than that obtained with bortezomib. In agreement, double calcein labeling demonstrated that PIs increased bone formation rate (Figure 5e). These data demonstrate that the epoxyketone-based PIs carfilzomib and oprozomib enhance bone volume in healthy mice through both anabolic and anti-catabolic properties that are equipotent to or even superior to that of bortezomib.

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Carfilzomib and oprozomib decrease MM tumor burden and protect mice from bone destruction [3]
To examine the combined anti-tumor and bone-preserving effects of carfilzomib and oprozomib for therapeutic treatment of established myeloma, we utilized two in vivo mouse models. Intravenous injection of 5TGM1-GFP murine myeloma cells into immunocompetent, syngeneic C57Bl/KaLwRij mice yields disseminated tumors with significant bone destruction within 28 days.51,52 5TGM1 tumors were established for 14 days after which bortezomib, carfilzomib, or oprozomib were administered on schedules correlating with each drug’s clinical dosing (see Materials and Methods). All PIs significantly decreased tumor burden as measured by serum levels of the clonotypic antibody IgG2b (Figure 6a) or by percentage of BM or spleen comprised of GFP-expressing tumor cells (Figures 6b and c). Protection from tumor-induced bone loss was evident by microCT in all PI-treated groups (Figures 6d and e), with serum markers of bone turnover showing significant anti-resorptive (Figure 6f) and bone anabolic (Figure 6g) effects. Notably, although differences within PIs were not statistically significant, a trend toward increased N-terminal propeptide of type-I procollagen activity with carfilzomib and oprozomib versus bortezomib was observed.

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In vivo activity of the Carfilzomib/vorinostat regimen in an in vivo Granta xenograft model [4]
To assess the in vivo activity of the carfilzomib/vorinostat regimen, a Granta-luciferace cell xenograft flank model was employed, analogous to the DLBCL model we have described (24). Animals were inoculated in the flank with 10 × 106 cells, and following the appearance of tumors, animals were treated with 2.0mg/kg carfilzomib (IV, BIW- day 1,2) ± 70 mg/kg vorinostat (IP, TIW- day1,2,3) after which tumor size was monitored twice weekly. Values represent the results of two separate experiments performed independently, and mean tumor volumes for each group was calculated by combining tumor growth data for the two experiments. As shown in Fig 6A, vorinostat alone had minimal effects whereas carfilzomib moderately reduced tumor growth by day 20. However, vorinostat/carfilzomib co-administration virtually abrogated tumor growth. Parallel studies were performed in animals inoculated with luciferase-expressing cells, and tumor progression was monitored by an IVIS bioimager. Combined treatment resulted in a pronounced reduction in bioluminescence compared to animals treated with single agents or controls (Fig 6B). Toxicity of combined treatment e.g., hair loss, weight reduction (< 10%) was minimal (Fig 6C). Finally, Western blot analysis obtained from proteins extracted from excised tumors revealed a clear increase in phospho-JNK, γH2A.X, and cleaved caspse-3 in tumor obtained from animals treated with both agents compared to single agents or controls (Fig 6D), consistent with in vitro results.

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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Enzyme-linked immunosorbent assay for subunit profiling of Carfilzomib [1]
ANBL-6 cells (2 × 106/well) were plated in 96-well plates and treated with carfilzomib doses from 0.001 to 10 μM for 1 hour. Cells were then lysed (20 mM Tris-HCl, 0.5 mM EDTA), and cleared lysates were transferred to polymerase chain reaction (PCR) plates. A standard curve was generated using untreated ANBL-6 cell lysates starting at a concentration of 6 μg protein/μL. The active site probe [biotin-(CH2)4-Leu-Leu-Leu-epoxyketone; 20 μM] was added and incubated at room temperature for 1 hour. Cell lysates were then denatured by adding 1% sodium dodecyl sulfate (SDS) and heating to 100°C, followed by mixing with 20 μL per well streptavidin-sepharose high-performance beads in a 96-well multiscreen DV plate and incubated for 1 hour.

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Competitive binding for subunit profiling of Carfilzomib [1]
The protocol used to determine carfilzomib subunit specificity via competitive binding was adapted from Berkers et al. Briefly, ANBL-6 cells were preincubated with increasing carfilzomib doses at 37°C, followed by addition of the hapten-labeled cell-permeant vinyl sulfone (VS) proteasome inhibitor VS-L3-AHx3-danysl. Western blots were then prepared as detailed in the next section and probed with polyclonal antidansyl antibodies.

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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Apoptotic DNA fragmentation assay [1]
For apoptosis experiments, cells were seeded onto 96-well plates, treated with a 1-hour pulse of 300 nM (RPMI 8226, ANBL-6) or 100 nM Carfilzomib (KAS-6/1, U266), and allowed to recover for 24 hours before analysis with the Cell Death Detection ELISAPLUS kit according to the manufacturer's specifications. The fold increase in DNA fragmentation is presented as the mean relative to vehicle-treated control cells.

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Mitochondrial membrane potential (ΔΨm) [1]
ANBL-6 cells pulsed with 100 nM Carfilzomib were washed and suspended in PBS containing 5 μg/mL of JC-1, which exhibits potential-dependent accumulation in mitochondria. Analysis of the mitochondrial membrane potential-dependent color shift from 525 to 590 nm was carried out on a FacScan, and the data were analyzed with CellQuest software.

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Viability assays [3]
A total of 5 × 104 cells/ml were plated and standard MTT assay was performed. For transient dosing experiments, cells were washed twice with phosphate-buffered saline and replaced with drug-free media after 1 h (bortezomib, Carfilzomib) or 4 h (oprozomib).

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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In vivo drug treatment [3]
PIs were administered to mice on the following weekly schedules: bortezomib (1 mg/kg intravenously days 1 and 4); Carfilzomib (5 mg/kg for C57Bl/6, 3 mg/kg for KaLwRij, intravenously days 1 and 2); oprozomib (30 mg/kg by oral gavage once daily for 5 consecutive days followed by 2 days of rest). Vehicle mice were administered both oral 1% carboxy-methylcellulose (oprozomib schedule) and intravenous 10% Captisol in 10 mM citrate buffer, pH 3.5 (Carfilzomib schedule). In Figure 5f, following 14 days of drug treatment, three doses of 1 mg/kg of RANKL were given intraperitoneally at 24 h intervals as described in Tomimori et al.34 Serum was collected 90 min after the final RANKL injection.

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Animal Studies [4]
Animal studies were performed utilizing Beige-nude-XID mice. 10×106 Granta514 cells were pelleted, washed twice with 1X PBS, injected subcutaneously into the right flank. Once the tumors were visible, 5 to 6 mice were treated with Carfilzomib ± vorinostat and progress of tumor growth or regression was monitored as described earlier. Stock vorinostat and Carfilzomib was dissolved in DMSO and 10% sulfobutylether betacyclodextrin in 10mM citrate buffer pH respectively. They were stored in −80°C in small aliquots and diluted before injection as described earlier.

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.

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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.

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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).

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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.

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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.

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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

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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.

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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.

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The ubiquitin-proteasome pathway (UPP) is an attractive chemotherapeutic target due to its intrinsically stringent regulation of cell cycle, pro-survival, and anti-apoptotic regulators that disproportionately favor survival and proliferation in malignant cells. A reversible first-in-class proteasome inhibitor, bortezomib, is Food and Drug Administration approved for multiple myeloma and relapsed/refractory mantle cell lymphoma and has proven to be extremely effective, both as a single agent and in combination. An irreversible second generation proteasome inhibitor, Carfilzomib, has shown preclinical effectiveness against hematological and solid malignancies both in vitro and in vivo. Carfilzomib, a peptidyl-epoxyketone functions similarly to bortezomib through primary inhibition of chymotrypsin-like (ChT-L) activity at the b5 subunits of the core 20S proteasome. Carfilzomib is also currently achieving successful response rates within the clinical setting. In addition to conventional proteasome inhibitors, a novel approach may be to specifically target the hematological-specific immunoproteasome, thereby increasing overall effectiveness and reducing negative off-target effects. The immunoproteasome-specific inhibitor, IPSI-001, was shown to have inhibitory preference over the constitutive proteasome, and display enhanced efficiency of apoptotic induction of tumor cells from a hematologic origin. Herein, we discuss the preclinical and clinical development of carfilzomib and explore the potential of immunoproteasome-specific inhibitors, like IPSI-001, as a rational approach to exclusively target hematological malignancies. [2]

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Proteasome inhibitors (PIs), namely bortezomib, have become a cornerstone therapy for multiple myeloma (MM), potently reducing tumor burden and inhibiting pathologic bone destruction. In clinical trials, Carfilzomib, a next generation epoxyketone-based irreversible PI, has exhibited potent anti-myeloma efficacy and decreased side effects compared with bortezomib. Carfilzomib and its orally bioavailable analog oprozomib, effectively decreased MM cell viability following continual or transient treatment mimicking in vivo pharmacokinetics. Interactions between myeloma cells and the bone marrow (BM) microenvironment augment the number and activity of bone-resorbing osteoclasts (OCs) while inhibiting bone-forming osteoblasts (OBs), resulting in increased tumor growth and osteolytic lesions. At clinically relevant concentrations, carfilzomib and oprozomib directly inhibited OC formation and bone resorption in vitro, while enhancing osteogenic differentiation and matrix mineralization. Accordingly, carfilzomib and oprozomib increased trabecular bone volume, decreased bone resorption and enhanced bone formation in non-tumor bearing mice. Finally, in mouse models of disseminated MM, the epoxyketone-based PIs decreased murine 5TGM1 and human RPMI-8226 tumor burden and prevented bone loss. These data demonstrate that, in addition to anti-myeloma properties, carfilzomib and oprozomib effectively shift the bone microenvironment from a catabolic to an anabolic state and, similar to bortezomib, may decrease skeletal complications of MM.[3]

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.

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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.=

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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.

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

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