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]

---

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]

---

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].

---

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]

---

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.

---

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.

---

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.

---

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.

---

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.

---

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.

---

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.

---

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.

---

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.

---

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
Following a single intravenous dose of 27 mg/m², Cmax = 4232 ng/mL; AUC = 379 ng•hr/mL; carfilzomib does not accumulate systemically. Drug exposure increases dose-dependently within the dose range of 20 to 36 mg/m². Steady-state volume of distribution (Vd, 20 mg/m²) = 28 L. Systemic clearance = 151 - 263 L/hour. Since this value exceeds hepatic blood flow, it suggests that carfilzomib is primarily cleared via extrahepatic pathways. Metabolism/Metabolites Carfilzomib is rapidly and extensively metabolized in the liver. The main metabolites are peptide fragments and diols of carfilzomib, suggesting that its primary metabolic pathways are peptidase cleavage and epoxide hydrolysis. The cytochrome P450 enzyme system plays a minimal role in the metabolism of carfilzomib. All metabolites are inactive.

---

Biological half-life
After intravenous injection of a dose ≥15 mg/m^2, carfilzomib is rapidly cleared from systemic circulation on day 1 of cycle 1, with a half-life ≤1 hour.

Hepatotoxicity
Elevated serum transaminase levels are common in large clinical trials of carfilzomib, with an incidence between 8% and 13%. However, transaminase levels exceeding the upper limit of normal (ULN) by 5 times are uncommon, occurring in 1% to 2% of patients. Some studies have reported clinically significant liver injury, including acute liver failure, in patients treated with carfilzomib; however, in most cases, patients are taking multiple other medications concurrently (e.g., lenalidomide), so the specific role of carfilzomib in causing liver injury is not always clear. Liver injury typically occurs within the first treatment cycle. The clinical characteristics and injury patterns of clinically significant liver injury induced by carfilzomib have not been described in published literature. Hepatotoxicity is listed as a warning on the carfilzomib product label, and monitoring of serum enzymes is recommended during treatment. Probability Score: D (May cause clinically significant liver injury).

---

Effects during pregnancy and lactation
◉ Overview of use during lactation
There is currently no information on the clinical use of carfilzomib during lactation. Because carfilzomib binds to plasma proteins at a rate of 97%, its concentration in breast milk may be very low. The manufacturer recommends discontinuing breastfeeding during carfilzomib treatment and for two weeks after the last dose.
◉ Effects on breastfed infants
As of the revision date, no relevant published information was found.
◉ Effects on lactation and breast milk
As of the revision date, no relevant published information was found.

---

Protein binding
Carfilzomib has a protein binding rate of 97% in the concentration range of 0.4–4 μmol.

[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 composed of morpholine-4-acetyl, L-2-amino-4-phenylbutyryl, L-leucyl, and L-phenylalanyl residues linked sequentially, with its C-terminus linked to the amino group of (2S)-2-amino-4-methyl-1-[(2R)-2-methylepoxyethylene-2-yl]-1-oxopentane-1-one via an amide bond. Carfilzomib is used to treat patients with multiple myeloma, exhibiting dual antitumor and proteasome inhibitor effects. It is a tetrapeptide, belonging to the morpholine class of compounds, and is also an epoxide. Carfilzomib is an injectable antitumor drug (intravenous injection only). Chemically, it is a modified tetrapeptide epoxide, an analogue of cyclooxygenin. It is also a selective proteasome inhibitor. In July 2012, the FDA approved carfilzomib for the treatment of adult patients with relapsed or refractory multiple myeloma, as a monotherapy or in combination therapy. Carfilzomib is a proteasome inhibitor. Carfilzomib's mechanism of action is as a proteasome inhibitor. Carfilzomib is an irreversible proteasome inhibitor and antitumor drug used to treat refractory multiple myeloma. The incidence of elevated serum enzymes during carfilzomib treatment is low, but a few case reports have shown clinically significant acute liver injury, some of which have even led to death. Carfilzomib is an cyclooxygenin derivative with potential antitumor 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 variety of cellular proteins. Proteasome-mediated inhibition of proteolytic hydrolysis leads to the accumulation of polyubiquitinated proteins, which may result in cell cycle arrest, induce apoptosis, and inhibit tumor growth.

---

Drug Indications
Carfilzomib is indicated for the treatment of adult patients with relapsed or refractory multiple myeloma who have received one to three lines of prior therapy, in combination with lenalidomide and dexamethasone; or dexamethasone; or daratumumab and dexamethasone; or daratumumab, hyaluronidase-fihj and dexamethasone; or ixartuximab and dexamethasone. It can also be used as monotherapy for the treatment of relapsed or refractory multiple myeloma patients who have received one or more prior therapies.
FDA Label
Carfilzomib (Kyprolis), in combination with daratumumab and dexamethasone, in combination with lenalidomide and dexamethasone, or alone in combination with dexamethasone, is indicated for the treatment of adult patients with multiple myeloma who have received at least one prior line of therapy.
Treatment of Acute Lymphoblastic Leukemia
Treatment of Multiple Myeloma

---

Mechanism of Action
Carfilzomib is a proteasome inhibitor composed of four modified peptides. Carfilzomib irreversibly and selectively binds to the N-terminal threonine-containing active site of the 20S proteasome (the proteolytic core particle in the 26S proteasome). This 20S core has three catalytically active sites: chymotrypsin site, trypsin site, and caspase-like site. Inhibition of the chymotrypsin site by carfilzomib (β5 and β5i subunits) is the most effective target for reducing cell proliferation, ultimately leading to cell cycle arrest and cancer cell apoptosis. At high doses, carfilzomib inhibits both the trypsin and caspase-like sites.

---

Pharmacodynamics
The proteasome chymotrypsin-like activity in the blood was measured 1 hour after the first dose following intravenous injection of carfilzomib, and the results showed inhibition of proteasome activity. On day 1 of the first treatment cycle, the inhibition rate of proteasomes in peripheral blood mononuclear cells (PBMCs) was 79% to 89% at a dose of 15 mg/m² and 82% to 83% at a dose of 20 mg/m². Furthermore, after carfilzomib administration, the inhibition rates of the LMP2 and MECL1 subunits of the immunoproteasome were 26% to 32% and 41% to 49%, respectively (at a dose of 20 mg/m²). During weekly dosing, proteasome inhibition persisted for ≥48 hours after the first dose. Resistance to carfilzomib has been observed, although the mechanism is not yet clear, but upregulation of P-glycoprotein is considered a contributing factor. Furthermore, studies have shown that carfilzomib is more potent than bortezomib. The ubiquitin-proteasome pathway (UPP) is a highly attractive chemotherapeutic target because it intrinsically and tightly regulates cell cycle, pro-survival, and anti-apoptotic regulators that disproportionately promote the survival and proliferation of malignant cells. Bortezomib is a reversible, first-in-class proteasome inhibitor approved by the U.S. Food and Drug Administration (FDA) for the treatment of multiple myeloma and relapsed/refractory mantle cell lymphoma, and has been shown to be effective both as a monotherapy and in combination therapy. Carfilzomib is an irreversible second-generation proteasome inhibitor that has shown efficacy against hematologic malignancies and solid tumors in both in vitro and preclinical in vivo studies. Carfilzomib is a peptidyl epoxide ketone compound with a mechanism of action similar to bortezomib, both acting by inhibiting chymotrypsin-like (ChT-L) activity on the β5 subunit of the 20S proteasome core. Currently, carfilzomib has also achieved good efficacy in clinical applications. In addition to traditional proteasome inhibitors, a new strategy may be to specifically target hematologic-specific immunoproteasomes, thereby improving overall efficacy and reducing off-target effects. The immunoproteasome-specific inhibitor IPSI-001 has been shown to have an inhibitory advantage against constitutive proteasomes and can more effectively induce apoptosis in hematologic tumor cells. This article will explore the preclinical and clinical development of carfilzomib and investigate the potential of immunoproteasome-specific inhibitors (such as IPSI-001) as a rational strategy for targeting hematologic malignancies. [2] Proteasome inhibitors (PIs), especially bortezomib, have become cornerstone therapies for multiple myeloma (MM) by effectively reducing tumor burden and inhibiting pathological bone destruction. In clinical trials, the next-generation epoxide ketone irreversible proteasome inhibitor carfilzomib has shown stronger anti-myeloma efficacy and fewer side effects compared to bortezomib. Carfilzomib and its more bioavailable oral analogue opezomib effectively reduce the viability of multiple myeloma (MM) cells after continuous or short-term treatment, mimicking in vivo pharmacokinetics. The interaction between myeloma cells and the bone marrow microenvironment increases the number and activity of osteoclasts (OCs) while inhibiting osteoblasts (OBs), leading to increased tumor growth and osteolytic lesions. At clinically relevant concentrations, carfilzomib and opezomib directly inhibit osteoclast formation and bone resorption in vitro while enhancing osteogenic differentiation and matrix mineralization. Correspondingly, in non-tumor mouse models, carfilzomib and opezomib increased trabecular bone volume, reduced bone resorption, and promoted bone formation. Finally, in disseminated MM mouse models, epoxetine-based protease inhibitors reduced the tumor burden of mouse 5TGM1 and human RPMI-8226 tumors and prevented bone loss. These data suggest that, in addition to their anti-myeloma properties, carfilzomib and opezomib can effectively change the bone microenvironment from a catabolic state to an anabolic state and, similar to bortezomib, can reduce skeletal complications of multiple myeloma. [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.

---

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.)