Caspase-like Research Articles

Abstract 2607: The benefits of irreversibility: Infusion administration of carfilzomib results in potent proteasome inhibition and improved safety in animals

Abstract Carfilzomib (CFZ) is a tetrapeptide epoxyketone-based inhibitor whose primary target is the chymotrypsin-like (CT-L) activity of the 20S proteasome. In clinical trials, intravenous (IV) bolus administration of carfilzomib has shown promising anti-tumor activity in multiple myeloma (MM), including patients relapsed and/or refractory to bortezomib. CFZ is a time-dependent and irreversible inhibitor, suggesting that target inhibition in vivo is likely a result of total dose administered and not steady state or maximum plasma concentration (Cmax). In order to determine if the method of delivery would affect the pharmacodynamics (PD) and toxicity of carfilzomib, we performed a series of experiments in rats. When delivered as an IV bolus or 30-minute infusion, a dose of 8 mg/kg resulted in ≥90% inhibition of the CT-L activity of the proteasome in whole blood and in all analyzed tissues (adrenal, lung, heart, and liver). Inhibition of the trypsin-like (T-L) and caspase-like (C-L) activity of the proteasome was ≥80% and ≥60%, respectively, was indistinguishable between the 2 groups. Despite an identical PD profile, infusion administration was significantly better tolerated than bolus. In the bolus group mortality was 44% (14 of 32) and the remaining animals displayed clinical signs of dyspnea, lethargy and poor blood perfusion to the extremities. None of the 24 animals receiving an infusion administration of CFZ died or were euthanized for humane reasons. A dose of 0.5 mg/kg bortezomib (BTZ), delivered as an IV bolus, produced similar clinical signs and mortality rates as bolus CFZ in rats. This result enabled us to determine which toxicity findings were target mediated. Interestingly, this dose of BTZ resulted in similar levels of inhibition of the CT-L (≥90%) but substantially less inhibition of T-L activity (≤20%) and slightly greater inhibition in C-L activity (≥80%) in blood and tissues. Bolus administration of either CFZ or BTZ resulted in 3 – 6-fold increases in blood urea nitrogen (BUN) and serum creatinine, while infusion administration of CFZ resulted in only a modest increase in BUN and no changes in creatinine levels. Other changes, such as a transient decrease in platelets, were not affected by the method of delivery of carfilzomib. These data suggest that bolus administration of either CFZ or BTZ in rats produced sequelae including pulmonary and cardiovascular function changes, renal azotemia and death and these toxicities are likely Cmax-related. However, because of its irreversible mechanism of proteasome inhibition, CFZ can be safely administered as an infusion resulting in profound proteasome inhibition in blood and tissues. The improved safety of infusion administration seen in these studies provides rationale for clinical application of infusion administration of CFZ in patients with MM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2607. doi:10.1158/1538-7445.AM2011-2607

Pharmacodynamic and Efficacy Studies of a Novel Proteasome Inhibitor NPI-0052 in Human Plasmacytoma Xenograft Mouse Model

Background: We recently characterized a novel proteasome inhibitor NPI-0052, a small molecule derived from the fermentation of a marine gram-positive actinomycete Salinispora tropica. NPI-0052 induces apoptosis in multiple myeloma (MM) cells resistant to conventional and bortezomib therapies. Importantly, NPI-0052 is distinct from bortezomib in its chemical structure, proteasome inhibition profiles, and mechanisms of action. In the present study, we utilized a human plasmacytoma xenograft mouse model to examine the effect of NPI-0052 on proteasome activity profiles in selected organs and tumors. Our results demonstrate that NPI-0052 rapidly leaves the vascular compartment in an active form after intravenous (IV) administration and inhibits the proteasome in extra-vascular tumors and other organs, excluding brain. NPI-0052 triggers a more sustained proteasome inhibition in tumors than in other organs examined. Importantly, we also confirmed the anti-tumor efficacy of NPI-0052.Methods and Model: Animal studies were approved by the DFCI Institutional Animal Care and Use Committee. Sixty CB-17 SCID-male mice were inoculated with 5.0 × 106 MM.1S cells in 100ul of serum free RPMI-1640 medium. The mice were divided into three different groups: Groups 1 and 2 (25 mice EA group) for pharmacodynamic studies (time course) and Group 3 (10 mice) for drug efficacy study. Tumor size was measured every third day in two dimensions using calipers, and tumor volume was calculated using the formula V = 0.5 a × b2, where a and b are the long and short diameter of the tumor respectively. When tumors were ~250 mm3 (~three-four weeks after injection), mice were treated with 0.15 mg/kg of NPI-0052 (IV) or vehicle control. Proteasome inhibition was assessed after either single NPI-0052 treatment (given at Day1) or three treatments (given at Day1, Day4 and Day8). Mice were euthanized at 10 mins, 1h, 4h, and 24h; and packed whole blood (PWB), liver, spleen, lung, kidney, brain and tumors were analyzed for chymotrypsin-like (CT-L), Caspase-like (C-L), and Trypsin-like (T-L) proteasome activities. For efficacy studies mice were treated with NPI-0052 twice a week for three weeks. Mice were sacrificed when their tumors reached ~1.5 cm3. NPI-0052 was dissolved in 100% DMSO to generate a 10 mg/ml stock solution, aliquoted, and stored frozen at − 80°C. The stock solution was serially diluted with 100% DMSO and for injection with 5% Solutol (Solutol HS, polyethylene glycol 660, 12 hydroxystearate; BASF, Shreveport, LA) yielding a final concentration of 2% DMSO and 98% (5% Solutol). The vehicle control was 2% DMSO and 98% (5% Solutol). The pH of the dosing solutions is between 6–7.Results:Inhibition of all three proteasome activities after a single treatment of NPI-0052 was detectable as early as 10 mins in the liver, lung, spleen, kidney and PWB;Within 24h after either a single or three IV treatments of NPI-0052, proteasome activity recovered in liver, lung, spleen and kidney, but not in tumor or PWB;No significant proteasome inhibition was noted in brain up to 24h after either a single or three IV treatments with NPI-0052;CT-L activity was inhibited within 1h post first dose, and 24h exposure triggered marked inhibition of CT-L, C-L and T-L activities in vivo in the xenografted MM.1S tumors.For example, in 1h CT-L activity was inhibited 34%, T-L activity 6% and C-L activity 16%. After 24h hours, CT-L activity was inhibited 60%, T-L activity 24% and C-L activity 49%; and finally, 6) Inhibition of CT-L, C-L and T-L activities increased in the tumor after the third NPI-0052 treatment compared to the first treatment. For example, at 1h post third dose all three activities were inhibited approximately 70–80%. Additionally, the anti-MM activity of NPI-0052 was associated with significant proteasome inhibition in tumors (P < 0.005).Conclusions: Our findings show that NPI-0052 induces a prolonged inhibition (>24h) of all three-20S proteasome activities in established MM.1S tumor xenografts which correlated with marked anti-tumor activity. In contrast, proteasome inhibition in normal tissues including liver, spleen, kidney and lung markedly recovered within 24h of administration after a single or three treatments with NPI-0052. In addition, no significant inhibition of proteasome activities was detectable in the brain after either treatment schedule.

Leaving Groups Prolong the Duration of 20S Proteasome Inhibition and Enhance the Potency of Salinosporamides

Salinosporamide A ( 1 (NPI-0052)) is a potent, monochlorinated 20S proteasome inhibitor in clinical trials for the treatment of cancer. To elucidate the role of the chlorine leaving group (LG), we synthesized analogues with a range of LG potentials and determined their IC 50 values for inhibition of chymotrypsin-like (CT-L), trypsin-like (T-L), and caspase-like (C-L) activities of 20S proteasomes. Proteasome activity was also determined before and after attempted removal of the inhibitors by dialysis. Analogues bearing substituents with good LG potential exhibited the greatest potency and prolonged duration of proteasome inhibition, with no recovery after 24 h of dialysis. In contrast, activity was restored after </=12 h in the case of non-LG analogues. Intermediate results were observed for fluorosalinosporamide, with poor LG potential. Kinetic studies indicate that 1 acts as a classical slow, tight inhibitor of the CT-L, T-L, and C-L activities and that inhibition occurs via a two-step mechanism involving reversible recognition followed by rate-limiting formation of a covalent enzyme-inhibitor complex.

Combination of proteasome inhibitors bortezomib and NPI-0052 trigger in vivo synergistic cytotoxicity in multiple myeloma

AbstractOur recent study demonstrated that a novel proteasome inhibitor NPI-0052 triggers apoptosis in multiple myeloma (MM) cells, and importantly, that is distinct from bortezomib (Velcade) in its chemical structure, effects on proteasome activities, and mechanisms of action. Here, we demonstrate that combining NPI-0052 and bortezomb induces synergistic anti-MM activity both in vitro using MM cell lines or patient CD138+ MM cells and in vivo in a human plasmacytoma xenograft mouse model. NPI-0052 plus bortezomib–induced synergistic apoptosis is associated with: (1) activation of caspase-8, caspase-9, caspase-3, and PARP; (2) induction of endoplasmic reticulum (ER) stress response and JNK; (3) inhibition of migration of MM cells and angiogenesis; (4) suppression of chymotrypsin-like (CT-L), caspase-like (C-L), and trypsin-like (T-L) proteolytic activities; and (5) blockade of NF-κB signaling. Studies in a xenograft model show that low dose combination of NPI-0052 and bortezomib is well tolerated and triggers synergistic inhibition of tumor growth and CT-L, C-L, and T-L proteasome activities in tumor cells. Immununostaining of MM tumors from NPI-0052 plus bortezomib–treated mice showed growth inhibition, apoptosis, and a decrease in associated angiogenesis. Taken together, our study provides the preclinical rationale for clinical protocols evaluating bortezomib together with NPI-0052 to improve patient outcome in MM.

Combination of Proteasome Inhibitors Bortezomib and NPI-0052 Trigger In Vivo Synergistic Cytotoxicity in Multiple Myeloma.

Bortezomib (Velcade™), the first in class proteasome inhibitor is FDA approved drug for the treatment of relapsed and relapsed/refractory multiple myeloma (MM). However, as with other agents, dose-limiting toxicities and the development of resistance limits its long-term utility. Our recent study demonstrated that a novel proteasome inhibitor NPI-0052 triggers apoptosis in MM cells; and importantly, is distinct from bortezomib in its chemical structure, effects on proteasome activities, and mechanisms of action. Here, we demonstrate that combining NPI-0052 and bortezomb induces synergistic anti-MM activity both in vitro using MM cell lines or patient CD138+ MM cells and in vivo in a human plasmacytoma xenograft mouse model. NPI-0052 + bortezomib-induced synergistic apoptosis is associated with:1.activation of caspase-8, caspase-9, caspase-3, and PARP;2.induction of ER-stress response and JNK;3.inhibition of migration of MM cells and angiogenesis;4.suppression of chymotrypsin-like (CT-L), caspase-like (C-L) and trypsin-like (T-L) proteolytic activities; and5.blockade of NF-kappa B signaling.Studies in a xenograft MM model show that low dose combination of NPI-0052 and bortezomib is well tolerated and triggers synergistic inhibition of tumor growth. Importantly, analysis of resected xenografted tumors show that 30–40% proteasome inhibition of all three (CT-L, C-L and T-L) proteasomal activities is sufficient to trigger significant MM cell apoptosis, confirming both the sensitivity of MM cells to proteasome inhibition and the importance of inhibiting all three proteolytic activities to obtain maximum response. Immunohistochemical analysis of MM tumors excised from NPI-0052 + bortezomib-treated mice showed growth inhibition, apoptosis, and a decrease in associated angiogenesis. The clinical observation that bortezomib therapy can be associated with toxicity and drug-resistance, coupled with our present preclinical findings demonstrating that low doses of bortezomib together with NPI-0052 trigger a potent anti-MM effect in vitro and in vivo, suggests the promise of combination treatment strategies to enhance anti-MM activity, reduce toxicity, overcome drug resistance, and improve outcome in MM patients. In addition to the above studies, data related to combination of NPI-0052 with lenalidomide (Revlimid™) and with histone deacetylase inhibitors such as MS-275, Tubacin or LBH589, will be presented.

In Vitro and In Vivo Proteasome Activity Profiles of Bortezomib and a Novel Proteasome Inhibitor NPI-0052.

Proteasome inhibition is an effective anti-cancer therapy. Proteasome function is mediated by three catalytic activities: chymotrypsin-like (CT-L), trypsin-like (T-L), and caspase-like (C-L). Kinetics of inhibition of catalytic activities may define the pharmacologic utility of proteasome inhibitors. Here we utilized two structurally distinct proteasome inhibitors Bortezomib, a dipeptide boronic acid; and a non-peptide proteasome inhibitor NPI-0052 to determine their effect on proteasome activities in vitro and in animal model. Examination of the proteasome activity using human erythrocyte 20S proteasomes and fluorogenic substrates shows that NPI-0052 and Bortezomib inhibit all three proteasome activities, albeit at different concentrations: NPI-0052 inhibits CT-L and T-L activities at lower concentrations than Bortezomib (NPI-0052: EC50 = 3.5 ± 0.3 nM versus Bortezomib: 7.9 ± 0.5 nM for CT-L activity; and NPI-0052: EC50 = 28 ± 2 nM versus Bortezomib: EC50 = 590 ± 67 nM for T-L activity); in contrast, higher concentrations of NPI-0052 than Bortezomib are required to inhibit C-L activity (NPI-0052 EC50 = 430 ± 34 nM versus Bortezomib: EC50 = 53 ± 10 nM for C-L activity). We next compared the effects of NPI-0052 and Bortezomib on all three proteasome activities in vivo. Mice were treated with a single MTD dose of NPI-0052 (0.15 mg/kg i.v) or Bortezomib (1 mg/kg i.v); blood samples were collected at 90 mins, 24h, 48h, 72h, or 168h; and whole blood cells were then analyzed for proteasome activity. NPI-0052 completely inhibited CT-L activity by 90 mins, which was recoverable by 168h; whereas Bortezomib-inhibited CT-L activity is recoverable at 24h. T-L activity is significantly inhibited by NPI-0052 at 90 mins, 24h, 48h, and 72h; and is recoverable by 168h; in contrast, Bortezomib enhances T-L activity. Finally, NPI-0052 inhibits C-L activity at 90 mins, 24h, 48h, and 72h; and this activity recovered at 168h, whereas Bortezomib significantly inhibits C-L activity at 90 mins, 24h, 48h, and 72h; and is similarly recoverable at 168h. We next utilized a novel methodology to measure proteasome activity by immunoblotting using dansylAhx3L3VS as a probe (Berkers et al., Nature Methods, 2005), which also allow for determining subunit specificity of a proteasome inhibitor. Multiple myeloma (MM) cells were cultured in the presence or absence of various concentrations of either NPI-0052 (2 nM; 7 nM: IC50; or 20 nM) or Bortezomib (2 nM; 5 nM: IC50; or 20 nM). Competition experiments between either NPI-0052 or Bortezomib and dansylAhx3L3VS revealed that NPI-0052 (7 nM) markedly inhibits the CT-L activity represented by beta-5 subunit of the proteasome and decreased the dansylAhx3L3VS-labeling of the beta-1 (C-L activity) and -2 (T-L activity) subunits. Slightly higher concentrations of Bortezomib are necessary to markedly inhibit beta-5 and -1 subunits, whereas beta-2 subunits are not inhibited. Importantly, both agents trigger apoptosis in MM cells; however, NPI-0052 is remarkably less toxic to normal lymphocytes than Bortezomib. Our data show that NPI-0052, like Bortezomib, targets the proteasome, but triggers a proteasome activity profile distinct from Bortezomib. The mechanistic insights gained from these studies will allow for improved drug design based on targeting specific proteasome subunits.