Abstract
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.
TNF-alpha inhibition ameliorates HDV-induced liver damage in a mouse model of acute severe infection
