Delanzomib

CEP-18770 demonstrates marginal prevention of the tryptic and peptidyl gultamyl activities of the protesome.^[1] CEP-18770 inhibits A2780 ovarian cancer cells, PC3 prostate cancer, H460, LoVo colon cancer, RPMI8226 multiple myeloma cancer and HS-Sultan anaplastic non-Hodgkin lymphoma with IC50 values of 13.7, 22.2, 34.2 11.3, 5.6 and 8.2 nM, respectively.^[1] CEP-18770 blocks the ubiquitin-proteasome pathway in several MM and in the chronic myelogenous leukemia cell line, K562. CEP-18770 causes an accumulation of ubiquitinated proteins over 4 to 8 hours.^[1] IκBα degradation is completely blocked by pretreatment with CEP-18770. CEP-18770 significantly inhibits high levels of NF-κB activity in both RPMI-8226 and U266 cells. The time- and concentration-dependent suppression of NF-kB DNA-binding activity in MM cell lines by CEP-18770 leads to a decrease of expression of several NF-κB-modulated genes mediating the growth and survival of tumor cells including IkBα itself, the X-chromosome-linked inhibitor-of-apoptosis protein (XIAP), the pro-inflammatory cytokines TNF-α and interleukin-1β (IL-1β), the intracellular adhesion molecule (ICAM1), and the pro-angiogeneic factor vascular endothelial growth factor. ^[1] The expression of these NF-κB–mediated genes are associated with more favorable clinical responsiveness to this agent, highlighting their potential prognostic value in response to CEP-18770 exposure. ^[1] The proapoptotic activity of CEP-18770 against MM is not limited solely to tumor-derived MM cell lines, but extends to primary MM explants from relapsed or refractory patients. ^[1] In addition, CEP-18770 in combination produces synergistic inhibition of MM cell viability in vitro.^[2]

CEP-18770 reveals sustained dose-related relative tumor weight inhibition. CEP-18770 leads to dose-related complete tumor regressions, which results in a 50% incidence of CR at its maximally tolerated dose (MTD) of 1.2 mg/kg intravenously. ^[1] CEP-18770 reveals dose-related increase in the incidence of tumor-free mice by the completion of these studies (120 days after tumor transplantation). Oral administration of CEP-18770 produces a marked decrease in tumor weight and notable dose-related incidence of complete tumor regression with minimal changes in animal body weight over the course of the 120 day studies. ^[1]_Equiactive doses of CEP-18770 reveal a greater and more sustained dose-related inhibition of tumor proteasome activity, corresponding temporarily with maximum induction of caspase-3 and 7 activity.^[1] The maximum apoptotic signal is 2.5 fold greater for CEP-18770. In contrast, proteasome inhibition profiles of CEP-18870 are comparable in the normal peripheral mouse tissues examined (liver, lungs, whole blood, and brain [no activity]) in both their magnitude and their duration.^[1] No proteasome inhibition is detected in brain tissue at any time point for either CEP-18770 or. ^[1] Single agent CEP-18770 PO also shows marked anti-MM effects in these xenograft models^[1]

• Probing proteasome activity in cell extracts

  Human multiple myeloma cells are washed twice with cold phosphate-buffered saline, pelleted and lysed with one volume of glass beads (<106 microns, acid-washed) and an equal volume of homogenization buffer (50 mM Tris (pH 7.4), 1 mM dithiothreitol, 5 mM MgCl₂, 2 mM ATP and 250 mM sucrose) by vortexing at high speed for 15-30 min at 4 °C. Beads, membrane fractions, nuclei and cell debris are then removed from the supernatant by centrifugation at 16,000g for 5 min. The protein content of extracts is quantitated using the Bradford assay. Proteasome activity is assayed as described below. Equal amounts (typically 60 g) of protein are denatured by boiling in reducing sample buffer, separated by 12.5% SDS-PAGE and electrotransferred onto polyvinylidene difluoride (PVDF) membranes. Immunoblotting is performed using a dansyl-sulfonamidohexanoyl polyclonal antibody (1:7,500, rabbit) and horseradish peroxidase–coupled goat or swine anti-rabbit secondary antibody followed by enhanced chemiluminescence.

• Cell lines

  HMEC and TEC cells

• Concentrations

  0-100 nM

• Incubation Time

  6 hours

• Method

  HMEC and TEC cells are seeded into 24-well plates at a density of 10⁴ cells/well in DMEM supplemented with 5% FCS. After incubation with proteasome inhibitors (48 hours), cells are washed, air dried, and stained with crystal violet as described. Cell number is determined, in duplicate samples, on the basis of a standard curve obtained with known cell numbers. All experiments are performed in triplicate. In vitro formation of capillary-like structures is studied on cells (4 × 10⁴ cells/well in DMEM supplemented with 5% FCS. After incubation with proteasome inhibitors (48 hours), cells are washed (cells/well in 24-well plates) and seeded onto Matrigel-coated wells in DMEM containing 0.25% BSA. HMEC and TEC cells (5 × 10³ per well), suspended in 200 μL DMEM with 5% FCS (positive control), serum-free medium (negative control), are layered onto the Matrigel surface in the presence or absence of proteasome inhibitor CEP-18770. Cells are observed with a microscope and experimental results are then recorded after a 6-hour incubation at 37 °C. Data is analyzed, as the mean (× 1 SD) of total length of capillary-like structures, by the Micro-Image system and is expressed as mm/field by the computer analysis system in 5 different fields at 100 × magnification in duplicated wells for 4 different experiments.

• Animal Models

  Human MM RPMI 8226 subcutaneous xenograft model in SCID mice

• Dosages

  From 1.5 to 4 mg/kg, twice for 7 days to 4 weeks.

• Administration

  Intravenously