Phase 1 Clinical Trial of the Novel Structure Proteasome Inhibitor NPI-0052.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2693-2693 ◽  
Author(s):  
Andrew Spencer ◽  
Michael Millward ◽  
Paul Mainwaring ◽  
Simon Harrison ◽  
Laurence Catley ◽  
...  
Keyword(s):  
Solid Tumor ◽  
Whole Blood ◽  
Proteasome Inhibitor ◽  
Mononuclear Cells ◽  
Proteasome Inhibitors ◽  
Proteasome Inhibition ◽  
Pharmacokinetic Data ◽  
Employment Equity ◽  
Cognitive Changes ◽  
Equity Ownership

Abstract Abstract 2693 Poster Board II-669 Background: NPI-0052 is a proteasome inhibitor with a novel bicyclic structure (other proteasome inhibitors in clinical use are peptide based). Preclinical studies indicate rapid, broad and prolonged inhibition of all 3 catalytic sites of the proteasome, and subsequently unique proteasome inhibition, signal transduction, toxicology and efficacy profiles. Taken together these suggest the potential for improvements in therapeutic ratio and activity in hematologic and solid tumor malignancies. Materials and Methods: Patients with solid tumor, lymphoma, leukemia or myeloma diagnoses without standard treatment options have been treated with IV NPI-0052 on one of two arms (weekly or twice weekly) in this 3+3 design dose escalation study. This is followed by 10 patient Recommended Phase 2 dose Cohorts of patients with lymphomas, CLL and myeloma respectively. Proteasome inhibition (pharmacodynamics) and pharmacokinetics are also assayed in whole blood, and proteasome inhibition in peripheral blood mononuclear cells (PBMC). Results: 44 patients have been treated with NPI-0052 at doses ranging from 0.075 mg/m2 to 0.9 mg/m2. Common adverse events include fatigue, parosmia/dysgeusia, transient peri-infusion site pain, lymphopenia, headaches, dizziness / unsteady gait, closed-eye visuals, cognitive changes. Incidence and grade of these events correlate with dose, being quite tolerable at the MTD of 0.7 mg/m2 on the weekly dosing arm. An MTD has not yet been determined for the twice weekly dosing arm. Pharmacokinetic data has demonstrated a rapid elimination half-life (<20 minutes) and relatively large volume of distribution. Assessment of proteasome inhibition has demonstrated increasing inhibition of chymotrypsin-like activity of up to 88% Day 1 and 100% Day 15. Inhibition of caspase-like and trypsin-like activity of up to 52% and 71% respectively has also been seen. Inhibition remains between doses in whole blood (principally RBC), but recovers between doses in PBMC. Clinical benefit, including stable disease, regression or response, was reported in patients with mantle cell lymphoma, myeloma, Hodgkin's lymphoma, cutaneous marginal zone lymphoma, follicular lymphoma, sarcoma, prostate carcinoma and melanoma. Conclusions: NPI-0052 produces dose-dependent pharmacologic effects through the predicted efficacious range, while producing a toxicity profile that is dissimilar to what is reported with other proteasome inhibitors (notably deficient in peripheral neuropathy, neutropenia and thrombocytopenia) in spite of producing equal or greater proteasome inhibition. These data indicate a broad range of potential uses, and led to additional studies in hematologic malignancies and solid tumors alone and in combination. Disclosures: Longenecker: Nereus Pharmaceuticals: Employment. Palladino:Nereus Pharmaceuticals: Employment, Equity Ownership. Lloyd:Nereus Pharmaceuticals: Employment, Equity Ownership. Neuteboom:Nereus Pharmaceuticals: Employment, Equity Ownership. Spear:Nereus Pharmaceuticals: Employment, Equity Ownership.

Phase 1 Clinical Evaluation of Twice-Weekly Marizomib (NPI-0052), a Novel Proteasome Inhibitor, in Patients with Relapsed/Refractory Multiple Myeloma (MM)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 302-302 ◽  
Author(s):  
Paul G. Richardson ◽  
Andrew Spencer ◽  
Paul Cannell ◽  
Simon J. Harrison ◽  
Laurence Catley ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Mononuclear Cells ◽  
Dose Range ◽  
Proteasome Inhibition ◽  
The United States ◽  
Employment Equity ◽  
Advisory Committees ◽  
Cognitive Changes ◽  
Safety And Efficacy ◽  
Equity Ownership

Abstract Abstract 302 Background: Marizomib has a novel, non-peptide based, bicyclic structure and compared to other proteasome inhibitors, unique properties of clinical relevance. Specifically, marizomib produces rapid, broad and prolonged inhibition of all 3 20S proteasome catalytic activities, and markedly different safety and efficacy profiles, including activity against MM resistant to bortezomib (BZ) both in vitro and in vivo. Materials and Methods: Marizomib was given IV over 1–120 minutes on days 1, 4, 8 and 11 of 21-day cycles in 2 separate and parallel dose escalation studies performed in Australia and the United States in patients with relapsed and refractory MM. In addition to standard safety and efficacy monitoring, pharmacokinetics (PK) and proteasome inhibition as part of pharmacodynamics (PD) were assessed. Dexamethasone (20 mg) was given the day prior to and day of treatment in one study and could be added for patients who did not achieve a minimal response (MR) or better after 2 cycles in the other study. Toxicity evaluation was performed using CTCAE v3.0 and response was assessed by modified European Group for Blood and Marrow Transplantation (EBMT) and Uniform Criteria (UC). Results: 34 patients (16 men and 18 women) have been treated at doses of 0.075 to 0.6 mg/m2/dose BIW with a median age of 62.5 years, in both studies. Patients received a median of 6 prior regimens; 30 patients (88%) had been exposed to prior BZ, including 24 (71%) who were BZ -refractory. The maximum tolerated dose of marizomib was found to be 0.4 mg/m2 over a 60 minute infusion time and 0.5 mg/m2 over a 120 minute infusion. Dose limiting toxicities included transient hallucinations, cognitive changes and loss of balance, all of which proved reversible. The most common drug-related adverse events were fatigue, nausea, vomiting, dizziness, headache, diarrhea, constipation, insomnia, anorexia, and dyspnea, which proved manageable with supportive care and/or dose reduction. Importantly, marizomib did not appear to induce myelosuppression, peripheral neuropathy (PN) or thrombocytopenia. PK analysis demonstrated a rapid elimination half-life (< 20 minutes) and large volume of distribution, with PD analyses of packed whole blood (PWB) and peripheral blood mononuclear cells (PBMC) confirming dose dependent proteasome inhibition. At interim analysis, of 22 patients with evaluable disease for best response to marizomib +/− dexamethasone, 3 had achieved partial response (PR) by EBMT/UC (14%). In the active dose range of 0.4–0.6 mg/m2, 15 pts were evaluable with PR in 3 pts (20%), all of whom were refractory to prior BZ. Median time on treatment was 1.5 months, with stable disease or better documented in 16 pts (73%). Conclusions: The safety profile of marizomib clearly differs from BZ, without significant treatment–emergent PN or myelosuppression described. Preliminary results suggest anti-myeloma activity, with responses seen in patients in whom BZ had previously failed, as well as interesting PK/PD characteristics and tissue distribution supporting a possible role in patients with different disease characteristics (such as extramedullary spread). The efficacy and safety of 0.5 mg/m2 of marizomib given twice weekly, alone or with low dose dexamethasone, warrants further study, and continues to be investigated. Future directions will include combination approaches with lenalidomide and dexamethasone. Disclosures: Richardson: Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees. Cannell:Nereus Pharmaceuticals: Investigator. Harrison:Nereus Pharmaceuticals: Research Funding. Jakubowiak:Ortho Biotech: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Millennium Pharmaceuticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Onyx Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Exelixis: Consultancy, Honoraria. Palladino:Nereus Pharmaceuticals, Inc: Employment, Equity Ownership. Longenecker:Nereus Pharmaceuticals, Inc: Employment, Equity Ownership. Lay:Nereus Pharmaceuticals, Inc: Employment, Equity Ownership. Lloyd:Nereus Pharmaceuticals, Inc: Employment, Equity Ownership. Hannah:Nereus Pharmaceuticals, Inc.: Consultancy. Reich:Nereus Pharmaceuticals: Consultancy. Spear:Nereus Pharmaceuticals, Inc: Employment, Equity Ownership. Anderson:Onyx: Consultancy; Merck: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Acetylon: Founder; Nereus Pharmaceuticals, Inc: Consultancy; Millennium: Consultancy; Celgene: Consultancy.

Potent Inhibition of Multiple Proteasome Subunits by Carfilzomib in Multiple Myeloma and Solid Tumor Patients

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5068-5068
Author(s):  
Susan J Lee ◽  
Tina M Woo ◽  
Shirin Arastu-Kapur ◽  
Alvin F Wong ◽  
Thomas E Renau ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Active Site ◽  
Whole Blood ◽  
Active Sites ◽  
Matrix Effects ◽  
Objective Response ◽  
Proteasome Inhibition ◽  
Reversible Inhibitor ◽  
Employment Equity ◽  
Equity Ownership

Abstract Abstract 5068 Proteasome inhibition has been validated as a therapeutic approach for multiple myeloma (MM) and mantle cell lymphoma, however the contributions of inhibiting individual active-site subunits of the constitutive proteasome (c20S) and the immunoproteasome (i20S) has not been fully explored due the lack of effective tools. A novel assay was developed, validated, and used to quantitatively measure the levels of individual proteasome active site subunits in vitro and in tissue samples from patients exposed to the proteasome inhibitor (PI) carfilzomib from 5 clinical trials, PX-171-003, PX-171-004, PX-171-005, PX-171-006 & PX-171-007. This assay, called ProCISE (proteasome constitutive/immune subunit ELISA), was shown to have good analytical recovery without interfering matrix effects and was used to measure the activity of c20S subunits (b5, b2 and b1) and i20S subunits (LMP7, MECL1 and LMP2) using subunit-specific antibodies in whole blood, PBMC, and bone marrow derived CD138+ MM cells. Following an initial dose of carfilzomib across doses of 15 – 45 mg/m2, ≥80% inhibition of the chymotrypsin-like (CT-L) active sites b5 and LMP7 as well as dose-dependent inhibition of MECL1 and LMP2 was observed. Additionally, neither renal function nor co-administration of the commonly used MM agents, lenalidomide or dexamethasone, had an effect on the pharmacodynamics of carfilzomib. Carfilzomib inhibited 63% of all active sites of the immunoproteasome at 45 mg/m2 and 78% at 56mg/m2. In tumor cells, which express a mixture of both proteasome types, inhibition of CT-L activity correlated with levels of inhibition in whole blood. Proteasome inhibition with carfilzomib was prolonged in both whole blood and PBMC. Cumulative and sustained proteasome inhibition was seen in whole blood while complete or near complete recovery was noted in PBMC by the start of a second cycle of administration. The depth and duration of proteasome inhibition with carfilzomib is greater than what has been reported with the reversible inhibitor bortezomib. In a limited analysis of MM patients, stratified by best response to carfilzomib, we did not detect a difference in the full proteasome inhibition profile in patients achieving an objective response and those that did not achieve clinical benefit. While these data do not currently demonstrate that proteasome inhibition alone is a predictive marker of clinical response, with larger sample sizes and further investigation regarding subunit-specific inhibition of the proteasome, we hope to be able to demonstrate a correlation between proteasome inhibition and patient response. Disclosures: Lee: Onyx Pharmaceuticals: Employment, Equity Ownership. Woo:Onyx Pharmaceuticals: Employment, Equity Ownership. Arastu-Kapur:Onyx Pharmaceuticals: Employment, Equity Ownership. Wong:Onyx Pharmaceuticals: Employment, Equity Ownership. Renau:Onyx Pharmaceuticals: Employment, Equity Ownership. Kirk:Onyx Pharmaceuticals: Employment, Equity Ownership.

Immunoglobulin Expression Is a Major Determinant Of Patient Sensitivity To Proteasome Inhibitors

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1903-1903
Author(s):  
Brian B Tuch ◽  
Andrea Loehr ◽  
Jeremiah D Degenhardt ◽  
Kevin A Kwei ◽  
Jonathan J Keats ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Strong Association ◽  
Single Agent ◽  
Proteasome Inhibitors ◽  
Proteasome Inhibition ◽  
Training Data ◽  
P Value ◽  
Tumor Cell Death ◽  
Employment Equity ◽  
Equity Ownership

Abstract Several therapies for multiple myeloma are now approved and many more are in development, promising improved outcomes for patients with this incurable cancer. With expanding treatment options, however, comes a pressing need to pair each patient with the most efficacious and safe treatment. Proteasome inhibitors, such as carfilzomib and bortezomib, have become a standard therapy across all lines of multiple myeloma treatment. Despite extensive study, the mechanism of selective tumor cell death following proteasome inhibition is poorly understood. However, the uniquely high sensitivity of myeloma cells to proteasome inhibition, the uniquely high burden of protein (immunoglobulin) secretion these cells experience, and the key role of the proteasome in maintaining protein homeostasis, together point toward a unifying model in which protein load drives proteasome inhibitor sensitivity. This simple model is supported by published studies of murine and human myeloma cell lines (e.g., Meister et al. & Bianchi et al.). As part of company-sponsored Phase II & III clinical trials of proteasome inhibitors, CD138+ tumor cells collected during patient screening were banked for comprehensive genomic analyses. Patient samples banked on bortezomib trials were utilized in now-published microarray-based RNA studies, while samples from carfilzomib trials are currently being used for NGS-based DNA and RNA studies. Here, examining our early carfilzomib data along with publically-available bortezomib data, we find a strong association between higher immunoglobulin expression and sensitivity to each compound (Wilcoxon P-value = 3x10-3 and P-value = 2x10-4, respectively). In fact, using IGH expression alone, we are able to classify response with 55% sensitivity and 91% specificity in our carfilzomib training data. As expected for a bona fide predictive biomarker of proteasome inhibition, an association between IG expression and response was not found in patients treated with single agent dexamethasone (Wilcoxon P-value = 0.82). Median time to progression for IGH-high carfilzomib patients was 6-fold longer than for IGH-low carfilzomib patients (7.6 months vs. 1.4 months; log-rank P-value = 0.003). This is the first report that high levels of IG expression correlate with response to proteasome inhibitors and therefore IG expression represents, to our knowledge, the first validated biomarker for this important class of anti-tumor agents. Disclosures: Tuch: Onyx Pharmaceuticals: Employment, Equity Ownership. Loehr:Onyx Pharmaceuticals: Employment, Equity Ownership. Degenhardt:Onyx Pharmaceuticals: Employment, Equity Ownership. Kwei:Onyx Pharmaceuticals: Employment, Equity Ownership. Kirk:Onyx Pharmaceuticals: Employment, Equity Ownership.

In-Vitro and in-Vivo Evaluation of the Orally Active Proteasome Inhibitor MLN9708 in Hematological Models of Human Cancer.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2709-2709
Author(s):  
Erik Kupperman ◽  
Edmund Lee ◽  
Yueying Cao ◽  
Bret Bannerman ◽  
Michael Fitzgerald ◽  
...  
Keyword(s):  
Overall Survival ◽  
Antitumor Activity ◽  
Proteasome Inhibitor ◽  
Cell Lymphoma ◽  
Proteasome Inhibition ◽  
Tumor Burden ◽  
Luciferase Reporter ◽  
Employment Equity ◽  
Xenograft Models ◽  
Equity Ownership

Abstract Abstract 2709 Poster Board II-685 Introduction: The ubiquitin-proteasome system processes the majority of cellular proteins and is the principal manner by which cells regulate protein homeostasis. The successful development of bortezomib for multiple myeloma and previously treated mantle cell lymphoma has validated the proteasome as a therapeutic target for hematological malignancies. MLN9708 was identified in screens for a proteasome inhibitor with greater antitumor activity than bortezomib in preclinical xenograft models. MLN9708 immediately hydrolyzes to MLN2238, the biologically active form, upon exposure to aqueous solutions or plasma. MLN2238 was used for all preclinical studies described below. Results: MLN2238 inhibited the 20S proteasome b5 site (IC50 of 3.4 nM), an engineered proteasome substrate (4x ubiquitin-luciferase reporter) and blocked TNFa-induced activation of the NFkB pathway. Cell viability studies confirmed that MLN2238 has potent activity against both myeloma and lymphoma cell lines. The proteasome dissociation half life of MLN2238 was determined to be approximately 6-fold faster than bortezomib, consistent with data generated from Proteasome-Glo wash out experiments where proteasome activity recovered more quickly in MLN2238-treated cells compared to bortezomib. In immunocompromised mice MLN2238 achieved exposures that resulted in significant blood and tumor proteasome inhibition and had increased plasma and tumor exposure compared to bortezomib (when dosed at MTD). In Sprague-Dawley rats, MLN2238 had improved plasma exposure, lower plasma clearance, higher blood Vdss, better oral F% and higher plasma protein binding than bortezomib. In WSU-DLCL2 xenografts, a model of diffuse large B cell lymphoma, greater antitumor activity was seen in mice treated with IV or SC doses of MLN2238 compared to bortezomib. For example, MLN2238 dosed SC QD at its MTD resulted in a T/C of 0.29 compared to bortezomib dosed SC QD at its MTD which resulted in a T/C of 0.79. Pharmacodynamic (PD) responses (in mice) were assessed by evaluating tumor 20S b5 site-specific activity and expression levels of GADD34, an unfolded protein response (UPR) pathway gene shown to be upregulated in response to proteasome inhibition. Consistent with the efficacy difference between MLN2238 and bortezomib in WSU-DLCL2 xenografts, MLN2238 demonstrated an improved PD response compared to bortezomib, showing higher levels of tumor proteasome inhibition and pathway marker elevation. MLN2238 and bortezomib were then evaluated for their ability to reduce tumor burden and improve overall survival in a disseminated model of lymphoma. Tumor burden was tracked over time via bioluminescent scans in NOD-SCID mice inoculated with OCI-Ly7-luciferase cells. The strongest antitumor response was seen in mice treated with MLN2238 SC QD at its MTD, and this dosing regimen also significantly prolonged overall survival compared to vehicle treated controls (median survival was 54 vs. 33 days). Weaker antitumor responses were seen following treatment with bortezomib (either at its SC QD MTD or weekly IV MTD) and these dose regimens did not significantly prolong survival in this study. Conclusions: MLN2238 is a potent, reversible and orally bioavailable proteasome inhibitor with improved pharmacokinetics, pharmacodynamics and antitumor activity in preclinical xenograft models compared to bortezomib. MLN9708 is currently in clinical development for a variety of oncology indications. Disclosures: Kupperman: Milllennium: Employment. Lee:Milllennium: Employment, Equity Ownership. Cao:Milllennium: Employment, Equity Ownership. Bannerman:Milllennium: Employment. Fitzgerald:Millennium Pharmaceuticals: Employment. Berger:Millennium Pharmaceuticals: Employment. Yu:Millennium Pharmaceuticals: Employment. Zhang:Millennium Pharmaceuticals: Employment. Hales:Millennium Pharmaceuticals: Employment. Bruzzese:Millennium Pharmaceuticals: Employment. Liu:Millennium Pharmaceuticals: Employment. Blank:Millennium Pharmaceuticals: Employment. Garcia:Millennium Pharmaceuticals: Employment. Tsu:Millennium Pharmaceuticals: Employment. Dick:Millennium Pharmaceuticals: Employment. Fleming:Millennium Pharmaceuticals: Employment. Yu:Millennium Pharmaceuticals: Employment. Manfredi:Millennium Pharmaceuticals: Employment. Rolfe:Millennium Pharmaceuticals: Employment. Bolen:Millennium Pharmaceuticals: Employment.

Preclinical Combination Of The Oral Investigational Agents ACY-1215, a Selective HDAC6 Inhibitor, and Ixazomib, a Proteasome Inhibitor, Demonstrates Combination Benefit In Multiple Myeloma Cell Lines and Xenograft Models

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4437-4437
Author(s):  
Allison J Berger ◽  
Bret Bannerman ◽  
Steven N Quayle ◽  
Jie Yu ◽  
Khristofer Garcia ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Pharmaceutical Company ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Hdac Inhibitors ◽  
Proteasome Inhibitors ◽  
Employment Equity ◽  
Equity Ownership

Introduction The combination of HDAC inhibitors and proteasome inhibitors has demonstrated preclinical benefit in several settings, including multiple myeloma and lymphoma, and is being explored in clinical trials testing various HDAC inhibitors in combination with proteasome inhibitors. ACY-1215 is an investigational, orally available HDAC6-selective inhibitor that has demonstrated preclinical combination benefit with bortezomib in vitro and in vivo (Santos et al, Blood 2012; 119: 2579). These preclinical studies also support the hypothesis that the improved selectivity of ACY-1215 for HDAC6 over class I HDACs (HDAC1,2,3) may provide an improved tolerability profile compared to pan-HDAC inhibitors, while still providing the anti-myeloma effect of other HDACi/proteasome inhibitor combinations. ACY-1215 is currently in a Phase I/II trial in multiple myeloma with bortezomib (VELCADE) and dexamethasone to test this hypothesis (NCT01323751). Ixazomib citrate (MLN9708) is an investigational oral proteasome inhibitor in Phase III clinical trials in multiple myeloma (NCT01850524, NCT01564537). To examine the potential efficacy of the all-oral combination of ixazomib citrate and ACY-1215, we evaluated the combination of these agents in cell lines and xenograft models of multiple myeloma. Results In vitro viability experiments in 2 multiple myeloma cell lines (RPMI-8226 and MM.1S) using a dose matrix format demonstrated a combination benefit of ACY-1215 and ixazomib over a range of concentrations, very similar to the previously reported benefit of ACY-1215 plus bortezomib. Likewise, the combination benefit of the selective HDAC6 inhibitor ACY-1215 with ixazomib was similar to the combination effect observed with the pan-HDAC inhibitor SAHA (vorinostat). Together, these in vitro studies support the hypothesis that the combination of ACY-1215 and ixazomib provides similar levels of benefit as do combinations including other HDACi/proteasome inhibitors. Furthermore, experiments in MM.1S xenograft-bearing mice demonstrated an in vivo combination benefit of ACY-1215 and ixazomib. An all-oral regimen was well tolerated when ACY-1215 was dosed at 100 mg/kg PO twice daily for 5 days per week in combination with ixazomib dosed at 5 mg/kg PO twice weekly, and the combination regimen demonstrated additive antitumor activity (Figure 1). The in vivo combination benefit of ACY-1215 and ixazomib was further demonstrated in MM.1S xenograft-bearing mice using alternate routes of administration (IV dosing of ixazomib and IP dosing of ACY-1215). The combination of ACY-1215 dosed at 30 mg/kg IP once daily for 5 days per week with ixazomib dosed IV at 1.5 mg/kg twice-weekly was also well tolerated and had striking antitumor activity. This combination regimen in fact caused regression of the MM.1S xenograft tumors below the starting volumes, and this level of activity was maintained throughout the entire 17 day dosing period (Figure 2). In an accompanying pharmacodynamic (PD) study of the PO and IP doses of ACY-1215, we confirmed selective HDAC6 inhibition in MM.1S xenograft tumors as evidenced by elevated acetylation levels of the HDAC6 substrate tubulin, with little if any change in the levels of acetylated histone H3, a class I HDAC substrate. In vivo experiments in a second xenograft model, RPMI-8226, also demonstrated a combination benefit of ACY-1215 (30 mg/kg IP for 5 days per week) with ixazomib (0.75 mg/kg IV twice-weekly). Conclusion The combination benefit of ACY-1215 and ixazomib observed here in preclinical experiments utilizing in vitro and in vivo models of multiple myeloma provides rationale for clinical evaluation of this first all-oral combination of a proteasome inhibitor with an HDAC inhibitor. Disclosures: Berger: Takeda Pharmaceutical Company Ltd: Employment. Bannerman:Takeda Pharmaceutical Company Ltd: Employment. Quayle:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Yu:Takeda Pharmaceutical Company Ltd: Employment. Garcia:Takeda Pharmaceutical Company Ltd: Employment. Ciavarri:Takeda Pharmaceutical Company Ltd: Employment. Tamang:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Yang:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Jones:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership.

scholarly journals Proteasome Inhibition Measurements: Clinical Application

2000 ◽  
Vol 46 (5) ◽  
pp. 673-683 ◽  
Author(s):  
Eric S Lightcap ◽  
Teresa A McCormack ◽  
Christine S Pien ◽  
Vincent Chau ◽  
Julian Adams ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Phase I ◽  
Whole Blood ◽  
Catalytic Mechanism ◽  
Proteasome Inhibitors ◽  
Proteasome Inhibition ◽  
Ratio Method ◽  
Phase I Clinical Trials ◽  
Model Studies ◽  
Multiple Phase

Abstract Background: PS-341, a selective inhibitor of the proteasome, currently is under evaluation as an anticancer agent in multiple phase I clinical trials. In animal-model studies, PS-341 was rapidly removed from the vascular compartment and distributed widely, quickly approaching the limits of detection. An accurate pharmacodynamic assay has been developed as an alternative or complement to pharmacokinetic measurements. Methods: Fluorogenic kinetic assays for both the chymotryptic and tryptic activities of the proteasome have been optimized for both whole blood and blood cells. Using the ratio of these activities and the catalytic mechanism of the proteasome, we developed a novel method of calculating percentage of inhibition, using two structurally unrelated inhibitors (PS-341 and lactacystin). Results: This ratio method was demonstrated to be sensitive (detection limit of 13% inhibition with 10 μg of cell lysate), specific to the proteasome (PS-341 provides &gt;98% inhibition), accurate (112% analyte recovery), and precise (0% ± 5% inhibition at 0 nmol/L PS-341 and 74.5% ± 1.7% inhibition at 200 nmol/L PS-341). Using these assays, we found that both erythrocytes and leukocytes contain proteasome at 3 μmol/L. Pharmacodynamic results for PS-341 obtained from the whole-blood ratio method were comparable to those using leukocytes determined by another method. Conclusions: The described assay provides a reliable method for studying the pharmacodynamics of proteasome inhibitors and is now in use in concurrent phase I clinical trials with PS-341.

scholarly journals Rapid induction of p62 and GABARAPL1 upon proteasome inhibition promotes survival before autophagy activation

2018 ◽  
Vol 217 (5) ◽  
pp. 1757-1776 ◽  
Author(s):  
Zhe Sha ◽  
Helena M. Schnell ◽  
Kerstin Ruoff ◽  
Alfred Goldberg
Keyword(s):  
Proteasome Inhibitor ◽  
Neuroblastoma Cells ◽  
Proteasome Inhibitors ◽  
Proteasome Inhibition ◽  
Nuclear Factor ◽  
Ubiquitinated Proteins ◽  
Rapid Induction ◽  
Independent Mechanism ◽  
Transcription Factor Nuclear Factor ◽  
Inhibitor Treatment

Proteasome inhibitors are used as research tools and to treat multiple myeloma, and proteasome activity is diminished in several neurodegenerative diseases. We therefore studied how cells compensate for proteasome inhibition. In 4 h, proteasome inhibitor treatment caused dramatic and selective induction of GABARAPL1 (but not other autophagy genes) and p62, which binds ubiquitinated proteins and GABARAPL1 on autophagosomes. Knockdown of p62 or GABARAPL1 reduced cell survival upon proteasome inhibition. p62 induction requires the transcription factor nuclear factor (erythroid-derived 2)-like 1 (Nrf1), which simultaneously induces proteasome genes. After 20-h exposure to proteasome inhibitors, cells activated autophagy and expression of most autophagy genes by an Nrf1-independent mechanism. Although p62 facilitates the association of ubiquitinated proteins with autophagosomes, its knockdown in neuroblastoma cells blocked the buildup of ubiquitin conjugates in perinuclear aggresomes and of sumoylated proteins in nuclear inclusions but did not reduce the degradation of ubiquitinated proteins. Thus, upon proteasome inhibition, cells rapidly induce p62 expression, which enhances survival primarily by sequestering ubiquitinated proteins in inclusions.

scholarly journals Mechanistic Insights into the Enhancement of Adeno-Associated Virus Transduction by Proteasome Inhibitors

2013 ◽  
Vol 87 (23) ◽  
pp. 13035-13041 ◽  
Author(s):  
Angela M. Mitchell ◽  
R. Jude Samulski
Keyword(s):  
Cysteine Protease ◽  
Proteasome Inhibitor ◽  
Proteasome Inhibitors ◽  
Proteasome Inhibition ◽  
Proteasome Activity ◽  
Protease Inhibition ◽  
Adeno Associated Virus

Proteasome inhibitors (e.g., bortezomib, MG132) are known to enhance adeno-associated virus (AAV) transduction; however, whether this results from pleotropic proteasome inhibition or off-target serine and/or cysteine protease inhibition remains unresolved. Here, we examined recombinant AAV (rAAV) effects of a new proteasome inhibitor, carfilzomib, which specifically inhibits chymotrypsin-like proteasome activity and no other proteases. We determined that proteasome inhibitors act on rAAV through proteasome inhibition and not serine or cysteine protease inhibition, likely through positive changes late in transduction.

Neurodegeneration Induced by Bortezomib Exposure in Vitro Occurs Via Proteasome Independent Mechanisms.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2859-2859
Author(s):  
Shirin Arastu-Kapur ◽  
Andrew J. Ball ◽  
Janet L. Anderl ◽  
Mark K Bennett ◽  
Christopher J Kirk
Keyword(s):  
Peripheral Nerve ◽  
Proteasome Inhibitor ◽  
Serine Proteases ◽  
Proteasome Inhibitors ◽  
Proteasome Inhibition ◽  
Nerve Degeneration ◽  
Differentiated Cells ◽  
Favorable Safety ◽  
Neurite Degeneration

Abstract Abstract 2859 Poster Board II-835 BACKGROUND: The dipeptide boronate proteasome inhibitor bortezomib (BTZ; Velcade®) is approved for the treatment of multiple myeloma and non-Hodgkin's lymphoma. Bortezomib-induced peripheral neuropathy (BIPN, Blood (2008)112:1593-1599) is seen in ∼30% of BTZ-treated patients and can result in dose reductions and discontinuations that may result in suboptimal levels of proteasome inhibition. Carfilzomib (CFZ), a tetrapeptide epoxyketone, is a selective and irreversible proteasome inhibitor that is structurally and mechanistically distinct from bortezomib. Single agent treatment with CFZ has demonstrated strong activity in relapsed and refractory myeloma and a favorable safety profile in Phase 2 trials (ASH2008:864 & 865). Importantly treatment-emergent PN was seen at low levels and did not result in dose modifications or discontinuations. The disparate safety data for these proteasome inhibitors suggest that non-proteasomal mechanisms may underlie BIPN. Using activity-based probes in peripheral blood mononuclear cell (PBMC) lysates, we previously demonstrated inhibition of non-proteasomal proteases by BTZ and other proteasome inhibitors with a boronate pharmacophore (EHA2009:0939). However, the involvement of the proteasome in the peripheral nerve degeneration and BIPN in BTZ-treated myeloma patients remains to be established. AIMS: To establish an in vitro model of peripheral nerve degeneration and to determine the effects of proteasome inhibition by BTZ and CFZ on neurite outgrowth and cell survival. METHODS: SH-SY5Y neuroblastoma cells were differentiated by long term culture in retinoic acid and brain derived nerve growth factor to induce neurite outgrowth. The effects of proteasome inhibitors were measured by high content image analysis of fluorescent images for cell survival (Hoechst nuclear counterstain) and neurite degeneration (FITC-mouse anti-beta-III-tubulin). Phase contrast images were also collected to observe morphological effects and gross cell death. Cell viability and proteasome inhibition was measured in undifferentiated and differentiated cells. The MEROPS (peptidase) database was mined for candidate serine proteases with a P1 selectivity of Leu/Phe/Tyr to identify candidate off-targets CFZ and BTZ and candidate proteases were validated by standard biochemical and cell biology techniques. RESULTS: In differentiated SH-SY5Y cells, the average neurite length decreased by 33% following 24 hr exposure to 10nM BTZ but was unaffected by the same concentration of CFZ. Proteasome inhibition as determined by a fluorescent substrate for the chymotrypsin-like activity was equivalent (∼70%) after a 24 hr exposure for both compounds in differentiated cells, suggesting that neurodegeneration involves non-proteasomal pathways. With 72 hrs continuous exposure, BTZ was 10-fold more potent than CFZ at inducing neurodegeneration. Furthermore, in both undifferentiated and differentiated SH-SY5Y cells, BTZ was 5-fold more cytotoxic than CFZ. Database mining for serine proteases with a selectivity for Leu/Phe/Tyr at P1 was used to identify other potential BTZ targets that might underlie neurotoxicity. One candidate is HtrA2 (also called Omi), an inducible mitochondrial serine protease whose activity protects neurons from stress induced apoptosis (Hum Mol Genet (2005) 14(5):2099-2111). HtrA2 levels increased 2-fold in SH-SY5Y cells treated with either BTZ or CFZ for 6 hrs at 40 nM. Using a gel based assay and purified enzyme preparations, BTZ inhibited HtrA2 activity with an IC50 ∼ 4 nM, equivalent to its activity against the proteasome. In contrast, Carfilzomib did not inhibit HtrA2 at the highest concentration tested (10 mM). CONCLUSIONS: These data demonstrate that BTZ induces neuronal cell death and neurite degeneration in vitro by proteasome-independent mechanisms. We propose that combined inhibition of the proteasome and HtrA2 by BTZ may underlie peripheral nerve toxicities in vitro and may be involved in BIPN in myeloma patients. In this model, CFZ, which mediates equivalent proteasome inhibition to BTZ in neurons, does not induce neurodegeneration due to inactivity against HtrA2. Future profiling of non-proteasomal targets of BTZ, including HtrA2 activity, in patient samples is merited. These results suggest that the favorable safety profile of CFZ in myeloma patients may be a result of its high selectivity for proteasomal proteases. Disclosures: Arastu-Kapur: Proteolix, Inc: Employment. Ball:Millipore Corp: Employment. Anderl:Millipore Corp: Employment. Bennett:Proteolix: Employment. Kirk:Proteolix, Inc: Employment.

Specimen Source (BM or PB) Does Not Affect Proteomic Signaling In Patients with AML and Circulating Blasts

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2693-2693
Author(s):  
Alessandra Cesano ◽  
David B. Rosen ◽  
Santosh Putta ◽  
Urte Gayko ◽  
Larry Cripe ◽  
...  
Keyword(s):  
Linear Regression ◽  
Single Cell ◽  
Intracellular Signaling ◽  
Goodness Of Fit ◽  
Kinase Inhibitors ◽  
Mononuclear Cells ◽  
Linear Regression Analysis ◽  
Employment Equity ◽  
Weak Response ◽  
Equity Ownership

Abstract Abstract 2693 Background: Single Cell Network Profiling (SCNP) is used to measure simultaneously the effects of multiple modulators (including drugs) on signaling cascades at the single cell level. Using this technology, ECOG in collaboration with Nodality is developing several novel biomarker assays with the aim to find blast functional signaling profiles predictive of response to induction therapy and risk of relapse in AML patients. To date, such assays utilized patient bone marrow (BM) as the sample source of blasts. However, in about 65% of patients with AML, circulating peripheral blasts are detected and peripheral blood (PB) sampling is easier and less invasive for patients than BM sampling. Objectives: The objective of this study was to compare by SCNP the functional effects of a panel of compounds simultaneously on different signaling pathways (such as the phosphoinositide 3-kinase (P13K )and the Janus Kinases (Jak) signal transducers and activators of transcription (Stat) pathway) relevant both to the biology of the disease and the development of new therapeutics, in paired, diagnostic, cryopreserved PB mononuclear cells (PBMC) and BMMC samples from 44 AML patients. A paired sample was defined as a BMMC and PBMC specimen collected from the same patient on the same day. Methods: Modulated SCNP using a multiparametric flow cytometry platform was used to evaluate the activation state of intracellular signaling molecules in leukemic blasts under basal conditions and after treatment with specific modulators (Table 1). The SCNP phosphoflow assay was performed on 88 BMMC/PBMC pairs from ECOG trial, E3999. The relationship between readouts of modulated intracellular proteins (“nodes”) between BMMC and PBMC was assessed using linear regression, Bland-Altman method or Lin's concordance correlation coefficient. Results: Table 1 shows the goodness of fit (R2) values from the linear regression analysis for both the basal levels and the modulated levels of intracellular signaling proteins. Most of the signaling nodes show strong correlations (R2 >0.64) with several of the exceptions belonging to nodes with weak response to modulation (e.g. SCF -> p-Akt) or antibodies with dim fluorphores (i.e. Alexa 647). The lack of response is however, consistent between the tissue types for the weak response nodes. Using a rank based metric that is less sensitive to the absolute intensity levels seem to perform better for the antibodies with dim fluorophores. Results from other methods; Bland Altman and Lin's Concordance also show good concordance between the tissue types. Conclusions: The data presented here demonstrate: 1) Specimen source (BM or PB) does not affect proteomic signaling in patients with AML and circulating blasts. 2) PB myeloblasts can be used as a sample source for Nodality SCNP assays to identify functionally distinct leukemic blats cell populations with distinct sensitivities to therapy. 3) The ability to use PB as a sample source will greatly improve the utility of these assays. In particular, our results will facilitate the monitoring of cellular signaling effects following the administration of targeted therapies, e.g., kinase inhibitors, at time-points when BM aspirates are not clinically justifiable. Disclosures: Cesano: Nodality Inc.: Employment, Equity Ownership. Rosen:Nodality Inc.: Employment, Equity Ownership. Putta:Nodality Inc.: Employment, Equity Ownership. Gayko:Nodality Inc.: Employment, Equity Ownership.

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