ALBERT

Description: The proteasome is a multi-subunit structure responsible for degrading the majority of proteins in the cell. The three catalytic subunits in the 20S core of the proteasome are defined by their preference for amino acid side chains at the P1 position: the beta5 subunit (chymotrypsin-like), the beta2 subunit (trypsin-like), and the beta1 subunit (caspase-like) hydrolyze substrates after hydrophobic, basic, and acidic residues, respectively. Bortezomib (Velcade®), currently an approved drug for the treatment of multiple myeloma, is a potent inhibitor of the beta5 subunit but also has activity against the beta1 subunit (9-fold less active) and the beta2 subunit (250-fold less active). PR-171, a peptide epoxy-ketone proteasome inhibitor currently in Phase I clinical trials in multiple myeloma and NHL patients, has a distinct profile of inhibition. Similar to bortezomib, PR-171 is most potent against the beta5 subunit, but differs in its activity against the beta2 and beta1 subunits (45-fold less active and 140-fold less active, respectively). Although both bortezomib and PR-171 are cytotoxic to a variety of tumor cell types, the connection between inhibition of specific proteasomal subunits and cellular consequences, including induction of apoptosis, is currently unclear. To investigate this relationship, we synthesized a series of PR-171 analogs and profiled them in our proteasome-specific active site ELISA binding assay to identify compounds with differential binding profiles for the three catalytic subunits. Using this approach, we have identified compounds that bind all three catalytic sites with approximately equivalent activity as well as compounds that have increased specificity for the beta5 subunit. We have extended this approach to search for compounds with differential binding profiles for the immunoproteasome catalytic subunits. The immunoproteasome is the predominant form of the proteasome expressed in cells of hematopoetic origin and in other cell types after interferon-g stimulation. In the 20S core of the immunoproteasome the beta5, beta2 and beta1 subunits of the “constitutive” proteasome are replaced by the homologous subunits LMP7, MECL1, and LMP2. We have successfully identified inhibitors with distinct active site profiles for the immunoproteasome as well as the constitutive proteasome. The impact of differential active site inhibition on tumor cell viability is under investigation in a variety of solid and hematological tumor cell lines expressing the constitutive proteasome or the immunoproteasome.

Description: The clinical development of the dipeptide boronate bortezomib (Velcade™) has validated proteasome inhibition as a treatment for multiple myeloma and non-Hodgkin’s lymphoma. In hematopoetically derived normal and malignant cells, the form of the proteasome expressed, termed the immunoproteasome, differs from that found in most tissues, referred to as the constitutive proteasome. Bortezomib inhibits the chymotrypsin-like activity of the immunoproteasome and the constitutive proteasome with equal potency and induces side effects such as peripheral neuropathy and gastrointestinal toxicity that may be related to proteasome inhibition in cells expressing the constitutive proteasome. This finding suggests that development of immunoproteasome-specific inhibitors would selectively target hematological malignances without affecting non-hematological cells. We have undertaken an effort to profile hematological tumor cell lines for immunoproteasome expression to determine which hematological cancers could be effectively targeted using an immunoproteasome selective inhibitor. We have observed that several cell lines derived from hematologic malignancies express predominantly the immunoproteasome while others express a mixture of both proteasome types. To develop selective inhibitors of the immunoproteasome, we generated analogs of PR-171, a peptide keto-epoxide that is currently being investigated in clinical trials for the treatment of myeloma and lymphoma. Purified 20S preparations were used to screen for compounds that selectively inhibit the chymotrypsin-like activity of the immunoproteasome (subunit LMP7) over the constitutive proteasome (subunit beta5). While the parental molecule PR-171 targets both forms of the proteasome with similar potency, several analogs have been identified with an IC50 below 50 nM on LMP7 and greater than 10-fold selectivity compared to beta5. To evaluate the immunoproteasome selectivity of our compounds in cells, an active site-specific ELISA was utilized. Cells expressing both constitutive and immunoproteasome were treated with the analogs and the occupancy of the LMP7 and beta5 active sites was determined. Several compounds retained their immunoproteasome selectivity in cells with an IC50 below 300 nM for LMP7 inhibition. In cell viability studies, some of these compounds demonstrated enhanced cytotoxicity in hematological tumor cell lines expressing the immunoproteasome as compared to solid tumor cell lines which express the constitutive proteasome. Finally, when pharmacodynamics was evaluated following intravenous administration to mice, LMP7 selectivity was retained in tissues expressing both the immunoproteasome and constitutive proteasome. The in vivo anti-tumor properties and toxicity profiles of the immunoproteasome-selective inhibitors are currently being evaluated.

Description: Clinical application of proteasome inhibitors in the treatment of hematologic malignancies such as myeloma and lymphoma is restricted in part by the necessity of frequent IV administration and would be improved by oral (PO) administration. Selective inhibitors of the protease subunits of the 20S proteasome can be generated from peptidyl aldehydes, boronates, vinyl sulfones, and epoxyketones. Many of these peptide based proteasome inhibitors are cell permeant and capable of systemic proteasome inhibition upon intravenous (IV) administration to experimental animals such as mice and rats. In the cases of the peptidyl boronate bortezomib (Velcade™) and the epoxyketone PR-171, proteasome inhibition can be achieved in patients with IV administration. However, systemic exposure following PO administration of these inhibitors may be limited by several factors including gastric pH, gastric and intestinal peptidases, efflux pumps, biliary excretion and intestinal and hepatic metabolic activities. We have tested over 80 peptide epoxyketones with potent (IC50 80%) proteasome inhibition in most tissues. The anti-tumor efficacy of these orally bioavailable proteasome inhibitors are being assessed in both human tumor xenograft and mouse syngeneic models. The results from these studies will enable further pre-clinical development of potent, orally bioavailable proteasome inhibitors for the treatment of malignant diseases.

Description: Clinical studies using the boronic acid-based proteasome inhibitor bortezomib (VelcadeTM) have validated the proteasome as a therapeutic intervention point for the treatment of multiple myeloma and non-Hodgkin’s Lymphoma. Despite encouraging clinical response rates with this drug, significant toxicities, including neutropenia, thrombocytopenia and peripheral neuropathy have restricted the intensity of bortezomib dosing. PR-171 is a novel peptide epoxyketone inhibitor that selectively and irreversibly inhibits the chymotryptic subunit of the 20S proteasome. We have characterized the pharmacokinetics, pharmacodynamics and anti-tumor efficacy of PR-171 in rodents and non-human primates. PR-171 is rapidly cleared from the plasma compartment following intravenous bolus administration, with a terminal half-life in rats and monkeys of 15 and 7.2 min, respectively. Despite this rapid clearance, PR-171 administration results in a prolonged dose-dependent inhibition of the 20S proteasome in all tissues examined with the exception of brain. Single doses of PR-171 that are tolerated in mice, rats and monkeys result in greater than 90% inhibition of proteasome activity in blood and many tissues. Recovery of proteasome activity following exposure to PR-171 is dependent upon synthesis of new proteasome subunits and occurs with a t1/2 of ~ 24 hr in nucleated cells. Recovery of proteasome activity in erythrocytes is dependent upon red blood cell turnover and occurs more slowly in animals. The safety of PR-171 has been assessed in rodents using multiple dosing schedules including weekly, biweekly, daily and BID administration. In rats, daily administration of PR-171 at doses that resulted in 〉80% inhibition of proteasome activity in whole blood and selected tissues were well tolerated. Recovery of proteasome activity following repeated daily administrations was unchanged from that seen after a single dose. PR-171 was also well tolerated when administered daily to monkeys at equivalent doses on a body surface area comparison. A transient thrombocytopenia was noted in both rats and monkeys but neutrophil and lymphocyte counts were not decreased in response to PR-171 administration. PR-171 induced a significant anti-tumor response in beige-nu-xid (BNX) triple immunodeficient mice bearing established HS-Sultan Burkitt’s lymphoma tumors and in Balb/c mice challenged with A20 lymphoma cells. We have also demonstrated anti-tumor activity of PR-171 in several mouse models of solid tumors including syngeneic and human tumor xenograft models of colorectal cancer. These studies demonstrate the tolerability, anti-tumor activity and dosing flexibility of PR-171 and provide validation for the clinical testing of PR-171 in the treatment of hematologic malignancies utilizing dose intensive schedules.

Description: INTRODUCTION: PR-171 is a novel, irreversible proteasome inhibitor under investigation for the treatment of hematological malignancies. In preclinical studies, PR-171 was well tolerated in rats and monkeys when given daily × 5 (QDx5), every two weeks. Proteasome inhibition determined in blood and other tissues one hour after dosing at the maximum tolerated dose (MTD) was 〉80%. Despite the irreversible mechanism of action, the half-life of recovery of proteasome inhibition in tissues was approximately 24 hours. PR-171 was effective in suppression of tumor growth in xenograft studies in mice when administered QDx2 every week for three weeks. Two phase I dose-escalation studies have been initiated, aimed at determining the safety, tolerability, and clinical response to PR-171. METHODS: Two different dose-intensive schedules were employed in these phase I studies. In PX-171-001, PR-171 was administered on a two week cycle, QDx5 with nine days rest, while in PX-171-002, PR-171 was administered on a four week cycle, QDx2 weekly for three weeks with 12 days rest. The objective of these studies was to evaluate the safety and effectiveness of single-agent PR-171 administered according to a modified Fibonacci dose escalation scheme in cohorts of three. Patients with multiple myeloma (MM), non-Hodgkin Lymphoma (NHL), Hodgkin disease, or Waldenstrom’s Macroglobulinemia who received two or more prior treatments were eligible. RESULTS: Both studies were initiated with a dose of 1.2 mg/m2. Thus far, a total of 15 and 23 subjects have been enrolled in PX-171–001 and -002, respectively. PR-171 has been well tolerated at the highest doses thus far, 8.4 and 15 mg/m2, respectively. Proteasome inhibition in whole blood at the highest dose levels exceeded 75%, and in peripheral blood mononuclear cells inhibition exceeded 65%, one hour after the first dose. There have been no dose-limiting toxicities, no adverse events (AEs) considered probably related to study medication, no dose-related increases in the incidence or severity of AEs, and no incidence of painful peripheral neuropathy on either study. Although the MTD has not yet been identified on either study, preliminary evidence of efficacy has been observed with reduction in myeloma paraprotein levels and symptomatic improvement in patients on both protocols. Eleven subjects remain on study with stable disease (SD), which has been observed in MM and NHL patients on both studies, lasting up to eight months. CONCLUSION: PR-171 is well-tolerated in relapsed and refractory MM and NHL patients at proteasome inhibition levels of more than 75%. Several subjects have achieved long-lasting SD, reduction in paraprotein levels or symptomatic improvement. Further data from these ongoing trials will be discussed at the meeting.

Description: 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.