Clinical studies with bortezomib have validated the proteasome as a therapeutic target for the treatment of multiple myeloma and non-Hodgkin's lymphoma. However, significant toxicities have restricted the intensity of bortezomib dosing.

The proteasome has emerged as an important target for cancer therapy with the approval of bortezomib, a first-in-class, reversible proteasome inhibitor, for relapsed/refractory multiple myeloma (MM). However, many patients have disease that does not respond to bortezomib, whereas others develop resistance, suggesting the need for other inhibitors with enhanced activity. We therefore evaluated a novel, irreversible, epoxomicin-related proteasome inhibitor, carfilzomib. In models of MM, this agent potently bound and specifically inhibited the chymotrypsin-like proteasome and immunoproteasome activities, resulting in accumulation of ubiquitinated substrates. Carfilzomib induced a dose-and time-dependent inhibition of proliferation, ultimately leading to apoptosis. Programmed cell death was associated with activation of c-Jun-N-terminal kinase, mitochondrial membrane depolarization, release of cytochrome c, and activation of both intrinsic and extrinsic caspase pathways. This agent also inhibited proliferation and activated apoptosis in patient-derived MM cells and neoplastic cells from patients with other hematologic malignancies. Importantly, carfilzomib showed increased efficacy compared with bortezomib and was active against bortezomib-resistant MM cell lines and samples from patients with clinical bortezomib resistance. Carfilzomib also overcame resistance to other conventional agents and acted synergistically with dexamethasone to enhance cell death. Taken together, these data provide a rationale for the clinical evaluation of carfilzomib in MM.

Carfilzomib is a proteasome inhibitor in clinical development that primarily targets the chymotrypsin-like (CT-L) subunits in both the constitutive proteasome (c20S) and the immunoproteasome (i20S). To investigate the impact of inhibiting the CT-L activity with carfilzomib, we set out to quantitate the levels of CT-L subunits ␤5 from the c20S and LMP7 from the i20S in normal and malignant hematopoietic cells. We found that the i20S is a major form of the proteasome expressed in cells of hematopoietic origin, including multiple myeloma (MM) CD138 ؉ tumor cells. Although specific inhibition of either LMP7 or ␤5 alone was insufficient to produce an antitumor response, inhibition of all proteasome subunits was cytotoxic to both hematologic tumor cells and peripheral blood mononuclear cells. However, selective inhibition of both ␤5 and LMP7 was sufficient to induce an antitumor effect in MM, non-Hodgkin lymphoma, and leukemia cells while minimizing the toxicity toward nontransformed cells. In MM tumor cells, CT-L inhibition alone was sufficient to induce proapoptotic sequelae, including proteasome substrate accumulation, Noxa and caspase 3/7 induction, and phospho-eIF2␣ suppression. These data support a hypothesis that hematologic tumor cells are uniquely sensitive to CT-L inhibition and provide a mechanistic understanding of the clinical safety profile and antitumor activity of proteasome inhibitors. (Blood.

Purpose: Bortezomib (Velcade), a dipeptide boronate 20S proteasome inhibitor and an approved treatment option for multiple myeloma, is associated with a treatment-emergent, painful peripheral neuropathy (PN) in more than 30% of patients. Carfilzomib, a tetrapeptide epoxyketone proteasome inhibitor, currently in clinical investigation in myeloma, is associated with low rates of PN. We sought to determine whether PN represents a target-mediated adverse drug reaction (ADR).Experimental Design: Neurodegenerative effects of proteasome inhibitors were assessed in an in vitro model utilizing a differentiated neuronal cell line. Secondary targets of both inhibitors were identified by a multifaceted approach involving candidate screening, profiling with an activity-based probe, and database mining. Secondary target activity was measured in rats and patients receiving both inhibitors.Results: Despite equivalent levels of proteasome inhibition, only bortezomib reduced neurite length, suggesting a nonproteasomal mechanism. In cell lysates, bortezomib, but not carfilzomib, significantly inhibited the serine proteases cathepsin G (CatG), cathepsin A, chymase, dipeptidyl peptidase II, and HtrA2/Omi at potencies near or equivalent to that for the proteasome. Inhibition of CatG was detected in splenocytes of rats receiving bortezomib and in peripheral blood mononuclear cells derived from bortezomib-treated patients. Levels of HtrA2/Omi, which is known to be involved in neuronal survival, were upregulated in neuronal cells exposed to both proteasome inhibitors but was inhibited only by bortezomib exposure.Conclusion: These data show that bortezomib-induced neurodegeneration in vitro occurs via a proteasome-independent mechanism and that bortezomib inhibits several nonproteasomal targets in vitro and in vivo, which may play a role in its clinical ADR profile. Clin Cancer Res; 17(9); 2734-43. Ó2011 AACR.

Proteasome inhibition has been validated as a therapeutic modality in the treatment of multiple myeloma and non-Hodgkin's lymphoma. Carfilzomib, an epoxyketone currently undergoing clinical trials in malignant diseases, is a highly selective inhibitor of the chymotrypsin-like (CT-L) activity of the proteasome. A chemistry effort was initiated to discover orally bioavailable analogues of carfilzomib, which would have potential for improved dosing flexibility and patient convenience over intravenously administered agents. The lead compound, 2-Me-5-thiazole-Ser(OMe)-Ser(OMe)-Phe-ketoepoxide (58) (PR-047), selectively inhibited CT-L activity of both the constitutive proteasome (beta5) and immunoproteasome (LMP7) and demonstrated an absolute bioavailability of up to 39% in rodents and dogs. It was well tolerated with repeated oral administration at doses resulting in >80% proteasome inhibition in most tissues and elicited an antitumor response equivalent to intravenously administered carfilzomib in multiple human tumor xenograft and mouse syngeneic models. The favorable pharmacologic profile supports its further development for the treatment of malignant diseases.