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

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.

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 Vd_ss, 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.

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.

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