Oprozomib is an oral proteasome inhibitor currently under investigation in patients with hematologic malignancies or solid tumors. Oprozomib elicits potent pharmacological actions by forming a covalent bond with the active site N-terminal threonine of the 20S proteasome. Oprozomib has a short half-life across preclinical species and in patients due to systemic clearance via metabolism. Potential for drug-drug interactions (DDIs) could alter the exposure of this potent therapeutic; therefore, a thorough investigation of pathways responsible for metabolism is required. In the present study, the major drug-metabolizing enzyme responsible for oprozomib metabolism was identified in vitro. A diol of oprozomib was found to be the predominant metabolite in human hepatocytes, which formed via direct epoxide hydrolysis. Using recombinant epoxide hydrolases (EHs) and selective EH inhibitors in liver microsomes, microsomal EH (mEH) but not soluble EH (sEH) was found to be responsible for oprozomib diol formation. Coincubation with 2-nonylsulfanyl-propionamide, a selective mEH inhibitor, resulted in a significant decrease in oprozomib disappearance (>80%) with concurrent complete blockage of diol formation in human hepatocytes. On the contrary, a selective sEH inhibitor did not affect oprozomib metabolism. Pretreatment of hepatocytes with the pan-cytochrome P450 (P450) inhibitor 1-aminobenzotriazole resulted in a modest reduction (∼20%) of oprozomib metabolism. These findings indicated that mEH plays a predominant role in oprozomib metabolism. Further studies may be warranted to determine whether drugs that are mEH inhibitors cause clinically significant DDIs with oprozomib. On the other hand, pharmacokinetics of oprozomib is unlikely to be affected by coadministered P450 and sEH inhibitors and/or inducers.

Bispecific T-cell engager (BiTE) molecules are biologic T cell-directing immunotherapies. Blinatumomab is approved for treatment of B-cell malignancies, but BiTE molecule development in solid tumors has been more challenging. Here, we employed intravital imaging to characterize exposure and pharmacodynamic response of an anti-muCD3/anti-huEGFRvIII mouse surrogate BiTE molecule in EGFR variant III (EGFRvIII)-positive breast tumors implanted within immunocompetent mice. Our study revealed heterogeneous temporal and spatial dynamics of BiTE molecule extravasation into solid tumors, highlighting physical barriers to BiTE molecule function. We also discovered that high, homogeneous EGFRvIII expression on cancer cells was necessary for a BiTE molecule to efficiently clear tumors. In addition, we found that resident tumor-infiltrating lymphocytes (TIL) were sufficient for optimal tumor killing only at high BiTE molecule dosage, whereas inclusion of peripheral T-cell recruitment was synergistic at moderate to low dosages. We report that deletion of stimulatory conventional type I DCs (cDC1) diminished BiTE molecule-induced T-cell activation and tumor clearance, suggesting that in situ antigen-presenting cell (APC) engagements modulate the extent of BiTE molecule efficacy. In summary, our work identified multiple requirements for optimal BiTE molecule efficacy in solid tumors, providing insights that could be harnessed for solid cancer immunotherapy development.