Hydroxylation near the epoxyketone of carfilzomib confers protection from microsomal epoxide hydrolase-mediated metabolism.

Carfilzomib (CFZ) is a tetrapeptide epoxyketone-based proteasome inhibitor (PI) drug approved for multiple myeloma therapy. Despite its improved efficacy and safety profiles over bortezomib (the first-in-class PI drug), CFZ has a short half-life in vivo (< 1 hour), possibly contributing to the lack of efficacy against solid cancers. Previous studies indicated that microsomal epoxide hydrolase (mEH) plays a predominant role in the metabolic degradation of CFZ. With that in mind, we synthesized a novel hydroxylated analog of CFZ (dubbed "CFZ-OH"), which was predicted to have a lower affinity to mEH than CFZ. Here, we assessed the metabolic stability of CFZ-OH under varying conditions in vitro: HEK293 cells expressing human mEH, rat liver homogenates, rat or human liver microsomes, and rat or human primary hepatocytes. In vitro, CFZ-OH showed protection from mEH-mediated metabolism and improved metabolic stability over CFZ. In rats receiving CFZ-OH or CFZ (4 mg/kg, intravenously), CFZ-OH exhibited 2.6-fold higher systemic exposure, consistent with the protection of CFZ-OH from mEH-mediated metabolism. However, CFZ-OH and CFZ displayed comparable terminal half-lives, suggesting that CFZ-OH may be subjected to metabolic degradation in vivo by enzymes other than mEH. CFZ-OH degradation was faster than CFZ in rat blood and lung homogenates but was partially inhibited by bortezomib (a PI) or N-ethylmaleimide (a broad-spectrum cysteine protease inhibitor). Together, our results indicate the need to assess the stability of epoxyketone-based PI drugs through multiple metabolic components, including cysteine proteases, and their relative contribution in developing next-generation PI drugs with prolonged circulation in vivo. SIGNIFICANCE STATEMENT: The hydroxylated analog of Carfilzomib (CFZ-OH) displayed enhanced proteasome target binding and greater in vitro metabolic stability against microsomal epoxide hydrolase, previously considered a main contributor to the in vivo instability of CFZ. In vivo, CFZ-OH exhibited a higher systemic exposure than CFZ, but their terminal half-lives were comparable. These findings suggest that enhancing the in vivo circulation of CFZ requires structural modifications that confer protection against microsomal epoxide hydrolase and other enzymes, such as cysteine proteases.