Pharmacokinetics, Target Engagement of an Immunoproteasome Subunit Low-Molecular-Mass Polypeptide‑2 (LMP2) Inhibitor AR-01, and Its Anti-Alzheimer's Effects in Rodents

Previously, we reported that AR-01, an irreversible macrocyclic peptide epoxyketone, selectively inhibits the immunoproteasome catalytic subunit LMP2 (low-molecular mass polypeptide-2). We also showed that LMP2 inhibition produces anti-Alzheimer's effects in animal models of Alzheimer's disease (AD) by suppressing microglia-mediated inflammation. As such, AR-01 is being developed as a potential treatment for AD. Typically, CNS drugs with peptide backbones face significant challenges due to unfavorable properties for crossing the blood-brain barrier. In this report, we evaluated the pharmacokinetic properties of AR-01, including brain permeability (i.e., brain-to-plasma partition coefficient), in healthy mice and rats as part of early stage drug development. We also verified AR-01 target engagement in the brain using two alternative approaches: (i) LMP2 activity assay and (ii) LMP2 band shift on Western blotting, which is caused by the formation of an irreversible LMP2:AR-01 adduct. The results confirmed that a single intravenous administration of AR-01 inactivates LMP2 in the mouse brain in a dose-dependent manner. Multiple doses of AR-01 led to cumulative LMP2 adduct formation in the mouse brain and improved cognitive function in 5xFAD mice, a widely used model of amyloidogenesis. Taken together, the results suggest that AR-01 has promising pharmaceutical properties as an AD therapeutic.