Project Summary/Abstract: Alzarrok Bio is developing a peptide, derived from the human defense protein cathepsin G, as a multitarget drug for Alzheimer’s disease (AD). Existing drugs can temporarily mitigate symptoms or marginally delay the progression of the disease in certain patients but absence of safe and effective pharmacological treatment for AD remains a huge unmet clinical need. AD drugs (existing or currently in development) usually engage a single specific target. This appears to be insufficient to significantly inhibit the complex pathogenesis driving the progression of AD. Our solution is a peptide entity (ALZ100) with inherent multitarget activity against 4 mediators of AD, which are driving a feed-forward loop between neurodegeneration and neuroinflammation. ALZ100 inhibits and reverses the formation of Aβ1-42 oligomers and fibrils; binds with high affinity the receptor for advanced glycation end-products (RAGE), the toll-like receptor 4 (TLR4) and the S100 calcium-binding protein A9 (S100A9); inhibits Aβ1-42 -induced neuronal cell death; and suppresses the activation of TLR4 by S100A9. The targets of ALZ100 (Aβ1-42, RAGE, TLR4, and S100A9) are overexpressed in the brain of AD patients and inhibition of each one of them was shown to protect against AD-like phenotype in mouse models. Building on our in vitro characterization of multitarget activity, our specific aim is to demonstrate the activity of ALZ100 in vivo, by showing target engagement and inhibition of neuroinflammation and neurodegeneration in an AD mouse model. We will use the double transgenic mouse model APP/PS1 that recapitulates salient features of the AD pathogenesis, including Aβ deposition, neuroinflammation, neurodegeneration, and upregulation of the 4 targets of ALZ100. We will first infuse wild-type (WT) mouse brain with low, medium, or high concentrations of ALZ100 and measure steady state concentrations of the drug in brain and blood. We will then infuse both APP/PS1 and WT mouse with saline alone, low, medium, or high concentrations of ALZ100 and measure the expression level of each target, the activation of TLR4 and RAGE receptors by measuring transcription of downstream target genes, as well as other markers of neuroinflammation and neurodegeneration. At the conclusion of phase 1, we expect to have demonstrated efficacy of ALZ100 to inhibit neuroinflammation and neurodegeneration in an AD mouse model and determined therapeutic concentration of the drug in the brain. In phase 2, we will formulate ALZ100 for intranasal administration and conduct preclinical efficacy studies in mouse models of AD, using extended intranasal treatment with ALZ100. Completion of this project will advance our long-term goal, which is the development of a new generation of drugs for AD, with game-changing multitarget activity and superior delivery method for safe and effective treatment of patients with AD.