Project Summary Over 2,200 Americans die annually of acute alcohol poisoning, and an additional tens of thousands die due to the direct results of binge drinking. Billions are spent annually treating alcohol poisoning cases. However, no FDA-approved pharmacological treatment for alcohol poisoning exists, nor are any means of blocking alcohol’s effects known. The long-term goal of this work is to develop a pharmaceutical that rapidly clears ethanol from the blood and functions as a therapeutic for alcohol poisoning and as a harm reduction tool following binge drinking. The goal of this project is to validate a lead pharmaceutical formulation comprising three enzymes encapsulated in liposomes for the ability to rapidly eliminate ethanol in vivo. The central hypothesis is that a high rate of ethanol degradation can be maintained in vivo by intravenous administration of this formulation, thereby ameliorating alcohol poisoning. The rationale underlying this proposal is that encapsulated enzyme systems are more active than free enzymes in blood serum. Encapsulation of the enzymes in liposomes physically separates them from serum proteases, the immune system, and off-target ligands. It also co-localizes the enzymes within the liposome, greatly improving enzyme pathway kinetics. The innovations that will enable this work are recently gained insights into enzyme biochemistry and co-localization as well as methodologies that gently encapsulate protein at high yields. Specific aims of this project are: Aim 1: Demonstrate that the encapsulated enzyme system eliminates ethanol in vivo. Aim 2: Use screening and directed evolution techniques to discover improved enzymes. Aim 3: Prototype a microfluidic system for high-efficiency protein encapsulation. The expected outcome of this project is a scalable liposomal formulation that can rapidly eliminate alcohol from the blood. The data from this work will also support raising of private capital for IND-enabling preclinical studies. The positive impact of the alcohol elimination treatment is twofold: First and foremost, it would make available an efficacious first-line treatment for acute alcohol poisoning. Secondly, the availability of such a treatment at ERs, urgent care centers, and mobile units would enable harm reduction strategies targeting binge drinking. The system described here will also serve as a prototype for a wider range of enzymes that target other drugs (e.g. fentanyl) or high concentrations of endogenous metabolites (e.g. phenylalanine).