| Summary: | In ulcerative colitis (UC), the inflammation is localized in the colon, and one of the successful strategies for colon-targeting drug delivery is the prodrug approach. In this work, we present a novel phospholipid (PL)-based prodrug approach, as a tool for colonic drug targeting in UC. We aim to use the phospholipase A<sub>2</sub> (PLA<sub>2</sub>), an enzyme that is overexpressed in the inflamed colonic tissues of UC patients, as the PL-prodrug activating enzyme, to accomplish the liberation of the parent drug from the prodrug complex at the specific diseased tissue(s). Different linker lengths between the PL and the drug moiety can dictate the rate of activation by PLA<sub>2</sub>, and subsequently determine the amount of free drugs at the site of action. The feasibility of this approach was studied with newly synthesized PL-Fmoc (fluorenylmethyloxycarbonyl) conjugates, using Fmoc as a model compound for testing our hypothesis. In vitro incubation with bee venom PLA<sub>2</sub> demonstrated that a 7-carbon linker between the PL and Fmoc has higher activation rate than a 5-carbon linker. 4-fold higher colonic expression of PLA<sub>2</sub> was demonstrated in colonic mucosa of colitis-induced rats when compared to healthy animals, validating our hypothesis of a colitis-targeting prodrug approach. Next, a novel molecular dynamics (MD) simulation was developed for PL-based prodrugs containing clinically relevant drugs. PL-methotrexate conjugate with 6-carbon linker showed the highest extent of PLA<sub>2</sub>-mediated activation, whereas shorter linkers were activated to a lower extent. In conclusion, this work demonstrates that for carefully designed PL-drug conjugates, PLA<sub>2</sub> overexpression in inflamed colonic tissues can be used as prodrug-activating enzyme and drug targeting strategy, including insights into the activation mechanisms in a PLA<sub>2</sub> binding site.
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