Prodrug-Based Targeting Approach for Inflammatory Bowel Diseases Therapy: Mechanistic Study of Phospholipid-Linker-Cyclosporine PLA2-Mediated Activation.

Milica Markovic,Aaron Aponick,Arik Dahan,Ellen M Zimmermann,Shimon Ben-Shabat,Yifat Miller,Clil Regev,Karina Abramov-Harpaz

doi:10.3390/ijms23052673

Abstract

Therapeutics with activity specifically at the inflamed sites throughout the gastrointestinal tract (GIT) would be a major advance in our therapeutic approach to inflammatory bowel disease (IBD). We aimed to develop the prodrug approach that can allow such site-specific drug delivery. Currently, using cyclosporine as a drug of choice in IBD is limited to the most severe cases due to substantial systemic toxicities and narrow therapeutic index of this drug. Previously, we synthesized a series of a phospholipid-linker-cyclosporine (PLC) prodrugs designed to exploit the overexpression of phospholipase A2 (PLA2) in the inflamed intestinal tissues, as the prodrug-activating enzyme. Nevertheless, the extent and rate of prodrug activation differed significantly. In this study we applied in-vitro and modern in-silico tools based on molecular dynamics (MD) simulation, to gain insight into the dynamics and mechanisms of the PLC prodrug activation. We aimed to elucidate the reason for the significant activation change between different linker lengths in our prodrug design. Our work reveals that the PLC conjugate with the 12-carbon linker length yields the optimal prodrug activation by PLA2 in comparison to shorter linker length (6-carbons). This optimized length efficiently allows cyclosporine to be released from the prodrug to the active pocket of PLA2. This newly developed mechanistic approach, presented in this study, can be applied for future prodrug optimization to accomplish optimal prodrug activation and drug targeting in various conditions that include overexpression of PLA2.

Highlights

Design and Activation of phospholipase A2 (PLA2) -Trigerred PLC Prodrug Depends on the Length of the Linker
We demonstrated that the PL-linker-cyclosporine with the longest linker exhibited optimal, fastest rate of activation through in-vitro PLA2 -mediated activation, at all 3 enzyme concentrations
We have shown by molecular dynamics (MD) simulations that the insufficient activation of the shorter

Summary

Introduction

Inflammatory bowel diseases (IBD) are a group of chronic inflammatory diseases of the gastrointestinal tract (GIT) and include Crohn’s disease and ulcerative colitis [1]. These diseases have emerged as a public health challenge worldwide [2,3]. Therapeutic strategies in IBD are largely based on anti-inflammatory drugs, steroids, and biological therapy [4–7]. Due to severe side effects in some patients with IBD, these therapies eventually need to be discontinued [8]

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