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Oral medication with activity specifically at the inflamed sites throughout the gastrointestinal tract and limited systemic exposure would be a major advance in our therapeutic approach to inflammatory bowel disease (IBD). For this purpose, we have designed a prodrug by linking active drug moiety to phospholipid (PL), the substrate of phospholipase A2 (PLA2). PLA2 expression and activity is significantly elevated in the inflamed intestinal tissues of IBD patients. Since PLA2 enzyme specifically hydrolyses the sn-2 bond within PLs, in our PL-based prodrug approach, the sn-2 positioned FA is replaced with cyclosporine, so that PLA2 may be exploited as the prodrug-activating enzyme, releasing the free drug from the PL-complex. Owing to the enzyme overexpression, this may effectively target free cyclosporine to the sites of inflammation. Four PL-cyclosporine prodrugs were synthesized, differing by their linker length between the PL and the drug moiety. To study the prodrug activation, a novel enzymatically enriched model was developed, the colonic brush border membrane vesicles (cBBMVs); in this model, tissue vesicles were produced from colitis-induced (vs. healthy) rat colons. PLA2 overexpression (3.4-fold) was demonstrated in diseased vs. healthy cBBMVs. Indeed, while healthy cBBMVs induced only marginal activation, substantial prodrug activation was evident by colitis-derived cBBMVs. Together with the PLA2 overexpression, these data validate our drug targeting strategy. In the diseased cBBMVs, quick and complete activation of the entire dose was obtained for the 12-carbon linker prodrug, while slow and marginal activation was obtained for the 6/8-carbon linkers. The potential to target the actual sites of inflammation and treat any localizations throughout the GIT, together with the extended therapeutic index, makes this orally delivered prodrug approach an exciting new therapeutic strategy for IBD treatment. [ABSTRACT FROM AUTHOR]

The enzyme phospholipase A 2 (PLA 2 ) is overexpressed in the inflamed intestine in inflammatory bowel disease (IBD) patients, and in this work we aimed to exploit PLA 2 as a prodrug-activating enzyme for a novel PL-drug conjugate, thereby liberating the free drug specifically in the targeted diseased tissue(s). The proposed prodrug contains a drug moiety covalently bound through a linker to the sn-2 position of a phospholipid (PL). The NSAID diclofenac was used as model molecule, and four different linker lengths (2, 4, 6 and 8 –CH 2 units) were studied. The four PL-diclofenac conjugates were synthesized and characterized by LC/MS and NMR. PLA 2 -mediated activation of the prodrugs was analyzed in-vitro , and the remaining intact complex and free drug liberation were assessed after incubation with PLA 2 . The rate and degree of PLA 2 -mediated activation were highly dependent on the linker length; 2- and 4-carbon linker conjugates were activated to lower extent than the 6-carbon conjugate, and longer linker again decreased the affinity towards PLA 2 . The 6-carbon linker conjugate was found to be the optimal and released ~ 95% of the free drug after incubation with PLA 2 , whereas only ~ 20% were delivered by the 2-carbon linker prodrug. The 6-carbon linker conjugate was shown to be stable in intestinal perfusate, fresh plasma, and pH 4.0 and 6.8 buffers, but not at pH 1.0. In conclusion, the results of this work confirm the feasibility of our general aim to exploit PLA 2 as a prodrug-activating enzyme of PL-drug conjugates. This may provide a novel oral drug targeting approach in IBD therapy. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Prodrugs are bioreversible, inactive drug derivatives, which have the ability to convert into a parent drug in the body. In the past, prodrugs were used as a last option; however, nowadays, prodrugs are considered already in the early stages of drug development. Optimal prodrug needs to have effective absorption, distribution, metabolism, and elimination (ADME) features to be chemically stable, to be selective towards the particular site in the body, and to have appropriate safety. Traditional prodrug approach aims to improve physicochemical/biopharmaceutical drug properties; modern prodrugs also include cellular and molecular parameters to accomplish desired drug effect and site-specificity. Here, we present recently investigated prodrugs, their pharmaceutical and clinical advantages, and challenges facing the overall prodrug development. Given examples illustrate that prodrugs can accomplish appropriate solubility, increase permeability, provide site-specific targeting (i.e., to organs, tissues, enzymes, or transporters), overcome rapid drug metabolism, decrease toxicity, or provide better patient compliance, all with the aim to provide optimal drug therapy and outcome. Overall, the prodrug approach is a powerful tool to decrease the time/costs of developing new drug entities and improve overall drug therapy. [ABSTRACT FROM AUTHOR]