Improving Brain Delivery of TRH: A Novel Prodrug Approach




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The goal of my research project was to validate a novel prodrug design concept for the brain-enhanced delivery of an important neuropeptide, thyrotropin-releasing hormone (TRH, pGlu-His-Pro-NH2). TRH has a variety of clinically relevant central effects that cannot be utilized with the direct administration of TRH, due to intrinsic characteristics that give rise to metabolic instability and insufficient transport through the blood-brain barrier (BBB). Consequently, large doses are required to generate central effects, which concomitantly induces unwanted hormonal liabilities in the periphery. To overcome these caveats, the prodrug design described herein proposes a novel brain-targeting approach that synergistically utilizes two enzymes, both preferentially expressed in the brain, for the enhanced brain-delivery of TRH. The conjugation of a lipoamino acid (LAA) transport moiety to the N-terminus of a TRH progenitor sequence (Gln-His-Pro-NH2) via a prolyl oligopeptidase (POP)-sensitive linker allows the POP enzyme to release the TRH progenitor sequence at the site-of-action upon crossing the BBB, where it is further transformed by glutaminyl cyclase (QC). QC catalyzes Gln to form pGlu at the N-terminus of the progenitor sequence, thereby releasing TRH. I tested the hypothesis that a representative molecule, developed according to this prodrug design approach, would exhibit adequate drug-likeness for BBB penetration and efficacious release of TRH within the brain. Immobilized artificial membrane chromatography was used to predict the BBB penetration of this experimental prodrug, labeled “PRODRUG 1,” in addition to its calculated logP. Next, PRODRUG (1) was compared to TRH (the “parent” peptide) in an in vitro metabolic stability assessment, followed by an in vivo neuro-pharmacodynamical evaluation in rodents. The Porsolt swim test, PST, an established animal behavioral model that detects depressive-like behavior was used to confirm brain-delivery of prodrug-derived TRH after systemic administration of the prototype prodrug. Capitalizing on TRH’s antidepressant-like effect, the PST results were also used to validate the tail suspension test (TST), a new technique that I implemented in our laboratory for the evaluation of neuroactive compounds with potential antidepressant-like activity. My findings support the extension of the TRH progenitor sequence from the N-terminus, through the conjugation of two LAA residues (each with a 10-carbon sidechain) via a single proline POP-sensitive linker, as a successful means to increase penetration across the BBB and sufficiently bind with cleaving and activating enzymes, POP and QC, respectively, for efficacious TRH release in the brain. Lastly, molecular modeling was used to create a library of similarly designed prodrugs to computationally assess their bindings with POP, the cleaving enzyme, to further explore the customizable prodrug design concept described here. Ultimately, this adaptable prodrug delivery model demonstrates the effectiveness of increased lipophilicity and site-of-action targeting to facilitate brain-enhanced delivery of TRH.