Metabolism-based brain-targeting system for a thyrotropin-releasing hormone analogue

Gln-Leu-Pro-Gly, a progenitor sequence for the thyrotropin-releasing hormon e (TRH) analogue [Leu(2)]TRH (pGlu-Leu-Pro-NH2), was covalently and bioreve rsibly modified on its N- and C-termini (by a 1,4-dihydrotrigonellyl and a cholesteryl group, respectively) to create lipoidal brain-targeting systems for the TRH analogue. The mechanism of targeting and the recovery of the p arent peptide at the target site involve several enzymatic steps, including the oxidation of the 1,4-dihydropyridine moiety. Due to the lipid insolubl ity of the peptide pyridinium conjugate obtained after this reaction, one o f the rudimentary steps of brain targeting (i.e., trapping in the central n ervous system) can be accomplished. Our design also included spacer amino a cid(s) inserted between the N-terminal residue of the progenitor sequence a nd the dihydrotrigonellyl group to facilitate the posttargeting removal of the attached modification. The release of the TRH analogue in the brain is orchestrated by a sequential metabolism utilizing esterase/lipase, peptidyl glycine alpha-amidating monooxygenase (PAM), peptidase cleavage, and gluta minyl cyclase. In addition to in vitro experiments to prove the designed me chanism of action, the efficacy of brain targeting for [Leu(2)]TRH administ ered in the form of chemical-targeting systems containing the embedded prog enitor sequence was monitored by the antagonistic effect of the peptide on the barbiturate-induced anesthesia (measure of the activational effect on c holinergic neurons) in mice, and considerable improvement was achieved over the efficacy of the parent peptide upon casing this paradigm.