Abstract
Using thyrotropin-releasing hormone (TRH) as a model, we explored whether synergistic combination of lipoamino acid(s) and a linker cleaved by prolyl oligopeptidase (POP) can be used as a promoiety for prodrug design for the preferential brain delivery of the peptide. A representative prodrug based on this design principle was synthesized, and its membrane affinity and in vitro metabolic stability, with or without the presence of a POP inhibitor, were studied. The in vivo formation of TRH from the prodrug construct was probed by utilizing the antidepressant effect of the peptide, as well as its ability to increase acetylcholine (ACh) synthesis and release. We found that the prototype prodrug showed excellent membrane affinity and greatly increased metabolic stability in mouse blood and brain homogenate compared to the parent peptide, yet a POP inhibitor completely prevented prodrug metabolism in brain homogenate. In vivo, administration of the prodrug triggered antidepressant-like effect, and microdialysis sampling showed greatly increased ACh release that was also antagonized upon a POP inhibitor treatment. Altogether, the obtained promising exploratory data warrant further investigations on the utility of the prodrug approach introduced here for brain-enhanced delivery of small peptides with neurotherapeutic potential.
Highlights
Thyrotropin-releasing hormone (TRH, pGlu-His-Pro-NH2) shown in Figure 1a, is an excellent representative of small neuropeptides with unfulfilled neurotherapeutic potential for the treatment of neurological and psychological disorders due to its inherent ability to alter brain chemistry and, subsequently, behavior and physiology [1,2,3,4,5]. This peptide is abundant in the central nervous system (CNS) and believed to act through two known (TRH-R1 and thyrotropin-releasing hormone (TRH)-R2) receptors [2]
As with peptides in general, and especially in the context of non-invasive brain delivery for potential neurotherapy, TRH suffers from numerous shortcomings including inadequate metabolic stability in the periphery [6,7]
To assess TRH’s modulation of ACh after its in vivo liberation from the prodrug, a pilot study utilizing microdialysis sampling of the extracellular space of rat frontal cortex was done according to our previously published protocol [16,35] with modifications. These modifications included ESI coupled with tandem mass spectrometry (MS/MS) instead of amperometry to measure ACh concentrations [36] after high-performance liquid chromatography (HPLC) separation of the analyte, which allowed us to avoid the use of the acetylcholinesterase inhibitor neostigmine in our experiment as an auxiliary agent
Summary
Thyrotropin-releasing hormone (TRH, pGlu-His-Pro-NH2) shown in Figure 1a, is an excellent representative of small neuropeptides with unfulfilled neurotherapeutic potential for the treatment of neurological and psychological disorders due to its inherent ability to alter brain chemistry and, subsequently, behavior and physiology [1,2,3,4,5]. Thyrotropin-releasing hormone (TRH, pGlu-His-Pro-NH2) shown, is an excellent representative of small neuropeptides with unfulfilled neurotherapeutic potential for the treatment of neurological and psychological disorders due to its inherent ability to alter brain chemistry and, subsequently, behavior and physiology [1,2,3,4,5]. This peptide is abundant in the central nervous system (CNS) and believed to act through two known (TRH-R1 and TRH-R2) receptors [2]. EpSoI msitoivdee-wioenrEe SdIomneoodne awTehreerdmoonQe uonesat LTCheQr-mDoeQcauieosnt tLrCapQm-Daescsaspioencttrroampemtears(sTshpeercmtroomFisehteerr(STchieenrmtifioc; WFaislthhearmS,cMienAt,ifUicS; AW) aclothuapmle,dMwAit,h Ua SSAur)vceoyuoprlMedSwLCithsyastSeumrv(eTyhoerrmMoSFLisChesrySscteiemnti(fiTch)e. rLmCo–EFSisIh-MerS foSrciiennvtiiftirco).sLtuCd–iEeSsIw-MaSs dfoornien ovnitraoTsStuQdQiesuawnatsudmonUeltorna atrTipSlQe-qQuuaadnrtuupmolUelmtraastrsipsplee-cqturoamdreuteprocleomupalsesd wsipthecatrSoumrevteeyrocroHupPlLeCd swyistthema S(Tuhrveerymoor FHisPhLeCr Sscyisetnemtifi(cT).hAernmoothFerisThhererSmcioenFtiinfinc)ig. aAnnTotShQerQTuhaenrtmuom UFlitnranitgriapnleTqSuQadQruupanotluemtanUdletmra MtriSpcleonqnueacdterduptoolae VtaanndqeumishMFSlexcoLnCne(cTthedermtooaFiVshaenrqSucisiehntFilfiecx) wLCas u(tTilhizeerdmfoorFitshheearnSacliyensetisfiocf) iwnavsivuotilmiziecdrofdoiratlhyesiasnsaalmyspelsesoftoinesvtiavbolimshicerxotdraiacleylsluislasramApClhesletvoeelsst.ablish extracellular ACh levels
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