Enkephalin analog prodrugs: Assessment of in vitro conversion, enzyme cleavage characterization and blood-brain barrier permeability

Diana L. Greene, Vincent S. Hau, Thomas J. Abbruscato, Hubert Bartosz, Aleksandra Misicka, Andrjej W. Lipkowski, Sharon Hom, Terrence J. Gillespie, Victor J. Hruby, Thomas P. Davis

Research output: Contribution to journalArticlepeer-review

44 Scopus citations


To improve the blood-brain barrier penetration of the δ-opioid receptor peptides [D-Pen2,D-Pen5]enkephalin (DPDPE) and [D-Pen2,L-Cys5]enkephalin (DPLCE), various prodrug forms were synthesized to increase lipophilicity and drug delivery to the brain. The aims of this study were 3-fold, 1) to assess the metabolic conversion of various DPDPE and DPLCE prodrugs in vitro using mouse brain homogenate and mouse serum, 2) to characterize the proteolytic enzymes responsible for cleaving prodrugs to the parent compounds using select peptidase inhibitors and 3) to assess the blood-brain barrier permeability of prodrugs, compared with their parent compounds, using the in vitro bovine brain microvessel endothelial cell culture model. The prodrugs with carboxyl-terminal phenylalanine residues (DPDPE-Phe and DPLCE-Phe) had significantly longer metabolic conversion times in both mouse serum and brain homogenates than did the prodrugs with amino-terminal phenylalanine residues. Inhibition of leucine aminopeptidase with bestatin in the serum increased the conversion time of Phe(o)-DPDPE from 6.8 min to 92.2 min. Inhibition of aminopeptidase M with amastatin in the brain homogenate increased the conversion time of Phe(o)-DPDPE from 3.9 min to >450 min. The long half-life of DPLCE-Arg-Pro-Ala in serum (317 min) vs. brain (9.2 min) can be explained by the high levels of the degradative endopeptidase 24.15 (EC in the central nervous system but not in plasma. The data also showed that, for specific prodrugs of DPDPE such as Phe(o)-DPDPE and DPDPE-Arg-Gly, the prodrug shows a significant improvement in permeability, compared with the parent compound. Therefore, these data provide evidence that prodrugs or prodrug-enzyme inhibitor combinations may optimize the delivery of peptide and/or protein drugs to the central nervous system.

Original languageEnglish (US)
Pages (from-to)1366-1375
Number of pages10
JournalJournal of Pharmacology and Experimental Therapeutics
Issue number3
StatePublished - Jun 1996

ASJC Scopus subject areas

  • Molecular Medicine
  • Pharmacology


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