ENKEPHALIN ANALOG PRODRUGS - ASSESSMENT OF IN-VITRO CONVERSION, ENZYME CLEAVAGE CHARACTERIZATION AND BLOOD-BRAIN-BARRIER PERMEABILITY

To improve the blood-brain barrier penetration of the delta-opioid rec eptor peptides [D-Pen(2),D-Pen(5)]enkephalin (DPDPE) and [D-Pen(2),L-C ys(5)]enkephalin (DPLCF), various prodrug forms were synthesized to in crease lipophilicity and drug delivery to the brain. The aims of this study were 3-fold, 1)to assess the metabolic conversion of various DPD PE and DPLCE prodrugs in vitro using mouse brain homogenate and mouse serum, 2) to characterize the proteolytic enzymes responsible for clea ving prodrugs to the parent compounds using select peptidase inhibitor s and 3) to assess the blood-brain barrier permeability of prodrugs, c ompared with their parent compounds, using the in vitro bovine brain m icrovessel endothelial cell culture model. The prodrugs with carboxyl- terminal phenylalanine residues (DPDPE-Phe and DPLCE-Phe) had signific antly longer metabolic conversion times in both mouse serum and brain homogenates than did the prodrugs with amino-terminal phenylala-nine r esidues. Inhibition of leucine aminopeptidase with bestatin in the ser um increased the conversion time of Phe(0)-DPDPE from 6.8 min to 92.2 min. Inhibition of aminopeptidase M with amastatin in the brain homoge nate increased the conversion time of Phe(0)-DPDPE from 3.9 min to >45 0 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 3.4.24.15) in the central nervous system but not in plasma. The data also showed that, for specific prodrugs of DPD PE such as Phe(0)-DPDPE and DPDPE-Arg-Gly, the prodrug shows a signifi cant improvement in permeability, compared with the parent compound. T herefore, these data provide evidence that prodrugs or prodrug-enzyme inhibitor combinations may optimize the delivery of peptide and/or pro tein drugs to the central nervous system.