IN-VIVO CLEAVABILITY OF A DISULFIDE-BASED CHIMERIC OPIOID PEPTIDE IN RAT-BRAIN

Brain delivery of systemically administered neuropeptide drugs may be achieved by the synthesis of chimeric peptides, wherein the peptide is coupled to transport vectors via avidin-biotin technology. The present study focuses on factors that optimize the linkage of drugs to transport vectors. The vector is the OX26 monoclonal antibody to the transferrin receptor, and the model peptide used in these studies is [Lys(7)]dermorphin (K7DA). The K7DA is monobiotinylated at the E-amino group of the Lys(7) residue with either a cleavable linker, e.g., disulfide, using NHS-SS-biotin, or a noncleavable linker, e.g., amide, using NHS-XX-biotin. Disulfide cleavage of the biotinylated derivative yields the desbiotinylated peptide, which is thiolated. Structures of the K7DA analogues were confirmed by secondary ion mass spectrometry. The biotinylated peptides were coupled to a thiol-ether conjugate of the OX26 antibody and either neutral avidin (NLA) or streptavidin. The binding constants (K-i) of the K7DA, the biotinylated K7DA (bio-XX-K7DA), the desbiotinylated K7DA, and the bio-XX-KD7A conjugated to NLA-OX26 were 0.62 +/- 0.14, 1.59 +/- 0.27, 1.24 +/- 0.24, and >10 nM, respectively, and were determined with a mu-opioid peptide radioreceptor assay. Comparable results were obtained with in vivo tail-flick analgesia testing following intracerebroventricular (icy) injection of opioid chimeric peptides. Reversibility of pharmacologic action of thiolated peptide was demonstrated by icy naloxone administration. The cleavability of the disulfide linker in vivo in rat plasma and brain was assessed with gel filtration HPLC and internal carotid artery perfusion of labeled opioid chimeric peptides. These studies are consistent with the following conclusions: (a) opioid peptides have minimal pharmacologic activity when bound to the transport vector, indicating the need for cleavable disulfide linker; (b) the disulfide linker is stable in plasma in vivo as well as brain capillary endothelial cells, but is rapidly cleaved in rat brain in vivo, indicating that disulfide cleavage occurs beyond the endothelial cells of brain capillaries; and (c) the thiolated peptide released following disulfide cleavage is pharmacologically active at the mu-opioid peptide receptor via a naloxone reversible mechanism, indicating the thiolated peptide is not likely covalently bound to the receptor.