Opioid peptides, in particular dynorphin peptides, may prove to be useful in the treatment of heroin withdrawal and tolerance, and have particular advantages over conventional drugs with respect to new drug development considerations. However, opioid peptides are not transported through the brain capillary wall, i.e., the blood-brain barrier (BBB). The proposed work develops a new strategy for neuropeptide delivery through the BBB which is the formation of chimeric peptides. Chimeric peptides are produced when a peptide, e.g., opioid peptide, that is normally not transported through the BBB, is covalently coupled to a BBB transport vector such as insulin, transferrin, or cationized albumin, i.e., proteins that normally enjoy receptor-mediated or absorptive-mediated transcytosis through the BBB. The chimeric peptides are joined by disulfide bonds which are stable in plasma but are labile in cells, including brain, and subsequent to the transport of the chimeric peptide into brain parenchyma free pharmacologically active opioid peptide is released. To ensure the release in brain of pharmacologically active peptide it is important to use optimal coupling strategies, and the proposed work will focus on developing two different coupling strategies for opioid peptides. [D-Ala2-Cys6] leucine enkephalin (DALCE) will be used as a model delta-receptor opioid peptide and will be covalently coupled to captionized albumin directly through the cysteine sulfhydryl. A model kappa-opioid peptide receptor analogue will be a dynorphin analogue, [D-Ala2, Arg11,13] dynorphin A (1.13) glycine-NH(CH2)5NH2, which is the D-Ala2 analogue of the dynorphin A analogue kappa-ligand (DAKLI) and the analogue is abbreviated DDAKLI. The Boc-alpha-amino protected form a DDAKLI will allow for selective coupling to the C-terminal extended amino group. While it is recognized that the formation of the D-Ala2 analogue causes loss of kappa-selectivity, the use of this analogue for the proposed in vivo studies is essential to prevent rapid peptide inactivation by brain capillary aminopeptidase (see Appendix 7). The following Specific Aims are proposed: (1) chemical synthesis of the DALCE- and DDAKLI-cationized albumin chimeric peptides; (2) studies of the binding and endocytosis of the opioid chimeric peptides by isolated brain capillaries used as an in vitro model system of the BBB; (3) transcytosis of the opioid chimeric peptides through the BBB in vivo using an internal carotid artery perfusion/capillary depletion method in anesthetized rats; (4) studies of the cleavage of the opioid chimeric peptide by brain disulfide reductases using both in vitro studies and in vivo studies and subcellular localization of the disulfide reductases to the vascular, synaptosomal, microsomal, or cytosol fractions; (5) pharmacologic assays using in vitro radioreceptor assays for the delta-, mu-, or kappa-receptor, in vivo pharmacokinetic studies and in vivo pharmacologic assays employing tail-flick experiments; (6) toxicity studies involving administration of opioid chimeric peptide to rats over four- and eight-week periods. The overall goal of these studies is to develop opioid chimeric peptides that are pharmacologically active in brain in vivo and that are capable of effective transport through the BBB following systemic administration.
Showing the most recent 10 out of 12 publications
