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.

Agency
National Institute of Health (NIH)
Institute
National Institute on Drug Abuse (NIDA)
Type
Research Project (R01)
Project #
5R01DA006748-03
Application #
2119048
Study Section
Drug Abuse Biomedical Research Review Committee (DABR)
Project Start
1992-04-01
Project End
1996-03-31
Budget Start
1994-04-01
Budget End
1996-03-31
Support Year
3
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of California Los Angeles
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
119132785
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Bickel, U; Yoshikawa, T; Pardridge, W M (2001) Delivery of peptides and proteins through the blood-brain barrier. Adv Drug Deliv Rev 46:247-79
Wu, D; Kang, Y S; Bickel, U et al. (1997) Blood-brain barrier permeability to morphine-6-glucuronide is markedly reduced compared with morphine. Drug Metab Dispos 25:768-71
Pardridge, W M; Kang, Y S; Buciak, J L et al. (1995) Human insulin receptor monoclonal antibody undergoes high affinity binding to human brain capillaries in vitro and rapid transcytosis through the blood-brain barrier in vivo in the primate. Pharm Res 12:807-16
Bickel, U; Kang, Y S; Pardridge, W M (1995) In vivo cleavability of a disulfide-based chimeric opioid peptide in rat brain. Bioconjug Chem 6:211-8
Bickel, U; Kang, Y S; Yoshikawa, T et al. (1994) In vivo demonstration of subcellular localization of anti-transferrin receptor monoclonal antibody-colloidal gold conjugate in brain capillary endothelium. J Histochem Cytochem 42:1493-7
Pardridge, W M; Kang, Y S; Buciak, J L (1994) Transport of human recombinant brain-derived neurotrophic factor (BDNF) through the rat blood-brain barrier in vivo using vector-mediated peptide drug delivery. Pharm Res 11:738-46
Samii, A; Bickel, U; Stroth, U et al. (1994) Blood-brain barrier transport of neuropeptides: analysis with a metabolically stable dermorphin analogue. Am J Physiol 267:E124-31
Bickel, U; Yamada, S; Pardridge, W M (1994) Synthesis and bioactivity of monobiotinylated DALDA: a mu-specific opioid peptide designed for targeted brain delivery. J Pharmacol Exp Ther 268:791-6
Kang, Y S; Bickel, U; Pardridge, W M (1994) Pharmacokinetics and saturable blood-brain barrier transport of biotin bound to a conjugate of avidin and a monoclonal antibody to the transferrin receptor. Drug Metab Dispos 22:99-105
Pardridge, W M; Yoshikawa, T; Kang, Y S et al. (1994) Blood-brain barrier transport and brain metabolism of adenosine and adenosine analogs. J Pharmacol Exp Ther 268:14-8

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