These peptide-based drugs normally exhibit very specific targeting to the desired receptor. However, in order to exert their therapeutic effects, they must also be able to cross into the brain and be enzymatically stable. In this proposal, the transport and metabolism of dynorphin A (Dyn A) at the blood-brain barrier (BBB) is investigated. Dyn A is a natural ligand of the kappa opioid receptor. Analogs of dynorphin and its metabolites have been investigated for the treatment of peripheral pain, drug addiction, and affective disorders. Paradoxically, Dyn A has also been shown to be neurotoxic. Elevated levels of this peptide in the CNS has been associated with neuropathic pain and Alzheimer's disease. Neuropeptides, such as dynorphin, can play multiple roles in vivo including that of a neurotransmitter, neuromodulator, and hormone. In contrast to small molecule neurotransmitters such as glutamate and dopamine, neuropeptides are not taken back up by neurons following their release but are metabolized in the extracellular space into smaller peptide fragments. These fragments can directly affect the cells in the extracellular space in the surrounding area, pass through the BBB, and exert effects at sites far removed from the site of their initial release. There are three main goals of this proposal. The first is to investigate the transport and metabolism of Dyn A 1-17, its metabolites, and synthetic analogs across the BBB. Information acquired through these experiments will lead to a better understanding of the mechanism of transport and will make it possible to produce better therapeutics targeted at the kappa opioid receptor. The second is to develop separationbased sensors that can be used to monitor the effects of dynorphin and its metabolites on neurotransmitter release in the CMS. The third objective is to develop a separation-based sensor that can be placed onanimal so that measurements of neurotransmitters and behavior can be made simultaneously, using awake, freely roaming animals. These techniques will be employed to study the pharmacological actions of Dyn A at the BBB and in the CNS. The methodology developed here will be generally applicable to all neurochemical studies in which the measurement of blood brain barrier transport or neurotransmitters is desired, especially those cases where monitoring behaviour is also of interest. Significance: Dynorphin is an important neuropeptide that is involved in pain, cocaine addiction, depression, and Alzheimer's disease. A better understanding of the transport and metabolism of this peptide across the BBB will assist scientists in producing better treatments for these diseases. The analytical methods developed in this proposal will also be of general utility to scientists involved in CNS research.
