The Pharmacology and Distribution of Serotonin 5-HT-1E Receptors in the Mammalian Brain & Drug Development for the Human 5-HT-1E Receptor.

摘要

The serotonin 5-HT1E receptor is highly expressed in the human brain, and its structure is conserved amongst humans, suggesting an important role for 5-HT1E receptors in human physiology. This receptor was originally discovered in human brain tissue via radioligand binging methodology and was subsequently cloned; homologous genes have been identified in several other species. However, an unusual finding was made: rodent species, including rats and mice, lack a 5-HT1E receptor gene. Thus, Rats and mice, the typical pre-clinical animal species, cannot be used to study the physiological functions of 5-HT1E receptors or test the efficacy of drugs that target 5-HT1E receptors. Because of this, little is known of the role 5-HT1E receptors play in mediating the effects of serotonin, a critical neurotransmitter in humans, and drug development for this receptor has been stalled. This is unfortunate, as many effective therapeutic agents have been developed that act through serotonin receptors.;This dissertation aims to establish the guinea pig, a species that possess a 5-HT1E receptor gene, as a pre-clinical animal model for 5-HT 1E receptor investigations and drug development. Brain regions that exhibit the characteristic pharmacology of the 5-HT1E receptor are identified (i.e. [3H]5-HT binding insensitive to the drugs 5-carboxyamidotryptamine (5-CT), ritanserin, and LY344864). Concentrations of these drugs were optimized to mask [3H]5-HT binding at all non-5-HT1E receptors. The affinities of 16 drugs were determined at the hippocampal-expressed 5-HT1E binding site and were found to be indistinguishable from the affinities determined at the cloned receptor (R2 = 0.97). These findings indicate that the guinea pig brain does express the 5-HT1E receptor and that signal transduction studies could be conducted if a brain region with sufficient 5-HT1E receptor density can be identified.;Using immunohistochemical techniques, this dissertation presents the first specific localization of 5-HT1E receptors in the mammalian brain and confirms a functional link between the stimulation of 5-HT 1E receptors and the inhibition of adenylate cyclase activity in brain tissue. High levels of 5-HT1E receptors are detected in olfactory bulb glomeruli. The molecular layer of the dentate gyrus displays a robust expression of 5-HT1E receptors. In dentate gyrus membranes, BRL54443, a 5-HT1E/5-HT1F-selective agonist, stimulates 5-HT 1E receptors and potently inhibits forskolin-dependent cAMP production (IC50 = 14nM). The staining pattern of 5-HT1E receptors in brain tissue suggests this receptor is expressed predominantly in neurons rather than in glia. Additionally, 5-HT1E receptors are detected in the adventitial layer of cerebral arteries but not in the microvasculature, venous tissue, or other brain arteries. These observations should help predict the clinical effects of 5-HT1E-selective drugs. For instance, the expression of Gi/o-coupled 5-HT1E receptors in the dentate gyrus suggests this receptor may mediate serotonin's regulation of hippocampal activity, making it a possible drug target for the treatment of psychiatric disorders characterized by memory deficits (e.g. Alzheimer's Disease) or as a target for the treatment of temporal lobe epilepsy.;There have been few reports on the development of drugs for the 5-HT 1E receptor. The data presented in this dissertation concerning the distribution of 5-HT1E receptors in the guinea pig brain will hopefully encourage drug discovery programs to explore the development of selective agonists and antagonists for the 5-HT1E receptor. This dissertation attempts to identify new avenues for future 5-HT1E receptor drug development with BRL54443 as a lead compound. This tryptamine-related agonist is one of very few agents that binds 5-HT1E receptors with high affinity and some selectively; unfortunately, it binds equally well to 5-HT 1F receptors (Ki ≈ 1 nM). The differences between tryptamine binding requirements of these two receptor populations have never been extensively explored, and this must be done in order to guide the design of analogs with greater selectivity for 5-HT1E receptors versus 5-HT1F receptors. 5-HT1E and 5-HT1F receptor binding affinities for a large series of tryptamine analogs are determined and found to be high correlated (R2 = 0.66). There is significant overlap between 5-HT1E and 5-HT1F receptor orthosteric binding properties; thus, identification of 5-HT1E-selective orthosteric ligands will be difficult and allosteric drug development may be a more tractable option for this receptor. The insights generated from this dissertation encourage further investigation of the physiological roles played by 5-HT1E receptors in the brain and will inform future 5-HT1E receptor drug development efforts.