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037.K The Role of Histamine in a Hypocretin orexin ; -Deficient Sleep Disorder, Narcolepsy Nishino S, 1 Honda K, 2 Fujiki N, 1 Wurts SW, 1 Chang M, 1 Sakurai E, 3 Kato M, 3 Watanabe T, 3 Mignot E, 1 Yanai K2 1 ; Center for Narcolepsy, Stanford Sleep Research Center, 2 ; Department of Biocybernetics, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 3 ; Department of Pharmacology, Tohoku University School of Medicine Introduction: Narcolepsy is a chronic sleep disorder which affects 0.030.18% of the general population. Studies in dogs and mice have led to the identification of genes preprohypocretin and hypocretin receptor 2 [Hcrtr-2] genes ; responsible for the disease. In humans, genetic mutations in hypocretin-related genes are rare 1 ; , but hypocretin deficiency is functionally involved in most cases 1, 2 ; . Hypocretins are excitatory neuropeptides located exclusively in the lateral hypothalamic area. Hypocretin-containing neurons send projections to most monoaminergic nuclei, such as adrenergic locus coeruleus, dopaminergic ventral tegmental area, and histaminergic tuberomammillary nucleus TM ; . It reported that intracerebroventricular ICV ; injection of hypocretin-1 enhances wakefulness in rats. Considering the fact that Hcrtr-2 is enriched in the TM 3 ; , coupled with the fact that the histaminergic system plays an important role in the control of vigilance, it is likely that a deficit in histaminergic neurotransmission plays an important role in the pathophysiology of narcolepsy. It is also possible that the wake-promoting action of hypocretin may be partially mediated by the histaminergic system. Methods: Brain tissue from 9 familial Hcrtr-2 mutated ; narcoleptic and 9 control Dobermans was used. The mean age of narcoleptic and control animals was 26.021 months old, and 27.722.1 months old, respectively. Histamine content, as well as monoamine content dopamine and norepinephrine and dopamine turnover ; , was measured in the cortex, thalamus and hippocampus with a fluorometric HPLC system and with an HPLC with an electrochemical detector, respectively. In order to evaluate whether the wake-promoting effect of hypocretin is mediated by the activation of the histaminergic neurotransmitter system, we also measured changes in histamine content in the brains cortex, brainstem, cerebellum ; of rats control n 5, hypocretin n 6 ; 2 hours after ICV injection of hypocretin-1 5 nmol ; . Results: Histamine content in the cortex 43667 [SE] pmol g ; and the thalamus 884100 pmol g ; was significantly lower in narcoleptic Dobermans compared to controls 73393 pmol g and 1749227 pmol g, respectively ; . Hippocampus levels 602132 pmol g and 793196 pmol g, respectively ; were also decreased, but this decrease did not reach statistical significance. In the rat ICV study, hypocretin-1 significantly enhanced wakefulness 169.9 % of the vehicle treatments ; , and increased histamine levels in the cortex 124 % of the vehicle treatments ; and brainstem 130 % ; , but did not significantly increase levels in the cerebellum 110% ; . Conclusions: In narcolepsy, a hypocretin-deficient sleep disorder, a decrease in global histaminergic neurotransmission is suggested. In contrast, DA and NE neurotransmission in the brain regions tested were not altered. This may be consistent with our pharmacological finding that H3 antagonists, such as GT-2331 and thioperamide, significantly reduce cataplexy and sleep in narcoleptic Dobermans. In rat experiments, it is also suggested that the wake-promoting effect of hypocretin may be partially mediated by the enhancement of histaminergic neurotransmission.These results therefore suggest involvement of a hypocretin-histamine interaction in the regulation of vigilance in both normal and pathological conditions. A23 SLEEP, Vol. 24, Abstract Supplement 2001. 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Company, on March 14, 2003, we completed the sale of our genomics services business to privately held Agencourt Bioscience Corporation Agencourt ; . We retain rights to our PathoGenomeTM Database product, including all associated intellectual property, subscriptions and royalty rights on products developed by subscribers. As part of the agreement with Agencourt, we transferred our sequencing operations, including certain equipment and personnel, to Agencourt for the following consideration: -- an up-front cash payment of $200, 000; -- cash payments over a two year period ending in March 2005 totaling approximately $784, 000 pursuant to a right to receive a percentage of revenues from our former commercial and government customers; and -- 500, 000 shares of Agencourt's common stock. As a result of the acquisition of Agencourt by Beckman Coulter, we received i ; a payment of approximately $2, 245, 000 during the quarter ended June 30, 2005, in exchange for the shares of Agencourt we held, ii ; a payment of approximately $143, 000 during the quarter ended September 30, 2005 due to a post-closing merger adjustment, and iii ; the right to receive additional consideration if certain milestones are achieved by Agencourt over the next three years. Internally Funded Early-Stage Target Research Program -- As part of our strategic decision to concentrate on development and commercialization of our pharmaceutical products, we adopted a plan in 2003 to substantially reduce our research effort in internally funded early-stage target discovery programs. Under this plan, we eliminated 44 full-time positions and paid out $609, 000 related to severance costs against the restructuring liability in 2004. This charge includes associated severance costs, outplacement services and a non-cash charge for the acceleration of vesting of previously granted stock options, as well as impairment charges related to the value of laboratory and computer equipment no longer used in operations. On May 16, 2005 we entered into a License and Contribution Agreement with MaxThera, Inc., whereby we granted an exclusive license to certain patent rights relating to certain of our legacy anti-infective technology. Title to these patent rights will vest in MaxThera upon meeting certain financial milestones. In consideration of the license and upon transfer of title to MaxThera, we will receive shares of MaxThera's common stock as well as a right to royalty payments if any products covered by the patent rights are commercialized. On May 31, 2005 we entered into a License and Sale Agreement with Azee, Inc., whereby we granted an exclusive license to certain patent rights relating to Genesoft's small molecule anti-genomic therapeutics legacy technology. Title to these patent rights will vest in Azee upon Azee meeting certain financial milestones. In consideration of the license, we received shares of Azee's common stock and are entitled to royalty payments if any products covered by the patent rights are commercialized. Upon transfer of title to the patents to Azee, we will receive additional shares of Azee's common stock. As a combined category, these research efforts represented 0% and 9% of total research and development expenses for the fiscal years ended December 31, 2005 and 2004, respectively and theo-dur.

Important structures and compelling synvisc medical mal rocephin paid multiple etodolac monoamine. Sera are diluted 1: 100, 1: and 1: 800 for IgG and IgM; 100 1.11 of each serum dilution is incubated in the plates on an ELISA shaker for 45 min at room temperature. Plates are washed and conjugates added: IgG conjugate, goat-anti-human IgG-peroxidaselabeled De Beer, Diessen, The Netherlands ; , IgM conjugate, goat-anti-human IgM-peroxidase-labeled Pasteur, Marnes la Coquettes, France ; . Plates with conjugates are incubated for 30 min at room temperature and are w ashed. Substrate, 100 l of 0.3 g L OPD in citratebuffered solution pH 5.05 ; with 0.0002% H202, is added and after 20 min 100 p, 1 of 1 H2SO4 is added to stop the reaction. Plates are read at 490 nm. In every assay, four EBV-negative control sera are tested to determine cutoff values mean + 3 x standard for IgG anti-EBNA-1 and anti-VCA reactivity, serum from a healthy seropositive volunteer is used. Standard for IgGanti-EA D ; was obtained from a chronic EBV patient with high anti-EA D ; titres and as a standard for IgM-anti-EA D ; and anti-VCA sera from a patient with infectious mononucleosis is used. The amount of antibody in patient serum is expressed as a percentage of the standard serum which is included in each plate as described earlier [van der Giessen et al., 1990].

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