S.13.03 Pharmacology of the human CRF1, CRF2α and CRF2β receptors and CRF binding protein

S.13 Neuropeptide antagonists as drugs in psychiatry tion and its pharmacoeconomic consequences present a complete profile directly from the OCD patient, including course of illness as well as psychosocial and economic consequences. Interim analysis of the findings were previously reported (3). Patients who received an incorrect diagnosis or ineffective treatment utilized a significantly greater mean number of providers and had greater mean outpatient provider costs than those receiving appropriate treatment. Approximately one in four patients were hospitalized at least once in their lifetime, with hospital costs ranging from $20,000 to over $10t3,000. The majority of OCD costs were indirect, stemming from work loss, early retirement, and absenteeism. The psychological and economic burden of OCD is enormous. Misdiagnosis and comorbidity must be considered, given the strong overlap between OCD spectrum disorders and other psychiatric illnesses. The treatment algorithms provided may serve as a direction for treatment and reduce economic and psychosocial burdens.

References [1] Karno M, Golding JM, Sorenson SB, Bumam A: The epidemiology of obsessive-compulsive disorder in five U.S. communities. Arch Gen Psychiatry 1988, 45:1094-1099 [2] Dupont RL, Rice DP, Shiraki S, Rowland CR: Pharmacoeconomics: Economic costs of obsessive-compulsive disorder. Medical Interface 1995; April: 102-109 [3] Hollander E, Kwon JH, Stein DJ, Broatch J, Rowland CT, Himelein CA: Obsessive--compulsiveand spectrum disorders: Overview and quality of life issues. J Clin Psychiatry 1996; 57 (suppl) 3-6

S.13 Neuropeptide antagonists as drugs in psychiatry ~

Interactions between CCK, dopamine, glutamate and aspartate in the rat striatum

T. H6kfelt 1, D. Blacker 1, C. Broberger 1, M. Herrera-Marschitz 2, S.-O. Ogren 1.1Department of Neuroscience, Department of Physiology,

2Department of Pharmacology, Karolinska Institute, S-171 77 Stockholm, Sweden The rat striatum receives innervation from, among others, dopamine (DA) neurons in the ventral mesencephalon and by cortical afferents mostly using glutamate as the principal transmitter. In both these systems at least part of the neurons also synthesize and release the peptide cholecystokinin (CCK). Thus a large population of the DA neurons in the ventral mescencephalon contain CCK mRNA and express CCK peptide. However, detectable levels of CCK in nerve terminals are only present in some of the forebrain areas, that is in parts of the nucleus accumbens, olfactory tubercle and in the medial aspects of the striatum. The projections of the cortico-striatal CCK neurons have been less well defined, but CCK neurons in the frontal cortex seem to participate in the formation of CCK patches in the medial half of the striatum (1). In vivo microdialysis experiments suggest interesting interactions between CCK and excitatory amino acids. Thus, administration of sulphated CCK-8 into the striatum via the microdiaiysis tube causes a dramatic increase in aspartate release (2). However, only minimal effects are seen on glutamate, dopamine and GABA. The origin of this aspartate is not known, but administration of DA D1 agonists to unilaterally 6-hydroxy-DA denervated rats causes the appearance of a population of aspartate positive neurons on the ipsilateral side (3). These neurons seem to represent a novel subpopulation of striatal intemeurons, but whether or not they are the source of aspartate released by CCK is not known. There is also evidence for interaction between CCK and glutamate. Thus, it has been shown that glutamate causes dephosphorylation and inactivation of the phosphatase inhibitor DARPP-32, that is has an antagonistic action to dopamine on the medium spiny striatal projection neurons, which express DARPP-32. More recently it has been shown that also CCK counteracts forskolin-induced DARPP-32 phosphorylation, and that this effect is dependent on NMDA receptors (4). It is therefore possible that CCK enhances glutamate release from cortico-striatal neurons. Alternatively, this CCK effect may be mediated by aspartate released from striatal interneurons in agreement with the above mentioned results. Finally, we have explored a possible role of endogenously released CCK with the help of CCKB antagonists. The results

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show that CCK may be involved in exploratory behaviour exhibited by rats in a novel environment (unhabituated rats). Furthermore, CCK seems to have a suppressant effect on locomotion elicited by phencyclidine and MK-801, an effect mediated via CCKB receptors. Taken together these results suggest multiple sites of involvement of CCK in basal ganglia and cortical functions, and support the view of antagonistic actions between the nigro-striatal DA and the cortico-striatal glutamatergic systems with CCK involved in both instances. It is possible that d~rugs acting on CCK-ergic transmission may be beneficial for treatment of diseases such as schizophrenia.

References [I] E Morino, F. Mascagni, A. McDonald and T. H~kfelt (1994) Cholecystokinin corticostdatal pathway in the rat: evidence for bilateral origin from medial prefrontal cortical areas. Neuroscience 4, 939-952. [12] Z-B. You, M. Herrera-Marscbitz, E. Penersson, I. Nylander, M. Goiny, H.-Z. Shou, J. Kehr, O. Godukhin et al. (1996) Modulation of neurotJransmitterrelease by cholecystokiuln in the neostriatum and substantia nigra of the rat: Regional and receptor specificity. Neuroscience 74, 793-804. [3] E. Penersson, M. Herrera-Marschitz, R. Rodrigues-Puertas, Z.-Q. Xu, Z.-B. You, J. Hughers, R. R Elde et al. (1996) Evidence for aspartate-immunoreactire neurons in the neostriatum of the rat: modulation by the mesencephalic dopamine pathway via D 1-subtype of receptor. Neuroscience 74, 51-66. [4] G.L. Snyder, G. Fisone, E Morino, V. Gunderson, O.E Onersen, T. H/Skfeltand E Greengard (1993) Regulation by the neuropeptide cholecystokinin (CCK-8S) of protein phosphorylation in the neostriatum. Proc. Natl. Acad. Sci. USA 90. l 1277-11281.

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Pharmacology of the human CRFI, CRF2~ and CRF2~ receptors and CRF binding protein

A. Ardati, J. Gottowik, O. Valdenaire, T. Giller, V. Breu, V. Goetschy, S. Henriot, U. Deuschle, J.-L. Moreau, G.J. Kilpatrick. Pharma Division,

F Hoffmann-La Roche, 4070 Basel, Switzerland Corticotropin releasing factor (CRF) and the related peptide, urocortin, are thought to play an important role in integrating the stress response. CRF has also been implicated in the pathophysiology of several diseases, including anxiety, depression and Alzheimer's disease (see e.g. Chalmers et al., 1996). Receptors for CRF and urocortin have been divided into CRF1 and CRF2 subtypes on the basis of molecular cloning. Additionally, a binding protein with a high affinity for CRF and urocortin exists whose main function is likely to be to terminate the effects of these peptides. In the rat, two isoforms of the CRF2 receptor, termed CRF2a and CRF2~, have been identified but attempts to clone the human CRF2~ receptor had failed (Liaw et al., 1996). However, we have recently been successful in cloning the human version of the CRF2t~ receptor (Valdenaire et al., 1997) and here report on the characterisation of its pharmacology along with the human CRFI and CRF2a receptors and human CRF binding protein. For this characterisation we have developed novel binding assays using [~H]-urocortin and (for the receptors) a luciferase gone reporter system. The human CRFI receptor was cloned from a human cerebellum cDNA library, the human C R F ~ and CRF2~ receptors from human skeletal muscle cDNA and the human CRF binding protein clone was obtained from Research Genetics, Huntsville, USA (IMAGE consortium clone ID #266336). Each of the above were subcloned into the pCEP4 expression vector and transfected independently into HEK293 cells and selected for stable expression. A stable HEK 293 cell iline expressing the luciferase gene under the transcriptional control of multiple cAMP responsive elements was also used to generate stable cell lines expressing the different receptor constructs. Binding assays were conducted using [3H]-urocortin (0,2 nM for the receptors and 0.6 nM for the binding protein). For the receptor assays, bound and free ligand were separated by filtration through glass fibre filters. For the binding protein, bound and free were separated by precipitation with "I~ble 1 Competition for [3H]-Urocortin binding hCRFI pKi hCRF z~ pKi hCRF2ppKi h Urocortin 8.3 8.8 8.6 r,11 CRF 7.9 7.1 7.0 l.lrotensin 1 8.3 7.3 7.3 Sauvagine 7.6 8.0 8.0 CP 154,526 7.9 <5 <5 SC 241 7.5 <5 <5

hCRF BP pICs0 8.0 8.4 8.4 7.4 <6 <6

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Neuropeptide antagonists as drugs in psychiatry

activated charcoal. Data are the means of 3 separate determinations. BP = binding protein. CP 154,526 (N-Butyl-N-[2,5-dimethyl-7-(2,4,6-trimethylphenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]-N-ethylamine); SC 241 (I3-(2-bromo-4-isopropyl-phenyl)-5-methyl-3H-[1,2,3]triazolo[4,5-d] pyrimidin-7-yl]-bis-(2-methoxy-ethyl)-amine). Table 2 Response of the luciferase reporter cells hCRFI pD2 hCRF2apD2 h Urocortin 10.1 10.0 r/hCRF 10.1 9.7 Urotensin 1 10.2 9.5 Sauvagine 10.2 10.3

hCRF2#pD2 9.5 9.2 9.6 10.8

Results are the means of 3 separate determinations. Similar levels of maximal response were obtained for each peptide. ND = not done [3H]-Urocortin proved to be a useful ligand for the CRF receptors and CRF binding protein. In each case specific binding was approximately 60-70% of total. The hCRF2# receptor had a very similar pharmacological profile to the h C R F ~ receptor with urocortin and sauvagine having the highest affinities and the non-l~eptide compounds, CP 154,526 and SC 241 having no activity (up to 10-aM). The four CRF-related peptides and the two non-peptide compounds, CP 154,526 and SC 241 had a similar high affinity for the hCRFI receptor. CRF and urotensin had the highest affinity for the hCRF binding protein The non-peptide compounds had no activity at this site. Maximal stimulation of luciferase production by CRF was approximately 5-7 fold basal levels for each receptor. The maximal stimulation by urocortin, urotensin and sauvagine was similar to that observed with CRF. The potency of the peptides was higher than observed in the binding assays. A similar rank order of activity was maintained although, in relative terms, sauvagine was more potent. The following conclusions can be drawn from these studies: (1) In a recombinant system, the human CRF2~ receptor is functionally active and has a pharmacological profile similar to the CRF2~ receptor. (2) Like the CRF2a receptor, urocortin may be the natural ligand for the CRF2a receptor since it has a higher affinity and potency than CRE (3) The non-peptide compounds, CP 154,526 and SC 241 are highly selective for the CRF1 receptor with no measurable activity at the CRF2 receptors or the CRF binding protein. (4) [3H]-Urocortin binding and the cAMP responsive luciferase reporter gene cell lines are useful tools for the characterisation of CRF receptors and binding protein.

References Chalmers, D.T., Lovenberg "KW.,Grigoriadis D+E.,Behan D.P. and De Souza E.B. (1996) Corticotropin-releasing factor receptors: from molecular biology to drug discovery. Trends Pharmacol. Sci. 12: 166-172. Liaw, C.W., Lovenberg, T.W., Barry, G., Oltersdoff, T., Grigoriadis, D.E. and De Souza, E.B. (1996) Cloning and characterisation of the human corticotropinreleasing factor-2 receptor complementary deoxyribonucleic acid. Endocrinol. 137: 72-77. Valdenaire, O., Giller, T., Breu, V., Gottowik, J. and Kilpatrick, G. (1997) A new functional splice variant of the human receptor for cordcotropin-releasing lhctor (CRF2). Biochem. Biophys. Acta (in press)

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Galanin receptors and their functional characterization

S.O. Ogren, J. Kehr, P.A. Schttt, K. Snitt. Department of Neuroscience, Karolinska Institute, S-171 77 Stockholm, Sweden Galanin (GAL), a 29 amino acid neuropeptide, has a widespread anatomical distribution in the central (CNS) and peripheral (PNS) nervous system. A variety of physiological actions in the CNS (feeding, analgesia, release of pituitary hormones) and in the PNS (insulin release, smooth muscle contraction) has been associated with GAL. These effects appear to be mediated by multiple types of GAL receptors. Recent cloning studies have established the deduced aminoacid sequence of a receptor named GAL-RI. In recent years evidence has been provided for a potential role for GAL in learning and memory. Several studies in the rat suggest that GAL may play an inhibitory role in various learning tasks by stimulation of GAL receptors which results in reduced acetylcholine (ACh) transmission (Crawley, 1996). It has been hypothesised that GAL may have

an inhibitory role in the basal cholinergic forebrain neurones which are damaged in Alzheimer's disease. The present paper will focus on the interactions between GAL and ACh in the basal forebraln neurones of the rat and the potential role of GAL receptors in cognition. GAL has been shown to coexist with ACh in the medial septum and diagonal band of Broca within a population of ACh neurones which project mainly to the ventral hippocampus (HPC) (the septohippocampal pathway). This projection is believed to play a role in spatial learning in the rat. A high concentration of GAL-binding sites is also found in the forebrain areas with a high distribution of GAL terminals (entorhinat cortex, septal nuclei, hippocampus, amygdala). The role of ventral hippocampal GAL in spatial learning has been investigated in the male rat using a spatial learning task, the Morris swim maze. In addition, the role of functional subtypes of GAL receptors was analyzed by the use of the putative GAL receptor antagonist M35 which is a chimeric peptide [galanin(l- 13)-hradykinin(2-9)-amide]. Daily bilateral microinjections of porcine GAL (1.5 nmol/side for 5 days) via chronic cannulae placed in the ventral HPC (i.v.h.) produced a significant impairment of acquisition of the spatial task, while the l nmol dose of GAL facilitated acquisition (Ogren et al., 1996). The 6 nmol dose of GAL failed to affect performance. A trend for an impairment of long-term memory retention (examined seven days after the last training session) was observed after 3 nmol of GAL, while the 1 nmol dose facilitated retention performance. These results indicate that the effects of GAL on cognition are not strictly dose-dependent. The acquisition impairment induced by GAL (3 nmol/rat) was completely blocked by M35 (1 nmol/rat) suggesting an involvement of functional GAL receptors. The effects of GAL on both basal and scopolamine-evoked release of ACh were assessed in the ventral HPC using in vivo microdialysis technique. In freely moving rats, GAL (3 nmol/10 /zl) microinjected into the lateral ventricle (i.c.v.) did not affect basal ACh release (0gren et al., 1996). In contrast, perfusion (100 min) with GAL (0.1 nmol or 0.3 nmol/1.25 ~l/min) through the ventral hippocampal probe resulted in a dose-dependent reduction of basal ACh release. The decrease in ACh release caused by GAL (0.3 nmol) was fully blocked by M35 (0.1 nmol//zl). GAL given i.c.v. (3 nmol/10 /zl) or through the probe (0.3 nmol/l+25 ~l/min) attenuated the increase in scopolamine (0.1 mg/kg, s.c.; 0.001 nmol/1.25 /zl/min through the probe) evoked ACh release. The GAL plus scopolamine combination produced a 50% lower increase of the extracellular ACh concerttrations than scopolamine alone. This suggests that the mechanism(s) behind the suppression of scopolamineand basal ACh release probably are the same (Ogren et at., 1996). Since the above results are based on the use of exogenously applied GAL the in vivo distribution and kinetics of injected GAL was compared with the effects on spatial learning. Such an information is critical since the GAL-R1 receptor type is mairdy found in the ventral HPC, while other GAL receptors may be found elsewhere, e.g. hypothalamus. The use of immunohistochemical techniques, with an antibody raised against porcine GAL to visualize GAL-like imnmnoreactivity (GAL-ir), displayed that GAL-ir following GAL i.v.h, injection wa~scontained mainly within the HPC and did not spread to the hypothalamus (Sch6tt et al., 1997). GAL-ir was rapidly cleared from the extracellular space between 5 and 20 min after the injection. Five min after infusion of G A L a number of cells in the ventral HPC both within and outside the zone of extracellularly located GAL showed a positive GAL-ir. These cells appear morphologically to be middle sized neurones with a similar position as cells showing NPY-like ir. At 20 and 60 min after infusion of GAL no cells with detectable levels of GAL-ir could be seen. Thus, also intracellular events must be considered when analysing the action of GAL. Bilateral microinjections of GAL (3 nmol/rat) i.v.h., as above, produced a significant impairment of acquisition of the spatial learning task, when injected 20 min hut not 5 min before the start of the daily training session. A trend for impairment was seen in animals injected 60 min before training. No impairment of memory retention (examined 24 h after the last training session) was observed in the GAL-treated animals. These findings indicate that the peak concentrations of extracelhilar GAL and the activation of putative GAL-membrane receptors are not strictly connected with the learning performance. These results suggest that GAL infused into the ventral HPC inhibits spatial learning and powerfully reduces ACh transmission via stimulation of GAL-receptors which probably are of the GAL-R1 type. The results also indicate that GAL-receptors within the ventral HPC have a powerful